Download Probiotic characteristics of Lactobacillus acidophilus and

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
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
i
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
ii
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. MATERIALS AND METHODS........................................................................ 63
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
iii
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. MATERIALS AND METHODS........................................................................ 96
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
iv
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
v
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.
vi
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, 115118.
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).
vii
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
th
system. 38 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).
viii
List of Tables
Table 1.1.
Table 1.2.
Table 2.1.
Table 2.2.
Table 3.1.
Table 3.2.
Table 3.3.
Table 3.4.
Table 5.1.
Table 5.2.
Table 5.3.
Table 5.4.
Table 5.5.
Table 5.6.
Table 5.7.
Microorganisms that are commonly considered as
probiotics.
Therapeutic properties of lactic acid bacteria.
(Adapted from Saxelin et al., 2005; Reid et al.,
2006).
Survival of Lactobacillus and Bifidobacterium
strains in simulated gastric conditions for 3 h.
Survival of Lactobacillus and Bifidobacterium
strains in milk-yeast medium with oxgall.
Proliferative responses of spleen cells stimulated
with ConA and LPS in mice fed with either L.
acidophilus L10 or L. paracasei L26.
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.
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.
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.
Effect of L. acidophilus L10 on gene expression
(Up- and down-regulated) in the small intestine of
normal mice.
Functional analysis of differentially expressed
genes (Up-regulated) after oral administration of
L. acidophilus L10 in normal mice.
Functional analysis of differentially expressed
genes (Down-regulated) after oral administration
of L. acidophilus L10 in normal mice.
Pathway analysis of differentially expressed
genes (Up- and down-regulated) after oral
administration of L. acidophilus L10 in normal
mice.
Effect of L. acidophilus L10 on gene expression
(Up- and down-regulated) in the small intestine of
cholera toxin mice model.
Functional analysis of differentially expressed
genes (Up-regulated) after oral administration of
L. acidophilus L10 in cholera toxin mice model.
Functional analysis of differentially expressed
genes (Down-regulated) after oral administration
of L. acidophilus L10 in cholera toxin mice
model.
16
24, 25
43,44
45
74
80
81
82
123,124,
125
126
127
128
131, 132,
133
134
135
ix
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
x
List of Figures
Figure 1.1.
Figure 1.2.
Figure 1.3.
Figure. 2.1.
Figure 2.2.
Figure 2.3.
Figure 3.1.
Figure 3.2.
Figure 3.3.
Figure 3.4.
Figure 3.5.
Figure 4.1.
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 - Tcell receptor. (Adapted from McCracken and
Lorenz, 2001).
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).
Schematic representation of gut immune system.
(Adapted from McCracken and Lorenz, 2001).
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.
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.
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.
Effect of A) L. acidophilus L10 and B) L.
paracasei L26 on immunoglobulin (Ig)-A
producing cells in the small intestine of mice.
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.
Estimation of cytokines A) interleukin (IL)-10
and B) interferon (IFN)- in serum of mice fed
with L. acidophilus L10 or L. paracasei L26.
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.
Effect of A) L. acidophilus L10 and B) L.
paracasei L26 on phagocytic activity of the
peritoneal macrophages.
Nitric oxide production in A) intestinal fluid and
B) serum of mice fed with L. acidophilus L10 or
L. paracasei L26.
6
10
13
48
49
50
72
73
75
76
79
101
xi
Figure 4.2.
Figure 4.3.
Figure 4.4.
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.
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.
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.
102
104,105
106
xii
List of Abbreviations
CD
CFU
ConA
CT
FITC
GALT
GI
GRAS
IBD
IBS
IFN
Ig
IL
LAB
LPS
MLN
MRS
NGYC
NK
NO
PBS
TNF
WC
Cluster of differentiation
Colony Forming Units
Concanavalin A
Cholera toxin
Fluorescein isothiocyanate
Gut-associated lymphoid tissue
Gastrointestinal
Generally Regarded As Safe
Inflammatory Bowel Disease
Irritable Bowel Syndrome
Interferon
Immunoglobulin
Interleukin
Lactic acid bacteria
Lipopolysaccharide
Mesenteric lymph node
de Man, Rogosa and Sharpe agar or broth
Non-fat skim milk, glucose, yeast extract and cysteine
medium
Natural Killer (Cell)
Nitric oxide
Phosphate buffered saline
Tumor necrosis factor
Wilkins-Chalgren agar
xiii
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
1
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 downregulation 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.
2
CHAPTER 1
Introduction
3
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
4
represents 90% of the bacterial population, whereas less than 0.01% consists of
diversified bacterial population such as pathogenic microorganisms (Tournut, 1993).
5
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).
6
The intestinal microflora influence wide variety of host functions such as synthesis
of biotin, folate, vitamin K and conversion of unabsorbed dietary sugars into shortchain 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
7
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
8
preventing the microbial adherence to epithelial cell surfaces (Magnusson and
Stjernstorm, 1982; Ouellette, 1999).
9
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).
10
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.
11
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.
12
Figure 1.3. Schematic representation of gut immune system. (Adapted from McCracken and Lorenz, 2001).
13
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 rodshaped 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
14
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).
15
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
16
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
17
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
18
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.
19
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)
20
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
21
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.
22
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.
23
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.
24
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.
25
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.
26
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.
27
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).
28
CHAPTER 2
Selection and characterisation of Lactobacillus and Bifidobacterium species for
potential probiotic strains
29
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.
30
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)
31
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,
32
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
33
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.
34
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%
35
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 milkyeast 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
36
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:
Hydrophobicity ( H %) = [( AO − A) / AO ] x 100
Where A0 and A represents the absorbance of aqueous phase before and after contact
with n-hexadecane.
37
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:
Autoaggreg ation (%) = 1 − ( At / Ao ) x 100
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.
38
The coaggregation percentage is expressed as:
Coaggregation (% ) =
(( Ax + Ay) / 2) − A( x + y)
x 100
( Ax + Ay ) / 2
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).
39
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
40
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
41
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.
42
Table 2.1. Survival of Lactobacillus and Bifidobacterium strains in simulated gastric conditions for 3 h.
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
Strains
43
Table 2.1. (Continued)
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
Strains
a
Initial bacterial cell concentration.
b
Values (Log10 CFU ml-1) are mean ± SEM (n = 6).
c
Expressed as Log10 values using the formula: (pH 6.5 – pH 4.0) + (pH 6.5 – pH 3.0) + (pH 6.5 – pH 2.0).
d
Not detected. Detection limit was 101 CFU/ml.
*
Mean values were significantly different (P < 0.01) from the pH 6.5 (Control).
44
Table 2.2. Survival of Lactobacillus and Bifidobacterium strains in milk-yeast medium with oxgall.
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
Strains
a
Initial bacterial cell concentration.
Values (Log10 CFU ml-1) are mean ± SEM (n = 6).
b
c
Expressed 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).
45
2.4.3. Surface hydrophobicity of Lactobacillus and Bifidobacterium strains
The cell surface hydrophobicity of Lactobacillus and Bifidobacterium strains to nhexadecane 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.
46
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).
47
Hydrophobicity %
30
20
10
ASCC 2400
ASCC 1912
Bb12
B94
L26
L10
0
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).
48
Autoaggregation %
40
30
ASCC 2400
ASCC 1912
Bb12
B94
L26
L10
20
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).
49
80
*
Coaggregation %
60
*
*
40
20
L26 + ASCC 2400
L10 + ASCC 2400
L26 + ASCC 1912
L10 + ASCC 1912
L26 + Bb12
L10 + Bb12
L26 + B94
L10 + B94
L10 + L26
0
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.
50
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
51
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
52
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
53
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
54
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).
55
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.
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
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.
58
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 Bcell 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
59
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
60
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
61
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.
62
3.3. MATERIALS AND METHODS
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
63
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.
64
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.
65
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 IFNsandwich 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
66
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
67
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).
3.3.12. 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).
68
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 IFNproducing 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.
69
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 IFNlevels 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.
70
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.
71
A
100
Control
IgA cells/10 fields
L10
#
*
80
60
40
B
Control
IgA cells/10 fields
70
L26
#
*
55
40
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.
72
A
Control
IL-10 cells/10 fields
75
L10
L26
#
*
#
*
45
15
B
75
#
*
Control
L10
45
#
*
IFN-
cells/10 fields
L26
15
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.
73
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
Control
3.28 ± 0.19
2.19 ± 0.07
5.04 ± 0.29 **, #
4.31 ± 0.10 *, #
L. acidophilus L10
L. paracasei L26
4.25 ± 0.32 *, #
LPS
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.
74
A
500
#
*
Control
L10
L26
IL-10 pg/ml
400
300
#
*
200
100
B
400
#
*
Control
L10
300
IFN-
pg/ml
L26
200
#
*
100
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.
75
A
IL-10 pg/ml
2500
#
**
Control
L10
L26
2000
#
*
1500
1000
B
600
*
*
pg/ml
IFN-
Control
L10
L26
400
200
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.
76
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
77
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).
78
A
60
Control
*
Phagocytosis %
L10
40
20
B
60
Control
Phagocytosis %
L26
*
40
20
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.
79
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
Peyer’s patchesa
MLNa
Spleena
Livera
Bloodb
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
Treatment
Control
Values are mean ± SEM (n = 6).
Significantly different from the control mice *P < 0.05; **P < 0.01.
a
Detection limit was 102 CFU g-1
b
Detection limit was 102 CFU ml-1
c
Not detected.
80
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
Peyer’s patchesa
MLNa
Spleena
Livera
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
Treatment
Values are mean ± SEM (n = 6).
Significantly different from the control mice *P < 0.01.
Significant differences between L. acidophilus and L. paracasei experimental groups #P < 0.01.
a
Detection limit was 102 CFU g-1
b
Detection limit was 102 CFU ml-1
c
Not detected.
81
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
Peyer’s patchesa
MLNa
Spleena
Livera
Bloodb
Control
NDc
ND
ND
ND
ND
L. acidophilus L10
ND
ND
ND
ND
ND
L. paracasei L26
ND
ND
ND
ND
ND
Treatment
Values are mean ± SEM (n = 6).
a
Detection limit was 102 CFU g-1
b
Detection limit was 102 CFU ml-1
c
Not detected.
82
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.
83
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. Tlymphocytes 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 Bcell 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
84
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.
85
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
86
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
87
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.
88
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 IFNcytokines 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.
89
CHAPTER 4
Immunostimulatory responses of L. acidophilus LAFTI L10 and L. paracasei
LAFTI L26 in cholera toxin mice
90
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.
91
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
92
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).
93
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
94
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.
95
4.3. MATERIALS AND METHODS
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.
96
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).
97
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 (SigmaAldrich, 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.
98
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 antirabbit 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.
4.3.9. 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).
99
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.
100
A)
Control
80
#
*
L10
Intestinal fluid NO (µmol/L)
L26
60
#
40
20
B)
#
*
Control
40
L10
Serum NO (µmol/L)
L26
#
*
30
20
10
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.
101
A)
20.0
Control
L10
Intestinal fluid IgA (µg/ml)
L26
*
*
17.5
15.0
B)
16
Control
L10
L26
#
**
#
**
Serum IgA (µg/ml)
12
8
4
0
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
experimental groups #P < 0.01.
102
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.
103
A
Control
75
L10
L26
**
60
Cells/10 fields
***
**
45
30
15
0
IFN -
TNF -
Figure 4.3. (Continued)
104
B
**
75
Cells/10 fields
Control
#
***
100
L10
L26
#
*
**
50
25
0
IL - 4
IL - 6
IL - 10
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
experimental groups #P < 0.05.
105
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.
106
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 IFNproducing 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.
107
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).
108
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).
109
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, IL12, 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).
110
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. paracaseifed 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.
111
CHAPTER 5
Microarray analysis of L. acidophilus LAFTI L10 induced gene expression in the
small intestine of mice
112
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 downregulation 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.
113
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).
114
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
115
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.
116
5.3. MATERIALS AND METHODS
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
117
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.
118
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
119
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 logratios 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.
120
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
121
(Starch and sucrose metabolism) and glutathione S-transferase A5 (Glutathione
metabolism) were also down-regulated in L. acidophilus-fed mice (Table 5.4).
122
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
AK014713
BC006756
NM_013778
NM_009669
NM_020581
NM_019870
NM_009730
AK007010
NM_009885
NM_009887
AK003088
NM_019696
NM_025583
AK007772
NM_023182
NM_008411
NM_011339
NM_009998
NM_010003
AK008688
AK018047
M27347
NM_026419
BC006944
NM_008003
ATP-binding cassette, sub-family A (ABC1), member 12 (ABCA12)
Ankyrin repeat and BTB (POZ) domain containing 2 (ABTB2)
Aldo-keto reductase family 1, member C13 (AKR1C13)
Amylase, alpha 2A (pancreatic) (AMY2A)
Angiopoietin-like 4 (ANGPTL4)
ARD1 homolog A, N-acetyltransferase (S. cerevisiae) (ARD1A)
Attractin (ATRN)
Antizyme inhibitor 1 (AZIN1)
Carboxyl ester lipase (bile salt-stimulated lipase) (CEL)
Cerberus 1, cysteine knot superfamily, homolog (Xenopus laevis) (CER1)
Carboxypeptidase A1 (pancreatic) (CPA1)
Carboxypeptidase X (M14 family), member 1 (CPXM1)
Chymotrypsinogen B1 (CTRB1)
Chymotrypsin C (CTRC)
Chymotrypsin-like (CTRL)
CUB and zona pellucida-like domains 1 (CUZD1)
Chemokine (C-X-C motif) ligand 15 (CXCL15)
Cytochrome P450, family 2, subfamily B, polypeptide 6 (CYP2B6)
Cytochrome P450, family 2, subfamily C, polypeptide 19 (CYP2C19)
Cytochrome P450, family 2, subfamily C, polypeptide 8 (CYP2C8)
EDAR-associated death domain (EDARADD)
Elastase 1, pancreatic (ELA1)
Elastase 3B, pancreatic (ELA3B)
Ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3)
Fibroblast growth factor 19 (FGF19)
Fold change
1.4
1.5
1.6
2.1
1.5
1.4
1.3
1.5
2.8
1.4
2.2
1.4
2.4
1.9
2.0
1.5
1.4
1.4
1.4
1.4
1.5
1.8
2.8
1.3
1.3
123
Table 5.1. (Continued)
GenBank
Gene name
NM_007989
NM_025989
NM_008182
NM_008210
M34857
M12571
BC006780
X53632
Y09010
NM_020575
NM_008610
NM_020276
NM_009513
NM_011864
NM_021543
NM_013750
NM_011107
NM_026925
NM_018874
AK004304
AB009661
NM_023333
NM_008941
D13904
NM_009042
Forkhead box H1 (FOXH1)
Glycoprotein 2 (zymogen granule membrane) (GP2)
Glutathione S-transferase A5 (GSTA5)
H3 histone, family 3A (H3F3A)
Homeobox B9 (HOXB9)
Heat shock 70kDa protein 1A (HSPA1A)
Keratin 5 (KRT5)
Lysozyme (renal amyloidosis) (LYZ)
Mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1)
Membrane-associated ring finger (C3HC4) 7 (MARCH7)
Matrix metallopeptidase 2 (MMP2)
Nasal embryonic LHRH factor (NELF)
Neurensin 1 (NRSN1)
3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2)
Protocadherin 8 (PCDH8)
Pleckstrin homology-like domain, family A, member 3 (PHLDA3)
Phospholipase A2, group IB (pancreas) (PLA2G1B)
Pancreatic lipase (PNLIP)
Pancreatic lipase-related protein 1 (PNLIPRP1)
Prickle-like 1 (Drosophila) (PRICKLE1)
Protease, serine, 2 (trypsin 2) (PRSS2)
Protease, serine, 3 (mesotrypsin) (PRSS3)
Protease, serine, 7 (enterokinase) (PRSS7)
Protein tyrosine phosphatase, receptor type, D (PTPRD)
Regenerating islet-derived 1 alpha (REG1A)
Fold change
1.4
1.5
1.7
1.4
1.4
1.6
1.4
1.3
1.6
1.5
1.6
1.4
1.7
1.4
1.4
1.4
1.4
1.9
2.5
1.4
2.5
1.9
1.5
1.3
1.6
124
Table 5.1. (Continued)
GenBank
Gene name
NM_011271
NM_009081
NM_009091
NM_009789
D83146
NM_019481
AF026489
AK003083
S76673
NM_009363
AK016603
BC006649
Ribonuclease, RNase A family, 1 (pancreatic) (RNASE1)
Ribosomal protein L28 (RPL28)
Ribosomal protein S15 (RPS15)
S100 calcium binding protein G (S100G)
Sine oculis-related homeobox 5 homolog (Drosophila) (SIX5)
Solute carrier family 13 (sodium/sulfate symporters), member 1 (SLC13A1)
Spectrin, beta, non-erythrocytic 2 (SPTBN2)
Syncollin (SYCN)
Transcription factor binding to IGHM enhancer 3 (TFE3)
Trefoil factor 2 (spasmolytic protein 1) (TFF2)
Transketolase-like 2 (TKTL2)
Ubiquitination factor E4A (UFD2 homolog, yeast) (UBE4A)
Fold change
1.6
1.4
1.4
1.7
1.4
1.4
1.4
2.1
1.4
1.6
1.6
1.5
125
Table 5.2. Functional analysis of differentially expressed genes (Up-regulated) after oral administration of L. acidophilus L10 in
normal mice.
Functions & Diseases
Small molecule biochemistry
Genetic disorder
Cell-cell signalling and interaction
Hematological system development and function
Cell signalling
Cellular assembly and organisation
Cellular development
Cellular growth and proliferation
Developmental disorder
DNA replication, recombination and repair
Embryonic development
Immune and lymphatic development and function
Lipid metabolism
Molecular transport
Post-translational modification
Genes, n Functions & Diseases
5
4
3
3
2
2
2
2
2
2
2
2
2
2
2
Tissue development
Amino acid metabolism
Carbohydrate metabolism
Cardiovascular disease
Cell death
Cell morphology
Connective tissue disorders
Endocrine system disorder
Gene expression
Immune response
Infectious disease
Inflammatory disease
Metabolic disease
Skeletal and muscular development and function
Viral function
Genes, n
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
126
Table 5.3. Functional analysis of differentially expressed genes (Down-regulated) after oral administration of L. acidophilus L10 in
normal mice.
Functions & Diseases
Small molecule biochemistry
Lipid metabolism
Molecular transport
Protein degradation
Drug metabolism
Cancer
Cellular movement
Organismal development
Vitamin and mineral metabolism
Amino acid metabolism
Carbohydrate metabolism
Cellular development
Developmental disorder
Embryonic development
Endocrine system development and function
Genetic disorder
Inflammatory disease
Organ morphology
Post-translation modification
Respiratory disease
Genes, n Functions & Diseases
14
6
6
5
4
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
Skeletal and muscular disorders
Tissue morphology
Cardiovascular disease
Cell cycle
Cell morphology
Cell-cell signalling and interaction
Cellular assembly and organisation
Cellular growth and proliferation
Connective tissue development and function
Connective tissue disorders
Digestive system development and function
Endocrine system disorders
Gastrointestinal disease
Hematological system development and function
Hepatic system disease
Organ development
Organismal injury and abnormalities
Skeletal and muscular development and function
Tissue development
Tumor morphology
Genes, n
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
127
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
128
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 upregulated 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
129
(MAN1B1), starch and sucrose metabolism (SI), cAMP-mediated signalling and Gprotein coupled receptor signalling pathways (ADORA1) (Table 5.8).
130
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
U05671
NM_013778
NM_007442
NM_007446
NM_009669
AK010761
NM_008486
X15191
AY033514
NM_007464
NM_007643
NM_009690
X00496
NM_011926
NM_013492
NM_007749
AK003088
NM_025583
NM_023182
AF197159
NM_010003
AF047725
Adenosine A1 receptor (ADORA1)
Aldo-keto reductase family 1, member C13 (AKR1C13)
Aristaless-like homeobox 4 (ALX4)
Amylase, alpha 1A (salivary) (AMY1A)
Amylase, alpha 2A (pancreatic) (AMY2A)
Ankyrin repeat domain 11 (ANKRD11)
Alanyl (membrane) aminopeptidase (ANPEP)
Apolipoprotein B (including Ag(x) antigen) (APOB)
Butyrobetaine (gamma), 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 (BBOX1)
Baculoviral IAP repeat-containing 3 (BIRC3)
CD36 molecule (thrombospondin receptor) (CD36)
CD5 molecule-like (CD5L)
CD74 molecule, major histocompatibility complex, class II invariant chain (CD74)
Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) (CEACAM1)
Clusterin (CLU)
Cytochrome c oxidase subunit VIIc (COX7C)
Carboxypeptidase A1 (pancreatic) (CPA1)
Chymotrypsinogen B1 (CTRB1)
Chymotrypsin-like (CTRL)
Cubilin (intrinsic factor-cobalamin receptor) (CUBN)
Cytochrome P450, family 2, subfamily C, polypeptide 19 (CYP2C19)
Cytochrome P450, family 2, subfamily C, polypeptide 38 (CYP2C38)
Fold change
1.4
1.6
1.4
1.6
1.7
1.7
1.4
1.6
1.9
1.4
1.4
1.3
1.3
1.4
1.3
1.3
1.8
2.4
1.6
1.4
2.1
1.9
131
Table 5.5. (Continued)
GenBank
Gene name
NM_007815
NM_017396
AY039762
NM_007876
NM_030238
M27347
BC004798
NM_008375
X93167
NM_008181
NM_013541
NM_008218
NM_016868
NM_015783
BC006645
NM_008585
AK016915
X82786
NM_025523
BC002097
NM_021543
NM_018874
NM_026438
AB009661
NM_023333
Cytochrome P450, family 2, subfamily C, polypeptide 9 (CYP2C9)
Cytochrome P450, family 3, subfamily A, polypeptide 5 (CYP3A5)
24-dehydrocholesterol reductase (DHCR24)
Dipeptidase 1 (renal) (DPEP1)
Dynein, cytoplasmic 1, heavy chain 1 (DYNC1H1)
Elastase 1, pancreatic (ELA1)
ELK4, ETS-domain protein (SRF accessory protein 1) (ELK4)
Fatty acid binding protein 6, ileal (gastrotropin) (FABP6)
Fibronectin 1 (FN1)
Glutathione S-transferase A5 (GSTA5)
Glutathione S-transferase pi (GSTP1)
Hemoglobin, alpha 2 (HBA2)
Hypoxia inducible factor 3, alpha subunit (HIF3A)
ISG15 ubiquitin-like modifier (ISG15)
Mannosidase, alpha, class 1B, member 1 (MAN1B1)
Meprin A, alpha (PABA peptide hydrolase) (MEP1A)
Mesoderm induction early response 1 homolog (Xenopus laevis) (MI-ER1)
Antigen identified by monoclonal antibody Ki-67 (MKI67)
NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1, 6kDa (NDUFC1)
NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2, 14.5kDa (NDUFC2)
Protocadherin 8 (PCDH8)
Pancreatic lipase-related protein 1 (PNLIPRP1)
Pyrophosphatase (inorganic) 1 (PPA1)
Protease, serine, 2 (trypsin 2) (PRSS2)
Protease, serine, 3 (mesotrypsin) (PRSS3)
Fold change
1.6
1.3
1.4
1.5
1.3
1.7
1.6
2.0
1.4
1.9
1.3
1.4
1.3
1.4
1.7
1.4
1.3
1.4
1.4
1.4
1.3
1.4
1.3
1.8
1.5
132
Table 5.5. (Continued)
GenBank
Gene name
NM_008941
M31441
NM_009042
NM_009789
AB030906
AK008441
NM_011388
NM_011402
NM_019754
NM_011609
AK003881
AK013986
NM_009509
Protease, serine, 7 (enterokinase) (PRSS7)
RAD52 homolog (S. cerevisiae) (RAD52)
Regenerating islet-derived 1 alpha (pancreatic stone protein, pancreatic thread protein) (REG1A)
S100 calcium binding protein G (S100G)
Sex comb on midleg homolog 1 (Drosophila) (SCMH1)
Sucrase-isomaltase (alpha-glucosidase) (SI)
Solute carrier family 10 (sodium/bile acid cotransporter family), member 2 (SLC10A2)
Solute carrier family 34 (sodium phosphate), member 2 (SLC34A2)
Transgelin 3 (TAGLN3)
Tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A)
Ubiquinol-cytochrome c reductase complex (7.2 kD) (UCRC)
Ubiquitin specific peptidase 45 (USP45)
Villin 1 (VIL1)
Fold change
1.5
1.5
2.1
1.5
1.3
1.5
1.6
1.6
1.4
1.5
1.3
1.3
1.3
133
Table 5.6. Functional analysis of differentially expressed genes (Up-regulated) after oral administration of L. acidophilus L10 in
cholera toxin mice model.
Functions & Diseases
Small molecule biochemistry
Drug metabolism
Cancer
Lipid metabolism
Cell death
Cell-cell signalling and interaction
Hematological system development and function
Carbohydrate metabolism
Cellular movement
Endocrine system development and function
Hematological diseases
Molecular transport
Skeletal and muscular development and function
Embryonic development
Immune response
Cardiovascular disease
Cardiovascular system development and function
Cell cycle
Cellular function and maintenance
Cellular growth and proliferation
Connective tissue development and functions
DNA replication, recombination and repair
Gene expression
Genes, n Functions & Diseases
11
6
6
5
4
4
4
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
Genetic disorder
Hepatic system disease
Immune and lymphatic development and function
Immunological disease
Skeletal and muscular disorder
Tissue development
Vitamin and mineral metabolism
Amino acid metabolism
Cell morphology
Cellular assembly and organisation
Cellular compromise
Cellular development
Connective tissue disorders
Developmental disorder
Digestive system development and functions
Infectious disease
Nucleic acid metabolism
Organ development
Organ morphology
Organismal injury and abnormalities
Protein trafficking
Tissue morphology
Tumor morphology
Genes, n
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
134
Table 5.7. Functional analysis of differentially expressed genes (Down-regulated) after oral administration of L. acidophilus L10 in
cholera toxin mice model.
Functions & Diseases
Small molecule biochemistry
Cancer
Cell death
Cellular movement
Lipid metabolism
Molecular transport
Cell-cell signalling and interaction
Cellular assembly and organisation
Cardiovascular disease
Cellular compromise
Hematological disease
Immune response
Tumor morphology
Cardiovascular system development and function
Cellular growth and proliferation
Genetic disorder
Hematological system development and function
Immunological disease
Inflammatory disease
Metabolic disease
Organismal injury and abnormalities
Tissue development
Cell morphology
Cellular function and maintenance
Genes, n Functions & Diseases
8
7
6
6
6
6
5
5
4
4
4
4
4
3
3
3
3
3
3
3
3
3
2
2
Digestive system development and function
Drug metabolism
Embryonic development
Endocrine system disorders
Immune and lymphatic development and function
Organ development
Organ morphology
Protein trafficking
Respiratory disease
Skeletal and muscular disorders
Carbohydrate metabolism
Cell cycle
Cell signalling
Connective tissue disorders
DNA replication, recombination and repair
Endocrine system development and function
Gastrointestinal disease
Infectious disease
Nucleic acid metabolism
Post-translation modification
Protein synthesis
Viral function
Viral infection
Vitamin and mineral metabolism
Genes, n
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
135
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
136
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 (CX-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
137
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 upregulated 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
138
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
139
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
140
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
141
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
142
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 actinbinding 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 factorinduced epithelial cell motility (Athman et al., 2003). The CD74 gene was also downregulated 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
143
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.
144
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.
145
CHAPTER 6
Concluding summary
146
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
147
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.
148
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 Mcells 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).
149
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.
150
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, IFNin 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
151
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 upregulated 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.
152
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.
153
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