Differential host immune responses in BALB c and C57BL 6 mice to burkholderia pseudomallei infection

DIFFERENTIAL HOST IMMUNE RESPONSES IN BALB/C AND C57BL/6 MICE TO BURKHOLDERIA PSEUDOMALLEI INFECTION KOO GHEE CHONG (B SC (HONS.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2006 i Acknowledgements I really would not have finished this project if not for the many people who have been always there to inspire, love, guide, teach, support and motivate me during the past few years of challenging journey that I have gone through I would like to express my heartfelt gratitude to my supervisor Dr Gan Yunn Hwen, for her constant guidance, advice, patience and understanding I would also like to thanks Ms Lim Soh Chan for her constant help, technical support, advice, prayers, comments, comforting and encouraging words I am grateful to all my friends and labmates, especially Sun Guang Wen, Lee Tien Huat, Liu Boping, Xie Chao, Ong Yong Mei, Chen Kang, Chan Ying Ying, Cheryl Lee, Justin Lee, Ng Kian Hong, Hu Huan, Wong Kok Lun, Ng Hui Ling, Hii Chung Shii, Chen Yahua, Tang Soong Yew, Lim Kok Siong, Lu Guodong, Dawn Koh, Chan Mann Yin, Bian Hao Sheng, Fei Wei Hua, Low Choon Pei, Clarence Kho, Jowett Wong, Lim Chih Gang, Joshua Lau and many others for their assistance, encouragement and friendship To my parents and family members for their love, support and understanding all these years To brothers and sisters from my church for constantly keeping me in prayers Most of all, I am eternally thankful to God for sustaining me and bringing me through all the difficulties, for constantly staying by my side and being the source of my strength, inspiration and hope ii Table of Contents Contents Page Title Page…………………………………………………………………………… .i Acknowledgements……………………………………………………………………….ii Table of Contents……………………………………………………………………… iii Summary………………………………………………………………………………….ix List of Tables…………………………………………………………………………… x List of Figures……………………………………………………………………………vii List of Abbreviations……………………………………………………………………viii Chapter Introduction………………………………………………………………….1 1.1 Melioidosis………………………………………………………………………… 1.2 Prevalence and epidemiology……………………………………………………… 1.3 Melioidosis in Singapore…………………………………………………………… 1.4 Modes of transmission…………………………………………………………… 1.5 Clinical manisfestations…………………………………………………………… 1.6 Diagnosis…………………………………………………………………………… 1.6.1 Identification of Burkholderia pseudomallei……………………………………7 1.6.2 Serological tests…………………………………………………………………8 1.6.3 Molecular identification techniques…………………………………………… 1.7 Management and treatment … …………………………………………………… 1.8 Bacterial pathogensis……………………………………………………………… 11 1.8.1 Gene and genome………………………………………………………………11 1.8.2 Virulence factors……………………………………………………………….11 iii 1.9 Animals model for melioidosis…………………………………………………… 13 1.10 Role of cytokines in immunity………………………………………………… ….15 1.11 Objectives of present study…………………………………………………………18 Chapter Characterization of B pseudomallei mucosal infection model…… … 20 2.1 Introduction…………………………………………………………………………21 2.2 Materials and methods………………………………………………………… 23 2.2.1 Animals……………………………………………………………………… 23 2.2.2 Bacteria ………………………………………………………………………23 2.2.3 LD50 determination… ……………………………………………………… 24 2.2.4 Infection of mice and preparation of organs………………………………… 24 2.2.5 RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)………………………………………………………………………25 2.2.6 Determination of cytokines concentration by ELISA…………………………26 2.2.7 Flow cytometric analysis………………………………………………………26 2.2.8 Tissue pathology……………………………………………………………….27 2.2.9 Statistical analysis…………………………………………………………… 27 2.3 Results… ………………………………………………………………………….28 2.3.1 LD50 of Burkholderia pseudomallei in BALB/c and C57BL/6 mice………….28 2.3.2 Bacterial loads in the infected organs……………………………… ……… 28 2.3.3 Cytokine Responses……………………………………………………………31 2.3.4 Kinetics of IFN-γ response upon low dose infection………………………… 32 2.3.5 IFN-γ response and bacterial loads in the blood of C57BL/6 mice upon high dose infection………………………………………………………………… 34 iv 2.3.6 Bacterial loads in organs of high dose and low dose challenged C57BL/6 mice…………………………………………………………………………….34 2.3.7 Pathology of infected organs………………………………………………… 36 2.3.8 Infiltration of immune cells during infection with B peudomallei……………39 2.4 Discussion ……………………………………………………………………… 41 Chapter Humoral immune response to Burkholderia pseudomallei………………47 3.1 Introduction………………………………………………………………………… 48 3.2 Materials and methods……………………………………………………………….53 3.2.1 Mice……………………………………………………………………………… 53 3.2.2 Bacteria…………………………………………………………………………….53 3.2.3 Expression of recombinant flagellin (r-FliC)……………… …………………….53 3.2.4 Purification of recombinant protein……………………………………………… 54 3.2.4.1 Preparation of cleared lysate under denaturing condition………………… 54 3.2.4.2 Purification of protein under denaturing condition………………………….55 3.2.4.3 Analysis of purified flagellin by SDS-PAGE……………………………….56 3.2.4.4 Determination of protein concentration by Bradford method …………… 56 3.2.4.5 Dialysis and concentration of purified flagellin…………………………… 56 3.2.5 Immunization of BALB/c mice …………………………………………… 57 3.2.6 Infection with live bacteria and protection study…………………………….57 3.2.7 Collection of serum………………………………………………………… 57 3.2.8 Detection of antigen specific antibodies by ELISA … …………………… 58 3.2.8.1 Flagellin specific antibodies.…………………………………………58 3.2.8.2 Burkholderia pseudomallei specific antibodies …………………… 58 v 3.2.9 Western blot………………………………………………………………… 59 3.3 Results……………………………………………………………………………… 60 3.3.1 Purication of flagellin protein (r-FliC)……………… …………………… 60 3.3.2 Immunization and protection study………………………………………… 61 3.3.3 Flagellin specific antibody responses (total IgG and IgG subclasses)……… 64 3.3.4 Western blot analysis of anti-sera ………………………………………… 66 3.3.5 Specificity of antibodies against whole bacteria…………………………… 66 3.3.6 Low dose-high dose infection in C57BL/6 mice …………………………….68 3.3.6.1 Kinetics of the antibody responses (total IgG, IgG1 and IgG2a)……… 68 3.3.6.2 Flagellin specific antibodies (total IgG, IgG1 and IgG2a)……………….70 3.4 Discussion……………………………………………………………………………71 Chapter Characterization of IFN-γ response in vitro………………………………77 4.1 Introduction… …………………………………………………………………….78 4.2 Materials and methods………………………………………………………………81 4.2.1 Mice……………………………………………………………………………81 4.2.2 Bacteria……………………………………………………………………… 81 4.2.3 Infection with Burkholderia pseudomallei…………………………………….81 4.2.4 Preparation and stimulation of splenocytes in vitro………………………… 82 4.2.5 Cell viability determination……………………………………………………82 4.2.6 Bacterial load determination…………………………… ……………….……83 4.2.7 Magnetic cell separation for cell-type purification………………………… 83 4.2.8 Cytokine determination by ELISA…………………………………………….84 4.2.9 Flow Cytometric analysis…………………………………………………… 84 vi 4.2.10 Intracellular cytokine staining ……………… ………………………………85 4.2.11 Cytokine capturing assay for IFN-γ………………………………………… 85 4.2.12 Isolation of human neutrophils……………………………………………….86 4.2.13 Statistical analyses……………………………………………………………86 4.3 Results……………………………………………………………………………….87 4.3.1 IFN-γ response in the splenocytes stimulated with B pseudomallei in vitro….87 4.3.2 Bacterial loads of the infected splenocytes……………………… ………… 88 4.3.3 Cell viability of infected splenocytes from BALB/c and C57BL/6 mice…… 90 4.3.4 Cytokine profiles in BALB/c and C57BL/6 mice after B pseudomallei infection……………………………………………………………………… 94 4.3.5 The effects of IL-12, IL-18 and IL-10 neutralizing antibodies on IFN-γ response…………………………………………………………………97 4.3.6 Cell types produce IFN-γ in response to bacteria in BALB/c and C57BL/6 mice……………………………………………………… ……… 97 4.3.6.1 Intracellular cytokine staining…………………………………………… 97 4.3.6.2 Cytokine capturing assay………… …………………………………… 97 4.3.6.3 Effect of cell depletion on the production of IFN-γ……………………… 99 4.3.7 Gr-1 expression populations in BALB/c and C57BL/6 splenocytes…………103 4.3.8 Role of T cells in IFN-γ response ……………………………………………107 4.3.9 Role of LPS in IFN-γ response to B pseudomallei………………………… 108 4.3.9.1 The effect of polymyxin B treatment on IFN-γ response to B pseudomallei………………………………………………………………107 4.3.9.2 TLR-4 signaling pathway is required for IFN-γ response to heat-killed Bacteria but dispensable for the IFN-γ response to live bacteria……… 107 3.10 IFN-γ response in human neutrophils……………………………………….110 vii 4.4 Discussion……………………………………………………………………….…110 Chapter Conclusion and future studies……………………………………………116 References……………… … ……………………………………………………… 122 Appendix I: Recipes …………………………………… ………………………… 148 Appendix II: Publications…………………………….…………………………… 151 viii Summary Burkholderia pseudomallei is the causative agent of melioidosis—an endemic disease in the Southeast Asia and Northern Australia Infections can result in clinical manifestations ranging from asymptomatic, chronic suppurative infection to potentially fatal septicemia The aim of this project is to study the host responses and factors which contribute to resistance or susceptibility in two strains of mice showing differential responses to B pseudomallei infection We found that BALB/c mice were highly susceptible to low dose intranasal B pseudomallei infection They developed acute disease and died within weeks, whereas C57BL/6 mice were relatively resistant Susceptibility of BALB/c mice correlates with high bacterial loads in the lung and spleen, infiltration of leukocytes (especially neutrophils), tissue pathology (in the lung and spleen), and hyper-inflammation A transient hyperproduction of IFN-γ was found at 48h post-infection in the serum of BALB/c, but not in the relatively resistant C57BL/6 mice C57BL/6 did not show a complete resistance to infection, as high dose B pseudomallei infected C57BL/6 mice died of septicemia resembling the characteristics of low dose infected BALB/c mice C57BL/6 mice which survived after an initial infection were found to be resistant to subsequent low dose or high dose challenge Resistance of low dose immunized C57BL/6 mice to high dose infection correlated to high serum IgG2a, which was indirect evidence of IFN-γ induced cell-mediated immunity, and with a low IgG1 response In contrast, BALB/c mice immunized with recombinant flagellin (r-FliC) induced high serum IgG1, which did not confer protection against B pseudomallei infecton However, some low dose infected and all high dose infected C57BL/6 mice eventually developed splenic abscesses and died at much later time points ix In vitro study showed that naïve BALB/c splenocytes produced higher IFN-γ than naïve C57BL/6 splenocytes in response to B pseudomallei infection, mirroring what was seen in vivo Splenocytes from BALB/c mice contained higher number of intracellular bacteria than C57BL/6, which could explain why they made more IFN-γ The IFN-γ response was IL-12 dependent and IL-18 could have a synergistic effect, while IL-10 ameliorated the IFN-γ response There was no obvious difference in the cell types making IFN-γ in BALB/c and C57BL/6 splenocytes In both strains of mice, Gr-1 positive cells (particularly the CD8+ T lymphocytes and DX5+ NK cells), and CD4+ cells were the major producers of the IFN-γ in response to B pseudomallei BALB/c splenocytes also contained higher numbers of Gr-1intermediate expressing NK and CD8+ cells Using splenocytes from nude mice, the redundancy of T cells in IFN-γ response of splenocytes to B pseudomallei was observed TLR-4 signaling was found to be essential for IFN-γ response of splenocytes to the heat-killed bacteria, but not for the response to live bacteria Besides hyperproduction of IFN-γ, BALB/c mice produced more TNF-α, IL-1β, IL-6, higher basal IL-18, and more anti-inflammatory cytokine (IL-10) compared to the splenocytes of C57BL/6 mice This study suggests that resistance to infection lies in the innate ability to control the bacterial growth that allows subsequent development 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S Sirisinha 2004 Immunostimulatory CpG oligodeoxynucleotide confers protection in a murine model of infection with Burkholderia pseudomallei Infect Immun 72(8): 4494-502 146 Woods, D E., A L Jones, and P J Hill 1993 Interaction of insulin with Pseudomonas pseudomallei Infect Immun 61(10): 4045-50 Wuthiekanun, V., M D Smith, and N J White 1995 Survival of Burkholderia pseudomallei in the absence of nutrients Trans R Soc Trop Med Hyg 89(5): 491 Wysocka, M., S Robertson, H Riemann, J Caamano, C Hunter, A Mackiewicz, L J Montaner, G Trinchieri, and C L Karp 2001 IL-12 suppression during experimental endotoxin tolerance: dendritic cell loss and macrophage hyporesponsiveness J Immunol 166:7504-7513 Yabuuchi, E., Y Kosako, H Oyaizu, I Yano, H Hotta, Y Hashimoto, T Ezaki, and M Arakawa 1992 Proposal of Burkholderia gen nov and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb nov Microbiol Immunol 36(12): 1251-75 Yahata, S N., K Santa, A Ohta, Y Ohmi, S Habu, and T Nishimura 1996 Functional characterization of NK1.1+ Ly-6C+ cells Immunol Lett 54:5-9 Yang, H., C D Kooi, and P A Sokol 1993 Ability of Pseudomonas pseudomallei malleobactin to acquire transferrin-bound, lactoferrin-bound, and cell-derived iron Infect Immun 61(2): 656-62 Yeaman, G R., J E Collins, J K Currie, P M Guyre, C R Wira, and M W Fanger 1998 IFN-γ is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils J Immunol 160:5145-5153 147 Appendix I: Recipes 1.1 Media for bacteria culture Tryptic Soy Agar (TSA): 15.0 g/L bacto-tryptone, g/L soya peptone, 5.0 g/L NaCl, 15.0 g/L agar, pH 7.3 Ashdown slective medium: 10 g/L TSA, % glycerol (w/v), mg/L crystal violet, 50 mg/L neutral red and mg/L gentamicin LB broth: 10 g/L bacto-tryptone, g/L bacto yeast extract, g/L NaCl, pH 7.0 LB agar: LB medium containing 15 g/L bactor agar • All the media used for bacteria culture were sterilized by autoclaving at 121 °C, 15 PSI for 15 1.2 Supplements Kanamycin stock solution: 25 mg/ml in H2O, sterile filter, store in aliquots at – 20 °C Ampicillin stock solution: 50 mg/ml in H2O, sterile filter, store in aliquots at – 20 °C Gentamicin stock solution: 100 mg / ml in H2O, sterile filter, store in aliquots at – 20 °C IPTG (1M): 238 mg/ml in H2O, sterile filter through 0.22 μm filter, store in aliquots at – 20 °C 1.3 Buffers for purification under denature conditions: Buffer B: 100mM NaH2PO4 , 10 mM Tris HCL, M Urea, pH 8.0 Buffer C: 100mM NaH2PO4 , 10 mM Tris HCL, 0.5 % Sodium deoxycholate, M Urea, pH 6.3 Buffer D: 100mM NaH2PO4 , 10 mM Tris HCL, M Urea, pH 5.9 Buffer E: 100mM NaH2PO4 , 10 mM Tris HCL, M Urea, pH 4.5 148 1.4 Reagents for SDS-PAGE Resolving Gel (8%) Gels Gel Solution D 10.5 ml ml ddH2O 4.2 ml ml Solution A 4.2 ml ml % SDD 2.1 ml ml 10% APS* 105 μl 50 μl TEMED* 10.5 μl μl Gels Gel Solution C 2.5 ml 1.25 ml ddH2O 1.5 ml 750 μl Solution A 0.5 ml 250 μl % SDD 0.5 ml 250 μl 10% APS* 12.5 μl 6.25 μl TEMED* 10 μl μl Stacking Gel (4%) Solution A (Bis-acrylamide): acrylamid 45 g, bis-acryalmide 1.2 g, H2O 100 ml Solution B (5x running buffer): 15.14 g Tris (125 mM), 72 g glycine (0.96 M), g SDS (0.5 %), top up with H2O to liter, pH 8.3 Solution C (Stacking buffer): 30.28 g Tris (0.25 M), top up with H2O to liter, pH 6.8 Solution D (Resolving buffer): 90.9 g Tris (0.75M), top up with top up with H2O to liter, pH 8.8 Solution F (Ammonium persulfate): 0.03 g APS in 300 μl H2O (10%) 149 SDS-PAGE running buffer: 25 mM Tris-base, 0.19 M glycine, 0.1 % (w/v) SDS, pH 8.3 Coomassie Blue Staining Fixing solution for SDS-PAGE: Glacial acetic acid: methanol: H2O = 10: 50: 40 Staining solution: 0.05 % Coomassie blue Glacial acetic acid: methanol: H2O = 10: 50: 40 Destaining solution: Glacial acetic acid: methanol: H2O = 7: 5: 88 1.5 Buffers for ELISA 10X PBS: % (w/v) NaCl, g/L 0.2 % (w/v) KCl, 0.2 g/L 1.44 % Na2HPO4, 1.44 g/L 0.24 % KH2PO4, 0.24 g/L Adjust to pH 7.4 Bicarbonate coating buffer: 1.59 g Na2CO3, 2.93 g NaHCO3, top up to L with H2O pH 9.5 Wash buffer PBST: 500 μl Tween 20 to liter 1×PBS Assay buffer (0.5 % BSA-PBST): 0.5 g of BSA was dissolved in 100 ml PBST 1.6 Buffer for flowcytometry staining Fixing buffer: % parafolmadehyde in x PBS Permeabilization/wash buffer: 0.1 % saponin- % FBS – x PBS 150 Appendix II: Publications Journal Papers Boping Liu, Ghee Chong Koo, Eu Hian Yap, Kim Lee Chua and Yunn-Hwen Gan A Model of differential Susceptibility to Mucosal Infection of Burkholderia pseudomallei Infect Immun 2002, 70(2): 504-511 Ghee Chong Koo, Yunn-Hwen Gan The innate interferon gamma response of BALB/c and C57BL/6 mice to in vitro to Burkholderia pseduomallei infection BMC Immunology 2006, 7:19 Poster presentations Ghee Chong Koo, Yunn-Hwen Gan 2004 The Roles of IFN-γ During Burkholderia pseudomallei Infection Poster 31 The 5th Combined Scientific Meeting, NUS (Merit Award) Ghee Chong Koo, Yunn-Hwen Gan 2004 Innate IFN-γ response to Burkholderia pseudomallei Poster 110 The 4th World Melioidosis Congress, Singapore Ghee Chong Koo, Yunn-Hwen Gan 2005 Innate IFN-γ response in mice showing differential susceptibility to Burkholderia pseudomallei infection Poster 209 The Keystone Symposia on Innate Immunity to Pathogens, Colorado, USA (Keystone Symposia Scholarship Winner) 151 ... resistant C5 7BL /6 mice C5 7BL /6 did not show a complete resistance to infection, as high dose B pseudomallei infected C5 7BL /6 mice died of septicemia resembling the characteristics of low dose infected... bacterial counts in C5 7BL /6 mice were decreased 12 h after infection in comparison to BALB/ c mice which suggests an innate immune mechanism against B pseudomallei in the early phase of infection contributing... Bacterial loads of the infected splenocytes……………………… ………… 88 4.3.3 Cell viability of infected splenocytes from BALB/ c and C5 7BL /6 mice? ??… 90 4.3.4 Cytokine profiles in BALB/ c and C5 7BL /6 mice