Epithelial Immunomodulation by Giardia

University of Calgary

PRISM: University of Calgary's Digital Repository

Graduate Studies The Vault: Electronic Theses and Dissertations

2015-09-25

Epithelial Immunomodulation by Giardia

Cotton, James

Cotton, J. (2015). Epithelial Immunomodulation by Giardia (Unpublished doctoral thesis).

University of Calgary, Calgary, AB. doi:10.11575/PRISM/25927

http://hdl.handle.net/11023/2507

doctoral thesis

University of Calgary graduate students retain copyright ownership and moral rights for their

thesis. You may use this material in any way that is permitted by the Copyright Act or through

licensing that has been assigned to the document. For uses that are not allowable under

copyright legislation or licensing, you are required to seek permission.

Downloaded from PRISM: https://prism.ucalgary.ca

UNIVERSITY OF CALGARY

Epithelial Immunomodulation by Giardia

By

James Anthony Cotton

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

GRADUATE PROGRAM IN BIOLOGICAL SCIENCES

CALGARY, ALBERTA

AUGUST, 2015

© James A. Cotton 2015

ii

ABSTRACT

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a non-invasive, protozoan parasite

of the upper small intestine of animals, including humans. Despite parasite loads exceeding 106

trophozoites per cm of small intestine, the intestinal mucosa in the majority of the Giardia-

infected individuals are devoid of signs of overt inflammation. Human studies also suggest

Giardia infections are capable of modulating the development of diarrheal disease in their host

via unknown mechanisms.

The first part of this study focused on whether Giardia infections were capable of

attenuating neutrophil (PMN) accumulation in different experimental models of inflammation.

Accumulation of PMNs is an archetypal event during many acute intestinal inflammatory

responses contributing to the development of diarrheal disease. Our findings demonstrated

Giardia infections attenuate granulocyte infiltration in an experimental model of colitis, in an

isolate-dependent manner. Giardia infections also decrease tissue expression of mediators

associated with PMN recruitment during in vivo colitis. Similar results were observed when

Giardia trophozoites were incubated ex vivo with inflamed mucosal biopsy tissues collected

from the descending colon of patients with Crohn’s disease.

The second part of this study focused on identifying mechanisms by which Giardia

trophozoites attenuate PMN accumulation. The intestinal epithelium is a single layer of polarized

cells separating the external environment from underlying host tissues. In response to pro-

inflammatory stimuli, this structure releases the PMN chemokine interleukin-8 (CXCL8) that

recruits PMNs to the basolateral membrane of the intestinal epithelium. We demonstrated that

Giardia trophozoites attenuate CXCL8 secretion from ex vivo small intestinal mucosal biopsy

tissues and in vitro Caco-2 monolayers, following administration of IL-1β or Salmonella sp.; this

involved parasite-mediated degradation of CXCL8 and attenuation of CXCL8- and C5a-induced

PMN chemotaxis. Genetic assemblages of Giardia capable of infecting humans contain unique

cathepsin proteases and Giardia trophozoties release cathepsin proteases into supernatants.

Inhibition of Giardia cathepsin B proteases prevented parasite-mediated degradation of CXCL8

and attenuation of CXCL8 and C5a-induced PMN chemotaxis.

Our findings demonstrate that Giardia infections attenuate PMN accumulation in an in vivo

model of colitis. Research done in vitro highlights a role for Giardia cathepsin B proteases in

degrading CXCL8 and attenuating CXCL8-induced PMN chemotaxis.

iii

PREFACE

Work from this thesis has contributed to the following publications:

A Bhargava, Cotton JA, Dixon BR, Yates RM, Buret AG. Giardia duodenalis surface cysteine

proteases induce cleavage of intestinal epithelial cytoskeletal villin via myosin light chain kinase.

PLoS One (in press)

Cotton J.A., J.P. Motta, L.P. Schenck, S.A. Hirota, P.L. Beck, A.G. Buret. 2014. Giardia

duodenalis infection reduces granulocyte infiltration in an in vivo model of bacterial toxin-

induced colitis and attenuates inflammation in human intestinal tissue. PLoS One. 9(10):e109087

Cotton J.A., A. Bhargava, J.G. Ferraz, R.M. Yates, P.L. Beck, A.G. Buret. 2014. Giardia

duodenalis cathepsin B proteases degrade interleukin-8 and attenuate interleukin-8-induced

neutrophil chemotaxis. Infection and Immunity. 82(7):2772-2787

Cotton J.A., J.K. Beatty, and A.G. Buret. 2011. Host parasite interactions and pathophysiology

in Giardia infections. International Journal for Parasitology, Volume 41, Issue 9, Pages 925-933

Buret, A.G., and Cotton, J. 2010. Pathophysiological processes and clinical manifestations of

giardiasis, IN: LUJAN, H.D., SVARD, S., Giardia and giardiasis, Springer-Verlag, Wien and

New York, Section IV, 301-318

iv

ACKNOWLEDGMENTS

The first person I need to thank is my supervisor Dr. Andre Buret. His dedication to his

students and their future success, and the boundless energy and enthusiasm he displays for

teaching and research have made him an exceptional role model. The mentorship and

opportunities Andre has provided led me to discover my own passion for research and realize my

own potential. It has been a life-changing experience. Andre, thank you.

I also need to thank Dr.’s Paul Beck, Wallace MacNaughton, and Lash Gedamu for

serving as my supervisory committee. Thank you all for your open door policy and willingness

to meet and discuss my research project. Your advice was always extremely helpful and greatly

appreciated. I would also like to thank Dr.’s Doug Morck, John Gilleard, and Patrick Hanington

for serving on PhD defence committee.

During my time in the lab, I’ve had the fortune of working with some exceptional people.

To all past and present Buret lab members, thank you for all the help and support you have

provided me over the years. Somehow my constant grumblings about being overly hungry or

sleepy did not drive you completely insane (maybe it did a little). I felt fortunate working with all

of you on a daily basis, and the lab felt like an extended family. Thanks to all of you both good

and bad days were made better by your company. I’m going to miss working with all of you.

You’re all outstanding people and I wish nothing but the best in your future endeavours. Several

of you will be joining me in July. I look forward to reminiscing about past lab experiences as we

start the next chapter of our lives.

I am extremely grateful of the support from several of my peers in the Leaders in

Medicine program. You’re all wonderful people and I look forward to seeing you in school in

July!

Thank you, to the Natural Sciences and Engineering Research Council of Canada,

Alberta Innovates Health Solutions, and the Crohn’s and Colitis Foundation of Canada for their

financial support.

Finally, I would like to thank everyone in my family. Their continued love and support

have led to me to where I am today.

v

TABLE OF CONTENTS

1.0 Introduction ....................................................................................................................1

1.1 GIARDIA DUODENALIS ..............................................................................................1 1.1.1 Giardia pathophysiology .........................................................................................2 1.1.2 Giardia assemblages ...............................................................................................2 1.1.3 Giardia parasite factors ...........................................................................................4 1.1.4 Host factors .............................................................................................................6

1.2 GIARDIA AND INFLAMMATION .............................................................................7

1.3 NEUTROPHILS AND ACUTE INTESTINAL INFLAMMATION............................9

1.3.1 Neutrophil chemoattractants .................................................................................10 1.3.2 Bone marrow egression ........................................................................................11

1.3.3 Transendothelial migration ...................................................................................12 1.3.4 Tissue migration ...................................................................................................13 1.3.5 Intestinal transepithelial migration .......................................................................14

1.3.6 Pathophysiology of neutrophils ............................................................................15

1.4 THE INTESTINAL EPITHELIUM AND NEUTROPHIL RECRUITMENT ...........18

1.4.1 Control of intestinal epithelial CXCL8 secretion .................................................19

1.5 PARASITE IMMUNOMODULATORY FACTORS .................................................20

1.5.1 Cathepsin cysteine proteases ................................................................................20 1.5.2 Parasite cathepsin cysteine proteases ....................................................................21

1.6 SUMMARY .................................................................................................................22

1.7 HYPOTHESIS AND OBJECTIVES ...........................................................................23 2.0 Materials and Methods .................................................................................................24

2.1 ETHICS STATEMENT ...............................................................................................24

2.2 REAGENTS .................................................................................................................24

2.3 HUMAN BIOPSY TISSUES AND CELL LINES .....................................................25

2.4 PARASITES ................................................................................................................26

2.5 SALMONELLA TYPHIMURIUM ...............................................................................26

2.6 GIARDIA TROPHOZOITE ISOLATION ...................................................................27

vi

2.7 GIARDIA-MEDIATED ATTENUATION OF INTESTINAL EPITHELIAL CXCL827 2.7.1 Cytokine analysis ..................................................................................................28

2.8 GIARDIA MODULATION OF CLOSTRIDIUM DIFFICILE TOXIN-INDUCED

COLITIS ...................................................................................................................29 2.8.1 Giardia in vivo infection .......................................................................................29

2.8.2 Clostridium difficile toxin-induced colitis ............................................................29 2.8.3 Assessment of colonic cytokine protein levels .....................................................30 2.8.4 Tissue MPO assay .................................................................................................30 2.8.5 Immunohistochemistry .........................................................................................31

2.9 INTESTINAL EPITHELIAL CXCL8 MRNA EXPRESSION ..................................32

2.10 PROTEIN ANALYSIS AND MODELING ..............................................................33

2.11 NUCLEOTIDE SEQUENCE GENBANK ACCESSION NUMBERS ....................33 2.11.1 Cathepsin B GenBank accession numbers ..........................................................33 2.11.2 Cathepsin L GenBank accession numbers ..........................................................34

2.12 VISUALIZATION OF GIARDIA CATHEPSIN PROTEASES ...............................34

2.13 CATHEPSIN CYSTEINE PROTEASE INHIBITION .............................................35

2.13.1 Caco-2 supernatant pre-treatment .......................................................................35 2.13.2 Inhibition of Giardia trophozoite cathepsin proteases ........................................35

2.14 GIARDIA VIABILITY ASSAYS ..............................................................................36

2.15 CATHEPSIN CYSTEINE PROTEASE ACTIVITY ASSAYS ................................36

2.16 GIARDIA MODULATION OF CXCL8-INDUCED PMN CHEMOTAXIS ............37 2.16.1 Chemotactic supernatant generation ...................................................................37 2.16.2 PMN isolation and chemotaxis assay .................................................................37

2.17 MYELOPEROXIDASE ASSAY ..............................................................................38

2.18 WESTERN BLOTTING ............................................................................................38

2.19 STATISTICS .............................................................................................................39 3.0 Results ..........................................................................................................................40

3.1 IN VIVO GIARDIA INFECTIONS ATTENUATE GRANULOCYTE INFILTRATION

IN AN ISOLATE-DEPENDENT MANNER ...........................................................40

3.2 GIARDIA NF INFECTIONS ATTENUATE COLONIC EXPRESSION OF SEVERAL

NEUTROPHIL-ASSOCIATED MEDIATORS .......................................................47

vii

3.3 GIARDIA INFECTIONS MODULATE COLONIC EXPRESSION OF SEVERAL PRO-

INFLAMMATORY MEDIATORS..........................................................................50

3.4 GIARDIA TROPHOZOITES ATTENUATE EXPRESSION OF INFLAMMATORY

MEDIATORS FROM INFLAMED EX VIVO HUMAN MUCOSAL BIOPSY

TISSUES ...................................................................................................................57

3.5 GIARDIA TROPHOZOITES ATTENUATE INTESTINAL EPITHELIAL CXCL8 90 3.5.1 Giardia NF trophozoites attenuate IL-1β-induced CXCL8 secretion from ex vivo

small intestinal mucosal biopsy tissues..................................................................90 3.5.2 Giardia trophozoites attenuate IL-1β-induced CXCL8 secretion from in vitro Caco-2

monolayers .............................................................................................................91

3.6 GIARDIA TROPHOZOITES ATTENUATE SALMONELLA TYPHIMURIUM-

INDUCED CXCL8 SECRETION FROM IN VITRO CACO-2 MONOLAYERS102

3.7 GIARDIA TROPHOZOITES ATTENUATE IL-1Β-INDUCED CXCL8 SECRETION

VIA A CASPASE-3 INDEPENDENT MECHANISM .........................................102

3.8 ATTENUATION OF CXCL8 BY GIARDIA TROPHOZOITES INVOLVES

PARASITE-MEDIATED DEGRADATION OF CXCL8 .....................................105

3.9 GIARDIA TROPHOZOITES RELEASE FACTORS THAT ATTENUATE PMN

CHEMOTAXIS ......................................................................................................113

3.9.1 Giardia trophozoites attenuate CXCL8-induced PMN chemotaxis ....................116 3.9.2 Giardia trophozoites attenuate C5a-induced PMN chemotaxis ..........................122

3.10 BIOINFORMATICS OF GIARDIA CATHEPSIN CYSTEINE PROTEASES ......127

3.11 VISUALIZATION OF GIARDIA INTRA-TROPHOZOITE CATHEPSIN

PROTEASES ..........................................................................................................132

3.12 ASSESSMENT OF SECRETED GIARDIA CATHEPSIN CYSTEINE PROTEASES

.................................................................................................................................138

3.13 APICAL TO BASOLATERAL MIGRATION OF GIARDIA CATHEPSIN CYSTEINE

PROTEASES ..........................................................................................................148

3.14 BROAD-SPECTRUM INHIBITION OF SUPERNATANT CATHEPSIN PROTEASES

.................................................................................................................................151

3.15 GIARDIA CATB PROTEASES DEGRADE CXCL8 .............................................158

3.16 INHIBITION OF GIARDIA CATHEPSIN B PROTEASES...................................158

viii

3.17 GIARDIA CATB PROTEASES ATTENUATE CXCL8- AND C5A-INDUCED PMN

CHEMOTAXIS ......................................................................................................161 4.0 Discussion ..................................................................................................................171

4.1 SUMMARY ...............................................................................................................171

4.2 GIARDIA ATTENUATES GRANULOCYTE INFILTRATION IN VIVO ..............172

4.2.1 Modulation of diarrheal disease by Giardia ........................................................173

4.3 GIARDIA INFECTIONS ATTENUATE EXPRESSION OF PRO-INFLAMMATORY

MEDIATORS .........................................................................................................176 4.3.1 Attenuation of acute phase response proteins .....................................................176

4.3.2 Attenuation of CCL chemokines and pro-inflammatory cytokines ....................177

4.4 GIARDIA ATTENUATES INTESTINAL EPITHELIAL CXCL8 ...........................178

4.5 GIARDIA CATHEPSIN PROTEASES .....................................................................181 4.5.1 Specificity of Giardia cathepsin proteases ..........................................................182

4.6 GIARDIA IMMUNOMODULATORY MOLECULES ................................................183

4.7 CONCLUSION ..........................................................................................................186

4.8 FUTURE DIRECTIONS ...........................................................................................187

5.0 References ..................................................................................................................188 6.0 Appendix ....................................................................................................................208

6.1 PURIFICATION OF CATHEPSIN CYSTEINE PROTEASE ACTIVITY .............208

6.2 ANALYSIS OF GIARDIA CATHEPSIN CYSTEINE PROTEASES ......................208

ix

List of Figures

Figure 1. Ad libitium administration of broad-spectrum antibiotics to mouse drinking water

resulted in effective small intestinal colonization by Giardia NF and GS/M trophozoites. . 41

Figure 2. Giardia NF infections reduce and GS/M infections do not affect colonic

myeloperoxidase activity following rectal administration of TcdAB. .................................. 43


myeloperoxidase activity following rectal administration of TcdAB. .................................. 45

Figure 4. In vivo Giardia NF infections attenuate the colonic expression of several PMN-

associated mediators following rectal administration of 100 µg TcdAB. ............................ 48

Figure 5. In vivo Giardia NF infections attenuate the colonic expression of several pro-

inflammatory mediators following rectal administration of 100 µg TcdAB. ....................... 51

Figure 6. In vivo Giardia NF infections do not attenuate colonic expression of several pro-

inflammatory mediators upregulated following rectal administration of 100 µg TcdAB. ... 53

Figure 7. In vivo Giardia NF infections do not modulate colonic expression of several

inflammatory mediators unaffected by i.r. administration of 100 µg TcdAB. ..................... 55

Figure 8. In vivo Giardia GS/M infections enhance colonic expression of several

inflammatory mediators upregulated by i.r. administration of 100 µg TcdAB. ................... 58



Figure 10. In vivo Giardia GS/M infections do not modulate colonic expression of two

inflammatory mediators upregulated by i.r. administration of 100 µg TcdAB. ................... 62

Figure 11. In vivo Giardia GS/M infections do not modulate colonic expression of


Figure 12. Giardia NF trophozoites attenuate supernatant levels of several PMN-associated

mediators from descending colon mucosal biopsy tissues. .................................................. 67

Figure 13. Giardia NF trophozoites attenuate CXCL8 tissue homogenate levels from

descending colon mucosal biopsy tissue homogenates......................................................... 69

Figure 14. Giardia NF trophozoites attenuate supernatant levels of several chemokines from

descending colon mucosal biopsy tissues. ............................................................................ 71

Figure 15. Giardia NF trophozoites do not attenuate tissue homogenate levels of several

chemokines from descending colon mucosal biopsy tissues. ............................................... 73

x

Figure 16. Giardia NF trophozoites attenuate supernatant levels of several inflammatory

mediators from descending colon mucosal biopsy tissues. .................................................. 76

Figure 17. Giardia NF trophozoites attenuate tissue homogenate levels of several pro-

inflammatory mediators from descending colon mucosal biopsy tissues. ............................ 79

Figure 18. Giardia NF trophozoites do not attenuate supernatant levels of several




Figure 20. Giardia NF trophozoites do not attenuate supernatant levels of several growth

factors from descending colon mucosal biopsy tissues. ....................................................... 86


growth factors released from descending colon mucosal biopsy tissues. ............................. 88

Figure 22. Giardia NF trophozoites attenuate CXCL8 from small intestinal mucosal biopsy

tissues. ................................................................................................................................... 92

Figure 23. Giardia NF trophozoites attenuate IL-1β-induced CXCL8 secretion from in vitro

Caco-2 monolayers in a dose-dependent manner. ................................................................ 95

Figure 24. Assemblage A and B Giardia trophozoites attenuate IL-1β-induced CXCL8

secretion from in vitro Caco-2 monolayers. ......................................................................... 97

Figure 25. Attenuation of supernatant CXCL8 induced by IL-1β in Caco-2 monolayers does

not require direct contact between Giardia NF trophozoites and in vitro Caco-2

monolayers. ......................................................................................................................... 100

Figure 26. Giardia NF trophozoites attenuate Salmonella-induced CXCL8 secretion from in

vitro Caco-2 monolayers. .................................................................................................... 103

Figure 27. Giardia NF trophozoites attenuate IL-1β-induced CXCL8 secretion via a caspase-

3-independent mechanism. ................................................................................................. 106

Figure 28. Giardia trophozoites attenuate IL-1β-induced CXCL8 secretion via a post-

transcriptional mechanism. ................................................................................................. 109

Figure 29. Giardia trophozoites attenuate CXCL8 via parasite-mediated degradation of

CXCL8. ............................................................................................................................... 111

Figure 30. Giardia NF and GS/M trophozoites do not degrade IL-1β. ...................................... 114

xi

Figure 31. Giardia NF trophozoites attenuate CXCL8-induced PMN chemotaxis and degrade

CXCL8. ............................................................................................................................... 117

Figure 32. Giardia WB trophozoites attenuate CXCL8-induced PMN chemotaxis and

degrade CXCL8. ................................................................................................................. 120

Figure 33. Giardia NF and GS/M trophozoite sonicates fail to attenuate CXCL8-induced

PMN chemotaxis. ................................................................................................................ 123

Figure 34. Giardia NF trophozoites attenuate C5a-induced PMN chemotaxis. ......................... 125

Figure 35. ClustalW alignment of catB proteases. ..................................................................... 128

Figure 36. Phylogenetic tree construction of parasite catB cysteine proteases. ......................... 130

Figure 37. ClustalW alignment of cathepsin L cysteine proteases. ............................................ 133

Figure 38. Phylogenetic tree construction of parasite catL cysteine proteases........................... 136

Figure 39. Giardia trophozoites express proteases capable of degrading cathepsin fluorogenic

substrate ZFR-AMC. ........................................................................................................... 139

Figure 40. Giardia NF trophozoites increase supernatant cathepsin B/L supernatant activity

in a dose-dependent manner. ............................................................................................... 142

Figure 41. Assemblage A and B Giardia trophozoite isolates release cysteine proteases into

cell supernatants in the presence or absence of in vitro Caco-2 monolayers that degrade

ZFR-AMC. .......................................................................................................................... 144



ZRR-AMC. ......................................................................................................................... 146

Figure 43. Giardia NF cathepsin cysteine protease activity translocates across in vitro

intestinal epithelial Caco-2 monolayers following exposure to Salmonella typhimurium. 149

Figure 44. Giardia trophozoites secrete cysteine proteases that are inhibited by E-64d. ........... 152

Figure 45. Pre-treatment of Caco-2 supernatants with the cathepsin B-specific inhibitor Ca-

074Me does not affect trophozoite viability. ...................................................................... 154

Figure 46. Giardia trophozoites secrete cathepsin cysteine proteases susceptible to inhibition

with Ca-074Me. .................................................................................................................. 156

Figure 47. Giardia cathepsin B proteases degrade interleukin-8. .............................................. 159

xii

Figure 48. Pre-treatment of Giardia NF trophozoites with Ca-074Me does not affect

trophozoite viability. ........................................................................................................... 162

Figure 49. Pre-treatment of Giardia NF trophozoites with Ca-074Me inhibits intra-

trophozoite cathepsin activity. ............................................................................................ 164

Figure 50. Inhibition of cathepsin B activity in Giardia NF trophozoites prevents attenuation

of interleukin-8-induced neutrophil chemotaxis. ................................................................ 167

Figure 51. Inhibition of cathepsin B activity in Giardia NF trophozoites prevents attenuation

of C5a-induced neutrophil chemotaxis. .............................................................................. 169

xiii

LIST OF ABBREVATIONS

5’-AMP 5’-adenosine monophosphate

ADI Arginine deiminase

AJC Apical junctional complex

AP-1 Activator protein-1

BLAST Basic local alignment search tool

Ca-074 L-3-trans-(Propylcarbamoyl)Oxirane-2-Carbonyl)-L-Isoleucyl-L-Proline

CAR Coxsackie adenovirus receptor

catB Cathepsin B

catL Cathepsin L

CCL2 Monocyte chemotactic protein-1

CCL3 Macrophage inflammatory protein 1α

CCL4 Macrophage inflammatory protein 1β

CCL5 Regulated on activated, normal T cell expressed and secreted

CCL7 Monocyte chemotactic protein 3

CCL10 Macrophage inflammatory protein 1γ

CCL11 Eotaxin-1

CCL22 Macrophage-derived chemokine

CD Crohn’s disease

CTX Cortical thymocyte

CRP C-reactive protein

CXCL1 KC/Growth related oncogene-α

CXCL2 Macrophage inflammatory protein 2α/Growth related oncogene β

CXCL3 Growth related oncogene γ

CXCL8 Interleukin-8

CXCL9 Monokine induced by γ interferon

CXCL10 Interferon gamma induced protein 10

CX3CL1 Fractalkine

DAPI 4’,6-diamidino-2-phenylindole

DC Dendritic cell

DMSO Dimethyl sulphoxide

DTT Dithiothreitol

E-64 (2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane

EDTA Ethylenediaminetetraacetic acid

FGF Fibroblast growth factor

Flt-3L FMS-like tyrosine kinase 3

fMLF N-formylmethionyl-leucyl-phenylalanine

FBS fetal bovine serum

G-CSF Granulocyte colony stimulating factor

GM-CSF Granulocyte monocyte colony stimulating factor

GRO Growth-related oncogene

HIF Hypoxia inducible factor

IBD Inflammatory bowel disease

xiv

ICAM Intracellular adhesion molecule

IgA Immunoglobulin A

IEC Intestinal epithelial cell

IFNα Interferon α

IFNγ Interferon γ

IL Interleukin

IL-1α Interleukin-1α

IL-1β Interleukin-1β

IL-1ra Interleukin-1 receptor antagonist

IL-2 Interleukin-2

IL-5 Interleukin-5

IL-6 Interleukin-6

IL-7 Interleukin-7

IL-9 Interleukin-9

IL-10 Interleukin-10





i.r. Intra-rectal

JAM Junctional adhesion molecule

kDa Kilodalton

LIF Leukocyte inhibitory factor

MAPK Mitogen activated protein kinase

MEME Minimum Essental Medium Eagle

MLCK Myosin light chain kinase

MPO Myeloperoxidase

MOI Multiplicity of infection

NF-κB Nuclear factor κB

NET Neutrophil extracellular trap

NO Nitric oxide

PAR Proteinase activated receptor

PDGF-AA Platelet-derived growth factor AA

PDGF-BB Platelet-derived growth factor BB

PBS Phosphate buffered saline

p.i. Post-infection

PI3K Phosphatidylinositol 3-kinase

PMN Polymorphonuclear leukocyte or neutrophil

RFU Reflective light unit

sCD40L Soluble CD40 ligand

SDS Sodium dodecyl sulphate

TBS Tris-buffered saline

TcdA Clostridium difficile toxin A

TcdB Clostridium difficile toxin B

xv

TEM transepithelial migration

TGFα Transforming growth factor α

Th1 Helper T type 1

Th2 Helper T type 2

TLR Toll-like receptor

TNBS 2,4,6-Trinitrobenzensulphonic acid

TNFα Tumor necrosis factor α

TNFβ Tumor necrosis factor β

VEGF Vascular endothelial growth factor

VSP Variant surface protein

ZFR-AMC Benzyloxycarbonyl-L-Phenylalanyl-L-Arginine 4-Methyl-Coumaryl-7-Amide

ZRR-AMC Benzyloxycarbonyl-L-Arginine-L-Arginine 4-Methyl-Coumaryl-7-Amide

1

1.0 Introduction

1.1 Giardia duodenalis

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a non-invasive protozoan parasite

of the upper small intestine and a common cause of waterborne diarrheal disease in a wide

variety of species, including humans (1, 2). The parasite is so common worldwide that it was

recently included in the World Health Organization (WHO) Neglected Disease Initiative (1).

Parasite transmission typically occurs following ingestion of infectious cysts via the

consumption of contaminated food or water, or through direct fecal-oral route (reviewed in (3)).

For reasons that remain obscure, Giardia-infected individuals present with a spectrum of

symptoms ranging from asymptomatic carriage to acute or chronic diarrheal disease. Host

immune status plays a key role in the variable manifestations of giardiasis (4). Giardia infections

tend to be self-limiting in individuals with competent immune systems. Immunocompromised

people are at increased risk of developing chronic giardiasis, and yet the latter is also common in

immunocompetent individuals. In the majority of human Giardia infections, symptoms typically

include diarrhea, bloating, abdominal pain, nausea, and vomiting. These symptoms may also be

associated with anorexia and failure to thrive. Indeed, Giardia infections may have detrimental

effects on body weight in humans, food-producing animals, and pets (1, 2, 5, 6). Although the

majority of individuals do not develop signs of overt intestinal inflammation, a rare subset of

chronically infected patients may develop “microscopic duodenal inflammation” (7-9). While

these patients may continue to have diarrheal disease, reports suggest that some individuals may

also become constipated (7, 8). In addition to the above-mentioned symptoms, there is now

evidence that suggests Giardia-infected individuals may also develop post-infectious disorders,

2

such as post-infectious irritable bowel syndrome or post-infectious chronic fatigue syndrome

(reviewed in (10)).

1.1.1 Giardia pathophysiology

The biology of Giardia infections remains incompletely understood. At the height of

Giardia infections, millions of trophozoites closely associate with the apical surface of the small

intestinal epithelium and induce pathophysiological responses that result in a largely

malabsorptive diarrheal disease (reviewed in (11)). Currently, it is unknown how Giardia

trophozoites transverse the intestinal mucus layer. Giardia trophozoites strongly adhere to the

epithelial surface of the intestine via a ventral adhesive disk. A number of parasitic surface

molecules are engaged in this tight interaction, including giardins (importantly α, β, δ, and γ

giardins), as well as a complex network of contractile proteins, which play key roles in

trophozoite attachment (12-16). While the implication of these molecules in pathogenesis and/or

immunity remains unknown, this tight attachment between Giardia trophozoites and intestinal

epithelial cells triggers a series of events that culminate in diarrheal disease. To date, these

pathophysiological stages are believed to involve heightened rates of enterocyte apoptosis, small

intestinal barrier dysfunction, activation of host lymphocytes, shortening of brush border

microvilli, disaccharidase deficiencies, small intestinal malabsorption, anion hypersecretion, and

increased intestinal transit rates (reviewed in (11)).

1.1.2 Giardia assemblages

Giardia duodenalis exists as eight different genetic “assemblages” that are designated as

assemblage A to H (17, 18). Assemblage A and B isolates are infective to humans and capable of

3

causing symptomatic infection (19-21). It has been proposed that the Giardia genome contains a

core set of genes and genomic differences confer antigenic variation and host specificity (18-21).

There is also ongoing discussion as to whether assemblage A and B Giardia isolates represent

distinct Giardia species. Moreover, completion of the Giardia WB (assemblage A) and Giardia

GS/M (assemblage B) genomes have revealed these isolates contain only 77% nucleotide and

78% amino acid complementarity within protein coding regions (22, 23). The difference between

assemblage A and B protein coding regions creates the potential for assemblage-specific proteins

or parasite factors that can contribute to assemblage-specific Giardia pathogenesis. For example,

α-2 giardin protein is an assemblage A specific protein (24), but the role of this protein in

pathogenesis has yet to be investigated. The potential existence of assemblage-dependent

pathogenic mechanisms in giardiasis represents an important avenue for future investigation. To

date, research in vitro (25, 26) and in vivo (27-29) has demonstrated differences in the

pathogenesis of Giardia isolates, but has failed to indicate whether these differences are

assemblage-specific. Moreover, isolate-dependent Giardia pathogenesis in vivo differs

depending on the selected animal model (27-29). Various reports from around the world have

also tried to correlate assemblage B infections with more severe symptomatology, while others

have correlated more severe symptoms with assemblage A infections. In addition, both

assemblage A and B infections have been reported to cause differing lengths of infection in

healthy individuals (20). Collectively, these results suggest that parasite genotype alone may not

explain differences in symptoms or lengths of infection in healthy individuals (7). Moreover,

additional experiments are required in order to elucidate potential assemblage-specific Giardia

pathology.

4

1.1.3 Giardia parasite factors

Many Giardia parasite factors have been suggested to contribute to various aspects of

disease pathogenesis, but the number of potential parasite factors involved and their mechanisms

of action remain incompletely understood. To date, Giardia parasite molecules are known to

cause pathophysiological events in intestinal epithelial cells (IECs) and modulate aspects of their

host’s immune responses (reviewed in (30)). These factors may be synthesized and/or released

following exposure to host cells. Indeed, exposure to epithelial cells results in significant

changes in Giardia trophozoite gene expression, and the release of several Giardia-produced

metabolic enzymes (31, 32). However, additional research is required in order to identify these

factors as well as determine how they can affect their host.

1.1.3.1 Intestinal epithelial pathophysiology

As Giardia trophozoites are non-invasive, the intestinal epithelium represents a primary

point of contact between parasite and host and, therefore, is also a target of Giardia parasite

factors. Giardia trophozoite attachment to the intestinal epithelium occurs via its ventral

adhesive disk and involves various parasitic factors including surface lectins, giardin proteins,

variant surface proteins (VSPs), and unknown cysteine proteases (15, 31, 33-37). In addition to

promoting intestinal epithelial attachment, several Giardia parasite factors induce further

pathophysiological responses within IECs. An unknown 58 kilodalton (kDa) “enterotoxin”

produced by Giardia trophozoites causes excessive ion secretion and intestinal fluid

accumulation via the induction of several signal transduction pathways in host enterocytes (38).

5

Intestinal epithelial disaccharidase deficiencies have been induced following exposure to parasite

soluble extracts ranging from 32 to 200 kDa in size (39). Activation of caspase-3 and -9, and

potentially myosin light chain kinase (MLCK), in enterocytes occurs following exposure to via

yet to be identified parasitic products (25, 26). In addition to inducing intestinal epithelial

apoptosis, consumption of exogenous arginine via Giardia arginine deiminase (ADI) reduces in

vitro intestinal epithelial cell proliferation (40). Based on these findings, identification of

additional Giardia parasite factors and their effects on intestinal epithelial pathophysiology

represents an important avenue for future Giardia research.

1.1.3.2 Effects on host immunity

In addition to inducing intestinal epithelial pathophysiology, several Giardia parasite

products modulate their host’s immune response or induce specific immune responses. Further to

their role in intestinal epithelial attachment, VSPs promote Giardia antigenic variation and,

resultingly, contribute to evasion from host humoral immune responses. VSPs may also confer

zoonotic infectivity (35) and can be further modified by Giardia ADI (41). Secretion of Giardia

thiol proteinases may also contribute evasion of host humoral immune responses as they have

been shown to cleave human immunoglobulin (IgA) using a proteolytic enzyme assay with

Giardia sonicate preparations (33). In addition to evasion of humoral immunity, Giardia

trophozoites and their products modulate aspects of their host’s cellular immune responses.

Consumption of exogenous arginine by Giardia ADI as an energy source also prevents intestinal

epithelial expression of nitric oxide (NO), a cystostatic molecule to Giardia trophozoites (32,

42). Giardia ADI has also been described to modulate in vitro DC responses to

6

lipopolysaccharide (43); however, these results contrast with LPS-induced cytokine profile of

DCs exposed to Giardia trophozoite sonicates (44). These results suggest additional research is

required in order to elucidate how Giardia trophozoites modulate DC immune responses.

Previous research has demonstrated that Giardia trophozoites induce host immune

responses (4), but, as these studies have produced conflicting results, additional research is

required to elucidate host immune responses to Giardia. Microarray analysis of in vitro human

intestinal epithelial cells co-incubated with Giardia trophozoites resulted in a chemokine profile

expected to chemoattract DC and lymphocyte populations (45); but as of yet, the factors

responsible for the induction of this specific chemokine profile have not been identified. A study

examining the oral administration of excretory/secretory products from Giardia trophozoites to

specific pathogen free BALB/c mice resulted in a Th2-dominant immune response and

eosinophil intestinal infiltration (46). Moreover, induction of Th2-dominant immune responses

during Giardia infections in vivo has been reported to occur in an isolate-dependent manner (28).

However, reports examining immune profiles of human patients infected with Giardia have

produced conflicting results (47, 48). Indeed, the roles that Giardia parasite factors may have in

either inducing or down-regulating specific host immune profiles have yet to be clarified.

1.1.4 Host factors

Although less is known on the role that host responses play in Giardia infections, it is now

established that host responses play a key role in the pathogenesis of giardiasis. Indeed, Giardia

infections result in decreased mucosal surface area for the absorption of nutrients, electrolytes,

and water by shortening microvilli in CD8+ lymphocyte-dependent manner (49, 50). Very few

7

reports have examined the involvement of host factors in the pathogenesis of infection, although

it may be postulated that several other host mechanisms are involved in the pathogenesis of

Giardia infections. For example, the increased activity of sodium glucose co-transporter 1

(SGLT-1) is protective against Giardia-induced apoptosis in vitro (51). As this endogenous

protective mechanism appears to be overridden in giardiasis, research is needed to determine

whether host factors may contribute to the inhibition of this protective SGLT-1 activation (52).

The identification of Giardia parasitic products and host factors implicated in pathophysiology

represents a key field for future Giardia research.

1.2 Giardia and inflammation

At the height of Giardia infections, millions of trophozoites closely associate with the

apical surface of the intestinal epithelium and induce pathophysiological responses that can

culminate in diarrheal disease (reviewed in (11)). With the exception of a small but significant

increase in numbers of intra-epithelial lymphocytes, acute infection with Giardia is not

associated with the infiltration of inflammatory cells, for reasons that remain obscure (49, 53).

This represents a counter-intuitive observation not only in view of the direct presence of large

numbers of parasites, but also because Giardia breaks the epithelial barrier via direct effects on

tight junctional proteins (25, 54-56). Therefore, Giardia infections likely facilitate the

translocation of potent pro-inflammatory luminal antigens to underlying host tissues. Giardia

infections can also occur concurrently with other pro-inflammatory gastrointestinal pathogens,

such as Cryptosporidium sp. (57), Helicobacter pylori (58), rotavirus (59, 60), and Salmonella

(61). To date, little research has focused on Giardia’s ability to modulate its host’s intestinal

8

pro-inflammatory responses following exposure to pro-inflammatory stimuli, and studies

examining host immune responses to Giardia have largely been performed in the absence of an

overt pro-inflammatory stimulus. Previous studies have demonstrated that mast-cell hyperplasia

occurs in the late stages of a Giardia infection or following parasite clearance (62) and that

eosinophil accumulation may occur in vivo in a isolate-dependent manner (28). Furthermore,

Giardia parasite products have been shown to modulate dendritic cell (DC) responses to

lipopolysaccharide (43, 44), while separate studies have demonstrated IECs exposed to Giardia

trophozoites produce a unique chemokine profile (45).

The need to investigate Giardia’s ability to modulate its host’s pro-inflammatory responses

is now apparent due to data collected from both in vivo animals and human studies. Microarray

analysis of jejunal tissues collected from assemblage E Giardia-infected calves revealed

decreased mRNA expression of several pro-inflammatory mediators and increased expression of

anti-inflammatory transcription factors (63). While this study did not examine whether intestinal

inflammatory responses in Giardia-infected cattle would be attenuated following exposure to a

separate pro-inflammatory stimulus, it did suggest that this parasite is capable of modulating

inflammatory responses within its host. Several human studies have also suggested that Giardia

infections in children may reduce the incidence or severity of diarrheal disease (59, 64, 65). One

study went on to demonstrate that Tanzanian children infected with Giardia had a reduced

likelihood of developing fever and had lower levels of serum C-reactive protein (CRP), a classic

marker of inflammation, when compared to their non-infected counterparts (64). A separate

study suggested children co-infected with rotavirus and Giardia displayed a marked reduction in

the severity of diarrheal disease when compared to children only infected with rotavirus (59).

9

However, findings from this study directly conflict with data from another study that was unable

to find a reduction in the severity of diarrheal disease when children were co-infected with

rotavirus and Giardia (60). These contrasting studies may suggest that Giardia-mediated

protection from diarrheal disease may be isolate-specific, reliant on host genetics, or other yet-to-

be determined factors. These studies also did not identify potential mechanisms via which

Giardia may be capable of attenuating the development of diarrheal disease within its host. As

acute intestinal inflammatory responses can contribute to the development of diarrheal disease,

we hypothesized that the ability of Giardia to modulate the development of diarrheal disease

within its host may stem from the parasites ability to attenuate pro-inflammatory responses

within the intestinal tract.

1.3 Neutrophils and acute intestinal inflammation

Acute intestinal inflammation in humans is comprised of a combination of cellular and

humoral effector responses with participation from a variety of cells. Tissue accumulation of

polymorphonuclear leukocytes (PMNs), or neutrophils, represents an archetypal event during

many acute intestinal inflammatory responses. These cells are myeloid-derived innate immune

cells heavily involved in acute intestinal inflammatory responses and essential to host defence

against many bacterial and fungal pathogens; this is exemplified by individuals highly

susceptible to life-threatening infections due to genetic mutations that result in defective PMN

function (66-68). PMNs eliminate microbes through several mechanisms including phagocytosis,

extrusion of granule contents, or release of neutrophil extracellular traps (NETs) (reviewed in

(69)). While essential to pathogen clearance, these mechanisms can also be highly deleterious

10

and injurious to self-tissue. Indeed, excessive PMN recruitment and activation contributes to the

disease pathology of several chronic gastrointestinal inflammatory disorders including

inflammatory bowel disease (IBD) and Clostridium difficile colitis (70-72). As a result, PMNs

are not recruited to host tissues en masse under homeostatic conditions, but largely confined to

the bone marrow and circulation in a quiescent state. Moreover, recruitment of PMNs to

intestinal tissues during intestinal inflammatory responses is highly regulated and occurs through

a series of steps including PMN bone marrow egression, extravasation, migration through host

tissues, and trans-epithelial migration.

1.3.1 Neutrophil chemoattractants

PMN chemoattractants are heavily involved in recruiting PMNs into host tissues and are

comprised of both host- and microbially-derived compounds. These factors include, but are not

limited to, bacterial formylated peptides (fMLF), complement fragments (ie. C5a), chemokines

(ie. interleukin-8), and lipid mediators (ie. LTB4). Although PMN chemoattractants represent a

structurally diverse collection of compounds, all bind seven transmembrane spanning serpentine

receptors that couple to heterotrimeric G proteins; in PMNs, this receptor-ligand binding results

in a multitude of processes, including the induction of chemotaxis, or migration along an

increasing concentration gradient (reviewed in (73, 74). Host-derived chemokines containing a

consecutive glutamate-leucine-arginine sequence (ELR+ chemokines) in their N-terminal region,

such as CXCL1, CXCL2, and CXCL8, are produced by a variety of cells within the intestinal

mucosa, including IECs (see below). These products bind CXC receptors (CXCRs). In humans,

CXCL8 binds both CXCR1 and CXCR2, while CXCL1 and CXCL2 only bind CXCR2 (75).

11

ELR+ chemokines are involved in multiple processes of PMN tissue recruitment, including bone

marrow egression, exit from the blood stream, and migration through host tissues (discussed

below).

1.3.2 Bone marrow egression

Healthy human adults contain approximately 6 x 1011 PMNs with 5.0 to 10.0 x 1010

PMNs being produced in the bone marrow on a daily basis (76, 77). Under homeostatic

conditions the majority of PMNs are retained in the bone marrow, with typically < 2% being

found in circulation (78-80). The mechanisms of PMN bone marrow egression remain

incompletely understood, but in vivo research has suggested this process involves antagonism

between CXCR2 and CXCR4. Under these conditions, the expression of the CXCR4 ligand

stromal cell derived factor 1α (SDF-1α/CXCL12) by stromal cells dominates over endothelial-

produced CXCR2 ligands and promotes PMN retention within the bone marrow until PMNs

reach maturity (78, 80). CXCL12, at least partially, retains PMNs within the bone marrow by

enhancing interactions between PMN integrins and surface receptors on bone marrow

endothelial and stromal cells (81). During an acute inflammatory response, the number of PMNs

within circulation greatly increases; this is, at least partially, mediated by elevated circulating

levels of granulocyte colony stimulating factor (G-CSF). This factor decreases stromal cell

expression of CXCL12 while simultaneously enhancing expression of CXCR2 ligands by

endothelial cells within the bone marrow; these two processes result in PMN bone marrow

egression and increased circulating numbers of PMNs (79, 82). In addition, the production of

interleukin-17A (IL-17A) by tissue-resident cells can increase circulating G-CSF levels and

12

promote subsequent PMN bone marrow egression (83, 84). Moreover, attenuation of PMN

chemokines at the inflammatory site has resulted in reduced numbers of circulating PMNs (82).

Therefore, local tissue responses can influence acute inflammatory responses by promoting

increased circulating numbers of PMNs.

1.3.3 Transendothelial migration

PMNs exit the vasculature and enter tissues via post-capillary venules in a series of

highly characterized steps that have been extensively reviewed elsewhere (85-87). In short, the

process of PMN extravasation is subdivided into five steps: tethering, rolling, adhesion,

crawling, and transmigration (85). Upon exposure to pro-inflammatory stimuli, tissue-resident

immune cells release an array of pro-inflammatory mediators, such as histamines, leukotrienes,

and cytokines, that induce endothelial cells to luminal expression of P- and E-selectin molecules;

these molecules bind glycosylated ligands, such as P-selectin glycoprotein 1 (PSGL-1), on the

PMN surface and induce PMN tethering and, subsequently, PMN rolling (88). PMN adhesion to

the endothelium is initiated as rolling PMNs contact ELR+ PMN chemokines immobilized on

the endothelial luminal surface by heparin sulfate (89). PMN contact with chemokines on the

luminal surface of the endothelium results in conformational changes and heightened expression

of surface PMN integrin molecules CD11a/CD18 and CD11b/CD18 and allows for interaction

with corresponding ligands, such as intracellular adhesion molecules (ICAMs), on the

endothelial surface (88, 90, 91). Interaction between PMNs and apical endothelial surface

chemokines results in PMN arrest and adhesion to the endothelial surface. Following adherence,

PMNs crawl along the apical surface of the endothelium looking for an appropriate place to

13

transmigrate across the endothelium. Preferential sites of PMN transmigration are believed to be

tricellular endothelial cell corners (92, 93) and is believed to preferentially occur through a

paracellular mechanism; however, transcellular migration of PMNs has also been reported (93).

1.3.4 Tissue migration

Following their exit from the vasculature, PMNs are capable of chemotaxis through

multiple and sequential PMN chemotactic gradients within host tissues (94). Their accumulation

and activation at an inflammatory or infection site involves migration through various spatial and

temporal chemotactic gradients (95, 96). PMN chemoattractants are organized into a hierarchy of

intermediate and end-target chemoattractants (85). Intermediate and end-target chemoattractants

differ not only in their site and source of production, but also in intracellular signaling pathways

activated in PMNs following receptor-ligand binding. In order to prioritize the importance of a

chemotactic signal, intracellular signaling pathways activated by end-target PMNs

chemoattractants override those activated by intermediate chemoattractants. As a result,

intermediate chemoattractant-induced PMN chemotaxis via activation of the phosphatidylinositol

3-kinase (PI3K) pathway is overridden by end-target chemoattractant signals transmitted via the

p38 mitogen activated protein kinase (p38 MAPK) pathway (97). The spatial expression and

hierarchy of signaling pathways activated by intermediate and end-target chemoattractants

promotes their accumulation at an inflammatory/infection site. As such, extravasated PMNs are

initially directed to an inflammatory site via intermediate chemoattractants, such as the lipid

mediator leukotriene B4 and ELR+ chemokines, which are produced by a variety of cells

surrounding the inflammatory site. As PMNs migrate through these gradients, chemotaxis will

14

shift and occur in response to end-target chemoattractants, such as the complement factor C5a

and bacterial formylated peptides, found in the immediate vicinity of the infection or

inflammatory site.

1.3.5 Intestinal transepithelial migration

Transepithelial migration (TEM) of PMNs is a multistep process initiated via contact with

the basolateral surface of the intestinal epithelium (reviewed in (98)), whereby interactions

between multiple surface factors on PMNs and IECs result in functional changes to both cells to

allow for its occurrence. PMN TEM is also dependent on chemoattractants, but not all appear to

promote this process. Experiments performed in vitro have shown that PMN TEM occurs in

response to apically applied bacterial formylated peptides, or the eicosanoid hepoxilin A3; while

apical administration of the ELR+ chemokine CXCL8 is less effective at inducing PMN TEM,

its release recruits PMNs to the basolateral membrane of the intestinal epithelium (99-101). The

initial contact between PMNs and the basolateral surface of the intestinal epithelium results in

enhanced intestinal paracellular permeability (102, 103); this contact is mediated, at least in part,

by PMN CD11b/CD18 and yet to be identified basolateral epithelial surface proteins (104, 105).

Basolateral activation of IEC proteinase activated receptor (PAR)-1 and PAR-2 by PMN serine

proteases also increases intestinal permeability (106). Following this initial contact, PMNs

subsequently traverse intestinal epithelial paracellular junctions, including desmosomes and the

apical junctional complex (AJC), en route to the apical surface of the intestinal epithelium.

Members of the junctional adhesion molecule (JAM) and cortical thymocyte (CTX) protein

(JAM/CTX) family serve as important mediators of PMN transepithelial migration at this stage

15

(107, 108). However, research has suggested that PMN surface adhesion molecules associated

with migration across the desmosome and AJC are reliant on the particular apical

chemoattractant (109). Following TEM, PMNs are retained on the apical surface of IECs via

CD11b/CD18 and ICAM-1 interactions, respectively (110); this interaction further increases

intestinal permeability via activation of myosin light chain kinase to enhance subsequent PMN

TEM (111), while PMN adhesion to the epithelial surface also delays the onset of PMN

apoptosis (112, 113).

1.3.6 Pathophysiology of neutrophils

Though PMNs are important to pathogen clearance during acute inflammatory responses,

their presence can be extremely detrimental during chronic gastrointestinal inflammatory

disorders. PMN accumulation and heightened expression of PMN mediators is observed during

IBD exacerbations (114-119), and fecal calprotectin levels can be successful at predicting

disease relapse (119, 120). Similarly, PMN recruitment appears to be heavily involved in causing

disease following infection with the opportunistic pathogen Clostridrium difficile. Individuals

with a common CXCL8 single nucleotide polymorphism are more susceptible to developing C.

difficile diarrhea and display elevated fecal levels of CXCL8 (121). Moreover, recruited PMNs

promote their own recruitment via the release of additional chemokines to enhance PMN tissue

accumulation (95), and PMNs are a major source of PMN chemokines, such as CXCL8, during

IBD exacerbation (115). Therefore, PMN recruitment during chronic gastrointestinal

inflammatory disorders, such as IBD, may result in a perpetuated inflammatory response due to

the ongoing recruitment of PMNs. Therefore, processes initiated by PMNs during their

16

recruitment and TEM likely contribute to the disease pathophysiology of chronic gastrointestinal

inflammatory disorders.

1.3.6.1 Intestinal barrier dysfunction

A dysfunctional epithelial barrier is observed during many chronic gastrointestinal

inflammatory diseases, including IBD and C. difficile colitis (122, 123), and accumulating

evidence suggests PMNs contribute to this state. A variety of PMN-derived molecules are

capable of increasing intestinal epithelial barrier permeability in order to promote TEM; these

factors likely also induce intestinal barrier dysfunction during chronic inflammatory disorders

involving PMN accumulation. Analysis of human biopsy tissues has found decreased expression

of numerous IEC AJC proteins in areas surrounding PMNs undergoing TEM, and this

downregulation is postulated to contribute to enhanced permeability during active IBD (124,

125). Excessive PMN TEM may also cause focal wounds during IBD exacerbations (126), as

PMN-derived JAML can bind to CAR and inhibit wound closure by inhibiting IEC proliferation

(127). This is postulated to result in improper healing of the intestinal epithelial barrier and

increased intestinal permeability. To summarize, during chronic intestinal inflammatory

disorders, PMN recruitment causes intestinal barrier dysfunction by disrupting junctional

proteins that control the paracellular flux of luminal contents or by damaging the integrity of the

intestinal epithelial barrier and impeding its subsequent repair. As a result, intestinal barrier

dysfunction induced by exaggerated PMN activity may also further enhance translocation of

luminal antigens and bacteria to underlying tissues, resulting in a perpetuated inflammatory

response and impaired gut healing. Experimental evidence in vivo demonstrates that intestinal

17

epithelial dysfunction results in extensive mucosal immune responses that accelerate the onset

and severity of experimental colitis (128), while preventing intestinal epithelial dysfunction

attenuates chronic experimental colitis (129). As a result, attenuation of PMN accumulation

and/or TEM may prevent intestinal barrier dysfunction that could, potentially, perpetuate

inflammatory responses and lead to chronic intestinal inflammation.

1.3.6.2 Induction of diarrheal disease

Diarrhea is a common symptom of many chronic gastrointestinal infections, including IBD

and C. difficile infection (130, 131). During chronic gastrointestinal inflammatory responses,

PMNs may be involved in the development of diarrheal disease. Individuals with a common

CXCL8 single nucleotide polymorphism are more susceptible to developing diarrheal disease

during C. difficile infection and display elevated fecal levels of CXCL8 (121). Development of

diarrheal disease due to PMN accumulation may occur via several mechanisms including

intestinal barrier dysfunction and active solute loss. Intestinal barrier dysfunction results in

augmented solute loss and, as a result of diffusion, enhanced water loss; therefore, a defective

intestinal epithelium may result in the development of diarrheal disease via a “leak-flux”

mechanism (reviewed in (132)). Therefore, PMN-mediated intestinal barrier dysfunction may

also, at least partially, promote the development of diarrhea via this mechanism. In addition,

PMNs may also promote diarrheal disease by inducing or promoting active ion secretion in IECs

that leads to water loss and, subsequently, diarrhea. PMNs on the apical surface of intestinal

epithelium release 5’-adenosine monophosphate (5’-AMP) that is converted into adenosine by

CD73 on the apical IEC surface; acting on the A2b receptor, adenosine can then induce

18

electrogenic ion secretion that results in luminal fluid accumulation, and, hence, diarrhea, (133,

134). PMNs can also facilitate the TEM of platelets across the intestinal epithelial barrier, where

platelet-derived ATP is converted into adenosine and, similarly, induces ion secretion and

luminal fluid accumulation (135). Collectively, these results demonstrate that PMNs can directly

contribute to diarrheal disease via multiple mechanisms. Therefore, treating excessive PMN

accumulation and TEM during chronic gastrointestinal inflammatory diseases may help to

alleviate patient symptoms.

1.4 The intestinal epithelium and neutrophil recruitment

The intestinal epithelium forms a biochemical and physical barrier that separates the

external environment of the intestinal lumen from underlying host tissues and is comprised of a

heterogeneous population of IECs (reviewed in (136, 137)). As Giardia is a non-invasive

parasite, this structure represents a primary point of contact between parasite and host, and

Giardia induce a plethora of pathophysiological responses within IECs (reviewed in (11, 30)).

The intestinal epithelium also participates in PMN recruitment via the secretion of pro-

inflammatory chemokines (reviewed in (138, 139)). In response to a variety of pro-inflammatory

stimuli, including direct exposure to translocated bacterial antigens, IECs secrete different

classes of chemokines including the potent PMN chemoattractant CXCL8 (140-142).

Experiments done in vivo have demonstrated continual secretion of CXCL8 from IECs without

additional pro-inflammatory signals results in PMN accumulation within the lamina propria, in

the absence of extensive tissue damage or TEM (143). Further experimental evidence in vitro has

indicated that secretion of CXCL8 from IECs exposed to pathogenic bacteria, such as Salmonella

19

enterica serovar typhimurium (S. typhimurium) or Shigella flexneri, promotes PMN

accumulation at the basolateral membrane, while apically released hepoxilin A3 promotes PMN

TEM (99, 144, 145). Collectively, these results suggest that secretion of CXCL8 by IECs plays

an important role in recruiting PMNs to the basolateral membrane of the intestinal epithelium,

but additional signals are required to promote their TEM. These data further indicate that PMN-

mediated pathophysiological responses are activated via separate signals that are preceded by

IEC-mediated CXCL8-induced PMN chemotaxis.

1.4.1 Control of intestinal epithelial CXCL8 secretion

CXCL8 is an immediate early gene product that is rapidly synthesized, transcribed into

protein, and, subsequently, released following exposure to pro-inflammatory stimuli (146); its

expression and secretion are controlled via multiple regulatory processes. In IECs, CXCL8

expression is inducible and occurs in response to a variety of host- and pathogen-derived pro-

inflammatory stimuli, including ligands associated with the Toll/interleukin-1 receptor (TLR/IL-

1R) pathway (141). Upon appropriate receptor-ligand binding, host- and pathogen-derived pro-

inflammatory stimuli trigger intracellular signaling cascades in IECs that activate pro-

inflammatory transcription factors, such as those associated with the classical nuclear factor κB

(NF-κB) and the activator protein-1 (AP-1) pathways, that culminate in CXCL8 mRNA

transcription (147-149). Following mRNA transcription, activation of the p38 MAPK pathway

promotes CXCL8 mRNA stabilization (149). Full length CXCL8 is considered to have 77 amino

acids (CXCL8 1-77); however, due to various post-translational modifications, at least 10

isoforms of CXCL8 have been identified (150). Alternative cleavage of the CXCL8 export signal

20

peptide results in an isoform containing 2 extra NH2-terminal amino acids: CXCL8 (CXCL8 -2-

77) (151). Various proteases, including cathepsin L, process full length CXCL8 (1-77) into

alternative isoforms; this protease-mediated cleavage also affects CXCL8’s chemotactic

potential towards PMNs. Indeed, PMN chemotaxis towards CXCL8 is greatly enhanced when

full length CXCL8 (1-77) is NH2-terminally truncated by five to eight amino acids (152-154).

Peptidylarginine deiminases may also citrullinate CXCL8 into isoforms that reduce the ability of

CXCL8 to promote PMN extravasation (155). Therefore, CXCL8 expression and release is

controlled by multiple regulatory processes, and, processes aimed at attenuating the CXCL8

expression in IECs could target multiple regulatory pathways involved with CXCL8 expression.

1.5 Parasite immunomodulatory factors

1.5.1 Cathepsin cysteine proteases

In humans, many different classes of proteases belong to the cathepsin protease family,

including serine proteases (cathepsin A and G), aspartic proteases (cathepsin D and E), and

cysteine cathepsins (cathepsin B, C, F, K, L, O, S, V, X and W) (reviewed in (156)). The term

“cathepsin” was initially used to describe a protease active in a lightly acidic environment, such

as a lysosome (157, 158); however, ongoing research has resulted in revision of this definition.

In particular, cathepsin cysteine proteases are defined by a catalytic dyad comprised of an active

site cysteine (Cys) and histidine (His) residue, whereby the His donates electrons to the Cys

residue to create a thiolate-imidazolium charge relay diad; this structure is an excellent

nucleophile capable of hydrolyzing a variety of substrates. Cathepsin cysteine proteases are

classified as clan CA cysteine proteases and further subdivided into superfamilies such as the

21

cathepsin L (catL)-like or cathepsin B (catB)-like superfamilies (reviewed in (156)). While the

majority of cathepsin cysteine proteases are exopeptidases, catB proteases contain an

approximate 20 amino acid insertion referred to as the occluding loop containing two

characteristic His residues that enables their function as an endo- or exopeptidase (159).

1.5.2 Parasite cathepsin cysteine proteases

Cathepsin cysteine proteases are important virulence factors for a variety of single-celled

parasites that can also be employed to modulate or evade their host’s immune system (reviewed

in (160, 161)). Indeed, cathepsin cysteine proteases of parasites, other than Giardia, have been

found to inhibit pro-inflammatory transcription factors (162), degrade host effector molecules,

such as immunoglobulins (163), induce specific host immune responses (164, 165), and modify

host chemokines (166-168). The Giardia genome contains genes for numerous cathepsin

cysteine proteases, the majority of which have no described function (169, 170). A role for a

specific Giardia catB protease in parasite encystation and excystation has been described (170),

but, apart from this, evidence that Giardia cathepsin cysteine proteases are involved in the

pathogenesis or modulation of host immune responses remains circumstantial. One report has

demonstrated that several Giardia cathepsin proteases are upregulated upon exposure to IECs

(171), while others have suggested that Giardia cysteine proteases can induce specific host

immune responses (172). As a result, the roles of Giardia cathepsin cysteine proteases during

infection remain largely unknown. Interestingly, previous research has demonstrated that

Entamoeba histolytica cysteine protease 2 cleaves CXCL8 into a more potent isoform that

22

enhances PMN chemotaxis (167). Therefore, we hypothesized that Giardia cathepsin cysteine

proteases may be involved in modulating PMN chemotaxis.

1.6 Summary

Even though parasite numbers can exceed 106 trophozoites per centimetre of gut, Giardia

infections rarely induce an overt intestinal inflammatory response within their host. In addition,

Giardia infections have been reported to occur concurrently with other pro-inflammatory

gastrointestinal pathogens that cause inflammation and diarrheal disease. Some of these studies

also demonstrated that Giardia infections can modulate the development of diarrheal disease in

their host via mechanisms that remain obscure. In particular, one study demonstrated that

Giardia infections reduced the likelihood of developing diarrhea and fever and also reduced

serum C-reactive protein levels (64). Acute intestinal inflammatory responses, including PMN

accumulation and TEM induce pathophysiological responses that result in diarrhea. Many cell

types are involved in recruiting PMNs into intestinal tissues, including IECs. In response to pro-

inflammatory stimuli, IECs secrete a variety of pro-inflammatory mediators including the potent

PMN chemokine CXCL8. This factor recruits PMNs to the basolateral membrane of the

intestinal epithelium so other factors can, if necessary, induce their TEM. As Giardia

trophozoites, are non-invasive the intestinal epithelium represents a primary point of contact

between parasite and host. Therefore, modulation of host inflammatory responses may involve

parasite modulation of intestinal epithelial CXCL8. The Giardia genome contains genes for

approximately 20 cathepsin proteases. However, their functions remain largely unknown.

Several single celled parasites use similar proteases during their infection to modulate their

23

host’s immune response. In particular, Entamoeba histolytica cysteine protease 2 has been shown

to cleave CXCL8 and, resultantly, enhance CXCL8-induced PMN chemotaxis. Therefore,

Giardia trophozoites may employ similar proteases to attenuate CXCL8 released from IECs

during an acute inflammatory response.

1.7 Hypothesis and objectives

The purpose of this study was to determine whether Giardia trophozoites modulate

inflammatory responses within their host and identify potential immunomodulatory molecules.

Our hypothesis for this study was that Giardia trophozoites release cathepsin cysteine proteases

that are capable of attenuating PMN chemotaxis. The first aim of this study was to determine

whether Giardia infections were capable of attenuating PMN accumulation and chemotaxis. We

hypothesized that Giardia infections would be capable of attenuating PMN accumulation during

an in vivo model of bacterial toxin-induced colitis. Furthermore, we believed that this attenuation

of PMN recruitment would result from parasite-mediated attenuation of PMN chemokines, and

this would be observed in multiple experimental models of inflammation. Finally, we postulated

that this modulation of host inflammatory responses involved Giardia-mediated modulation of

intestinal epithelial CXCL8 expression. The second aim of this study was to determine whether

Giardia cathepsin proteases modulated intestinal epithelial CXCL8 and whether this modulation

affected PMN chemotaxis. Therefore, we set-out to classify Giardia cathepsin cysteine

proteases. As differences between assemblage A and B isolates have been demonstrated, we

hypothesized cathepsin proteases may differ between Giardia isolates and/or assemblages. We

also postulated that Giardia trophozoites were capable of secreting cathepsin cysteine proteases

24

that could be inhibited via the use of pharmacological inhibitors, whereby inhibition of these

proteases would prevent modulation of intestinal epithelial CXCL8-mediated PMN chemotaxis.

2.0 Materials and Methods

2.1 Ethics statement

All studies involving human small intestinal mucosal biopsy tissues were approved by the

Conjoint Health Research Ethics Board (CHREB) at the University of Calgary and the Calgary

Health Region. In accordance with CHREB guidelines, adult subjects used in this study provided

informed, written consent and a parent or guardian of any child participant provided informed,

written consent on their behalf. Animal experiments were approved by the Life and

Environmental Sciences Animal Care Committee of the University of Calgary, conducted in

compliance with that approval, and followed guidelines established by the Canadian Council of

Animal Care.

2.2 Reagents

Recombinant human interleukin-1 (IL-1), CXCL8, C5a, and corresponding ELISAs

were purchased from R&D Systems (Minneapolis, MN, USA). The broad-spectrum, clan CA

membrane permeable cysteine protease inhibitor (2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-

methylbutane ethyl ester (E-64d) (173) was purchased from Sigma-Aldrich (Oakville, ON,

Canada). The membrane permeable, catB-specific inhibitor L-3-trans-

(Propylcarbamoyl)Oxirane-2-Carbonyl)-L-Isoleucyl-L-Proline Methyl Ester (Ca-074Me) (159),

the catB/L fluorgenic substrate benzyloxycarbonyl-L-Phenylalanyl-L-Arginine 4-Methyl-

25

Coumaryl-7-Amide (ZFR-AMC), and the catB fluorogenic substrate benzyloxycarbonyl-L-

Arginine-L-Arginine 4-Methyl-Coumaryl-7-Amide (ZRR-AMC) were purchased from Peptides

International (Louisville, KY, USA) (174, 175). QIAZol, RNEasy RNA extraction kits,

QuantiTect Reverse Transcription Kit, QuantiFast SYBR Green PCR kits, and human CXCL8

and -2 microglobulin (2M) primers were purchased from Qiagen (Toronto, ON, Canada).

Mouse monoclonal interleukin-8 (1:500) was purchased from Santa Cruz Biotechnology (Dallas,

TX, USA), and mouse horseradish peroxidase conjugated secondary antibody (1:1000) was

purchased from Cell Signaling Technology (Beverly, MA, USA).

2.3 Human biopsy tissues and cell lines

Adapting a previous protocol (176), small intestinal mucosal biopsy tissues were obtained

from the terminal ileum of patients with Crohn’s disease (CD) in remission or from CD patients

with areas of active disease in the terminal ileum or descending colon. Samples were washed

once in Dulbecco’s PBS (Sigma-Aldrich) contained 0.016% 1,4-Diothioerythritol (Sigma-

Aldrich) to remove loosely adherent mucous and bacteria followed by three washes with PBS.

Washed biopsy tissues were placed into 96-well plates and incubated in 300 µL of OptiMEM

(Life Technologies) at 370C, 5% CO2, and 96% humidity. The human adenocarcinoma Caco-2

cell (ATCC) was grown in Minimum Essential Medium Eagle (MEME) (Sigma-Aldrich)

supplemented with 100 g/mL streptomycin, 100 U/mL penicillin, 200 mM L-glutamine, 5mM

sodium pyruvate, and 20% heat-inactivated FBS (VWR). Cells were passaged at 80% confluence

with 2x Trypsin-EDTA and seeded onto 6-well plates or small petri dishes pre-treated with poly-

26

L-ornithine (Sigma-Aldrich). Cells were maintained at 370C, 5% CO2, and 96% humidity and

media replaced every 2 to 3 days. Cells were used between passage 22 and 34.

2.4 Parasites

All Giardia trophozoite isolates used in this study were previously obtained and isolated.

Giardia NF trophozoites were obtained from a water sample during an outbreak of giardiasis in

Newfoundland, Canada (177), S2 trophozoites were initially isolated from a sheep (178), WB

trophozoites were obtained from a symptomatic patient with chronic giardiasis (179), PB was

obtained from a symptomatic patient from Portland, OR (180), and GS/M trophozoites were

isolated during a previous study (181). The WB and PB isolates have been previously

characterized as assemblage A isolates, while the GS/M isolate is an assemblage B isolate (182).

Trophozoites were grown axenically at 370C in Diamond’s TYI-S-33 medium (183)

supplemented with piperacillin (Sigma-Aldrich) in 15 ml polystryene tubes (Becton-Dickinson

Falcon) until confluence.

2.5 Salmonella typhimurium

Salmonella enterica serovar typhimurium 14028 was a gift from Dr. Kenneth Sanderson,

University of Calgary. A non-agitated microaerophilic culture of log-phase S. typhimurium was

generated by inoculating 10 L of an overnight stationary phase culture into 10 mL of Luria

Broth and incubated at 370C until log-phase was attained. The number of CFUs per mL was

determined by measuring the optical density at 600 nm (OD600) on. Cultures were subsequently

centrifuged at 1000 x g and re-suspended in a volume of Caco-2 growth medium without

27

antibiotics that corresponded to a multiplicity of infection (MOI) of 100:1 when 10 L of the

suspension was applied to cell supernatants.

2.6 Giardia trophozoite isolation

Confluent tubes of Giardia trophozoites were harvested by cold shock on ice for 30

minutes and subsequently pooled into 50 mL polypropylene tubes (Falcon) and centrifuged at

500 x g for 10 minutes. Resulting pellets were collectively re-suspended in 10 mL of ice cold

PBS (Sigma-Aldrich) and centrifuged at 500 x g for 10 minutes. The pellet was re-suspended in

3 mL of fresh PBS and trophozoites were enumerated with a hemocytometer and adjusted to the

appropriate concentration. For ex vivo human biopsy experiments, Giardia trophozoites were

adjusted to a concentration of 5.0x106 trophozoites/well, while trophozoites were co-incubated

with in vitro Caco-2 monolayers at a multiplicity of infection of 1:1, 10:1, or 50:1. For all

experiments involving co-incubation of ex vivo human small intestinal biopsy tissues or in vitro

Caco-2 monolayers with Giardia trophozoites, cells were maintained at 370C, 5% CO2, and 96%

humidity for the experimental duration. For sonication, trophozoites were re-suspended in

appropriate solution and sonicated three times on ice with three bursts of 30 seconds on power 4

with 30 seconds rest on ice between each round of sonication (550 Versonic Dismembranator,

Fisher Scientific).

2.7 Giardia-mediated attenuation of intestinal epithelial CXCL8

Ex vivo human terminal ileal mucosal biopsy tissues from patients with CD in remission

were co-incubated with Giardia NF trophozoites in OptiMEM (Life Technologies) for 2 hours

28

and subsequently administered 1.0 ng/mL of pro-inflammatory IL-1 or vehicle control for 4

hours. Similarly, ex vivo inflamed human terminal ileal mucosal or descending colon biopsy

tissues collected from patients with CD were co-incubated with Giardia NF trophozoites in

OptiMEM (Life Technologies) for 6 hours. Giardia trophozoites (NF, WB, PB, S2 or GS/M)

were also co-incubated in the presence or absence of in vitro Caco-2 monolayers in Caco-2

growth medium for 2 or 24 hours and subsequently administered IL-1 (1.0 ng/mL) or CXCL8

(1.0 ng/mL) for 4 hours. Giardia NF trophozoites were also co-incubated with Caco-2

monolayers for 2 hours and subsequently administered S. typhimurium (MOI 100:1).

2.7.1 Cytokine analysis

Supernatants from ex vivo human biopsy tissue experiments and in vitro Caco-2

experiments were collected and centrifuged at 500 x g for 15 minutes at 40C. Resulting

supernatants were decanted and stored at -700C. Biopsy tissues were homogenized in lysis buffer

(20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Tween-20 and a Complete Minitab protease

inhibitor cocktail (Roche)) using a Fast-Prep24 device (MP Biochemicals) at speed 6.0 for 40

seconds. The resulting homogenate solution was collected into pyrogen-free 1.5mL Eppendorf

tubes and centrifuged at 10,000 x g for 15 minutes at 40C. In vitro Caco-2 supernatant cytokine

levels were assessed via CXCL8 or IL-1β ELISAs (R&D Systems). Ex vivo biopsy cell-culture

supernatants and tissue cytokine levels were assessed using a Luminex XMap assay according to

manufacturer’s instructions (Luminex Corp.). CXCL8 levels in ex vivo biopsy cell-culture

supernatants and tissue cytokine levels were also assessed via CXCL8 ELISA (R&D Systems).

29

2.8 Giardia modulation of Clostridium difficile toxin-induced colitis

2.8.1 Giardia in vivo infection

Using a previously described in vivo Giardia infection model (27, 184), male C57BL/6

mice aged 4 to 6 weeks (Charles River Laboratories, Sherbrooke, QC) were acclimatized for 1

week prior to infection with Giardia trophozoites. Forty-eight hours prior to infection, mice were

administered broad-spectrum antibiotics to their drinking water ad libitum (1.4 mg/mL neomycin

(Alfa Aesar), 1.0 mg/mL ampicillin (Alfa Aesar) and 1.0 mg/mL vancomycin (Alfa Aesar). This

regimen was maintained for the experimental duration. After 48 hours, mice were gavaged with

107 Giardia NF or GS/M trophozoites in 0.1 mL of TYI-S-33 medium. After 7 days, small

intestinal parasite loads were quantified. Mice were euthanized and the first 3 centimetres of the

small intestine distal to the ligament of Treitz were opened longitudinally, placed in 1.5 mL

Eppendorf tubes containing 1mL of PBS, and kept on ice for 15 minutes. After 15-minute

incubation, tubes were vortexed and trophozoite numbers were enumerated with a

hemocytometer.

2.8.2 Clostridium difficile toxin-induced colitis

Following a previously described model of C. difficile toxin-induced colitis (185), mice

previously infected with Giardia NF or GS/M trophozoites for 7 days were rectally administered

a 100 µg solution of C. difficile A/B toxin (kindly provided by Dr. P.L. Beck) for 3 hours.

Briefly, a 5F infant feeding tube catheter containing side ports (Mallinckrodt Inc., St. Louis, MO,

USA) was lubricated with a water-soluble personal lubricant and inserted 2.5 cm up the colon.

With pressure applied to the anal area to prevent leakage, 100 µL of a 1.0 µg/µL C. difficile toxin

30

A/B solution diluted in PBS was slowly administered over a period of 30 seconds. Following

this, the tube was slowly removed and pressure was maintained for another 30 seconds.

Appropriate control animal groups were administered PBS.

2.8.3 Assessment of colonic cytokine protein levels

Colonic tissue samples were weighed and collected in 2 mL Fast-Prep tubes (MP

Biomedicals, Solon, OH, USA) containing a mixture of 0.9-2.0 mm stainless steel beads

(NextAdvance, Averill Park, NY, USA). Tissue samples were suspended in lysis buffer (20 mM

Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Tween-20 and a Complete Minitab protease inhibitor

cocktail (Roche, Laval, QC, Canada)) at a ratio of 50 mg tissue per 1 mL lysis buffer. Samples

were then homogenized using a Fast-Prep24 device (MP Biochemicals) at speed 6.0 for 40

seconds. The resulting homogenate solution was collected into pyrogen-free 1.5mL Eppendorf

tubes and centrifuged at 10,000x g for 15 minutes at 40C. Supernatants were collected and

protein levels assessed using a Luminex XMap assay according to manufacturer’s instructions

(Luminex Corp.).

2.8.4 Tissue MPO assay

Colonic tissue samples were weighed and collected in 2 mL Fast-Prep tubes (MP

Biomedicals) containing a mixture of 0.9-2.0 mm stainless steel beads (NextAdvance). Tissue

samples were suspended in 50 mM potassium phosphate buffer containing 5 mg/mL

hexadecyltrimethylammonium bromide (Sigma-Aldrich) at a ratio of 50 mg tissue per 1 mL lysis

buffer. Samples were then homogenized using a Fast-Prep24 device (MP Biochemicals) at speed

31

6.0 for 40 seconds. The resulting homogenate solution was collected into pyrogen-free 1.5mL

Eppendorf tubes and centrifuged at 10,000x g for 15 minutes at 40C. 7 µL of the resulting

supernatant was added to a standard 96-well plate along with 200 µL of the reaction mixture

(comprised of 0.005 g O-dianisidine (Sigma-Aldrich), 30 mL of distilled H2O, 3.33 mL of

potassium phosphate buffer, and 17 L of 1% H2O2). Using a microplate scanner (SpectraMax

M2e, Molecular Devices, Sunnyvale, CA, USA), three absorbance readings at 450 nm were

recorded every 30 seconds. MPO activity was measured as units of activity per milligram of

tissue, with 1 unit of MPO being defined as the amount required to degrade 1 µmol of H2O2 per

minute at room temperature.

2.8.5 Immunohistochemistry

Samples were embedded in paraffin wax, cut on a cryostat into 8 μm sections, and

mounted on poly-D-lysine coated slides. For antigen retrieval, tissues were successively washed

in xylene (2 x 3 minutes) and 95% ethanol (2 x 3 minutes) followed by 5 minutes under running

tap water. Following this, slides were sequentially incubated in a boiling Tris/EDTA, pH 9.0

solution for 30 minutes, cooled under running tap water for 5 minutes, incubated in 1% sodium

borohydride in PBS, and washed in tap water for an additional 5 minutes. Slides were then

blocked in tissue blocking buffer (1% BSA, 0.1% Triton X-100 in PBS) for 1 hour (3 x 20

minute washes) and incubated with an anti-MPO antibody (abcam ab9535) (1 in 200 dilution)

overnight at 40C. At room temperature, slides were washed for 1 hour (3 x 20 minutes),

incubated for 1 hour with secondary antibody (1 in 1000), and then washed for 1 hour (3 x 20

minutes). After this, slides were mounted with FluoroshieldTM containing 4’,6-diamidino-2-

32

phenylindole (DAPI) (Sigma-Aldrich) and visualized using a Leica DMR fluorescence

microscope at 40X magnification.

2.9 Intestinal epithelial CXCL8 mRNA expression

Caco-2 monolayers were co-incubated with Giardia NF or GS/M trophozoites at an MOI

of 10:1 for 2 hours in Caco-2 growth medium and subsequently administered 1.0 ng/mL IL-1.

After 1 and 4 hours post IL-1 administration, cell monolayers were washed with PBS, lysed in

1 mL of QIAZol, and stored at -700C in RNAse free tubes. mRNA was isolated using a modified

RNEasy protocol from Qiagen. Briefly, 0.2 mL of chloroform was added to tubes and samples

were shaken for 15 seconds. After 3-minute incubation at room temperature, samples were

centrifuged at 12,000x g for 15 minutes at 40C. The top mRNA-containing layer was mixed with

an equal volume of 70% ethanol and applied to RNEasy spin columns; at this point, the Qiagen

RNEasy mRNA isolation protocol was followed. Samples were assessed with a NanoDrop to

determine mRNA concentration and ensure that 260/280 ratios were greater than 1.8. Following

this, 1 g of mRNA from each sample was reverse-transcribed into cDNA via a QuantiTect

Reverse Transcription Kit and a PCR thermal cycler (BioRad). qPCR was run on cDNA samples

using QuantiFast SYBR Green PCR Kit using a RotorGeneQ qPCR machine (Qiagen). A

positive control sample was used to generate a relative standard curve that consisted of six 1 in

10 dilutions. All samples were diluted 1 in 10 to fit within the standard curve. Levels of CXCL8

were normalized against loading control -2 microglobulin (2M). All primers were pre-

designed and purchased from Qiagen.

33

2.10 Protein analysis and modeling

Multi-sequence amino-acid alignment and phylogenetic tree construction of catB and catL

cysteine proteases was performed with CLC Sequence Viewer 7.0.2 (http://www.clcbio.com).

Sequences were aligned using the ClustalW option (186). Phylogenetic tree construction used

bootstrapped-confirmed neighbor joining trees (100 replicates). Giardia theoretical molecular

weights and isoelectric points were obtained from the Giardia genome (169).

2.11 Nucleotide sequence GenBank accession numbers

2.11.1 Cathepsin B GenBank accession numbers

Homo sapiens (AAH95408.1), Mus musculus (AAA37375.1), Rattus norvegicus

(NP_072119.2), Bos taurus (NP_776456.1), Trypanosoma brucei (AAR88085.1), Trypanosoma

cruzi (AAD03404.1), Leishmania donovani (AAG44365.1), Leishmania chagasi (AAG44098.1),

Leishmania major (AAB48119.1), Schistosoma mansonii (AAA29865.1), Giardia GS 438

(EET02294.1), Giardia WB 114165 (EDO82645.1), Giardia WB 16468 (EDO80409.1), Giardia

GS 2309 (EET00465.1), Giardia WB 29304 (EDO78911.1), Giardia GS 78 (EET02635.1),

Giardia WB 16779 (EDO76908.1), Giardia WB 14019 (EDO77203.1), Giardia GS 2946

(EES99819.1), Giardia WB 16160 (EDO76677.1), Giardia GS 2036 (EET00718.1), Giardia

WB 15564 (EDO79609.1), Giardia GS 3619 (EES99155.1), Giardia GS 159 (EET02549.1),

Giardia WB 10217 (EDO78699.1), Giardia GS 2318 (EET00443.1), Giardia WB 17516

(EDO81359.1), Giardia GS 3635 (EES99137.1).

http://www.ncbi.nlm.nih.gov/protein/AAH95408.1

http://www.ncbi.nlm.nih.gov/protein/AAA37375.1

http://www.ncbi.nlm.nih.gov/protein/NP_072119.2

http://www.ncbi.nlm.nih.gov/protein/NP_776456.1

http://www.ncbi.nlm.nih.gov/protein/AAR88085.1

http://www.ncbi.nlm.nih.gov/protein/AAD03404.1

http://www.ncbi.nlm.nih.gov/protein/AAG44365.1


http://www.ncbi.nlm.nih.gov/protein/AAB48119.1


http://www.ncbi.nlm.nih.gov/protein/EET02294.1

http://www.ncbi.nlm.nih.gov/protein/EDO82645.1







http://www.ncbi.nlm.nih.gov/protein/EES99819.1










34

2.11.2 Cathepsin L GenBank accession numbers

Homo sapiens (AAA66974.1), Mus musculus (EDL16242.1), Rattus norvegicus

(BAM14518.1), Bos taurus (CAA62870.1), Leishmania donovani (AAW80538.1), Leishmania

chagasi (AAG45727.1), Leishmania major (AAB48120.1), Schistosoma mansoni

(CAA83538.1), Giardia WB 3099 (EDO78598.1), Giardia WB 3169 (EDO80941.1), Giardia

GS 3178 (EES99584.1), Giardia WB 9548 (EDO76746.1), Giardia WB 137680 (EDO77330.1),

Giardia WB 11209 (EDO79154.1), Giardia GS 3714 (EES99039.1), Giardia WB 17607

(EDO82224.1), Giardia GS 259 (EET02454.1), Giardia WB 14983 (EDO76750.1), Giardia WB

16380 (EDO76749.1), Giardia GS 4331 (EES98507.1), Giardia GS 4332 (EES98508.1), and

Giardia GS 4531 (EES98268.1).

2.12 Visualization of Giardia cathepsin proteases

A previously described protocol (187) was adapted to visualize Giardia intratrophozoite

cathepsin cysteine protease activity. To visualize intratrophozoite cathepsin cysteine protease

activity, isolated Giardia NF or GS/M trophozoites were adjusted to a concentration of 1.0 x 108

trophozoites/mL in cathepsin assay buffer (100 mM sodium acetate, 10 mM DTT, 0.1% Triton

X-100, 1 mM EDTA, 0.5% DMSO, pH 7.2) and sonicated (see above). Samples were

subsequently centrifuged at 10,000x g for 10 minutes at 40C. Following centrifugation, resulting

supernatants from both secreted products or trophozoite sonicates were decanted and incubated

with an equal volume of non-denaturing electrophoresis buffer for 15 minutes at room

temperature. At 40C and protected from light, 50 µL of this solution was then added to 7% SDS-

PAGE gels containing 200 µM Z-Phe-Arg-AMC or Z-Arg-Arg-AMC and run at 100V. To


http://www.ncbi.nlm.nih.gov/protein/EDL16242.1

http://www.ncbi.nlm.nih.gov/protein/BAM14518.1

http://www.ncbi.nlm.nih.gov/protein/CAA62870.1

http://www.ncbi.nlm.nih.gov/protein/AAW80538.1


http://www.ncbi.nlm.nih.gov/protein/AAB48120.1

http://www.ncbi.nlm.nih.gov/protein/CAA83538.1















35

remove SDS, gels were protected from light and washed 4 times for 15 minutes in a cold 2.5%

Triton X-100 distilled water solution followed by the same number of washes in cold distilled

water. Gels were subsequently incubated in cathepsin assay buffer overnight at 370C. Bands

were visualized using a ChemiDoc XRS System (Bio-Rad).

2.13 Cathepsin cysteine protease inhibition

2.13.1 Caco-2 supernatant pre-treatment

Previous reports have suggested that 1 M E-64 is the maximal inhibitory dose that does

not affect Giardia trophozoite viability (36). The minimal inhibitory concentration of Ca-074Me

required for inhibiting cathepsin cysteine protease activity without affecting trophozoite viability

was determined by performing experiments with increasing concentrations of Ca-074Me.

Therefore, Caco-2 supernatants were pre-treated with 1 M E-64d, increasing concentrations of

Ca-074Me (1, 10, or 50 M), or vehicle control (DMSO) for ~10-15 minutes. After this

incubation period, Giardia trophozoites were added to supernatants and incubated for 6 hours.

Cell supernatants were collected and assayed for cathepsin cysteine protease activity (see below).

2.13.2 Inhibition of Giardia trophozoite cathepsin proteases

Confluent tubes of Giardia trophozoites were pre-treated with 1 M E-64, increasing

concentrations of Ca-074Me (1, 10, or 50 M), or vehicle control (DMSO) for 30 minutes.

Following this, trophozoites were harvested via cold shock on ice for 30 minutes. Giardia

trophozoites were then co-incubated with Caco-2 monolayers for 6 hours, or sonicated and 1 in

10 dilutions of sonicates were assayed for cathepsin cysteine protease activity.

36

2.14 Giardia viability assays

Giardia trophozoite motility was used as an indicator of viability (188). After a 6-hour co-

incubation with Caco-2 monolayers, supernatants were collected, vortexed, and 10 L plated on

to a hemocytometer. The ratio of the number of motile trophozoites to total trophozoites was

determined and expressed as percent control.

2.15 Cathepsin cysteine protease activity assays

Cathepsin cysteine protease activity was determined by measuring the liberation of 7-

aminomethylcoumarin (AMC) from fluorogenic substrates. Cathepsin protease activity

corresponds to a change in reflective light units (RFUs) over time (174, 175). In vitro cell

supernatants and trophozoite sonicates (diluted 1:10) were plated on a 96-well microplate along

with cathepsin assay buffer (100 mM sodium acetate, 10 mM DTT, 0.1% Triton X-100, 1 mM

EDTA, 0.5% DMSO) containing 200 M ZFR-AMC or ZRR-AMC at a 1:2 ratio of supernatant

to assay buffer. Assay buffer was adjusted to pH 7.2; this value was chosen since maximal

Giardia cathepsin cysteine protease activity is observed at ≈ pH 7.0 and, also, to mimic the

luminal pH of the upper small intestine. Microplates were incubated at 370C for 5 minutes, and

subsequently measured kinetically using a microplate reader (SpectraMax M2e, Molecular

Devices) at 370C with excitation and emission wavelengths of 354nm and 445nm, respectively.

Measurements were recorded every 30 seconds for 5 minutes.

37

2.16 Giardia modulation of CXCL8-induced PMN chemotaxis

2.16.1 Chemotactic supernatant generation

Caco-2 monolayers grown to confluence on poly-L-ornithine (Sigma-Aldrich) pre-treated

small petri dishes and were co-incubated with Giardia trophozoites in OptiMEM for 2 hours and

subsequently administered CXCL8 (100 ng/mL) for 4 hours. Cell supernatants were collected

and centrifuged at 500x g for 10 minutes and the resulting supernatant decanted and stored at -

700C until further use.

2.16.2 PMN isolation and chemotaxis assay

Human blood was obtained from healthy volunteers according to standard techniques in

accordance with protocols approved by the University of Calgary. Human neutrophils were

isolated by dextran sedimentation and Percoll density-gradient separation, as described

previously (189). In short, blood was collected in acid-citrate-dextrose Vacutainers (Becton

Dickinson), pooled, and centrifuged at 350x g for 20 minutes. The resulting platelet-rich plasma

was discarded and leukocytes were separated from erythrocytes via 0.6% dextran (Sigma-

Aldrich) sedimentation. The leukocyte rich upper layer was fractionated using isotonic Percoll

(Sigma-Aldrich). PMNs were collected from the 70%-81% interface and subsequently washed

twice in Hanks Balanced Salt Solution (Sigma-Aldrich) without Ca2+/Mg2+. PMNs were

enumerated using a hemocytometer and cell viability assessed via trypan blue exclusion.

Chemotactic supernatants were thawed and 600 L was added to the lower chamber of 8.0 m

transwells (Costar), while 5.0x105 PMNs in OptiMEM were added in 150 L of OptiMEM to the

upper chamber (see Figure 1). Plates were allowed to incubate for 1 hour at 370C, 5% CO2, 96%

38

humidity before upper and lower supernatants were collected and assessed via myleoperoxidase

to quantify PMN chemotaxis.

2.17 Myeloperoxidase assay

PMN chemotaxis was assessed via myeloperoxidase (MPO) activity assays using the O-

dianisidine method (190). Briefly, PMNs in the upper and lower chambers of 8.0 m transwells

were lysed in 150 L of a 1:1 ratio of 1M sodium citrate and 10% Triton-X 100 (Sigma-

Aldrich), where upper chambers were adjusted to a final volume of 600 L prior to lysis.

Following addition of lysis buffer, samples were incubated for 15 minutes at 40C with shaking.

After 15-minute incubation, 100 L of supernatant was added to a 96-well microplate along with

150 L of the reaction mixture (comprised of 0.005 g O-dianisidine (Sigma-Aldrich), 30 mL of

distilled H2O, 3.33 mL of potassium phosphate buffer, and 17 L of 1% H2O2). Using a

microplate scanner (SpectraMax M2e, Molecular Devices, Sunnyvale, CA), three absorbance

readings at 450 nm were recorded every 30 seconds. PMN chemotaxis was determined by taking

the bottom to top ratio of myeloperoxidase activity.

2.18 Western blotting

Samples were separated on 15% SDS-PAGE and transferred to nitrocellulose membranes

(Whatman). Membranes were blocked for 1 hour in 5% nonfat dry milk in Tris-buffered saline

(TBS) with 0.1% Tween (TBS-T) and probed with primary antibodies (1 in 300) in 5% milk

TBS-T overnight at 40C. Membranes were washed 4 times for 15 minutes and probed with the

appropriate horseradish peroxidase-conjugated secondary antibody in 5% milk TBS-T (1 in

39

1000), and washed again for 4 times for 15 minutes each. Bands were visualized using the

Amersham ECL Prime Western blotting detection reagent (GE Healthcare).

2.19 Statistics

Statistical analysis was performed using GraphPad Prism 6 software where the normality

of the data was assessed prior to analysis. Parametric values are represented as mean ± SEM and

non-parametric values are represented as min to max. All parametric comparisons were made

using one-way ANOVA with Tukey’s post hoc analysis, while non-parametric comparisons were

made using a Mann Whitney test. Statistical significance was established at p < 0.05 (*).

40

3.0 Results

3.1 In vivo Giardia infections attenuate granulocyte infiltration in an isolate-dependent

manner

Isolate-specific differences in in vivo Giardia-mediated pathology that may relate to

assemblage of the selected isolates have been reported previously (27, 28). Therefore,

experiments were performed to determine whether in vivo assemblage A Giardia NF or

assemblage B Giardia GS/M infections were capable of attenuating acute intestinal

inflammatory responses to i.r. administration of Clostridrium difficile toxin A (TcdA) and toxin

B (TcdB) bacterial toxin. In vivo administration of C. difficile TcdAB mimics the pathology of C.

difficile colitis and is associated with rapid and acute intestinal inflammatory responses; this

includes increased colonic accumulation of pro-inflammatory cells, such as PMNs, and

heightened expression of PMN chemokines (185). Administration of broad-spectrum antibiotics

to mouse drinking water ad libitium resulted in effective small intestinal colonization by Giardia

NF and GS/M trophozoites, while 3-hour administration of C. difficile TcdAB had no effect on

trophozoite numbers (Figure 1). Small intestinal trophozoites numbers in Giardia GS/M-infected

animals were higher than numbers from NF-infected animals (Figure 1). These results are

consistent with previous observations that assemblage B Giardia isolates, such as GS/M, have

higher parasite burdens compared to assemblage A isolates, such as NF trophozoites (27, 28).

MPO activity was used as a marker of granulocyte infiltration (190). As previously

demonstrated (185), i.r. administration of 100 μg C. difficile TcdAB significantly increased

colonic tissue MPO activity (Figure 2A and B) and qualitatively increased numbers of MPO-

positive cells within colonic tissues (Figure 3), compared to uninfected PBS control animals. In

Giardia NF-infected animals administered i.r. 100 μg TcdAB, reduced colonic MPO activity

41

Figure 1. Ad libitium administration of broad-spectrum antibiotics to mouse drinking water

resulted in effective small intestinal colonization by Giardia NF and GS/M trophozoites.

Male C57BL/6 mice, aged 4 to 6 weeks, were administered broad-spectrum antibiotics to their

drinking water ad libitum (1.4 mg/mL neomycin, 1.0 mg/mL ampicillin and 1.0 mg/mL

vancomycin) 48 hours prior to infection with Giardia GS/M or NF trophozoites. After 7 days,

trophozoite numbers in were quantified in upper small intestine. All data are representative of

two independent experiments (n = 7-11/group) and represented as mean ± SEM. * p < 0.05

42

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

2

4

6

8

*

*

*

log

tro

ph

oz

oit

es

/cm

gu

t

43


myeloperoxidase activity following rectal administration of TcdAB.

Male C57BL/6 mice, aged 4 to 6 weeks, were infected with (A) Giardia NF or (B) GS/M

trophozoites for 7 days and subsequently administered 100 µg TcdAB. Colonic myeloperoxidase

activity was assessed after 3-hour instillation with TcdAB. All data are representative of two

independent experiments (n = 7-11/group) and represented as mean ± SEM. n.s. = not significant

* p < 0.05

44

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

1

2

3

4

5

MP

O u

nit

s /

mg

tis

su

e * *

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

1

2

3

4

5

MP

O u

nit

s /

mg

tis

su

e *n .s .A B

45


myeloperoxidase activity following rectal administration of TcdAB.

Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia NF or GS/M trophozoites

for 7 days and subsequently administered 100 µg TcdAB. Colonic myeloperoxidase activity was

visualized via immunohistochemistry. All data are representative of two independent

experiments (n = 7-11/group).

46

Uninfected

Giardia NF

Giardia GS/M

Toxin

Giardia NF + Toxin

Giardia GS/M + Toxin

47

(Figure 2A) and numbers of MPO-positive cells were observed (Figure 3) when compared

against uninfected TcdAB-administered animals. Conversely , MPO activity levels (Figure 3B)

and numbers of MPO-positive cells (Figure 3) in Giardia GS/M-infected animals intra-rectally

instilled with 100 μg TcdAB were not significantly different from TcdAB-administered controls.

Collectively, these results demonstrated in vivo i.r. instillation of 100 μg TcdAB induced a rapid

accumulation of granulocytes within colonic tissues, and that was attenuated in animals infected

with Giardia NF trophozoites and not in animals infected with Giardia GS/M trophozoites.

3.2 Giardia NF infections attenuate colonic expression of several neutrophil-associated

mediators

A bead-based cytokine assay on colonic tissue samples was performed to determine if

Giardia NF infections attenuated protein levels of pro-inflammatory cytokines and chemokines

associated with granulocyte tissue recruitment. Compared to uninfected animals given PBS, i.r

instillation of 100 μg TcdAB into uninfected animals significantly increased tissue levels of

several PMN-associated mediators, including CXCL1, CXCL2, and IL-17 (Figure 4). Colonic

tissues collected from Giardia NF infected-animals administered TcdAB i.r. demonstrated

significantly decreased protein levels of CXCL1, CXCL2, and IL-17 (Figure 4A to C). Notably,

colonic protein levels of CXCL1, CXCL2, and IL-17 in Giardia GS/M-infected animals were

not significantly different from uninfected TcdAB animals (Figure 4D to F). These results

support above observations that granulocyte infiltration is not attenuated in Giardia GS/M

infected animals instilled with TcdAB, and suggest attenuation of granulocyte infiltration in

Giardia NF-infected animals given TcdAB resulted from decreased expression of PMN-

48

Figure 4. In vivo Giardia NF infections attenuate the colonic expression of several PMN-

associated mediators following rectal administration of 100 µg TcdAB.

Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia NF (A to C) or (D to F)

GS/M trophozoites for 7 days and subsequently administered 100 µg TcdAB for 3 hours.

Colonic cytokine expression levels were assessed via a mouse Luminex XMap assay for (A and

D) CXCL1, (B and E) CXCL2, and (C and F) IL-17. All data are representative of two


* p < 0.05

49

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

CX

CL

1 (

pg

/mL

)* *

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

CX

CL

1 (

pg

/mL

)

* n .s .

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

CX

CL

2 (

pg

/ml)

* *

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

1 0 0 0

2 0 0 0

3 0 0 0

CX

CL

2 (

pg

/ml)

* n .s .

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

IL-1

7 (

pg

/mL

)

* *

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

1

2

3

IL-1

7 (

pg

/mL

)

*

A

B

C

E

F

D

50

assassociated mediators. These results also indicated that Giardia-mediated attenuation of

granulocyte infiltration induced via TcdAB occurs in an isolate-dependent manner.

3.3 Giardia infections modulate colonic expression of several pro-inflammatory mediators

We also investigated whether Giardia infections attenuated colonic expression of other

inflammatory mediators. Compared to uninfected animals given PBS, i.r. instillation of TcdAB

to uninfected animals resulted in heightened protein levels of CCL2, IL-6, and leukocyte

inhibitory factor (LIF) (Figure 5) and IL-1β, IL-5, CXCL10, and CCL11 (Figure 6). Colonic

protein levels of CCL2, IL-6, and LIF were significantly reduced in Giardia NF-infected animals

given i.r. TcdAB (Figure 5A to C). In addition, colonic IL-12p70 levels were significantly

greater in uninfected TcdAB controls when compared to Giardia NF infected animals given i.r.

TcdAB (Figure 5D). Conversely, CCL2, IL-6, LIF, and IL-12p70 levels in Giardia GS/M-

infected animals administered TcdAB were not significantly different from uninfected animals

given TcdAB (Figure 5E to H). These results demonstrate that Giardia NF infections attenuate

colonic levels of CCL2, IL-6, LIF, and, potentially, IL-12p70 induced by 100 μg i.r. TcdAB in

an isolate-dependent manner. Giardia NF-infections were unable to attenuate heightened colonic

expression levels of IL-1β, IL-5, CXCL10 and CCL11 (Figure 6). Protein levels of several other

inflammatory mediators remained unchanged between all animal groups (Figure 7). Therefore,

Giardia NF infections specifically decreased colonic expression of PMN-associated mediators

(CXCL1, CXCL2, and IL-17) and other pro-inflammatory mediators (CCL2, IL-6, and LIF)

increased following TcdAB instillation.

51

Figure 5. In vivo Giardia NF infections attenuate the colonic expression of several pro-

inflammatory mediators following rectal administration of 100 µg TcdAB.

Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia (A to D) NF or (E to H)

GS/M trophozoites for 7 days and subsequently administered 100 µg TcdAB for 3 hours.

Colonic chemokine expression levels were assessed via a mouse Luminex XMap assay for (A

and E) CCL2 (B and F) IL-6, (C and G) leukocyte inhibitory factor (LIF), and (D and H) IL-

12p70. All data are representative of two independent experiments (n = 7-11/group) and

represented as mean ± SEM. n.s. = not significant * p < 0.05

52

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

5 0 0

1 0 0 0

1 5 0 0C

CL

2 (

pg

/ml)

* *

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

CC

L2

(p

g/m

l)

* n .s .

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

IL-6

(p

g/m

l)

* *

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

1 0 0 0

2 0 0 0

3 0 0 0

IL-6

(p

g/m

l)

*n .s .

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

1 0

2 0

3 0

4 0

5 0

LIF

(p

g/m

l)

* *

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

2 0

4 0

6 0

LIF

(p

g/m

l)

* n .s .

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

5

1 0

1 5

IL-1

2p

70

(p

g/m

L) *

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

5

1 0

1 5

IL-1

2p

70

(p

g/m

L)

A

B F

E

C G

D H

53

Figure 6. In vivo Giardia NF infections do not attenuate colonic expression of several pro-

inflammatory mediators upregulated following rectal administration of 100 µg TcdAB.

Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia NF trophozoites for 7 days

and subsequently administered 100 µg TcdAB for 3 hours. Colonic cytokine expression levels

were assessed via a mouse Luminex XMap assay for (A) IL-1β, (B) IL-5, (C) CXCL10, and (D)

CCL11. All data are representative of two independent experiments (n = 7-11/group) and


54

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

2 0

4 0

6 0

8 0

IL-1

(pg

/mL

)

* n .s .

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

2

4

6

8

1 0

IL-5

(p

g/m

L)

*n .s .

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

1 0 0

2 0 0

3 0 0

4 0 0

CX

CL

10

(p

g/m

L) *

n .s .

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

1 0 0

2 0 0

3 0 0

4 0 0

CC

L1

1 (

pg

/mL

)

*n .s .

A

C D

B

55

Figure 7. In vivo Giardia NF infections do not modulate colonic expression of several

inflammatory mediators unaffected by i.r. administration of 100 µg TcdAB.

Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia NF trophozoites for 7 days

and subsequently administered 100 µg TcdAB for 3 hours. Colonic cytokine expression levels

were assessed via a mouse Luminex XMap assay for (A) IL-1α, (B) IL-2, (C) IL-7, (D) IL-10,

(E) IL-12p40, (F) IL-15, (G) IFN-γ, and (H) VEGF. All data are representative of two


* p < 0.05

56

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

2 0

4 0

6 0

8 0

1 0 0

IL-1

(pg

/mL

)

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

5

1 0

1 5

2 0

2 5

IL-2

(p

g/m

L)

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

2

4

6

8

1 0

IL-7

(p

g/m

L)

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

1 0

2 0

3 0

IL-1

0

(pg

/mL

)

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

5 0

1 0 0

1 5 0

IL-1

2p

40

(p

g/m

L)

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

2 0

4 0

6 0

IL-1

5

(pg

/mL

)

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

1 0

2 0

3 0

4 0

5 0

IFN

(p

g/m

L)

Co

ntr

ol

To

xin

Gia

rdia

NF

Gia

rdia

NF

+T

oxin

0

5

1 0

1 5

2 0

VE

GF

(p

g/m

L)

A

D

G

B

E

H

C

F

57

Bead-based cytokine analysis of colonic tissues indicated Giardia GS/M infections

upregulated colonic expression levels of a subset of pro-inflammatory mediators, following

TcdAB. In uninfected animals, i.r. administration of TcdAB significantly increased colonic

expression levels of IL-1β and CXCL10 (Figure 8), and protein levels of these mediators were

significantly increased in Giardia GS/M-infected animals given TcdAB compared to uninfected

TcdAB animals (Figure 8). Giardia GS/M-infected animals given TcdAB also demonstrated

increased colonic protein levels of several inflammatory mediators not initially increased via i.r.

instillation of TcdAB (Figure 9). This did not appear to be a global increase in expression of

inflammatory mediators, as CCL3 and CCL11 concentrations were not significantly different

between Giardia GS/M-infected animals given TcdAB and uninfected TcdAB controls (Figure

10). Several inflammatory mediators also remained unchanged between all experimental groups

(Figure 11). These results suggest Giardia GS/M infections enhance expression of certain pro-

inflammatory cytokines and chemokines following i.r. instillation of TcdAB. These data

indicated Giardia infections enhanced or decreased the expression of inflammatory mediators

following administration of a bacterial toxin, and these events appeared to be isolate-specific.

3.4 Giardia trophozoites attenuate expression of inflammatory mediators from inflamed ex

vivo human mucosal biopsy tissues

We next studied whether live Giardia parasites could attenuate inflammatory mediators

from inflamed human colonic tissue. Therefore, Giardia NF trophozoites were co-incubated ex

vivo with colonic mucosal biopsy tissues collected from the descending colon of patients with

active CD, where supernatants and biopsy tissue homogenates were analyzed via a bead-based

58


inflammatory mediators upregulated by i.r. administration of 100 µg TcdAB.

Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia GS/M trophozoites for 7

days and subsequently administered 100 µg TcdAB for 3 hours. Colonic cytokine expression

levels were assessed via a mouse Luminex XMap assay for (A) IL-1β and (B) CXCL10. All data

are representative of two independent experiments (n = 7-11/group) and represented as mean ±

SEM. n.s. = not significant * p < 0.05

59

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

IL-1

(pg

/mL

)

* *

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

5 0 0

1 0 0 0

1 5 0 0

CX

CL

10

(p

g/m

L) * *

A B

60





levels were assessed via a mouse Luminex XMap assay for (A) IL-2, (B) IL-5, (C) IL-15, (D)

IFN-γ, (E) CXCL9, (F) CCL4, and (G) VEGF. All data are representative of two independent

experiments (n = 7-11/group) and represented as mean ± SEM. n.s. = not significant * p < 0.05

61

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

1 0

2 0

3 0

4 0

5 0IL

-2

(pg

/mL

)*

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

5

1 0

1 5

IL-5

(p

g/m

L)

*

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

2 0

4 0

6 0

8 0

1 0 0

IL-1

5

(pg

/mL

)

*

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

2 0

4 0

6 0

8 0

IFN

(p

g/m

L)

*

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

CX

CL

9 (

pg

/mL

)

*

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

5

1 0

1 5

2 0

2 5

CC

L4

(p

g/m

L)

*

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

1 0

2 0

3 0

VE

GF

(p

g/m

L) *

A

D

G

B

E

C

F

62

Figure 10. In vivo Giardia GS/M infections do not modulate colonic expression of two

inflammatory mediators upregulated by i.r. administration of 100 µg TcdAB.



levels were assessed via a mouse Luminex XMap assay for (A) CCL3, and (B) CCL11. All data

are representative of two independent experiments (n = 7-11/group) and represented as mean ±


63

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

1 0

2 0

3 0

CC

L3

(p

g/m

L)

* n .s .

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

CC

L1

1 (

pg

/mL

)

*n .s .A B

64

Figure 11. In vivo Giardia GS/M infections do not modulate colonic expression of




levels were assessed via a mouse Luminex XMap assay for (A) IL-1α, (B) IL-7, (C) IL-9, (D)

IL-10, (E) IL-12p40, and (F) TNFα. All data are representative of two independent experiments

(n = 7-11/group) and represented as mean ± SEM. n.s. = not significant * p < 0.05

65

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

5 0

1 0 0

1 5 0

IL-1

(pg

/mL

)

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

5

1 0

1 5

IL-7

(p

g/m

L)

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

2 0

4 0

6 0

8 0

IL-9

(p

g/m

L)

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

1 0

2 0

3 0

IL-1

0 (

pg

/mL

)

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

5 0

1 0 0

1 5 0

IL-1

2p

40

(p

g/m

L)

Co

ntr

ol

To

xin

Gia

rdia

GS

M

Gia

rdia

GS

M+T

oxin

0

2

4

6

8

TN

F

(p

g/m

l)

A

D

B

E

C

F

66

cytokine assay. Supernatants collected from co-incubation of Giardia NF trophozoites with

inflamed biopsy tissues displayed significantly reduced supernatant levels of several PMN-

associated mediators, including CXCL8, growth-related oncogene (GRO) family proteins

(CXCL1-3), IL-17A, G-CSF, and GM-CSF (Figure 12). Interestingly, decreased tissue

expression of CXCL8 was observed in tissue homogenates collected from samples co-incubated

with Giardia NF trophozoites, while levels of CXCL1-3, IL-17A, G-CSF, and GM-CSF were

not significantly different (Figure 13). Therefore, with the exception of CXCL8, attenuation of

PMN-related inflammatory mediators by Giardia NF trophozoites appeared to largely occur

following their release into supernatants. Furthermore, these results demonstrate that Giardia NF

trophozoites are capable of attenuating the expression of a variety of PMN-associated mediators

in various models of inflammation. As in vivo Giardia NF infections attenuated colonic levels of

CCL2 (Figure 5A), we investigated whether live trophozoites could attenuate protein expression

of other chemokines. Supernatant levels of CCL2 to CCL5, CCL7, CCL11, CCL22, CXCL10,

and CX3CL1 were significantly reduced in biopsy tissues co-incubated with Giardia NF

trophozoites compared against supernatant levels collected from biopsy tissues incubated in the

absence of the parasite (Figure 14). Tissue homogenate levels of these mediators were not

significantly different when biopsy tissues were incubated in the presence or absence of Giardia

NF trophozoites (Figure 15). Therefore, attenuation of mediators by Giardia NF trophozoites

occurred following release into supernatants. These results also suggested Giardia NF

trophozoites attenuated CCL2 in different experimental models of inflammation.

67

Figure 12. Giardia NF trophozoites attenuate supernatant levels of several PMN-associated

mediators from descending colon mucosal biopsy tissues.

Human descending colon mucosal biopsy tissues obtained from areas of active inflammation

from patients with active Crohn’s disease (CD) were incubated with 5.0x106 Giardia NF

trophozoites for 6 hours. Supernatant levels of (A) CXCL8, (B) CXCL1-3, (C) IL-17A (D) G-

CSF, and (E) GM-CSF were determined via a bead-based cytokine assay. All data are

representative of three independent experiments (n = 6/group) and represented as min to max. * p

< 0.05

68

Co

ntr

ol

Gia

rdia

NF

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

CX

CL

8 (

pg

/mL

)

*

Co

ntr

ol

Gia

rdia

NF

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

CX

CL

1-3

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

5

1 0

1 5

2 0

2 5

IL-1

7A

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

G-C

SF

( p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

4 0

5 0

GM

-CS

F (

pg

/mL

)

*

A B

C D

E

69

Figure 13. Giardia NF trophozoites attenuate CXCL8 tissue homogenate levels from

descending colon mucosal biopsy tissue homogenates.



trophozoites for 6 hours. Tissue homogenate levels of (A) CXCL8, (B) CXCL1-3, (C) IL-17A,

(D) G-CSF, and (E) GM-CSF were determined via a bead-based cytokine assay. All data are

representative of three independent experiments (n = 6/group) and represented as min to max.

n.s. = not significant * p < 0.05

70

Co

ntr

ol

Gia

rdia

NF

0

5 0 0

1 0 0 0

1 5 0 0

CX

CL

8 (

pg

/mL

)

*

Co

ntr

ol

Gia

rdia

NF

0

1 0 0 0

2 0 0 0

3 0 0 0

CX

CL

1-3

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

4 0

IL-1

7A

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2 0

4 0

6 0

G-C

SF

( p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

4 0

GM

-CS

F (

pg

/mL

)

n .s .

A B

C D

E

71

Figure 14. Giardia NF trophozoites attenuate supernatant levels of several chemokines

from descending colon mucosal biopsy tissues.



trophozoites for 6 hours. Supernatant levels of (A) CCL2, (B) CCL3, (C) CCL4, (D) CCL5, (E)

CCL7, (F) CCL11, (G) CCL22, (H) CXCL10, and (I) CX3CL1 were determined via a bead-

based cytokine assay. All data are representative of three independent experiments (n = 6/group)

and represented as min to max. * p < 0.05

72

Co

ntr

ol

Gia

rdia

NF

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

CC

L2

(p

g/m

l)

*

Co

ntr

ol

Gia

rdia

NF

0

1 0 0

2 0 0

3 0 0

4 0 0

CC

L3

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

CC

L4

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

5 0

1 0 0

1 5 0

2 0 0

CC

L5

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

2 0

4 0

6 0

CC

L7

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

4 0

CC

L1

1 (

pg

/mL

)

*

Co

ntr

ol

Gia

rdia

NF

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

CC

L2

2 (

pg

/mL

)

*

Co

ntr

ol

Gia

rdia

NF

0

2 0 0

4 0 0

6 0 0

8 0 0

CX

CL

10

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

CX

3C

L1

(p

g/m

L)

*

A

D

B

E

C

F

G H I

73


chemokines from descending colon mucosal biopsy tissues.



trophozoites for 6 hours. Tissue homogenate levels of (A) CCL2, (B) CCL3, (C) CCL4, (D)

CCL5, (E) CCL7, (F) CCL11, (G) CCL22, (H) CXCL10, and (I) CX3CL1 were determined via a

bead-based cytokine assay. All data are representative of three independent experiments (n =

6/group) and represented as min to max. n.s. = not significant

74

Co

ntr

ol

Gia

rdia

NF

0

5 0 0

1 0 0 0

1 5 0 0

CC

L2

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0 0

2 0 0

3 0 0

4 0 0

CC

L3

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2 0 0

4 0 0

6 0 0

CC

L4

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

CC

L5

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2 0

4 0

6 0

CC

L7

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2 0 0

4 0 0

6 0 0

8 0 0

CC

L1

1 (

pg

/mL

)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0 0

2 0 0

3 0 0

4 0 0

CC

L2

2 (

pg

/mL

)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

CX

CL

10

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0 0

2 0 0

3 0 0

4 0 0

CX

3C

L1

(p

g/m

L)

n .s .

A

D

B

E

C

F

G H I

75

Compared against supernatant levels collected from biopsy tissues incubated in the absence of

the parasite, co-incubation of Giardia NF trophozoites with inflamed biopsy tissues ex vivo

resulted in significant attenuation of IL-6, IL-7, IL-10, IL-12p40, IL-12p70, IFN-α, and vascular

endothelial growth factor (VEGF) (Figure 16). Tissue homogenate levels of IL-1α, IL-6, IL-

12p40, and TNF-α were significantly reduced in biopsy tissue homogenates co-incubated with

Giardia NF trophozoites, compared to biopsy tissue homogenates incubated in the absence of

trophozoites (Figure 17). These results suggest that Giardia NF trophozoites attenuate secreted

IL-7, IL-10, IL-12p70, IFN-α, and VEGF from inflamed mucosal biopsy tissues ex vivo, while

also attenuating tissue levels of IL-1α, IL-6, IL-12p40, and TNF-α. These results also suggest

that Giardia infections are capable of attenuating IL-6 and IL-12p70 in several experimental

models of inflammation. Our data indicated Giardia NF trophozoites did not attenuate the

expression of several pro-inflammatory cytokines (Figure 18 and Figure 19) or growth factors

(Figure 20 and Figure 21) within supernatants or biopsy tissue homogenates. These results

suggest Giardia NF attenuated several pro-inflammatory mediators when co-incubated with

inflamed colonic mucosal biopsy tissues ex vivo, but the inability to decrease expression of all

mediators suggests this may be targeted towards certain inflammatory mediators. Observations

that some inflammatory mediators are preferentially degraded within supernatants while others

within tissues suggests that Giardia NF trophozoites possess multiple mechanisms capable of

attenuating expression of inflammatory mediators in inflamed intestinal mucosal biopsy tissues.

76

Figure 16. Giardia NF trophozoites attenuate supernatant levels of several inflammatory

mediators from descending colon mucosal biopsy tissues.



trophozoites for 6 hours. Supernatant levels of (A) IL-1α, (B) IL-6, (C) IL-7, (D) IL-10, (E) IL-

12p40, (F) IL-12p70, (G) IFNα, (H) IFNγ, (I) TNFα, (J) sCD40L, and (K) VEGF were

determined via a bead-based cytokine assay. All data are representative of three independent

experiments (n = 6/group) and represented as min to max. n.s. = not significant * p < 0.05

77

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

4 0

5 0

IL-1

(pg

/mL

)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

IL-6

(p

g/m

l)

*

Co

ntr

ol

Gia

rdia

NF

0

1

2

3

4

5

IL-7

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

2

4

6

8

IL-1

0

(pg

/mL

)

*

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

4 0

IL-1

2p

40

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

5

1 0

1 5

IL-1

2p

70

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

2 0

4 0

6 0

IFN

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

5

1 0

1 5

2 0

2 5

IFN

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2 0

4 0

6 0

8 0

1 0 0

TN

F

(p

g/m

L)

n .s .

A

D

B

E

C

F

G H I

78

Co

ntr

ol

Gia

rdia

NF

0

1 0 0

2 0 0

3 0 0

4 0 0

sC

D4

0L

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

2 0

4 0

6 0

8 0

VE

GF

(pg

/mL

)*J K

79

Figure 17. Giardia NF trophozoites attenuate tissue homogenate levels of several pro-

inflammatory mediators from descending colon mucosal biopsy tissues.



trophozoites for 6 hours. Tissue homogenate levels of (A) IL-1α, (B) IL-6, (C) IL-7, (D) IL-10,

(E) IL-12p40, (F) IL-12p70, (G) IFNα, (H) IFNγ, (I) TNFα, (J) sCD40L, and (K) VEGF were

determined via a bead-based cytokine assay. All data are representative of three independent

experiments (n = 6/group) and represented as min to max. n.s. = not significant * p < 0.05

80

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

IL-1

(pg

/mL

)

*

Co

ntr

ol

Gia

rdia

NF

0

5 0

1 0 0

1 5 0

2 0 0

IL-6

(p

g/m

L)

*

Co

ntr

ol

Gia

rdia

NF

0

2

4

6

8

IL-7

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0 .0

0 .5

1 .0

1 .5

2 .0

IL-1

0

(pg

/mL

)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

4 0

5 0

IL-1

2p

40

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

5

1 0

1 5

2 0

IL-1

2p

70

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2 0

4 0

6 0

8 0

IFN

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

IFN

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

4 0

TN

F

(p

g/m

L)

*

A

D

B

E

C

F

G H I

81

Co

ntr

ol

Gia

rdia

NF

0

1 0 0

2 0 0

3 0 0

4 0 0

sC

D4

0L

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

VE

GF

(pg

/mL

)n .s .J K

82

Figure 18. Giardia NF trophozoites do not attenuate supernatant levels of several




trophozoites for 6 hours. Supernatant levels of (A) IL-1ra, (B) IL-1β, (C) IL-2, (D) IL-13, (E) IL-

15, (F) IFNγ, and (G) TNFβ were determined via a bead-based cytokine assay. All data are


n.s. = not significant

83

Co

ntr

ol

Gia

rdia

NF

0

1 0 0

2 0 0

3 0 0

4 0 0

IL-1

ra

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

IL-1

(pg

/ml)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2

4

6

8

1 0

IL-2

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

IL-1

3

(pg

/mL

)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

5

1 0

1 5

2 0

IL-1

5 (

pg

/mL

)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2

4

6

8

1 0

TN

F

(p

g/m

l)

n .s .

A

D

B

E

C

F

84





trophozoites for 6 hours. Tissue homogenate levels of (A) IL-1ra, (B) IL-1β, (C) IL-2, (D) IL-13,

(E) IL-15, (F) TNFβ were determined via a bead-based cytokine assay. All data are



85

Co

ntr

ol

Gia

rdia

NF

0

2 0 0

4 0 0

6 0 0

8 0 0

IL-1

ra

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

4 0

IL-1

(pg

/ml)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2

4

6

8

IL-2

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2

4

6

IL-1

3

(pg

/mL

)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

5

1 0

1 5

IL-1

5 (

pg

/mL

)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2

4

6

8

1 0

TN

F

(p

g/m

l)n .s .

A

D

B

E

C

F

86

Figure 20. Giardia NF trophozoites do not attenuate supernatant levels of several growth

factors from descending colon mucosal biopsy tissues.



trophozoites for 6 hours. Supernatant levels of (A) FGF, (B) Flt-3L, (C) PDGF-AA, (D) PDGF-

BB, and (E) TGFα were determined via a bead-based cytokine assay. All data are representative

of three independent experiments (n = 6/group) and represented as min to max. n.s. = not

significant

87

Co

ntr

ol

Gia

rdia

NF

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

FG

F (

pg

/mL

)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2 0

4 0

6 0

8 0

1 0 0

Flt

-3L

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

5

1 0

1 5

2 0

PD

GF

-AA

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

5

1 0

1 5

PD

GF

-BB

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2 0

4 0

6 0

8 0

TG

F

(p

g/m

L)

n .s .

A

D

B

E

C

88


growth factors released from descending colon mucosal biopsy tissues.



trophozoites for 6 hours. Tissue homogenate levels of (A) FGF, (B) Flt-3L, (C) PDGF-AA, (D)

PDGF-BB, and (E) TGFα were determined via a bead-based cytokine assay. All data are



89

Co

ntr

ol

Gia

rdia

NF

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

FG

F (

pg

/mL

)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

4 0

Flt

-3L

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

1 0

2 0

3 0

4 0

5 0

PD

GF

-AA

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

2 0

4 0

6 0

8 0

1 0 0

PD

GF

-BB

(p

g/m

L)

n .s .

Co

ntr

ol

Gia

rdia

NF

0

5 0

1 0 0

1 5 0

TG

F

(p

g/m

L)

n .s .

A

D

B

E

C

90

3.5 Giardia trophozoites attenuate intestinal epithelial CXCL8

The intestinal mucosa in the overwhelming majority of Giardia-infected individuals is

devoid of signs of overt intestinal inflammation (53). Recent reports also suggest that Giardia

infections attenuate their host’s pro-inflammatory response via unknown mechanisms (63, 64).

The intestinal epithelium represents the primary point of contact between Giardia trophozoites

and its host and previous reports from our lab and others have demonstrated that Giardia

trophozoites induce pathophysiological events within IECs (25, 26, 50, 177). IECs also

participate in the induction of acute intestinal inflammatory responses by secreting chemokines

such as CXCL8 (140-142). However, previous studies have demonstrated that Giardia

trophozoites do not induce CXCL8 secretion from in vitro IEC monolayers (42, 140). Therefore,

initial experiments focused on ascertaining whether co-incubation of Giardia trophozoites with

IECs resulted in the attenuation of CXCL8 secretion induced by the administration of a pro-

inflammatory stimulus.

3.5.1 Giardia NF trophozoites attenuate IL-1β-induced CXCL8 secretion from ex vivo small

intestinal mucosal biopsy tissues

Initial experiments were performed to determine if Giardia assemblage A NF

trophozoites were capable of attenuating IL-1β-induced CXCL8 secretion from human small

intestinal mucosal biopsy tissues. This isolate has previously been used in our lab to characterize

the pathophysiological effects of Giardia infections (25, 51). Adapted from previous studies

(176), we developed a novel method of modeling Giardia interactions with intestinal tissues,

whereby ex vivo human small intestinal mucosal biopsies were co-incubated with 5.0x106

Giardia NF trophozoites for 2 hours, with or without subsequent administration of 1.0 ng/mL IL-

91

1β, and incubated for an additional 4 hours. As determined via ELISA, IL-1 administration to

biopsy tissues significantly increased the levels of CXCL8 detected within supernatants, while

CXCL8 supernatant levels in biopsy tissues co-incubated with Giardia NF trophozoites and

subsequently administered IL-1 were significantly attenuated compared to IL-1β-stimulated

specimens (Figure 22A). These results demonstrated that assemblage A Giardia NF isolate

trophozoites attenuated IL-1-induced CXCL8 secretion from small intestinal mucosal biopsy

tissues. Similar results were observed in the biopsy tissues homogenates: CXCL8 concentrations

in groups containing Giardia NF trophozoites in the presence or absence of IL-1β were

significantly lower than biopsy tissues only administered IL-1β (Figure 22B). These results

demonstrated that assemblage A Giardia NF isolate trophozoites attenuated IL-1-induced

CXCL8 secretion from small intestinal mucosal biopsy tissues.

3.5.2 Giardia trophozoites attenuate IL-1β-induced CXCL8 secretion from in vitro Caco-2

monolayers

Similar experiments to those performed above were repeated in vitro using the human

Caco-2 intestinal epithelial cell line; this cell line has previously been used to delineate

pathophysiological events induced by Giardia and other enteropathogens in human enterocytes

(40, 42) and has well defined CXCL8 signaling pathways in response to host- and pathogen-

induced pro-inflammatory stimuli such as IL-1 and Salmonella typhimurium (142, 191). In

order to determine if attenuation of IL-1β-induced CXCL8 secretion was dependent on the

concentration of Giardia trophozoites, Caco-2 monolayers were co-incubated with Giardia NF

trophozoites at a multiplicity of infection (MOI) of 1:1, 10:1, or 50:1 for 2 hours, administered

92

Figure 22. Giardia NF trophozoites attenuate CXCL8 from small intestinal mucosal biopsy

tissues.

Human small intestinal biopsy tissues obtained from the terminal ileum of patients with Crohn’s

disease in remission were incubated with 5.0x106 Giardia NF trophozoites for 2 hours and

subsequently administered 1.0 ng/mL of recombinant IL-1 for 4 hours. Biopsy tissues

administered IL-1β in the absence of Giardia trophozoites was used as a positive control for

supernatant CXCL8 levels. CXCL8 levels in (A) supernatants and (B) biopsy tissues were

determined via ELISA. (C) Human small intestinal biopsy tissues obtained from areas of active

inflammation in the terminal ileum of patients with active Crohn’s disease were incubated with

5.0x106 Giardia NF trophozoites for 6 hours. Supernatant CXCL8 levels were determined by

ELISA. All data are representative of at least seven independent experiments (n = 3-9/group) and

represented as mean ± SEM. * p < 0.05.

93

Co

ntr

ol

IL-1

Gia

rdia

NF

Gia

rdia

NF

+ IL

-1

0

5 0

1 0 0

1 5 0

2 0 0

CX

CL

8 (

pg

/mL

)

* *

Co

ntr

ol

IL-1

Gia

rdia

NF

Gia

rdia

NF

+ IL

-1

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

CX

CL

8 (

pg

/mL

)

*

Co

ntr

ol

Gia

rdia

NF

0

1 0 0 0

2 0 0 0

3 0 0 0

CX

CL

8 (

pg

/mL

)

*

A B

C

94

1.0 ng/mL IL-1β, and incubated for an additional 4 hours. Our results demonstrate that CXCL8

supernatant levels from Caco-2 monolayers co-incubated with all MOI’s of Giardia NF isolate

trophozoites administered IL-1β were significantly reduced compared to control monolayers only

administered IL-1 (Figure 23). Levels of CXCL8 detected in IL-1-stimulated Caco-2

supernatants co-incubated with Giardia NF isolate trophozoites at an MOI of 50:1 were also

significantly reduced from IL-1-stimulated Caco-2 monolayers incubated with trophozoites at

an MOI of 1:1 and 10:1 (Figure 23). Therefore, attenuation of IL-1β-induced CXCL8 secretion

in Caco-2 monolayers occurs in a Giardia trophozoite dose-dependent manner. As our results

demonstrated an attenuation of CXCL8 secretion in ex vivo small intestinal mucosal biopsy

tissues and in vitro Caco-2 monolayers, Giardia-mediated attenuation of IL-1-induced CXCL8

secretion is, at least partially, mediated via attenuation of IEC CXCL8 secretion. Moreover, the

experiments in vitro using Caco-2 cells validated this model to further investigate the mechanism

via which Giardia trophozoites attenuate CXCL8 secretion in IECs.

Experiments were performed to determine if different Giardia trophozoite isolates were

capable of attenuating supernatant CXCL8 levels, induced via administration of IL-1β, from in

vitro Caco-2 monolayers. Therefore, Caco-2 monolayers were co-incubated with Giardia NF,

WB, or GS/M trophozoites at an MOI of 10:1 for 2 hours and then incubated with IL-1β for an

additional 4 hours. After the 6-hour incubation period, CXCL8 supernatant levels collected from

Caco-2 monolayers co-incubated with Giardia NF or WB trophozoites and administered IL-1β

were significantly reduced compared to monolayers administered IL-1β alone (Figure 24A).

Interestingly, CXCL8 supernatant levels collected from Caco-2 monolayers co-incubated with

Giardia GS/M trophozoites and administered IL-1β were not significantly different from groups

95

Figure 23. Giardia NF trophozoites attenuate IL-1β-induced CXCL8 secretion from in vitro

Caco-2 monolayers in a dose-dependent manner.

Caco-2 monolayers were co-incubated with Giardia NF isolate trophozoites at an MOI of 1, 10,

or 50:1 for 2 hours and incubated with IL-1β for 4 hours. CXCL8 supernatant levels were

determined via ELISA. All data are representative of at least three independent experiments (n =

3/group) and represented as mean ± SEM. * p < 0.05

96

CX

CL

8 (

pg

/mL

)

Co

ntr

ol

IL-1

Gia

rdia

NF

(M

OI:

1)

Gia

rdia

NF

(M

OI:

1)

+ IL

-1

Gia

rdia

NF

(M

OI:

10)

Gia

rdia

NF

(M

OI:

10)

+ IL

-1

Gia

rdia

NF

(M

OI:

50)

Gia

rdia

NF

(M

OI:

50)

+ IL

-1

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

**

*

97

Figure 24. Assemblage A and B Giardia trophozoites attenuate IL-1β-induced CXCL8

secretion from in vitro Caco-2 monolayers.

(A) Caco-2 monolayers were co-incubated with Giardia NF, WB, or GS/M trophozoite isolates

(MOI 10:1) for 2 hours and incubated with IL-1β for 4 hours. (B) Caco-2 monolayers were co-

incubated with Giardia NF, WB, or GS/M trophozoite isolates (MOI 10:1) for 24 hours and

incubated with IL-1β for another 4 hours. CXCL8 levels were determined via ELISA. All data

are representative of at least two independent experiments (n = 2-3/group) and represented as

mean ± SEM. n.s. = not significant * p < 0.05

98

CX

CL

8 (

pg

/mL

)

Co

ntr

ol

IL-1

Gia

rdia

NF

Gia

rdia

NF

+ IL

-1

Gia

rdia

WB

Gia

rdia

WB

+ IL

-1

Gia

rdia

GS

M

Gia

rdia

GS

M +

IL

-1

0

5 0 0

1 0 0 0

1 5 0 0

**

n .s .

Co

ntr

ol

IL-1

Gia

rdia

NF

Gia

rdia

NF

+ IL

-1

Gia

rdia

WB

Gia

rdia

WB

+ IL

-1

Gia

rdia

GS

/M

Gia

rdia

GS

/M +

IL

-1ß

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

CX

CL

8 (

pg

/mL

)*

*

*A B

99

given IL-1 alone (Figure 24A). As Giardia GS/M trophozoites replicate slower within in vitro

culture tubes, we repeated our IL-1β experiments after a longer initial incubation period.

Therefore, Caco-2 monolayers were co-incubated with Giardia NF, WB, GS/M trophozoites for

24 hours and then incubated with IL-1β for an additional 4 hours. Supernatant levels of CXCL8

were significantly reduced in supernatants collected from Caco-2 monolayers co-incubated with

Giardia NF, WB, or GS/M trophozoites compared to monolayers only administered IL-1β and

these levels were not significantly different from each other (Figure 24B). Therefore, multiple

Giardia isolates were capable of attenuating supernatant levels of CXCL8 released by Caco-2

monolayers following administration of pro-inflammatory IL-1β. Follow-up experiments sought

to determine if direct contact between Giardia trophozoites and in vitro Caco-2 monolayers was

required for attenuation of supernatant CXCL8 to occur. As a result, Caco-2 monolayers were

co-incubated with Giardia NF trophozoites in direct contact with monolayers or separated via

0.4 µm transwells for 24 hours and exposed to IL-1β for 4 hours. ELISA analysis demonstrated

that CXCL8 supernatant levels were significantly reduced compared to control monolayers only

administered IL-1β when Giardia NF trophozoites were directly contacting Caco-2 monolayers

or when separated by 0.4 µm transwells and subsequently administered IL-1β, and these groups

were not significantly different from each other (Figure 25). Therefore, Giardia-mediated

attenuation of IL-1β-induced supernatant CXCL8 levels did not require direct contact between

Caco-2 monolayers Giardia trophozoites.

100

Figure 25. Attenuation of supernatant CXCL8 induced by IL-1β in Caco-2 monolayers

does not require direct contact between Giardia NF trophozoites and in vitro Caco-2

monolayers.

Caco-2 monolayers were co-incubated with Giardia NF trophozoites (MOI 10:1) in direct

contact with monolayers (Giardia NF) or separated by 0.4 µm transwells (Giardia NF (T)) for

24 hours and incubated with IL-1β for another 4 hours. CXCL8 levels were determined via

ELISA. All data are representative of at least three independent experiments (n = 3/group) and

represented as mean ± SEM. * p < 0.05

101

Co

ntr

ol

IL-1

Gia

rdia

NF

Gia

rdia

NF

+ IL

-1

Gia

rdia

NF

(T

)

Gia

rdia

NF

(T

) + IL

-1

0

5 0 0

1 0 0 0

1 5 0 0

CX

CL

8 (

pg

/mL

) **

102

3.6 Giardia trophozoites attenuate Salmonella typhimurium-induced CXCL8 secretion

from in vitro Caco-2 monolayers

CXCL8 secretion also occurs in response to a variety of pathogens and pathogen-derived

pro-inflammatory stimuli. Previous reports have demonstrated that administration of S.

typhimurium 14028 to intestinal epithelial monolayers results in CXCL8 secretion (144).

Therefore, experiments were performed to determine if assemblage A Giardia NF trophozoites

were capable of attenuating CXCL8 secretion in epithelial cells following exposure to the

enteropathogen S. typhimurium. Caco-2 monolayers were co-incubated with Giardia NF

trophozoites at an MOI of 10:1 for 2 hours, administered S. typhimurium at an MOI of 100:1,

and incubated for an additional 5 hours. As determined via ELISA, Giardia NF trophozoites

significantly inhibited the production of CXCL8 induced by S. typhimurium (Figure 26A). The

number of S. typhimurium CFUs associated with Caco-2 monolayers was not significantly

different whether experiments were performed in the presence or absence of Giardia NF

trophozoites, excluding the possibility that the observations resulted from a reduced microbial

load (Figure 26B). Therefore, Giardia NF trophozoites directly attenuated CXCL8 secretion

from IECs in response to the enteropathogen S. typhimurium and this did not occur via Giardia

anti-microbial effects.

3.7 Giardia trophozoites attenuate IL-1β-induced CXCL8 secretion via a caspase-3

independent mechanism

Giardia infections are associated with heightened rates of caspase-3-dependent IEC

apoptosis (25, 26, 56). As induction of caspase-3 has been shown to inactivate elements of the

NF-κB pathway (192-194) and consequently inhibit CXCL8 production (195), we investigated

103

Figure 26. Giardia NF trophozoites attenuate Salmonella-induced CXCL8 secretion from in

vitro Caco-2 monolayers.

(A) Caco-2 monolayers were co-incubated with Giardia NF isolate trophozoites (MOI 10:1) for

2 hours and subsequently administered S. typhimurium (MOI 100:1) for 5 hours. (B) Caco-2

monolayers were lysed in sterile RIPA and spot-plated onto LB agar to determine the number of

S. typhimurium-associated CFUs. CXCL8 levels were determined via ELISA. All data are

representative of at least two independent experiments (n = 2-3/group) and represented as mean

± SEM. n.s. = not significant * p < 0.05

104

Co

ntr

ol

Salm

onella

Gia

rdia

NF

Gia

rdia

NF

+ S

alm

onella

0

5 0

1 0 0

1 5 0

CX

CL

8 (

pg

/mL

)

*

*

*

Co

ntr

ol

Salm

onella

Gia

rdia

NF

Gia

rdia

NF

+ S

alm

onella

0

2

4

6

8

Sa

lmo

ne

lla

pla

tec

ou

nts

(lo

g

CF

U/m

L)

n .s .A B

105

whether attenuation of IL-1β-induced CXCL8 secretion by Giardia trophozoites was mediated

by induction of caspase-3 within in vitro Caco-2 monolayers. Therefore, pro-apoptotic Giardia

NF trophozoites were co-incubated with Caco-2 monolayers for 24 hours in the presence of a

caspase-3 inhibitor (Z-DEVD-FMK) and subsequently administered IL-1β for 4 hours. A 24-

hour incubation period was chosen because previous experiments have demonstrated caspase-3

activation at this timepoint (25). In the presence or absence of a caspase-3 inhibitor, CXCL8

supernatant levels from the co-incubation of Giardia NF trophozoites, Caco-2 monolayers, and

IL-1β were significantly reduced from control monolayers only administered IL-1β (Figure 27);

moreover, CXCL8 supernatant levels between these two groups were not significantly different

from each other. Therefore, attenuation of IL-1β-induced CXCL8 from in vitro Caco-2

monolayers occurred via a caspase-3-independent mechanism.

3.8 Attenuation of CXCL8 by Giardia trophozoites involves parasite-mediated degradation

of CXCL8

As our results demonstrated that induction of caspase-3 by Giardia trophozoites did not

appear to be involved in attenuation of IL-1β-induced CXCL8 secretion, follow-up experiments

sought to determine if attenuation of CXCL8 secretion by Giardia trophozoites resulted from

decreased CXCL8 mRNA transcription. Therefore, Caco-2 monolayers were co-incubated with

assemblage A (NF) or assemblage B (GS/M) Giardia trophozoites for 2 hours and then

administered 1.0 ng/mL IL-1β. CXCL8 mRNA levels relative to loading control β-2

microglobulin (2M) were determined via real-time reverse transcriptase PCR after 1 and 4

hours post-IL-1β administration to determine if assemblage A and/or B Giardia trophozoites

106

Figure 27. Giardia NF trophozoites attenuate IL-1β-induced CXCL8 secretion via a

caspase-3-independent mechanism.

Caco-2 monolayers were co-incubated with Giardia NF isolate trophozoites for 24-hours in the

presence or absence of a caspase-3 inhibitor (Z-DEVD-FMK : 50 µM) and subsequently

administered IL-1β (1.0 ng/mL) for 4 hours. CXCL8 levels were determined via ELISA. All data

are representative of three independent experiments (n = 3/group) and represented as mean ±


107

Co

ntr

ol

IL-1

Gia

rdia

NF

Gia

rdia

NF

+ IL

-1

Co

ntr

ol (Z

-DE

VD

)

IL-1 (

Z-D

EV

D)

Gia

rdia

NF

(Z

-DE

VD

)

Gia

rdia

NF

+ IL

-1 (Z

-DE

VD

)

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

CX

CL

8 (

pg

/mL

)

*

*

*

n .s .

108

modulated CXCL8 mRNA transcription and/or stability. After 1-hour incubation with IL-1β,

CXCL8 mRNA levels from Caco-2 monolayers given IL-1 alone were not significantly

different from levels found in monolayers that had been initially co-incubated with Giardia NF

or GS/M trophozoites (Figure 28A). After 4-hour incubation with IL-1β, CXCL8 mRNA levels

in monolayers co-incubated with Giardia NF trophozoites were significantly greater than control

monolayers administered IL-1 and monolayers initially co-incubated with Giardia GS/M

trophozoites prior to IL-1β administration; moreover, CXCL8 mRNA levels in Caco-2

monolayers co-incubated with Giardia GS/M trophozoites were not significantly different from

control monolayers administered IL-1β (Figure 28B). As Caco-2 monolayers co-incubated with

Giardia NF trophozoites did not have decreased levels of CXCL8 mRNA relative to respective

controls, we concluded that attenuation of IL-1β-induced CXCL8 secretion by Giardia

trophozoites occurred via a post-transcriptional mechanism.

Having demonstrated that Giardia trophozoites attenuate CXCL8 secretion via a post-

transcriptional mechanism, additional experiments sought to determine if Giardia trophozoites

directly degraded CXCL8 protein. Assemblage A (NF or WB) or B (GS/M) Giardia live

trophozoites were co-incubated in the presence of Caco-2 monolayers at an MOI of 10:1 for 2

hours, the co-culture was given 1.0 ng/mL CXCL8, and incubated for an additional 4 hours.

ELISA analysis determined that the remaining levels of CXCL8 detected in supernatants of

assemblage A Giardia trophozoites (NF and WB) co-incubated with Caco-2 monolayers were

significantly reduced compared to control CXCL8 groups (Figure 29A). Moreover, supernatants

levels of CXCL8 from the co-incubation of assemblage B (GS/M) Giardia trophozoites with

Caco-2 monolayers were not significantly different from control groups spiked with CXCL8

109

Figure 28. Giardia trophozoites attenuate IL-1β-induced CXCL8 secretion via a post-

transcriptional mechanism.

Caco-2 monolayers were co-incubated with Giardia NF or GS/M trophozoite isolates (MOI

10:1) for 2 hours and IL-1β was subsequently administered to supernatants and incubated for (A)

1 hour or (B) 4 hours to induce CXCL8 secretion. Levels of CXCL8 mRNA relative to loading

control -2 microglobulin (2M) were determined. All data are representative of at least two


* p < 0.05

110

CX

CL

8/

2M

mR

NA

Co

ntr

ol

IL-1

Gia

rdia

NF

Gia

rdia

NF

+ IL

-1

Gia

rdia

GS

/M

Gia

rdia

GS

/M +

IL

-1

0 .0

0 .5

1 .0

1 .5

n .s .

n .s .

CX

CL

8/

2M

mR

NA

Co

ntr

ol

IL-1

Gia

rdia

NF

Gia

rdia

NF

+ IL

-1

Gia

rdia

GS

/M

Gia

rdia

GS

/M +

IL

-1

0

2

4

6

8

1 0

* *

n .s .A B

111

Figure 29. Giardia trophozoites attenuate CXCL8 via parasite-mediated degradation of

CXCL8.

(A) Giardia NF, WB, or GS/M trophozoites (MOI 10:1) were incubated in the presence of Caco-

2 monolayers for 2 hours. CXCL8 (1.0 ng/ml) was administered to supernatants and incubated

for 4 hours. Supernatant levels of CXCL8 were determined via ELISA. (B) Giardia NF or WB

trophozoites (MOI 10:1) were incubated in Caco-2 growth media without Caco-2 monolayers for

2 hours and CXCL8 (1.0 ng/ml) was administered to supernatants and incubated for 4 hours.

Supernatant levels of CXCL8 were determined via ELISA. All data are representative of at least

two independent experiments (n = 2-3/group) and represented as mean ± SEM. n.s. = not

significant * p < 0.05

112

CX

CL

8 (

pg

/mL

)

Co

ntr

ol

CX

CL

8

Gia

rdia

NF

Gia

rdia

NF

+ C

XC

L8

Gia

rdia

WB

Gia

rdia

WB

+ C

XC

L8

Gia

rdia

GS

/M

Gia

rdia

GS

/M +

CX

CL

8

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0 **

n .s .

Co

ntr

ol

CX

CL

8

Gia

rdia

NF

Gia

rdia

NF

+ C

XC

L8

Gia

rdia

WB

Gia

rdia

WB

+ C

XC

L8

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

CX

CL

8 (

pg

/mL

)

**

A B .

C a c o -2 n o C a c o -2

113

(Figure 29A). These results corroborate previous data from our study demonstrating only

assemblage A (NF or WB) isolates were capable of attenuating IL-1β-induced CXCL8 secretion

from Caco-2 monolayers following a 6-hour incubation (Figure 24A). To determine if these

effects required the presence of host cells, or whether Giardia trophozoites were directly

responsible for degrading CXCL8, assemblage A Giardia trophozoites (NF and WB) were

incubated in Caco-2 growth medium in the absence of Caco-2 monolayers for 2 hours and

subsequently given CXCL8 for 4 hours. As determined by ELISA, assemblage A Giardia

trophozoites significantly decreased CXCL8 levels in the absence of Caco-2 monolayers (Figure

29). Based on these results, experiments were performed to determine if Giardia trophozoites

promoted the degradation of other pro-inflammatory mediators, such as IL-1β. Therefore, the

above experiments were repeated by replacing CXCL8 with IL-1β. As determined via ELISA,

Giardia NF, WB or GS/M trophozoites did not alter the levels of IL-1β (Figure 30). Therefore,

Giardia assemblage A (NF and WB) but not assemblage B (GS/M) trophozoite isolates released

factors into cell supernatants that degraded CXCL8 but not IL-1β at 6 hours.

3.9 Giardia trophozoites release factors that attenuate PMN chemotaxis

Chemotaxis of extravasated PMNs is a multi-step process involving cellular migration

through a sequential cascade of chemotactic gradients, whereby chemokines constituting these

gradients can be subdivided into intermediate and end-target chemoattractants (reviewed in (85,

196)). PMN chemotaxis is prioritized to ensure cellular processes promoting migration towards

an intermediate chemokine, such as CXCL8, are overridden by those of an end-target

chemoattractant, such as C5a (97, 197). As previous reports have demonstrated that other

114

Figure 30. Giardia NF and GS/M trophozoites do not degrade IL-1β.

Giardia NF or GS/M trophozoites (MOI 10:1) were incubated in the presence of Caco-2

monolayers for 2 hours. IL-1β (1.0 ng/ml) was administered to supernatants and incubated for 4

hours. Supernatant levels of IL-1β were determined via ELISA. All data are representative of at

least two independent experiments (n = 2-3/group) and represented as mean ± SEM. n.s. = not

significant

115

IL-1

(pg

/ml)

Co

ntr

ol

IL-1

Gia

rdia

NF

Gia

rdia

NF

+ IL

-1

Gia

rdia

GS

M

Gia

rdia

GS

M +

IL

-1

0

5 0 0

1 0 0 0

1 5 0 0

n .s .

n .s .

116

parasites are capable of cleaving or inactivating chemokines, such as CXCL8 and C5a, into

alternative isoforms that can result in either enhanced or decreased chemotactic potential towards

PMNs, and hence modulate local inflammatory cell infiltrates (167, 168, 198, 199), experiments

sought to assess the effects of Giardia on CXCL8- and C5a-induced PMN chemotaxis.

Therefore, Caco-2 monolayers were co-incubated with Giardia NF trophozoites at an MOI of 10

or 50:1 for 2 hours and administered CXCL8 or C5a for 4 hours. Adapting a well-established

PMN chemotaxis assay (200), supernatants from these experiments were collected and applied to

the bottom chamber of 8 m transwells and PMNs isolated from healthy donors were applied to

the top chamber. PMN chemotaxis was assessed after 1 hour by determining the bottom to top

MPO ratio.

3.9.1 Giardia trophozoites attenuate CXCL8-induced PMN chemotaxis

PMN chemotaxis was significantly reduced when supernatants collected from the co-

incubation of Caco-2 monolayers and Giardia trophozoites at an MOI of 50:1 and CXCL8 were

used (Figure 31A). Western blotting analysis of supernatants collected from the co-incubation of

Caco-2 monolayers, Giardia NF trophozoites at an MOI of 50:1, and CXCL8 demonstrated the

loss of a detectable CXCL8 band when compared against control groups not co-incubated with

Giardia trophozoites (Figure 31B). Supernatants collected from the co-incubation of Caco-2

monolayers with Giardia NF trophozoites at an MOI of 10:1 and CXCL8 had a slight but non-

significant decrease in CXCL8-induced PMN chemotaxis when compared against control

(Figure 31A). Furthermore, analysis of these groups using Western blotting demonstrated the

presence of a detectable CXCL8 band (Figure 31B). Collectively, these results indicated that live

117

Figure 31. Giardia NF trophozoites attenuate CXCL8-induced PMN chemotaxis and

degrade CXCL8.

Caco-2 monolayers were co-incubated with Giardia NF trophozoites at an MOI of 10, or 50:1

for 2 hours and then CXCL8 (100 ng/mL) was administered to supernatants and incubated for an

additional 4 hours. (A) Supernatants were collected and applied to the bottom chamber of 8 M

transwells and human PMNs were applied to the top chamber and incubated for 1 hour. PMN

chemotaxis was quantified by determining the bottom to top myeloperoxidase ratio. (B)

Supernatants were collected and analyzed via Western blotting for CXCL8 expression levels. All

data are representative of at least three independent experiments (n = 3-4/group) and represented

as mean ± SEM. n.s. = not significant * p < 0.05

118

Co

ntr

ol

CX

CL

8

Gia

rdia

NF

(10:1

)

Gia

rdia

NF

+ C

XC

L8 (

10:1

)

Gia

rdia

NF

(50:1

)

Gia

rdia

NF

+ C

XC

L8 (

50:1

)

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

PM

N m

igra

tio

n

(Bo

tto

m

: T

op

M

PO

) **

n .s .

A

B

119

Giardia NF trophozoites attenuated CXCL8-induced PMN chemotaxis and degraded CXCL8 in

a dose-dependent manner.

Having established that supernatants collected from the co-incubation of 100 ng/mL of

CXCL8 with Caco-2 monolayers and Giardia NF trophozoites at an MOI of 50:1 were less

effective at inducing PMN chemotaxis, experiments were repeated using an assemblage A (WB)

and assemblage B (GS/M) isolate. CXCL8-induced PMN chemotaxis was again significantly

reduced when supernatants were used from the co-incubation containing Caco-2 monolayers,

Giardia WB trophozoites, and CXCL8, when compared to positive control supernatants (Figure

32A); conversely, PMN chemotaxis was not altered by Giardia GS/M trophozoites (Figure 32A).

Western blotting analysis demonstrated the loss of a detectable CXCL8 band in supernatants

collected from the co-incubation of Caco-2 monolayers, Giardia WB trophozoites at an MOI of

50:1, and CXCL8; conversely, Western blotting demonstrated the presence of a detectable band

in supernatants collected from the co-incubation of Caco-2 monolayers, Giardia GS/M

trophozoites at an MOI of 50:1, and CXCL8 (Figure 32B). These results are consistent with

observations above that, of the isolates tested, only assemblage A Giardia isolates (NF and WB)

were capable of degrading CXCL8 at a 6-hour timepoint (Figure 29) and indicated that

degradation of CXCL8 by Giardia trophozoites results in attenuation of CXCL8-induced PMN

chemotaxis.

Additional experiments were performed to determine if Giardia parasite products were

sufficient to attenuate CXCL8-induced PMN chemotaxis. Therefore, Giardia NF or GS/M

trophozoite sonicates at an MOI of 50:1 were co-incubated with Caco-2 monolayers for 2 hours,

120

Figure 32. Giardia WB trophozoites attenuate CXCL8-induced PMN chemotaxis and

degrade CXCL8.

Caco-2 monolayers were co-incubated with Giardia WB or GS/M trophozoites at an MOI of

50:1 for 2 hours and subsequently administered CXCL8 (100 ng/mL) to supernatants and

incubated for an additional 4 hours. (A) Supernatants were collected and applied to the bottom

chamber of 8 M transwells and human PMNs were applied to the top chamber and incubated

for 1 hour. PMN chemotaxis was quantified by determining the bottom to top myeloperoxidase

ratio. (B) Supernatants were collected and analyzed via Western blotting for CXCL8 expression

levels. All data are representative of at least three independent experiments (n = 3-4/group) and


121

Co

ntr

ol

CX

CL

8

Gia

rdia

WB

Gia

rdia

WB

+ C

XC

L8

Gia

rdia

GS

M

Gia

rdia

GS

M +

CX

CL

8

0

2

4

6

PM

N m

igra

tio

n

(Bo

tto

m

: T

op

M

PO

) *

n .s .A

B

122

and subsequently administered 100 ng/mL of CXCL8 for 4 hours. As above, these supernatants

from these experiments were collected and assessed for their chemotactic potential towards

PMNs. Interestingly, the co-incubation of Giardia NF or GS/M sonicates and Caco-2

monolayers with CXCL8 did not result in attenuation of CXCL8-induced PMN chemotaxis

(Figure 33). Together the data indicated that at least some Giardia trophozoites released factors

capable of attenuating CXCL8-induced PMN chemotaxis and degrading CXCL8.

3.9.2 Giardia trophozoites attenuate C5a-induced PMN chemotaxis

As PMN chemokines are subdivided into intermediate and end-target chemokines (85),

additional experiments were performed to determine if Giardia trophozoites are capable of

attenuating PMN chemotaxis induced by the end-target chemokine C5a (97). Therefore, the

PMN chemotaxis experiments were performed whereby Giardia NF trophozoites and Caco-2

monolayers were co-incubated with C5a. Similar to above results, C5a-induced PMN chemotaxis

was significantly reduced in supernatants collected from the co-incubation of Giardia NF

trophozoites at an MOI of 50:1, Caco-2 monolayers, and C5a when compared to control

supernatants collected from the co-incubation of Caco-2 monolayers and C5a (Figure 34). These

results demonstrated that Giardia trophozoites attenuated C5a-induced PMN chemotaxis, and,

with above results, indicate that Giardia trophozoites attenuated PMN chemotaxis induced by

intermediate and end-target PMN chemokines.

123

Figure 33. Giardia NF and GS/M trophozoite sonicates fail to attenuate CXCL8-induced

PMN chemotaxis.

Caco-2 monolayers were co-incubated with Giardia NF or GS/M trophozoite sonicates at an

MOI of 10, or 50:1 2 hours and then CXCL8 (100 ng/mL) was administered to supernatants and

incubated for an additional 4 hours. Supernatants were collected and applied to the bottom

chamber of 8 M transwells and human PMNs were applied to the top chamber and incubated

for 1 hour. PMN chemotaxis was quantified by determining the bottom to top myeloperoxidase

ratio. All data are representative of at least three independent experiments (n = 3-4/group) and

represented as mean ± SEM.

124

Co

ntr

ol

CX

CL

8

Gia

rdia

NF

(so

n)

Gia

rdia

NF

(so

n)

+ C

XC

L8

Gia

rdia

GS

M (

so

n)

+ C

XC

L8

0

2

4

6

8

PM

N m

igra

tio

n

(Bo

tto

m

: T

op

M

PO

)

125

Figure 34. Giardia NF trophozoites attenuate C5a-induced PMN chemotaxis.

Caco-2 monolayers were co-incubated with Giardia NF trophozoites at an MOI of 10, or 50:1 2

hours and then C5a (400 ng/mL) was administered to supernatants and incubated for an

additional 4 hours. Supernatants were collected and applied to the bottom chamber of 8 M

transwells and human PMNs were applied to the top chamber and incubated for 1 hour. PMN

chemotaxis was quantified by determining the bottom to top myeloperoxidase ratio. All data are

representative of at least three independent experiments (n = 3-4/group) and represented as mean

± SEM. n.s. = not significant * p < 0.05

126

PM

N m

igra

tio

n

(Bo

tto

m

: T

op

M

PO

)

Co

ntr

ol

C5a

Gia

rdia

NF

(10)

Gia

rdia

NF

(10)

+ C

5a

Gia

rdia

NF

(50)

Gia

rdia

NF

(50)

+ C

5a

0

1 0

2 0

3 0

4 0

*

127

3.10 Bioinformatics of Giardia cathepsin cysteine proteases

Using the completed Giardia duodenalis genome (169), catB and catL cysteine proteases

from several mammalian and parasite proteases were aligned with the sequenced genomes from

assemblage A WB and assemblage B GS/M proteases in CLC Sequence Viewer 7.0.2

(http://www.clcbio.com) using the ClustalW method (186). Following ClustalW alignment,

active site and S2 subsite residues were identified by using the NCBI Protein database and

highlighting for sequence features (201). According to the Giardia genome, both Giardia WB

and GS/M contain 9 catB proteases. Alignment of catB proteases indicated the majority Giardia

WB and GS/M catB proteases contain an active site Cys residue (black arrow), but also

demonstrated that catB Giardia WB 17516 and Giardia GS 2318 contain Ser residues in their

active site (Figure 35). Similarly, active site-associated residues (white arrows) for the majority

of Giardia catB proteases are identical to mammalian and other parasite catB proteases, with the

exception of the Giardia WB 17516 and Giardia GS 2318 (Figure 35). Greater variability in S2

subsite residues (grey arrows) was observed. Amino acids associated with certain S2 subsite

residues displayed significant conservation between Giardia isolates, other parasites, and

mammalian catB proteases, while others displayed a higher degree of variability (Figure 35).

CatB proteases contain an approximately 20 amino acid insertion referred to as an occluding

loop that allows their function as C-terminal carboxypeptidases (159, 202). ClustalW alignment

indicated that Giardia catB proteases contain a shortened occluding loop and amino acid

residues associated with the shortened loop are not conserved when compared against

mammalian and other parasite catB proteases (Figure 35). A phylogenetic tree of ClustalW

aligned catB proteases was subsequently generated using bootstrapped-confirmed neighbour

http://www.clcbio.com/

128

Figure 35. ClustalW alignment of catB proteases.

Giardia cathepsin-B cysteine proteases were aligned in CLC Sequence Viewer 7.0.2

(http://www.clcbio.com) using the ClustalW option. Active-site (black), active-site associated

(white), and S2 subsite residues (red) were identified using the NCBI Protein database and

highlighting for sequence features.


129

130

Figure 36. Phylogenetic tree construction of parasite catB cysteine proteases.

Giardia cathepsin-B cysteine proteases were generated in CLC Sequence Viewer 7.0.2

(http://www.clcbio.com) using the ClustalW option and phylogenetic trees of catB proteases was

generated using bootstrapped-confirmed neighbor joining trees.


131

132

joining trees in CLC Sequence Viewer 7.0.2 (http://www.clcbio.com). Phylogenetic tree

construction demonstrated that assemblage A and B Giardia contain five orthologous catB

proteases, with the exception of Giardia WB 114165 and Giardia GS 3635 that do not contain

an orthologous catB protease in the other Giardia genome (Figure 36). Collectively, these results

demonstrate that Giardia catB proteases contain several similarities and differences to human

and other parasite catB proteases. They also suggest that assemblage A and B Giardia

trophozoites may contain a unique proteases.

A ClustalW alignment and phylogenetic tree of Giardia and several mammalian and

parasite catL proteases was also constructed. According to the Giardia genome, Giardia WB

contains 8 catL proteases while Giardia GS/M contains 6 catL proteases (169). Furthermore,

Giardia catL proteases contain sequences of amino acids that do not overlap with mammalian or

other parasite proteases that were used for this alignment (Figure 37). Similar to Giardia catB

proteases, the majority of catL proteases contain active site Cys residues with the exception of

Giardia WB 11209 and Giardia GS 3714 that contain active site Ser residues (black arrow)

(Figure 37). According to this alignment active-site associated amino acids (white arrow)

between Giardia and several mammalian and parasite proteases are identical, while amino acid

variability is observed in S2 subsite residues (Figure 37). Similar to catB phylogenetic trees,

assemblage A and B Giardia contain a number of highly similar catL proteases (Figure 38).

3.11 Visualization of Giardia intra-trophozoite cathepsin proteases

As ClustalW alignments and phylogenetic tree construction demonstrated differences

between Giardia WB and GS/M, experiments were performed to determine if we could visualize


133

Figure 37. ClustalW alignment of cathepsin L cysteine proteases.

Giardia cathepsin-L cysteine proteases were aligned in CLC Sequence Viewer 7.0.2

(http://www.clcbio.com) using the ClustalW option. Active-site (black), active-site associated

(white), and S2 subsite residues (red) were identified using the NCBI Protein database and

highlighting for sequence features.


134

135

136

Figure 38. Phylogenetic tree construction of parasite catL cysteine proteases.

Alignment of Giardia cathepsin-L cysteine proteases were generated in CLC Sequence Viewer

7.0.2 (http://www.clcbio.com) using the ClustalW option and phylogenetic trees of catL

proteases was generated using bootstrapped-confirmed neighbor joining trees.


137

138

differences in cathepsin cysteine protease activity within Giardia trophozoites. This was done by

modifying protocols used to visualize serine protease activity within an SDS-PAGE gel (187). In

humans and other animal species, catB and catL proteases are capable of hydrolysing

benzyloxycarbonyl-L-Phenylalanyl-L-Arginine 4-Methyl-Coumaryl-7-Amide (ZFR-AMC),

while only catB proteases are capable of processing benzyloxycarbonyl-L-Arginine-L-Arginine

4-Methyl-Coumaryl-7-Amide (ZRR-AMC) (174, 175). Therefore, Giardia trophozoites were run

under non-denaturing conditions through SDS-PAGE gels copolymerized with ZFR-AMC.

Different banding patterns were observed when Giardia NF or GS/M sonicates were incubated

in SDS-PAGE gels co-polymerized with ZFR-AMC (Figure 39). These results support the above

bioinformatics data that cathepsin cysteine protease activity can vary between Giardia isolates.

3.12 Assessment of secreted Giardia cathepsin cysteine proteases

Having demonstrated that Giardia trophozoites contain cathepsin cysteine protease

activity, follow-up experiments sought to determine if Giardia trophozoites secrete cathepsin

cysteine proteases that degrade CXCL8. As previous studies have demonstrated that Giardia

trophozoites secrete cysteine proteases following exposure to IECs (171), we hypothesized that

these factors may be involved in degrading CXCL8. Initial experiments focused on

characterizing cathepsin cysteine protease activity in Caco-2 supernatants exposed to Giardia

trophozoites for 6 hours. Therefore, supernatants collected from the co-incubation of Caco-2

monolayers and Giardia trophozoites were collected and incubated with catB/L or catB

fluorogenic substrates mentioned above. As previous reports have demonstrated that Giardia

cathepsin cysteine protease activity is optimal at a neutral pH (203), cysteine protease was

139

Figure 39. Giardia trophozoites express proteases capable of degrading cathepsin

fluorogenic substrate ZFR-AMC.

Giardia NF or GS/M sonicates were run in non-denaturing conditions through a 7% SDS-PAGE

gel containing 200 uM ZFR-AMC. Proteolytic activity was visualized with a ChemiDoc (Bio-

Rad) where bands are indicative of proteolytic activity towards the polymerized substrate.

140

141

assayed with cathepsin fluorogenic substrates in assay buffer at a pH of 7.2 to represent the

lumenal pH within the upper small intestine. This was done by measuring the liberation of 7-

aminomethylcoumarin (AMC) from fluorogenic substrates following hydrolysis by cysteine

proteases. The calculated slope from the change in reflective light units (RFUs) over time is

indicative of cysteine protease activity, as described previously (174, 175).

A dose-dependent increase in the hydrolysis of ZFR-AMC over time was observed in

supernatants collected from the co-incubation of Caco-2 monolayers and increasing

concentrations of Giardia NF trophozoites; this was also corroborated by increasing slope values

(Figure 40). Therefore, the level of supernatant cathepsin activity increases in a trophozoite dose-

dependent manner. Hydrolysis of ZFR-AMC was also observed in supernatants collected from

the co-incubation of Caco-2 monolayers with any of the three Giardia trophozoites isolates (NF,

WB, or GS/M) and was further corroborated by increased slope values compared to controls

(Figure 41A). Hydrolysis of ZFR-AMC was also observed in supernatants collected when

Giardia trophozoite isolates were incubated in the absence of Caco-2 monolayers and

corroborated by increased slope values versus controls (Figure 41B). Therefore, both assemblage

A and B Giardia trophozoite isolates secreted cathepsin cysteine proteases that hydrolysed ZFR-

AMC.

Hydrolysis of ZRR-AMC was observed in supernatants incubated with all Giardia

trophozoite isolates in the presence or absence of Caco-2 monolayers compared with controls

(Figure 42). However, hydrolysis of ZRR-AMC in assemblage A (NF and WB) Giardia groups

was significantly reduced when slope values were compared between groups incubated in the

presence (Figure 42A) or absence (Figure 42B) of Caco-2 monolayers. In contrast, hydrolysis of

142

Figure 40. Giardia NF trophozoites increase supernatant cathepsin B/L supernatant

activity in a dose-dependent manner.

Caco-2 monolayers were co-incubated with Giardia NF trophozoites at an MOI of 1:1, 10:1,

50:1. Supernatants were incubated with the cathepsin B & L fluorogenic substrate ZFR-AMC

(200 µM : 5 minutes : 370C : pH 7.2). Proteolytic activity of catB and catL proteases was

measured by recording the change in reflective lights units over time and slopes were

determined. All data are representative of at least two independent experiments (n = 3/group) and

represented as mean ± SEM.

143

0 1 0 0 2 0 0 3 0 0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

T im e (s e c o n d s )

Re

fle

cti

ve

lig

ht

un

its

( R

FU

s)

G ia rd ia N F (M O I 1 )

G ia rd ia N F (M O I 1 0 )

G ia rd ia N F (M O I 5 0 )

C o n tro l

Ca

the

ps

in B

/L a

cti

vit

y

( R

FU

/se

co

nd

)

Co

ntr

ol

Gia

rdia

NF

(M

OI :

1)

Gia

rdia

NF

(M

OI :

10)

Gia

rdia

NF

(M

OI :

50)

0

5

1 0

1 5

2 0

144



ZFR-AMC.

Assemblage A (NF and WB) and assemblage B (GS/M) Giardia trophozoites were incubated in

the presence (A) or absence (B) of Caco-2 monolayers for 6 hours. Supernatants were incubated

with the cathepsin B & L fluorogenic substrate ZFR-AMC (200 µM : 5 minutes : 370C : pH 7.2).

Proteolytic activity of catB and catL proteases was measured by recording the change in

reflective lights units over time and slopes were determined. All data are representative of at

least two independent experiments (n = 2-3/group) and represented as mean ± SEM. * p < 0.05

145

0 1 0 0 2 0 0 3 0 0

7 0 0 0

8 0 0 0

9 0 0 0

1 0 0 0 0

1 1 0 0 0

1 2 0 0 0

1 3 0 0 0


Re

fle

cti

ve

lig

ht

un

its

( R

FU

s)

C o n tro l

G ia rd ia N F

G ia rd ia G S /M

G ia rd ia W B

Ca

the

ps

in B

/L a

cti

vit

y

( R

FU

/se

co

nd

)

Co

ntr

ol

Gia

rdia

NF

Gia

rdia

WB

Gia

rdia

GS

/M

0

2

4

6

8

*

*

0 1 0 0 2 0 0 3 0 0

7 0 0 0

8 0 0 0

9 0 0 0

1 0 0 0 0

1 1 0 0 0

1 2 0 0 0

1 3 0 0 0


Re

fle

cti

ve

lig

ht

un

its

( R

FU

s) G ia rd ia W B

G ia rd ia N F

G ia rd ia G S /M

C o n tro l

Co

ntr

ol

Gia

rdia

NF

Gia

rdia

WB

Gia

rdia

GS

/M

-2

0

2

4

6

Ca

the

ps

in B

/L a

cti

vit

y

( R

FU

/se

co

nd

)

*

A

B

C a c o -2

n o C a c o -2

146



ZRR-AMC.

Assemblage A (NF and WB) and assemblage B (GS/M) Giardia trophozoites were incubated in

the presence (B) or absence (C) of Caco-2 monolayers for 6 hours. Supernatants were incubated

with the cathepsin B & L fluorogenic substrate ZRR-AMC (200 µM : 5 minutes : 370C : pH 7.2).

Proteolytic activity of catB and catL proteases was measured by recording the change in

reflective lights units over time and slopes were determined. All data are representative of at

least two independent experiments (n = 2-3/group) and represented as mean ± SEM. * p < 0.05

147

0 1 0 0 2 0 0 3 0 0

8 0 0 0

9 0 0 0

1 0 0 0 0

1 1 0 0 0

1 2 0 0 0

1 3 0 0 0

1 4 0 0 0


Re

fle

cti

ve

lig

ht

un

its

( R

FU

s)

C o n tro l

G ia rd ia W B

G ia rd ia N F

G ia rd ia G S /M

Ca

the

ps

in B

ac

tiv

ity

( R

FU

/se

co

nd

)

Co

ntr

ol

Gia

rdia

NF

Gia

rdia

WB

Gia

rdia

GS

/M

-2

0

2

4

6

*

**

0 1 0 0 2 0 0 3 0 0

8 0 0 0

9 0 0 0

1 0 0 0 0

1 1 0 0 0

1 2 0 0 0


Re

fle

cti

ve

lig

ht

un

its

( R

FU

s)

C o n tro l

G ia rd ia W B

G ia rd ia N F

G ia rd ia G S /M

Co

ntr

ol

Gia

rdia

NF

Gia

rdia

WB

Gia

rdia

GS

/M

-2

0

2

4

6

Ca

the

ps

in B

ac

tiv

ity

( R

FU

/se

co

nd

)

*

A

B

C a c o -2

n o C a c o -2

148

ZRR-AMC from supernatants collected from Giardia GS/M trophozoites incubated in the

presence or absence of Caco-2 monolayers was the same (Figure 42A and B). Therefore,

consistent with assemblage-specific differences in cathepsin proteases, assemblage A (NF and

WB) Giardia trophozoites reduced their overall expression of proteases that hydrolysed ZRR-

AMC when exposed to enterocytes, while assemblage B (GS/M) trophozoites do not.

3.13 Apical to basolateral migration of Giardia cathepsin cysteine proteases

Basolateral intestinal epithelial CXCL8 recruits PMNs to the basolateral membrane of the

epithelium so other signals can, if necessary, promote PMN transepithelial migration (97, 143,

144). Experiments sought to determine whether apically produced Giardia cathepsin cysteine

proteases may translocate to the basolateral side of the epithelium. Results indicate that,

concurrently with exposure to S. tyhpmurium, Giardia proteases may translocate to the

basolateral side in a time- and dose-dependent manner (Figure 43). A significant increase in both

apical (Figure 43A) and basolateral (Figure 43B) cysteine protease activity was observed in

supernatants collected from the co-incubation of polarized Caco-2 monolayers and Giardia NF

trophozoites at an MOI of 50:1 administered S. typhimurium for 7 hours. In these same groups

we observed attenuation of apical (Figure 43C) and basolateral (Figure 43D) supernatant

CXCL8. These results suggest basolateral translocation of Giardia cathepsin cysteine proteases

occurred following exposure to S. typhimurium and resulted in attenuated basolateral supernatant

CXCL8.

149

Figure 43. Giardia NF cathepsin cysteine protease activity translocates across in vitro

intestinal epithelial Caco-2 monolayers following exposure to Salmonella typhimurium.

Caco-2 monolayers were co-incubated with Giardia NF trophozoites for 2 hours at an MOI of

10:1 and then administered S. typhimurium (MOI 100:1) for 7 hours. (A) Apical and (B)

basolateral supernatants were incubated with the cathepsin B & L fluorogenic substrate ZFR-

AMC (200 µM : 5 minutes : 370C : pH 7.2). Proteolytic activity of catB/L proteases was

measured by recording the change in reflective lights units over time and slopes were

determined. (C) Apical and (D) basolateral supernatant CXCL8 levels were analyzed via ELISA.

All data are representative of at least three independent experiments (n = 3/per group) and

represented as mean ± SEM. * p < 0.05

150

Co

ntr

ol

Gia

rdia

NF

(M

OI :

10)

Gia

rdia

NF

(M

OI :

50)

Salm

onella

Gia

rdia

NF

(M

OI :

10)

+ S

alm

onella

Gia

rdia

NF

(M

OI :

50)

+ S

alm

onella

-5

0

5

1 0

1 5

Ca

the

ps

in B

/L a

cti

vit

y

( R

FU

/se

co

nd

)

*

*

Co

ntr

ol

Gia

rdia

NF

(M

OI :

10)

Gia

rdia

NF

(M

OI :

50)

Salm

onella

Gia

rdia

NF

(M

OI :

10)

+ S

alm

onella

Gia

rdia

NF

(M

OI :

50)

+ S

alm

onella

-0 .5

0 .0

0 .5

1 .0

1 .5

Ca

the

ps

in B

/L a

cti

vit

y

( R

FU

/se

co

nd

)

*

*

*

*A B

Co

ntr

ol

Salm

onella

Gia

rdia

NF

(10)

Gia

rdia

NF

(10)

+ S

alm

onella

Gia

rdia

NF

(50)

Gia

rdia

NF

(50)

+ S

alm

onella

0

2 0

4 0

6 0

8 0

1 0 0

CX

CL

8

(pg

/mL

)

* *

*

Co

ntr

ol

Salm

onella

Gia

rdia

NF

(10)

Gia

rdia

NF

(10)

+ S

alm

onella

Gia

rdia

NF

(50)

Gia

rdia

NF

(50)

+ S

alm

onella

0

1 0

2 0

3 0

4 0

5 0

CX

CL

8

(pg

/mL

)

* *

*C D

151

3.14 Broad-spectrum inhibition of supernatant cathepsin proteases

To determine if Giardia cathepsin cysteine proteases were involved in the degradation of

CXCL8, initial experiments sought to elucidate if supernatant proteolytic activity was sensitive

to the broad-spectrum cysteine protease inhibitor E-64d (173) or to the catB-specific inhibitor

Ca-074Me (204). A previous report demonstrated that 1 M E-64 is the maximal inhibitory dose

that does not affect Giardia trophozoite viability (36). Our experiments confirmed that pre-

treatment of Caco-2 supernatants with 1 M E-64d significantly decreased the hydrolysis of

ZFR-AMC when Caco-2 monolayers were co-incubated with Giardia NF trophozoites, and was

corroborated by slopes not significant from control supernatants (Figure 44). To our knowledge,

the effects Ca-074Me on Giardia trophozoites have not been previously evaluated. Supernatants

pre-treated with increasing concentrations of Ca-074Me (1, 10, and 50 M) did not affect

Giardia NF trophozoite viability after the 6-hour incubation period. Indeed, the number of

trophozoites detected within cell supernatants (Figure 45A) or the ratio of motile to total

trophozoites (Figure 45B) was unchanged compared against vehicle control pre-treated

supernatants. Slope values calculated from the supernatant hydrolysis of ZFR-AMC (Figure

46A) and ZRR-AMC (Figure 46B) were significantly decreased when Giardia NF trophozoites

were co-incubated with Caco-2 monolayers pre-treated with 10 or 50 µM of Ca-074Me. As a

result, follow-up experiments examining supernatant inhibition of catB were performed with

Caco-2 supernatants pretreated with 10 M Ca-074Me.

152

Figure 44. Giardia trophozoites secrete cysteine proteases that are inhibited by E-64d.

Caco-2 monolayers were pre-treated with 1 M E-64d and subsequently co-incubated with

Giardia NF trophozoites (MOI 10:1) for 6 hours. Supernatants were incubated with the cathepsin

B & L fluorogenic substrate ZFR-AMC (200 µM : 5 minutes : 370C : pH 7.2). Proteolytic

activity was determined by graphing the change in reflective light units over time. All data are

representative of at three two independent experiments (n = 3 /group) and represented as mean ±


153

0 1 0 0 2 0 0 3 0 0

5 5 0 0

6 0 0 0

6 5 0 0

7 0 0 0

7 5 0 0

8 0 0 0

8 5 0 0


Re

fle

cti

ve

lig

ht

un

its

( R

FU

s)

C o n tro l

E -6 4 d

G ia rd ia N F

G ia rd ia N F (E -6 4 d )

Ca

the

ps

in B

/L a

cti

vit

y

( R

FU

/se

co

nd

)

Co

ntr

ol

Gia

rdia

NF

E64d

Gia

rdia

NF

(E

64d

)

0

1

2

3

4 *

n .s .

154

Figure 45. Pre-treatment of Caco-2 supernatants with the cathepsin B-specific inhibitor

Ca-074Me does not affect trophozoite viability.

Caco-2 monolayers were pre-treated with increasing concentrations of Ca-074Me (1, 10, or 50

M and subsequently co-incubated with Giardia NF trophozoites (MOI 10:1) for 6 hours.

Supernatants were collected and the log total of supernatant trophozoites (A) and the ratio of

motile : non motile trophozoites (B) was determined via enumeration on a hemocytometer. All

data are representative of at least two independent experiments (n = 2-3 /group) and represented

as mean ± SEM.

155

Veh

icle

1

M

10

M

50

M

0

2

4

6

8

Su

pe

rn

ata

nt

tro

ph

oz

oit

es

(lo

g t

ro

ph

oz

oit

es

/ml)

Veh

icle

1

M

10

M

50

M

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

Tro

ph

oz

oit

e v

iab

ilit

y

(mo

tile

: t

ota

l)

A B

156

Figure 46. Giardia trophozoites secrete cathepsin cysteine proteases susceptible to

inhibition with Ca-074Me.

Caco-2 monolayers were pre-treated with increasing concentrations of Ca-074Me (1, 10, or 50

M) and subsequently co-incubated with Giardia NF trophozoites (MOI 10:1) for 6 hours.

Supernatants were incubated with the cathepsin B & L fluorogenic substrate ZFR-AMC (A) or

the catB fluorogenic substrate ZRR-AMC (B) (200 µM : 5 minutes : 370C : pH 7.2). Proteolytic

activity was determined by graphing the change in reflective light units over time. All data are

representative of at least two independent experiments (n = 2-3 /group) and represented as mean

± SEM.

157

0 1 0 0 2 0 0 3 0 0

6 0 0 0

7 0 0 0

8 0 0 0

9 0 0 0

1 0 0 0 0

1 1 0 0 0


Re

fle

cti

ve

lig

ht

un

its

( R

FU

s)

V e h ic le

1 M

1 0 M

5 0 M

Veh

icle

1

M

10

M

50

M

0

2

4

6

Ca

the

ps

in B

/L a

cti

vit

y

( R

FU

/se

co

nd

)

*

0 1 0 0 2 0 0 3 0 0

9 0 0 0

1 0 0 0 0

1 1 0 0 0

1 2 0 0 0

1 3 0 0 0


Re

fle

cti

ve

lig

ht

un

its

( R

FU

s)

V e h ic le

1 M

1 0 M

5 0 M

Veh

icle

1

M

10

M

50

M

0

1

2

3

4

Ca

the

ps

in B

ac

tiv

ity

( R

FU

/se

co

nd

)

*

A

B

158

3.15 Giardia catB proteases degrade CXCL8

As previously shown, levels of CXCL8 secreted by Caco-2 monolayers administered IL-

1β are significantly reduced when monolayers are initially co-incubated with Giardia NF

trophozoites; however, when Caco-2 monolayers are co-incubated with Giardia NF trophozoites

in the presence of 1 µM E-64d and administered IL-1β, the inhibitory effect was abolished, and

CXCL8 levels detected within supernatants were not significantly different from respective IL-

1β-induced controls (Figure 47A). Therefore, Giardia-mediated attenuation of IL-1β-induced

CXCL8 secretion in Caco-2 monolayers occurs via the secretion of a cysteine protease sensitive

to inhibition with E-64d. Similarly, levels of recombinant CXCL8 administered to supernatants

were significantly reduced when Caco-2 monolayers were initially co-incubated with Giardia

NF trophozoites; these effects were also reversed when experiments were performed in the

presence of 1M E-64d or 10 M Ca-074Me (Figure 47B). Based on these observations, we

concluded that Giardia trophozoites secreted catB-like proteases that degraded CXCL8.

3.16 Inhibition of Giardia cathepsin B proteases

In order to implicate Giardia catB-like activity in the attenuation of CXCL8-induced PMN

chemotaxis, experiments were modified to specifically inhibit these proteases in Giardia

trophozoites. This was essential as preliminary experiments indicated that the presence of E-64d

or Ca-074Me in PMN chemotactic supernatants negatively affected CXCL8-induced PMN

chemotaxis. Initial experiments sought to determine if administration of E-64d or Ca-074Me to

confluent tubes of Giardia trophozoites would result in inhibition of cysteine protease activity.

Therefore, confluent tubes of assemblage A Giardia NF trophozoites were pre-

159

Figure 47. Giardia cathepsin B proteases degrade interleukin-8.

(A) Caco-2 monolayers were pre-treated with 1 M E-64d and subsequently co-incubated with

Giardia NF trophozoites (MOI 10:1) for 2 hours. After this, IL-1β (1.0 ng/ml) was administered

to supernatants and incubated for 4 hours. (B) Caco-2 monolayers were pre-treated with 1 M E-

64d or 10 M Ca-074Me and subsequently co-incubated with Giardia NF trophozoites (MOI

10:1) for 2 hours. After this, CXCL8 (1.0 ng/ml) was administered to supernatants and incubated

for 4 hours. CXCL8 supernatant levels were determined via ELISA. All data are representative

of at least three independent experiments (n = 3-4/group) and represented as mean ± SEM. n.s. =

not significant * p < 0.05

160

CX

CL

8 (

pg

/mL

)

Co

ntr

ol

IL-1

Gia

rdia

NF

Gia

rdia

NF

+ IL

-1

E64d

E64d

+ IL

-1

Gia

rdia

NF

(E

64d

)

Gia

rdia

NF

(E

64d

) + IL

-1

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0 *

n .s .

CX

CL

8 (

pg

/mL

)

Co

ntr

ol

CX

CL

8

Gia

rdia

NF

Gia

rdia

NF

+ C

XC

L8

E64d

E64d

+ C

XC

L8

Gia

rdia

NF

(E

64d

)

Gia

rdia

NF

(E

64d

) + C

XC

L8

Gia

rdia

NF

(C

a074M

e)

Gia

rdia

NF

(C

a074M

e)

+ C

XC

L8

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0*

n .s .

n .s .

A

B

161

treated with increasing concentrations of Ca-074Me (1, 10, or 50 M), 1 M E-64d, or vehicle

control for 30 minutes. After this incubation period, trophozoites sonicates were obtained and

assayed with ZFR-AMC and ZRR-AMC. Pre-treatment of Giardia NF trophozoites with

increasing concentrations of Ca-074Me did not appear to affect trophozoite viability, as

supernatant numbers of trophozoites (Figure 48A) and the ratio of motile to non-motile

trophozoites (Figure 48B) were not significantly different from vehicle-treated controls. Cysteine

protease activity assays and corresponding slopes demonstrated that hydrolysis of ZFR-AMC

was significantly reduced in groups where Giardia trophozoites were pre-treated with Ca-074Me

at concentrations of 10 and 50 M and 1M E-64d and resulted in decreased slope values

(Figure 49A). Similarly, hydrolysis of ZRR-AMC and corresponding slope values were

significantly reduced in groups when Giardia trophozoites were pre-treated with 10 and 50 M

Ca-074Me; interestingly, E-64d pre-treatment did not affect hydrolysis of ZRR-AMC (Figure

49B). Based on these results, it was determined that Giardia trophozoites would be pre-treated

with 10 M Ca-074Me to study its effects on PMN chemotaxis, as it resulted in inhibition of the

hydrolysis of ZFR-AMC and ZRR-AMC.

3.17 Giardia catB proteases attenuate CXCL8- and C5a-induced PMN chemotaxis

As described above, confluent tubes of Giardia trophozoites were pre-treated with

vehicle control (DMSO) or Ca-074Me 30 minutes prior to isolation and subsequently co-

incubated with Caco-2 monolayers and CXCL8. Compared to supernatants containing vehicle

control-treated Giardia NF trophozoites, hydrolysis of ZFR-AMC and corresponding slopes

162

Figure 48. Pre-treatment of Giardia NF trophozoites with Ca-074Me does not affect

trophozoite viability.

Confluent tubes of Giardia NF trophozoites were treated with increasing concentrations of Ca-

074Me (1, 10, or 50 M) or 1 M E-64d for 30 minutes. (A to D). After 30 minutes, trophozoites

were isolated and sonicated. Trophozoite sonciates were incubated with the cathepsin B & L

fluorogenic substrate ZFR-AMC (A) or the catB fluorogenic substrate ZRR-AMC (B) (200 µM :

5 minutes : 370C : pH 7.2). Proteolytic activity was determined by graphing the change in

reflective light units over time. Supernatants were collected and the log total of supernatant

trophozoites (C) and the ratio of motile : non motile trophozoites (D) was determined via

enumeration on a hemocytometer. All data are representative of at least two independent

experiments (n = 2-3/group) and represented as mean ± SEM.

163

Co

ntr

ol

1

M

10

M

50

M

0

2

4

6

8

Su

pe

rn

ata

nt

tro

ph

oz

oit

es

(lo

g t

ro

ph

oz

oit

es

/ml)

Co

ntr

ol

1

M

10

M

50

M

0 .0

0 .2

0 .4

0 .6

Tro

ph

oz

oit

e v

iab

ilit

y

(mo

tile

: t

ota

l)

A B

164

Figure 49. Pre-treatment of Giardia NF trophozoites with Ca-074Me inhibits intra-

trophozoite cathepsin activity.

Confluent tubes of Giardia NF trophozoites were treated with increasing concentrations of Ca-

074Me (1, 10, or 50 M) or 1 M E-64d for 30 minutes. (A and B) After 30 minutes,

trophozoites were isolated and sonicated. Trophozoite sonciates were incubated with the

cathepsin B & L fluorogenic substrate ZFR-AMC (A) or the catB fluorogenic substrate ZRR-

AMC (B) (200 µM : 5 minutes : 370C : pH 7.2). Proteolytic activity was determined by graphing

the change in reflective light units over time. All data are representative of at least two

independent experiments (n = 2-3/group) and represented as mean ± SEM. * p < 0.05

165

0 1 0 0 2 0 0 3 0 0

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

2 5 0 0 0 0


Re

fle

cti

ve

lig

ht

un

its

( R

FU

s)

D M S O

1 M C a 0 7 4 M e

1 0 M C a 0 7 4 M e

5 0 M C a 0 7 4 M e

E 6 4 d

Veh

icle

1

M

10

M

50

M

E64d

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

Ca

the

ps

in B

/L a

cti

vit

y

( R

FU

/se

co

nd

)

*

0 1 0 0 2 0 0 3 0 0

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0


Re

fle

cti

ve

lig

ht

un

its

( R

FU

s)

D M S O

1 M C a 0 7 4 M e

1 0 M C a 0 7 4 M e

5 0 M C a 0 7 4 M e

E 6 4 d

Veh

icle

1

M

10

M

50

M

E-6

4d

0

1 0 0

2 0 0

3 0 0

Ca

the

ps

in B

ac

tiv

ity

( R

FU

/se

co

nd

)

*

A

B

C a -0 7 4 M e

C a -0 7 4 M e

166

indicated a significant reduction in cathepsin activity within supernatants when trophozoites

were pre-treated with Ca-074Me (Figure 50A). These results suggest that Ca-074Me pre-

treatment of Giardia NF trophozoites significantly reduced their ability to secrete active

cathepsin cysteine proteases into supernatants. For PMN chemotaxis assays, supernatants

collected from the co-incubation of vehicle control-treated Giardia NF trophozoites, Caco-2

monolayers, and CXCL8 displayed significantly less chemotactic potential for PMNs compared

to control supernatants (Figure 50B). More importantly, the inhibitory effect of Giardia was

abolished with Ca-074Me (Figure 50A). These results suggest that Ca-074Me pre-treatment of

Giardia NF trophozoites significantly reduced their ability to secrete active cathepsin cysteine

proteases into supernatants. More importantly, the inhibitory effect of Giardia was abolished by

Ca-074Me (Figure 50B). Similar experiments were also performed whereby Caco-2 monolayers

were co-incubated with vehicle control or Ca-074Me-treated Giardia NF trophozoites for 2

hours and subsequently incubated with C5a for 4 hours. Supernatants collected from the co-

incubation of Caco-2 monolayers, vehicle control-treated Giardia NF trophozoites, and C5a

displayed significantly reduced chemotactic potential towards PMNs (Figure 51). However, the

chemotactic potential towards PMNs of supernatants collected from the co-incubation of Caco-2

monolayers, Ca-074Me-treated Giardia NF trophozoites, and C5a was not significantly different

from control groups administered C5a in the absence of Giardia NF trophozoites (Figure 51).

These results demonstrated that Giardia catB-like cysteine protease activity attenuated CXCL8-

and C5a-induced PMN chemotaxis.

167

Figure 50. Inhibition of cathepsin B activity in Giardia NF trophozoites prevents

attenuation of interleukin-8-induced neutrophil chemotaxis.

Confluent tubes of Giardia NF trophozoites were treated with 10 M Ca-074Me for 30 minutes.

After 30-minute incubation, trophozoites were co-incubated with Caco-2 monolayers at an MOI

of 50:1 for 2 hours, administered CXCL8 (100 ng/mL) and then incubated for an additional 4

hours. (A) Supernatants were collected and incubated with the cathepsin B & L fluorogenic

substrate ZFR-AMC (200 µM : 5 minutes : 370C : pH 7.2). Proteolytic activity was determined

by graphing the change in reflective light units over time. (B) Supernatants were collected and

applied to the bottom chamber of 8 M transwells and human PMNs were applied to the top

chamber and incubated for 1 hour. PMN chemotaxis was quantified by determining the bottom

to top myeloperoxidase ratio. (C) Supernatants were collected and assayed via Western blotting

for supernatant CXCL8. All data are representative of at least three independent experiments (n

= 3-4/group) and expressed are mean ± SEM. n.s = not significant * p < 0.05

168

Co

ntr

ol

Gia

rdia

NF

(V

eh

icle

)

Gia

rdia

NF

(C

a074M

e)

-5 0

0

5 0

1 0 0

1 5 0

Ca

the

ps

in B

/L a

cti

vit

y

( R

FU

/se

co

nd

)

** *

Co

ntr

ol

CX

CL

8

Gia

rdia

NF

(V

eh

icle

)

Gia

rdia

NF

(V

eh

icle

) + C

XC

L8

Gia

rdia

NF

(C

a074M

e)

Gia

rdia

NF

(C

a074M

e)

+ C

XC

L8

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

PM

N m

igra

tio

n

(Bo

tto

m

: T

op

M

PO

) *

n .s .

*

A

C

B

169

Figure 51. Inhibition of cathepsin B activity in Giardia NF trophozoites prevents

attenuation of C5a-induced neutrophil chemotaxis.

Confluent tubes of Giardia NF trophozoites were treated with 10 M Ca-074Me for 30 minutes.

After 30-minute incubation, trophozoites were co-incubated with Caco-2 monolayers at an MOI

of 50:1 for 2 hours, administered C5a (400 ng/mL) and then incubated for an additional 4 hours.

Supernatants were collected and applied to the bottom chamber of 8 M transwells and human

PMNs were applied to the top chamber and incubated for 1 hour. PMN chemotaxis was

quantified by determining the bottom to top myeloperoxidase ratio. All data are representative of

at least two independent experiments (n = 2-4/group) and expressed are mean ± SEM. n.s. = not

significant * p < 0.05

170

Co

ntr

ol

C5a

Gia

rdia

NF

(DM

SO

)

Gia

rdia

NF

(DM

SO

) + C

5a

Gia

rdia

NF

(Ca-0

74M

e)

Gia

rdia

NF

(Ca-0

74M

e)

+ C

5a

0

1

2

3

4

PM

N m

igra

tio

n

(Bo

tto

m

: T

op

M

PO

) *

n .s .

*

171

4.0 Discussion

4.1 Summary

Results from the present study demonstrate novel observations that Giardia infections in

mice attenuate pro-inflammatory responses elicited by an inflammatory bacterial toxin in vivo or

from inflamed human intestinal tissues ex vivo in an isolate-dependent manner. In vivo Giardia

NF infections attenuate granulocyte infiltration via reducing expression of several inflammatory

mediators, including those associated with granulocyte tissue recruitment. Supporting our in vivo

observations, several of these factors were also decreased following co-incubation of Giardia NF

trophozoites and ex vivo inflamed colonic mucosal biopsy tissues. Conversely, Giardia GS/M-

infected animals did not display reduced granulocyte infiltration nor decreased expression of

inflammatory mediators. Instead, Giardia GS/M infections further enhanced expression of

several factors initially upregulated following TcdAB administration and, also, increased

expression of several factors not initially increased following i.r. instillation. Therefore, Giardia

infections attenuate intestinal pro-inflammatory responses in an isolate-dependent manner, and

attenuation of such responses are associated with reduced tissue granulocyte infiltration.

Results from the present study also reveal a hitherto unrecognized immune-modulatory

effect by a parasite, in which Giardia trophozoites attenuate supernatant levels of CXCL8, a

potent PMN chemokine, that have been released from inflamed ex vivo CD small intestinal

mucosal biopsy tissues or uninflamed tissues administered pro-inflammatory IL-1 and in vitro

Caco-2 monolayers administered IL-1β or S. typhimurium. Attenuation of supernatant CXCL8

by Giardia GS/M trophozoites occurred with different kinetics than other isolates. This

attenuation of CXCL8 by Giardia trophozoites was the result of secreted Giardia catB proteases

172

that degraded CXCL8. Degradation of CXCL8 by Giardia trophozoites resulted in an end

product less effective at promoting PMN chemotaxis. The inhibition of Giardia catB proteases

prevented the parasite-mediated degradation of CXCL8 and subsequent attenuation of CXCL8-

and C5a-induced PMN chemotaxis. Therefore, Giardia trophozoites secrete catB proteases

capable of degrading CXCL8 and attenuating CXCL8- and C5a-induced chemotaxis.

Collectively, these results demonstrate that Giardia trophozoites possess immunomodulatory

factors that can inhibit pro-inflammatory intestinal responses in Giardia-infected individuals.

4.2 Giardia attenuates granulocyte infiltration in vivo

PMN tissue accumulation represents an archetypal event during many acute intestinal

inflammatory responses. Although essential to pathogen clearance, PMNs can also be highly

deleterious and injurious to self-tissue. Excessive PMN recruitment and activation contributes to

disease pathology in several chronic gastrointestinal inflammatory disorders, such as IBD and C.

difficile colitis (70, 72, 98). Therefore, PMNs are kept from entering host tissues and confined to

the bone marrow and circulation during tissue homeostasis. This study demonstrated that in vivo

Giardia NF infections attenuate granulocyte tissue recruitment induced by a pro-inflammatory

bacterial toxin by reducing tissue expression of mediators that contribute to the tissue

recruitment of PMNs. Release of ELR+ chemokines, such as CXCL1, 2, and 8, from a variety of

intestinal tissue-resident cells promotes PMN accumulation and chemotaxis through intestinal

tissues. In mice, these chemokines include CXCL1 and CXCL2, while in humans this also

includes CXCL8 (205-208). Separately, enhanced expression of G-CSF promotes the release of

PMNs into the bloodstream (79, 82), in a process that can be initiated via IL-17 production (83,

173

84). PMN chemokines locally released within tissues may affect PMN bone marrow egression

(82), while G-CSF and GM-CSF can also delay PMN apoptosis (209, 210). Our results

demonstrate in vivo Giardia NF infections attenuate granulocyte infiltration into colonic tissues

following i.r. TcdA/B by reducing colonic expression levels of CXCL1, CXCL2, and IL-17;

these results are supported by observations that Giardia GS/M infections failed to attenuate

colonic granulocyte infiltration and expression of PMN-associated mediators. In addition,

Giardia NF parasites attenuated supernatant levels of CXCL8, GRO-family proteins (CXCL1-3),

IL-17, G-CSF, and GM-CSF when co-incubated with ex vivo descending colon mucosal biopsy

tissues. Therefore, we have demonstrated the novel observation that Giardia infections attenuate

the release of a variety of mediators associated with PMN recruitment in different experimental

models of inflammation, in an isolate-dependent manner. Although these results demonstrate

Giardia isolates are capable of attenuating intestinal inflammation induced by a pro-

inflammatory bacterial toxin in vivo, they do not identify a potential mechanism for this

attenuation. Recently, luminal administration of CXCL1 or fMLF to small intestinal loops in

vivo has been demonstrated to induce tissue PMN accumulation and TEM (127, 211). This in

vivo model of inflammation would be extremely beneficial in elucidating the ability of Giardia

trophozoites to attenuate CXCL1 and/or fMLF-induced PMN chemotaxis and TEM.

4.2.1 Modulation of diarrheal disease by Giardia

Results from this study may explain observations that Giardia infections can decrease

(60, 64) or increase (59) diarrheal disease incidence rates. It is possible that certain Giardia

isolates or assemblages may attenuate pro-inflammatory responses elicited by other co-infecting

174

pathogens that cause diarrheal disease, while others may enhance its development. Following

their recruitment to tissues, PMNs induce multiple pathophysiological events capable of causing

diarrheal within intestinal tissues (19, 22-24). Therefore, Giardia NF infections may protect

against the development of diarrheal disease by attenuating PMN recruitment during intestinal

pro-inflammatory responses. Indeed, diarrhea caused by experimental infection with

enterohemorrhagic Escherichia coli was found to be directly induced by neutrophilic infiltration,

independently of toxin production by the bacteria (212). Similarly, Giardia GS/M infections did

not enhance granulocyte infiltration following i.r. TcdAB; however, these infections resulted in

enhanced expression of several pro-inflammatory mediators that could also enhance the

development of diarrheal disease. For example, IL-1β and IFNγ have been shown to modulate

intestinal barrier dysfunction and ion secretion, and, resultantly, cause diarrhea (213-216).

Inhibition of PMN recruitment to intestinal tissues during Giardia NF infections may also

enhance the development of diarrheal disease. PMNs are heavily involved in pathogen clearance

and, also, induce protective immune responses that limit the extent of intestinal inflammation.

For example, in vivo CXCR2-dependent PMN influx protects against the development of

diarrhea and pathogen burden following infection with the attaching/effacing pathogen

Citrobacter rodentium (205). Moreover, inhibition of PMN tissue accumulation in vivo has

resulted in enhanced disease pathology in experimental models of colitis (217), and the

consumption of oxygen by transmigrating PMNs has been shown to activate protective hypoxia

inducible factor (HIF) signaling in IECs and enhance resolution of intestinal inflammation (218).

Furthermore, HIF signaling within IECs has been shown to mediate intestinal damage during C.

difficile colitis (176). Therefore, attenuation of PMN accumulation may also result in enhanced

175

intestinal inflammatory responses that could, potentially, culminate in elevated incidence of

diarrheal disease. Our studies also demonstrated that Giardia NF trophozoites decreased tissue

expression of IL-6 in several experimental models. As intestinal epithelial release of IL-6 has

been shown to enhance PMN-mediated killing of S. typhimurium (219), it is possible that

Giardia infections prevent PMNs from performing anti-microbial functions associated with

pathogen clearance; this may also enhance the development of diarrheal disease in Giardia-

infected hosts. Similarly, increased intestinal expression of pro-inflammatory mediators induced

during Giardia GS/M infections may protect against the development of diarrhea by enhancing

pathogen clearance. Intestinal production of IL-1β is important in discriminating between

commensal and pathogenic bacteria and induces protective immune responses (220), and

microbiota-mediated production of IL-1β during in vivo C. difficile infection is required for PMN

recruitment that results in pathogen elimination (221). Similarly, T-cell-mediated production of

IFN-γ induces immune responses during C. rodentium infection that promote pathogen clearance

(222). In addition, modulation of diarrheal disease during Giardia infections may also be reliant

on the co-infecting species. In vivo experiments have shown that animals infected with

Trichinella spiralis were more susceptible to infection with Giardia GS/M trophozoites and

demonstrate enhanced mast cell accumulation and degranulation (223). As a result, future studies

examining how modulation of inflammatory responses during Giardia infections affects the

development of diarrheal disease in the host are sorely needed. Human studies examining

Giardia co-infections where the infecting agent and the assemblage of the infecting Giardia

isolate are recorded would be extremely helpful.

176

4.3 Giardia infections attenuate expression of pro-inflammatory mediators

4.3.1 Attenuation of acute phase response proteins

Data from this study demonstrated Giardia infections in mice reduce expression of pro-

inflammatory mediators not typically associated with PMN recruitment, in an isolate-dependent

manner. Giardia NF infections attenuated colonic expression of IL-6 and the related cytokine

LIF induced following i.r. TcdAB, whereas Giardia GS/M infection did not modulate expression

of these factors. Moreover, co-incubation of Giardia NF trophozoites with inflamed intestinal

mucosal biopsy tissues ex vivo also resulted in attenuation of supernatant and tissue IL-6. The

ability of Giardia sp. to modulate IL-6 during in vivo infection remains incompletely understood.

Animals infected with Giardia muris display decreased IL-6 levels in jejunal tissues 5 days p.i.

(49), while mice infected with Giardia GS/M display elevated IL-6 intestinal tissue levels of 15

days p.i. (224). Although these differences in intestinal IL-6 expression may be due to different

time-points analyzed, they may also result from the infecting Giardia species used. Indeed,

results from our study suggest that Giardia NF and GS/M infections modulate intestinal

expression of IL-6 differently. In addition, the release of IL-6 and the related cytokine LIF has

been shown to induce expression and synthesis of acute phase response proteins from

hepatocytes, including C-reactive protein (CRP) (225, 226). As Tanzanian children infected with

Giardia were found to have lower serum CRP than their non-infected counterparts (64), future

studies should examine whether Giardia infections modulate activation of the acute phase

response and establish whether parasite-mediated attenuation of IL-6 expression is involved in

these processes; these experiments should also take into consideration the infecting Giardia

isolate.

177

4.3.2 Attenuation of CCL chemokines and pro-inflammatory cytokines

Results from this study indicated Giardia NF infections in mice attenuate colonic

expression of CCL2 and IL-12p70, and similar results were demonstrated in parasites co-

incubated with inflamed ex vivo intestinal mucosal biopsy tissues. CCL2 is an important

chemokine for monocytes and macrophages (reviewed in (227)). Therefore, data from our study

suggest that Giardia infections may be capable of attenuating the recruitment of these immune

cells. Conversely, co-incubation of Giardia trophozoites with in vitro intestinal epithelial Caco-2

monolayers results in increased mRNA expression of CCL2 (45). Therefore, additional research

is required in order to understand how Giardia infections modulate monocyte/macrophage

recruitment. IL-12p70 is composed of IL-12p35 and IL-12p40 and is responsible for inducing

helper T1 (Th1) adaptive immune responses (reviewed in (228)). Previous research has

demonstrated Giardia trophozoite products are capable of attenuating IL-12p70 expression from

DCs stimulated with bacterial lipopolysaccharide (44). However, these results contrast with other

studies suggesting that certain Giardia parasite products upregulate DC IL-12p70 expression (43,

229). Therefore, additional research is required in order to determine how Giardia infections

modulate CCL2 and IL-12p70 expression during intestinal inflammatory responses. Moreover,

future studies should examine whether modulation of these cytokines is, in fact, isolate- and,

potentially, assemblage-dependent.

Co-incubation of Giardia NF trophozoites with ex vivo intestinal mucosal biopsy tissues

resulted in attenuation of large number of inflammatory mediators, in addition to those observed

during in vivo Giardia NF infections. Moreover, expression of certain inflammatory mediators

178

was reduced in supernatants while others in biopsy tissues. These results suggest that Giardia NF

trophozoites attenuate mediators expressed by inflamed intestinal biopsy tissues via multiple

mechanisms. Additional experiments are required in order to determine whether Giardia

trophozoites differently modulate the expression of inflammatory mediators from mice versus

humans, and/or whether Giardia infections alter the expression of inflammatory mediators

depending on their location to their expression. Indeed, it is possible that Giardia infections

attenuate a greater variety of inflammatory mediators if they are in the immediate vicinity to

their expression in host tissues.

4.4 Giardia attenuates intestinal epithelial CXCL8

As our study demonstrated certain Giardia infections were capable of attenuating PMN

accumulation and expression of PMN-associated mediators in an in vivo model of colitis and

following co-incubation with inflamed colonic mucosal biopsy tissues, we hypothesized that this

may, at least partially, involve parasite-mediated attenuation of intestinal epithelial CXCL8.

Since Giardia trophozoites are non-invasive, the intestinal epithelium is one of few structures

directly in contact with the parasite. Following exposure to various pro-inflammatory stimuli,

IECs secrete the potent PMN chemoattractant CXCL8 (140, 141). Moreover, in IECs, the

secretion of CXCL8 occurs rapidly during acute intestinal inflammatory responses and recruits

extravasated PMNs to the basolateral surface of the intestinal epithelium (97, 144). In order to

investigate whether Giardia trophozoites attenuated intestinal epithelial CXCL8 secretion, we

developed a novel model to investigate Giardia : human intestinal mucosal interactions. Human

small intestinal biopsy tissues were co-incubated with Giardia trophozoites ex vivo, and

179

administered pro-inflammatory IL-1β to mimic an inflamed intestinal tissue milieu. This study

demonstrated that co-incubation of small intestinal mucosal biopsy with Giardia trophozoites

resulted in attenuation of supernatant CXCL8 induced via administration of IL-1β. Similar

results were obtained in vitro when Caco-2 monolayers were co-incubated with Giardia

trophozoites. Finally, using a well-known PMN chemotaxis assay (200) and freshly isolated

human neutrophils, we demonstrated that Giardia catB proteases degraded CXCL8 into a

product less effective at promoting CXCL8-induced PMN chemotaxis. Results from our study

confirm previous observations that Giardia trophozoites do not induce CXCL8 secretion from

IECs (42, 140) and this lack of induction of IEC CXCL8 mRNA or protein supports the notion

that human Giardia infections occur in the absence of overt intestinal inflammation (53). The

present study also demonstrates that Giardia catB proteases degrade CXCL8 produced by human

enterocytes, and reduce its chemotactic potential towards neutrophils, thereby suggesting a role

for these parasitic proteases in preventing neutrophil recruitment and accumulation. Receptors

for CXCL8 are also expressed on a variety of pro-inflammatory cells, including mast cells and

eosinophils (230, 231). In addition, the apical administration of CXCL8 has been shown to

induce gene transcription within IECs in vitro via CXCR1 (232). Therefore, attenuation of

CXCL8 by Giardia trophozoites and its role in affecting other pro-inflammatory cells, such as

mast cells, eosinophils, and IEC gene transcription requires further study.

The intestinal epithelium separates the external environment of the intestinal lumen from

underlying host tissues; this structure is comprised of a single layer of polarized intestinal

epithelial cells joined via AJCs and desmosomes (reviewed in (136)). Impairment of the

intestinal epithelium occurs during many intestinal disease states and gastrointestinal infections

180

and results in enhanced permeability of the barrier to luminal contents. During infection, many

gastrointestinal pathogens enhance paracellular permeability to luminal contents via the

disruption of AJC proteins (reviewed in (233)). Gastrointestinal pathogens can also facilitate

transcellular translocation of luminal contents to underlying host tissues (234). In our study, co-

incubation of polarized Caco-2 monolayers with Giardia NF trophozoites and S. typhimurium

resulted in the basolateral translocation of cathepsin cysteine proteases and attenuation of

supernatant CXCL8. In the absence of S. typhimurium, basolateral cysteine protease activity was

not detected in groups where polarized Caco-2 monolayers were co-incubated with Giardia NF

trophozoites. As S. typhimurium invades IECs and disrupts AJC proteins during its infection

(235, 236), it is possible that the translocation of Giardia cathepsin cysteine proteases to the

basolateral compartment may have occurred via a transcellular or paracellular route, during our

experiments. Since Giardia infections can occur with various gastrointestinal pathogens (57-60,

65), additional research is required in order to determine how Giardia cathepsin cysteine

proteases translocate across intestinal epithelial barriers during co-infection. In addition, previous

reports from our lab and others have demonstrated that Giardia infections disrupt many AJC

proteins (25, 26, 50, 56, 177). Consequently, it remains to be determined whether intestinal

epithelial barrier disruption mediated by Giardia trophozoites is sufficient to promote basolateral

translocation of cathepsin cysteine proteases without the presence of another gastrointestinal

pathogen. A recent report has also demonstrated that the co-incubation of polarized Caco-2

monolayers, Giardia trophozoites, and the IC-21 macrophage cell line in vitro results in

basolateral attenuation of CXCL8 and GRO family proteins via an unknown mechanism (237).

As macrophages have been shown to increase permeability of intestinal epithelial barriers (238),

181

basolateral translocation of Giardia cathepsin cysteine proteases require interaction between

trophozoites, the intestinal epithelium, and tissue-resident macrophages. However, additional

experiments are required in order to confirm this hypothesis.

4.5 Giardia cathepsin proteases

Cathepsin cysteine proteases of single-celled parasites have been well described, but their

function remains incompletely understood (reviewed in (160, 161)). Parasite cysteine proteases

have been shown to prevent activation of pro-inflammatory transcription factors (162), degrade

host effector molecules (239), promote specific host immune responses (240, 241), and cleave

host chemokines (167, 168). Prior to this study, reports had described a role for a Giardia catB in

trophozoite encystation and excystation (170), but the role of these proteases in disease

pathogenesis and host immunity has remained largely speculative (172, 242). The results

presented herein demonstrated that catB proteases secreted by Giardia trophozoites might be

associated with the attenuation of aspects of their host’s pro-inflammatory immune response.

Our study further demonstrates isolate-dependent differences in Giardia catB and catL cysteine

proteases. Construction of phylogenetic trees for Giardia catB proteases indicated the presence

of unique catB proteases within Giardia WB and GS/M isolates. Similarly, Giardia WB was

found to contain several additional catL proteases not found within Giardia GS/M. To date, only

two human Giardia isolates have been sequenced (169), and whole genome sequencing of

several assemblage A and B Giardia isolates are required in order to determine whether the

difference in Giardia cathepsin cysteine proteases are isolate-specific or assemblage-specific.

182

As the Giardia genome contains genes for several cathepsin cysteine proteases, including

9 catB proteases (169), it remains to be seen whether Giardia cathepsin cysteine proteases may

modulate various aspects of their host’s immune response, and target a variety of cells within the

intestinal mucosa. Results from in vivo and ex vivo experiments demonstrated that Giardia

trophozoites are capable of attenuating a wide variety of inflammatory mediators. Therefore,

additional experiments are required in order to determine whether Giardia cathepsin proteases

attenuate expression of these mediators. As analysis of the coding region of Giardia WB and

Giardia GS/M genomes has indicated an amino acid identity of only 78% (22), ongoing research

needs to assess whether cathepsin cysteine proteases in assemblage A and B Giardia isolates

play different roles in modulating or attenuating their host’s immune response. Moreover,

differences in these proteases may contribute to different pathophysiological events within

Giardia-infected hosts. The kinetics of cathepsin cysteine protease secretion by assemblage A

and B Giardia trophozoites also requires further study. This research may explain results in this

study demonstrating that Giardia GS/M trophozoites only attenuated IL-1β-induced supernatant

CXCL8 when incubated with Caco-2 monolayers for 24 hours prior to addition of IL-1β.

4.5.1 Specificity of Giardia cathepsin proteases

This study demonstrated that a 10 µM Ca-074Me treatment was sufficient to inhibit

degradation of CXCL8 by Giardia NF trophozoites while also having a more significant impact

on limiting hydrolysis of the catB/L substrate ZFR-AMC than processing of the catB substrate

ZRR-AMC by trophozoite sonicates. Therefore, certain Giardia catB proteases appear to possess

structural features that prevent them from hydrolyzing ZFR-AMC while still being capable of

183

hydrolyzing ZRR-AMC following treatment with Ca-074Me. Similar studies have shown

Leishmania major catB is unable to hydrolyse ZRR-AMC but is susceptible to inhibition with

Ca-074 (243). Additionally, as the Giardia genome contains genes for numerous cathepsin

proteases, it is possible that additional cathepsins or other proteases are capable of cleaving

CXCL8. Indeed, a basic local alignment search tool (BLAST) analysis (244) of the protein

responsible for cleaving CXCL8 in E. histolytica (167) against the Giardia genome has indicated

structural similarity to several catL proteases suggesting that multiple Giardia proteases may be

capable of degrading or cleaving CXCL8. Additionally, Giardia cathepsin cysteine proteases

may require activation via separate proteases. For example, Toxoplasma gondii catB proteases

require proteolytic activation by the parasite’s catL cysteine proteases (245). Additional research

is required to examine the structure and substrate specificities of Giardia catB proteases to

determine whether these factors exhibit unique substrate specificities and, furthermore, if other

proteases participate in the degradation of CXCL8. Regardless, our study is the first to

demonstrate an anti-inflammatory role for Giardia trophozoites while specifically identifying the

host cells targeted and the parasite factors involved.

4.6 Giardia immunomodulatory molecules

This study demonstrated that Giardia catB proteases degrade intestinal epithelial CXCL8

and attenuate CXCL8 and C5a-induced PMN chemotaxis. However, it remains to be seen

whether Giardia catB proteases are involved in attenuating granulocyte tissue recruitment during

in vivo infections. In particular, a new model of in vivo intestinal inflammation induced via intra-

luminal instillation of CXCL1 or fMLF may help elucidate whether Giardia infections attenuate

184

in vivo PMN accumulation in response to these chemokines (127, 211). Pre-treatment of small

intestinal loops with catB inhibitors would also help elucidate a role for Giardia catB proteases

in attenuating CXCL1-induced PMN accumulation. It has also recently been demonstrated that

Giardia DNA can be modified via the Cre/loxP system (246). As a result, it may be possible to

delete catB genes within Giardia trophozoites. This system would be extremely useful in

identifying the particular catB protease involved in degrading CXCL8. Furthermore, microarray

analysis of Giardia assemblage E-infected cattle demonstrated a decrease in genes associated

with lipid metabolism (63). Therefore, additional research is required to determine whether

Giardia trophozoites may also modulate the expression of pro-inflammatory lipid mediators. The

present findings also revealed that Giardia catB failed to have a similar proteolytic effect on IL1-

β, but it remains to be seen whether Giardia is capable of degrading or modulating other PMN

chemokines. Indeed, our studies demonstrated that Giardia infections modulated the expression

of various inflammatory mediators from inflamed mucosal biopsy tissues. Some of these

mediators were attenuated within supernatants, while others were attenuated within biopsy tissue

homogenates. Therefore, it is entirely possible that Giardia trophozoites modulate expression of

various inflammatory mediators via different mechanisms.

Apart from Giardia catB proteases, the number of immunomodulatory compounds

identified in Giardia remains relatively unknown. Giardia ADI has been shown to attenuate

nitric oxide production from intestinal epithelial cells (32, 40, 42), inhibit intestinal epithelial

proliferation (40), induce expression of IL-12p70 from DCs (229), and inhibit T-cell

proliferation (247). However, all these studies have been performed in vitro and none of these

effects have been demonstrated during in vivo Giardia infections. Additional studies are needed

185

to identify other potential Giardia immunomodulatory molecules and elucidate whether these

factors are involved in modulating host responses in vivo. Giardia trophozoites express the ecto-

5’-nucleotidase CD73 on their cell surface, and is hypothesized to participate in salvaging

purines (248). It is possible Giardia CD73 may also modulate host immune responses. Genetic

deletion of CD73 in mice has been shown to enhance the severity of TNBS colitis (249), and its

expression is mediated by HIF transcription factors (250). Therefore, Giardia CD73 may also

protect in the development of intestinal inflammatory responses. Moreover, conversion of ATP

into adenosine via CD73 activates the A2b receptor on IECs and induces IL-6 expression (251).

Therefore, consumption of exogenous ATP by Giardia CD73 may explain the observed decrease

in tissue IL-6. However, additional research is required in order to confirm these hypotheses.

The treatment and management of chronic gastrointestinal disorders such as IBD and C.

difficile colitis represent significant economic burdens (252-254). PMNs are capable of causing

pathophysiological responses in chronic gastrointestinal disorders, such as IBD and C. difficile

infection, via multiple mechanisms. Moreover, these diseases are also associated with increased

expression levels of various PMN chemoattractants, such as CXCL8 and C5a (116, 255-258).

Therefore, strategies aimed at blocking PMN accumulation and TEM are being investigated as

potential therapeutics for the treatment of chronic gastrointestinal inflammatory disorders (259-

261). Specifically, blockade of PMN chemokines represents a viable strategy for attenuating the

intestinal accumulation of PMNs during chronic intestinal inflammatory disorders. Food-grade

bacteria genetically engineered to express anti-inflammatory mediators have been shown to

attenuate intestinal inflammation in vivo has recently been described (262). The identification of

specific Giardia catB proteases that degrade PMN chemokines, such as CXCL8, and their

186

genetic insertion into similar organisms represents an exciting future avenue for this project that

may have significant therapeutic potential for individuals with chronic gastrointestinal

inflammatory disorders.

4.7 Conclusion

In conclusion, the results from this study demonstrate the novel observation that Giardia

infections, in an isolate-dependent manner, attenuate granulocyte infiltration and expression of

PMN chemokines, in an in vivo model of colitis. Moreover, Giardia trophozoites reduced

expression of a variety of inflammatory mediators released from inflamed colonic mucosal

biopsy tissues from CD patients. Our findings are the first to demonstrate an anti-inflammatory

effect for Giardia infections in several experimental models of inflammation and suggest this

mechanism is dependent on the infecting Giardia isolate. In conclusion, our data reveal a novel

role for Giardia catB cysteine proteases. Giardia catB proteases can degrade CXCL8 secreted

by human intestinal epithelial cells in response to host- and pathogen-derived pro-inflammatory

stimuli, such as IL-1β and S. typhimurium. The degradation of CXCL8 by Giardia catB cysteine

proteases directly attenuated CXCL8-induced PMN chemotaxis. Our findings are the first to

demonstrate that Giardia trophozoites are capable of attenuating acute inflammatory responses

within the intestinal mucosa that promote the recruitment of pro-inflammatory PMNs and assign

a role for Giardia catB cysteine proteases in this process.

187

4.8 Future directions

Findings from this study open the avenue for future research examining the

immunomodulatory effects of Giardia and may also lead to identification of novel anti-

inflammatory compounds that could be used for the treatment of chronic gastrointestinal

inflammatory disorders. Future studies could focus on:

1. Identifying the specific catB protease(s) responsible for degrading CXCL8 and

attenuating CXCL8-induced PMN chemotaxis by generating Giardia trophozoite

knockouts

2. Determining whether the identified Giardia catB protease(s) are capable of attenuating

gastrointestinal inflammation in vivo

3. Examining whether attenuation of PMN accumulation in vivo enhances or impedes the

resolution of inflammation

4. Determine whether Giardia infections modulates the expression of pro-inflammatory

lipid mediators

5. Assess whether Giardia co-infections modulate inflammatory responses in humans

188

5.0 References

1. Savioli L, Smith H, Thompson A. 2006. Giardia and Cryptosporidium join the

'Neglected Diseases Initiative'. Trends in parasitology 22:203-208.

2. Geurden T, Vercruysse J, Claerebout E. 2010. Is Giardia a significant pathogen in

production animals? Experimental parasitology 124:98-106.

3. Ankarklev J, Jerlstrom-Hultqvist J, Ringqvist E, Troell K, Svard SG. 2010. Behind

the smile: cell biology and disease mechanisms of Giardia species. Nature reviews.

Microbiology 8:413-422.

4. Eckmann L. 2003. Mucosal defences against Giardia. Parasite immunology 25:259-270.

5. Aloisio F, Filippini G, Antenucci P, Lepri E, Pezzotti G, Caccio SM, Pozio E. 2006.

Severe weight loss in lambs infected with Giardia duodenalis assemblage B. Veterinary

parasitology 142:154-158.

6. Kohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD, Sears CL, Lima AA,

Guerrant RL. 2008. Giardia duodenalis assemblage, clinical presentation and markers of

intestinal inflammation in Brazilian children. Transactions of the Royal Society of

Tropical Medicine and Hygiene 102:718-725.

7. Hanevik K, Hausken T, Morken MH, Strand EA, Morch K, Coll P, Helgeland L,

Langeland N. 2007. Persisting symptoms and duodenal inflammation related to Giardia

duodenalis infection. The Journal of infection 55:524-530.

8. Morken MH, Nysaeter G, Strand EA, Hausken T, Berstad A. 2008. Lactulose breath

test results in patients with persistent abdominal symptoms following Giardia lamblia

infection. Scandinavian journal of gastroenterology 43:141-145.

9. Morken MH, Valeur J, Norin E, Midtvedt T, Nysaeter G, Berstad A. 2009.

Antibiotic or bacterial therapy in post-giardiasis irritable bowel syndrome. Scandinavian

journal of gastroenterology 44:1296-1303.

10. Halliez MC, Buret AG. 2013. Extra-intestinal and long term consequences of Giardia

duodenalis infections. World journal of gastroenterology : WJG 19:8974-8985.

11. Cotton JA, Beatty JK, Buret AG. 2011. Host parasite interactions and pathophysiology

in Giardia infections. International journal for parasitology 41:925-933.

12. Peattie DA, Alonso RA, Hein A, Caulfield JP. 1989. Ultrastructural localization of

giardins to the edges of disk microribbons of Giarida lamblia and the nucleotide and

deduced protein sequence of alpha giardin. The Journal of cell biology 109:2323-2335.

13. Peattie DA. 1990. The giardins of Giardia lamblia: genes and proteins with promise.

Parasitology today 6:52-56.

14. Nohria A, Alonso RA, Peattie DA. 1992. Identification and characterization of gamma-

giardin and the gamma-giardin gene from Giardia lamblia. Molecular and biochemical


15. Jenkins MC, O'Brien CN, Murphy C, Schwarz R, Miska K, Rosenthal B, Trout JM.

2009. Antibodies to the ventral disc protein delta-giardin prevent in vitro binding of

Giardia lamblia trophozoites. The Journal of parasitology 95:895-899.

16. Jimenez-Cardoso E, Eligio-Garcia L, Cortes-Campos A, Flores-Luna A, Valencia-

Mayoral P, Lozada-Chavez I. 2009. Changes in beta-giardin sequence of Giardia

189

intestinalis sensitive and resistant to albendazole strains. Parasitology research 105:25-

33.

17. Monis PT, Caccio SM, Thompson RC. 2009. Variation in Giardia: towards a taxonomic

revision of the genus. Trends in parasitology 25:93-100.

18. Lasek-Nesselquist E, Welch DM, Sogin ML. 2010. The identification of a new Giardia

duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis

population biology in marine systems. International journal for parasitology 40:1063-

1074.

19. Caccio SM, Thompson RC, McLauchlin J, Smith HV. 2005. Unravelling

Cryptosporidium and Giardia epidemiology. Trends in parasitology 21:430-437.

20. Robertson LJ, Hanevik K, Escobedo AA, Morch K, Langeland N. 2010. Giardiasis--

why do the symptoms sometimes never stop? Trends in parasitology 26:75-82.

21. Thompson RC, Monis PT. 2004. Variation in Giardia: implications for taxonomy and

epidemiology. Advances in parasitology 58:69-137.

22. Franzen O, Jerlstrom-Hultqvist J, Castro E, Sherwood E, Ankarklev J, Reiner DS,

Palm D, Andersson JO, Andersson B, Svard SG. 2009. Draft genome sequencing of

giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different

species? PLoS pathogens 5:e1000560.

23. Jerlstrom-Hultqvist J, Ankarklev J, Svard SG. 2010. Is human giardiasis caused by

two different Giardia species? Gut microbes 1:379-382.

24. Steuart RF, O'Handley R, Lipscombe RJ, Lock RA, Thompson RC. 2008. Alpha 2

giardin is an assemblage A-specific protein of human infective Giardia duodenalis.

Parasitology 135:1621-1627.

25. Chin AC, Teoh DA, Scott KG, Meddings JB, Macnaughton WK, Buret AG. 2002.

Strain-dependent induction of enterocyte apoptosis by Giardia lamblia disrupts epithelial

barrier function in a caspase-3-dependent manner. Infection and immunity 70:3673-3680.

26. Panaro MA, Cianciulli A, Mitolo V, Mitolo CI, Acquafredda A, Brandonisio O,

Cavallo P. 2007. Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced

by the parasite Giardia intestinalis. FEMS immunology and medical microbiology

51:302-309.

27. Solaymani-Mohammadi S, Singer SM. 2011. Host immunity and pathogen strain

contribute to intestinal disaccharidase impairment following gut infection. Journal of

immunology 187:3769-3775.

28. Bartelt LA, Roche J, Kolling G, Bolick D, Noronha F, Naylor C, Hoffman P,

Warren C, Singer S, Guerrant R. 2013. Persistent G. lamblia impairs growth in a

murine malnutrition model. The Journal of clinical investigation 123:2672-2684.

29. Cevallos A, Carnaby S, James M, Farthing JG. 1995. Small intestinal injury in a

neonatal rat model of giardiasis is strain dependent. Gastroenterology 109:766-773.

30. Buret AG. 2007. Mechanisms of epithelial dysfunction in giardiasis. Gut 56:316-317.

31. Ringqvist E, Avesson L, Soderbom F, Svard SG. 2011. Transcriptional changes in

Giardia during host-parasite interactions. International journal for parasitology 41:277-

285.

32. Ringqvist E, Palm JE, Skarin H, Hehl AB, Weiland M, Davids BJ, Reiner DS,

Griffiths WJ, Eckmann L, Gillin FD, Svard SG. 2008. Release of metabolic enzymes

190

by Giardia in response to interaction with intestinal epithelial cells. Molecular and

biochemical parasitology 159:85-91.

33. Parenti DM. 1989. Characterization of a thiol proteinase in Giardia lamblia. The Journal

of infectious diseases 160:1076-1080.

34. Byrd LG, Conrad JT, Nash TE. 1994. Giardia lamblia infections in adult mice.

Infection and immunity 62:3583-3585.

35. Singer SM, Elmendorf HG, Conrad JT, Nash TE. 2001. Biological selection of

variant-specific surface proteins in Giardia lamblia. The Journal of infectious diseases

183:119-124.

36. Rodriguez-Fuentes GB, Cedillo-Rivera R, Fonseca-Linan R, Arguello-Garcia R,

Munoz O, Ortega-Pierres G, Yepez-Mulia L. 2006. Giardia duodenalis: analysis of

secreted proteases upon trophozoite-epithelial cell interaction in vitro. Memorias do

Instituto Oswaldo Cruz 101:693-696.

37. Prucca CG, Lujan HD. 2009. Antigenic variation in Giardia lamblia. Cellular

microbiology 11:1706-1715.

38. Shant J, Ghosh S, Bhattacharyya S, Ganguly NK, Majumdar S. 2005. Mode of action

of a potentially important excretory--secretory product from Giardia lamblia in mice

enterocytes. Parasitology 131:57-69.

39. Mohammed SR, Faubert GM. 1995. Purification of a fraction of Giardia lamblia

trophozoite extract associated with disaccharidase deficiencies in immune Mongolian

gerbils (Meriones unguiculatus). Parasite 2:31-39.

40. Stadelmann B, Merino MC, Persson L, Svard SG. 2012. Arginine consumption by the

intestinal parasite Giardia intestinalis reduces proliferation of intestinal epithelial cells.

PloS one 7:e45325.

41. Touz MC, Ropolo AS, Rivero MR, Vranych CV, Conrad JT, Svard SG, Nash TE.

2008. Arginine deiminase has multiple regulatory roles in the biology of Giardia lamblia.

Journal of cell science 121:2930-2938.

42. Eckmann L, Laurent F, Langford TD, Hetsko ML, Smith JR, Kagnoff MF, Gillin

FD. 2000. Nitric oxide production by human intestinal epithelial cells and competition

for arginine as potential determinants of host defense against the lumen-dwelling

pathogen Giardia lamblia. Journal of immunology 164:1478-1487.

43. Banik S, Renner Viveros P, Seeber F, Klotz C, Ignatius R, Aebischer T. 2013.

Giardia duodenalis arginine deiminase modulates the phenotype and cytokine secretion of

human dendritic cells by depletion of arginine and formation of ammonia. Infection and

immunity 81:2309-2317.

44. Kamda JD, Singer SM. 2009. Phosphoinositide 3-kinase-dependent inhibition of

dendritic cell interleukin-12 production by Giardia lamblia. Infection and immunity

77:685-693.

45. Roxstrom-Lindquist K, Ringqvist E, Palm D, Svard S. 2005. Giardia lamblia-induced

changes in gene expression in differentiated Caco-2 human intestinal epithelial cells.


46. Jimenez JC, Fontaine J, Grzych JM, Dei-Cas E, Capron M. 2004. Systemic and

mucosal responses to oral administration of excretory and secretory antigens from

Giardia intestinalis. Clinical and diagnostic laboratory immunology 11:152-160.

191

47. Bayraktar MR, Mehmet N, Durmaz R. 2005. Role of IL-2, IL-4 and IL-10 in patients

infected with Giardia lamblia. Turkiye parazitolojii dergisi / Turkiye Parazitoloji Dernegi

= Acta parasitologica Turcica / Turkish Society for Parasitology 29:160-162.

48. Matowicka-Karna J, Dymicka-Piekarska V, Kemona H. 2009. IFN-gamma, IL-5, IL-

6 and IgE in patients infected with Giardia intestinalis. Folia histochemica et

cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical

Society 47:93-97.

49. Scott KG, Logan MR, Klammer GM, Teoh DA, Buret AG. 2000. Jejunal brush border

microvillous alterations in Giardia muris-infected mice: role of T lymphocytes and

interleukin-6. Infection and immunity 68:3412-3418.

50. Scott KG, Meddings JB, Kirk DR, Lees-Miller SP, Buret AG. 2002. Intestinal

infection with Giardia spp. reduces epithelial barrier function in a myosin light chain

kinase-dependent fashion. Gastroenterology 123:1179-1190.

51. Yu LC, Huang CY, Kuo WT, Sayer H, Turner JR, Buret AG. 2008. SGLT-1-

mediated glucose uptake protects human intestinal epithelial cells against Giardia

duodenalis-induced apoptosis. International journal for parasitology 38:923-934.

52. Yu LC, Flynn AN, Turner JR, Buret AG. 2005. SGLT-1-mediated glucose uptake

protects intestinal epithelial cells against LPS-induced apoptosis and barrier defects: a

novel cellular rescue mechanism? FASEB journal : official publication of the Federation

of American Societies for Experimental Biology 19:1822-1835.

53. Oberhuber G, Kastner N, Stolte M. 1997. Giardiasis: a histologic analysis of 567 cases.

Scandinavian journal of gastroenterology 32:48-51.

54. Koh WH, Geurden T, Paget T, O'Handley R, Steuart RF, Thompson RC, Buret AG.

2013. Giardia duodenalis assemblage-specific induction of apoptosis and tight junction

disruption in human intestinal epithelial cells: effects of mixed infections. The Journal of


55. Humen MA, Perez PF, Lievin-Le Moal V. 2011. Lipid raft-dependent adhesion of

Giardia intestinalis trophozoites to a cultured human enterocyte-like Caco-2/TC7 cell

monolayer leads to cytoskeleton-dependent functional injuries. Cellular microbiology

13:1683-1702.

56. Troeger H, Epple HJ, Schneider T, Wahnschaffe U, Ullrich R, Burchard GD,

Jelinek T, Zeitz M, Fromm M, Schulzke JD. 2007. Effect of chronic Giardia lamblia

infection on epithelial transport and barrier function in human duodenum. Gut 56:328-

335.

57. Wang L, Xiao L, Duan L, Ye J, Guo Y, Guo M, Liu L, Feng Y. 2013. Concurrent

infections of Giardia duodenalis, Enterocytozoon bieneusi, and Clostridium difficile in

children during a cryptosporidiosis outbreak in a pediatric hospital in China. PLoS

neglected tropical diseases 7:e2437.

58. Ankarklev J, Hestvik E, Lebbad M, Lindh J, Kaddu-Mulindwa DH, Andersson JO,

Tylleskar T, Tumwine JK, Svard SG. 2012. Common coinfections of Giardia

intestinalis and Helicobacter pylori in non-symptomatic Ugandan children. PLoS

neglected tropical diseases 6:e1780.

192

59. Bilenko N, Levy A, Dagan R, Deckelbaum RJ, El-On Y, Fraser D. 2004. Does co-

infection with Giardia lamblia modulate the clinical characteristics of enteric infections in

young children? European journal of epidemiology 19:877-883.

60. Bhavnani D, Goldstick JE, Cevallos W, Trueba G, Eisenberg JN. 2012. Synergistic

effects between rotavirus and coinfecting pathogens on diarrheal disease: evidence from a

community-based study in northwestern Ecuador. American journal of epidemiology

176:387-395.

61. Oberhelman RA, Flores-Abuxapqui J, Suarez-Hoil G, Puc-Franco M, Heredia-

Navarrete M, Vivas-Rosel M, Mera R, Gutierrez-Cogco L. 2001. Asymptomatic

salmonellosis among children in day-care centers in Merida, Yucatan, Mexico. The

Pediatric infectious disease journal 20:792-797.

62. Hardin JA, Buret AG, Olson ME, Kimm MH, Gall DG. 1997. Mast cell hyperplasia

and increased macromolecular uptake in an animal model of giardiasis. J Parasitol

83:908-912.

63. Dreesen L, Rinaldi M, Chiers K, Li R, Geurden T, Van den Broeck W, Goddeeris B,

Vercruysse J, Claerebout E, Geldhof P. 2012. Microarray analysis of the intestinal host

response in Giardia duodenalis assemblage E infected calves. PloS one 7:e40985.

64. Veenemans J, Schouten LR, Ottenhof MJ, Mank TG, Uges DR, Mbugi EV, Demir

AY, Kraaijenhagen RJ, Savelkoul HF, Verhoef H. 2012. Effect of preventive

supplementation with zinc and other micronutrients on non-malarial morbidity in

Tanzanian pre-school children: a randomized trial. PloS one 7:e41630.

65. Muhsen K, Cohen D, Levine MM. 2013. Can Giardia lamblia infection lower the risk of

acute diarrhea among preschool children? Journal of tropical pediatrics.

66. Kostman R. 1975. Infantile genetic agranulocytosis. A review with presentation of ten

new cases. Acta paediatrica Scandinavica 64:362-368.

67. Zeidler C, Germeshausen M, Klein C, Welte K. 2009. Clinical implications of ELA2-,

HAX1-, and G-CSF-receptor (CSF3R) mutations in severe congenital neutropenia.

British journal of haematology 144:459-467.

68. Kuijpers T, Lutter R. 2012. Inflammation and repeated infections in CGD: two sides of

a coin. Cellular and molecular life sciences : CMLS 69:7-15.

69. Amulic B, Cazalet C, Hayes GL, Metzler KD, Zychlinsky A. 2012. Neutrophil

function: from mechanisms to disease. Annual review of immunology 30:459-489.

70. Kumar NB, Nostrant TT, Appelman HD. 1982. The histopathologic spectrum of acute

self-limited colitis (acute infectious-type colitis). The American journal of surgical

pathology 6:523-529.

71. Chin AC, Parkos CA. 2006. Neutrophil transepithelial migration and epithelial barrier

function in IBD: potential targets for inhibiting neutrophil trafficking. Annals of the New

York Academy of Sciences 1072:276-287.

72. Kelly CP, Becker S, Linevsky JK, Joshi MA, O'Keane JC, Dickey BF, LaMont JT,

Pothoulakis C. 1994. Neutrophil recruitment in Clostridium difficile toxin A enteritis in

the rabbit. The Journal of clinical investigation 93:1257-1265.

73. Van Haastert PJ, Devreotes PN. 2004. Chemotaxis: signalling the way forward. Nature

reviews. Molecular cell biology 5:626-634.

74. Thelen M. 2001. Dancing to the tune of chemokines. Nature immunology 2:129-134.

193

75. Wolf M, Delgado MB, Jones SA, Dewald B, Clark-Lewis I, Baggiolini M. 1998.

Granulocyte chemotactic protein 2 acts via both IL-8 receptors, CXCR1 and CXCR2.

European journal of immunology 28:164-170.

76. Cartwright GE, Athens JW, Wintrobe MM. 1964. The Kinetics of Granulopoiesis in

Normal Man. Blood 24:780-803.

77. Summers C, Rankin SM, Condliffe AM, Singh N, Peters AM, Chilvers ER. 2010.

Neutrophil kinetics in health and disease. Trends in immunology 31:318-324.

78. Semerad CL, Liu F, Gregory AD, Stumpf K, Link DC. 2002. G-CSF is an essential

regulator of neutrophil trafficking from the bone marrow to the blood. Immunity 17:413-

423.

79. Eash KJ, Greenbaum AM, Gopalan PK, Link DC. 2010. CXCR2 and CXCR4

antagonistically regulate neutrophil trafficking from murine bone marrow. The Journal of

clinical investigation 120:2423-2431.

80. Suratt BT, Petty JM, Young SK, Malcolm KC, Lieber JG, Nick JA, Gonzalo JA,

Henson PM, Worthen GS. 2004. Role of the CXCR4/SDF-1 chemokine axis in

circulating neutrophil homeostasis. Blood 104:565-571.

81. Petty JM, Lenox CC, Weiss DJ, Poynter ME, Suratt BT. 2009. Crosstalk between

CXCR4/stromal derived factor-1 and VLA-4/VCAM-1 pathways regulates neutrophil

retention in the bone marrow. Journal of immunology 182:604-612.

82. Wengner AM, Pitchford SC, Furze RC, Rankin SM. 2008. The coordinated action of

G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute

inflammation. Blood 111:42-49.

83. Mei J, Liu Y, Dai N, Hoffmann C, Hudock KM, Zhang P, Guttentag SH, Kolls JK,

Oliver PM, Bushman FD, Worthen GS. 2012. Cxcr2 and Cxcl5 regulate the IL-17/G-

CSF axis and neutrophil homeostasis in mice. The Journal of clinical investigation

122:974-986.

84. Schwarzenberger P, Huang W, Ye P, Oliver P, Manuel M, Zhang Z, Bagby G,

Nelson S, Kolls JK. 2000. Requirement of endogenous stem cell factor and granulocyte-

colony-stimulating factor for IL-17-mediated granulopoiesis. Journal of immunology

164:4783-4789.

85. Kolaczkowska E, Kubes P. 2013. Neutrophil recruitment and function in health and

inflammation. Nature reviews. Immunology 13:159-175.

86. Phillipson M, Kubes P. 2011. The neutrophil in vascular inflammation. Nature medicine

17:1381-1390.

87. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. 2007. Getting to the site of

inflammation: the leukocyte adhesion cascade updated. Nature reviews. Immunology

7:678-689.

88. Mantovani A, Cassatella MA, Costantini C, Jaillon S. 2011. Neutrophils in the

activation and regulation of innate and adaptive immunity. Nature reviews. Immunology

11:519-531.

89. Massena S, Christoffersson G, Hjertstrom E, Zcharia E, Vlodavsky I, Ausmees N,

Rolny C, Li JP, Phillipson M. 2010. A chemotactic gradient sequestered on endothelial

heparan sulfate induces directional intraluminal crawling of neutrophils. Blood 116:1924-

1931.

194

90. Phillipson M, Heit B, Colarusso P, Liu L, Ballantyne CM, Kubes P. 2006.

Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process

from adhesion in the recruitment cascade. The Journal of experimental medicine

203:2569-2575.

91. Jones DH, Anderson DC, Burr BL, Rudloff HE, Smith CW, Krater SS, Schmalstieg

FC. 1988. Quantitation of intracellular Mac-1 (CD11b/CD18) pools in human

neutrophils. Journal of leukocyte biology 44:535-544.

92. Burns AR, Bowden RA, MacDonell SD, Walker DC, Odebunmi TO, Donnachie EM,

Simon SI, Entman ML, Smith CW. 2000. Analysis of tight junctions during neutrophil

transendothelial migration. Journal of cell science 113 ( Pt 1):45-57.

93. Woodfin A, Voisin MB, Beyrau M, Colom B, Caille D, Diapouli FM, Nash GB,

Chavakis T, Albelda SM, Rainger GE, Meda P, Imhof BA, Nourshargh S. 2011. The

junctional adhesion molecule JAM-C regulates polarized transendothelial migration of

neutrophils in vivo. Nature immunology 12:761-769.

94. Foxman EF, Campbell JJ, Butcher EC. 1997. Multistep navigation and the

combinatorial control of leukocyte chemotaxis. The Journal of cell biology 139:1349-

1360.

95. Chou RC, Kim ND, Sadik CD, Seung E, Lan Y, Byrne MH, Haribabu B, Iwakura

Y, Luster AD. 2010. Lipid-cytokine-chemokine cascade drives neutrophil recruitment in

a murine model of inflammatory arthritis. Immunity 33:266-278.

96. McDonald B, Pittman K, Menezes GB, Hirota SA, Slaba I, Waterhouse CC, Beck

PL, Muruve DA, Kubes P. 2010. Intravascular danger signals guide neutrophils to sites

of sterile inflammation. Science 330:362-366.

97. Heit B, Tavener S, Raharjo E, Kubes P. 2002. An intracellular signaling hierarchy

determines direction of migration in opposing chemotactic gradients. The Journal of cell

biology 159:91-102.

98. Chin AC, Parkos CA. 2007. Pathobiology of neutrophil transepithelial migration:

implications in mediating epithelial injury. Annual review of pathology 2:111-143.

99. Mrsny RJ, Gewirtz AT, Siccardi D, Savidge T, Hurley BP, Madara JL, McCormick

BA. 2004. Identification of hepoxilin A3 in inflammatory events: a required role in

neutrophil migration across intestinal epithelia. Proceedings of the National Academy of

Sciences of the United States of America 101:7421-7426.

100. McCormick BA, Hofman PM, Kim J, Carnes DK, Miller SI, Madara JL. 1995.

Surface attachment of Salmonella typhimurium to intestinal epithelia imprints the

subepithelial matrix with gradients chemotactic for neutrophils. The Journal of cell

biology 131:1599-1608.

101. McCormick BA, Parkos CA, Colgan SP, Carnes DK, Madara JL. 1998. Apical

secretion of a pathogen-elicited epithelial chemoattractant activity in response to surface

colonization of intestinal epithelia by Salmonella typhimurium. Journal of immunology

160:455-466.

102. Nash S, Stafford J, Madara JL. 1987. Effects of polymorphonuclear leukocyte

transmigration on the barrier function of cultured intestinal epithelial monolayers. The

Journal of clinical investigation 80:1104-1113.

195

103. Edens HA, Levi BP, Jaye DL, Walsh S, Reaves TA, Turner JR, Nusrat A, Parkos

CA. 2002. Neutrophil transepithelial migration: evidence for sequential, contact-

dependent signaling events and enhanced paracellular permeability independent of

transjunctional migration. Journal of immunology 169:476-486.

104. Parkos CA, Delp C, Arnaout MA, Madara JL. 1991. Neutrophil migration across a

cultured intestinal epithelium. Dependence on a CD11b/CD18-mediated event and

enhanced efficiency in physiological direction. The Journal of clinical investigation

88:1605-1612.

105. Zen K, Liu DQ, Li LM, Chen CX, Guo YL, Ha B, Chen X, Zhang CY, Liu Y. 2009.

The heparan sulfate proteoglycan form of epithelial CD44v3 serves as a CD11b/CD18

counter-receptor during polymorphonuclear leukocyte transepithelial migration. The

Journal of biological chemistry 284:3768-3776.

106. Chin AC, Lee WY, Nusrat A, Vergnolle N, Parkos CA. 2008. Neutrophil-mediated

activation of epithelial protease-activated receptors-1 and -2 regulates barrier function

and transepithelial migration. Journal of immunology 181:5702-5710.

107. Zen K, Liu Y, McCall IC, Wu T, Lee W, Babbin BA, Nusrat A, Parkos CA. 2005.

Neutrophil migration across tight junctions is mediated by adhesive interactions between

epithelial coxsackie and adenovirus receptor and a junctional adhesion molecule-like

protein on neutrophils. Molecular biology of the cell 16:2694-2703.

108. Zen K, Babbin BA, Liu Y, Whelan JB, Nusrat A, Parkos CA. 2004. JAM-C is a

component of desmosomes and a ligand for CD11b/CD18-mediated neutrophil

transepithelial migration. Molecular biology of the cell 15:3926-3937.

109. Hurley BP, Sin A, McCormick BA. 2008. Adhesion molecules involved in hepoxilin

A3-mediated neutrophil transepithelial migration. Clinical and experimental immunology

151:297-305.

110. Parkos CA, Colgan SP, Diamond MS, Nusrat A, Liang TW, Springer TA, Madara

JL. 1996. Expression and polarization of intercellular adhesion molecule-1 on human

intestinal epithelia: consequences for CD11b/CD18-mediated interactions with

neutrophils. Molecular medicine 2:489-505.

111. Sumagin R, Robin AZ, Nusrat A, Parkos CA. 2013. Transmigrated neutrophils in the

intestinal lumen engage ICAM-1 to regulate the epithelial barrier and neutrophil

recruitment. Mucosal immunology.

112. Hu M, Lin X, Du Q, Miller EJ, Wang P, Simms HH. 2005. Regulation of

polymorphonuclear leukocyte apoptosis: role of lung endothelium-epithelium bilayer

transmigration. American journal of physiology. Lung cellular and molecular physiology

288:L266-274.

113. Ginis I, Faller DV. 1997. Protection from apoptosis in human neutrophils is determined

by the surface of adhesion. The American journal of physiology 272:C295-309.

114. Pullman WE, Elsbury S, Kobayashi M, Hapel AJ, Doe WF. 1992. Enhanced mucosal

cytokine production in inflammatory bowel disease. Gastroenterology 102:529-537.

115. Grimm MC, Elsbury SK, Pavli P, Doe WF. 1996. Interleukin 8: cells of origin in

inflammatory bowel disease. Gut 38:90-98.

196

116. Nielsen OH, Rudiger N, Gaustadnes M, Horn T. 1997. Intestinal interleukin-8

concentration and gene expression in inflammatory bowel disease. Scandinavian journal

of gastroenterology 32:1028-1034.

117. Hasegawa M, Yamazaki T, Kamada N, Tawaratsumida K, Kim YG, Nunez G,

Inohara N. 2011. Nucleotide-binding oligomerization domain 1 mediates recognition of

Clostridium difficile and induces neutrophil recruitment and protection against the

pathogen. Journal of immunology 186:4872-4880.

118. Savidge TC, Pan WH, Newman P, O'Brien M, Anton PM, Pothoulakis C. 2003.

Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine.

Gastroenterology 125:413-420.

119. Tibble JA, Sigthorsson G, Bridger S, Fagerhol MK, Bjarnason I. 2000. Surrogate

markers of intestinal inflammation are predictive of relapse in patients with inflammatory

bowel disease. Gastroenterology 119:15-22.

120. Gisbert JP, Bermejo F, Perez-Calle JL, Taxonera C, Vera I, McNicholl AG, Algaba

A, Lopez P, Lopez-Palacios N, Calvo M, Gonzalez-Lama Y, Carneros JA, Velasco

M, Mate J. 2009. Fecal calprotectin and lactoferrin for the prediction of inflammatory

bowel disease relapse. Inflammatory bowel diseases 15:1190-1198.

121. Jiang ZD, DuPont HL, Garey K, Price M, Graham G, Okhuysen P, Dao-Tran T,

LaRocco M. 2006. A common polymorphism in the interleukin 8 gene promoter is

associated with Clostridium difficile diarrhea. The American journal of gastroenterology

101:1112-1116.

122. Munkholm P, Langholz E, Hollander D, Thornberg K, Orholm M, Katz KD, Binder

V. 1994. Intestinal permeability in patients with Crohn's disease and ulcerative colitis and

their first degree relatives. Gut 35:68-72.

123. Teahon K, Smethurst P, Levi AJ, Menzies IS, Bjarnason I. 1992. Intestinal

permeability in patients with Crohn's disease and their first degree relatives. Gut 33:320-

323.

124. Kucharzik T, Walsh SV, Chen J, Parkos CA, Nusrat A. 2001. Neutrophil

transmigration in inflammatory bowel disease is associated with differential expression of

epithelial intercellular junction proteins. The American journal of pathology 159:2001-

2009.

125. Zeissig S, Burgel N, Gunzel D, Richter J, Mankertz J, Wahnschaffe U, Kroesen AJ,

Zeitz M, Fromm M, Schulzke JD. 2007. Changes in expression and distribution of

claudin 2, 5 and 8 lead to discontinuous tight junctions and barrier dysfunction in active

Crohn's disease. Gut 56:61-72.

126. Nusrat A, Parkos CA, Liang TW, Carnes DK, Madara JL. 1997. Neutrophil

migration across model intestinal epithelia: monolayer disruption and subsequent events

in epithelial repair. Gastroenterology 113:1489-1500.

127. Weber DA, Sumagin R, McCall IC, Leoni G, Neumann PA, Andargachew R, Brazil

JC, Medina-Contreras O, Denning TL, Nusrat A, Parkos CA. 2014. Neutrophil-

derived JAML inhibits repair of intestinal epithelial injury during acute inflammation.

Mucosal immunology.

128. Su L, Shen L, Clayburgh DR, Nalle SC, Sullivan EA, Meddings JB, Abraham C,

Turner JR. 2009. Targeted epithelial tight junction dysfunction causes immune

197

activation and contributes to development of experimental colitis. Gastroenterology

136:551-563.

129. Arrieta MC, Madsen K, Doyle J, Meddings J. 2009. Reducing small intestinal

permeability attenuates colitis in the IL10 gene-deficient mouse. Gut 58:41-48.

130. Sandle GI. 2005. Pathogenesis of diarrhea in ulcerative colitis: new views on an old

problem. Journal of clinical gastroenterology 39:S49-52.

131. Hookman P, Barkin JS. 2007. Review: Clostridium difficile-associated

disorders/diarrhea and Clostridium difficile colitis: the emergence of a more virulent era.

Digestive diseases and sciences 52:1071-1075.

132. Schulzke JD, Ploeger S, Amasheh M, Fromm A, Zeissig S, Troeger H, Richter J,

Bojarski C, Schumann M, Fromm M. 2009. Epithelial tight junctions in intestinal

inflammation. Annals of the New York Academy of Sciences 1165:294-300.

133. Madara JL, Patapoff TW, Gillece-Castro B, Colgan SP, Parkos CA, Delp C, Mrsny

RJ. 1993. 5'-adenosine monophosphate is the neutrophil-derived paracrine factor that

elicits chloride secretion from T84 intestinal epithelial cell monolayers. The Journal of


134. Strohmeier GR, Lencer WI, Patapoff TW, Thompson LF, Carlson SL, Moe SJ,

Carnes DK, Mrsny RJ, Madara JL. 1997. Surface expression, polarization, and

functional significance of CD73 in human intestinal epithelia. The Journal of clinical

investigation 99:2588-2601.

135. Weissmuller T, Campbell EL, Rosenberger P, Scully M, Beck PL, Furuta GT,

Colgan SP. 2008. PMNs facilitate translocation of platelets across human and mouse

epithelium and together alter fluid homeostasis via epithelial cell-expressed ecto-

NTPDases. The Journal of clinical investigation 118:3682-3692.

136. Turner JR. 2009. Intestinal mucosal barrier function in health and disease. Nature

reviews. Immunology 9:799-809.

137. Shen L. 2009. Functional morphology of the gastrointestinal tract. Current topics in

microbiology and immunology 337:1-35.

138. Mumy KL, McCormick BA. 2005. Events at the host-microbial interface of the

gastrointestinal tract. II. Role of the intestinal epithelium in pathogen-induced

inflammation. American journal of physiology. Gastrointestinal and liver physiology

288:G854-859.

139. Eckmann L, Kagnoff MF. 2005. Intestinal mucosal responses to microbial infection.

Springer seminars in immunopathology 27:181-196.

140. Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, Morzycka-Wroblewska E,

Kagnoff MF. 1995. A distinct array of proinflammatory cytokines is expressed in human

colon epithelial cells in response to bacterial invasion. The Journal of clinical

investigation 95:55-65.

141. Eckmann L, Jung HC, Schurer-Maly C, Panja A, Morzycka-Wroblewska E,

Kagnoff MF. 1993. Differential cytokine expression by human intestinal epithelial cell

lines: regulated expression of interleukin 8. Gastroenterology 105:1689-1697.

142. Yang SK, Eckmann L, Panja A, Kagnoff MF. 1997. Differential and regulated

expression of C-X-C, C-C, and C-chemokines by human colon epithelial cells.

Gastroenterology 113:1214-1223.

198

143. Kucharzik T, Hudson JT, 3rd, Lugering A, Abbas JA, Bettini M, Lake JG, Evans

ME, Ziegler TR, Merlin D, Madara JL, Williams IR. 2005. Acute induction of human

IL-8 production by intestinal epithelium triggers neutrophil infiltration without mucosal

injury. Gut 54:1565-1572.

144. McCormick BA, Colgan SP, Delp-Archer C, Miller SI, Madara JL. 1993. Salmonella

typhimurium attachment to human intestinal epithelial monolayers: transcellular

signalling to subepithelial neutrophils. The Journal of cell biology 123:895-907.

145. Mumy KL, Bien JD, Pazos MA, Gronert K, Hurley BP, McCormick BA. 2008.

Distinct isoforms of phospholipase A2 mediate the ability of Salmonella enterica

serotype typhimurium and Shigella flexneri to induce the transepithelial migration of

neutrophils. Infection and immunity 76:3614-3627.

146. Schluger NW, Rom WN. 1997. Early responses to infection: chemokines as mediators

of inflammation. Current opinion in immunology 9:504-508.

147. Jobin C, Panja A, Hellerbrand C, Iimuro Y, Didonato J, Brenner DA, Sartor RB.

1998. Inhibition of proinflammatory molecule production by adenovirus-mediated

expression of a nuclear factor kappaB super-repressor in human intestinal epithelial cells.

Journal of immunology 160:410-418.

148. Jobin C, Holt L, Bradham CA, Streetz K, Brenner DA, Sartor RB. 1999. TNF

receptor-associated factor-2 is involved in both IL-1 beta and TNF-alpha signaling

cascades leading to NF-kappa B activation and IL-8 expression in human intestinal

epithelial cells. Journal of immunology 162:4447-4454.

149. Jijon HB, Panenka WJ, Madsen KL, Parsons HG. 2002. MAP kinases contribute to

IL-8 secretion by intestinal epithelial cells via a posttranscriptional mechanism. American

journal of physiology. Cell physiology 283:C31-41.

150. Walz A, Baggiolini M. 1990. Generation of the neutrophil-activating peptide NAP-2

from platelet basic protein or connective tissue-activating peptide III through monocyte

proteases. The Journal of experimental medicine 171:449-454.

151. Van Damme J, Rampart M, Conings R, Decock B, Van Osselaer N, Willems J,

Billiau A. 1990. The neutrophil-activating proteins interleukin 8 and beta-

thromboglobulin: in vitro and in vivo comparison of NH2-terminally processed forms.

European journal of immunology 20:2113-2118.

152. Clark-Lewis I, Schumacher C, Baggiolini M, Moser B. 1991. Structure-activity

relationships of interleukin-8 determined using chemically synthesized analogs. Critical

role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis,

exocytosis, and receptor binding activities. The Journal of biological chemistry

266:23128-23134.

153. Van den Steen PE, Proost P, Wuyts A, Van Damme J, Opdenakker G. 2000.

Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminoterminal processing,

whereas it degrades CTAP-III, PF-4, and GRO-alpha and leaves RANTES and MCP-2

intact. Blood 96:2673-2681.

154. Ohashi K, Naruto M, Nakaki T, Sano E. 2003. Identification of interleukin-8

converting enzyme as cathepsin L. Biochimica et biophysica acta 1649:30-39.

155. Proost P, Loos T, Mortier A, Schutyser E, Gouwy M, Noppen S, Dillen C, Ronsse I,

Conings R, Struyf S, Opdenakker G, Maudgal PC, Van Damme J. 2008.

199

Citrullination of CXCL8 by peptidylarginine deiminase alters receptor usage, prevents

proteolysis, and dampens tissue inflammation. The Journal of experimental medicine

205:2085-2097.

156. Turk V, Stoka V, Vasiljeva O, Renko M, Sun T, Turk B, Turk D. 2012. Cysteine

cathepsins: from structure, function and regulation to new frontiers. Biochimica et

biophysica acta 1824:68-88.

157. de Duve C. 1983. Lysosomes revisited. European journal of biochemistry / FEBS

137:391-397.

158. Willstätter R, Bamann E. 1929. Über die Proteasen der Magenschleimhaut. Erste

Abhandlung über die Enzyme der Leukocyten, p. 127, Hoppe-Seyler´s Zeitschrift für

physiologische Chemie, vol. 180.

159. Musil D, Zucic D, Turk D, Engh RA, Mayr I, Huber R, Popovic T, Turk V,

Towatari T, Katunuma N, et al. 1991. The refined 2.15 A X-ray crystal structure of

human liver cathepsin B: the structural basis for its specificity. The EMBO journal

10:2321-2330.

160. McKerrow JH, Caffrey C, Kelly B, Loke P, Sajid M. 2006. Proteases in parasitic

diseases. Annual review of pathology 1:497-536.

161. Sajid M, McKerrow JH. 2002. Cysteine proteases of parasitic organisms. Molecular

and biochemical parasitology 120:1-21.

162. Doyle PS, Zhou YM, Hsieh I, Greenbaum DC, McKerrow JH, Engel JC. 2011. The

Trypanosoma cruzi protease cruzain mediates immune evasion. PLoS pathogens

7:e1002139.

163. Kelsall BL, Ravdin JI. 1993. Degradation of human IgA by Entamoeba histolytica. The

Journal of infectious diseases 168:1319-1322.

164. Denise H, McNeil K, Brooks DR, Alexander J, Coombs GH, Mottram JC. 2003.

Expression of multiple CPB genes encoding cysteine proteases is required for

Leishmania mexicana virulence in vivo. Infection and immunity 71:3190-3195.

165. Pollock KG, McNeil KS, Mottram JC, Lyons RE, Brewer JM, Scott P, Coombs GH,

Alexander J. 2003. The Leishmania mexicana cysteine protease, CPB2.8, induces potent

Th2 responses. Journal of immunology 170:1746-1753.

166. Benitez-Hernandez I, Mendez-Enriquez E, Ostoa P, Fortoul T, Ramirez JA,

Stempin C, Cerban F, Soldevila G, Garcia-Zepeda EA. 2010. Proteolytic cleavage of

chemokines by Trypanosoma cruzi's cruzipain inhibits chemokine functions by

promoting the generation of antagonists. Immunobiology 215:413-426.

167. Pertuz Belloso S, Ostoa Saloma P, Benitez I, Soldevila G, Olivos A, Garcia-Zepeda

E. 2004. Entamoeba histolytica cysteine protease 2 (EhCP2) modulates leucocyte

migration by proteolytic cleavage of chemokines. Parasite immunology 26:237-241.

168. Reed SL, Ember JA, Herdman DS, DiScipio RG, Hugli TE, Gigli I. 1995. The

extracellular neutral cysteine proteinase of Entamoeba histolytica degrades

anaphylatoxins C3a and C5a. Journal of immunology 155:266-274.

169. Aurrecoechea C, Brestelli J, Brunk BP, Carlton JM, Dommer J, Fischer S, Gajria

B, Gao X, Gingle A, Grant G, Harb OS, Heiges M, Innamorato F, Iodice J,

Kissinger JC, Kraemer E, Li W, Miller JA, Morrison HG, Nayak V, Pennington C,

Pinney DF, Roos DS, Ross C, Stoeckert CJ, Jr., Sullivan S, Treatman C, Wang H.

200

2009. GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist

pathogens Giardia lamblia and Trichomonas vaginalis. Nucleic acids research 37:D526-

530.

170. DuBois KN, Abodeely M, Sakanari J, Craik CS, Lee M, McKerrow JH, Sajid M.

2008. Identification of the major cysteine protease of Giardia and its role in encystation.

The Journal of biological chemistry 283:18024-18031.

171. Ma'ayeh SY, Brook-Carter PT. 2012. Representational difference analysis identifies

specific genes in the interaction of Giardia duodenalis with the murine intestinal

epithelial cell line, IEC-6. International journal for parasitology 42:501-509.

172. Jimenez JC, Fontaine J, Grzych JM, Capron M, Dei-Cas E. 2009. Antibody and

cytokine responses in BALB/c mice immunized with the excreted/secreted proteins of

Giardia intestinalis: the role of cysteine proteases. Annals of tropical medicine and


173. Barrett AJ, Kembhavi AA, Brown MA, Kirschke H, Knight CG, Tamai M, Hanada

K. 1982. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its

analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. The

Biochemical journal 201:189-198.

174. Barrett AJ. 1980. Fluorimetric assays for cathepsin B and cathepsin H with

methylcoumarylamide substrates. The Biochemical journal 187:909-912.

175. Tchoupe JR, Moreau T, Gauthier F, Bieth JG. 1991. Photometric or fluorometric

assay of cathepsin B, L and H and papain using substrates with an

aminotrifluoromethylcoumarin leaving group. Biochimica et biophysica acta 1076:149-

151.

176. Hirota SA, Fines K, Ng J, Traboulsi D, Lee J, Ihara E, Li Y, Willmore WG, Chung

D, Scully MM, Louie T, Medlicott S, Lejeune M, Chadee K, Armstrong G, Colgan

SP, Muruve DA, MacDonald JA, Beck PL. 2010. Hypoxia-inducible factor signaling

provides protection in Clostridium difficile-induced intestinal injury. Gastroenterology

139:259-269 e253.

177. Teoh DA, Kamieniecki D, Pang G, Buret AG. 2000. Giardia lamblia rearranges F-actin

and alpha-actinin in human colonic and duodenal monolayers and reduces transepithelial

electrical resistance. The Journal of parasitology 86:800-806.

178. Buret A, denHollander N, Wallis PM, Befus D, Olson ME. 1990. Zoonotic potential of

giardiasis in domestic ruminants. The Journal of infectious diseases 162:231-237.

179. Smith PD, Gillin FD, Spira WM, Nash TE. 1982. Chronic giardiasis: studies on drug

sensitivity, toxin production, and host immune response. Gastroenterology 83:797-803.

180. Meyer EA. 1976. Giardia lamblia: isolation and axenic cultivation. Experimental


181. Aggarwal A, Merritt JW, Jr., Nash TE. 1989. Cysteine-rich variant surface proteins of

Giardia lamblia. Molecular and biochemical parasitology 32:39-47.

182. Monis PT, Andrews RH, Mayrhofer G, Ey PL. 1999. Molecular systematics of the

parasitic protozoan Giardia intestinalis. Molecular biology and evolution 16:1135-1144.

183. Diamond LS, Harlow DR, Cunnick CC. 1978. A new medium for the axenic

cultivation of Entamoeba histolytica and other Entamoeba. Transactions of the Royal

Society of Tropical Medicine and Hygiene 72:431-432.

201

184. Singer SM, Nash TE. 2000. T-cell-dependent control of acute Giardia lamblia infections

in mice. Infection and immunity 68:170-175.

185. Hirota SA, Iablokov V, Tulk SE, Schenck LP, Becker H, Nguyen J, Al Bashir S,

Dingle TC, Laing A, Liu J, Li Y, Bolstad J, Mulvey GL, Armstrong GD,

MacNaughton WK, Muruve DA, MacDonald JA, Beck PL. 2012. Intrarectal

instillation of Clostridium difficile toxin A triggers colonic inflammation and tissue

damage: development of a novel and efficient mouse model of Clostridium difficile toxin

exposure. Infection and immunity 80:4474-4484.

186. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H,

Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23:2947-2948.

187. Zhao Z, Raftery MJ, Niu XM, Daja MM, Russell PJ. 2004. Application of in-gel

protease assay in a biological sample: characterization and identification of urokinase-

type plasminogen activator (uPA) in secreted proteins from a prostate cancer cell line PC-

3. Electrophoresis 25:1142-1148.

188. Upcroft JA, Upcroft P. 2001. Drug susceptibility testing of anaerobic protozoa.

Antimicrobial agents and chemotherapy 45:1810-1814.

189. Lapointe TK, Buret AG. 2012. Interleukin-18 facilitates neutrophil transmigration via

myosin light chain kinase-dependent disruption of occludin, without altering epithelial

permeability. American journal of physiology. Gastrointestinal and liver physiology

302:G343-351.

190. Arndt H, Kubes P, Grisham MB, Gonzalez E, Granger DN. 1992. Granulocyte

turnover in the feline intestine. Inflammation 16:549-559.

191. Jobin C, Haskill S, Mayer L, Panja A, Sartor RB. 1997. Evidence for altered

regulation of I kappa B alpha degradation in human colonic epithelial cells. Journal of


192. Barkett M, Xue D, Horvitz HR, Gilmore TD. 1997. Phosphorylation of IkappaB-alpha

inhibits its cleavage by caspase CPP32 in vitro. The Journal of biological chemistry

272:29419-29422.

193. Reuther JY, Baldwin AS, Jr. 1999. Apoptosis promotes a caspase-induced amino-

terminal truncation of IkappaBalpha that functions as a stable inhibitor of NF-kappaB.

The Journal of biological chemistry 274:20664-20670.

194. Levkau B, Scatena M, Giachelli CM, Ross R, Raines EW. 1999. Apoptosis overrides

survival signals through a caspase-mediated dominant-negative NF-kappa B loop. Nature

cell biology 1:227-233.

195. Fischer CD, Beatty JK, Zvaigzne CG, Morck DW, Lucas MJ, Buret AG. 2011. Anti-

Inflammatory benefits of antibiotic-induced neutrophil apoptosis: tulathromycin induces

caspase-3-dependent neutrophil programmed cell death and inhibits NF-kappaB signaling

and CXCL8 transcription. Antimicrobial agents and chemotherapy 55:338-348.

196. Sadik CD, Kim ND, Luster AD. 2011. Neutrophils cascading their way to

inflammation. Trends in immunology 32:452-460.

197. Heit B, Robbins SM, Downey CM, Guan Z, Colarusso P, Miller BJ, Jirik FR, Kubes

P. 2008. PTEN functions to 'prioritize' chemotactic cues and prevent 'distraction' in

migrating neutrophils. Nature immunology 9:743-752.

202

198. Mortier A, Berghmans N, Ronsse I, Grauwen K, Stegen S, Van Damme J, Proost P.

2011. Biological activity of CXCL8 forms generated by alternative cleavage of the signal

peptide or by aminopeptidase-mediated truncation. PloS one 6:e23913.

199. Mortier A, Gouwy M, Van Damme J, Proost P. 2011. Effect of posttranslational

processing on the in vitro and in vivo activity of chemokines. Experimental cell research

317:642-654.

200. Harvath L, Falk W, Leonard EJ. 1980. Rapid quantitation of neutrophil chemotaxis:

use of a polyvinylpyrrolidone-free polycarbonate membrane in a multiwell assembly.

Journal of immunological methods 37:39-45.

201. Cooper P, Landrum M, Mizrachi I, Weisemann J 2011 Jul 20 2010 Jun 29, posting

date. Entrez Sequences Quick Start. National Center for Biotechnology Information.

[Online.]

202. Illy C, Quraishi O, Wang J, Purisima E, Vernet T, Mort JS. 1997. Role of the

occluding loop in cathepsin B activity. The Journal of biological chemistry 272:1197-

1202.

203. DuBois KN, Abodeely M, Sajid M, Engel JC, McKerrow JH. 2006. Giardia lamblia

cysteine proteases. Parasitology research 99:313-316.

204. Murata M, Miyashita S, Yokoo C, Tamai M, Hanada K, Hatayama K, Towatari T,

Nikawa T, Katunuma N. 1991. Novel epoxysuccinyl peptides. Selective inhibitors of

cathepsin B, in vitro. FEBS letters 280:307-310.

205. Spehlmann ME, Dann SM, Hruz P, Hanson E, McCole DF, Eckmann L. 2009.

CXCR2-dependent mucosal neutrophil influx protects against colitis-associated diarrhea

caused by an attaching/effacing lesion-forming bacterial pathogen. Journal of


206. Ranganathan P, Jayakumar C, Manicassamy S, Ramesh G. 2013. CXCR2 knockout

mice are protected against DSS-colitis-induced acute kidney injury and inflammation.

American journal of physiology. Renal physiology 305:F1422-1427.

207. Jamieson T, Clarke M, Steele CW, Samuel MS, Neumann J, Jung A, Huels D, Olson

MF, Das S, Nibbs RJ, Sansom OJ. 2012. Inhibition of CXCR2 profoundly suppresses

inflammation-driven and spontaneous tumorigenesis. The Journal of clinical investigation

122:3127-3144.

208. Buanne P, Di Carlo E, Caputi L, Brandolini L, Mosca M, Cattani F, Pellegrini L,

Biordi L, Coletti G, Sorrentino C, Fedele G, Colotta F, Melillo G, Bertini R. 2007.

Crucial pathophysiological role of CXCR2 in experimental ulcerative colitis in mice.

Journal of leukocyte biology 82:1239-1246.

209. Derouet M, Thomas L, Cross A, Moots RJ, Edwards SW. 2004. Granulocyte

macrophage colony-stimulating factor signaling and proteasome inhibition delay

neutrophil apoptosis by increasing the stability of Mcl-1. The Journal of biological

chemistry 279:26915-26921.

210. Zahran N, Sayed A, William I, Mahmoud O, Sabry O, Rafaat M. 2013. Neutrophil

apoptosis: impact of granulocyte macrophage colony stimulating factor on cell survival

and viability in chronic kidney disease and hemodialysis patients. Archives of medical

science : AMS 9:984-989.

203

211. Brazil JC, Liu R, Sumagin R, Kolegraff KN, Nusrat A, Cummings RD, Parkos CA,

Louis NA. 2013. alpha3/4 Fucosyltransferase 3-dependent synthesis of Sialyl Lewis A on

CD44 variant containing exon 6 mediates polymorphonuclear leukocyte detachment from

intestinal epithelium during transepithelial migration. Journal of immunology 191:4804-

4817.

212. Li Z, Bell C, Buret A, Robins-Browne R, Stiel D, O'Loughlin E. 1993. The effect of

enterohemorrhagic Escherichia coli O157:H7 on intestinal structure and solute transport

in rabbits. Gastroenterology 104:467-474.

213. Utech M, Ivanov AI, Samarin SN, Bruewer M, Turner JR, Mrsny RJ, Parkos CA,

Nusrat A. 2005. Mechanism of IFN-gamma-induced endocytosis of tight junction

proteins: myosin II-dependent vacuolarization of the apical plasma membrane. Molecular

biology of the cell 16:5040-5052.

214. Wang F, Schwarz BT, Graham WV, Wang Y, Su L, Clayburgh DR, Abraham C,

Turner JR. 2006. IFN-gamma-induced TNFR2 expression is required for TNF-

dependent intestinal epithelial barrier dysfunction. Gastroenterology 131:1153-1163.

215. Al-Sadi R, Guo S, Ye D, Dokladny K, Alhmoud T, Ereifej L, Said HM, Ma TY.

2013. Mechanism of IL-1beta modulation of intestinal epithelial barrier involves p38

kinase and activating transcription factor-2 activation. Journal of immunology 190:6596-

6606.

216. Paul G, Marchelletta RR, McCole DF, Barrett KE. 2012. Interferon-gamma alters

downstream signaling originating from epidermal growth factor receptor in intestinal

epithelial cells: functional consequences for ion transport. The Journal of biological

chemistry 287:2144-2155.

217. Kuhl AA, Kakirman H, Janotta M, Dreher S, Cremer P, Pawlowski NN,

Loddenkemper C, Heimesaat MM, Grollich K, Zeitz M, Farkas S, Hoffmann JC. 2007. Aggravation of different types of experimental colitis by depletion or adhesion

blockade of neutrophils. Gastroenterology 133:1882-1892.

218. Campbell EL, Bruyninckx WJ, Kelly CJ, Glover LE, McNamee EN, Bowers BE,

Bayless AJ, Scully M, Saeedi BJ, Golden-Mason L, Ehrentraut SF, Curtis VF,

Burgess A, Garvey JF, Sorensen A, Nemenoff R, Jedlicka P, Taylor CT, Kominsky

DJ, Colgan SP. 2014. Transmigrating neutrophils shape the mucosal microenvironment

through localized oxygen depletion to influence resolution of inflammation. Immunity

40:66-77.

219. Nadeau WJ, Pistole TG, McCormick BA. 2002. Polymorphonuclear leukocyte

migration across model intestinal epithelia enhances Salmonella typhimurium killing via

the epithelial derived cytokine, IL-6. Microbes and infection / Institut Pasteur 4:1379-

1387.

220. Franchi L, Kamada N, Nakamura Y, Burberry A, Kuffa P, Suzuki S, Shaw MH,

Kim YG, Nunez G. 2012. NLRC4-driven production of IL-1beta discriminates between

pathogenic and commensal bacteria and promotes host intestinal defense. Nature


221. Hasegawa M, Kamada N, Jiao Y, Liu MZ, Nunez G, Inohara N. 2012. Protective role

of commensals against Clostridium difficile infection via an IL-1beta-mediated positive-

feedback loop. Journal of immunology 189:3085-3091.

204

222. Shim EJ, Bang BR, Kang SG, Ma J, Otsuka M, Kang J, Stahl M, Han J, Xiao C,

Vallance BA, Kang YJ. 2013. Activation of p38alpha in T cells regulates the intestinal

host defense against attaching and effacing bacterial infections. Journal of immunology

191:2764-2770.

223. von Allmen N, Christen S, Forster U, Gottstein B, Welle M, Muller N. 2006. Acute

trichinellosis increases susceptibility to Giardia lamblia infection in the mouse model.

Parasitology 133:139-149.

224. Zhou P, Li E, Zhu N, Robertson J, Nash T, Singer SM. 2003. Role of interleukin-6 in

the control of acute and chronic Giardia lamblia infections in mice. Infection and

immunity 71:1566-1568.

225. Pepys MB, Hirschfield GM. 2003. C-reactive protein: a critical update. The Journal of


226. Mayer P, Geissler K, Ward M, Metcalf D. 1993. Recombinant human leukemia

inhibitory factor induces acute phase proteins and raises the blood platelet counts in

nonhuman primates. Blood 81:3226-3233.

227. Deshmane SL, Kremlev S, Amini S, Sawaya BE. 2009. Monocyte chemoattractant

protein-1 (MCP-1): an overview. Journal of interferon & cytokine research : the official

journal of the International Society for Interferon and Cytokine Research 29:313-326.

228. Gee K, Guzzo C, Che Mat NF, Ma W, Kumar A. 2009. The IL-12 family of cytokines

in infection, inflammation and autoimmune disorders. Inflammation & allergy drug

targets 8:40-52.

229. Obendorf J, Renner Viveros P, Fehlings M, Klotz C, Aebischer T, Ignatius R. 2013.

Increased expression of CD25, CD83, and CD86, and secretion of IL-12, IL-23, and IL-

10 by human dendritic cells incubated in the presence of Toll-like receptor 2 ligands and

Giardia duodenalis. Parasites & vectors 6:317.

230. Petering H, Gotze O, Kimmig D, Smolarski R, Kapp A, Elsner J. 1999. The biologic

role of interleukin-8: functional analysis and expression of CXCR1 and CXCR2 on

human eosinophils. Blood 93:694-702.

231. Nilsson G, Mikovits JA, Metcalfe DD, Taub DD. 1999. Mast cell migratory response to

interleukin-8 is mediated through interaction with chemokine receptor

CXCR2/Interleukin-8RB. Blood 93:2791-2797.

232. Rossi O, Karczewski J, Stolte EH, Brummer RJ, van Nieuwenhoven MA, Meijerink

M, van Neerven JR, van Ijzendoorn SC, van Baarlen P, Wells JM. 2013. Vectorial

secretion of interleukin-8 mediates autocrine signalling in intestinal epithelial cells via

apically located CXCR1. BMC research notes 6:431.

233. O'Hara JR, Buret AG. 2008. Mechanisms of intestinal tight junctional disruption during

infection. Frontiers in bioscience : a journal and virtual library 13:7008-7021.

234. Kalischuk LD, Inglis GD, Buret AG. 2009. Campylobacter jejuni induces transcellular

translocation of commensal bacteria via lipid rafts. Gut pathogens 1:2.

235. Raffatellu M, Wilson RP, Chessa D, Andrews-Polymenis H, Tran QT, Lawhon S,

Khare S, Adams LG, Baumler AJ. 2005. SipA, SopA, SopB, SopD, and SopE2

contribute to Salmonella enterica serotype typhimurium invasion of epithelial cells.


205

236. Boyle EC, Brown NF, Finlay BB. 2006. Salmonella enterica serovar Typhimurium

effectors SopB, SopE, SopE2 and SipA disrupt tight junction structure and function.

Cellular microbiology 8:1946-1957.

237. Fisher BS, Estrano CE, Cole JA. 2013. Modeling long-term host cell-Giardia lamblia

interactions in an in vitro co-culture system. PloS one 8:e81104.

238. Zareie M, Singh PK, Irvine EJ, Sherman PM, McKay DM, Perdue MH. 2001.

Monocyte/macrophage activation by normal bacteria and bacterial products: implications

for altered epithelial function in Crohn's disease. The American journal of pathology

158:1101-1109.

239. Tran VQ, Herdman DS, Torian BE, Reed SL. 1998. The neutral cysteine proteinase of

Entamoeba histolytica degrades IgG and prevents its binding. The Journal of infectious

diseases 177:508-511.

240. Que X, Kim SH, Sajid M, Eckmann L, Dinarello CA, McKerrow JH, Reed SL. 2003.

A surface amebic cysteine proteinase inactivates interleukin-18. Infection and immunity

71:1274-1280.

241. Somanna A, Mundodi V, Gedamu L. 2002. Functional analysis of cathepsin B-like

cysteine proteases from Leishmania donovani complex. Evidence for the activation of

latent transforming growth factor beta. The Journal of biological chemistry 277:25305-

25312.

242. de Carvalho TB, David EB, Coradi ST, Guimaraes S. 2008. Protease activity in

extracellular products secreted in vitro by trophozoites of Giardia duodenalis.

Parasitology research 104:185-190.

243. Chan VJ, Selzer PM, McKerrow JH, Sakanari JA. 1999. Expression and alteration of

the S2 subsite of the Leishmania major cathepsin B-like cysteine protease. The

Biochemical journal 340 ( Pt 1):113-117.

244. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment

search tool. Journal of molecular biology 215:403-410.

245. Dou Z, Coppens I, Carruthers VB. 2013. Non-canonical maturation of two papain-

family proteases in Toxoplasma gondii. The Journal of biological chemistry 288:3523-

3534.

246. Wampfler PB, Faso C, Hehl AB. 2014. The Cre/loxP system in Giardia lamblia: genetic

manipulations in a binucleate tetraploid protozoan. International journal for parasitology.

247. Stadelmann B, Hanevik K, Andersson MK, Bruserud O, Svard SG. 2013. The role of

arginine and arginine-metabolizing enzymes during Giardia - host cell interactions in

vitro. BMC microbiology 13:256.

248. Russo-Abrahao T, Cosentino-Gomes D, Daflon-Yunes N, Meyer-Fernandes JR.

2011. Giardia duodenalis: biochemical characterization of an ecto-5'-nucleotidase

activity. Experimental parasitology 127:66-71.

249. Louis NA, Robinson AM, MacManus CF, Karhausen J, Scully M, Colgan SP. 2008.

Control of IFN-alphaA by CD73: implications for mucosal inflammation. Journal of


250. Synnestvedt K, Furuta GT, Comerford KM, Louis N, Karhausen J, Eltzschig HK,

Hansen KR, Thompson LF, Colgan SP. 2002. Ecto-5'-nucleotidase (CD73) regulation

206

by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. The

Journal of clinical investigation 110:993-1002.

251. Sitaraman SV, Merlin D, Wang L, Wong M, Gewirtz AT, Si-Tahar M, Madara JL.

2001. Neutrophil-epithelial crosstalk at the intestinal lumenal surface mediated by

reciprocal secretion of adenosine and IL-6. The Journal of clinical investigation 107:861-

869.

252. van der Valk ME, Mangen MJ, Leenders M, Dijkstra G, van Bodegraven AA,

Fidder HH, de Jong DJ, Pierik M, van der Woude CJ, Romberg-Camps MJ,

Clemens CH, Jansen JM, Mahmmod N, van de Meeberg PC, van der Meulen-de

Jong AE, Ponsioen CY, Bolwerk CJ, Vermeijden JR, Siersema PD, van Oijen MG,

Oldenburg B, group Cs, the Dutch Initiative on C, Colitis. 2014. Healthcare costs of

inflammatory bowel disease have shifted from hospitalisation and surgery towards anti-

TNFalpha therapy: results from the COIN study. Gut 63:72-79.

253. Kappelman MD, Rifas-Shiman SL, Porter CQ, Ollendorf DA, Sandler RS, Galanko

JA, Finkelstein JA. 2008. Direct health care costs of Crohn's disease and ulcerative

colitis in US children and adults. Gastroenterology 135:1907-1913.

254. Zimlichman E, Henderson D, Tamir O, Franz C, Song P, Yamin CK, Keohane C,

Denham CR, Bates DW. 2013. Health care-associated infections: a meta-analysis of

costs and financial impact on the US health care system. JAMA internal medicine

173:2039-2046.

255. Darkoh C, Turnwald BP, Koo HL, Garey KW, Jiang ZD, Aitken SL, Dupont HL.

2014. Colonic Immunopathogenesis of Clostridium difficile Infections. Clinical and

vaccine immunology : CVI.

256. Banks C, Bateman A, Payne R, Johnson P, Sheron N. 2003. Chemokine expression in

IBD. Mucosal chemokine expression is unselectively increased in both ulcerative colitis

and Crohn's disease. The Journal of pathology 199:28-35.

257. Izutani R, Loh EY, Reinecker HC, Ohno Y, Fusunyan RD, Lichtenstein GR,

Rombeau JL, Macdermott RP. 1995. Increased expression of interleukin-8 mRNA in

ulcerative colitis and Crohn's disease mucosa and epithelial cells. Inflammatory bowel

diseases 1:37-47.

258. Steiner TS, Flores CA, Pizarro TT, Guerrant RL. 1997. Fecal lactoferrin, interleukin-

1beta, and interleukin-8 are elevated in patients with severe Clostridium difficile colitis.

Clinical and diagnostic laboratory immunology 4:719-722.

259. Boppana NB, Devarajan A, Gopal K, Barathan M, Bakar SA, Shankar EM,

Ebrahim AS, Farooq SM. 2014. Blockade of CXCR2 signalling: A potential therapeutic

target for preventing neutrophil-mediated inflammatory diseases. Experimental biology

and medicine.

260. Brazil JC, Louis NA, Parkos CA. 2013. The role of polymorphonuclear leukocyte

trafficking in the perpetuation of inflammation during inflammatory bowel disease.

Inflammatory bowel diseases 19:1556-1565.

261. Kelly CP, Kyne L. 2011. The host immune response to Clostridium difficile. Journal of

medical microbiology 60:1070-1079.

262. Motta JP, Bermudez-Humaran LG, Deraison C, Martin L, Rolland C, Rousset P,

Boue J, Dietrich G, Chapman K, Kharrat P, Vinel JP, Alric L, Mas E, Sallenave

207

JM, Langella P, Vergnolle N. 2012. Food-grade bacteria expressing elafin protect

against inflammation and restore colon homeostasis. Science translational medicine

4:158ra144.

208

6.0 Appendix

6.1 Purification of cathepsin cysteine protease activity

Two rounds of ion-exchange chromatography were used to purify Giardia secreted or

intratrophozoite cathepsin cysteine proteases. For intratrophozoite cysteine proteases, Giardia

NF or GS/M were isolated (see above), adjusted to a concentration of 1.0 x 108 trophozoites/mL

in a cation exchange buffer (50 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.7) and

sonicated (see above). Following this, intratrophozoite cysteine proteases were subject to SP

Sepharose (Sigma-Aldrich) cation exchange chromatography. Elutions were assayed for

cathepsin cysteine protease activity (see above) and proteolytically active fractions underwent

subsequent diafiltration to replace 50 mM MES (pH 6.7) with anion exchange buffer (50 mM L-

Histidine, pH 6.5). Following this, samples underwent DEAE Sepharose (Sigma-Aldrich) anion

exchange chromatography. Elutions were subsequently assayed for cysteine protease activity

(see above).

6.2 Analysis of Giardia cathepsin cysteine proteases

Giardia sonicate elutions collected from SP Sepharose columns were collected and

incubated with catB/L and catB fluorogenic substrates. Hydrolysis of ZFR-AMC and ZRR-AMC

was observed within column washes, where no NaCl was added to the cation exchange buffer

(Appendix Figure 1). Therefore, cathepsin cysteine proteases capable of hydrolyzing ZFR-AMC

and ZRR-AMC were only detected in column flow through samples and did not bind the SP

Sepharose column in this assay. Following this, flow-through samples containing active

cathepsin cysteine proteases underwent diafiltration to replace the SP Sepharose cation exchange

209

buffer for the DEAE Sepharose anion exchange buffer and subsequently underwent anion

exchange chromatography; these elutions were collected and also incubated with catB/L and

catB fluorgenic substrates. Interestingly, elutions 14 to 16, or in the 300 mM NaCl group, were

capable of hydrolyzing ZRR-AMC (Appendix Figure 2). Elutions 17 to 20, or the 500 mM NaCl

group, were able to hydrolyze ZFR-AMC (Appendix Figure 2). These results suggest that

Giardia cathepsin cysteine proteases were capable of binding the DEAE anion exchange

chromatography column. Moreover, our data demonstrate that Giardia cathepsin cysteine

proteases capable of hydrolyzing ZFR-AMC and ZRR-AMC elute from DEAE anion exchange

chromatography columns at different NaCl concentrations. These findings may help in the future

isolation and purification of Giardia cathepsin cysteine proteases.

210

Appendix Figure 1. Giardia trophozoite cathepsin cysteine proteases do not bind a cation

exchange chromatography column.

Giardia NF sonicates were separated via a SP Sepharose chromatography column via increasing

concentrations of NaCL (triangle). Elutions were collected and incubated with ZFR-AMC

(circle) or ZRR-AMC (box).

211

0 5 1 0 1 5 2 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

0

5 0 0

1 0 0 0

1 5 0 0

E lu tio n

R

FU

/se

co

nd

Na

Cl (m

Mo

l/L)

Z -P h e -A rg -A M C

Z -A rg -A rg -A M C

N a C l

212

Appendix Figure 2. Giardia trophozoite cathepsin cysteine proteases bind an anion

exchange chromatography column.

Giardia NF sonicates were separated via a DEAE Sepharose chromatography column via

increasing concentrations of NaCL (triangle). Elutions were collected and incubated with ZFR-

AMC (circle) or ZRR-AMC (square).

213

0 5 1 0 1 5 2 0

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

0

5 0 0

1 0 0 0

1 5 0 0

E lu tio n

R

FU

/se

co

nd

Na

Cl (m

Mo

l/L)

Z -P h e -A rg -A M C

Z -A rg -A rg -A M C

N a C l

Epithelial Immunomodulation by Giardia

Documents