Molecular mechanisms of bacteria-host interactions at the ...€¦ · What are the molecular mechanisms of bacteria-host interactions Inflammation meets metabolism: cellular stress

Molecular mechanisms of bacteria-hostinteractions at the epithelial cell level

Chair for BiofunctionalityDepartment of Food and Nutrition Science

Department of Medicine(www.wzw.tum.de/bflm)

Technical University of MunichProf. Dr. Dirk Haller

Robert Koch (1843 – 1910)

Theory about germs that cause infectious disease (1876)

Disease due to infectious microbes

Rudolf Virchow (1821 – 1902)

Theory about the cellular pathology of the diseased organ (1858)

Disease due to organ failure

Lessons from the past: germ theory vs. organ pathology

Normal gut

Ulcerative colitis

Crohn ׳s disease

Environmentaltriggers

Geneticpredisposition

• CARD15, NCF4, ATG16L1• TNFSF15, PTPN2, IL-23R

Microbial defenseImmune function

• ABCB1, PTGER4, SLC22AEpithelial function

Etiology of chronic inflammatory bowel diseases (IBD):a paradigm for bacteria-host interactions

Microorganisms in the gut (Dysbiosis) •

Infections, Smoking, Diet, NSAIDs •

Normal Gut

Enteric bacteria(SPF)

Enteric bacteria(SPF)

Wild type Wild type

Enteric bacteria(SPF)

Enteric bacteria(SPF)

Bacteroidesvulgatus

IL-10-/-

Enterococcusfaecalis

Chronic Inflammation

HLA-B27 IL-10-/- HLA-B27

Bacteroidesvulgatus

Enterococcusfaecalis

Enteric bacteria are not all equal in their capability to trigger chronic intestinal inflammation

Kim et al. 2007 Inflam. Bowel Dis. and 2005 Gastroenterology

Normal gutChronic

inflammation

SIgA(Tr1) IL-10

(Th3) TGF-βIL-11IL-15

IL-12 - IFN-γ (Th1)IL-23 - IL-17 (Th17)IL-13 (Th2)Smad7

Protective Colitogenic• Flagellin• GelE

CpG DNA •p40/70 •

RelA

U UUPP

Defensinesand

mucus

Epithelial cellintegrity

Failure of the epithelial cell homeostasis facilitates the loss of barrier function and chronic inflammation

Microbial and nutritional

signals

Diseasemechanisms

Ligand

Receptor

Signaltransduction

Gene- and protein-expression

Cell isolation

Cellular extractand fractions

Protein

2D-PAGEand

MALDI-TOF MS

Proteomeanalysis

Interface function of intestinal epithelial cells: from the proteome analysis to molecular mechanisms

Hostsignals

Clavel and Haller 2007 Inflam. Bowel. Dis.Werner and Haller 2007 Mutation Res.

What are the molecular mechanisms of bacteria-host interactions

Inflammation meets metabolism: cellular stress in the pathogenesis of chronic inflammation

Microbial determinants for pro-and anti-inflammatory effects at the epithelial cell level

IL-10-/-

IL-10-/-

Wild type

E. faecalis

Germ-free

Protective effects

Signalintegration

Colitogenic effects

Germ-free (sterile) mouse models for chronic intestinal inflammation: modeling bacteria-host cross-talk

Wild type

Germ-free

E. faecalis

IP-1

0 m

RN

A e

xpre

ssio

n(fo

ld in

crea

se)

0

5

10

15

20

25

GF 1 14 GF 1 14 time (weeks)

Histopathology(Score ± SD) Distal colon

E. faecalis OG1RF− + + − + +

0.5(0.1)

3.6(0.2)

Wild type IL-10-/-

0.6(0.1)

0.8(0.1)

0.3(0.1)

0.2(0.1)

*

*

*

Intestinal epithelial cell activation at early and late stages of bacterial colonization in wild type and IL-10-/- mice

Ruiz et al. 2005 J. Immunol.

TGF-βR2TGF-βR1

Enterococcus faecalis

TLR2

IP-10NF-κB

CBP/p300

P-Ac-Histone

IKK-β

IκB/NF-κBP-Smad2/3

TGF-β

Smad4

Ruiz et al. 2005 J. Immunol.

2

Haller et al. 2003 J.BC

1

Haller et al. 2002 JBC

15-deoxy-Δ12,14-prostaglandin J2

PP2A

3

Ruiz et al. 2004 JBC

Host-derived anti-inflammatory feed-back mechanisms in epithelial cells: signals from a complex environment

Inflammation

Time

Infla

mm

atio

n

Homeostasis

Chronic inflammation

Healing

Chronicinflammation

Enteric bacteria

Microbial-host cross-talk: from physiological inflammation to chronic inflammation

time post inoculation

Histoscore (±std) L.reuteri count (cfu) 1 x 108 per gram

Ileum Jejunum Ileum RLF-mice 0,7 (±0,24) - - 2d RLF-mice + 0,5 (±0,0) 3,9 5,4 RLF-mice 0,7 (±0,24) - - 6d RLF-mice + 1,1 (±0,2)* 6,5 7,8 RLF-mice 0,38 (±0,13) - - 21d RLF-mice + 0,55 (±0,24) 6,7 7,9

Reconstituted lactobacilli-free (RLF) mice as a model of bacteria-host interaction in the presence of a microbiota

J. Nutr. 2008

inoculation 2 6 21Num

ber o

f reg

ulat

ed /

iden

tifie

d pr

otei

ns

0

2

4

6

8

10

12

days

pH 3 pH 10

MasskDa

100

15

25

50

pH 3 pH 10

6 days after inoculation with L.reuteriCTRL

6

78

5 5

3

4 4

3

1

2 2

1

8 7

6

pH 3 pH 10

MasskDa

100

15

25

50

pH 3 pH 10

6 days after inoculation with L.reuteriCTRL

6

78

66

7788

5 55 5

3

4 4

333

44 44

33

1

2 2

111

22 22

11

8 7

6

88 77

66

β-Actin

I-FABP

grp58

- + + + L. reuteri2 6 21 days after inoculation

2 days 6 days 21 days 1 2 days 6 days 21 days 2 2 days 6 days 21 days 3 2 days 6 days 21 days

fold

cha

nge

-20

-10

0

10

20

30

40

50 IL-6 IL-1α MIP-2 A20

L.reuteri L.reuteri L.reuteri L.reuteri

**

**

*

*

IL-6 IL-1β MIP-2 A20

Reconstituted lactobacilli-free (RLF) mice as a model of bacteria-host interaction in the presence of a microbiota

What are the molecular mechanisms of bacteria-host interactions

Inflammation meets metabolism: cellular stress in the pathogenesis of chronic inflammation

Microbial determinants for pro-and anti-inflammatory effects at the epithelial cell level

Gel-basedprotein separation

1541

.868

1649

.903

1086

.862

1459

.719

1776

.714

1034

.818

1579

.791

2836

.480

1420

.752

1907

.632

2063

.611

1224

.763

832.

510

1148

.731

1352

.755

934.

586

2209

.628

0

1

2

3

4

5

6

4x10

Inte

ns. [

a.u.

]

1000 1250 1500 1750 2000 2250 2500 2750 3000m/z

Inte

nsity

m/z

2D-SDS-PAGE

Quantification and identification of protein expression changes in complex samples: from health to disease

Spot intensityProteolyticdigestion

Mass spectrometry (MS)

MALDI-TOFESI

WT

IL-10-/-

A Proteome analysis

× 3,6Grp-78

B Western Blot

Grp-78

β-actin

IL-10-/-WT

× 4,6

Induction of the ER stress response protein (Grp)-78 in the epithelium: from animal models to human IBD

Shkoda et al. 2007 Gastroenterology

Grp-78

β-actin

NI I

Western Blot

C Validation in IBD patients

NI I

ATF-6IRE-1α/β

PERK

Grp-78

Ribosome

Endoplasmic reticulum stress

+ ADP

S-S

Endoplasmic reticulum (ER) stress contributes to the pathogenesis of chronic degenerative diseases

ApoptosisAdaptation

Chronic inflammation

Epithelial cells are susceptible for ER-associated stress mechanisms in IBD: what is the evidence?

Bertolotti et al. 2001 J. Clin. Invest. ER-stress increased sensitivity to DSS colitis in IRE1β-deficient mice

Shkoda et al. 2007 GastroenterologyATF-6-mediated ER-stress in the epithelium of IL-10-deficient mice and IBD patients: protective role for IL-10

Heazlewood et al. 2008 PLOS MedicineAbberent mucin assemly causes ER stress-associated colitisand goblet cell loss in the epithelium

Kaser et al. 2008 CellXBP-1 variants are associated with ER-stress in Paneth cells and human IBD

Nucleus

TNF

RelA

P P PRelA

NF-κB

Inflammation

U

Bacteria

TNFR

U UP

P

P

TLR IKKα/β/γ

Stress responseATF6 XBP1s CHOPAP-1

UPRE

IL-10 IL-10

mtUPR

ROS

Cpn60

Cpn60

Cpn60

ATF6

Grp-78

IRE-1

PERKUPR

Cellular mechanisms for the regulation of stress responses: inflammation meets metabolism

17/9* 8/4* 38/38*

CRC UC UCCD

N=6 N=6 N=6 N=2

3/3*

Differential protein expression profile in primary IEC from colon and ileum of IBD patients: a pilot study

Shkoda et al. 2007 J. Proteome Res.

Non-inflamed (NI)

Inflamed (I)

Up

26 12UC Patient (No 1)

UC Patient (No 2)

Down

3 0

Comparison of inflamed versus non-inflamed tissue regions: the better-matched controls?

Highly interrelated protein networks indicate metabolic changes in primary IEC under chronic inflammation

Tissue pathology

Protektivesignals

Inflammatorysignals

Homeostasis

• Increase in glycolosyis• Increase in β-oxidation• Increase in apoptotic mechanisms• Increase in anti-oxidative responses• Increase in cell stress responses• Decrease in mitochondrial function

Shkoda et al. 2007 J. Proteome Res.Werner et al. 2007 J. Proteome Res.

Bibliometric co-citation analysiswith 66 proteins

Inflammatory Bowel Diseasesand Colorectal Cancer

Autoimmunityand Allergy

NeurodegenerativeDiseases

Obesity, insulin resistanceand Type 2 Diabetes

Inflammation meet metabolism: common disease mechanisms for chronic degenerative pathologies?

Inflammatory stressresponse

Metabolic stressresponse

Elias Metchnikoff

Gut bacteria contribute to diseasedevelopment (1907)

Disease due to microbial dysbalance

Rudolf Virchow

Theory about the cellular pathology of the diseased organ (1858)

Disease due to organ failure

Lessons from the past may be valid in the future

University of ZürichGerhard Rogler

FundingDFG, BMBF, Danone StiftungIndustry, EU, NuGo

Charité BerlinUlf GöbelStefan Bereswill

Biofunctionality, TUMDirk HallerIngrid SchmöllerSigrid KieslingThomas ClavelSusan ChangMicha HoffmannAnja MesslikTanja WernerEva RathGabriele HörmannspergerNatalie SteckEmanuel BergerMarie Anne von SchilldeStephan WagnerNico GebhardtBenjamin Tiemann

UNC, Chapel Hill, USABalfour R. SartorSandra C. Kim

DIfEMichael BlautGunnar Loh

Acknowledgements

Rowett InstituteDenise KellyCaroline Reiff