INVESTIGATING THE HOST IMMUNE RESPONSE TO MYCOBACTERIUM TUBERCULOSIS THE ROLES OF ANNEXIN a1 PROTEIN AND CLEC9A+ DENDRITIC CELLS

INVESTIGATING THE HOST IMMUNE RESPONSE TO MYCOBACTERIUM TUBERCULOSIS: THE ROLES OF ANNEXIN A1 PROTEIN AND CLEC9A+ DENDRITIC CELLS KOH HUI QI VANESSA NATIONAL UNIVERSITY OF SINGAPORE 2014 INVESTIGATING THE HOST IMMUNE RESPONSE TO MYCOBACTERIUM TUBERCULOSIS: THE ROLES OF ANNEXIN A1 PROTEIN AND CLEC9A+ DENDRITIC CELLS KOH HUI QI VANESSA (B. Sc. (Life Sciences, Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that the thesis is my original work and it has been written by my in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _________________________________ Koh Hui Qi Vanessa 30 Dec 2014 Acknowledgements I must express my utmost gratitude to my supervisor, A/P Sylvie Alonso, for her guidance, patience and trust. I feel truly fortunate to have the opportunity to learn as much as I did from you. I am grateful to our collaborators A/P Christiane Ruedl and A/P Lina Lim, and the members of their respective labs, for generously sharing their scientific expertise, as well as my Thesis Advisory Committee members, A/P Veronique Angeli and A/P Herbert Schwarz, for their invaluable advice and insight. I would also like to thank Lay Tin, Li Li, Joe and Siva from DSO National Laboratories for operational support and contributing to my BSL training, Dr. Paul Hutchinson and Guo Hui of the IP Flow Lab for their kind assistance regarding use of the flow cytometers, and Benson from the Kemeny Lab for accommodating my requests for animals. To the members of the Alonso Lab, past and present (and honorary)— Aakanksha, Adrian, Annabelle, Emily, Eshele, Fiona, Grace, Huimin, Issac, Jian Hang, Jie Ling, Jowin, Julia, Li Ching, Michelle, Peixuan, Per, Ran, Regina, Sze Wai, Weixin, Weizhen, Wenwei, Yok Hian and Zarina—thank you for the camaraderie. Our lab may not have windows, but with you all, it was and is always a sunny day indoors. Special thanks to the TB group with whom I shared many long hours in the BSL 3; we had a nice view, but most comforting has been the buddy behind the mask. i To my kindred spirits Weixin and Zarina, Team Omnom and the Gluttons, thank you for the unforgettable adventures and deliciousness. To the Sisterhood—Amanda, Joyce, Mingxian, Valerie and Velda—and Yen Han, thank you for the many, many good years of friendship. To my dear friends Bean, Emily, Kenrick, Marcus, Natascha, Wei Ting, Xiao Xuan and Yi Kang, I am thankful for your constant virtual chatter and for the warmth of your company around (ideally) round tables and on those blue-lit nights. Last but not least, to my family—Mom, Dad, Kenneth and Max—and my fiancé, Alvin, thank you for your unwavering encouragement and unconditional love. ii List of Publications Ang, M. L., Siti, Z. Z., Shui, G., Dianiškova, P., Madacki, J., Lin, W., Koh, V.H., Martínez Gómez, J.M., Sudarkodi, S., Bendt, A., Wenk, M., Mikušova, K., Korduláková, J, Pethe, K., & Alonso, S. (2014). An etha-ethr-deficient Mycobacterium bovis BCG mutant displays increased adherence to mammalian cells and greater persistence in vivo, which correlate with altered mycolic acid composition. Infection and Immunity, 82(5), 1850-9. doi:10.1128/IAI.01332-13 Lin, W., Mathys, V., Ang, E. L., Koh, V. H., Martínez Gómez, J. M., Ang, M. L., Zainul Rahim, S.Z., Tan, M.P., Pethe, K. & Alonso, S. (2012). Urease activity represents an alternative pathway for Mycobacterium tuberculosis nitrogen metabolism. Infection and Immunity, 80(8), 2771-9. doi:10.1128/IAI.06195-11 Martínez Gómez, J. M., Koh, V. H., Yan, B., Lin, W., Ang, M. L., Rahim, S. Z., Pethe K., Schwarz, H. , & Alonso, S. (2014). Role of the CD137 ligand (CD137L) signalling pathway during Mycobacterium tuberculosis infection. Immunobiology, 219(1), 78-86. doi:10.1016/j.imbio.2013.08.009 Tan, K. S., Lee, K. O., Low, K. C., Gamage, A. M., Liu, Y., Tan, G. Y., Koh, H.Q., Alonso, S., & Gan, Y. H. (2012). Glutathione deficiency in type diabetes impairs cytokine responses and control of intracellular bacteria. The Journal of Clinical Investigation, 122(6), 2289-300. doi:10.1172/JCI57817 Vanessa, K. H., Julia, M. G., Wenwei, L., Michelle, A. L., Zarina, Z. R., Lina, L. H., & Sylvie, A. (2014). Absence of annexin A1 impairs host adaptive immunity against Mycobacterium tuberculosis in vivo. Immunobiology. in press. doi:10.1016/j.imbio.2014.12.001 iii Table of Contents CHAPTER 1: GENERAL INTRODUCTION 1.1. Epidemiology of TB .   1.2. Aetiology and transmission of TB   1.2.1. M. tuberculosis is the main cause of TB in humans .   1.2.2. Airborne transmission of TB   1.3. Pathogenesis of TB: the spectrum of active and latent TB   1.3.1. Pathogenesis depends on environmental, host and microbial factors .   1.3.2. Active TB is complex and difficult to diagnose .   1.3.3. Latent TB: a heterogeneous state with diverse clinical outcomes   1.4. Prevention and treatment of TB . 11   1.4.1. Protection by BCG immunisation and vaccines under development 11   1.4.2. Treatment of TB 12   1.4.3. Drug-resistant TB 12   1.5. Initiation of infection and the innate immune response . 14   1.5.1. Macrophages are the first to be infected by M. tuberculosis and fail to restrict an initial phase of exponential bacterial growth 14   1.5.2. Neutrophil accumulation in the lungs is associated with pathology . 16   1.5.3. DCs deliver antigen from the lungs to the LN and initiate T cell responses 17   1.6. Granuloma formation 20   1.6.1. Granulomas are the characteristic pathological feature of TB 20   1.6.2. Macrophages initiate granuloma formation by secreting critical soluble factors 20   iv 1.6.3. Containment of infection through cellular recruitment and remodelling of the site of infection 21   1.6.4. Heterogeneity in granuloma morphology . 23   1.6.5. Collapse of the granuloma 25   1.6.6. Extracellular life of M. tuberculosis within the granuloma 26   1.7. Adaptive immunity to tuberculosis 28   1.7.1. Acquired cellular immunity to TB is T cell-dominated . 28   1.7.1.1. CD4+ Th cells are the predominant protective T cell subset 28   1.7.1.2. CD8+ Th cells may play a role in immune surveillance 28   1.7.1.3. Th2, Th17 and Treg cells . 29   1.7.2. Key cytokines balance the immune response between bacterial eradication and host survival . 30   1.7.2.1. TNF 30   1.7.2.2. IL-12 and IFNγ . 31   1.7.2.3. IL-10 . 32   1.7.3. Possible roles for DCs during chronic infection . 33   1.8. Mouse model of tuberculosis . 34   1.8.1. Infection profile of M. tuberculosis in the mouse . 34   1.8.2. Limitations of the mouse model in recapitulating latency and granuloma formation in humans 35   1.8.3. Advantages of the mouse model in studying host immune factors 35   1.9. General objectives and significance of this Thesis 37   CHAPTER 2: MATERIALS & METHODS 2.1. Microbiology . 38   v 2.1.1. Biosafety . 38   2.1.2. Mycobacterial strains 38   2.1.3. Mycobacterial culture . 39   2.2. Animal Work 40   2.2.1. Bioethics . 40   2.2.2. Mouse strains 40   2.2.3. Infection of live animals . 40   2.2.4. DT treatment . 41   2.2.5. Collection of BALF 41   2.2.6. Processing of organs for quantification of bacterial load in vivo 41   2.2.7. Histology . 42   2.3. Cell Biology . 43   2.3.1. Preparation and culture of BMMΦs 43   2.3.2. Preparation and culture of BMDCs . 43   2.3.3. Isolation and culture of primary splenocytes 44   2.3.4. Infection of BMMΦs for quantification of bacterial load in vitro 44   2.3.5. Infection of BMDCs and splenocytes for quantification of cytokine production in vitro 45   2.4. Immunology 46   2.4.1. Cytokine quantification . 46   2.4.2. Allogeneic mixed lymphocyte reaction 46   2.4.3. Preparation of samples for flow cytometry . 46   2.4.4. T cell re-stimulation 48   2.5. Statistical analysis 50   vi Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rössner, S., Koch, F., Romani, N., & Schuler, G. (1999). An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. Journal of Immunological Methods, 223(1), 77-92. doi:10037236 Ma, D. Y., & Clark, E. A. (2009). The role of CD40 and CD154/CD40L in dendritic cells. 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FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 14(3), 572-80. doi:10698973 END 169 [...]... understanding of the immune mechanisms associated with protection against M tuberculosis infection In this Thesis, we report and discuss our experimental findings on two aspects of host immunity to M tuberculosis infection: the role of Annexin A1 (ANXA1), a protein expressed endogenously by a variety of immune cells, and the role of CLEC9A+ dendritic cells (DCs), which includes CD103+ migratory DCs in the. .. 3: THE ROLE OF ANNEXIN A1 3.1 Introduction 51   3.1.1 ANXA1 and its receptor are expressed on immune cells 51   3.1.2 Counter-regulatory role of ANXA1 in the inflammatory response 52   3.1.3 ANXA1 expression is modulated by glucocorticoids and in turn mediates their anti-inflammatory effects 53   3.1.4 Diverse roles for ANXA1 in the regulation of cell proliferation 53   3.1.5 Roles. .. the lung and CD8+ DCs in the lymphoid organs First, using an ANXA1−/− mouse model of pulmonary M tuberculosis infection, we describe how ANXA1 influences the immune response to M tuberculosis infection A link between ANXA1 and TB was first established only recently; virulent M tuberculosis was shown in vitro to inhibit the ANXA1-dependent apoptotic pathway in order to promote dissemination via host necrosis... WT and ANXA1−/− M tuberculosis- infected mice Figure 3.4 Local cytokine profile in the BALF from WT and ANXA1−/− M tuberculosis- infected mice Figure 3.5 T cell populations in the lungs from WT and ANXA1−/− M tuberculosis- infected mice Figure 3.6 Infection profile macrophages Figure 3.7 Cytokine secretion by M tuberculosis- infected ANXA1−/− dendritic cells Figure 3.8 Cytokine secretion by ANXA1−/− splenocytes... Structure and cellular components of the TB granuloma Figure 1.6 Heterogeneity of granuloma morphology Figure 1.7 Typical M tuberculosis infection profile in a mouse model of low-dose aerosol infection Figure 3.1 Model of glucocorticoid modulation of the ANXA1 pathway in immune regulation Figure 3.2 Infection profile in WT and ANXA1−/− mice Figure 3.3 Histological analysis of lungs and spleens from WT and. .. granulomatous inflammation in the lungs 59   3.2.3 M tuberculosis- infected ANXA1−/− mice display reduced TNF and IFNγ production in the lungs during the early chronic phase of infection 61   3.2.4 M tuberculosis- infected ANXA1−/− mice display increased infiltration of activated CD4+ and CD8+ T cells in their lungs 62   3.2.5 Cytokine production, maturation and ability to activate naïve T cells. .. during the late chronic phase in ANXA1−/− mice is associated with higher frequencies of activated CD4 and CD8 T cells 74   3.3.5 ANXA1 is involved in the regulation of DC function in terms of cytokine production, maturation and T cell activation 75   3.4 Future Work 78   CHAPTER 4: THE ROLE OF CLEC9A+ DENDRITIC CELLS 4.1 Introduction 80   4.1.1 Heterogeneity of DCs... distribution of M tuberculosis in the lungs and mediastinal LN Figure 4.5 Effects of DT injection Figure 4.6 Bacterial load in the lungs, mediastinal LN and spleen in control vs CLEC9A+ DC-depleted mice Figure 4.7 Local cytokine responses in the lung Figure 4.8 T cell activation in the lungs and mediastinal LN Figure 4.9 Re-stimulation of T cells in vitro Figure 4.10 Schematic illustrating the proposed role of. .. individual is simply too localised or too weak to be detected systemically (O'Garra et al., 2013; Barry et al., 2009) (Figure 1.3) 9 Chapter 1: General Introduction Figure 1.3 The heterogeneous consequences of M tuberculosis infection The implications of the heterogeneity of the host immune response to M tuberculosis infection are becoming increasingly recognised The active and latent states simplistically... programmes, the worldwide burden of disease remains enormous, and the problem is compounded by the emergence of drug-resistant strains and synergistic coinfection with HIV Efforts to discover novel drugs have largely been focused on targeting the bacterium directly Alternatively, manipulating the host immune response may represent a valuable approach to enhance immunological clearance of the bacilli, . NATIONAL UNIVERSITY OF SINGAPORE 2014 INVESTIGATING THE HOST IMMUNE RESPONSE TO MYCOBACTERIUM TUBERCULOSIS: THE ROLES OF ANNEXIN A1 PROTEIN AND CLEC9A + DENDRITIC CELLS KOH HUI. INVESTIGATING THE HOST IMMUNE RESPONSE TO MYCOBACTERIUM TUBERCULOSIS: THE ROLES OF ANNEXIN A1 PROTEIN AND CLEC9A + DENDRITIC CELLS KOH HUI. and discuss our experimental findings on two aspects of host immunity to M. tuberculosis infection: the role of Annexin A1 (ANXA1), a protein expressed endogenously by a variety of immune cells,