Gunaranjan Paturi - ResearchDirect

Probiotic characteristics of Lactobacillus acidophilus and

Lactobacillus paracasei and their effects on immune response and

gene expression in mice

By

Gunaranjan Paturi

A thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Centre for Plant and Food Science

University of Western Sydney

Australia

March 2007

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Statement of Authentication

The work presented in this thesis is, to the best of my knowledge and belief, original

except as acknowledged in the text. I hereby declare that I have not submitted this

material, either in full or in part, for a degree at this or any other institution.

Gunaranjan Paturi

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Table of contents

Statement of Authentication ...................................................................................... i

Acknowledgements.................................................................................................... vi

List of Publications...................................................................................................vii

List of Tables ............................................................................................................. ix

List of Figures ............................................................................................................ xi

List of Abbreviations ..............................................................................................xiii

ABSTRACT ................................................................................................................ 1

CHAPTER 1. Introduction ....................................................................................... 3

1.1. GUT MICROFLORA ........................................................................................... 4

1.2. GUT IMMUNE SYSTEM .................................................................................. 11

1.3. PROBIOTICS ..................................................................................................... 14

1.4. IMMUNE RESPONSES OF LACTIC ACID BACTERIA ............................... 17

1.5. THERAPEUTIC EFFECTS OF LACTIC ACID BACTERIA .......................... 20

1.5.1. Diarrhoeal diseases.................................................................................. 20

1.5.2. Inflammatory bowel disease ................................................................... 21

1.5.3. Irritable bowel syndrome ........................................................................ 21

1.5.4. Helicobacter pylori infections................................................................. 22

1.6. FUNCTIONAL GENOMICS AND LACTIC ACID BACTERIA .................... 26

1.7. AIMS................................................................................................................... 28

CHAPTER 2. Selection and characterisation of Lactobacillus and

Bifidobacterium species for potential probiotic strains......................................... 29

2.1. ABSTRACT........................................................................................................ 30

2.2. INTRODUCTION .............................................................................................. 31

2.3. MATERIALS AND METHODS........................................................................ 35

2.3.1. Microorganisms and growth conditions.................................................. 35

2.3.2. Acid tolerance ......................................................................................... 35

2.3.3. Bile tolerance .......................................................................................... 36

2.3.4. Hydrophobicity assay.............................................................................. 37

2.3.5. Autoaggregation and coaggregation assays ............................................ 38

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2.3.6. Statistical analysis ................................................................................... 39

2.4. RESULTS ........................................................................................................... 40

2.4.1. Tolerance to simulated gastric juice........................................................ 40

2.4.2. Tolerance to bile salts.............................................................................. 41

2.4.3. Surface hydrophobicity of Lactobacillus and Bifidobacterium strains... 46

2.4.4. Autoaggregation and coaggregation of Lactobacillus and

Bifidobacterium strains ..................................................................................... 46

2.5. DISCUSSION ..................................................................................................... 51

2.6. CONCLUSIONS................................................................................................. 56

CHAPTER 3. Effect of L. acidophilus LAFTI L10 and L. paracasei LAFTI L26

on gut and systemic immunity and bacterial translocation in mice .................... 57

3.1. ABSTRACT........................................................................................................ 58

3.2. INTRODUCTION .............................................................................................. 59


3.3.1. Animals ................................................................................................... 63

3.3.2. Microorganisms ...................................................................................... 63

3.3.3. Feeding procedure................................................................................... 63

3.3.4. Histological samples ............................................................................... 64

3.3.5. Immunofluorescent detection of IgA cells .............................................. 64

3.3.6. Detection of cytokine producing cells in small intestine ........................ 65

3.3.7. Splenocytes ............................................................................................. 65

3.3.8. Lymphocyte proliferation assay.............................................................. 66

3.3.9. Estimation of cytokines in blood and splenocytes .................................. 66

3.3.10. Phagocytosis.......................................................................................... 67

3.3.11. Bacterial translocation assay................................................................. 67

3.3.12. Statistical analysis ................................................................................. 68

3.4. RESULTS ........................................................................................................... 69

3.4.1. Determination of IgA producing cells..................................................... 69

3.4.2. Effect of LAB on cytokines in gut .......................................................... 69

3.4.3. Lymphocyte proliferative responses ....................................................... 70

3.4.4. Effect of LAB on cytokines in blood serum and splenocytes................. 70

3.4.5. Phagocytic activity .................................................................................. 77

3.4.6. Bacterial translocation............................................................................. 77

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3.5. DISCUSSION ..................................................................................................... 83

3.6. CONCLUSIONS................................................................................................. 89

CHAPTER 4. Immunostimulatory responses of L. acidophilus LAFTI L10 and

L. paracasei LAFTI L26 in cholera toxin mice ...................................................... 90

4.1. ABSTRACT........................................................................................................ 91

4.2. INTRODUCTION .............................................................................................. 92


4.3.1. Mice ........................................................................................................ 96

4.3.2. Lactobacillus strains and feeding procedure........................................... 96

4.3.3. Cholera toxin immunisation.................................................................... 97

4.3.4. Intestinal fluid and serum preparation..................................................... 97

4.3.5. Determination of nitric oxide.................................................................. 97

4.3.6. Estimation of Immunoglobulin A ........................................................... 98

4.3.7. Histological samples ............................................................................... 98

4.3.8. Immunofluorescent detection of cytokine producing cells ..................... 99

4.3.9. Statistical analysis ................................................................................... 99

4.4. RESULTS ......................................................................................................... 100

4.4.1. Nitric oxide production in intestinal fluid and serum ........................... 100

4.4.2. Immunoglobulin A antibodies in intestinal fluid and serum................. 100

4.4.3. Detection of cytokine producing cells in small intestine ...................... 103

4.5. DISCUSSION ................................................................................................... 107

4.6. CONCLUSIONS............................................................................................... 111

CHAPTER 5. Microarray analysis of L. acidophilus LAFTI L10 induced gene

expression in the small intestine of mice .............................................................. 112

5.1. ABSTRACT...................................................................................................... 113

5.2. INTRODUCTION ............................................................................................ 114

5.3. MATERIALS AND METHODS...................................................................... 117

5.3.1. Animals ................................................................................................. 117

5.3.2. Lactic acid bacteria ............................................................................... 117

5.3.3. Experimental design.............................................................................. 117

5.3.4. RNA isolation ....................................................................................... 118

5.3.5. Microarray analysis............................................................................... 119

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5.3.6. Synthesis of cRNA probes .................................................................... 119

5.3.7. Hybridisation of probes to oligonucleotide microarray ........................ 119

5.3.8. Data Analysis ........................................................................................ 120

5.4. RESULTS ......................................................................................................... 121

5.5. DISCUSSION ................................................................................................... 137

5.6. CONCLUSIONS............................................................................................... 145

CHAPTER 6. Concluding summary .................................................................... 146

REFERENCES ....................................................................................................... 154

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Acknowledgements

I am sincerely thankful to A/Prof. Kasipathy Kailasapathy for his supervision,

guidance and enthusiastic encouragement throughout this study. I am also thankful

for sharing his knowledge and time.

My deepest gratitude to Dr. Michael Phillips for his guidance and constructive

comments. I am especially thankful for sharing his knowledge in microbiology

during the progression of this study.

I am thankful to A/Prof. Shelley Burgin for the friendship and support I received

during this study. Many thanks to Dr. Mark Jones for his help during the animal

experiments.

I am grateful to Prof. Gabriela Perdigon for sharing her expertise in immunology

during this study at University of Western Sydney, Australia and also at Centro de

Referencias para Lactobacilos, Tucuman, Argentina. My sincere thanks to Prof.

Gerald Tannock for giving me the opportunity to visit his laboratory at University of

Otago, Dunedin, New Zealand to enhance my skills in gut microbiology.

I wish to thank all my colleagues for their friendship, advice and support during this

study at University of Western Sydney, Australia. I am grateful to all the technical

and administrative staff of Centre for Plant and Food Science and School of Natural

Sciences for their assistance. I am also thankful to Commonwealth of Australia and

University of Western Sydney for providing me the scholarship and resources to

pursue this study.

Last but not the least, I am thankful to my wife, parents, family members and friends

for their continued support, love and encouragement.

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List of Publications

Journal Papers

1. Paturi G., Phillips, M., Jones, M., Kailasapathy, K., 2007. Immune enhancing

effects of Lactobacillus acidophilus LAFTI L10 and Lactobacillus paracasei

LAFTI L26 in mice. International Journal of Food Microbiology 115, 115-

118.

2. Paturi G., Phillips, M., Kailasapathy, K. Effect of probiotic strains

Lactobacillus acidophilus LAFTI L10 and Lactobacillus paracasei LAFTI

L26 on systemic immune functions and bacterial translocation in mice.

Journal of Food Protection (Manuscript accepted).

3. Paturi G., Phillips, M., Kailasapathy, K. Oral administration of Lactobacillus

acidophilus LAFTI L10 modulate immune responses and gene expression in

the small intestine of cholera toxin mice model. (Manuscript submitted).

4. Paturi G., Phillips, M., Kailasapathy, K. Microarray analysis of Lactobacillus

acidophilus LAFTI L10 induced gene expression in the small bowel of mice.

(Manuscript submitted).

Conference Presentations

1. Paturi G., Kailasapathy, K., Phillips, M., Jones, M., 2006. Unravelling the

effects of Lactobacillus acidophilus in murine cholera toxin model.

Proceedings of the Annual Australian Society for Microbiology Conference,

Gold Coast, Australia. (Oral Presentation).

2. Paturi G., Kailasapathy, K., Phillips, M., Jones, M., 2006. Microarray

analysis of gene expression in murine small intestine modulated by

Lactobacillus acidophilus. The International Conference on Nutrigenomics

and Gut Health, Auckland, New Zealand. (Oral Presentation).

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3. Paturi G., Kailasapathy, K., Phillips, M., Jones, M., 2005. Immunological

effects of probiotic bacteria with reference to Lactobacillus acidophilus and

Lactobacillus paracasei. Proceedings of the Annual Australian Society for

Microbiology Conference, Canberra, Australia. (Poster Presentation).

4. Paturi G., Kailasapathy, K., Phillips, M., Jones, M., Perdigon, G., 2005.

Studies of probiotic bacteria on immunostimulating properties in gut immune

system. 38th

Annual Australian Institute of Food Science and Technology

Convention, Sydney, Australia. (Poster Presentation).

5. Paturi G., Kailasapathy, K., Phillips, M., Perdigon, G., Jones, M., 2005.

Potential immunostimulating properties of Lactobacillus acidophilus (LAFTI

L10) and Lactobacillus paracasei (LAFTI L26) in mice. Beneficial Microbes,

American Society for Microbiology Conference, Nevada, USA. (Poster

Presentation).

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List of Tables

Table 1.1. Microorganisms that are commonly considered as probiotics.

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Table 1.2. Therapeutic properties of lactic acid bacteria. (Adapted from Saxelin et al., 2005; Reid et al., 2006).

24, 25

Table 2.1. Survival of Lactobacillus and Bifidobacterium strains in simulated gastric conditions for 3 h.

43,44

Table 2.2. Survival of Lactobacillus and Bifidobacterium strains in milk-yeast medium with oxgall.

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Table 3.1. Proliferative responses of spleen cells stimulated with ConA and LPS in mice fed with either L.

acidophilus L10 or L. paracasei L26.

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Table 3.2. Translocation of Lactobacillus spp. to Peyer’s patches, mesenteric lymph nodes (MLN), spleen, liver and blood in mice orally fed with either L.

acidophilus L10 or L. paracasei L26 for 14-days.

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Table 3.3. Translocation of total anaerobes to Peyer’s patches, mesenteric lymph nodes (MLN), spleen, liver and blood in mice orally fed with either L.


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Table 3.4. Translocation of enteric bacteria to Peyer’s patches, mesenteric lymph nodes (MLN), spleen, liver and blood in mice orally fed with either L.


82

Table 5.1. Effect of L. acidophilus L10 on gene expression (Up- and down-regulated) in the small intestine of normal mice.

123,124,

125

Table 5.2. Functional analysis of differentially expressed genes (Up-regulated) after oral administration of L. acidophilus L10 in normal mice.

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Table 5.3. Functional analysis of differentially expressed genes (Down-regulated) after oral administration of L. acidophilus L10 in normal mice.

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Table 5.4. Pathway analysis of differentially expressed genes (Up- and down-regulated) after oral administration of L. acidophilus L10 in normal mice.

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Table 5.5. Effect of L. acidophilus L10 on gene expression (Up- and down-regulated) in the small intestine of cholera toxin mice model.

131, 132,

133

Table 5.6. Functional analysis of differentially expressed genes (Up-regulated) after oral administration of L. acidophilus L10 in cholera toxin mice model.

134

Table 5.7. Functional analysis of differentially expressed genes (Down-regulated) after oral administration of L. acidophilus L10 in cholera toxin mice model.

135

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Table 5.8. Pathway analysis of differentially expressed genes (Up- and down-regulated) after oral administration of L. acidophilus L10 in cholera toxin mice model.

136

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List of Figures

Figure 1.1. Development of human and murine gastrointestinal ecosystems. Abbreviations in the figure: IELs - intraepithelial lymphocytes; LP - lamina propria; sIgM - surface immunoglobulin M; sIgA - surface immunoglobulin A; TCR - T-cell receptor. (Adapted from McCracken and Lorenz, 2001).

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Figure 1.2. Overview of gastrointestinal (GI) tract. (A) Microbial distribution (Anaerobic and aerobic genera) in various compartments of the GI tract. (B) Functional properties of intestinal microflora influencing intestinal mucosa. (Adapted from O’Hara and Shanahan, 2006).

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Figure 1.3. Schematic representation of gut immune system. (Adapted from McCracken and Lorenz, 2001).

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Figure. 2.1. Surface hydrophobicity of L. acidophilus LAFTI L10, L. paracasei LAFTI L26, B. lactis LAFTI B94, B. lactis Bb12, B. infantis ASCC 1912 and L. acidophilus ASCC 2400.

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Figure 2.2. Autoaggregation abilities of L. acidophilus LAFTI L10, L. paracasei LAFTI L26, B. lactis LAFTI B94, B. lactis Bb12, B. infantis ASCC 1912 and L. acidophilus ASCC 2400.

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Figure 2.3. Coaggregation abilities of L. acidophilus LAFTI L10, L. paracasei LAFTI L26, B. lactis LAFTI B94, B. lactis Bb12, B. infantis ASCC 1912 and L. acidophilus ASCC 2400.

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Figure 3.1. Effect of A) L. acidophilus L10 and B) L. paracasei L26 on immunoglobulin (Ig)-A producing cells in the small intestine of mice.

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Figure 3.2. Effect of orally administered L. acidophilus L10 or L. paracasei L26 on A) interleukin (IL)-10 and B) interferon (IFN)-� cytokine producing cells in the small intestine of mice.

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Figure 3.3. Estimation of cytokines A) interleukin (IL)-10 and B) interferon (IFN)-� in serum of mice fed with L. acidophilus L10 or L. paracasei L26.

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Figure 3.4. Production of A) interleukin (IL)-10 and B) interferon (IFN)-� by spleen cells from mice orally fed with either L. acidophilus L10 or L.

paracasei L26.

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Figure 3.5. Effect of A) L. acidophilus L10 and B) L.

paracasei L26 on phagocytic activity of the peritoneal macrophages.

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Figure 4.1. Nitric oxide production in A) intestinal fluid and B) serum of mice fed with L. acidophilus L10 or L. paracasei L26.

101

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Figure 4.2. Immunoglobulin (Ig)-A antibody response to cholera toxin in A) intestinal fluid and B) serum of mice fed with L. acidophilus L10 or L.

paracasei L26.

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Figure 4.3. Effect of L. acidophilus L10 and L. paracasei L26 on (A) interferon (IFN)-� and tumor necrosis factor (TNF)-�; (B) interleukin (IL)-4, IL-6 and IL-10 in the small intestine of mice.

104,105

Figure 4.4. Histological sections of the small intestine in mice fed with L. acidophilus L10 showing A) interleukin (IL)-10 and B) interferon (IFN)-� producing cells labelled with rabbit anti-mouse IL-10 and IFN-� respectively and secondary antibody as goat anti-rabbit conjugated with FITC, which fluoresces. Magnification 400X.

106

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List of Abbreviations

CD Cluster of differentiation

CFU Colony Forming Units

ConA Concanavalin A

CT Cholera toxin

FITC Fluorescein isothiocyanate

GALT Gut-associated lymphoid tissue

GI Gastrointestinal

GRAS Generally Regarded As Safe

IBD Inflammatory Bowel Disease

IBS Irritable Bowel Syndrome

IFN Interferon

Ig Immunoglobulin

IL Interleukin

LAB Lactic acid bacteria

LPS Lipopolysaccharide

MLN Mesenteric lymph node

MRS de Man, Rogosa and Sharpe agar or broth

NGYC Non-fat skim milk, glucose, yeast extract and cysteine medium

NK Natural Killer (Cell)

NO Nitric oxide

PBS Phosphate buffered saline

TNF Tumor necrosis factor

WC Wilkins-Chalgren agar

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ABSTRACT

Probiotic bacteria such as Lactobacillus and Bifidobacterium species are normal

inhabitants of healthy gastrointestinal (GI) tract, which may promote beneficial

effects on host through limiting the growth of undesirable microorganisms and

modulating the immune system. In the present study, Lactobacillus and

Bifidobacterium strains were screened for their in vitro acid and bile tolerance,

autoaggregation, coaggregation and hydrophobic abilities to identify potential

probiotic bacteria. Lactobacillus acidophilus LAFTI L10 and Lactobacillus

paracasei LAFTI L26 were selected based on their overall tolerance to in vitro acidic

conditions to further investigate their influence on various immune functions and

gene expression in mice.

Immunofluorescent analysis of small intestine in mice fed with L. acidophilus or L.

paracasei demonstrated an increase of immunoglobulin (Ig)-A, interleukin (IL)-10

and interferon (IFN)-� producing cells compared to control mice. In systemic

immune response, proliferative responses of splenocytes stimulated with mitogens,

concanavalin A (ConA) and lipopolysaccharide (LPS) showed differences between

L. acidophilus and L. paracasei strains. In comparison to control mice, IL-10 and

IFN-� levels in blood serum and splenocytes stimulated with ConA were enhanced in

mice fed with either L. acidophilus or L. paracasei. Increased phagocytic activity of

peritoneal macrophages against Candida albicans was determined in mice fed with

L. acidophilus or L. paracasei. Translocation of Lactobacillus spp. and total

anaerobes to Peyer’s patches as well as mesenteric lymph nodes were modulated in

L. acidophilus or L. paracasei-fed mice. Furthermore, there was no bacterial

2

translocation to spleen, liver or blood in mice fed with L. acidophilus or L.

paracasei.

In cholera toxin (CT) mice model, L. acidophilus-fed mice increased the nitric oxide

(NO) levels in serum, whereas L. paracasei-fed mice enhanced the NO levels in

serum and intestinal fluid. Mice fed with L. acidophilus or L. paracasei showed an

increased IgA response to CT in intestinal fluid and serum compared to control mice.

The analysis of cytokine producing cells in small intestine of L. acidophilus or L.

paracasei-fed mice showed the regulation of Th1 and Th2 cytokines such as IFN-�,

tumor necrosis factor (TNF)-�, IL-4, IL-6 and IL-10.

Microarray analysis of L. acidophilus induced gene expression in small intestine of

normal mice demonstrated an up- and down-regulation of 27 and 35 genes, which are

involved in cell-cell signalling, cell growth and proliferation, cell death and various

metabolic functions. In CT mice model, L. acidophilus influenced an up- and down-

regulation of 28 and 32 genes in small intestine that are involved in cell functions,

drug metabolism and immune responses.

In summary, L. acidophilus and L. paracasei showed tolerance to various gastric

conditions and bile salts. Lactobacillus acidophilus and L. paracasei enhanced gut

and systemic immune functions, particularly non-specific and specific immune

responses in normal and CT mice. Moreover, L. acidophilus regulated the genes

involved in various biological functions in small bowel of normal and CT mice,

which provided a basis in understanding the pathways through which these bacteria

are beneficial to the host.

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CHAPTER 1

Introduction

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1.1. GUT MICROFLORA

The gastrointestinal (GI) tract of human consists of diversified microflora ranging

between 300 - 500 bacterial species. The microflora of GI tract is about 10 times

higher than eukaryotic cells in the human body (Simon and Gorbach, 1986;

Bengmark, 1998). In a healthy host, enteric bacteria that were acquired from birth

colonise the alimentary tract and their composition remains mostly constant

thereafter. Aerobic and facultative microorganisms such as coliforms, lactobacilli

and streptococci are first to colonise the GI tract, which are considered to be

important in reducing oxidation process in the intestine that lead to the colonisation

of anaerobic bacteria such as Bacteroides and Bifidobacterium species.

The consumption of solid food gradually transforms the intestinal microflora into

stable microbial communities in adults, where anaerobic microbes prevail (Fig. 1.1).

The microflora in adult GI tract comprises of 99.9% obligate anaerobes, where

gastric secretions and rapid motility in the upper small intestine restrict them to 103

organisms per millilitre of luminal fluids at this site and between 1011 - 1012

organisms per gram or millilitre of colon contents (Xu and Gordon, 2003; Backhed et

al., 2005; Fig. 1.2A).

Bacterial composition in GI tract differ based on anatomical properties of the

interacting sites and other conditions such as pH, oxygen availability and transit time

of food contents. Bacterial species belonging to genera Bacteroides, Bifidobacterium,

Fusobacterium, Butyrivibrio, Clostridium, Eubacterium and Lactobacillus are some

of the resident microflora in GI tract. Predominantly, bifidobacteria and lactobacilli

5

represents 90% of the bacterial population, whereas less than 0.01% consists of

diversified bacterial population such as pathogenic microorganisms (Tournut, 1993).

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Figure 1.1. Development of human and murine gastrointestinal ecosystems. Abbreviations in the figure: IELs - intraepithelial lymphocytes; LP -

lamina propria; sIgM - surface immunoglobulin M; sIgA - surface immunoglobulin A; TCR - T-cell receptor. (Adapted from McCracken and

Lorenz, 2001).

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The intestinal microflora influence wide variety of host functions such as synthesis

of biotin, folate, vitamin K and conversion of unabsorbed dietary sugars into short-

chain fatty acids, which can be used as energy source by colonic mucosa (O’Hara

and Shanahan, 2006; Fig. 1.2B). The ability of commensal microflora differs in

promoting the development of gut associated lymphoid tissues (GALT). However,

commensal microflora play an important role in maintaining immune homeostasis in

developing and adult gut. Fermentation processes of colonic microflora enable to

salvage energy from food components that have not been digested in the upper GI

tract. Gibson et al. (1999) suggested that colonic bacterial fermentation provide

approximately 7% - 8% of total dietary energy that was required daily by the host.

Backhed et al. (2004) demonstrated that germ free mice require higher uptake of

calories to maintain normal body mass in comparison to normal mice.

Gut microflora also interacts with the host immune system in order to protect from

pathogenic bacteria and for the overall development and function of GI tract (Fig.

1.2B). Several gnotobiotic studies demonstrated the beneficial role of microflora in

influencing the physiological functions of gut. In comparison to normal animals,

reduction of digestive enzyme activity and local cytokine production were detected

in germ-free animals. Earlier studies suggested that the absence of indigenous

microflora modified the intestinal epithelial cells, which lead to the changes in

morphological and functional properties of germ free animals (Alam et al., 1994;

Wostmann, 1996). In germ free animals, development of GALT, lamina propria

cellularity and mucosal vascularity was also less effective. However, introducing

intestinal microflora into germ free mice helped the restoration of specific mucosal

immune system (Umesaki et al., 1995). Furthermore, germ-free mice demonstrated

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the inhibition of macrophage chemotaxis and phagocytic activity and less number of

spleen-derived macrophage precursors (Wostmann, 1996; Nicaise et al., 1998).

Earlier studies reported that commensal bacterial ligands are important in the

development and function of gut mucosal immune system (Rakoff-Nahoum et al.,

2004; Mazmanian et al., 2005). These commensal bacteria are also capable of

influencing the regulation of T-helper (Th) cell type-1 or type-2 cytokines (Cebra,

1999; Shanahan, 2002a). Cytokines such as interleukin (IL)-1�, IL-2, tumor necrosis

factor (TNF)-� and transforming growth factor (TGF)-� increased the proliferation of

intestinal stromal cells (Fritsch et al., 1999).

Intestinal mucosal surfaces are vulnerable to various harmful microorganisms from

outside environment that transit through the intestine, which may be harmful to the

host. In order to protect the host from these harmful microorganisms, active secretory

gut immune system has evolved. The release of secretory immunoglobulin (sIg)-A

into the intestinal fluid occurs in healthy host, which is important in preventing the

adherence of foreign antigens to epithelial cells. This process is known as immune

exclusion, which provides protection to mucosal surfaces without causing

inflammatory responses. The gut microflora also acts as a barrier in protecting the

host from pathogenic bacteria by producing regulatory factors such as short-chain

fatty acids and bacteriocins. Cebra, (1999) suggested the antagonistic activity of

intestinal microflora in inhibiting the colonisation of pathogenic bacteria in gut. The

innate immune defence mechanisms of intestinal epithelial cells include mucins and

antimicrobial compounds such as lysozyme and defensins, which are important in

9

preventing the microbial adherence to epithelial cell surfaces (Magnusson and

Stjernstorm, 1982; Ouellette, 1999).

10

Figure 1.2. Overview of gastrointestinal (GI) tract. (A) Microbial distribution (Anaerobic and aerobic genera) in various compartments of the GI

tract. (B) Functional properties of intestinal microflora influencing intestinal mucosa. (Adapted from O’Hara and Shanahan, 2006).

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1.2. GUT IMMUNE SYSTEM

In humans, gut is considered as a major immune organ. Gut mucosal surfaces are

constantly exposed to antigens derived from both diet and intestinal microflora. Gut

defence mechanisms have the capability of discriminating non-pathogenic

commensal bacteria and antigens from enteropathogens. Specifically, gut immune

responses towards antigens derived from commensal bacteria are non-harmful to the

organism, which further induce systemic tolerance against intestinal antigens. This

process can be expressed as immunological tolerance, which is important for gut

integrity. Earlier studies reported the mechanisms that are involved in immunological

tolerance (Schwartz, 1989; Garside et al., 1999). It was also suggested that the failure

of immunological tolerance towards luminal bacteria could lead to inflammatory

bowel disease (Duchmann et al., 1995).

The mucosal immune system is separated from gut lumen by a thin layer of intestinal

epithelial cells that lines the mucosal intestinal surface. These intestinal epithelial

cells are important in providing first line defence by preventing lumen antigens from

accessing the host immune system. However, antigens constantly interact with

immunosensory cells of the intestinal epithelium through different mechanisms (Fig.

1.3). The sampling of luminal antigens is performed through M-cell, which resides in

specialised epithelium that overlies the Peyer’s patches (Neutra et al., 1996). The

antigen presentation and affinity maturation of the transported luminal antigens occur

in Peyer’s patches (McGhee et al., 1999; Owen, 1999). In Peyer’s patches, activated

antigen-specific lymphocytes migrate to mesenteric lymph nodes and re-enter the

circulation through thoracic duct. Perdue, (1999) suggested that the transport of

antigen molecules can occur either through transcellular or paracellular pathways.

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The other route of sampling antigens could be through the dendrites of intestinal

dendritic cells (Rescigno et al., 2001). The intestinal dendritic cells are also

important in inducing local immune responses towards commensal bacteria through

ingestion and retaining live commensal bacteria, which are further transported to

mesenteric lymph nodes (Macpherson and Uhr, 2004). This process suggests the role

of mesenteric lymph nodes as potential gatekeepers by preventing the access of

commensal bacteria to internal environment.

Lamina propria and intraepithelial spaces are considered as main effector sites in the

intestine. The lamina propria mainly consists of over 60% immune cells, which

include immunoglobulin (Ig)-A producing B-cells, T-cells and macrophages

(McGhee et al., 1999). In the lamina propria, B-cells undergo differentiation into

plasmacytes in order to secrete dimeric IgA. The intestinal epithelial cells are capable

of producing polymeric Ig receptors, which transport dimeric IgA into the intestinal

lumen (Underdown and Schiff, 1986). It is often described that plasma cells on the

mucosal surfaces as well as in the lamina propria are capable of secreting upto 40 mg

of IgA per day, which is an important characteristic for an active gut immune system.

In lamina propria, T-cells have the capability of interacting with B-cell

immunoglobulin isotype (Lebman et al., 1990; Shockett and Stavnezer, 1991). The

T-cells in lamina propria are mainly CD4+ T-helper lymphocytes, which express

CD40 ligands and release cytokines such as IL-4, IL-5 and IL-10 (Liu and Arpin,

1997). Furthermore, cytokines released from CD4+ T-cells and intestinal epithelial

cells modulate IgA producing B-cells, which are known to be important in mucosal

defence mechanism.

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Figure 1.3. Schematic representation of gut immune system. (Adapted from McCracken and Lorenz, 2001).

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1.3. PROBIOTICS

The term probiotic, meaning ‘for life’ was first coined in 1960s in order to describe

“substances secreted by one microorganism that stimulate the growth of another”

(Lilly and Stillwell, 1965). However, the role of these beneficial bacteria in

promoting positive health effects upon host has been fully recognised only in the last

two decades. Fuller, (1989) redefined the word probiotics as “viable microorganisms

that contribute to intestinal microbial balance and have the potential to improve the

health of their human host”. Recently, probiotics have been defined as “live

microorganisms which when administered in adequate amounts confer a health

benefit on the host” (Reid et al., 2003).

Probiotics are live microbial food supplements that have been used for many years in

animal feed industry. However, probiotics are now widely used in the manufacturing

of fermented dairy products. Of the numerous microbes that are beneficial

inhabitants of GI tract, microorganisms that have been considered as probiotics

include yeast (Guslandi et al., 2000), Escherichia coli (Tromm et al., 2004) and

enterococci (Lund and Edlund, 2001). The most commonly used have been

bifidobacteria and lactic acid bacteria (LAB) such as lactobacilli, lactococci and

streptococci (Madsen, 2001; Isolauri et al., 2002; Table 1.1). Lactobacilli are rod-

shaped bacteria, which are capable of producing short-chain fatty acids, acetate and

lactate through carbohydrate fermentation process. Several Lactobacillus species are

known to inhabit the intestinal tracts of humans and animals. In large intestine,

bifidobacteria constitute in large numbers among other beneficial microorganisms,

which are capable of synthesising vitamins and digestive enzymes (Gibson et al.,

1995). Generally, Lactobacillus and Bifidobacterium strains are used in the

15

development of probiotic products intended for human consumption due to the belief

that these bacteria are members of intestinal microflora and also considered as

“generally regarded as safe” (GRAS) (Berg, 1998; Klein et al., 1998).

The probiotic effects are strain-specific, therefore it is difficult to generalise the

beneficial effects of a probiotic strain even comparing to strains of the same species.

Recent Food and Agriculture Organisation (FAO) and World Health Organisation

(WHO) guidelines suggested that probiotic bacteria incorporated in food products

must demonstrate their tolerance to gastric juices and bile salts in order to survive

during their transit in GI tract (FAO/WHO, 2001; 2002). Additionally, probiotic

bacteria must be capable of colonising the GI tract and have the potential in

maintaining their efficacy during shelf-life of the product. Potential probiotic bacteria

possess some or all of the functional properties such as: (i) tolerance to acid, bile,

enzyme and oxygen; (ii) ability to adhere host epithelial cells; (iii) colonisation in the

GI tract; (iv) pathogen exclusion; (v) production of antimicrobial substances towards

pathogens; (vi) non-pathogenic and non-carcinogenic and (vii) favourable towards

well balanced microbial gut ecosystem. Several studies reported the influence of

probiotic bacteria on synthesis of various energy sources such as folic acid

production (Deeth and Tamime, 1981; Alm, 1982) and short-chain fatty acids

(Mallett et al., 1989). Furthermore, studies also showed that probiotic bacteria are

also capable of influencing various immune responses such as humoral, cellular and

non-specific immunity (Matsuzaki et al., 1998; Chiang et al., 2000; Cross et al.,

2002; Elahi et al., 2005; Takahashi et al., 2006).

16

Table 1.1. Microorganisms that are commonly considered as probiotics.

Lactobacillus spp.

Bifidobacterium spp.

Others

L. acidophilus B. adolescentis Enterococcus faecalis

L. brevis B. animalis Enterococcus faecium

L. casei B. breve Escherichia coli Nissle

L. crispatus B. bifidum Saccharomyces boulardii

L. curvatus B. infantis Streptococcus cremoris

L. delbrueckii subsp.

bulgaricus

B. lactis Streptococcus diacetylactis

L. fermentum B. longum Streptococcus intermedius

L. gasseri B. thermophilum Streptococcus salivarius

L. johnsonii Streptococcus thermophilus

L. lactis

L. paracasei

L. plantarum

L. reuteri

L. rhamnosus

17

1.4. IMMUNE RESPONSES OF LACTIC ACID BACTERIA

Certain strains of LAB have the potential in contributing towards host defence

mechanisms of GI tract (Deplancke and Gaskins, 2001) through interactions with the

immune system (Lu and Walker, 2001). Several factors such as survival, adherence,

colonisation and sites of interaction in GI tract influence the ability of immune

enhancing properties of LAB. Earlier studies reported that probiotic bacteria are

capable of influencing host cell functions such as immune responses and intestinal

barrier integrity (Rosenfeldt et al., 2004; Sartor, 2005). The capability of dendritic

cells in recognising bacterial antigens also helps in regulating T-cell responses. In

vitro studies demonstrated the differential cytokine expression of murine dendritic

cells influenced by LAB and probiotic cocktail VSL#3, which favoured Th1, Th2 or

Th3 immune responses (Christensen et al., 2002; Hart et al., 2004). Particularly,

Lactobacillus strains demonstrated the regulation of IL-10 producing T-cells through

interaction with dendritic cell-specific intracellular adhesion molecule 3-grabbing

nonintegrin (Smits et al., 2005).

Epithelial cells of the intestine and colon also play an important role in immune

mechanisms. Zhang et al. (2005) reported that Caco-2 cells cultured with L.

rhamnosus Goldin-Gorbach (GG) regulated NF-�B signalling pathway to reduce

TNF-� induced IL-8 production. Lactobacillus reuteri and VSL#3 also demonstrated

their influence on NF-�B signalling pathway (Ma et al., 2004; Petrof et al., 2004).

Probiotic bacteria are also capable of strengthening the intestinal barrier. For

example, probiotic E. coli Nissle 1917 influenced the regulation of NF-�B signalling

pathway by inducing the expression of antimicrobial peptide �-defensin-2 (Wehkamp

18

et al., 2004), which could be important in enhancing intestinal barrier functions such

as inhibiting the growth of pathogenic bacteria.

Several reports have suggested that early exposure of live microorganisms with

dietary antigens could be helpful in developing gut barrier functions (Helgeland et

al., 1996; Sudo et al., 1997). Moreau et al. (1978) reported the development of gut

barrier through increased duodenal IgA plasmocyte cells. Peyer’s patches are known

to be important in sampling luminal bacterial antigens where they interact with

antigen-presenting cells carrying major histocompatibility complex (MHC) class II

(Weiner et al., 1994). The antigens that are transported through Peyer’s patches are

considered important towards the overall development of local secretory immune

responses. Subsequently, immune responses initiated in GALT can influence

immune responses at other mucosal sites. In vitro cell culture models have been used

to demonstrate the immune responses induced by non-pathogenic bacteria. As an

example, Haller et al. (2000) reported the effects of non-pathogenic bacteria in

inducing cytokines such as IL-8, IL-10 and TNF-� by Caco-2 cells.

Many investigators reported the influence of LAB in inducing sIgA responses in

GALT. Orally administered L. acidophilus and L. casei increased the sIgA levels in

small intestine of mice (Perdigon et al., 1995). In mice, L. acidophilus and

Bifidobacterium species modulated the mucosal and systemic immunity through

increased IgA responses to cholera toxin, which was used as an oral immunogen

(Tejada-Simon et al., 1999a). Lactobacillus GG also showed the capability in

increasing IgA response in children with Crohn’s disease (Malin et al., 1996) and

acute rotavirus diarrhoea (Kaila et al., 1992). Moreover, several studies also reported

19

the capability of probiotic bacteria in modulating IgE production. For example,

decrease in the production of IgE in serum was detected in mice orally fed with L.

casei Shirota (Matsuzaki et al., 1998). Similar results related to the reduction of IgE

production was also observed in IL-10 deficient mice fed with L. plantarum 229v

(Schultz et al., 2002).

The regulation of both innate and acquired immune responses was mediated through

cytokines. Several studies reported the influence of LAB in regulating T-cell

functions. As an example, cell wall and cytoplasmic components of LAB

demonstrated the capability of stimulating cytokines such as IL-6 and TNF-� and

nitric oxide (NO) (Tejada-Simon and Pestka, 1999b). Maassen et al. (2000) also

reported an increase of TNF-�, IL-2 and IL-1� in gut villi of mice fed with L. reuteri

or L. brevi. Furthermore, Lactobacillus strains demonstrated the enhancement of

TNF-� and IFN-� producing cells in the lamina propria of gut (Perdigon et al., 2002).

The cytokine, IFN-� is capable of enhancing nitric oxide production, activating

macrophages and NK cells. The phagocytic activity of macrophages plays an

important role in preventing microbial infections, whereas NK cells are pivotal in

defence against viral infections and cancer. Schiffrin et al. (1995) demonstrated the

supplementation of L. johnsonii La1 or B. lactis Bb12 for 3 weeks, which increased

the phagocytic activity of peripheral blood leukocytes in humans. Interestingly, the

influence of probiotic bacteria differ in the modulation of phagocytic activity in

healthy and allergic subjects (Pelto et al., 1998). Furthermore, Donnet-Hughes et al.

(1999) also reported the potential of LAB on phagocytic activity of the peripheral

blood leukocytes in healthy adult volunteers.

20

In general, probiotic bacteria can be regarded as a dietary adjuvant, which

beneficially affect the host physiology through modulating mucosal and systemic

immune functions. Lactic acid bacteria have been considered as potential candidates

for oral vaccination purposes due to their various functional properties and long

history of usage as safe food grade organisms. Maassen et al. (2000) demonstrated

the differences between Lactobacillus strains in inducing cytokines and their

adjuvant properties in gut. Although several studies reported the adjuvant effects of

LAB with various antigens, adjuvant characteristics of Lactobacillus strains often

vary due to the differences in bacterial strains, experimental designs, animals and

antigens (Tejada-Simon et al., 1999a; Cano et al., 2002; Plant and Conway, 2002; He

et al., 2005; Kim et al., 2005).

1.5. THERAPEUTIC EFFECTS OF LACTIC ACID BACTERIA

Lactic acid bacteria have the potential to induce several beneficial effects on human

health, which are listed in Table 1.2. The insights into the therapeutic effects of LAB

in GI tract and their effects on host immune functions are discussed below.

1.5.1. Diarrhoeal diseases

Certain LAB are proven to be beneficial in the prevention or amelioration of various

diarrhoeal diseases. Several human intervention studies reported the probiotic effects

of L. rhamnosus GG against rotavirus diarrhoea in infants (Isolauri et al., 1991; Kaila

et al., 1992; Majamaa et al., 1995). Szajewska et al. (2001) demonstrated the

capability of L. rhamnosus GG in reducing the duration of diarrhoea in comparison

to placebo. Additionally, L. rhamnosus GG prevented the urease-producing bacterial

growth during rotavirus diarrhoea (Isolauri et al., 1994). Recently, Paton et al. (2005)

21

demonstrated the treatment and prevention of E. coli diarrhoea through a toxin

binding recombinant probiotic. Probiotic bacteria also showed the capability of

producing bacteriocins such as nisin in order to inhibit the growth of pathogenic

bacteria (Dodd and Gasson, 1994; Miraglia del Giudice and De Luca, 2004).

1.5.2. Inflammatory bowel disease

Inflammatory bowel disease (IBD) has a complex etiology, which was characterised

by chronic or recurrent intestinal inflammation. Ulcerative colitis and Crohn’s

disease are the best known forms of IBD. The mechanisms responsible for the cause

of IBD still remain unclear. Several studies suggested that certain probiotic bacteria

are capable of treating mild or moderately active IBD (Fedorak and Madsen, 2004;

Sartor, 2004). Particularly, Pathmakanthan et al. (1999) reported the decrease of

lactobacilli in the inflamed mucosal tissues of ulcerative colitis patients. Therefore,

restoration of microflora in GI tract through selected probiotic bacteria could be

considered as a therapeutic strategy for ameliorating intestinal inflammation.

1.5.3. Irritable bowel syndrome

Irritable bowel syndrome (IBS) is associated with manifestation of abdominal pain,

bloating, flatulence and diarrhoea. Several studies suggested the role of colonic

microflora in pathogenesis of IBS (Bradley et al., 1987; Madden et al., 2001).

However, the cause of IBS is still unknown. In double blind and placebo controlled

studies, orally administered L. plantarum reduced flatulence and abdominal pain

(Nobaek et al., 2000; Niedzielin et al., 2001). Brigidi et al. (2001) also reported the

beneficial effects of VSL#3 by improving the clinical picture and altering the faecal

22

microflora composition with an increase of lactobacilli, bifidobacteria and S.

thermophilus in patients having IBS. In another study, L. plantarum did not either

modify the colonic fermentation process or improve symptoms in patients with IBS

(Sen et al., 2002). Recently, O’Mahony et al. (2005) suggested the

immunomodulatory role of B. infantis by improving the symptoms in patients with

IBS. However, further studies are needed to define the mechanisms of probiotic

bacterial influence on IBS.

1.5.4. Helicobacter pylori infections

Helicobacter pylori are responsible for causing chronic gastritis and idiopathic peptic

ulcer disease. Previous studies showed the potential of probiotic bacteria in inhibiting

the growth of H. pylori (Midolo et al., 1995; Kabir et al., 1997). Aiba et al. (1998)

showed the capability of L. salivarius in inhibiting the growth of H. pylori by

producing high amounts of lactic acid. It was postulated that higher levels of lactic

acid production could be helpful in reducing the urease activity of H. pylori. It has

also been reported that the combination of probiotic cultures such as L. acidophilus

La5 and B. latics Bb12 showed significant decrease in the urease activity after 6

weeks of oral administration in humans with H. pylori (Wang et al., 2004). In

contrast, a probiotic combination of L. rhamnosus GG, L. rhamnosus LC705, B.

breve Bb99 and Propionibacterium freudenreichii subsp. shermanii JS were not

effective in improving intestinal inflammation symptoms in humans with H. pylori

(Myllyluoma et al., 2005). Further studies are needed to determine the etiology of the

disease and well defined experimental methodologies could possibly substantiate the

efficacy of probiotic bacteria.

23

The mechanisms of probiotic bacterial interaction with the host need to be

investigated to elucidate their potential role especially in promoting human health

and well-being. Earlier studies attempted to define the cellular targets of probiotic

bacteria, whereas the molecular basis of the probiotic functions remains unknown.

The emerging demand of probiotic bacteria in functional food applications require

fundamental understanding of the molecular structures that are involved in

microbial-host interactions. Moreover, genomic approaches are considered necessary

in identifying the key gene targets of probiotic functions.

24

Table 1.2. Therapeutic properties of lactic acid bacteria. (Adapted from Saxelin et al., 2005; Reid et al., 2006).

Strain

Probiotic effect

Reference

L. acidophilus La5

Suppression of Helicobacter pylori with use of La5 and B. lactis BB12.

Wang et al., 2004.

L. acidophilus L1 Lower serum cholesterol – this would translate to 6–10% reduction in risk for coronary heart disease.

Anderson and Gilliland, 1999.

L. brevis CD2 Decreases H. pylori colonisation, thus reducing polyamine biosynthesis. Linsalata et al., 2004.

L. casei Shirota Improvement in treatment of constipation. Koebnick et al., 2003.

L. gasseri OLL 2716 Suppressed H. pylori and reduced gastric mucosal inflammation. Sakamoto et al., 2001.

L. rhamnosus 19070-2 and L. reuteri DSM 12246

Reduction in acute diarrhoea in children. Rosenfeldt et al., 2002.

L. rhamnosus GR-1 and L. fermentum RC-14

Reduction in, and better treatment of, urogenital infections in women taking oral lactobacilli daily for 2 months.

Reid et al., 2004.

L. johnsonii La1 A moderate but significant difference in H. pylori colonisation was detected in children.

Cruchet et al., 2003.

L. plantarum 299v Decreased incidence of infections in liver transplant patients. Rayes et al., 2002.

L. plantarum 299v Reduction of the recurrence of Clostridium difficile enterocolitis. Wullt et al., 2003; Plummer et al., 2004.

25

Table 1.2. (Continued)

Strain

Probiotic effect

Reference

L. plantarum 299v

Relief of irritable bowel syndrome.

Niedzielin et al., 2001.

L. reuteri ATCC 55730 For treatment of diarrhoea and to produce CD4-positive T-lymphocytes in the ileal epithelium.

Valeur et al., 2004.

L. rhamnosus GG Improved treatment of diarrhoea and management of atopy. Szajewska et al., 2001; Kirjavainen et al., 2003.

VSL#3 Effective for the management of remission of pouchitis and colitis. Mimura et al., 2004.

26

1.6. FUNCTIONAL GENOMICS AND LACTIC ACID BACTERIA

The word “functional genomics” constitute genomics, transcriptomics, proteomics,

metabolomics and bioinformatics, which can also be termed as systems biology. The

incorporation of functional genomics in understanding the complex intestinal ecosystem

is gaining momentum to elucidate the relationship between diet and human health.

However, understanding the role of nutrients in modulating gene and protein expression

and subsequently their influence on cellular and metabolic functions are necessary in

preventing diet-related diseases.

Molecular approaches have demonstrated that lactobacilli was able to survive through its

intestinal passage and altered the microbial population and diversity of Lactobacillus

species, but did not induce any adverse effects on dominant microflora (de Vos et al.,

2004; Zoetendal et al., 2004). Similarly, molecular techniques also showed that L. casei

and L. plantarum are active during the transit from stomach to colon in mice and played

an important role in protein synthesis (Bron et al., 2004; Oozeer et al., 2004).

Mechanisms such as autoaggregation and adhesion were considered as potential

properties of LAB in colonising the GI tract. Roos et al. (1999) reported that genes

coding for aggregation helicase and maltose-binding proteins are necessary in increasing

the autoaggregation ability of L. reuteri 1063. Earlier studies demonstrated that

lipoteichoic acid was involved in the adherence of L. johnsonii La1 to intestinal Caco-2

cells (Granato et al., 1999). The genome sequencing of L. johnsonii La1 uncovered the

genes that were involved in lipoteichoic acid synthesis and cell surface molecules

(Vaughan and Mollet, 1999). Furthermore, Mack et al. (1999) hypothesised the role of

L. plantarum 299v and L. rhamnosus GG in inhibiting the adherence of pathogenic E.

27

coli to HT-29 intestinal cells through increased expression of intestinal mucin genes

such as MUC2 and MUC3.

Several in vivo studies reported the influence of diet on gene expression. As an example,

Rao et al. (2001) showed that mice fed with low diet in selenium decreased the

expression of genes involved in detoxification and increased the expression of genes that

were involved in DNA damage processing, oxidative stress and cell-cycle control.

Similarly, magnesium deficiency up-regulated the genes involved in protection and

repair of oxidative stress in thymocytes of rat (Petrault et al., 2002). Blanchard et al.

(2001) demonstrated the potential of cDNA microarray analysis in revealing the

intestinal gene expression changes that occurred in early stages of zinc deficiency in

rats. Furthermore, Hooper et al. (2001) demonstrated the capability of commensal

bacteria in modulating gene expression that are involved in various intestinal functions

such as nutrient absorption, mucosal barrier fortification and xenobiotic metabolism.

The use of conventional methods restricted the understanding to a particular gene

function, whereas the DNA microarray technique was capable of unravelling the overall

gene expression patterns of the cell (Brown and Botstein, 1999; Khan et al., 1999).

Whole genome analysis of gene expression in GI tract could further elucidate the

microbial-host interactions. Earlier, several studies reported the beneficial effects of

LAB on various biological functions of host, however applications of novel molecular

approaches such as microarrays in combination with functional assays is necessary to

unravel new target’s that were influenced by the probiotic bacteria.

28

1.7. AIMS

The overall aim of this study was to characterise the in vitro probiotic properties of L.

acidophilus LAFTI L10 and L. paracasei LAFTI L26 and evaluate their influence on

mucosal and systemic immune responses and gene expression in mice.

The specific aims of this study were:

1. Characterisation of Lactobacillus and Bifidobacterium species for potential

probiotic strains (Chapter 2).

2. Examination of L. acidophilus LAFTI L10 and L. paracasei LAFTI L26

influence on mucosal and systemic immunity and bacterial translocation in mice

(Chapter 3).

3. Determination of L. acidophilus LAFTI L10 and L. paracasei LAFTI L26 effects

on mucosal and systemic immune responses in cholera toxin mice model

(Chapter 4).

4. Investigation of L. acidophilus LAFTI L10 influence on gene expression in the

small bowel of normal and cholera toxin mice (Chapter 5).

29

CHAPTER 2

Selection and characterisation of Lactobacillus and Bifidobacterium species for

potential probiotic strains

30

2.1. ABSTRACT

The present study aimed to investigate the in vitro probiotic properties of Lactobacillus

and Bifidobacterium strains. A total of 12 strains were screened towards their tolerance

to various acidic conditions. Following an initial screening, 6 strains were selected to

further characterise their probiotic properties such as tolerance to bile salts,

hydrophobicity, autoaggregation and coaggregation. The overall reduction in cell

survivability of L. acidophilus LAFTI L10 and L. paracasei LAFTI L26 in different pH

conditions was less compared to other bacterial strains. Particularly, these strains

showed high tolerance to pH 2.0 with more than 105 CFU/ml in viability. Lactobacillus

acidophilus LAFTI L10 and L. paracasei LAFTI L26 demonstrated high survivability

especially in 1.0% bile salts when compared with other bacterial strains. A correlation

between hydrophobicity and autoaggregation ability was observed among the bacterial

strains. In contrast, B. lactis Bb12, demonstrated high degree of hydrophobicity, whereas

autoaggregation ability was lower compared to other bacterial strains. Furthermore,

coaggregation of L. acidophilus LAFTI L10 with other bacterial strains showed

significant differences in their aggregation capabilities compared to L. paracasei LAFTI

L26. Overall, L. acidophilus LAFTI L10 and L. paracasei LAFTI L26 showed better

tolerance to acid and bile conditions and also their surface adherence characteristics

could be considered for their development as potential probiotic strains.

31

2.2. INTRODUCTION

Probiotic bacteria are commonly referred to as beneficial microbes, which are frequently

used as active ingredients in fermented milk products. The use of probiotic bacteria in

food products has been recently increased due to their potential attributes such as

contributing to host defence mechanisms and improving the microbial balance of the GI

tract. In order to exert beneficial effects on the host, probiotic bacteria are required to

survive in enough numbers while transiting through constantly changing gastric and bile

conditions of GI tract. The general acidity of stomach ranges from pH 2.5 to 3.5

(Holzapfel et al., 1998), however the pH of stomach could also be as low as pH 1.5

(Lankaputhra and Shah, 1995) or as high as pH 6.0 or higher after the intake of food

(Johnson, 1977). There were no specific pH conditions or in vitro methods set to screen

potential probiotic strains, however a wide range of pH conditions and in vitro methods

were used to screen the acid tolerance of Lactobacillus and Bifidobacterium strains

(Conway et al., 1987; Lankaputhra and Shah, 1995; Charteris et al., 1998; Chou and

Weimer, 1999; Chung et al., 1999; Zarate et al., 2000).

Several studies reported the screening of Lactobacillus and Bifidobacterium strains

tolerance to bile salts (Lankaputhra and Shah, 1995; Prasad et al., 1998; Chung et al.,

1999; Truelstrup Hansen et al., 2002). In the small intestine, transit of food may take

between 1 – 4 h (Smith, 1995), which can have a pH of around 8.0 (Keele and Neil,

1965). Bile concentrations ranging from 0.3% – 1.0% were used for in vitro screening to

identify potential probiotic strains that were resistant to bile salts (Prasad et al., 1998;

Jacobsen et al., 1999; Truelstrup Hansen et al., 2002). Goldin and Gorbach (1992)

32

suggested that bile salts of 0.15% – 0.3% concentrations were suitable for screening

Lactobacillus and Bifidobacterium strains for human consumption.

Microbial adhesion to intestinal cells is considered as a potential characteristic of

probiotic bacteria, which could enable them to colonise the GI tract and induce potential

health effects (Pedersen and Tannock, 1989; Alander et al., 1997). In order to

demonstrate a potential probiotic strain, adhesion ability is considered as one of the

screening criteria for selecting probiotic bacteria (Fogh et al., 1977). The in vivo

bacterial adhesion studies involve difficulties to characterise the interactions especially

in humans, which led to the development of several in vitro methods as model systems

for screening potential adherent probiotic bacteria (Mayra-Makinen et al., 1983; Conway

and Kjellberg, 1989; Ouwehand et al., 2001). Earlier studies showed the in vitro

adherence capability of probiotic bacteria to Caco-2 cells (Kimoto et al., 1999; Todoriki

et al., 2001; Fernandez et al., 2003; Lee et al., 2003). However, several studies suggested

indirect in vitro methods to characterise the adhering properties of probiotic bacteria that

demonstrated the correlation between cell adhesion ability, autoaggregation and cell

surface hydrophobicity (Wadstrom et al., 1987; Marin et al., 1997; Del Re et al., 1998;

Perez et al., 1998; Del Re et al., 2000; Kos et al., 2003; Pan et al., 2006).

The adhesion of bacterial cells is a multi-step process that involves physical contact with

cell surface, which could be influenced by structural composition of the cell wall

membrane and interacting surfaces. The overall adherence of microorganisms also

depends upon non-specific physical interactions between the two surfaces, which enable

them to interact specifically between adhesins and complementary receptors (Freter,

33

1992; Rojas and Conway, 1996; Perez et al., 1998). Previous studies suggested that

several factors contribute the adhesion of probiotic bacteria to host epithelial cells, such

as cell surface hydrophobicity and autoaggregation (Cesena et al., 2001; Ehrmann et al.,

2002; Ventura et al., 2002; Kos et al., 2003), lipoteichoic acids (Sherman and Savage,

1986; Granato et al., 1999) and cell wall proteins (Walter et al,., 2005). Autoaggregation

demonstrated its importance in adhesion to intestinal cell wall, whereas coaggregation

was crucial to form as a barrier to prevent the colonisation of pathogenic

microorganisms (Boris et al., 1997; Reid et al., 1988; Del Re et al., 2000).

Autoaggregation and coaggregation properties were considered to be important for

probiotic bacteria to persist in different locations of mucosal surfaces (Jankovic et al.,

2003). Castagliuolo et al. (2005) reported the importance of aggregation characteristics

of L. crispatus in exerting protective effects in colitis mice model.

It was reported previously, that cell surface properties such as hydrophobicity may affect

autoaggregation and adhesion ability of the bacteria (Wadstrom et al., 1987; Perez et al.,

1998; Del Re et al., 2000). It is well known that attractive forces between non-polar

surfaces in water solutes control the hydrophobic interactions. However, cell surface

structures of bacteria may have the potential to undergo physiochemical changes to alter

the hydrophobic ability in response to environmental conditions such as pH, bile salts or

ionic strength.

In the present study, tolerance to low pH and high bile conditions, hydrophobicity,

autoaggregation and coaggregation ability were considered as potential probiotic

characteristics (Prasad et al., 1998; Del Re et al., 2000; Kos et al., 2003; Castagliuolo et

34

al., 2005; Otero et al., 2006; Bujalance et al., 2007). Initially, 12 strains of Lactobacillus

and Bifidobacterium species were screened for their tolerance to simulated gastric juice

at various pH levels (pH 2.0, 3.0, 4.0 and 6.5). Based on acid tolerance, 6 strains were

selected to further investigate their probiotic properties such as tolerance to bile salts

(0.5% and 1.0%), hydrophobicity, autoaggregation and coaggregation.

35

2.3. MATERIALS AND METHODS

2.3.1. Microorganisms and growth conditions

The bacterial strains used in this study were obtained from various suppliers in

Australia. Lactobacillus acidophilus ASCC 2400, Lactobacillus casei subsp. casei

ASCC 2603, Lactobacillus paracasei subsp. paracasei ASCC 5437, Lactobacillus

rhamnosus ASCC 2625, Bifidobacterium breve ASCC 1900, Bifidobacterium bifidum

ASCC 1903, Bifidobacterium infantis ASCC 1912 and Bifidobacterium longum ASCC

5188 were obtained from Australian Starter Culture Research Centre (Werribee, VIC,

Australia). DSM Food Specialties (Moorebank, NSW, Australia) provided Lactobacillus

acidophilus LAFTI L10, Lactobacillus paracasei LAFTI L26 and Bifidobacterium

animalis subsp. lactis LAFTI B94. Bifidobacterium animalis subsp. lactis Bb12 was

supplied by Chr. Hansen, Bayswater, VIC, Australia.

All bacterial strains were grown anaerobically in gas jars using GasPak System (Oxoid,

Adelaide, Australia) for 24 h at 37°C in de Man, Rogosa and Sharpe (MRS) broth

(Oxoid, Adelaide, Australia). The bacterial cells were harvested at 4000 x g for 15 min

at 4°C and washed twice with sterile 0.01 M phosphate buffered saline (PBS) solution.

The cell viability of the bacterial cultures was determined by spread plate count on MRS

agar, which was incubated under anaerobic conditions at 37°C for 48 h.

2.3.2. Acid tolerance

The acid tolerance of Lactobacillus and Bifidobacterium strains was determined by

using milk-based medium (NGYC medium: 12% non-fat skim milk, 2% glucose, 1%

36

yeast extract and 0.05% L-cysteine) as described by Lankaputhra and Shah, (1995). The

NGYC medium was adjusted to pH 2.0, 3.0, 4.0 or 6.5 (Control) with 5 M HCL or 1 M

NaOH. The bacterial cell suspensions were added to the pH adjusted NGYC medium,

which were then incubated anaerobically for 3 h at 37°C. The bacterial survivability was

determined by spread plate count on MRS agar after incubated anaerobically at 37°C for

48 h. The average and standard error were estimated from 6 independent experiments.

The overall reduction in the viability of bacterial strains in different pH conditions was

calculated using the formula:

Overall reduction of viability = (pH 6.5 – pH 4.0) + (pH 6.5 – pH

3.0) + (pH 6.5 – pH 2.0)

2.3.3. Bile tolerance

The bile tolerance of Lactobacillus and Bifidobacterium strains was determined by using

milk-yeast extract medium as described by Truelstrup Hansen et al. (2002). The milk-

yeast extract medium (pH 6.9) consists of 10% non-fat skim milk powder, 0.5% yeast

extract, 0.05% L-cysteine and 0% (Control), 0.5% or 1.0% (w/v) bile salts (Oxgall,

Sigma, Australia). The bacterial cell suspensions were inoculated into the milk-yeast

extract medium and incubated anaerobically for 6 h at 37°C.

The survival of the bacterial cells was determined by spread plate count on MRS agar

after anaerobically incubated at 37°C for 48 h. The average and standard error were

37

estimated from 6 independent experiments. The overall reduction in the viability of the

bacterial strains in different bile concentrations was calculated using the formula:

Overall reduction of viability = (0% - 0.5%) + (0% - 1.0%)

2.3.4. Hydrophobicity assay

The adhesion of bacterial strains to hydrocarbons was determined according to Perez et

al. (1998). Briefly, bacterial cultures were grown anaerobically for 24 h at 37°C in MRS

broth and harvested at 4000 x g for 15 min at 4°C followed by washing twice with

sterile 0.01 M PBS solution. Bacterial cells were resuspended in 0.01 M PBS and

absorbance (A0) was read at 600 nm. A volume of 0.6 ml of n-hexadecane (Merck,

Melbourne, Australia) was added to 3-ml aliquots of bacterial cell suspension. The

samples were mixed thoroughly by vortexing for 2 min. The 2-phase separation was

observed by incubating the samples at 37°C for 1 h. Aqueous phase was removed and

absorbance (A) was measured at 600 nm. Hydrophobicity assay was repeated 6 times to

estimate the average and standard error. The percentage of cell surface hydrophobicity is

expressed as:

[ ] 100x/)(%)(cityHydrophobi OO AAAH −=

Where A0 and A represents the absorbance of aqueous phase before and after contact

with n-hexadecane.

38

2.3.5. Autoaggregation and coaggregation assays

The bacterial strains were grown anaerobically for 24 h at 37°C in MRS broth and

harvested at 4000 x g for 15 min at 4°C followed by washing twice with sterile 0.01 M

PBS solution. The bacterial cells were resuspended in 0.01 M PBS, which were used to

perform autoaggregation and coaggregation assays.

Autoaggregation assay was performed according to Kos et al. (2003). The bacterial cell

suspensions of 4-ml aliquots were mixed thoroughly for 10 s and incubated at 37°C for 6

h. The absorbance (A) of the upper suspension (0.1 ml) was measured at 600 nm.

Autoaggregation assay was repeated 6 times to estimate the average and standard error.

The autoaggregation percentage is expressed as:

100x)/(1(%)ationAutoaggreg ot AA−=

Where At represents the absorbance at 6 h and A0 represents the absorbance at 0 h.

Coaggregation assay was performed according to Handley et al. (1987). Briefly, equal

volumes (2-ml aliquots) of different bacterial cell suspensions were mixed together by

vortexing for 10 s, while the control tubes consist of 4-ml aliquots of individual bacterial

cell suspensions. The mixed bacterial cell suspensions and the control tubes were

incubated at 37°C for 6 h and absorbance (A) of the upper suspensions (0.1 ml) was

measured at 600 nm. Coaggregation assay was repeated 6 times to estimate the average

and standard error.

39

The coaggregation percentage is expressed as:

( )( )

100x2/yx

)yx()2/)yx((%ionCoaggregat

AA

AAA

+

+−+=

Where Ax and Ay represent individual bacterial strains in the control tubes and A(x + y)

represents the mixture of bacterial strains.

2.3.6. Statistical analysis

The results were expressed as mean ± standard error of mean (SEM). The data were

analysed using the one-way analysis of variance procedure of SPSS (Version 12.0.1).

Significant differences between groups were identified by Duncan’s multiple range test

(SPSS, Version 12.0.1).

40

2.4. RESULTS

2.4.1. Tolerance to simulated gastric juice

The effect of various gastric conditions on Lactobacillus and Bifidobacterium strains is

shown in Table 2.1. All the bacterial strains showed differences in cell viability in

various pH conditions for 3 h. Lactobacillus acidophilus LAFTI L10, L. paracasei

LAFTI L26 and B. lactis LAFTI B94 strains were tolerant to acid at pH 2.0 with a

reduction of 2.95 to 3.89 log colony-forming units (CFU)/ml. Particularly, L.

acidophilus LAFTI L10 and L. paracasei LAFTI L26 were tolerant to pH 2.0 with high

survival at 105 CFU/ml, whereas B. lactis LAFTI B94, B. infantis ASCC 1912, B. lactis

Bb12 and L. acidophilus ASCC 2400 showed the capability of surviving at 104 CFU/ml

after 3 h of incubation. At pH 2.0, L. rhamnosus ASCC 2625, B. bifidum ASCC 1903, B.

longum ASCC 5188, L. paracasei ASCC 5437 and B. breve ASCC 1900 showed less

survival ranging from 101 to 103 CFU/ml. Furthermore, there was no survival of L. casei

ASCC 2603 after incubating for 3 h at pH 2.0.

The ability of L. acidophilus LAFTI L10, L. paracasei LAFTI L26, L. lactic LAFTI

B94, B. infantis ASCC 1912, B. lactis Bb12 and L. acidophilus ASCC 2400 to survive at

pH 3.0 was more than 106 CFU/ml after incubation for 3 h. All other Lactobacillus and

Bifidobacterium strains also showed tolerance to pH 3.0 with survival ranging from 103

to 105 CFU/ml. Particularly at pH 3.0, L. acidophilus LAFTI L10 demonstrated greater

tolerance with less reduction in cell viability by 1.95 CFU/ml, which was followed by L.

paracasei LAFTI L26 and B. infantis ASCC 1912 by a decrease of 2.5 CFU/ml. In pH

4.0, L. acidophilus LAFTI L10 showed higher tolerance with a decrease of 1.25

41

CFU/ml. Similarly, B. infantis ASCC 1912, B. lactis Bb12 and L. acidophilus ASCC

2400 also showed high tolerance to pH 4.0 with more than 107 CFU/ml, whereas L.

paracasei LAFTI L26, L. lactic LAFTI B94 and L. casei ASCC 2603 survived at 106

CFU/ml. The other Lactobacillus and Bifidobacterium strains showed survival of 105

CFU/ml at pH 4.0 (Table 2.1). All the Lactobacillus and Bifidobacterium strains that

were screened for their tolerance to pH 2.0, pH 3.0 and pH 4.0 showed significant

variations (P < 0.01) in their cell viability when compared with pH 6.5 (Control).

Based on the survival of Lactobacillus and Bifidobacterium strains at various pH

conditions after incubating for 3 h, overall reduction of cell viability was calculated to

determine the potential probiotic strains. Lactobacillus acidophilus LAFTI L10, L.

paracasei LAFTI L26, L. lactic LAFTI B94, B. infantis ASCC 1912, B. lactis Bb12 and

L. acidophilus ASCC 2400 exhibited greater tolerance to various pH conditions,

particularly at pH 2.0 with survival more than 104 CFU/ml. As shown in Table 2.1, top 6

bacterial strains were selected to further investigate their probiotic properties.

2.4.2. Tolerance to bile salts

The effect of bile salts on the growth of Lactobacillus and Bifidobacterium strains are

shown in Table 2.2. The exposure of bile salts at 0.5% and 1.0% for 6 h was less

detrimental to L. paracasei LAFTI L26, which demonstrated less reduction in overall

cell viability. All Lactobacillus and Bifidobacterium strains in 1.0% bile salts showed

less than 1.0 CFU/ml reduction in survival with exception to L. acidophilus ASCC 2400,

which exhibited a decrease of 1.15 CFU/ml in cell viability. The resistance of

42

Lactobacillus and Bifidobacterium strains towards 0.5% bile salts also showed reduction

in cell survival, which was not higher than 1.0 CFU/ml. The overall reduction of cell

viability in different bile salts demonstrated that L. paracasei LAFTI L26 was a

potential probiotic strain with high tolerance to bile salts, whereas B. lactis LAFTI B94

and L. acidophilus LAFTI L10 showed similar overall reduction of survival at different

bile salts concentrations. However, L. acidophilus LAFTI L10 showed better tolerance

to 1.0% bile salts compared to B. lactis LAFTI B94.

43

Table 2.1. Survival of Lactobacillus and Bifidobacterium strains in simulated gastric conditions for 3 h.

Strains

Cell count a,b

pH 6.5b

pH 4.0b

pH 3.0b

pH 2.0b

Overall

reductionc

L. acidophilus LAFTI L10

8.78 ± 0.24

8.62 ± 0.13

7.37 ± 0.16*

6.67 ± 0.18*

5.67 ± 0.21*

6.15

L. paracasei LAFTI L26

8.96 ± 0.11 8.82 ± 0.21 6.78 ± 0.10* 6.30 ± 0.04* 5.11 ± 0.07* 8.27

B. lactis LAFTI B94 8.88 ± 0.26 8.76 ± 0.17 6.72 ± 0.19* 6.11 ± 0.11* 4.87 ± 0.16* 8.58

B. infantis ASCC 1912

8.98 ± 0.18 8.87 ± 0.15 7.22 ± 0.11* 6.37 ± 0.41* 4.36 ± 0.05* 8.66

B. lactis Bb12

9.21 ± 0.14 9.14 ± 0.27 7.13 ± 0.16* 6.44 ± 0.25* 4.76 ± 0.17* 9.09

L. acidophilus ASCC 2400

9.62 ± 0.19 9.45 ± 0.42 7.22 ± 0.33* 6.59 ± 0.17* 4.26 ± 0.21* 10.28

L. rhamnosus ASCC 2625

8.46 ± 0.22 8.37 ± 0.14 5.54 ± 0.24* 4.85 ± 0.04* 3.79 ± 0.14* 10.93

B. bifidum ASCC 1903

8.41 ± 0.12 8.29 ± 0.11 5.24 ± 0.16* 4.22 ± 0.19* 3.65 ± 0.24* 11.76

B. longum ASCC 5188 8.57 ± 0.29 8.48 ± 0.33 5.74 ± 0.32* 5.11 ± 0.24* 2.32 ± 0.21* 12.27

L. paracasei ASCC 5437

8.23 ± 0.14 8.12 ± 0.46 5.58 ± 0.61* 4.09 ± 0.32* 1.32 ± 0.14* 13.37

44


Strains

Cell count a,b

pH 6.5b

pH 4.0b

pH 3.0b

pH 2.0b

Overall

reductionc

L. casei ASCC 2603

8.30 ± 0.08

8.19 ± 0.21

6.10 ± 0.28*

4.15 ± 0.16*

NDd

14.23

B. breve ASCC 1900 9.39 ± 0.12 9.27 ± 0.41 5.26 ± 0.19* 3.85 ± 0.21* 1.58 ± 0.15* 17.12

aInitial bacterial cell concentration.

bValues (Log10 CFU ml-1) are mean ± SEM (n = 6).

cExpressed as Log10 values using the formula: (pH 6.5 – pH 4.0) + (pH 6.5 – pH 3.0) + (pH 6.5 – pH 2.0).

dNot detected. Detection limit was 101 CFU/ml.

*Mean values were significantly different (P < 0.01) from the pH 6.5 (Control).

45

Table 2.2. Survival of Lactobacillus and Bifidobacterium strains in milk-yeast medium with oxgall.

Strains

Cell count a,b

0%b

0.5%b

1.0%b

Overall

reductionc

L. paracasei LAFTI L26

8.93 ± 0.16

8.80 ± 0.10

8.61 ±0.18

8.31 ± 0.02

0.68

B. lactis LAFTI B94

8.91 ± 0.13 8.79 ± 0.08 8.47 ± 0.11 8.04 ± 0.06* 1.07

L. acidophilus LAFTI L10

8.99 ± 0.25 8.93 ± 0.13 8.57 ± 0.02 8.21 ± 0.04* 1.08

B. infantis ASCC 1912

8.68 ± 0.19 8.55 ± 0.05 8.04 ± 0.14* 7.88 ± 0.06* 1.18

B. lactis Bb12

9.20 ± 0.27 9.06 ± 0.09 8.72 ± 0.09 8.16 ± 0.17* 1.24

L. acidophilus ASCC 2400

9.39 ± 0.28 9.24 ± 0.15 8.51 ± 0.06* 8.09 ± 0.11* 1.88

aInitial bacterial cell concentration.

bValues (Log10 CFU ml-1) are mean ± SEM (n = 6).

cExpressed as Log10 values using the formula: (0% - 0.5%) + (0% - 1.0%).

*Mean values were significantly different (P < 0.01) from the 0% bile salts (Control).

46

2.4.3. Surface hydrophobicity of Lactobacillus and Bifidobacterium strains

The cell surface hydrophobicity of Lactobacillus and Bifidobacterium strains to n-

hexadecane was investigated as shown in Figure 2.1. The results indicate that B. lactis

Bb12 (25%) showed higher surface hydrophobicity compared to less hydrophobic B.

infantis ASCC 1912 (10%). Specifically, LAFTI strains, such as L. acidophilus L10, L.

paracasei L26 and B. lactis B94 demonstrated similar cell surface hydrophobicity

ranging between 14% – 16%. Furthermore, L. acidophilus ASCC 2400 also exhibited

similar hydrophobic levels by 14%.

2.4.4. Autoaggregation and coaggregation of Lactobacillus and Bifidobacterium

strains

The autoaggregation of bacterial strains was investigated on the basis of their

sedimentation properties after 6 h as shown in Figure 2.2. All Lactobacillus and

Bifidobacterium strains demonstrated the differences in their autoaggregation abilities.

Particularly, L. paracasei LAFTI L26, B. lactis LAFTI B94 and L. acidophilus ASCC

2400 showed higher autoaggregation ability by 33%, whereas L. acidophilus LAFTI L10

and B. infantis ASCC 1912 indicated similar autoaggregation profiles by 30%.

Particularly, B. lactis Bb12 exhibited autoaggregation ability by 29%, which was less in

comparison to other Lactobacillus and Bifidobacterium strains that were screened in this

study.

47

Coaggregation of L. acidophilus LAFTI L10 and L. paracasei LAFTI L26 and

individually with other potential probiotic strains such as, B. lactis LAFTI B94, B. lactis

Bb12, B. infantis ASCC 1912 and L. acidophilus ASCC 2400 was examined (Fig. 2.3).

The coaggregation ability was expressed as a percentage reduction in the absorbance of

mixed bacterial suspension compared to individual microbial suspension. Lactobacillus

acidophilus LAFTI L10 and L. paracasei LAFTI L26 indicated higher coaggregation

ability by 75% compared to their individual coaggregation ability with other potential

probiotic strains. The coaggregation ability of L. acidophilus LAFTI L10 and B. lactis

LAFTI B94 was increased by 54%, which was significantly different (P < 0.01)

compared to L. paracasei LAFTI L26 coaggregation with B. lactis LAFTI B94 (24%).

Simultaneously, L. acidophilus LAFTI L10 demonstrated increased (P < 0.01)

coaggregation by 53% with B. lactis Bb12 compared to L. paracasei LAFTI L26 with B.

lactis Bb12 (18%). The coaggregation of L. acidophilus LAFTI L10 or L. paracasei

LAFTI L26 with B. infantis ASCC 1912 showed no differences (P > 0.05) in their

aggregation capabilities. However, coaggregation of L. acidophilus LAFTI L10 or L.

paracasei LAFTI L26 with L. acidophilus ASCC 2400 demonstrated significant

differences (P < 0.01).

48

0

10

20

30

L10

L26

B94

Bb

12

AS

CC

1912

AS

CC

2400

Hyd

rop

ho

bic

ity %

Figure 2.1. Surface hydrophobicity of L. acidophilus LAFTI L10, L. paracasei LAFTI L26, B. lactis LAFTI B94, B. lactis Bb12, B.

infantis ASCC 1912 and L. acidophilus ASCC 2400. Values are mean ± SEM (n = 6).

49

20

30

40

L10

L26

B94

Bb

12

AS

CC

1912

AS

CC

2400

Au

toag

gre

gati

on

%

Figure 2.2. Autoaggregation abilities of L. acidophilus LAFTI L10, L. paracasei LAFTI L26, B. lactis LAFTI B94, B. lactis Bb12, B.

infantis ASCC 1912 and L. acidophilus ASCC 2400. Values are mean ± SEM (n = 6).

50

0

20

40

60

80

L10 +

L26

L10 +

B94

L26 +

B94

L10 +

Bb

12

L26 +

Bb

12

L10 +

AS

CC

1912

L26 +

AS

CC

1912

L10 +

AS

CC

2400

L26 +

AS

CC

2400

Co

ag

gre

gati

on

%

Figure 2.3. Coaggregation abilities of L. acidophilus LAFTI L10, L. paracasei LAFTI L26, B. lactis LAFTI B94, B. lactis Bb12, B.

infantis ASCC 1912 and L. acidophilus ASCC 2400. Values are mean ± SEM (n = 6). *Mean values were significantly different (P <

0.01) between the coaggregation of L. acidophilus LAFTI L10 or L. paracasei LAFTI L26 individually with other bacterial strains.

* *

*

51

2.5. DISCUSSION

In order to exert beneficial effects, probiotic bacteria must be tolerant to harsh gastric

conditions and high bile salt concentrations in the GI tract. In humans, pH of normal

gastric juice can be below 3.0, which can significantly prevent all bacterial growth.

Although the pH of stomach could be as low as 1.0, several studies reported the

preferred pH as 3.0 for in vitro assays (Garriga et al., 1998; Suskovic et al., 1997).

Usman and Hosono, (1999) also suggested the survival of probiotic bacteria at pH

3.0 for 2 h as an optimal screening condition for characterising probiotic strains

towards acid tolerance.

In the present study, overall reduction in cell viability of bacterial strains at various

pH conditions was considered as selection criteria, which lead to the identification of

6 Lactobacillus and Bifidobacterium strains. Particularly, these 6 Lactobacillus and

Bifidobacterium strains showed better survival at pH 2.0 with cell viability higher

than 104 CFU/ml (Table 2.1). Lactobacillus acidophilus LAFTI L10 remained highly

tolerant to various pH conditions with less overall decrease in cell viability. In pH

2.0, L. acidophilus LAFTI L10 and L. paracasei LAFTI L26 survived by more than

105 CFU/ml, whereas other bacterial strains that were selected remained at 104

CFU/ml. Strain differences at various pH conditions were observed, which were

consistent to the earlier studies that reported similar strain-specific variations to

simulated gastric conditions (Truelstrup Hansen et al., 2002; Mishra and Prasad,

2005).

Several studies reported the effects of various bile salt concentrations on the growth

of probiotic bacteria (Clark and Martin, 1994; Lankaputhra and Shah, 1995; Prasad

52

et al., 1998; Truelstrup Hansen et al., 2002). There were no reports suggesting the

precise concentration of bile salts that can be used as selection criteria towards

probiotic characterisation. The bile concentration varies accordingly in the human GI

tract; however it was believed to be 0.3% w/v (Sjovall, 1959, Gilliland et al., 1984).

In the current study, bile salt concentrations of 0.5% and 1.0% were used for

screening bacterial strains towards bile tolerance.

Lactobacillus and Bifidobacterium strains used in this study survived by more than

108 CFU/ml in 1.0% bile salts with exception to B. infantis ASCC 1912, which

survived at 107 CFU/ml. Lactobacillus paracasei LAFTI L26 showed an overall

higher tolerance to various bile salt concentrations, whereas L acidophilus LAFTI

L10 and B. lactis LAFTI B94 showed similar reductions in the overall cell

survivability. In contrast, L. acidophilus LAFTI L10 demonstrated higher tolerance

to 1.0% bile salts compared to B. lactis LAFTI B94. Interestingly, LAFTI strains

such as L. acidophilus L10, L. paracasei L26 and B. lactis B94 demonstrated high

tolerance to simulated gastric conditions and bile salt concentrations. These

characteristics demonstrate that LAFTI strains are likely to survive in varying GI

tract conditions, where they could exert positive health effects on the host.

Adhesion of bacterial strains to GI tract is necessary to colonise and induce

beneficial effects on the host. Several direct and indirect in vitro methods were used

to study the adherence ability of probiotic bacteria, but still there was no consensus

about defining an optimal method that could predict the in vivo microbial adhesion.

In the current study, microbial adhesion to n-hexadecane was used to predict the

adherence ability of Lactobacillus and Bifidobacterium strains. Bifidobacterium

53

lactis Bb12 showed higher hydrophobicity when compared with other bacterial

strains. The LAFTI strains, L. acidophilus L10, L. paracasei L26 and B. lactis B94

demonstrated similar hydrophobic abilities ranging between 14% – 16%.

Particularly, B. infantis ASCC 1912 showed less hydrophobicity compared to other

bacterial strains screened in the current study. The differences in hydrophobic ability

of bacterial strains observed in this study could be due to variations in the chemical

composition of cell surface components (Pelletier et al., 1997), particularly

lipoteichoic acid-protein complex (Sherman and Savage, 1986) or presence of cell

surface hydrophobic protein moiety (Ronner et al., 1990). Furthermore, Gomez

Zavaglia et al. (2002) demonstrated the influence of bile salts, which decreased the

autoaggregation ability of Bifidobacterium strains.

Earlier, several studies reported the composition, structure and forces of interaction

that are related to bacterial adherence with intestinal epithelial cells (Greene and

Klaenhammer, 1994; Pelletier et al., 1997; Perez et al., 1998). Moreover, the ability

of aggregation is related to cell surface characteristics of bacterial strains

(Vandevoorde et al., 1992; Del Re et al., 2000). Microbial aggregation is considered

as a desirable characteristic of probiotic bacteria (Drago et al., 1997; Mastromarino

et al., 2002; Castagliuolo et al., 2005). In the present study, autoaggregation ability of

bacterial strains was examined, which could be considered as an indication of

adherence characteristics. All the strains used in this study showed differences in

their autoaggregation abilities. Interestingly, LAFTI strains, L. acidophilus L10, L.

paracasei L26 and B. lactis B94 showed similarities in their autoaggregation and

hydrophobic abilities. These results were consistent with the earlier reports, which

suggested similarities in bacterial autoaggregation and hydrophobicity (Gomez

54

Zavaglia et al., 2002; Aslim et al., 2007). However, B. lactis Bb12 demonstrated

higher hydrophobicity and lower autoaggregation ability compared to other bacterial

strains used in the current study. Similar differences in autoaggregation and

hydrophobicity of bacterial strains was also reported earlier (Del Re et al., 2000).

The coaggregation ability of bacterial strains isolated from human dental plaque was

the first study to suggest the process of adhesion between genetically distinct

bacterial strains. However, coaggregation is now being investigated among the

bacteria that were isolated from human oral cavity, mammalian GI tracts and human

urogenital tract (Rickard et al., 2003). Drago et al. (1997) demonstrated the

coaggregation ability of Lactobacillus strains with enterotoxigenic E. coli,

Salmonella enteritidis and Vibrio cholerae. Earlier studies suggested the

phenomenon of coaggregation between bacterial strains isolated from dental plaque

of healthy individuals (Kolenbrander, 2000) and beneficial effects of cell aggregation

in promoting the colonisation of lactobacilli in GI and vaginal tracts (Vandevoorde et

al., 1992; Kmet and Lucchini, 1997; Cesena et al., 2001; Jankovic et al., 2003).

In the present study, coaggregation of L. acidophilus LAFTI L10 with B. lactis

LAFTI B94, B. lactis Bb12 and L. acidophilus ASCC 2400 showed significant

differences compared to L. paracasei LAFTI L26. Vizoso Pinto et al. (2007)

demonstrated higher degree of coaggregation ability of L. johnsonii compared to L.

casei Shirota and L. rhamnosus GG with human pathogens such as E. coli, L.

monocytogenes, Staphylococcus aureus and Salmonella Typhimurium. In the current

study, coaggregation of L. acidophilus LAFTI L10 with L. paracasei LAFTI L26

demonstrated higher coaggregating ability compared to their individual

55

coaggregation abilities with other bacterial strains. Especially, coaggregation abilities

of L. acidophilus LAFTI L10 and L. paracasei LAFTI L26 with pathogenic bacteria

need to be investigated, which may constitute as a protective mechanism against

infection (Reid et al., 1988; Schachtsiek et al., 2004).

56

2.6. CONCLUSIONS

Lactobacillus acidophilus LAFTI L10 and L. paracasei LAFTI L26 were selected

for subsequent in vivo studies based on their overall tolerance to various pH

conditions, especially due to their higher survival at pH 2.0. These strains also

showed better tolerance to bile conditions particularly to 1.0% bile salts. All bacterial

strains demonstrated the correlation of adherence abilities such as hydrophobicity

and autoaggregation with exception to B. lactis Bb12. Lactobacillus acidophilus

LAFTI L10 showed higher coaggregation abilities with L. paracasei LAFTI L26 and

also with other probiotic bacterial strains.

57

CHAPTER 3

Effect of L. acidophilus LAFTI L10 and L. paracasei LAFTI L26 on gut and

systemic immunity and bacterial translocation in mice

58

3.1. ABSTRACT

The immune enhancing properties of Lactobacillus acidophilus LAFTI L10 and

Lactobacillus paracasei LAFTI L26 in mice were investigated. Each mouse

(BALB/c) was orally fed with cultures of either L. acidophilus or L. paracasei at 108

CFU/50µl per day for 14-days. Immunofluorescence assay of the small intestine in

mice demonstrated the capability of both Lactobacillus strains in enhancing

immunoglobulin (Ig)-A, interleukin (IL)-10 and interferon (IFN)-� producing cells.

In systemic immune response, proliferative responses of splenocytes to concanavalin

A (ConA; T-cell mitogen) and lipopolysaccharide (LPS; B-cell mitogen) were

significantly higher in mice fed with L. acidophilus. ConA-induced splenocyte

proliferative responses were increased significantly in mice fed with L. paracasei,

however there was no significant difference of splenocyte proliferative responses to

LPS compared to control mice. In mice fed with either L. acidophilus or L.

paracasei, IL-10 and IFN-� levels in blood serum and splenocytes stimulated with

ConA were increased compared to the control group. Both Lactobacillus strains

showed similar patterns in activating the phagocytic activity of peritoneal

macrophages. Lactobacillus acidophilus or L. paracasei-fed mice modulated the

translocation of Lactobacillus spp. and total anaerobes to Peyer’s patches and

mesenteric lymph nodes when compared to control mice. Furthermore, there was no

indication of disruption of intestinal mucosal integrity and thus no bacterial

translocation to spleen, liver or blood in mice fed with L. acidophilus or L.

paracasei. The results of this study indicate that L. acidophilus and L. paracasei are

potential enhancers of gut and systemic immunity and are non-pathogenic, as

suggested by their bacterial translocation profiles in healthy mice.

59

3.2. INTRODUCTION

The mucous covering of gastrointestinal (GI) tract has various important functions

such as providing a well balanced habitat for beneficial intestinal microflora and

protecting the host from pathogenic bacterial infection (Corfield et al., 2001). Lactic

acid bacteria (LAB) are known to stimulate immune responses of gut associated

lymphoid tissues (GALT) through lymphoid cells of GI tract (Herias et al., 1999;

Perdigon et al., 1994; Perdigon et al., 1999). Evidence is accumulating to

demonstrate that certain LAB promote health benefits by stimulating immune

responses through phagocytosis (Perdigon et al., 1986), enhanced release of

cytokines (Marin et al., 1998; Miettinen et al., 1998; Kato et al., 1999; Tejada-Simon

et al., 1999) and increased mucosal and secretory antibody levels in response to

Escherichia coli 0157:H7 infection (Shu and Gill, 2002) and Shigella sonnei (Nader

de Macias et al., 1992). Several reports have shown that LAB influences the balance

of Th1/Th2 cytokines (Borruel et al., 2003; Morita et al., 2002; Wallace et al., 2003).

Stimulation of cells to produce cytokines and elimination of toxic compounds are

related to the surface adherence properties of LAB (Gomez Zavaglia et al., 2002).

The effect of probiotic bacteria on immune responses is often evaluated through in

vitro and ex vivo measurements of cytokine or immunoglobulin production, T- or B-

cell proliferation. Gill et al. (2000) demonstrated that orally administered LAB

enhances proliferative responses of splenocytes to concanavalin A (ConA; T-cell

mitogen) and lipopolysaccharide (LPS; B-cell mitogen). However, Kirjavainen et al.

(1999) reported that oral administration of LAB inhibits lymphocyte proliferation.

Several scientific studies reported the influence of LAB in inducing cytokines such

as interferon (IFN)-�, tumor necrosis factor (TNF)-�, interleukin (IL)-6, IL-10, IL-12

60

and IL-18 in human blood mononuclear cells (Miettinen et al., 1996, Solis-Pereyra et

al., 1997; Miettinen et al., 1998). Furthermore, LAB influenced the enhancement of

IFN-� levels in ConA stimulated human peripheral blood lymphocytes (De Simone et

al., 1986).

Macrophages are regulatory cells that play an important role in inflammation and

host defence-related functions such as phagocytosis, production of cytokines and

mediators, antigen presentation, antimicrobial and tumoricidal activity. Phagocytosis

is a critical function of mononuclear phagocytes and neutrophils. The ability of

lactobacilli to induce cytokine production by macrophages (Marin et al., 1998;

Hessle et al., 1999) and stimulation of macrophages through NF-kappa B and STAT

transcription factor has been demonstrated (Mestecky et al., 1999). Human

volunteers fed with either L. acidophilus La1 or B. bifidum Bb12 enhanced the

phagocytosis of peripheral blood leukocytes, which persisted for 6 weeks after oral

ingestion of these strains (Schiffrin et al., 1997).

Intestinal bacterial translocation is the passage of viable indigenous bacteria from

gastrointestinal lumen to lymphatic organs (Peyer’s patches and mesenteric lymph

nodes), spleen, liver, peritoneal cavity and blood stream (Berg, 1992). Translocation

of microorganisms includes E. coli and certain members of Enterobacteriaceae and

rarely anaerobes (Wells et al., 1988). Bacterial translocation in healthy mice occurs

continuously at a low rate, which is a highly regulated biological process (Ma et al.,

1990). The disruption of intestinal barrier or incompetent host immune system that is

unable to confine an infection could lead the pathogenic bacteria to bloodstream and

cause septicemia (Berg, 1999). However, translocation of LAB from gut to GALT is

61

considered as a normal and beneficial physiological process associated with the

stimulation of immune responses (Bengmark and Jeppsson, 1995). Furthermore,

scientific studies also reported the potential role of LAB in protecting gut barrier

functions in animal models (Luyer et al., 2005; Daniel et al., 2006; Zareie et al.,

2006).

Earlier reports showed the influence of probiotic bacteria on host immune functions

in a strain dependent manner and therefore it is inappropriate to generalise the

probiotic beneficial effects that are specific to genus or species (Perdigon et al.,

2001; Schiffrin and Blum, 2002). As shown in Chapter 2, Lactobacillus and

Bifidobacterium strains were screened to select bacterial strains that are tolerant to

simulated gastric juices and bile salts. After subsequent screening of the bacterial

strains, L. acidophilus LAFTI L10 and L. paracasei LAFTI L26 were identified as

potential probiotic strains based on their tolerance to low pH and high bile salts.

Lactobacillus acidophilus L10 and L. paracasei L26 also demonstrated better

adhesive abilities such as autoaggregation and coaggregation, which are known to be

important in bacterial adhesion to intestinal mucosa in order to influence host

immune functions. The commercial probiotic strains may lose their potential

probiotic properties and also change their physiological processes due to rigorous

industrial processing conditions and repeated subculturing. Earlier in vitro studies

reported that L. acidophilus L10 and L. paracasei L26 inhibited the growth of

Listeria monocytogenes and E. coli 0111 (Pidcock et al., 2002). Further studies

conducted using L. acidophilus L10 in mice showed the ability to inhibit the growth

of L. monocytogenes in the GI tract (Mahoney and Henriksson, 2003). Elahi et al.

(2005) demonstrated that mice fed with L. acidophilus L10 enhanced the clearance of

62

Candida albicans from oral cavities, which are correlated with increased levels of

IFN-� and nitric oxide in saliva.

There were no scientific reports that suggested the ability of L. acidophilus L10 or L.

paracasei L26 in stimulating in vivo gut immune responses. However, little was

known about the capability of L. acidophilus L10 or L. paracasei L26 in stimulating

distant immune responses at systemic levels (Cayzer et al., 2001; Clancy et al., 2006)

and furthermore no scientific studies reported the influence of these strains on

bacterial translocation. Therefore, this study investigated the effects of L. acidophilus

L10 and L. paracasei L26 on various indices of immune functions in gut and

systemic immunity and their influence on bacterial translocation in healthy mice.

63


3.3.1. Animals

Male 6 - 8 wks old BALB/c mice were obtained from Biological Resources Centre,

The University of New South Wales, Sydney, Australia. Control and experimental

groups consisted of six mice each, which were caged separately at 22 ± 1°C and

housed under 12 h light-dark photoperiod. All mice were fed with standard mouse

chow (Gordon's Specialty Stock Feeds, Sydney, Australia) and provided ad libitum

access to water. The University of Western Sydney (Sydney, Australia) Animal Care

and Ethics Committee approved all protocols for the animal experiment.

3.3.2. Microorganisms

Lactobacillus acidophilus LAFTI L10 and L. paracasei LAFTI L26 strains were

obtained from DSM culture collection (DSM Food Specialties Ltd., Sydney,

Australia). Lactobacillus acidophilus and L. paracasei were grown anaerobically in

gas jars using GasPak System (Oxoid, Adelaide, Australia) for 24 h at 37°C in de

Man, Rogosa and Sharpe (MRS) broth (Oxoid, Adelaide, Australia). The bacterial

cells were harvested at 4000 x g for 15 min at 4°C and washed twice with sterile 0.01

M phosphate buffered saline (PBS) solution.

3.3.3. Feeding procedure

After acclimatisation for 1 week, mice were fed daily by oral gavage with either L.

acidophilus or L. paracasei of 108 colony-forming units (CFU) in 50µl of 10% (w/v)

skimmed milk powder (SMP) for 14-days. Control group mice received 50µl of SMP

without lactic acid bacteria. The viability of bacteria was determined by spread plate

64

count of bacterial serial dilutions on MRS agar (Oxoid, Adelaide, Australia), which

were incubated anaerobically in gas jars using GasPak System at 37°C for 48 h.

3.3.4. Histological samples

At the end of 14-day feeding trail, mice were euthanized by carbon dioxide

inhalation and small intestines were removed, dissected into segments and stored in

95% ethanol (v/v). The tissues were then embedded in paraffin blocks and the

sections were processed for immunofluorescence assays.

3.3.5. Immunofluorescent detection of IgA cells

The number of immunoglobulin (Ig)-A secreting cells was determined in histological

sections of the mice small intestine by direct immunofluorescence assay.

Histological sections (4-µm) were deparaffinised by immersion in xylene and

rehydrated in ethanol, which were incubated with 1% blocking solution of bovine

serum albumin (Sigma-Aldrich, Sydney, Australia) for 30 min at room temperature.

The sections were then incubated with 1/100 dilution of anti-IgA mono-specific

antibody (�–chain specific) conjugated with fluorescein isothiocyanate (FITC)

(Sigma-Aldrich, Sydney, Australia) in 0.01 M PBS solution for 30 min at 37°C. The

sections were washed three times with 0.01 M PBS solution and the immune cells

were observed by using a fluorescence light microscope. Results were expressed as

number of positive fluorescent cells in 10 fields of vision at 1000x magnification.

65

3.3.6. Detection of cytokine producing cells in small intestine

Interleukin–10 and IFN-� producing cells were detected in histological sections of

the mice small intestine by indirect immunofluorescence assay. After

deparaffinization and rehydration in ethanol, histological sections (4-µm) were

incubated with 1/100 dilution of rabbit anti-mouse IL-10 and IFN-� polyclonal

antibodies (Peprotech Inc., Rocky Hill, USA) for 60 min at room temperature. The

incubation was followed by washing twice with 0.01 M PBS solution. The sections

were then incubated with 2/100 dilution of goat anti-rabbit antibody conjugated with

FITC (Jackson ImmunoResearch Laboratories Inc., West Grove, USA) for 45 min at

room temperature. The number of fluorescent cells was counted using a fluorescence

light microscope and expressed as positive fluorescent cells in 10 fields of vision at

1000x magnification.

3.3.7. Splenocytes

Spleens from mice were removed aseptically and placed individually in complete

RPMI-1640 medium containing 10% fetal calf serum (FCS), 100 U/ml penicillin and

100 µg/ml streptomycin. Splenocytes were isolated by chopping the spleen tissue

into small pieces with sterile scissors and forcing the spleen cell suspension through

a 5 ml syringe. The resulting spleen cell suspension was then transferred into a tube

containing 5 ml of complete RPMI-1640 medium and centrifuged at 300 x g for 10

min. To lyse erythrocytes, cells were resuspended in ACK lysis buffer (Tris-NH4Cl)

and incubated at room temperature for 5 min followed by washing the cells twice in

complete RPMI-1640 medium. The splenic lymphocyte suspensions were adjusted to

a final concentration of 2 x 106 cells/ml in complete RPMI-1640 medium. Trypan

blue exclusion method was used to determine the cell viability.

66

3.3.8. Lymphocyte proliferation assay

A 50 µl of complete RPMI-1640 medium with 2 x 106 cells ml-1 were added in

triplicates to the wells of a 96-well tissue culture plate and cultured in the presence or

absence of T- and B-cell mitogens. A 50 µl/well of pre-optimised concentrations of

mitogens, concanavalin A (ConA, 2.5 µg/ml; Sigma, Sydney, Australia) and

lipopolysaccharide (LPS, 5 µg/ml, derived from E. coli; Sigma, Sydney, Australia)

were added to the wells. Control wells received 50 µl of complete RPMI-1640

medium. The cells were cultured for 72 h at 37°C in a humidified incubator with CO2

and air (5: 95, v/v). The cell proliferation over the final 4 h of culture was determined

by using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega,

Madison, USA). The absorbance of each well was read at 492nm using a microtitre

plate reader (Multiskan Titertek, Huntsville, USA).

3.3.9. Estimation of cytokines in blood and splenocytes

Blood was collected upon euthanizing the mice after 14-day feeding trial and left to

coagulate overnight at 4°C. Serum was then processed from blood by centrifugation

at 2000 x g for 10 min and stored at -20°C until cytokines were assayed. Serum was

used to estimate the presence of IL-10 and IFN-� using mouse IL-10 and mouse IFN-

� sandwich ELISA kits (Chemicon International Inc., Temecula, USA).

Spleen cell suspensions (2 x 106 cells ml-1) were added in triplicates to the wells of a

96-well tissue culture plate and cultured in the presence of ConA (2.5 µg/ml) for 48 h

at 37°C in a humidified incubator with CO2 and air (5: 95, v/v). The supernatant

fractions were harvested from cell suspensions and stored at -80°C until cytokine

67

analysis. Estimation of IL-10 and IFN-� in the supernatants was determined using

mouse IL-10 (Chemicon International Inc., Temecula, USA) and mouse IFN-�

(Assay Designs Inc., Ann Arbor, USA) sandwich ELISA kits.

3.3.10. Phagocytosis

Peritoneal macrophages were isolated from the peritoneal cavity of each mouse by

flushing with 5 ml of RPMI-1640 medium. Peritoneal macrophages were washed

twice and adjusted to a final concentration of 106 cells/ml in RPMI-1640 medium.

Heat killed (100°C, 15 min) Candida albicans ATCC 10231 suspension (106 cells

ml-1) of 0.2 ml opsonised with 10% mouse autologous serum for 15 min at 37°C was

added to 0.2 ml of each macrophage suspension at a concentration of 106 cells/ml

and incubated for 30 min at 37°C. The results were expressed as percentage of

phagocytosis that was determined by counting over 100 macrophages using a Nikon

optical microscope.

3.3.11. Bacterial translocation assay

Bacterial translocation to Peyer’s patches, mesenteric lymph nodes (MLN), spleen,

liver and blood was determined in control and experimental group mice. The Peyer’s

patches, MLN, spleen and a sample of liver tissue were aseptically removed from

each animal and washed twice with 0.01 M PBS solution, which were transferred to

pre-weighed tubes containing 0.01 M PBS. The organs were weighed, homogenised

and serially diluted in 0.01 M PBS. Blood collected through cardiac puncture was

also serially diluted in 0.01 M PBS. Tissue homogenates and blood suspensions from

each mouse were plated separately in triplicates on MRS agar for detection of

68

Lactobacillus spp., Wilkins-Chalgren (WC) Anaerobe agar (Oxoid, Adelaide,

Australia) containing 5% horse blood (Oxoid, Adelaide, Australia) for detection of

total anaerobes and MacConkey agar (Oxoid, Adelaide, Australia) for detection of

enteric bacteria. Plates were incubated under anaerobic (MRS and WC agar plates)

and aerobic (MacConkey agar plates) conditions at 37°C for 48 h. Colonies were

enumerated on appropriate agar plates and expressed as either colony forming units

per g (CFU g-1) or per ml (CFU ml-1).




Significant differences between groups were identified by Duncan’s multiple range

test (SPSS, Version 12.0.1).

69

3.4. RESULTS

3.4.1. Determination of IgA producing cells

The effect of orally administered LAB on IgA producing cells in lamina propria of

the small intestine was determined by direct immunofluorescence assay (Fig. 3.1A,

B). In comparison to control mice, mice orally fed with L. acidophilus or L.

paracasei significantly enhanced (P < 0.001) the IgA producing cells in the small

intestine. Lactobacillus acidophilus-fed mice showed an increase of 68% in IgA cells

compared to control mice, whereas L. paracasei-fed mice demonstrated 20%

increase in IgA cells. The differences between L. acidophilus and L. paracasei

experimental groups were significant (P < 0.001) in enhancing the number of IgA

cells.

3.4.2. Effect of LAB on cytokines in gut

The effect of oral administration of LAB in enhancing cytokine producing cells in

lamina propria of the small intestine was determined by indirect immunofluorescence

assay (Fig. 3.2A, B). Significant increase (P < 0.001) of IL-10 and IFN-� producing

cells were detected in the small intestine of mice fed with L. acidophilus or L.

paracasei when compared to the control mice. Specifically, L. acidophilus-fed mice

showed an increase of 63% of IL-10 producing cells and more than 200% of IFN-�

producing cells compared to control group. Simultaneously, L. paracasei

demonstrated an increase of 18% of IL-10 and 95% of IFN-� producing cells,

however the increase was lesser compared to mice fed with L. acidophilus.

70

3.4.3. Lymphocyte proliferative responses

The proliferative responses of spleen cells stimulated with ConA and LPS were

estimated to determine the effect of orally administered LAB on T- and B-cell

functions in each experimental group (Table 3.1). The proliferative responses of

splenocytes stimulated with ConA were significantly higher in mice fed with L.

acidophilus (P < 0.01) or L. paracasei (P < 0.05) compared to control mice. In

comparison to control mice, LPS induced proliferative responses were increased (P <

0.05) in mice fed with L. acidophilus. The splenocyte proliferative responses induced

by LPS were higher in mice fed with L. paracasei but were not significantly different

(P > 0.05) compared to control mice. In contrast, L. acidophilus-fed mice

demonstrated higher (P < 0.01) proliferative responses when stimulated with ConA

and LPS compared to mice fed with L. paracasei.

3.4.4. Effect of LAB on cytokines in blood serum and splenocytes

The effects of L. acidophilus and L. paracasei in systemic immunity were

determined by estimating the serum cytokines. Lactobacillus acidophilus or L.

paracasei demonstrated a significant increase (P < 0.001) of IL-10 and IFN-� levels

in serum compared to control mice (Fig. 3.3A, B). The increase of IL-10 and IFN-�

levels detected in serum of mice fed with L. acidophilus were higher (P < 0.001)

compared to mice fed with L. paracasei.

The effects of ConA induced cytokine production in spleen cells from mice fed with

L. acidophilus or L. paracasei were estimated (Fig. 3.4A, B). In mice fed with L.

acidophilus, production of IL-10 (P < 0.01) and IFN-� (P < 0.05) by splenocytes

were enhanced when compared to control mice. Splenocytes from mice fed with L.

71

paracasei also demonstrated significantly higher levels of IL-10 and IFN-� (P <

0.05) compared to control mice. The differences between mice fed with L.

acidophilus and L. paracasei in enhancing IL-10 production was significant (P <

0.01). However, there were no differences (P > 0.05) between L. acidophilus and L.

paracasei in inducing IFN-� levels.

72

A

40

60

80

100

IgA

cell

s/1

0 f

ield

s

Control

L10

B

40

55

70

IgA

cell

s/1

0 f

ield

s

Control

L26

Figure 3.1. Effect of A) L. acidophilus L10 and B) L. paracasei L26 on

immunoglobulin (Ig)-A producing cells in the small intestine of mice. Values are

mean ± SEM (n = 4). *Mean values were significantly different (P < 0.001) from the

control group. #Significant differences (P < 0.001) between L. acidophilus L10 and

L. paracasei L26 experimental groups.

# *

# *

73

A

15

45

75

IL-1

0 c

ell

s/1

0 f

ield

s

Control

L10

L26

B

15

45

75

IFN

- � c

ell

s/1

0 f

ield

s

Control

L10

L26

Figure 3.2. Effect of orally administered L. acidophilus L10 or L. paracasei L26 on

A) interleukin (IL)-10 and B) interferon (IFN)-� cytokine producing cells in the

small intestine of mice. Values are mean ± SEM (n = 6). *Mean values were

significantly different (P < 0.001) from the control group. #Significant differences (P

< 0.001) between L. acidophilus L10 and L. paracasei L26 experimental groups.

# *

# *

# *

# *

74

Table 3.1. Proliferative responses of spleen cells stimulated with ConA and LPS in

mice fed with either L. acidophilus L10 or L. paracasei L26.

Mitogen stimulated splenocytes (Absorbance – OD 492 nm)

Group ConA LPS

Control

3.28 ± 0.19 2.19 ± 0.07

L. acidophilus L10

5.04 ± 0.29 **, # 4.31 ± 0.10 *, #

L. paracasei L26

4.25 ± 0.32 *, # 2.71 ± 0.08 #

Values are mean ± SEM (n = 6).

Significantly different from the control mice *P < 0.05; **P < 0.01.

Significant differences between L. acidophilus L10 and L. paracasei L26

experimental groups #P < 0.01.

75

A

100

200

300

400

500

IL-1

0 p

g/m

l

Control

L10

L26

B

100

200

300

400

IFN

- � p

g/m

l

Control

L10

L26

Figure 3.3. Estimation of cytokines A) interleukin (IL)-10 and B) interferon (IFN)-�

in serum of mice fed with L. acidophilus L10 or L. paracasei L26. Values are mean

± SEM (n = 6). *Mean values were significantly different (P < 0.001) from the

control group. #Significant differences (P < 0.001) between L. acidophilus L10 and

L. paracasei L26 experimental groups.

# *

# *

# *

# *

76

A

1000

1500

2000

2500

IL-1

0 p

g/m

l

Control

L10

L26

B

200

400

600

IFN

- � p

g/m

l

Control

L10

L26

Figure 3.4. Production of A) interleukin (IL)-10 and B) interferon (IFN)-� by spleen

cells from mice orally fed with either L. acidophilus L10 or L. paracasei L26.

Values are mean ± SEM (n = 6). Significantly different from the control mice *P <

0.05; **P < 0.01. Significant differences between L. acidophilus L10 and L.

paracasei L26 experimental groups #P < 0.01.

*

*

# *

# **

77

3.4.5. Phagocytic activity

The phagocytic activity of peritoneal macrophages in mice fed with either L.

acidophilus or L. paracasei was determined (Fig. 3.5A, B). Macrophages isolated

from the peritoneal cavity of mice fed with L. acidophilus or L. paracasei showed

significantly increased (P < 0.01) phagocytic activity against Candida albicans when

compared with the control mice. In contrast, L. acidophilus and L. paracasei strains

showed no differences (P > 0.05) in inducing phagocytic activity of the peritoneal

macrophages.

3.4.6. Bacterial translocation

Bacterial translocation to Peyer’s patches, MLN, spleen, liver and blood was

determined in mice fed with L. acidophilus or L. paracasei to evaluate possible

adverse effects that could lead to systemic dysfunction. Translocation of

Lactobacillus spp. to Peyer’s patches was significantly different in mice fed with L.

acidophilus (P < 0.01) or L. paracasei (P < 0.05) compared to control mice (Table

3.2). In mice fed with L. acidophilus or L. paracasei, Lactobacillus spp. translocated

to MLN was also increased (P < 0.01) compared to control mice. There were no

differences (P > 0.05) between mice fed with L. acidophilus and L. paracasei in

relation to the increased translocation of Lactobacillus spp. to Peyer’s patches and

MLN. Furthermore, there was no incidence of translocation of Lactobacillus spp. to

spleen, liver and blood in mice fed with either L. acidophilus or L. paracasei and

control group (Table 3.2).

Total anaerobes translocated to Peyer’s patches in L. acidophilus-fed mice was

higher (P < 0.01) compared to control mice (Table 3.3). Translocation of total

78

anaerobes to Peyer’s patches was also increased in mice fed with L. paracasei but

not significantly different (P > 0.05) when compared with control mice. Mice fed

with either L. acidophilus or L. paracasei showed increased (P < 0.01) translocation

of total anaerobes to MLN compared to control mice (Table 3.3). There were no

significant differences (P > 0.05) between mice fed with L. acidophilus and L.

paracasei in relation to increased translocation of total anaerobes to Peyer’s patches.

However, L. acidophilus and L. paracasei strains demonstrated the differences in

translocation of total anaerobes to MLN. There was no evidence of translocation of

total anaerobes to spleen, liver and blood in mice fed with either L. acidophilus or L.

paracasei and control group (Table 3.3).

In mice fed with L. acidophilus or L. paracasei and control group, translocation of

enteric bacteria to Peyer’s patches, MLN, spleen, liver and blood was not detected

(Table 3.4).

79

A

20

40

60

Ph

ag

ocyto

sis

%

Control

L10

B

20

40

60

Ph

ag

ocyto

sis

%

Control

L26

Figure 3.5. Effect of A) L. acidophilus L10 and B) L. paracasei L26 on phagocytic

activity of the peritoneal macrophages. Results are the mean ± SEM (n = 6).

Significantly different from the control mice *P < 0.01.

*

*

80

Table 3.2. Translocation of Lactobacillus spp. to Peyer’s patches, mesenteric lymph nodes (MLN), spleen, liver and blood in mice orally fed

with either L. acidophilus L10 or L. paracasei L26 for 14-days.

Organ

Treatment Peyer’s patchesa MLNa Spleena Livera

Bloodb

Control

4.61 ± 0.08 4.08 ± 0.06 NDc ND

ND

L. acidophilus L10

4.93 ± 0.16** 4.42 ± 0.14** ND ND

ND

L. paracasei L26

4.77 ± 0.06* 4.49 ± 0.10** ND ND

ND


Significantly different from the control mice *P < 0.05; **P < 0.01.

aDetection limit was 102 CFU g-1

bDetection limit was 102 CFU ml-1

cNot detected.

81

Table 3.3. Translocation of total anaerobes to Peyer’s patches, mesenteric lymph nodes (MLN), spleen, liver and blood in mice orally fed with

either L. acidophilus L10 or L. paracasei L26 for 14-days.

Organ


Bloodb

Control

5.01 ± 0.12 4.24 ± 0.04 NDc ND

ND

L. acidophilus L10

5.36 ± 0.19* 4.51 ± 0.02*, # ND ND

ND

L. paracasei L26

5.15 ± 0.12 4.69 ± 0.03*, # ND ND

ND


Significantly different from the control mice *P < 0.01.

Significant differences between L. acidophilus and L. paracasei experimental groups #P < 0.01.



cNot detected.

82

Table 3.4. Translocation of enteric bacteria to Peyer’s patches, mesenteric lymph nodes (MLN), spleen, liver and blood in mice orally fed with

either L. acidophilus L10 or L. paracasei L26 for 14-days.

Organ


Bloodb

Control

NDc ND ND ND

ND

L. acidophilus L10

ND ND ND ND

ND

L. paracasei L26

ND ND ND ND

ND




cNot detected.

83

3.5. DISCUSSION

The external secretions of IgA in GI tract play an active role in protecting the

surfaces of mucus membrane from pathogenic infections and carcinogens (Mestecky

and McGhee, 1987). The formation of immune complexes in intestinal lumen to bind

antigens was the protective effect of antigen-specific secretory IgA, which interferes

with the adherence of pathogenic microorganisms to intestinal epithelial cells and

also helps in neutralising toxins and viruses (Mestecky et al., 1999). In the current

study, L. acidophilus and L. paracasei strains enhanced the number of IgA producing

cells in the small intestine of mice (Fig. 3.1A, B), which suggest that these strains

could protect the GI tract from pathogenic microorganisms through increased IgA

response. In comparison to L. paracasei, L. acidophilus showed an increased number

of IgA producing cells in the lamina propria that could be beneficial to the host in

conferring greater protection against pathogenic microorganisms. Earlier, it was

reported that peptides derived from L. helveticus fermented milk enhanced the IgA

secretion in gut and systemic immunity in mice challenged with E. coli O157:H7

(Leblanc et al., 2004).

The Th1 cells produce important factors such as IL-2, IFN-� and tumor necrosis

factor for cell-mediated immunity. Cytokines like IL-4, IL-5 and IL-10 associated

with humoral immunity and allergic responses are produced by Th2 cells (Kidd,

2003). The Th1 cytokine, IFN-� is a multifunctional pro-inflammatory cytokine that

triggers innate immune responses such as phagocytosis and antimicrobial activity,

whereas Th2 cytokine, IL-10 regulates a number of events including IFN-� and

antibody production (Kidd, 2003). Both IL-10 and IFN- � were considered as pivotal

cytokines in inducing Th1 and Th2 responses (Perdigon et al., 2002). In this study, L.

84

acidophilus and L. paracasei strains were effective in activating mucosal and

systemic immune responses through the release of IL-10 and IFN-� cytokines (Fig.

3.2; 3.3; 3.4). Perdigon et al. (1990) reported that mice fed with fermented milk

consisting of LAB were protected against Salmonella Typhimurium infection.

Furthermore, Gould and Sonnenfeld, (1987) demonstrated that mice pre-treated with

IFN-� were also protected against Salmonella Typhimurium strain LT-2. In the

present study, increase of IFN-� producing cells in the intestinal mucosa of mice fed

with L. acidophilus or L. paracasei could therefore contribute to the protection of

mucosal surfaces against harmful pathogenic microorganisms.

Lymphocytes play an important role in the regulation of immune responses. T-

lymphocytes are the mediators of cellular immunity, whereas B-lymphocytes

develop into antibody producing cells in response to foreign antigens. In the current

study, mitogen-stimulated lymphocyte proliferative responses were used to asses the

T- and B-cell functions. Mice fed with L. acidophilus or L. paracasei exhibited the

enhancement of splenocyte proliferative responses to T-cell mitogen (ConA) and B-

cell mitogen (LPS). Although, LPS stimulated B-cell proliferative responses in L.

paracasei-fed mice was higher, but not significantly different from the control mice

(Table 3.1). The present study demonstrated the differences in mitogen-stimulated

proliferative responses of splenocytes, which could be due to strain-specific

variations. Kirjavainen et al. (1999) demonstrated similar strain-specific effects of

mitogen-stimulated splenocyte proliferative responses of T- and B-lymphocytes.

It is widely known that the balance between Th1 and Th2 responses was important to

elevate immunological responses against various diseases. Lactic acid bacteria are

85

known to modulate the balance of Th1 and Th2 immune responses (Maassen et al.,

2000; Perdigon et al., 2002). Human volunteers fed with lyophilised lactobacilli

showed an increased production of IFN-� in serum (De Simone et al., 1993). Schultz

et al. (2003) reported that oral administration of L. rhamnosus GG to healthy human

volunteers stimulated an increased secretion of anti-inflammatory cytokines (IL-10

and IL-4) and decreased secretion of pro-inflammatory cytokines (IFN-�, TNF-� and

IL-6). In the present study, IL-10 was significantly increased in serum as well as in

splenocytes stimulated with ConA of mice fed with L. acidophilus or L. paracasei.

The magnitude in the increment of IL-10 was higher than Th1 cytokine IFN-�, which

may be necessary to control the increase of the pro-inflammatory cytokine IFN-�

(Fig. 3.3; 3.4).

In the current study, splenocytes from mice fed with L. acidophilus or L. paracasei

significantly increased the production of IL-10 and IFN-� cytokines (Fig. 3.4A, B).

Roller et al. (2004) reported similar enhancement of IL-10 and IFN-� production in

Peyer’s patches of rats fed with synbiotics (Probiotics and prebiotics). In another

study, CD4+ T-cells from Peyer’s patches of mice fed with dietary

fructooligosaccharides showed dose-dependent increased production of IL-10 and

IFN-� levels (Hosono et al., 2003). The current study demonstrates that L.

acidophilus and L. paracasei were able to influence the regulation of Th1 and Th2

cytokines in gut and systemic immunity through increased production of IL-10 and

IFN-� (Fig. 3.2; 3.3; 3.4). In contrast, L. acidophilus-fed mice stimulated higher

levels of IL-10 and IFN-� compared to the increase detected in mice fed with L.

paracasei.

86

The influence of LAB on phagocytosis could be strain-specific, dose dependent or

survivability in the GI tract (Gill et al., 2000; Gill and Rutherfurd, 2001; Medici et

al., 2005). The differences in cell surface structures of Lactobacillus strains lead to

strain-specific variations in modulating immune responses (Sato et al., 1988). The

ability of LAB in stimulating phagocytic cells also depends on its survival and

persistence in GI tract and their adherence to intestinal epithelial cells (Perdigon and

Alvarez, 1992; Schiffrin et al., 1997). In the present study, orally administered L.

acidophilus or L. paracasei strains enhanced the phagocytic activity of peritoneal

macrophages (Fig. 3.5A, B), which suggests that these strains are tolerant to gastric

conditions of GI tract and can persist through the GI passage to induce host

immunological responses. Mice fed with L. acidophilus showed an increase of 21%

activation of peritoneal macrophages compared to control mice, whereas L.

paracasei-fed mice showed only 6% activation. However, there were no significant

differences observed between L. acidophilus and L. paracasei in inducing

macrophage activity.

Gut mucosal integrity is crucial in host immune defence mechanisms, such as

protection against pathogenic bacteria through interfering their adherence to

intestinal epithelial cells (Mestecky et al., 1999). Translocation of indigenous

microflora to distant intestinal sites such as spleen, liver and blood could occur due

to intestinal inflammation that causes the disruption of intestinal mucosal surfaces.

Vinderola et al. (2004) reported the translocation of various doses of different LAB

to liver, which activated gut and systemic immune responses. In the present study,

there was no evidence of translocation of intestinal microflora to spleen, liver or

blood in both the experimental group mice (Table 3.2; 3.3), which suggests that oral

87

administration of L. acidophilus or L. paracasei at 108 CFU/day did not induce

adverse effects such as disruption of immune defence mechanisms that leads to

inflammation of intestinal mucosal layers and bacterial translocation to sterile

organs.

The absence of bacterial translocation to spleen, liver or blood also demonstrates the

active role of immune defence mechanisms that could have inhibited the

translocation beyond MLN. Additionally, enteric bacteria were not detected in

Peyer’s patches, MLN, spleen, liver and blood either in control or experimental

group mice (Table 3.4).

Systemic dissemination of bacteria in mice has been correlated with the adhesion of

probiotic bacteria to intestinal epithelial cells (Wagner et al., 1997). Earlier reports

suggested that adhesion of bacterial strains to gut epithelial cells is necessary to

modulate immune responses (Perdigon and Alvarez, 1992; Schiffrin et al., 1997). In

the present study, L. acidophilus-fed mice showed increased translocation of

Lactobacillus spp. to Peyer’s patches (7%) and MLN (8%) compared to control

mice, whereas L. paracasei-fed mice demonstrated the translocation of Lactobacillus

spp. with an increase of 3% to Peyer’s patches and 10% to MLN (Table 3.2).

However, increased translocation of Lactobacillus spp. in mice fed with L.

acidophilus or L. paracasei were statistically significant compared to control mice

but showed very small differences. Similar small differences were also observed in

the translocation of total anaerobes to Peyer’s patches and MLN in both the

experimental groups with respect to control mice (Table 3.3). The results of this

study suggest that L. acidophilus and L. paracasei have potential adhesive properties

88

that can lead to the translocation of Lactobacillus spp. to Peyer’s patches and MLN,

which may contribute to the enhancement of various systemic immune functions.

89

3.6. CONCLUSIONS

Orally administered L. acidophilus and L. paracasei demonstrated the enhancement

of gut mucosal immunity in relation to the increase of IgA and cytokines (IL-10 and

IFN-�). In systemic immunity, L. acidophilus and L. paracasei enhanced the

phagocytic activity of peritoneal macrophages and release of IL-10 and IFN-�

cytokines from serum and splenocytes. In contrast, L. acidophilus showed higher

stimulation of gut and systemic immune responses compared to L. paracasei.

Additionally, there was no evidence of bacterial translocation to spleen, liver or

blood in mice fed with either L. acidophilus or L. paracasei. Therefore, L.

acidophilus and L. paracasei strains are considered non-pathogenic, as indicated by

their effects on bacterial translocation in healthy mice.

90

CHAPTER 4

Immunostimulatory responses of L. acidophilus LAFTI L10 and L. paracasei

LAFTI L26 in cholera toxin mice

91

4.1. ABSTRACT

Mice were orally fed with either L. acidophilus or L. paracasei for 14-days, during

which they were immunized twice with 10 �g of cholera toxin (CT). Lactobacillus

acidophilus-fed mice showed an increase (P < 0.01) in serum nitric oxide (NO)

levels. The increment of NO levels in intestinal fluid and serum of L. paracasei-fed

mice was significantly different compared to control mice. Immunoglobulin (Ig)-A

response to CT in intestinal fluid and serum were significantly enhanced by L.

acidophilus or L. paracasei. Cytokine producing cells in small intestine of mice fed

with either L. acidophilus or L. paracasei demonstrated the regulation of Th1 and

Th2 cytokines such as interferon (IFN)-�, tumor necrosis factor (TNF)-�, interleukin

(IL)-4, IL-6 and IL-10. The results of this study demonstrate that L. acidophilus and

L. paracasei are capable of enhancing gut immune responses as well as serum NO

and IgA in systemic levels of CT immunized mice.

92

4.2. INTRODUCTION

Gastrointestinal (GI) tract is considered to be a complex ecosystem consisting of

various resident microorganisms such as Escherichia coli, Clostridium spp.,

Streptococcus spp., Lactobacillus spp., Bacteroides spp., and Bifidobacterium spp.,

which are involved in the development of gut microflora and maintains host health

by exerting specific gut functions (McCracken and Lorenz, 2001). The microbial

population in GI tract, specifically, Lactobacillus and Bifidobacterium spp. were

considered as probiotic due to their beneficial health effects such as inhibiting the

growth of Escherichia coli (Forestier et al., 2001), Helicobacter pylori (Wang et al.,

2004) and resistance to diseases (Shanahan, 2002; de Waard et al., 2003).

Microorganisms of GI tract have the potential to influence immunoglobulin (Ig)-A

production for the development and activation of gut immune system (Dogi and

Perdigon, 2006). Furthermore, commensal bacteria also help in promoting the

development of B-cell population in Peyer’s patches that were not developed in

germ-free animals. In comparison to germ-free animals, IgA producing cells were

higher in intestinal lamina propria of animals possessing normal intestinal microflora

(Crabbe et al., 1968; Jiang et al., 2004). Due to the significance of IgA in host

immune functions, it is important to screen the capability of probiotic bacteria in

enhancing antibody response in gut and systemic immunity.

Oral administration of appropriate microbial strains could potentially restore and

maintain oral tolerance and host immune functions (Sudo et al., 1997; Kalliomaki et

al., 2001). Korhonen et al. (2001) reported that L. rhamnosus GG induced the

production of nitric oxide (NO), which played a functional role in protective

mechanisms of intestinal mucosa and regulating immune functions. In rats, dietary

93

supplementation of food with lactobacilli and nitrate increased the NO levels in small

intestine and caecum (Sobko et al., 2006). The endogenous NO was known to have

potential role in regulating various host functions including water and electrolyte

transport (Mourad et al., 1999) and motility (Mashimo et al., 2000). Scientific reports

speculated that some of the beneficial effects exerted by probiotic bacteria were

mediated through NO release in intestinal lumen (Xu and Verstraete, 2001; Lamine

et al., 2004; Sobko et al., 2005). Sobko et al. (2005) suggested the relationship

between increased number of intestinal microflora with higher NO levels in the

intestine of healthy new born infants. The source of NO detected in the lumen of new

born infants however remains unclear.

Gastrointestinal epithelial cells act as physical and chemical barriers by protecting

the host from harmful microorganisms that could hijack the regulation of cellular

molecules and signalling pathways (Cossart, 1997; Finlay and Falkow, 1997).

Epithelial cells of GI tract were considered important in mucosal immune system due

to their interdependency on adjacent lymphoid cells (Shanahan, 1999). Cytokines

such as interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-� were few of the

several cytokines secreted by intestinal epithelial cells (McCracken and Gaskins,

1999). Enhanced secretory antibodies and variations in mucous layers along the

intestine could be crucial in strengthening the intestinal barrier of GI tract (Yuan and

Walker, 2004). Earlier studies reported the capability of probiotic bacteria in

normalising and reinforcing gut barrier functions and protecting the host from

pathogenic bacteria (Terpend et al., 1999; Madsen et al., 2001).

94

Cholera toxin (CT), the enterotoxin of Vibrio cholerae, which affects epithelial cells

by inducing massive salt and water secretion results in severe diarrhoea. The toxin is

considered as a potent adjuvant and immunogen that can influence mucosal and

systemic immune functions (Elson and Ealding, 1984). Several scientific reports

demonstrated CT as a mucosal adjuvant by enhancing IgA and IgG immune

responses, increasing T-cell antigen priming and inhibiting oral tolerance to

unrelated antigens (Elson and Ealding, 1984; Lycke et al., 1985; Hornqvist et al.,

1991). Mucosal adjuvants are also known to activate immune responses by

stimulating cytokines that are involved in innate and acquired immunity (Elson and

Dertzbaugh, 1994). The degradation of orally administered antigens could occur in

GI tract due to constant changes such as low gastric pH and high bile salts, which

further may lead to either ineffective or minimal immune responses. Considerable

attention was given to lactic acid bacteria as an adjuvant due to their general use as

safe food grade microorganisms in the manufacturing of fermented dairy products.

Several studies have shown the differences between LAB strains in inducing

adjuvant effects when delivered orally with antigens (McCracken and Gaskins, 1999;

Tejada-Simon et al., 1999a; Plant and Conway, 2002).

As shown in chapter 3, L. acidophilus LAFTI L10 and L. paracasei LAFTI L26

demonstrated the stimulation of various immune functions in gut and systemic

immunity. Particularly, L. acidophilus L10 demonstrated higher stimulation of gut

and systemic immune responses with respect to L. paracasei L26. However, it is

unclear whether L. acidophilus L10 and L. paracasei L26 have the potential to alter

the regulation of B- and T-cell functions after oral immunisation with an antigen in

BALB/c mice. Therefore, present study aimed to use CT as an oral antigen to

95

determine the immune responses enhanced by oral administration of L. acidophilus

L10 and L. paracasei L26 in mice. The effects of L. acidophilus L10 and L.

paracasei L26 on various indices of immune functions such as NO, CT-specific IgA

response and Th1/Th2 cytokine producing cells in the small intestine and serum NO

and CT-specific IgA levels were evaluated.

96


4.3.1. Mice

Male 6-week old BALB/c mice were purchased from Biological Resources Centre,

The University of New South Wales, Sydney, Australia. Mice were housed at 22 ±

1°C under 12 h light-dark photoperiod and fed with standard mouse chow (Gordon’s

Speciality Stock Feeds, Sydney, Australia) and provided ad libitum access to water.

Control and experimental groups consisted of six mice each, which were randomly

allocated. All experiments were performed with the approval from Animal Care and

Ethics Committee of University of Western Sydney, Sydney, Australia.

4.3.2. Lactobacillus strains and feeding procedure

Lactobacillus acidophilus LAFTI L10 and Lactobacillus paracasei LAFTI L26 used

in this study were obtained from DSM culture collection (DSM Food Specialties

Ltd., Sydney, Australia). The bacterial strains were grown under anaerobic

conditions in gas jars using GasPak System (Oxoid, Adelaide, Australia) at 37°C for

24 h in de Man, Rogosa and Sharpe (MRS) broth (Oxoid, Adelaide, Australia) and

washed twice with sterile 0.01 M phosphate buffered saline (PBS; Sigma-Aldrich,

Sydney, Australia) solution following centrifugation at 4000 x g for 15 min at 4°C.

The viability of bacteria was determined by spread plate count of bacterial serial

dilutions on MRS agar (Oxoid, Adelaide, Australia), which were incubated

anaerobically for 48 h at 37°C. After acclimatisation for 1 week, mice (six per group)

were fed daily by oral gavage with 108 colony-forming units (CFU) of either L.

acidophilus or L. paracasei in 50 �l of 10% (w/v) skimmed milk powder (SMP) for

14-days. Control group mice received 50 �l of SMP without lactic acid bacteria.

97

4.3.3. Cholera toxin immunisation

Control and experimental group mice were orally immunized with 10 �g of cholera

toxin (Sigma-Aldrich, Sydney, Australia) in 25 �l of 0.1 M sodium bicarbonate

(Sigma-Aldrich, Sydney, Australia) on day 0 and 7. Mice were sacrificed by carbon

dioxide inhalation after 14-day feeding trail and intestinal fluid, blood and small

intestines were collected.

4.3.4. Intestinal fluid and serum preparation

Intestinal fluid of small intestine in each mouse was collected by flushing the

intestinal contents with 2 ml of 0.01 M PBS and centrifuged at 10,000 x g for 15 min

at 4°C. The supernatant was collected and stored at -20°C until use.

Blood was collected upon euthanizing the mice and left to coagulate overnight at 4°C

and centrifuged at 2000 x g for 10 min (4°C). The serum was collected and stored at

-20°C until use.

4.3.5. Determination of nitric oxide

Nitric oxide was determined by measuring nitrite accumulation in intestinal fluid and

serum using Griess reagent system (Promega, Sydney, Australia). Briefly, 50 µl of

intestinal fluid supernatant or serum were added in triplicates to the wells, which

were incubated with 50 µl of sulfanilamide solution for 10 min at room temperature

while protected from light. After adding 50 µl of N-1-napthylethylenediamine

dihydrochloride solution to all the wells, absorbance was read at 540 nm using a

microtitre plate reader (Multiskan Titertek, Huntsville, USA).

98

4.3.6. Estimation of Immunoglobulin A

The CT-specific IgA antibodies in intestinal fluid and serum were detected by

ELISA. Briefly, each well of the 96-well microtitre plate was coated with 100 �l of

CT (10 �g/ml) in 0.05 M carbonate-bicarbonate buffer, pH 9.6. For standard, 100 �l

of goat anti-mouse IgA (Bethyl Laboratories Inc., Montgomery, USA) was added to

the wells in triplicates at 10 �g/ml in 0.05 M carbonate-bicarbonate buffer, pH 9.6.

The plates were incubated overnight at 4°C and washed three times with 0.01 M PBS

containing 0.05% Tween 20 (PBS-T; Sigma-Aldrich, Sydney, Australia). Then the

plates were incubated for 2 h at 37°C with either 100 �l of mouse IgA-kappa (Sigma-

Aldrich, Sydney, Australia) as a standard or 100 �l of intestinal fluid or serum, which

were added in triplicates. The plates were washed three times with PBS-T and

incubated with 100�l of goat anti-mouse IgA horseradish peroxidase conjugate

(Sigma-Aldrich, Sydney, Australia) for 90 min at 37°C. Incubation was followed by

washing the plates three times with PBS-T and 200�l of o-phenylenediamine

dihydrochloride peroxide substrate (Sigma-Aldrich, Sydney, Australia) was added to

the wells and incubated for 30 min at room temperature. The absorbance was read at

450 nm using a microtitre plate reader (Multiskan Titertek, Huntsville, USA).

4.3.7. Histological samples

Mice small intestines were removed, dissected into segments and stored in 95%

ethanol (v/v). The tissues were then embedded in paraffin blocks and the sections

were processed for immunofluorescence assays.

99

4.3.8. Immunofluorescent detection of cytokine producing cells

The number of cytokine secreting cells in small intestine of mice was determined by

indirect immunofluorescence assay. The paraffin sections (4-�m) were

deparaffinised in xylene and rehydrated in ethanol, which were then washed twice

with 0.01 M PBS solution and incubated with a blocking solution (0.01 M PBS

containing 1% bovine serum albumin) for 30 min at room temperature. The sections

were incubated with 0.2 �g/ml (diluted in 0.01 M PBS) of rabbit anti-mouse IL-4,

IL-6, IL-10, interferon (IFN)-� and TNF-� (Peprotech Inc., Rocky Hill, USA)

polyclonal antibodies for 75 min at 37°C. The incubation was followed by washing

the sections twice with 0.01 M PBS. The sections were then incubated with goat anti-

rabbit antibody conjugated with fluorescein isothiocyanate (FITC; Jackson Immuno

Research Labs Inc., West Grove, USA) for 45 min at room temperature and washed

twice with 0.01 M PBS. The number of fluorescent cells was counted by using

fluorescence light microscope (Olympus Optical Co., Tokyo, Japan) and the results

were expressed as number of positive fluorescent cells in 10 fields of vision.




Significant differences between groups were identified by Duncan’s multiple range

test (SPSS, Version 12.0.1).

100

4.4. RESULTS

4.4.1. Nitric oxide production in intestinal fluid and serum

The effect of orally administered Lactobacillus strains on NO levels was determined

by Griess reagent system. As shown in Fig. 4.1A, L. paracasei showed significant

increase (P < 0.01) of NO levels in intestinal fluid compared to control mice. Nitric

oxide levels in intestinal fluid of mice fed with L. acidophilus were also increased

but not significantly different when compared to control mice. The serum NO levels

were increased significantly (P < 0.01) in mice fed with either L. acidophilus or L.

paracasei (Fig. 4.1B). The differences between L. acidophilus and L. paracasei were

significant (P < 0.01) in enhancing NO levels in intestinal fluid and serum.

4.4.2. Immunoglobulin A antibodies in intestinal fluid and serum

The IgA antibodies specific to CT in mice fed with L. acidophilus or L. paracasei

were estimated by ELISA (Fig. 4.2). In intestinal fluid, CT-specific IgA antibodies

were increased significantly (P < 0.01) in mice fed with L. acidophilus or L.

paracasei compared to control mice (Fig. 4.2A). In systemic response, strong serum

IgA response to CT was detected in mice fed with L. acidophilus or L. paracasei,

which differed significantly (P < 0.001) from control mice (Fig. 4.2B). There were

no significant differences (P > 0.05) between L. acidophilus and L. paracasei in

inducing CT-specific IgA response in intestinal fluid. In contrast, L. acidophilus and

L. paracasei demonstrated the differences (P < 0.01) in enhancing CT-specific IgA

antibodies in serum.

101

A)

20

40

60

80

Inte

sti

nal

flu

id N

O (

µm

ol/

L)

Control

L10

L26

B)

10

20

30

40

Seru

m N

O (

µm

ol/

L)

Control

L10

L26

Figure 4.1. Nitric oxide production in A) intestinal fluid and B) serum of mice fed

with L. acidophilus L10 or L. paracasei L26. The results shown are mean ± standard

error of mean (n = 6). Mean values were significantly different in comparison with

the control mice *P < 0.01. Significant differences between L. acidophilus L10 and

L. paracasei L26 experimental groups #P < 0.01.

#

*

#

#

*

#

*

102

A)

15.0

17.5

20.0

Inte

sti

nal

flu

id I

gA

(µ

g/m

l)

Control

L10

L26

B)

0

4

8

12

16

Seru

m I

gA

(µ

g/m

l)

Control

L10

L26

Figure 4.2. Immunoglobulin (Ig)-A antibody response to cholera toxin in A)

intestinal fluid and B) serum of mice fed with L. acidophilus L10 or L. paracasei

L26. The results shown are mean ± standard error of mean (n = 6). Mean values were

significantly different in comparison with the control mice *P < 0.01; **

P < 0.001.

Significant differences between L. acidophilus L10 and L. paracasei L26


* *

#

** #

**

103

4.4.3. Detection of cytokine producing cells in small intestine

Cytokine producing cells in small intestine of mice fed with L. acidophilus or L.

paracasei were shown in Figures 4.3; 4.4. Th1 cytokine, IFN-� producing cells were

significantly increased in mice fed with L. acidophilus (P < 0.001) or L. paracasei (P

< 0.01) compared to control mice (Fig. 4.3A). The TNF-� was increased in mice fed

with L. acidophilus, but not significantly different from control mice. However,

TNF-� producing cells in L. paracasei-fed mice were significantly increased (P <

0.01) with respect to control mice (Fig. 4.3A).

In L. paracasei-fed mice, Th2 cytokine, IL-4 producing cells were enhanced (P <

0.01) when compared with control mice. Significant differences (P > 0.05) were not

detected in IL-4 producing cells when compared between mice fed with L.

acidophilus and control group (Fig. 4.3B). In comparison to control mice, IL-6

producing cells were increased in mice fed with either L. acidophilus (P < 0.05) or L.

paracasei (P < 0.001) (Fig. 4.3B). The IL-10 producing cells were also enhanced (P

< 0.01) in mice fed with L. acidophilus compared to control mice. The increase of

IL-10 producing cells of mice fed with L. paracasei did not show significant

differences (P > 0.05) when compared to control mice (Fig. 4.3B). There were no

significant differences (P > 0.05) between L. acidophilus and L. paracasei in

enhancing cytokines such as IFN-�, TNF-�, IL-4, IL-10 with exception to the

significant difference (P < 0.05) between L. acidophilus and L. paracasei in

increasing IL-6 cytokine producing cells in small intestine of mice.

104

A

0

15

30

45

60

75

IFN - � TNF - �

Ce

lls

/10

fie

lds

Control

L10

L26

Figure 4.3. (Continued)

*** **

**

105

0

25

50

75

100

IL - 4 IL - 6 IL - 10

Cell

s/1

0 f

ield

sControl

L10

L26

Figure 4.3. Effect of L. acidophilus L10 and L. paracasei L26 on (A) interferon (IFN)-� and tumor necrosis factor (TNF)-�; (B) interleukin (IL)-

4, IL-6 and IL-10 in the small intestine of mice. The results shown are mean ± standard error of mean (n = 6). Mean values were significantly

different in comparison with the control mice *P < 0.05; **P < 0.01; ***

P < 0.001. Significant differences between L. acidophilus and L. paracasei


B

#

*

#

***

**

**

106

A B

Figure 4.4. Histological sections of the small intestine in mice fed with L. acidophilus L10 showing A) interleukin (IL)-10 and B) interferon

(IFN)-� producing cells labelled with rabbit anti-mouse IL-10 and IFN-� respectively and secondary antibody as goat anti-rabbit conjugated with

FITC, which fluoresces. Magnification 400X.

107

4.5. DISCUSSION

The NO in human body acts as a mediator in regulating various physiological and

pathophysiological processes (Moncada and Higgs, 1995). In physiological levels, NO

was essential in protecting intestinal mucosa through regulating mucus, bicarbonate

secretion and maintaining mucosal blood flow (Hutcheson et al., 1990; Alican and

Kubes, 1996; Lefer and Lefer, 1999). McCafferty et al. (1997) suggested that NO was

important in resolving gut inflammation and may also protect the GI tract in

inflammatory mechanisms. Several scientific reports indicated the involvement of NO in

protecting against intestinal mucosal permeability associated with reperfusion of

postischemic intestine (Payne and Kubes, 1993), preventing mucosal damage (Conforti

et al., 1993), and regulating GI tract protective mechanisms (Korhonen et al., 2001;

Lamine et al., 2004).

Elahi et al. (2005) correlated the increase of NO and IFN-� in saliva with enhanced

clearance of Candida albicans from oral cavities of L. acidophilus L10-fed mice.

Interestingly, results of the current study showed the capability of L. acidophilus and L.

paracasei in inducing NO levels in intestinal fluid and serum with increased IFN-�

producing cells in small intestine (Fig. 4.1; Fig. 4.3A). However, L. acidophilus-fed

mice showed no significant differences in increasing NO levels in intestinal fluid

compared to control mice. In relation to inducing NO levels in GI tract, L. paracasei

could be considered as a potential enhancer compared to L. acidophilus. Moreover, both

Lactobacillus strains showed significant enhancement of serum NO levels in systemic

immune response.

108

Gastrointestinal immune system consists of Peyer’s patches, mesenteric lymph nodes,

immunocompetent cells of lamina propria and mucosal epithelium. Lamina propria and

intraepithelial spaces are the main effector sites in the intestine. In lamina propria,

Peyer’s patch derived B-cells differentiate into dimeric IgA secreting plasma cells,

which were transported across epithelial cells and released as secretory IgA. The

secretory IgA is necessary in host defence mechanisms such as antigen binding and

preventing the adherence of pathogenic bacteria to intestinal epithelial cells (Hudault et

al., 1997; Gopal et al., 2001; Shu and Gill, 2002). In this study, L. acidophilus and L.

paracasei significantly enhanced CT-specific IgA response in intestinal fluid and serum

(Fig. 4.2). Intestinal lymphoid organ, Peyer’s patches were located in small intestine that

was considered as principal inductive sites of immune responses upon oral

administration of an antigen. In contrast to large intestine, small intestine is anatomically

connected to systemic immune system through lymphatic and blood circulation by

which the immune responses induced in small intestine could further lead to distant

mucosal sites in systemic immunity.

In the current study, increase of CT-specific IgA response was observed in intestinal

fluid of small intestine and blood serum of mice fed with L. acidophilus or L. paracasei,

which suggests that the release of CT-specific IgA antibodies in small intestine could

possibly lead into blood circulation. Therefore, blood serum could be regarded as a

potential marker to study the immunomodulation of orally administered antigens.

Furthermore, enhancement of CT-specific IgA response in intestinal fluid also suggests

the potential capability of L. acidophilus and L. paracasei strains, which could protect

the GI tract towards pathogenic microbial infections (Macpherson et al., 2001).

109

Goodrich and McGee, (1999) reported the ability of IL-6 in inducing terminal

differentiation of B-lymphocytes to IgA secreting cells. However, B-cells switching and

differentiation leading to plasmocyte secreting IgA also occurs in high IL-4, IL-5 and

transforming growth factor (TGF)-� environment (Blum et al., 1999). Despite the

capability of macrophages and T-cells to produce IL-6, intestinal epithelial cells are also

known to produce IL-6 (Vitini et al., 2000). In the present study, L. acidophilus and L.

paracasei increased IL-6 producing cells in small intestine (Fig. 4.3B). The

enhancement of IL-6 producing cells is considered to have a potential role in

augmenting the CT-specific IgA response in intestinal fluid and serum (Fig. 4.2). The

increase of IL-6 and IgA levels observed in this study is consistent with the earlier

studies that reported similar effects of lactic acid bacteria in enhancing IL-6 as well as

IgA responses (Miettinen et al., 1996; Galdeano and Perdigon, 2006).

Regulatory cytokines such as IL-4 and IL-10 are associated with humoral and allergic

immune responses. The IL-4 cytokine plays an important role in cell growth and

regulation of immune response particularly in inhibiting cytokines such as IL-1, IL-6,

IL-8 and TNF-� (Feghali and Wright, 1997). In the current study, differences in

stimulating IL-6 cytokine producing cells in mice fed with L. acidophilus or L.

paracasei were observed (Fig. 4.3B). The analysis of cytokine producing cells in small

intestine of mice showed the capability of L. acidophilus and L. paracasei strains in

modulating Th2 regulatory cytokines, such as IL-10 that plays an important role in

chronic inflammatory bowel disease (Braat et al., 2003) and amelioration of colitis (Di

Giacinto et al., 2005).

110

In the present study, pro-inflammatory cytokines such as IFN-� and TNF-� were

increased in mice fed with L. acidophilus or L. paracasei (Fig. 4.3A). In comparison to

control mice, L. acidophilus induced TNF-� cytokine producing cells were not

significantly different. Despite the role of IFN-� in inflammatory response, it can also

act as an effector molecule in immune responses against solid cancers (Numata et al.,

1991; Belardelli and Ferrantini, 2002). Macrophages and dendritic cells of lamina

propria are known to be crucial in producing various cytokines such as IL-6, IL-10, IL-

12, IFN-� and TNF-� (Husband et al., 1999). However, mast cells are also considered

necessary in releasing cytokines such as IL-4 and TNF-� that are important in initiating

immune and inflammatory responses (Feger et al., 2002). These various cell populations

from innate immune system might have been the source of cytokine producing cells in

lamina propria of small intestine that were determined in this study. Additionally,

current study also showed simultaneous stimulation of pro-inflammatory and regulatory

cytokines in mice fed with L. acidophilus or L. paracasei, which could be considered

necessary for maintaining a chronic and immunological balanced intestinal

inflammatory response termed as physiological inflammation (Cebra et al., 2005).

111

4.6. CONCLUSIONS

The current study demonstrates that L. acidophilus and L. paracasei were capable of

inducing NO levels in intestinal fluid and serum. Particularly, L. paracasei-fed mice

showed higher NO levels in intestinal fluid compared to mice fed with L. acidophilus.

Lactobacillus acidophilus and L. paracasei strains were demonstrated as potential

inducers of CT-specific IgA antibodies in gut and systemic immunity. Especially, L.

acidophilus induced higher CT-specific IgA levels in serum compared to L. paracasei-

fed mice. The analysis of cytokines in small intestine of mice reported the capability of

L. acidophilus and L. paracasei in regulating Th1 and Th2 cytokines. Particularly,

differences between L. acidophilus and L. paracasei were observed in inducing IL-6

cytokine producing cells in small intestine of mice.

112

CHAPTER 5

Microarray analysis of L. acidophilus LAFTI L10 induced gene expression in the

small intestine of mice

113

5.1. ABSTRACT

The microflora of gastrointestinal (GI) tract is known to be involved in promoting health

and physiological functions of the host. The influence of L. acidophilus in regulating

gene expression in the small intestine of normal and cholera toxin (CT) mice was

examined in this study. In the first experiment, mice were fed daily with L. acidophilus

at 108 CFU for 14-days, whereas in the second experiment, mice were fed with L.

acidophilus at 108 CFU/day for 14-days, during which they were immunized on day 0

and 7 with 10 �g of cholera toxin. In both the experiments, mice were euthanized after

14-day feeding trail and small intestines were excised to determine the gene expression

patterns using mouse oligonucleotide microarray. Microarray data was generated by

comparing the influence of L. acidophilus in regulating gene expression with the

respective control group mice. In the first experiment, normal mice fed with L.

acidophilus demonstrated the regulation of genes with an up-regulation of 27 and down-

regulation of 35 genes involved in various biological and molecular functions such as

cell-cell signalling, cell growth and proliferation, cell death and various metabolic

functions. In CT mice model, L. acidophilus influenced the up- and down-regulation of

28 and 32 genes that are involved in various cell functions, drug metabolism, immune

responses and metabolic functions. The present study demonstrated the influence of L.

acidophilus in regulating gene expression in the small bowel of normal and CT mice.

Furthermore, this study also provides a basis to identify the gene targets that may

represent a reference for further studies to define the pathways through which these

bacteria influence the host.

114

5.2. INTRODUCTION

Probiotic bacteria are considered as beneficial microbes due to their potential with

respect to different ways in which they can contribute to the positive health of GI tract.

Probiotic strains such as Lactobacillus and Bifidobacterium spp. are predominantly

incorporated into fermented dairy products. The interactions of probiotic bacteria in GI

tract are believed to be necessary in promoting host biological functions. Several reports

also suggested the probiotic effects on respiratory, subcutaneous and urogenital tract

functions (Tagg and Dierksen, 2003; Sheil et al., 2004; Reid, 2005). The microflora of

GI tract interacts with mucosal cell population, which includes epithelial cells (Hooper

et al., 2001). Commensal microbial population that colonises the GI tract acts as a

barrier in protecting the intestinal mucosa against pathogens through various

mechanisms, such as occupation of niches, antimicrobial production and competition for

essential nutrients.

The probiotic responses of microorganisms induced on host could be due to either

microbe-microbe or microbial-host interactions. Probiotic bacteria play an important

role in host defence mechanisms through activation of mucosal immune responses

(Shanahan, 2002a). Previous studies reported the capability of probiotic bacteria in

promoting gut barrier functions, balance of Th1/Th2 cytokines, enhancement of host

immune responses and interactions with gut associated lymphoid tissues (Erickson and

Hubbard, 2000; Isolauri et al., 2001). Furthermore, several studies also reported the

beneficial effects of probiotic bacteria in breast cancer model (de Moreno de LeBlanc et

al., 2005), respiratory infections (Racedo et al., 2006; Villena et al., 2006) and

inflammatory bowel diseases (Madsen et al., 2001; Schultz et al., 2002).

115

Probiotic bacteria especially Lactobacillus strains were characterised due to their

production of lactic acid, where most of them are indigenous inhabitants of human GI

tract and colonise in higher populations in the small intestine. Orally administered

probiotic bacteria are inducers of immune responses through potential inductive sites

such as M cells or follicle associated epithelial cells of Peyer’s patches on small

intestine. The immune responses induced in small intestine can effectively reach

systemic immune response due to its anatomical connection through lymphatic and

blood circulation, whereas immune responses induced in large intestine were mostly

limited to this environment. Vinderola et al. (2006) suggested the importance of small

intestine in understanding the immunomodulatory properties of orally administered kefir

microflora in mice.

The molecular interactions of gut microflora with host specifically in relation to

probiotic bacteria were not well understood. Microarray technology is a powerful tool,

which offers a comprehensive view of biological systems through monitoring the

expression of thousands of genes simultaneously and rapidly (Wu et al., 2001;

Sepulveda et al., 2002). Microarray analysis of the gene expression in host is necessary

to define these probiotic microorganisms as beneficial bacteria, which also provides

further insights into the interactions of these microbes with epithelial cells of GI tract.

In the current study, L. acidophilus L10 demonstrated its potential probiotic properties

such as tolerance to simulated gastric juices particularly at pH 2.0, better survival at

various bile salt concentrations and coaggregation abilities with other bacterial strains

(Chapter 2). Lactobacillus acidophilus L10 also showed its potential role in stimulating

116

various gut and systemic immune functions, especially non-specific and specific

immune responses in normal and cholera toxin (CT) mice (Chapters 3 & 4). Therefore,

L. acidophilus L10 was chosen to elucidate its influence on gene expression in the small

intestine of mice and also to provide further insights on genes that were involved in

regulating various biological and molecular functions of the host. This study was aimed

to investigate the effects of L. acidophilus L10 on gene expression patterns in the small

intestine of normal and CT mice.

117


5.3.1. Animals

Male 6 - 8 wks old mice (BALB/c) were obtained from Biological Resources Centre,

The University of New South Wales, Sydney, Australia. Control and experimental

groups consisted of six mice each, which were caged separately at 22 ± 1°C and housed

under 12 h light-dark photoperiod. All mice were fed with standard mouse chow

(Gordon's Specialty Stock Feeds, Sydney, Australia) and provided ad libitum access to

water. All the protocols for the animal experiment were approved by the University of

Western Sydney Animal Care and Ethics Committee.

5.3.2. Lactic acid bacteria

Lactobacillus acidophilus LAFTI L10 used in this study was obtained from DSM

culture collection (DSM Food Specialties Ltd., Sydney, Australia). Lactobacillus

acidophilus was grown anaerobically in gas jars using GasPak System (Oxoid, Adelaide,

Australia) for 24 h at 37°C in de Man, Rogosa and Sharpe (MRS) broth (Oxoid,

Adelaide, Australia). The bacterial cells were harvested at 4000 x g for 15 min at 4°C

and washed twice with sterile 0.01 M phosphate buffered saline (PBS) solution.

5.3.3. Experimental design

Two experiments were conducted. In the first experiment, mice (n = 6) were fed daily by

oral gavage with 108 CFU of L. acidophilus in 50 �l of 10% (w/v) skimmed milk

118

powder (SMP) for 14-days. Control group mice received 50 �l of SMP without lactic

acid bacteria.

In the second experiment, mice (n = 6) were fed daily by oral gavage with 108 CFU of L.

acidophilus in 50 �l of 10% (w/v) SMP for 14-days. Control group mice received 50 �l

of SMP without lactic acid bacteria. Experimental and control group mice were orally

immunized with 10 �g of cholera toxin (Sigma-Aldrich, Sydney, Australia) in 25 �l of

0.1 M sodium bicarbonate (Sigma-Aldrich, Sydney, Australia) on day 0 and 7.

The viability of bacteria was determined by spread plate count of bacterial serial

dilutions on MRS agar (Oxoid, Adelaide, Australia), which were incubated

anaerobically in gas jars using GasPak System at 37°C for 48 h. In both the experiments,

mice were euthanized by carbon dioxide inhalation after 14-day feeding trail and small

intestines were removed for gene expression analysis.

5.3.4. RNA isolation

Total RNA was isolated from the small intestine of each mouse using TRIzol reagent

(Invitrogen, Melbourne, Australia) according to the manufacturer’s instructions. The

total RNA was further purified by RNeasy mini kit (Qiagen, Melbourne, Australia). The

purity and concentration of RNA was determined by spectrophotometer readings at 260

and 280 nm. The RNA integrity was confirmed by agarose gel electrophoresis. For

microarray analysis, equal amounts of RNA was pooled from all the mice (n = 6) of

experimental and control groups.

119

5.3.5. Microarray analysis

Microarray analysis was performed with RNA extracted from the experimental and

control groups using a compugen mouse oligonucleotide microarray consisting of

22,000 probes. A list of oligonucleotides immobilized on the array can be obtained from

the web-site: http://www.microarray.adelaide.edu.au/libraries.

5.3.6. Synthesis of cRNA probes

The total RNA pooled from individual experimental and control group mice was

amplified using a SuperScript Indirect RNA Amplification System (Invitrogen,

Melbourne, Australia) according to the manufacturer’s instructions. The purified cRNA

was dried under reduced pressure, dissolved in 9 µl of 0.1 M NaHCO3 (pH 9.0), mixed

with Cy3 or Cy5 and left in the dark to couple at room temperature for 60 min. The

labelled cRNA was mixed with 41 µl of Milli Q water and purified using a QIAquick

PCR purification kit (Qiagen, Melbourne, Australia). The purified fluorescent cRNA

samples were eluted into a clean tube with 90 µl of Milli Q water and dried under

reduced pressure.

5.3.7. Hybridisation of probes to oligonucleotide microarray

Prior to hybridization, each microarray slide was immersed in 50 ml of hot (60-95˚C)

Milli-Q water for 1 min, then each array was dried by centrifugation at 650 x g for 5

min. The labelled cRNA probes were then mixed with 0.64 µl of 25 mg/ml yeast tRNA,

4 µl of 2 mg/ml poly A and 20 µl of 1 mg/ml mouse Cot-1 DNA, dried under reduced

120

pressure, resuspended in 16 µl of formamide and 16 µl of 6.25 X SSC, denatured by

heating to 100˚C for 3 min and transferred directly to ice, followed by addition of 0.5 µl

of 10% SDS. The probes were then applied to the array and incubated at 42˚C overnight

in a humidified chamber. The arrays were washed in 0.5 X SSC containing 0.01% SDS

for 5 min, 0.5 X SSC for 3 min and rinsed in 0.2 X SSC for 3 min. The slides were dried

by centrifugation at 650 x g for 5 min and were then scanned using an Axon 4000B

microarray scanner. Data analysis was performed by using the single image tif files.

5.3.8. Data Analysis

The Cy5 and Cy3 fluorescent signal intensity of each gene on the array was extracted

using SPOT software (CSIRO, Australia). After the subtraction of morphological

background fluorescence, ratio of signal intensities (Cy5/Cy3) was calculated. Each

probe ratio was log 2 transformed and normalised using a LOWESS algorithm (Locally

weighted scatterplot smoothing) within each slide using LIMMA (Linear models for

microarray analysis). Between the arrays, normalisation was performed to have the log-

ratios with same median-absolute-deviation (MAD) across all arrays. Linear modelling

combined with Bayesian statistical analysis of the normalised data produced a ranked

list of genes that were likely to be consistently differentially expressed on all 4 arrays.

121

5.4. RESULTS

In both the experiments, microarray analysis of gene expression in the small bowel of

mice was analysed using oligonucleotide microarray consisting of 22,000 genes.

Analysis of data from arrays ranked the genes in order of likelihood of being

differentially expressed. In both the experiments, top 100 genes that were likely to be

differentially expressed were selected and their functional and pathway analysis were

determined through Ingenuity Pathways Analysis (Ingenuity systems,

www.ingenuity.com).

In the first experiment, 62 of the top 100 genes were mapped to Ingenuity Pathways

Knowledge Base associated with known biological functions. In 62 genes, 27 and 35

genes were up- and down-regulated (Table 5.1). Orally administered L. acidophilus in

normal mice altered the expression of genes that are involved in various biological and

molecular functions such as cell-cell signalling, cell growth, molecular transport, tissue

development, immune response and various metabolic functions (Table 5.2; 5.3). Genes

such as MAP4K1, H3 histone family 3A and HSPA1A, which are involved in p38

MAPK signalling, TGF-� signalling and ERF/MAPK signalling pathways were up-

regulated in L. acidophilus-fed mice (Table 5.4).

Genes related to cytochrome P450 family (CYP2B6, CYP2C8, CYP2C19) that are

involved in linoleic acid, fatty acid and arachidonic acid metabolic pathways were

down-regulated by L. acidophilus. Several other genes such as fibroblast growth factor

19 (FGF signalling), carboxyl ester lipase (Bile acid metabolism), AMY2A and ENPP3

122

(Starch and sucrose metabolism) and glutathione S-transferase A5 (Glutathione

metabolism) were also down-regulated in L. acidophilus-fed mice (Table 5.4).

123

Table 5.1. Effect of L. acidophilus L10 on gene expression (Up- and down-regulated) in the small intestine of normal mice.

GenBank Gene name

Fold change

AK014713 ATP-binding cassette, sub-family A (ABC1), member 12 (ABCA12) 1.4 �

BC006756 Ankyrin repeat and BTB (POZ) domain containing 2 (ABTB2) 1.5 �

NM_013778 Aldo-keto reductase family 1, member C13 (AKR1C13) 1.6 �

NM_009669 Amylase, alpha 2A (pancreatic) (AMY2A) 2.1 �

NM_020581 Angiopoietin-like 4 (ANGPTL4) 1.5 �

NM_019870 ARD1 homolog A, N-acetyltransferase (S. cerevisiae) (ARD1A) 1.4 �

NM_009730 Attractin (ATRN) 1.3 �

AK007010 Antizyme inhibitor 1 (AZIN1) 1.5 �

NM_009885 Carboxyl ester lipase (bile salt-stimulated lipase) (CEL) 2.8 �

NM_009887 Cerberus 1, cysteine knot superfamily, homolog (Xenopus laevis) (CER1) 1.4 �

AK003088 Carboxypeptidase A1 (pancreatic) (CPA1) 2.2 �

NM_019696 Carboxypeptidase X (M14 family), member 1 (CPXM1) 1.4 �

NM_025583 Chymotrypsinogen B1 (CTRB1) 2.4 �

AK007772 Chymotrypsin C (CTRC) 1.9 �

NM_023182 Chymotrypsin-like (CTRL) 2.0 �

NM_008411 CUB and zona pellucida-like domains 1 (CUZD1) 1.5 �

NM_011339 Chemokine (C-X-C motif) ligand 15 (CXCL15) 1.4 �

NM_009998 Cytochrome P450, family 2, subfamily B, polypeptide 6 (CYP2B6) 1.4 �

NM_010003 Cytochrome P450, family 2, subfamily C, polypeptide 19 (CYP2C19) 1.4 �

AK008688 Cytochrome P450, family 2, subfamily C, polypeptide 8 (CYP2C8) 1.4 �

AK018047 EDAR-associated death domain (EDARADD) 1.5 �

M27347 Elastase 1, pancreatic (ELA1) 1.8 �

NM_026419 Elastase 3B, pancreatic (ELA3B) 2.8 �

BC006944 Ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) 1.3 �

NM_008003 Fibroblast growth factor 19 (FGF19) 1.3 �

124


GenBank Gene name

Fold change

NM_007989 Forkhead box H1 (FOXH1) 1.4 �

NM_025989 Glycoprotein 2 (zymogen granule membrane) (GP2) 1.5 �

NM_008182 Glutathione S-transferase A5 (GSTA5) 1.7 �

NM_008210 H3 histone, family 3A (H3F3A) 1.4 �

M34857 Homeobox B9 (HOXB9) 1.4 �

M12571 Heat shock 70kDa protein 1A (HSPA1A) 1.6 �

BC006780 Keratin 5 (KRT5) 1.4 �

X53632 Lysozyme (renal amyloidosis) (LYZ) 1.3 �

Y09010 Mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) 1.6 �

NM_020575 Membrane-associated ring finger (C3HC4) 7 (MARCH7) 1.5 �

NM_008610 Matrix metallopeptidase 2 (MMP2) 1.6 �

NM_020276 Nasal embryonic LHRH factor (NELF) 1.4 �

NM_009513 Neurensin 1 (NRSN1) 1.7 �

NM_011864 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2) 1.4 �

NM_021543 Protocadherin 8 (PCDH8) 1.4 �

NM_013750 Pleckstrin homology-like domain, family A, member 3 (PHLDA3) 1.4 �

NM_011107 Phospholipase A2, group IB (pancreas) (PLA2G1B) 1.4 �

NM_026925 Pancreatic lipase (PNLIP) 1.9 �

NM_018874 Pancreatic lipase-related protein 1 (PNLIPRP1) 2.5 �

AK004304 Prickle-like 1 (Drosophila) (PRICKLE1) 1.4 �

AB009661 Protease, serine, 2 (trypsin 2) (PRSS2) 2.5 �

NM_023333 Protease, serine, 3 (mesotrypsin) (PRSS3) 1.9 �

NM_008941 Protease, serine, 7 (enterokinase) (PRSS7) 1.5 �

D13904 Protein tyrosine phosphatase, receptor type, D (PTPRD) 1.3 �

NM_009042 Regenerating islet-derived 1 alpha (REG1A) 1.6 �

125


GenBank Gene name

Fold change

NM_011271 Ribonuclease, RNase A family, 1 (pancreatic) (RNASE1) 1.6 �

NM_009081 Ribosomal protein L28 (RPL28) 1.4 �

NM_009091 Ribosomal protein S15 (RPS15) 1.4 �

NM_009789 S100 calcium binding protein G (S100G) 1.7 �

D83146 Sine oculis-related homeobox 5 homolog (Drosophila) (SIX5) 1.4 �

NM_019481 Solute carrier family 13 (sodium/sulfate symporters), member 1 (SLC13A1) 1.4 �

AF026489 Spectrin, beta, non-erythrocytic 2 (SPTBN2) 1.4 �

AK003083 Syncollin (SYCN) 2.1 �

S76673 Transcription factor binding to IGHM enhancer 3 (TFE3) 1.4 �

NM_009363 Trefoil factor 2 (spasmolytic protein 1) (TFF2) 1.6 �

AK016603 Transketolase-like 2 (TKTL2) 1.6 �

BC006649 Ubiquitination factor E4A (UFD2 homolog, yeast) (UBE4A) 1.5 �

126

Table 5.2. Functional analysis of differentially expressed genes (Up-regulated) after oral administration of L. acidophilus L10 in

normal mice.

Functions & Diseases Genes, n Functions & Diseases Genes, n

Small molecule biochemistry 5 Tissue development 2 Genetic disorder 4 Amino acid metabolism 1 Cell-cell signalling and interaction 3 Carbohydrate metabolism 1 Hematological system development and function 3 Cardiovascular disease 1 Cell signalling 2 Cell death 1 Cellular assembly and organisation 2 Cell morphology 1 Cellular development 2 Connective tissue disorders 1 Cellular growth and proliferation 2 Endocrine system disorder 1 Developmental disorder 2 Gene expression 1 DNA replication, recombination and repair 2 Immune response 1 Embryonic development 2 Infectious disease 1 Immune and lymphatic development and function 2 Inflammatory disease 1

Lipid metabolism 2 Metabolic disease 1 Molecular transport 2 Skeletal and muscular development and function 1 Post-translational modification 2 Viral function 1

127

Table 5.3. Functional analysis of differentially expressed genes (Down-regulated) after oral administration of L. acidophilus L10 in

normal mice.


Small molecule biochemistry 14 Skeletal and muscular disorders 2 Lipid metabolism 6 Tissue morphology 2 Molecular transport 6 Cardiovascular disease 1 Protein degradation 5 Cell cycle 1 Drug metabolism 4 Cell morphology 1 Cancer 3 Cell-cell signalling and interaction 1 Cellular movement 3 Cellular assembly and organisation 1 Organismal development 3 Cellular growth and proliferation 1 Vitamin and mineral metabolism 3 Connective tissue development and function 1 Amino acid metabolism 2 Connective tissue disorders 1 Carbohydrate metabolism 2 Digestive system development and function 1 Cellular development 2 Endocrine system disorders 1

Developmental disorder 2 Gastrointestinal disease 1 Embryonic development 2 Hematological system development and function 1 Endocrine system development and function 2 Hepatic system disease 1 Genetic disorder 2 Organ development 1 Inflammatory disease 2 Organismal injury and abnormalities 1 Organ morphology 2 Skeletal and muscular development and function 1 Post-translation modification 2 Tissue development 1 Respiratory disease 2 Tumor morphology 1

128

Table 5.4. Pathway analysis of differentially expressed genes (Up- and down-regulated) after oral administration of L. acidophilus L10

in normal mice.

Canonical Pathway Analysis

Up-regulation

Genes, n Down-regulation Genes, n

p38 MAPK signalling 2 Linoleic acid metabolism 4

TGF-� signalling 1 Xenobiotic metabolism signalling 4

SAPK/JNK signalling 1 Arachidonic acid metabolism 3

ERF/MAPK signalling 1 Fatty acid metabolism 3

Wnt/�-catenin signalling 1 Glycerolipid metabolism 3

Tryptophan metabolism 3

Starch and sucrose metabolism 2

Bile acid metabolism 1

FGF signalling 1

Glutathione metabolism 1

Glycerophospholipid metabolism 1

Pentose phosphate metabolism 1

Protein ubiquitination pathway 1

Riboflavin metabolism 1

Sulphur metabolism 1

129

In the second experiment, regulation of gene expression by L. acidophilus in the small

intestine of CT mice model was examined. Sixty of the top 100 genes were mapped to

the Ingenuity Pathways Knowledge Base associated with known biological functions. In

60 genes, 28 and 32 genes were up- and down-regulated (Table 5.5). Lactobacillus

acidophilus influenced the expression of genes that are involved in various cell functions

such as cell death, cellular movement, cell cycle, cellular growth and proliferation.

Several other genes regulated are also involved in functions related to molecular

transport, immune responses, immunological and inflammatory diseases, gastrointestinal

diseases and various metabolic functions (Table 5.6; 5.7).

Genes related to cytochrome P450 family (CYP2C9, CYP2C19, CYP2C38, CYP3A5)

that are involved in various metabolic pathways such as arachidonic acid metabolism,

fatty acid metabolism, linoleic acid metabolism and tryptophan metabolism were up-

regulated in the small bowel of L. acidophilus-fed mice (Table 5.8). Additionally, genes

involved in oxidative phosphorylation such as COX7C, NDUFC1, NDUFC2, PPA1 and

UCRC were up-regulated in mice fed with L. acidophilus. Fibronectin 1, which is

involved in integrin and actin cytoskeleton signalling pathways was also up-regulated in

mice fed with L. acidophilus.

Orally administered L. acidophilus down-regulated genes such as BIRC3, TNFRSF1A,

CD74 that are involved in apoptosis, death receptor signalling, antigen presenting

pathway, IL-6 and p38 MAPK signalling pathways. Furthermore, L. acidophilus also

down-regulated several genes that are involved in various functions related to eicosanoid

signalling (DPEP1), glutathione metabolism (ANPEP), N-Glycan biosynthesis

130

(MAN1B1), starch and sucrose metabolism (SI), cAMP-mediated signalling and G-

protein coupled receptor signalling pathways (ADORA1) (Table 5.8).

131

Table 5.5. Effect of L. acidophilus L10 on gene expression (Up- and down-regulated) in the small intestine of cholera toxin mice

model.

GenBank Gene name

Fold change

U05671 Adenosine A1 receptor (ADORA1) 1.4 �

NM_013778 Aldo-keto reductase family 1, member C13 (AKR1C13) 1.6 �

NM_007442 Aristaless-like homeobox 4 (ALX4) 1.4 �

NM_007446 Amylase, alpha 1A (salivary) (AMY1A) 1.6 �

NM_009669 Amylase, alpha 2A (pancreatic) (AMY2A) 1.7 �

AK010761 Ankyrin repeat domain 11 (ANKRD11) 1.7 �

NM_008486 Alanyl (membrane) aminopeptidase (ANPEP) 1.4 �

X15191 Apolipoprotein B (including Ag(x) antigen) (APOB) 1.6 �

AY033514 Butyrobetaine (gamma), 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1) 1.9 �

NM_007464 Baculoviral IAP repeat-containing 3 (BIRC3) 1.4 �

NM_007643 CD36 molecule (thrombospondin receptor) (CD36) 1.4 �

NM_009690 CD5 molecule-like (CD5L) 1.3 �

X00496 CD74 molecule, major histocompatibility complex, class II invariant chain (CD74) 1.3 �

NM_011926 Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) (CEACAM1) 1.4 �

NM_013492 Clusterin (CLU) 1.3 �

NM_007749 Cytochrome c oxidase subunit VIIc (COX7C) 1.3 �

AK003088 Carboxypeptidase A1 (pancreatic) (CPA1) 1.8 �

NM_025583 Chymotrypsinogen B1 (CTRB1) 2.4 �

NM_023182 Chymotrypsin-like (CTRL) 1.6 �

AF197159 Cubilin (intrinsic factor-cobalamin receptor) (CUBN) 1.4 �


AF047725 Cytochrome P450, family 2, subfamily C, polypeptide 38 (CYP2C38) 1.9 �

132


GenBank Gene name

Fold change


NM_017396 Cytochrome P450, family 3, subfamily A, polypeptide 5 (CYP3A5) 1.3 �

AY039762 24-dehydrocholesterol reductase (DHCR24) 1.4 �

NM_007876 Dipeptidase 1 (renal) (DPEP1) 1.5 �

NM_030238 Dynein, cytoplasmic 1, heavy chain 1 (DYNC1H1) 1.3 �

M27347 Elastase 1, pancreatic (ELA1) 1.7 �

BC004798 ELK4, ETS-domain protein (SRF accessory protein 1) (ELK4) 1.6 �

NM_008375 Fatty acid binding protein 6, ileal (gastrotropin) (FABP6) 2.0 �

X93167 Fibronectin 1 (FN1) 1.4 �

NM_008181 Glutathione S-transferase A5 (GSTA5) 1.9 �

NM_013541 Glutathione S-transferase pi (GSTP1) 1.3 �

NM_008218 Hemoglobin, alpha 2 (HBA2) 1.4 �

NM_016868 Hypoxia inducible factor 3, alpha subunit (HIF3A) 1.3 �

NM_015783 ISG15 ubiquitin-like modifier (ISG15) 1.4 �

BC006645 Mannosidase, alpha, class 1B, member 1 (MAN1B1) 1.7 �

NM_008585 Meprin A, alpha (PABA peptide hydrolase) (MEP1A) 1.4 �

AK016915 Mesoderm induction early response 1 homolog (Xenopus laevis) (MI-ER1) 1.3 �

X82786 Antigen identified by monoclonal antibody Ki-67 (MKI67) 1.4 �

NM_025523 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1, 6kDa (NDUFC1) 1.4 �

BC002097 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2, 14.5kDa (NDUFC2) 1.4 �

NM_021543 Protocadherin 8 (PCDH8) 1.3 �

NM_018874 Pancreatic lipase-related protein 1 (PNLIPRP1) 1.4 �

NM_026438 Pyrophosphatase (inorganic) 1 (PPA1) 1.3 �

AB009661 Protease, serine, 2 (trypsin 2) (PRSS2) 1.8 �

NM_023333 Protease, serine, 3 (mesotrypsin) (PRSS3) 1.5 �

133


GenBank Gene name

Fold change

NM_008941 Protease, serine, 7 (enterokinase) (PRSS7) 1.5 �

M31441 RAD52 homolog (S. cerevisiae) (RAD52) 1.5 �

NM_009042 Regenerating islet-derived 1 alpha (pancreatic stone protein, pancreatic thread protein) (REG1A) 2.1 �

NM_009789 S100 calcium binding protein G (S100G) 1.5 �

AB030906 Sex comb on midleg homolog 1 (Drosophila) (SCMH1) 1.3 �

AK008441 Sucrase-isomaltase (alpha-glucosidase) (SI) 1.5 �

NM_011388 Solute carrier family 10 (sodium/bile acid cotransporter family), member 2 (SLC10A2) 1.6 �

NM_011402 Solute carrier family 34 (sodium phosphate), member 2 (SLC34A2) 1.6 �

NM_019754 Transgelin 3 (TAGLN3) 1.4 �

NM_011609 Tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) 1.5 �

AK003881 Ubiquinol-cytochrome c reductase complex (7.2 kD) (UCRC) 1.3 �

AK013986 Ubiquitin specific peptidase 45 (USP45) 1.3 �

NM_009509 Villin 1 (VIL1) 1.3 �

134

Table 5.6. Functional analysis of differentially expressed genes (Up-regulated) after oral administration of L. acidophilus L10 in

cholera toxin mice model.


Small molecule biochemistry 11 Genetic disorder 2 Drug metabolism 6 Hepatic system disease 2 Cancer 6 Immune and lymphatic development and function 2 Lipid metabolism 5 Immunological disease 2 Cell death 4 Skeletal and muscular disorder 2 Cell-cell signalling and interaction 4 Tissue development 2 Hematological system development and function 4 Vitamin and mineral metabolism 2 Carbohydrate metabolism 3 Amino acid metabolism 1 Cellular movement 3 Cell morphology 1 Endocrine system development and function 3 Cellular assembly and organisation 1 Hematological diseases 3 Cellular compromise 1 Molecular transport 3 Cellular development 1

Skeletal and muscular development and function 3 Connective tissue disorders 1 Embryonic development 3 Developmental disorder 1 Immune response 3 Digestive system development and functions 1 Cardiovascular disease 2 Infectious disease 1 Cardiovascular system development and function 2 Nucleic acid metabolism 1 Cell cycle 2 Organ development 1 Cellular function and maintenance 2 Organ morphology 1 Cellular growth and proliferation 2 Organismal injury and abnormalities 1 Connective tissue development and functions 2 Protein trafficking 1 DNA replication, recombination and repair 2 Tissue morphology 1 Gene expression 2 Tumor morphology 1

135

Table 5.7. Functional analysis of differentially expressed genes (Down-regulated) after oral administration of L. acidophilus L10 in

cholera toxin mice model.


Small molecule biochemistry 8 Digestive system development and function 2 Cancer 7 Drug metabolism 2 Cell death 6 Embryonic development 2 Cellular movement 6 Endocrine system disorders 2 Lipid metabolism 6 Immune and lymphatic development and function 2 Molecular transport 6 Organ development 2 Cell-cell signalling and interaction 5 Organ morphology 2 Cellular assembly and organisation 5 Protein trafficking 2 Cardiovascular disease 4 Respiratory disease 2 Cellular compromise 4 Skeletal and muscular disorders 2 Hematological disease 4 Carbohydrate metabolism 1 Immune response 4 Cell cycle 1

Tumor morphology 4 Cell signalling 1 Cardiovascular system development and function 3 Connective tissue disorders 1 Cellular growth and proliferation 3 DNA replication, recombination and repair 1 Genetic disorder 3 Endocrine system development and function 1 Hematological system development and function 3 Gastrointestinal disease 1 Immunological disease 3 Infectious disease 1 Inflammatory disease 3 Nucleic acid metabolism 1 Metabolic disease 3 Post-translation modification 1 Organismal injury and abnormalities 3 Protein synthesis 1 Tissue development 3 Viral function 1 Cell morphology 2 Viral infection 1 Cellular function and maintenance 2 Vitamin and mineral metabolism 1

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Table 5.8. Pathway analysis of differentially expressed genes (Up- and down-regulated) after oral administration of L. acidophilus L10

in cholera toxin mice model.

Canonical Pathway Analysis

Up-regulation

Genes, n Down-regulation Genes, n

Oxidative phosphorylation 5 Apoptosis signalling 2

Xenobiotic metabolism signalling 5 Death receptor signalling 2

Arachidonic acid metabolism 4 Protein ubiquitination pathway 2

Fatty acid metabolism 4 Amyotrophic lateral sclerosis signalling 1

Linoleic acid metabolism 4 Antigen presentation pathway 1

Tryptophan metabolism 4 cAMP-mediated signalling 1

Glutathione metabolism 2 Eicosanoid signalling 1

Starch and sucrose metabolism 2 Glutathione metabolism 1

Ubiquinone biosynthesis 2 G-Protein coupled receptor signalling 1

Actin cytoskeleton signalling 1 IL-6 signalling 1

Glycerolipid metabolism 1 NF-�B signalling 1

Integrin signalling 1 N-Glycan biosynthesis 1

Lysine degradation 1 p38 MAPK signalling 1

PPAR signalling 1

Starch and sucrose metabolism 1

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5.5. DISCUSSION

The GI tract harbours a well balanced microflora, which is in close and continuous

contact with epithelial and immune cells that are essential for a functionally balanced

immune system. Earlier studies suggested that complex modifications of epithelial cells

could occur due to the interactions between probiotic bacteria and enterocytes (Lu and

Walker, 2001). The cross-talk between microorganisms and enterocytes is required to

influence the mucosal barrier functions, which could favour positive health or cause

disease (Kohler et al., 2003). Lactobacilli remain as a major component of human

intestinal microflora that are frequently considered as good candidates in the preparation

of functional foods. The ability of several LAB strains in modulating host innate and

acquired immune responses has been demonstrated in many in vivo animal models.

However, influence of LAB on gene expression of the intestinal epithelial cells remains

unclear.

The present study investigated the effect of orally administered L. acidophilus on gene

expression in the small intestine of mice. Compugen mouse oligonucleotide microarray

was used to identify genes that were influenced by L. acidophilus through comparing the

gene expression profiles in the small intestine of L. acidophilus-fed mice with the

control group. Orally administered L. acidophilus induced the expression of genes that

are involved in immune and lymphatic development such as attractin and chemokine (C-

X-C motif) ligand 15. Attractin is a normal serum glycoprotein of 175 kDa, which is

expressed on activated T-cells. Attractin gene encoded protein is involved in initial

immune cell clustering during inflammatory responses, which may also regulate

chemotactic activity of chemokines. Attractin protein is also crucial in mediating the

138

clustering of non-proliferative T-lymphocytes and spreading of monocytes (Duke-Cohan

et al., 1998). Furthermore, Nagle et al. (1999) reported the potential role of attractin in

decreasing the obesity that was increased by diet in mice. Chemokine receptors are

suggested to be involved in allergic diseases (Dulkys et al., 2004). Chemokine CXCL12

and its receptor are known to be important in the migration of intestinal epithelial cells,

development of enterocytes and promoting mucosal barrier integrity (Smith et al., 2005).

The expression of chemokine (C-X-C motif) ligand 15 (CXCL15) was enhanced by L.

acidophilus, which is also known to be regulated by the influence of CD14 gene in

immune cells (Benhnia et al., 2005).

Several genes involved in various cellular functions such as cell-cell signalling, cell

development, cell death and cell morphology were up-regulated by L. acidophilus. As an

example, Protocadherin 8 (PCDH8) gene involved in cell signalling was enhanced after

oral administration of L. acidophilus. Protocadherin 8 belongs to protocadherin gene

family, a subfamily of cadherin superfamily that are crucial in cell interactions and

morphogenesis of epithelial tissue (Yagi and Takeichi, 2000; Rhee et al., 2003). Heat

shock protein 1A (HSPA1A) gene involved in cell signalling mechanism was up-

regulated by L. acidophilus. The heat shock proteins are known to be capable of

increasing tumor necrosis factor (TNF)-� protein in macrophages (Zheng et al., 2004).

Additionally, genes involved in cell signalling mechanism such as protein tyrosine

phosphatase, receptor type D (PTPRD) and mitogen-activated protein kinase kinase

kinase kinase 1 (MAP4K1) were up-regulated by L. acidophilus. The PTPRD gene is a

member of protein-tyrosine phosphatase (PTP) family, which are signalling molecules

139

involved in variety of cellular processes including cell growth, differentiation, mitotic

cycle, and oncogenic transformation. The protein-tyrosine phosphatases are also

involved in dephosphorylation of amino acids (Pulido et al., 1995). The MAPK family is

a major signalling system, which is considered as an intracellular mediator of

inflammation and also known to play a functional role in c-Jun amino-terminal kinases

(JNKs)/stress-activated protein kinases (SAPKs) pathway (Hu et al., 1996; van den

Blink et al., 2002).

Other genes up-regulated by L. acidophilus are involved in lipid metabolism such as

angiopoietin-like 4 (ANGPTL4) and ATP-binding cassette, sub-family A, member 12

(ABCA12). The ANGPTL4 is a member of angiopoietin/angiopoietin-like gene family,

which is a blood-borne hormone directly involved in regulating glucose homeostasis,

lipid metabolism, and insulin sensitivity. Human clinical trials reported the decrease of

ANGPTL4 serum levels compared to healthy subjects, which suggested that lower

ANGPTL4 levels could be a causative factor for type 2 diabetes (Xu et al., 2005).

Furthermore, Kaneda et al. (2002) reported that aberrant methylation of this gene is

associated with human gastric cancers. The ABCA12 gene encoded protein is a member

of ATP-binding cassette transporters superfamily, which is involved in transporting

various molecules across extra- and intracellular membranes.

Another gene enhanced by oral administration of L. acidophilus is lysozyme, which is

generally regarded as an anti-microbial agent found in human milk, spleen, white blood

cells and plasma. Several studies reported the importance of lysozyme expression in

140

human B-lymphocytes and gastric adenocarcinoma tissues (Hogerkorp et al., 2003; Oien

et al., 2003).

Lactobacillus acidophilus down-regulated genes involved in lipid metabolism such as

pancreatic lipase (PNLIP), carboxyl ester lipase (CEL) and phospholipase A2, group IB

(PLA2G1B). Pancreatic lipase (PNLIP) is a member of lipase gene family, which

encodes a carboxyl esterase that is essential for efficient digestion of dietary fats.

Pancreatic triglyceride lipase deficiency was shown to have minimal affect on dietary fat

absorption, however dietary cholesterol absorption was decreased in mice (Huggins et

al., 2003). The CEL is a glycoprotein secreted into digestive tract from pancreas, which

is necessary in the catabolism of cholesterol (Ikeda et al., 2002) and also influence the

secretion and assembly of lipoprotein through ceramide hydrolysis in mice intestine

(Kirby et al., 2002). Moreover, PLA2G1B gene involved in the catalyses of releasing

fatty acids from glycero-3-phosphocholines was down-regulated by L. acidophilus.

Several forms of PLA2 are known to be present in spleen, macrophages, leukocytes and

erythrocytes. In vitro studies using HL-60 cells reported the influence of interferon

(IFN)-� protein in increasing PLA2G1B protein activity (Visnjic et al., 1997).

Lactobacillus acidophilus down-regulated trefoil factor 2 (TFF2) gene, which is a

member of trefoil family that are commonly known as stable secretory proteins

expressed in gastrointestinal mucosa. Trefoil family secreted proteins play an active role

in protecting the mucosa by stabilising mucus layer and healing epithelial cells. In vitro

studies using human bronchial epithelial cell line, BEAS-2B demonstrated the capability

of TFF-peptides in modulating the inflammatory responses by regulating TNF-� induced

141

secretion of interleukin (IL)-6 and IL-8 (Graness et al., 2002). Furthermore, matrix

metallopeptidase 2 (MMP2) gene was also down-regulated by L. acidophilus. The

proteins that belong to MMP family are involved in tissue remodelling, arthritis and

metastasis disease processes. Several studies reported the role of MMP activity in

apoptosis of epithelial cells (Wiseman et al., 2003) and cancer (Egeblad and Werb,

2002).

In the second experiment, L. acidophilus induced gene expression in the small intestine

was determined in cholera toxin mice using compugen mouse oligonucleotide

microarray. Lactobacillus acidophilus induced gene expression profiles in the small

intestine of mice were identified by comparing with the control group. Mice fed with L.

acidophilus demonstrated the up-regulation of CD36 gene, which is involved in cell-cell

signalling mechanism. The CD36 encoded protein acts as a receptor for thrombospondin

in various cell lines. The CD36 protein also involves in various adhesive processes due

to its functional role as cell adhesion molecule. Recent study reported the role of CD36

in proinflammatory responses, particularly as a recognition receptor that mediates

microglial and macrophage response to beta-amyloid (El Khoury et al., 2003).

Several genes involved in cell death such as glutathione S-transferase pi (GSTP1),

fibronectin 1 (FN1) and ISG15 ubiquitin-like modifier (ISG15) were up-regulated by L.

acidophilus. The GSTP1 is a polymorphic gene, which belongs to GST family of

enzymes that are crucial in catalysing the conjugation of many hydrophobic and

electrophilic compounds through reduced glutathione. The GSTP1 gene is known to be

functional in xenobiotic metabolism and is also involved in the regulation of cell

142

signalling pathways (Gate et al., 2004). The FN1 gene is a glycoprotein, which involves

in cell adhesion and migration processes including host defence functions. Earlier it was

reported that fibronectin type III A1A2 domain was capable of regulating T-lymphocyte

proliferation and cytokine production (Puente Navazo et al., 2001). Furthermore,

fibronectin protein also showed the influence in wound healing process (Rybarczyk et

al., 2003). Moreover, ISG15 gene was up-regulated, which is involved in cell death

mechanism. The ISG15 gene, which is a member of cytokine cascade mechanism, plays

an important role in several host immune functions such as innate immune response and

regulating interferon signalling. D'Cunha et al. (1996) also suggested the role of ISG15

gene in immunomodulatory effects of IFN-� or IFN-�.

Lactobacillus acidophilus down-regulated genes involved in cell growth and

proliferation such as carcinoembryonic antigen-related cell adhesion molecule 1

(CEACAM1), clusterin (CLU) and tumor necrosis factor receptor superfamily, member

1A (TNFRSF1A). The CEACAM1 gene belongs to carcinoembryonic antigen (CEA)

gene family, which is a cell surface molecule known to have regulatory effects on T-cell

functions. Earlier studies reported the involvement of CEACAM1 protein in decreasing

tumor development and oxazolone colitis (Fournes et al., 2001; Iijima et al., 2004).

Furthermore, CEACAM1 protein also acts as a signal-transducing receptor, which could

regulate early maturation and activation of dendritic cells (Kammerer et al., 2001). The

CLU is a glycoprotein of 75-80 kDa in size secreted in physiological fluids, which is

known to be involved in apoptosis and colorectal cancer (Scaltriti et al., 2004; Andersen

et al., 2007). Moreover, L. acidophilus also down-regulated TNFRSF1A gene, which is

a member of TNF-receptor superfamily that are potential regulators of apoptosis, a

143

process considered to be important in maintaining immune homeostasis. Particularly,

TNFRSF1A protein is known to have a functional role as a regulator in inflammatory

responses. Several studies also reported the involvement of TNF-receptor proteins in

decreasing obesity, liver inflammation and tumor formation (Chan et al., 2003; Francis

et al., 2003; Arnott et al., 2004).

Oral administration of L. acidophilus down-regulated villin 1 (VIL1), which is an actin-

binding protein localises to the brush borders of intestine. Villin 1 gene is a member of

gelsolin family of proteins, which acts as a regulator in hepatocyte growth factor-

induced epithelial cell motility (Athman et al., 2003). The CD74 gene was also down-

regulated by L. acidophilus, which is considered to be important in the activation of

ERK-1/2 MAP kinase cascade and cell proliferation (Leng et al., 2003). Furthermore, L.

acidophilus down-regulated apolipoprotein B (APOB), which occurs in two forms in

plasma known as APOB-48 and APOB-100. Interestingly, Farese et al. (1995) reported

the potential role of APOB gene in diet-induced hypercholesterolemia mice model.

The present study generated a list of genes that were influenced by L. acidophilus in

normal and cholera toxin mice. To gain further insights into the potential value of this

data, it is important to analyse the gene expression in various segments of GI tract as

well as in distant mucosal site such as spleen in animal models. This study evaluated the

effects of a specific probiotic strain in the small intestine of mice for a specific period.

Therefore, it is also reasonable to predict that various factors such as strain variations,

dosage or duration of the probiotic feeding may affect in modulating the gene

144

expression. Furthermore, human trials with different clinical settings are necessary to

understand the clinical value of the microarray data reported in this study.

Nevertheless, this study represents a basic methodology to understand the influence of L.

acidophilus in regulating gene expression that was involved in various biological

functions.

145

5.6. CONCLUSIONS

Oral administration of L. acidophilus influenced the regulation of genes involved in

various biological functions of the small bowel in normal and cholera toxin mice. The

current study generated a comprehensive list of genes that were affected by L.

acidophilus in small intestine of mice, which lay a basis for further understanding the

interactions between probiotic bacteria and the host.

146

CHAPTER 6

Concluding summary

147

The current study examined the probiotic properties of L. acidophilus LAFTI L10 and L.

paracasei LAFTI L26 using various in vitro methods. The influence of orally

administered L. acidophilus and L. paracasei on gut and systemic immune responses in

normal and cholera toxin (CT) mice were investigated. Furthermore, L. acidophilus

induced gene expression was examined in the small intestine of normal and CT mice.

The intestinal microflora play a significant role in influencing the structural and

functional development of gut immune system. Lactic acid bacteria are non-pathogenic

food grade microorganisms that are natural components of intestinal microflora. Lactic

acid bacteria are important in a well balanced intestinal microbial ecosystem, which are

also known for their health-promoting attributes. Earlier studies suggested that probiotic

bacteria must be tolerant to adverse conditions of GI tract, especially in stomach that has

lower pH and high bile salt concentrations (Prasad et al., 1998; Mishra and Prasad,

2005). Furthermore, adhesive ability of probiotic bacteria to intestinal mucosa is also

considered as a prerequisite for colonisation and modulation of host immune functions

(Castagliuolo et al., 2005; Aslim et al., 2007).

The initial work in this study was focused in characterising the probiotic properties of

Lactobacillus and Bifidobacterium species to identify potential probiotic strains for

further in vivo mice studies. Lactobacillus acidophilus LAFTI L10 and L. paracasei

LAFTI L26 showed higher tolerance to simulated gastric conditions and bile salts

(Chapter 2). Lactobacillus acidophilus and L. paracasei demonstrated the correlation

between in vitro adhesive properties such as hydrophobicity and autoaggregation.

Coaggregation abilities of L. acidophilus and L. paracasei with other bacterial strains

148

showed significant variations, which could be due to the differences in their cell surface

proteins. Particularly, L. acidophilus demonstrated higher level of coaggregation with L.

paracasei and other probiotic strains. The tolerance of L. acidophilus and L. paracasei

to gastric pH 2.0 and 1.0% bile salts demonstrated the potential of these strains to

survive and colonise especially in the small intestine to induce beneficial effects on the

host.

Orally administered L. acidophilus and L. paracasei at 108 CFU/day for 14-days in

BALB/c mice stimulated the host immune functions (Chapter 3). Immunofluorescent

examination of small intestine in mice demonstrated the capability of L. acidophilus and

L. paracasei in enhancing immunoglobulin (Ig)-A producing B-cells, which are

necessary in mucosal defence mechanisms. The T-cells in small intestine of mice fed

with either L. acidophilus or L. paracasei showed an increase of anti-inflammatory

cytokine, interleukin (IL)-10 and pro-inflammatory cytokine, interferon (IFN)-�.

Lactobacillus acidophilus and L. paracasei were capable of enhancing IL-10 cytokine,

which is known to have a beneficial role in preventing immune hypersensitivity/atopy

(Majamaa and Isolauri, 1997; Kalliomaki et al., 2001) and alleviating inflammatory

bowel disease (Madsen et al., 1999; Matsumoto et al., 2001). In systemic immunity,

proliferative responses of splenocytes stimulated with concanavalin A (ConA) and

lipopolysaccharide (LPS) were increased in mice fed with L. acidophilus or L.

paracasei. Particularly, LPS induced proliferative responses of splenocytes in mice fed

with L. paracasei were not significantly higher compared to control mice.

149

Mice fed with either L. acidophilus or L. paracasei regulated the systemic immune

response by increasing Th1 cytokine, IFN-� and Th2 cytokine, IL-10 in serum and

spleen cells (Chapter 3). In the current study, stimulation of systemic immune responses

by probiotic bacteria might have occurred through the bacterial antigen uptake by M-

cells of Peyer’s patches in the small intestine. In Peyer’s patches, probiotic bacterial

antigens interact with lymphocytes that lead to the activation of antigen-specific

lymphocytes, which could have migrated through mesenteric lymph nodes and then

entered into blood stream via thoracic duct to stimulate systemic immune functions.

Lactobacillus acidophilus and L. paracasei also showed the capability in stimulating

non-specific immune functions such as macrophage activity (Chapter 3). The peritoneal

macrophages of mice fed with L. acidophilus or L. paracasei demonstrated the

enhancement of phagocytic activity against C. albicans compared to control mice.

Vinderola et al. (2004) suggested that lack of influence of probiotic bacteria in activating

systemic immune functions such as macrophage activity could be due to the inhibitory

effect of IL-10. Interestingly, inhibitory effect of IL-10 on macrophage activation was

not observed in the current study, where L. acidophilus and L. paracasei enhanced the

Th2 cytokine, IL-10 in serum and spleen cells (Chapter 3). As shown in chapter 2,

tolerance of L. acidophilus and L. paracasei to simulated acid and bile conditions and

their better adherence capabilities may have favoured them to persist, colonise and

adhere in GI tract of mice to stimulate the macrophage activity (Perdigon and Alvarez,

1992; Schiffrin et al., 1997).

150

The differences in translocation of Lactobacillus spp. and total anaerobes to Peyer’s

patches and MLN were observed in mice fed with L. acidophilus or L. paracasei

(Chapter 3). Particularly, L. acidophilus-fed mice showed higher translocation of

Lactobacillus spp. and total anaerobes to Peyer’s patches compared to L. paracasei-fed

mice and control group. Mice fed with L. acidophilus or L. paracasei showed increased

bacterial translocation to Peyer’s patches and MLN, which could be due to their

adhesive properties (Wagner et al., 1997) that may have contributed to the enhancement

of various gut and systemic immune functions. Furthermore, oral administration of L.

acidophilus or L. paracasei at 108 CFU/day for 14-days in BALB/c mice did not cause

intestinal inflammation, as there was no indication of translocation of Lactobacillus spp.

and total anaerobes to sterile organs such as spleen, liver and blood.

The influence of orally administered L. acidophilus and L. paracasei on host immune

responses was investigated in CT mice (Chapter 4). Lactobacillus acidophilus and L.

paracasei showed the differences in inducing nitric oxide (NO) production in intestinal

fluid and serum. Particularly, L. paracasei demonstrated as a potential inducer of NO

levels in intestinal fluid and serum compared to L. acidophilus. The potential of L.

paracasei in inducing the release of NO in intestinal fluid may be necessary to play a

protective role in colitis (Wallace et al., 1999; Lamine et al., 2004). Both, L. acidophilus

and L. paracasei increased the IgA response to CT in intestinal fluid and serum, which

could be considered as a potential attribute necessary in host immune defence

mechanisms.

151

As shown in chapter 4, immunofluorescent studies demonstrated the potential of L.

acidophilus and L. paracasei in regulating Th1- and Th2-cytokines in the small intestine

of CT mice. Both L. acidophilus and L. paracasei enhanced the IL-6 producing cells in

small intestine, which may have contributed to the stimulation of IgA response to CT in

intestinal fluid and serum. Lactobacillus acidophilus and L. paracasei showed the

regulation of anti-inflammatory cytokine, IL-10 and pro-inflammatory cytokine, IFN-�

in the small intestine of CT mice. Similar effects of L. acidophilus and L. paracasei in

regulating IL-10 and IFN-� was also observed in the small intestine of normal mice

(Chapter 3). In the current study, stimulation of IFN-� producing cells in small intestine

of normal and CT mice fed with L. acidophilus or L. paracasei may have contributed to

the increase in uptake of bacterial antigens into Peyer’s patches (Sutas et al., 1997),

which lead to the stimulation of gut and systemic immune functions (Chapters 3 & 4).

Moreover, mice fed with L. acidophilus or L. paracasei also enhanced the stimulation of

tumor necrosis factor (TNF)-� and IL-4 cytokines in small intestine of CT mice. Further

studies on dose-dependent oral administration of L. acidophilus and L. paracasei are

needed to understand the cytokine profiles at different time intervals.

Considering the in vitro probiotic characteristics of L. acidophilus and its immune

enhancing properties in normal and CT mice, L. acidophilus induced gene expression in

the small intestine of normal and CT mice was investigated using mouse oligonucleotide

microarray (Chapter 5). In normal mice, L. acidophilus altered the gene expression in

the small intestine through up- and down-regulation of 27 and 35 genes. Orally

administered L. acidophilus affected the genes that are important in cell signalling such

as HSPA1A and MAP4K1. The up-regulation of HSPA1A by L. acidophilus showed its

152

potential in interacting with intestinal epithelial cells to maintain cytoskeletal integrity.

Particularly, L. acidophilus-fed mice up-regulated MAP4K1 gene, which is an

intracellular signalling molecule that may have influenced the expression of HSPA1A in

intestinal epithelial cells (Tao et al., 2006). Moreover, L. acidophilus-fed mice up-

regulated the genes involved in immune responses (attractin and CXCL15) and cell

signalling (PCDH8), whereas the down-regulated genes are functional in lipid

metabolism (CEL, PLA2G1B and PNLIP) and apoptosis (MMP2).

In CT mice model, L. acidophilus modulated the gene expression in small intestine

through an up- and down-regulation of 28 and 32 genes. Orally administered L.

acidophilus up-regulated CD36 gene, which is involved in cell-cell signalling and

known to have a significant role in pro-inflammatory responses (El Khoury et al., 2003).

Other genes up-regulated by L. acidophilus are involved in cell death such as FN1,

GSTP1 and ISG15. Particularly, genes that belong to cytochrome P450 family

(CYP2C9, CYP2C19, CYP2C38 and CYP3A5) were also up-regulated by L.

acidophilus. Interestingly, L. acidophilus down-regulated TNFRSF1A gene, which is

considered to have a regulatory role in inflammatory mechanisms (Chan et al., 2003).

Additionally, L. acidophilus also down-regulated genes such as VIL1, CD74 and APOB.

The present study investigated the influence of L. acidophilus on gene expression in the

small intestine of mice, since it is considered as an important site for probiotic bacterial

interactions with intestinal epithelial cells. The analysis of L. acidophilus induced gene

expression in distant mucosal site such as spleen could be useful in further elucidating

the mechanisms through which these probiotic bacteria are beneficial to the host.

153

Moreover, the current study generated the pool of genes in small intestine of normal and

CT mice that were modified by oral administration of L. acidophilus. Lactobacillus

acidophilus induced gene expression was analysed in normal and CT mice. Therefore, it

is reasonable to suggest that the analysis of gene expression modulated by L. acidophilus

was limited and could possibly differ in various animal models. Furthermore, validating

the influence of L. acidophilus on gene expression in human clinical trails is necessary

to unravel the beneficial role of probiotic bacteria.

154

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