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

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 my graduate study. I 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 Introduction………………………………………………………………1 1.1 History of bacteria diseases……………………………………………………1 1.1.1 Vaccination as a strategy against bacterial disease……………………… 1.1.2 Criteria for an effective vaccine……………………………………………….2 1.2 Escherichia coli diseases………………………………………………… .7 1.2.1 Strategies in preventing E.coli infections………………………………… 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 II 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 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 Naïve 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 III 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 IV Chapter 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 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 V 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 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 Discussion……………………………………………………………95 References………………………………………………………………………… 107 Appendix……………………………………………………………………………140 VI 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, IL12p40, IL-10 and IL-23 induction, heat-killed E.coli was as potent as live and UVkilled 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 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 VII 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) 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. VIII LIST OF FIGURES Pages 1.1 The life cycle of DCs……………………………………………………… .12 1.2 TLR signaling pathway………………………………………………………23 1.3 Role of TLRs in the control of adaptive immunity………………………… 24 3.1 Phenotypic analysis of DCs stimulated with live and killed E.coli………….56 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 IX 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 X 151. 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Media was sterilized as above. 50X TAE buffer Tris base Acetic acid EDTA pH 7.8 M M 0.1 M 10X DNA loading buffer Ficoll4000 EDTA, pH 8.0 Bromophenol blue 20%(w/v) 0.1M 0.25%(w/v) 1xPBS KH2PO4 Na2HPO4 NaCl KCl 1.76 mM 10.4 mM 137 mM 2.7 mM SDS-PAGE stacking gel dH2O 0.5M Tris-HCL,pH 6.8 10%(w/v) SDS 30% Acrylamide/Bis solution 29:1 (3.3%) 10% APS TEMED 3.05 ml 1.25 ml 50 l 0.65 ml 25 l l 140 SDS-PAGE separating gel (12.5%) dH2O 1.5M Tris-HCL,pH 8.8 10% SDS 30% Acrylamide/Bis solution 29:1 (3.3%) 10% APS TEMED 3.17 ml 2.5 ml 100 l 4.16 ml 50 l l 10x SDS-PAGE electrophoresis buffer Tris base Glycine SDS pH 8.3 25 mM 192 mM 1%(w/v) 5x reducing sample loading buffer Tris-HCL,pH 6.8 Glycerol SDS Bromophenol Blue -mercaptoethanol 1M 50%(w/v) 10%(w/v) 1%(w/v) 0.5ml in 10ml of sample loading buffer 10x Western blot transfer buffer Tris base Glycine Methanol 25 mM 192 mM 15%(w/v) 10x TBS Tris,pH 7.5 NaCl 50 mM 150 mM TBST buffer 1x TBS buffer with 0.05% Tween 20 Blocking buffer 1x TBS with 5% non-fat milk 141 142 [...]... 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. .. 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... 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... 16 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... 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... 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 11 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... 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... 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,... pathway include: adaptor 19 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... B and T cells, could potentially leads to the development of more effective or novel vaccines 9 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 . 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 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. IL-12p35 promoter in live, heat -killed E.coli and LPS/rmIFN-γ response……………………………………………………….79 X 5.1 Live and heat -killed E.coli are stronger activators of TLR2 and TLR4 compared to TLR5……………………………………………