Dendritic cells respond differently to live and killed bacteria molecular mechanisms of pathogen recognition by dendritic cells and implications for vaccine development

Although heat-killed E.coli induced low IL-12p70 expression from DCs, MLR result indicated that like live and UV-killed E.coli, it was able to induce Th1 response.. Disparate IL-12 induc

ACKNOWLEDGEMENTS

Many people contributed to this study in ways large and small I would like to thank them all especially the following: A/P Lu Jinhua for accepting me as a part-time graduate student and for his many advice and suggestions throughout this study; Elaine, Boon King and Jinyao for the Dendritic cells cultures; Jason for his help in the transfection studies; Stephanie and Yen Seah for ensuring this study proceed smoothly with minimum delays by faithfully replenishing all reagents and pipette tips that I used up; and last, but not least, my boss, Dr Tai E Shyong for willing to support

my decision to do my graduate study

TABLE OF CONTENTS

Contents Pages

Acknowledgements……….I Table of contents……… II Summary……….VII List of Figures……… IX List of Tables……… XI Publications……….XII Abbreviations……….XV Chapter 1 Introduction………1

1.1 History of bacteria diseases………1

1.1.1 Vaccination as a strategy against bacterial disease……… 2

1.1.2 Criteria for an effective vaccine……….2

1.2 Escherichia coli diseases……… 7

1.2.1 Strategies in preventing E.coli infections……… 8

1.2.2 Future prospects………9

1.3 Adaptive and Innate Immune System……… 10

1.3.1 Dendritic cells……… 11

1.3.2 Antigen uptake and processing………13

1.3.3 Dendritic Cells in immune regulation……… 14

1.3.4 Clinical applications of DCs……… 15

1.3.5 Future Prospects……… 16

1.4 Toll like receptors (TLRs)……… 17

1.4.1 TLRs ligands………18

1.4.2 TLRs ligands as adjuvants………19

1.4.3 TLR signaling pathway………19

1.4.4 Role of TLRs in protection from infections……….20

1.4.5 TLR expression………22

1.4.6 Medical implications of TLRs……… 25

1.4.7 Future Prospects……… 27

1.5 Aims of Study……… ………29

Chapter 2 Materials and Methods………32

2.1 Bacteria culture and preparation……… 32

2.2 DC culture………32

2.3 DC activation……… 33

2.4 Enzyme linked Immunosorbent Assay (ELISA)……….33

2.5 Flow Cytometry……… 35

2.6 Total RNA isolation……….36

2.7 Reverse transcription (RT)……… 36

2.8 Nạve CD4+ T cells isolation……… 37

2.9 Generation of anti-CD3 and anti-CD28 antibody latex beads……….38

2.10 Mixed Lymphocyte Reaction (MLR)……… 38

2.11 Nuclear extract preparation……….39

2.12 Protein concentration assay……… 40

2.13 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)……… 40

2.14 Western Blot………40

2.15 Isolation of human genomic DNA……… 42

2.16 DNA Primer Synthesis……….42

2.17 Polymerase Chain Reaction (PCR)……… 43

2.18 DNA agarose gel electrophoresis………44

2.19 Isolation and purification of DNA from agarose gels……….44

2.20 Restriction endonuclease digestion……….45

2.21 DNA ligation………45

2.22 Transformation of competent cells……… 45

2.23 Identification of positive clones by PCR……….46

2.24 Plasmid purification for transfection………46

2.25 DNA Sequencing……….47

2.26 Electroporation of RAW264.7……….47

2.27 Mammalian Cell Culture……… 48

2.28 Liposome-based transfection of HEK293T cells……….49

2.29 Treatment of HEK293T with PAMPs……… 50

2.30 Dual Luciferase Assay……….51

2.31 Expression constructs……… ………51

2.32 Databases and sequence analyses……….52

2.33 Codon optimization of the TLR2 coding sequence……… 52

2.34 Expression of CD14 using prevalent TLR codons……… 54

Chapter 3 E.coli induces maturation and cytokine production in human

dendritic cells……… 55

3.1 Surface phenotype of E.coli infected DCs……… ……55

3.2 Killed E.coli infection induces the production of cytokines in human

DCs……… 55 3.3 Induction and regulation of p35, p40 and p19 mRNA expression………… 57 3.4 IL-12p70 production in heat killed E.coli infected DCs is partly dependent on

LPS ……… 63 3.5 IL-12p70 induction in response to heat-killed E.coli is dominant over the

stimulus of live bacteria……… 64 3.6 Exogeneous IFN- restores IL-12p70 production in heat-killed E.coli

infected DCs……….64 3.7 Live, UV- and heat-killed E.coli infected DCs induces IFN- production

from CD4+ T cells……… 66

Chapter 4 Heat-killed E.coli infection inhibits the nuclear translocation of

NF-B p65, c-Rel, RelB and IRF-1 in DCs, but not p50 and p52……… 68

4.1 Heat-killed E.coli inhibits the nuclear translocation of p65, c-Rel and

RelB proteins………68 4.2 Heat-killed E.coli does not block nuclear translocation of NF-B p50

and p52……….69 4.3 Heat-killed E.coli blocks IRF-1 but not IRF-3 localization in the nucleus… 73

4.4 Cloning and construction of human IL-12p35 luciferase reporter gene

vectors……… 75

4.5 Regulatory regions of the IL-12p35 promoter in live, heat-killed E.coli and LPS/rmIFN-γ response ………78

Chapter 5 Deviation from major codons in the TLR genes is associated with low TLR expression………80

5.1 Heat-killed E.coli engages both TLR2 and TLR4………80

5.2 Most human TLR genes exhibit low frequencies of major codons………… 82

5.3 More CD14 expression is detected on monocytes than TLR1 and TLR2… 85

5.4 The TLR9-coding sequence is more effectively expressed than that of TLR1, TLR2 and TLR7……… …87

5.5 TLR2 expression is markedly increased upon partial codon optimization… 87

5.6 Partial modification of CD14 sequence using prevalent TLR codons markedly reduces CD14 expression……….90

5.7 Over-expression of TLR2 constitutively activates NF-B……… 90

Chapter 6 Discussion………95

References……… 107

Appendix………140

SUMMARY

Escherichia coli (E.coli) is the most common enteric gram-negative bacteria to cause

extraintestinal infections in the ambulatory, long term-care and hospital settings (1-3)

as well as severe food poisoning Although vaccines against E.coli are being developed, with regard to the pathophysiology of E.coli infections, the involvement of

dendritic cells (DCs) has not been clarified DCs prime T cell activation and how DCs are activated affect T cell stimulation Immune responses to live and killed organisms can differ markedly, and studies have shown that DCs respond differently to live and

killed Neisseria meningitidis and Salmonella typhimurium In this study, we compared the response of DC to live and killed E.coli Heat-killed E.coli was

indistinguishable from live and UV-killed bacteria in promoting DC expression of MHC II, CD40, CD54, CD83, CD80 and CD86 With respect to TNF-, IL-6, IL-

12p40, IL-10 and IL-23 induction, heat-killed E.coli was as potent as live and killed E.coli However, with respect to IL-12p70, heat-killed E.coli was found to be a poor inducer as compared to live and UV-killed E.coli Interestingly, heat-killed E.coli induction of IL-12p70 is dominant over live or UV-killed E.coli stimulations in

UV-co-infections study Investigation of the IL-10 profiles suggested that IL-10 could not

be the cause of the poor IL-12p70 induction in response to heat-killed E.coli Instead,

results suggested that the cause of the poor IL-12p70 induction could be due to low IL-12p35 gene expression In addition, results indicated that the low IL-12p70

production in heat killed E.coli infected DCs is LPS dependent and IFN-γ can restore IL-12p70 level to a level comparable to live or UV-killed E.coli Although heat-killed E.coli induced low IL-12p70 expression from DCs, MLR result indicated that like live and UV-killed E.coli, it was able to induce Th1 response An analysis of nuclear

translocation of NF-B family members as well as IRF-1 and IRF-3 showed

inhibition of p65, c-Rel, RelB and IRF-1 nuclear translocation in heat-killed E.coli

infected cells This was not observed with live bacteria infection In addition, we have

also determined that both live and heat-killed E.coli strongly activates Toll-like

receptors (TLRs) 2 and 4 Low TLR expression is frequently observed in published work and codon usage may explain the global expression of TLRs, thus, an analysis

of the TLRs codons against the major human codons were carried out Results showed that all TLRs except TLR9 have a reduced number of major codons in their genes Replacement of the 302bp at the 5’ end of the TLR2 gene sequence with the major human codons showed that TLR2 expression could be increased

Collectively, this study showed that the differential induction of IL-12p70 by live, UV

and heat-killed E.coli could be due to differential regulation NF-κB and IRFs nuclear

localization that affects IL-12p35 gene expression This study also showed that

despite low induction of IL-12p70 by heat-killed E.coli, they were able to induce a Th1 response similar to live and UV-killed E.coli Finally, this study showed that the

deviation of TLR sequences from using major codons dictates the low TLR expression and this may protect the host against excessive inflammation and tissue damage

3.2 Cytokines profile in human DCs by E.coli……… 61

3.3 RT-PCR analysis of IL-12p35, IL-12p40 and p19 subunit……… 62 3.4 Effects of LPS on IL-12p70 secretion from DCs……….63 3.5 Co-stimulation of Live and heat-killed E.coli does not fully restores IL-12p70

production in DCs ……… ……….65 3.6 Effects of IFN- on IL-12p70 secretion from DCs in response to heat-killed

E.coli………65

3.7 IFN-, IL-4 and IL-17 production by CD4+ T cells in response to E.coli

infected DCs……… ……… 67 4.1 Nuclear translocation of NF-B family members p65, c-Rel and RelB…….71 4.2 Nuclear translocation of NF-B familty members p50 and p52……… 72 4.3 Nuclear localization of IRF-1 and IRF-3……….74 4.4 Cloning and construction of human IL-12p35 promoter luciferase reporter

gene vectors……….76 4.5 Generation of deletion mutants………77 4.6 Regulatory regions of the IL-12p35 promoter in live, heat-killed E.coli and

LPS/rmIFN-γ response……….79

5.1 Live and heat-killed E.coli are stronger activators of TLR2

and TLR4 compared to TLR5……… 81 5.2 Expression of CD14, TLR1, TLR2 and TLR9………86 5.3 Partial optimization of TLR2 codons increases TLR2 expression………… 89 5.4 Partial replacement of CD14 sequence with prevalent TLR codons

reduces CD14 expression……….91 5.5 Over-expression of TLR2, but not TLR1, TLR4, TLR7 or TLR9,

causes constitutive NF-B activation……… 93 5.6 Constitutive NF-B activation upon over-expression of TLR2……… 94

LIST OF TABLES

Pages

1.1 Examples of vaccines avaliable against bacteria infections……… 3

1.2 Criteria for an effective vaccine……….3

1.3 Commonly used adjuvants in humans ………7

1.4 TLRs ligands……… …….19

1.5 Codon usage in E.coli and S.cerevisiae………31

2.1 Sequences and annealing temperature for primers used in RT-PCR analysis……….37

2.2 Primers to generate the IL-12p35 promoter fragments………43

5.1 Codon usage in the human genome and the CD14 and TLR genes………….84

PUBLICATION

1 Fei Zhong, Weiping Cao, Edmund Chan, Puei Nam Tay, Florence Feby Cahya,

Haifeng Zhang and Jinhua Lu (2005) Deviation from major codons in the Toll-like receptor genes is associated with low Toll-like receptor expression

Immunology, 114, 83-93

2 Edmund Chan, Elaine Min Chern Lai, Boon King Teh and Jinhua Lu (2009)

Disparate IL-12 induction from dendritic cells by live and killed bacteria

through distinct NF-κB and IRF-1 activation Manuscript in preparation

Other Publications

1 Tai ES, Koay ES, Chan E, Seng TJ, Loh LM, Sethi SK, Tan CE

Compound heterozygous familial hypercholesterolemia and familial defective apolipoprotein B-100 produced exaggerated hypercholesterolemia

Clin Chem 2001 Mar;47(3):438-43

2 ES Tai, Ordavas JM, Corella D, Deurenberg-Yap M, Chan E, Adiconis X,

Chew SK, Loh LM, Tan CE

TaqIB and –629C>A polymorphisms at the cholesteryl ester transfer protein locus: associations with lipid levels in a multiethnic population

The 1998 Singapore National Health Survey

Clin Genet 2003 Jan;63(1):19-30

3 Chan E, Tan CS, Deurenberg-Yap M, Chia KS, Chew SK, Tai ES

The V227A polymorphism at the PPARA locus is associated with serum lipid concentrations and modulates the association between dietary polyunsaturated fatty acid intake and serum high density lipoprotein concentrations in Chinese woman

Atherosclerosis 2006 Aug;187(2):309-15

Poster Presentations

1 E Chan, LM Loh, ES Tai, ESC Koay, JM Ordavas, CE Tan

Single Strand Conformation polymorphism mutation detection at the LDLR locus in Singaporean patients with familial hypercholesterolemia

72nd Congress of the European Atherosclerosis Society, Glasgow

2 ES Tai, E Chan, LM Loh, BS Chew, CE Tan

Determinants of HDL cholesterol in Singaporean men with and without diabetes mellitus

2nd Congress of the Asian Pacific Society of Atherosclerosis and Vascular Disease Chiang Mai, Thailand Jan 2000

3 ES Tai, E Chan, BS Chew, Y Zhao, CE Tan

Genetic Analysis of familial hypercholesterolemia in Singapore Initial experience from the MED PED program

Asian Pacific Society of Atherosclerosis and Vascular Disease 1999 Workshop in Osaka May 2001

4 E.Chan, ES Tai, CS Tan, CE Tan, KS Chia, SK Chew, JM Ordavas

A novel polymorphism at the PPARA locus (V227A) interacts with dietary factors to determine lipid profiles in Chinese

XIIIth International Symposium on Atherosclerosis, Kyoto, Japan, Sep 2003

ABBREVIATIONS

AP alkaline phosphatase

BCRs B-cell receptors

BSA bovine serum albumin

DMEM Dulbecco’s modified Eagle’s medium

DNA deoxyribonucleic acids

E.coli Escherichia coli

EDTA ethylene diamine tetra acetic acid

FCS fetal calf serum

FITC fluorescein isothiocyanate

GM-CSF ganulocyte macrophage-colony stimulating factor

mRNA messenger RNA

MyD88 myeloid differentiation factor

NF-B nuclear factor kappa B

OD optical density

PBS phosphate buffered saline

PCR polymerase chain reaction

RNA ribonucleic acid

RPMI RPMI-1640 medium developed by Rosell Park Memorial Institute RT-PCR reverse transcription polymerase chain reaction

SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Chapter 1 Introduction

1.1 History of bacteria diseases

Bacteria are a large group of unicellular microorganisms that are typically 0.5-5 micrometers in length They are of various shapes, ranging from spheres to rods and spirals They can be found in every habitat on earth such as soil, water, plants and animals as well as in our stomach and intestines In addition, bacteria has also been used for centuries in the preparation of fermented food such as cheese, pickles, soy sauce, sauerkraut, vinegar, wine and yoghurt (4,5)

The vast majority of bacteria in our body are rendered harmless by the protective effects of the immune system, and a few are beneficial However, a few species of bacteria are pathogenic and cause infectious diseases History has shown that such pathogenic bacteria can cause widespread epidemics of human civilization Infectious diseases such as tuberculosis, typhus, plague, diphtheria, typhoid, cholera, dysentery and pneumonia have taken a large toll on humanity (6) At the beginning of the twentieth century, pneumonia, tuberculosis and diarrhea were the three leading causes of death Water purification, immunization and antibiotic treatment have reduced the morbidity and the mortality of bacterial diseases in the twenty-first century, at least in the developed world where these are acceptable cultural practices However, in developing countries with little money to spend on health care, such deadly infectious diseases have been allowed to gain ground Unfortunately, in these modern times where people travel fast and frequent, the bacteria travel with them (6) This can result in an epidemic or pandemic if the population is not immune to that disease

Bacteria have many different strategies to make us ill Understanding how the innate immune system respond to bacteria infection and how this can lead to adaptive immunity can result in better treatment, vaccination or prevention of that infectious disease

1.1.1 Vaccination as a strategy against bacterial disease

Our immune cells have the ability to specifically recognize and destroy bacteria Bacteria or bacteria toxins that enter our body will encounter the cells and mechanisms of the innate immune system which results in antibody production against the bacteria after infection This information is stored in the memory of antibody-producing B cells as well as in T cells, and, during a second infection, the bacteria will be destroyed before it can cause disease

Vaccination involves the induction of protective immune responses against common microbial pathogens in humans Table 1.1 list some of the bacterial diseases for which vaccination has become a commonly used strategy for prevention This immune mechanism allows us to induce immunity with a vaccine made from components of the infecting bacteria or the toxin that the bacteria produced so as to prevent future infections with the natural, full-strength bacteria

1.1.2 Criteria for an effective vaccine

An effective vaccine needs to meet several criteria (Table 1.2) The first of these

is that it must be safe, i.e it does not cause illness or death Several strategies have been utilized in designing vaccines against microbes to ensure their safety These are typically based on fundamental information about the microbe, such as how it infects cells and how the immune system responds to it These different

strategies were used to reduce the risk of illness, while retaining the ability to induce a beneficial immune response

Table 1.1 Examples of vaccines available against bacteria infections

Haemophilus influenzae B Streptococcus pneumoniae

Table 1.2 Criterias for an effective vaccine

Features of effective vaccines

Safe Vaccine must not itself cause illness or death Protective Vaccine must protect against illness resulting

from exposure to live pathogen Gives sustained protection Protection against illness must last for several

years Induces neutralizing antibody Neutralizing antibody is essential to prevent

infection Induces protective T cells Some pathogens are more effectively dealt with

by cell-mediated responses Practical considerations Low cost per dose; Biological stability; Ease of

administration and Few side effects Adapted from Janeway 6th edition

(1) Live attenuated microbes

This contains living microbes that have been weakened in the laboratory so that it cannot cause disease As these vaccines are closest in mimicking a natural infection, they elicit strong cellular and antibody responses and often confer life-long immunity with only one or two doses Unfortunately, a possibility exists that

an attenuated vaccine could revert to a virulent form by mutation Also, live attenuated vaccines are not suitable for people with damaged or weakened immune system In addition, live attenuated vaccines are more difficult to create for bacteria as compared to virus Bacteria have thousands of genes that are in contrast to the small number of genes in the virus genome This means that it is much harder to find the optimal gene mutations that render the bacteria non-infectious yet live and immunogenic The Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis is an example of a live attenuated bacteria vaccine

(2) Inactivated microbes

These vaccines are based on pathogens that are killed through chemical fixation, heat-inactivation, or radiation This leads to more stable or safer vaccines that cannot mutate back to their disease-causing state However, these vaccines stimulate a weaker immune response than live vaccines, thus requiring several additional doses The whole cell/recombinant B subunit (WC/rBS) vaccine against cholera is an example of inactivated bacteria vaccine (7)

(3) Subunits of the microbe

These vaccines include only the antigens that are best recognized by the immune system Since subunit vaccines contain only the essential antigens, chances of

adverse reactions to the vaccine are reduced The WC/rBS vaccine against cholera

is also considered a subunit vaccine

(4) Inactivated toxins

Toxoid vaccines are derived from bacteria that secrete toxins that cause illness They are usually inactivated with formalin When a toxoid vaccine challenges the immune system, antibodies will be produced that bind to and neutralize the toxin

A vaccine against tetanus is an example of toxoid vaccines The tetanus toxoid

vaccine is prepared from the tetanospasmin toxin produced by Clostridium tetani

that causes tetanus

Beside safety, an important criterion in vaccine development is the ability to induce long-lived immunological memory This requires the priming of both B and T lymphocytes Methods which attempt to enhance the immunogenicity of a vaccine include conjugation with carrier antigens and the use of adjuvant

(1) Conjugation of polysaccharide vaccine with carrier proteins

Conjugate vaccines are often made when the protective antigens are polysaccharides and these antigens are usually components on bacterium outer membranes These polysaccharides disguise other bacterium antigens from the immune system In a conjugate vaccine, carrier proteins, which are often toxoids from a different microbe, are linked to the polysaccharide antigens The carrier proteins are important because the polysaccharide antigens, which can be recognized by B cell antigen receptors (BCR), cannot be recognized by T cell receptors (TCR) because TCRs bind to peptide but not carbohydrate epitopes This linkage provide TCR binding sites on the antigen and therefore helps the

immune system recruit T cell help to induce isotype switching in B cells for the production of high affinity antibodies that react to the polysaccharide coatings and

defend against the disease causing bacterium The vaccine against Haemophilus influenzae type B is an example of a conjugate vaccine (8)

(2) Adjuvant

A vaccine’s immunogenicity often depends on accompanying adjuvant that is included with it Some adjuvant can enhance immunogenicity by converting soluble antigens into particulate materials that is more readily ingested by antigen-presenting cells (APC) such as dendritic cells (DCs) and macrophages Some adjuvants are bacterial components that augment the effects of a vaccine by stimulating the immune system for more vigorous responses As a result, these adjuvant increase host immunity to infections These microbial components are mostly ligands of Toll-like receptors (TLRs) which are abundantly expressed by APCs The immune system have evolved to recognize bacteria and bacterial components with these receptors, the incorporation of adjuvant in conjunction with the vaccine can greatly increase the innate immune response to the antigen

by augmenting the activities of DCs, lymphocytes and macrophages by mimicking

a natural infection They also induce the production of inflammatory cytokines and potent local inflammatory responses For example, purified constituents of

Bordetella pertusis is used as both antigen and adjuvant in the triplex DPT

(diphtheria, pertusis, tetanus) vaccine against these diseases

Table 1.3 Commonly used adjuvants in humans

Microbial natural and synthetic

derivatives

Cholera toxin and CpG DNA respectively

Adapted from Silvia et al 2010 FEMS Immunol Med Microbiol 58: 75-84

1.2 Escherichia coli diseases

Escherichia coli (E.coli) is a gram-negative bacterium commonly found in the lower intestines of mammals Most E.coli strains are harmless, but some, such as

serotype O157:H7 (9) are known to cause severe food poisoning in humans

Numerous types of diarrhoeagenic E.coli strains such as enteropathogenic (EPEC),

enterohaemorragic (EHEC) and enterotoxigenic (ETEC) have been identified worldwide Of these, ETEC strains are an important cause of infantile diarrhoea in developing countries where there is no adequate clean water and poor sanitation (10) They are the most commonly isolated bacterial enteropathogen in children below 5 years of age in developing countries, and account for several hundred million cases of diarrhoea and several ten of thousand deaths each year (11) ETEC was thought to account for approximately 200 million diarrhoea episodes and 380 000 deaths annually (12) ETEC are also the most common cause of travellers' diarrhoea that affects individuals from industrialized countries travelling to developing regions of the world (13,14) Each year, there are an estimated 10 million cases of ETEC travellers' diarrhoea worldwide (15)

Besides causing food poisoning, there are also pathogenic strains of E.coli known

to cause extraintestinal infections (extraintestinal pathogens E.coli [ExPEC]) (16)

These strains can be part of the normal intestinal flora and are isolated in 11% of healthy individuals (17) They do not cause gastroenteritis, but their main feature

is their ability to colonize extraintestinal sites and to induce infections in organs They are involved in urinary tract infections (UTI), septicemia, diverse abdominal infections, meningitis (18-20), bacteremia and sepsis (21-23) In the United States

(US), the annual costs of extraintestinal infections syndromes due to E.coli ranges

from 1 million to 1 billion US dollars (24) Therefore, from both a medical and an economical point of view, prevention of ExPEC infections is desirable

1.2.1 Strategies in preventing E.coli infections

Vaccines represent a rational approach for the prevention of E.coli infections

Studies have shown that ETEC infections in children are immunizing (25,26), as reflected by the declining rates of ETEC diarrhea with increasing age This suggests that immunizing against ETEC can be an effective preventive strategy Various types of vaccines being developed against ETEC infections shows varying degrees of success The oral killed WC/rBS cholera vaccine consisting of

4 batches of heat- or formalin-killed whole-cell Vibrio cholerae O1 and

supplemented with purified recombinant cholera toxin B-subunit was found to

prevent 52% of episodes due to ETEC infections as the heat-labile toxin of E.coli

cross-reacts with cholera toxin, but, this protection does not last more than a few months (27) In contrast, a vaccine based on recombinant cholera toxin B combined with 4 formalin-killed ETEC strains has been the most successful (28,29) It has shown a 80% protection against ST (heat-stable toxin)-ETEC diarrhea (30) Besides inactivated and subunit vaccines, live attenuated ETEC vaccine approach is also being pursued (31,32) The live attenuated ETEC strain

ACAM 2010, was found to be well tolerated and 73% immunogenic when fed to

human volunteers A polysaccharide conjugate vaccine against E.coli strain

O157:H7 has been developed and a phase 2 trial has been completed in children (33) It was found to be safe and immunogenic in young children The usual strategy against ExPEC infections are the use of antibiotics However, ExPEC strains are showing resistance to various classes of antibiotics (34,35) This has become a major concern in hospitals as the antimicrobial resistance will make the future management of ExPEC infections more difficult Therefore, like ETEC infections, vaccines also represent a rational approach for the prevention of ExPEC infections (36,37)

1.2.2 Future prospects

The diversity, frequency, potential severity and economic impact of E.coli infections are as great as for any bacterial pathogens Furthermore, E.coli strains have shown different infectious potential The diversity of E.coli strains and the requirement to induce immunity makes the development of vaccines against E.coli

infections a challenge Since, one of the criteria for an effective vaccine is the generation of long-lived immunological memory by the priming of both B and T

cells, an understanding of how E.coli interacts with APCs such as Dendritic cells

which secretes cytokines that primes both B and T cells, could potentially leads to the development of more effective or novel vaccines

1.3 Adaptive and Innate Immune System

The generation of long-lived immunological memory by the priming of both B and T cells is part of the adaptive immune system This system is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogenic challenges The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens to generate immunity, and to mount stronger attacks each time the pathogen is encountered Adaptive immunity also provides specific immune response by producing antibodies against a particular pathogen The adaptive immune system

is composed of B and T cells that recognise antigens through their antigen receptors expressed on the surface ie the B and T cell receptors (BCR and TCR) This system is highly adaptable and can respond to a wide range of antigens by generating random and highly diverse BCRs and TCRs by somatic gene recombination The interaction of the BCR and TCR with antigens activates the B and T cells leading to proliferation and the generation of antibodies (for B cells) and the generation of armed effector T cells such as cytotoxic T cells and helper T cells (Th) (for T cells) Some of these B and T cells will eventually become memory cells The immunological memory created is the basis of vaccination However, adaptive immunity would not occur without co-stimulatory signals from the innate immune system Innate immunity is an essential prerequisite for the adaptive immune response This is because the co-stimulatory molecules that are induced on cells of the innate immune system during their interaction with pathogens activate the antigen-specific lymphocytes of the adaptive immune response The cytokines produced as a result of the interaction between innate

immune cells and pathogens have an important role in stimulating the subsequent adaptive immune response and shaping its development

The innate immune system is an ancient system comprising of cells and mechanisms that defend the host from infection with broad specificity It is acquired at birth and is phylogenetically conserved and is present in almost all multicellular organisms, including invertebrates and plants (38) It acts as the first line of defense against pathogens This immune response is usually triggered when pattern recognition receptors (PRRs) on innate immune cells recognizes components (eg lipopolysaccharide) that are conserved among broad groups of microorganisms The response to pathogens in the innate immune system is immediate, mediated by neutrophils, natural killer cells (NK), macrophages and dendritic cells, cells that phagocytose and kill the pathogens Among the innate immune cells, dendritic cells are the most potent activators of adaptive immunity

1.3.1 Dendritic cells

Dendritic cells (DCs) are the key cell types involved in the connection between the innate and adaptive immune responses (Fig 3) They are bone marrow-derived leukocytes capable of initiating primary immune responses upon maturation (39) They are widely distributed in tissues such as the epidermis, intestines, heart, kidneys, blood and lymph DC precursors migrate from the bone marrow to the peripheral tissues where they differentiate into immature DCs, which are characterized by the capacity to uptake antigens and to phagocytose macroparticles In response to infectious agents and inflammatory products, immature DCs undergo a maturation process involving phenotypic and functional changes (39-43) DC maturation can be characterized by upregulation of MHC

and costimulatory molecules such as CD80, CD86 and CD40 (44-47), by cytokine production (eg IL-12, IL-10, TNF-α and IL-6) and by the ability to activate T cells (39,48-50) During the process of maturation, DCs migrate towards secondary lymphoid organs where they provide antigens and inflammatory signals

to promote the activation of naive T cells, B cells and Natural killer (NK) cells (39,48)

Figure 1.1 The life cycle of DCs (a) Circulating precursor DCs enter tissues as immature DCs

They can directly encounter pathogens (e.g a virus is shown here), which induce secretion of cytokines such as IFN-α, which in turn activate effector cells of innate immunity such as

eosinophils, macrophages and NK cells (b) Following antigen capture, immature DCs migrate to

lymphoid organs where, after maturation, they display peptide–MHC complexes, which allow

antigen presentation and selection of rare circulating antigen-specific lymphocytes (c) These

activated T cells help DCs in terminal maturation, which allows lymphocyte expansion and

differentiation to mediate adaptive immunity (d) Activated T lymphocytes (cytotoxic T

lymphocytes [CTLs], helper T cells and Tr cells) traverse inflamed epithelia and reach the

injured tissue, where they eliminate microbes and/or microbe-infected cells (e) Activated B cells

migrate into various areas where they mature into plasma cells that produce antibody (Ab) that

neutralizes the initial pathogen Karolina P and Jacques B 2002, Current Opinion in

Immunology 14:420-431

1.3.2 Antigen uptake and processing

In most tissues, DCs are present in the immature state where they are unable to stimulate T cells as they lack the co-receptors necessary for T cell activation, such

as CD40, CD54 and CD86 However, they have antigens presentation ability as they are (1) able to take up particles and microbes by phagocytosis (51-53), (2) able to form large pinocytic vesicles in which extracellular fluid and solutes are sampled, a process called macropinocytosis (54) and (3) express receptors such as macrophage mannose receptor (54), Fc and Fc receptors (55) that mediate adsorptive endocytosis Both the process of macropinocytosis and receptor-mediated antigen uptake makes immature DCs a very efficient antigen-presenting cell (APC) as picomolar and nanomolar levels of antigens are sufficient (54) Immature DCs constantly sample the surrounding environment for pathogens such

as viruses and bacteria This is done through pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs) (Fig 1.3) TLRs recognize specific pathogen-associated molecular patterns (PAMPs) found on pathogens Once the immature DCs have captured an antigen, its’ skills to capture antigen declines In addition, the antigen would also provide a signal for the immature DCs to mature and migrate to the lymph node The ingested antigen proteins would be degraded into small pieces and their fragments would be presented on the DCs surface using MHC molecules Simultaneously, cell-surface receptors that act as co-receptors in T-cell activation such as CD80, CD86, and CD40 greatly enhancing their ability

to activate T-cells were also upregulated CCR7, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to a lymph node were upregulated too At the lymph node, mature DCs as antigen-presenting cells: they activate helper T-cells and

killer T-cells as well as B-cells by presenting them with antigens derived from the pathogen, alongside non-antigen specific costimulatory signals

1.3.3 Dendritic Cells in immune regulation

DCs maturation plays a critical role in determining the type of induced immune response Upon maturation, DCs secrete cytokines Studies have shown the important role of DC-derived cytokines in determining the polarity of the immune response, Th1 or Th2 or Th17 immune response (42) DC can be induced to secrete cytokines by exposure to bacteria, viruses, and protozoa (56-59) or their derivatives such as Lipolysaccharide (LPS), double stranded RNA, or unmethylated DNA containing CpG motifs (60-62) Among the cytokines secreted by DCs is interleukin-12 (IL-12), a heterodimeric protein consisting of the p40 and p35 subunits encoded on separate chromosomes that is known to bias CD4+ T cells toward Th (T helpher) 1 differentiation (63,64) In addition, DCs were reported to show differential response to distinct pathogens For example, different bacteria LPS were reported to induce different classes of immune

responses E.coli LPS induces a Th1 response via IL-12 whereas Porphyromonas gingivalis LPS induces a Th2 response (65) Furthermore, experiments have also

shown that different forms of the same microbe can modulate DCs to induce

different responses The yeast form of the fungus Candida albicans induces a Th1

response via induction of IL-12 in DCs, whereas its hyphal form inhibits IL-12 and stimulates IL-4 production in DCs (66)

Studies have shown that cytokines released by DCs following virus infection play

a role in the development of anti-viral immunity Infection with influenza virus or measles virus has been demonstrated to induce large amounts of Interferon (IFN)

/, in addition to TNF- and IL-12 (56,57,67) IFN-/ can inhibit viral replication in infected cells and, together with IL-12 support the development of

an antiviral Th1/CTL (Cytotoxic lymphocyte) response (68-70)

DCs are also known to have a major effect on B cell growth and immunoglobulin secretion They stimulate the production of antibodies and the proliferation of B cells that have been stimulated by CD40L on activated T cells In addition, DCs also induce immunoglobulin class switching of these T cell activated B cells IL-

10 and TNF- can induce the secretion of IgA1 and IgA2 (71)

1.3.4 Clinical applications of DCs

The knowledge on DC biology, functions and the interactions between DCs and microbes can be used as the basis for the design of DC-based immunoprevention and immunotherapy against infectious diseases and cancer Vaccination with DCs holds the promise of generating long-lasting immunity Studies have shown that injection of pathogen loaded DCs can lead to the development of protective

immunity in models of infectious diseases, such as Chamydia trachomatis and Borrelia burgdorderi DCs pulsed with dead Chlamydia trachomatis produced IL-

12p40 and, when administered intravenously; provided protection to the female genital tract equivalent to that seen following live infection (72) Similar protective effects after immunization with microbe pulsed DCs have also been

observed in infection by Borrelia burgdorferi (73)

In cancer, phase I and II clinical trials of antigen-pulsed DCs have been carried out for different types of tumors, such as non-Hodgkin lymphoma, multiple myeloma, prostate cancer, melanoma, colorectal cancer and certain types of lung

cancer These clinical trials have shown that vaccination with antigen-loaded DCs

is safe and promising in the treatment of cancer (74)

DCs have also been tested as therapeutic agents in animal models of autoimmune disease, such as autoimmune (type I) diabetes, experimental allergic encephalomyelitis and multiple sclerosis and allergic asthma (75)

of certain vaccines preparations in vivo also lacks the capacity to stimulate DCs in vitro (80) However, vaccines that are able to engage multiple TLR ligands on

DCs have been shown to be successful (81) DCs have been reported to elicit different host responses against live versus killed bacteria in previous studies (82-85) In addition, killed bacteria or lysates/fractions are often used as vaccines Protocols used in bacteria killing also varied, including UV irradiation, chemical fixation and heating (82-85), and molecular markers were used to assess DC

activation Therefore, further understanding on how bacteria interacts with DCs in terms of induction of DC maturation and cytokines production may provide a strategy for improving the efficacy of vaccines against infectious diseases In addition, DC responses to live and killed bacteria may also provide important insights into the molecular mechanisms of how DC senses microbial pathogens These may help in targeting of vaccines to DCs for specific protections

1.4 Toll like receptors (TLRs)

TLRs are evolutionarily conserved, germline encoded receptors that recognize PAMPs in microorganisms They are type I transmembrane glycoproteins that serve as a key part of the innate immune system They are characterized by an extracellular leucine-rich repeats motifs and an intracellular Toll/IL-1 receptor (TIR) domain (86-88) The number of LRRs varies in the different TLRs, and is presumably involved in ligand binding or for dimerization (89) The TIR domain

is the motif of the TLR/interleukin-1 receptor superfamily, which includes receptors for cytokines such as interleukin (IL)-1 and IL-18 (90) It is about 200 amino acids long and contains three highly conserved regions The TIR domain mediates protein-protein interactions between the TLR and signal transduction components

The first identified member of the Toll family, Drosophila Toll, was discovered as

a maternal-effect gene that functions in a pathway that controls dorsoventral axis formation in fruitfly embryos (91,92) It was later found to be involved in anti-fungal defence (93) which senses fungal infection indirectly and is activated by the cleavage product of Spatzle Activation of Toll results in the recruitment of adaptor Tube and protein kinase Pelle to the membrane, leading to the degradation

of Cactus with the release of NF-B (nuclear factor B) like protein Human homologues for these Drosophila signaling components (eg MyD88 for tube, IRAK for Pelle and IB for Cactus) were reported (94) However, no human homologue of Spatzle has been found

In mammalin species, there are ten identified TLRs, and each have a distinct function in innate immune response The first mammalian TLR identified was TLR4 Its identification was based on its sequence similarity to Toll and IL-1R (Interleukin 1 receptor) (95) Functionally, it was shown to activate NF-B upon dimerization Similarly, the other nine TLRs were also identified based on their sequence features (96-100)

1.4.1 TLRs ligands

Most ligands recognize by TLRs can be classified as PAMPs, but non-PAMP ligands have also been shown to act via TLRs (eg heat shock proteins, extracellular matrix degradation products and anti-viral drugs) TLR recognition

of some of these ligands has been identified using in vitro systems such as ligand

binding assays or knockout mice Some of the known TLR ligands are listed in table 1.4

1.4.3 TLR signaling pathway

Activation of signal transduction pathways by TLRs leads to the induction of various genes that function in host defence, including inflammatory cytokines, chemokines, major histocompatibility complex (MHC) and co-stimulatory molecules (104) The essential components of this pathway include: adaptor

molecule myeloid differentiation factor 88 (MyD88), IL-1 receptor-associated kinase (IRAK), TNF receptor-associated factor (TRAF) 6, mitogen-activated protein (MAP) kinases, Toll-interacting protein (TOLLIP) and nuclear factor (NF)-B (Fig 1.2) Activation of TLRs results in the activation of NF-B by dissociation and phosphorylation of an inhibitor IB and its subsequent degradation Upon released from its inhibitor, NF-B moves from the cytoplasmic region to the nucleus where it binds to the promoters of genes of several cytokines, chemokines, and adhesion molecules and activates the production of all the molecules

However, not all TLRs transduce signals via this pathway TLR3 and TLR4 are known to transduce signals by a MyD88-independent pathway They mediate the activation of IRF-3 and the induction of IFN- IFN- activates signal transducer and activator of transcription 1 (STAT 1) leading to the production of several IFN-induced genes that are protective against viral infections (105,106)

1.4.4 Role of TLRs in protection from infections

The primary function of TLRs is to provide immediate protection from invading pathogens (107,108) The pivotal role of TLRs in innate host defenses was first indicatedby the finding that C3H/HeJ mice with nonfunctional TLR4 areresistant

to LPS-mediated shock (109) Besides the activation of complement pathway and phagocytosis, TLRs also induces anti-microbial proteins and peptides in various cell types such as cells of myeloid origin and Paneth cells of the gut epithelium (110) TLRs also induce activation of cytokines such as IL-1β, IL-6, TNF and chemokines such as KC-1 and MCP-1 that collectively induce acute inflammatory responses to pathogens In macrophages, TLRs induces enhanced phagocytosis

and killings by the macrophages In response to viral DNA or RNA, TLRs induce activation of type I interferon which leads to the transcription of anti-viral proteins necessary to clear viral infections TLR3, TLR7, TLR8 and TLR 9 are known to participate in viral recognition (111-115)

Evidence has suggested that functional activation of the adaptive immune system depends on signals from TLRs Adaptive immune responses are initiated when T cells recognize foreign peptides bound to self-MHC molecules on APCs The priming of nạve CD4+ T cells is a critical event in induction of adaptive immunity as differentiation of these cells into Th1 or Th2 cells, determine the nature of adaptive immune response (116) T cell activation requires not only recognition of foreign-peptide-MHC complex on APC but also stimulation by costimulatory molecules such as CD80/CD86 DCs are the major APCs responsible for priming nạve CD4+ T cells (39) The priming of T cells is controlled by TLRs (Fig 1.3) TLRs ability to bind products of microbial origin allows them to signal the presence of infection and to direct the adaptive immune response against antigens of microbial origins The role of TLR mediated recognition in the control of adaptive immune response was studied using MyD88-deficient mice These mice showed a block in antigen specific T-cell proliferation, the production of interferon- (IFN-) and the generation of ovalbumin specific antibodies when they are immunized with ovalbumin mixed with complete Freund’s adjuvant (CFA) (117) This result shows the crucial requirement of TLR mediated recognition in the generation of antigen specific Th1 responses

T cells by the DC subsets (124) In addition, different TLRs show distinct expression patterns during DC maturation For example, TLR3 is only expressed

in mature DCs (125)

Figure 1.2 TLR signaling pathway The Toll/interleukin-1 (IL-1)-receptor (TIR)-domain-

containing adaptor molecule MyD88 (myeloid differentiation primary-response protein 88) mediates the Toll-like receptor (TLR)-signalling pathway that activates IRAKs (IL-1-receptor- associated kinases) and TRAF6 (tumour-necrosis-factor-receptor-associated factor 6), and leads to the activation of the IKK complex (inhibitor of nuclear factor-B (IB)- kinase complex), which consists of IKK-, IKK- and IKK- (also known as IKK1, IKK2 and nuclear factor-B (NF-B) essential modulator, NEMO, respectively) This pathway is used by TLR1, TLR2, TLR4, TLR5, TLR6, TLR7 and TLR9 and releases NF-B from its inhibitor so that it translocates to the nucleus and induces expression of inflammatory cytokines TIRAP (TIR-domain-containing adaptor protein), a second TIR-domain-containing adaptor protein, is involved in the MyD88-dependent signalling pathway through TLR2 and TLR4 By contrast, TLR3- and TLR4-mediated activation

of interferon (IFN)-regulatory factor 3 (IRF3) and the induction of IFN- are observed in a independent manner A third TIR-domain-containing adaptor, TRIF (TIR-domain-containing adaptor protein inducing IFN-), is essential for the MyD88-independent pathway The non-typical IKKs IKK- and TBK1 (TRAF-family-member-associated NF-B activator (TANK)-binding kinase 1) mediate activation of IRF3 downstream of TRIF A fourth TIR-domain-containing adaptor, TRAM (TRIF-related adaptor molecule), is specific to the TLR4-mediated, MyD88-

MyD88-independent/TRIF-dependent pathway Akira, S., and K Takeda 2004 Nat Rev Immunol 4:499-511

Figure 1.3 Role of TLRs in the control of adaptive immunity TLRs sense the presence of

infection through recognition of PAMPs (pathogen-associated molecular patterns)

Recognition of PAMPs by Toll-like receptors (TLRs) expressed on antigen-presenting cells

(APC), such as dendritic cells, upregulates cell-surface expression of co-stimulatory (CD80

and CD86) molecules and major histocompatibility complex class II (MHC II) molecules

TLRs also induce expression of cytokines, such as interleukin-12 (IL)-12, and chemokines and

their receptors, and trigger many other events associated with dendritic cell maturation

Induction of CD80/86 on APCs by TLRs leads to the activation of T cells specific for

pathogens that trigger TLR signalling IL-12 induced by TLRs also contributes to the

differentiation of activated T cells into T helper (TH)1 effector cells It is not yet known

whether TLRs have any role in the induction of T H 2 responses IFN-γ; interferon-γ; PRR,

pattern-recognition receptor Ruslan Medzhitov 2004, Nature Reviews 1:135-145