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UNIVERSITY OF CALIFORNIA Los Angeles Mycobacterium tuberculosis tRNA triggers TLR8 to induce a pathway for Th1 cell instruction A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Microbiology, Immunology and Molecular Genetics by Caroline Keegan 2016 ã Copyright by Caroline Keegan 2016 ABSTRACT OF THE DISSERTATION Mycobacterium tuberculosis tRNA triggers TLR8 to induce a pathway for Th1 cell instruction by Caroline Keegan Doctor of Philosophy in Microbiology, Immunology and Molecular Genetics University of California, Los Angeles, 2016 Professor Robert L. Modlin, Chair Mycobacterium tuberculosis, the etiologic agent of tuberculosis, has infected one third of the world’s population and is one of the leading global infectious disease threats. Treatment is challenging, lasting upwards of six months even for drug sensitive infections, and the proliferation of multi and extensively drug resistant forms of tuberculosis underscore the urgent need for knowledge of the mechanisms driving pathogenesis of this disease. The ability of the innate immune system to combat infection involves activation of pattern recognition receptors (PRRs) that detect evolutionarily conserved pathogen-associated molecular patterns, including nucleic acids and lipoproteins. Activation of PRRs, including Toll-like receptors (TLRs), induces secretion of inflammatory and immunomodulatory cytokines that instruct adaptive ii immune pathways for T cell differentiation. For intracellular pathogens like M. tuberculosis, a Th1 response is required, whereas a Th2 response is beneficial for the control of extracellular parasites, but is associated with active tuberculosis disease. This dissertation seeks to understand modulators of the initial immune response to M. tuberculosis through characterization of innate immune pathways induced by distinct bacterial ligands. The first study explores a mechanism by which Th1 or Th2 cytokines alter the vitamin D-dependent antimicrobial pathway in response to M. tuberculosis 19kD lipoprotein, and illustrates the importance of the Th1 cytokine IFN-g in guiding an antimicrobial response. Next, is a comparison of the immune response induced by the M. tuberculosis-derived ligands: 19kD lipoprotein and purified tRNA. M. tuberculosis tRNA was found to induce a gene network inducing secretion of key Th1 cytokines including IL-12p70 and IFNg, which are necessary for host defense against TB. iii The dissertation of Caroline Keegan is approved. Linda Baum Genhong Cheng Stephen Smale Robert L. Modlin, Committee Chair University of California, Los Angeles 2016 iv TABLE OF CONTENTS List of Figures and Tables vi Acknowledgements viii Vita x Chapter 1 Introduction – Innate and adaptive immune response to Mycobacterium tuberculosis 1 References 26 Chapter 2 T-cell cytokines differentially control human monocyte antimicrobial responses by regulating vitamin D metabolism 41 References 46 Chapter 3 Mycobacterium tuberculosis tRNA triggers a gene network for instruction of Th1 cells 49 References 98 v LIST OF FIGURES AND TABLES FIGURES CHAPTER 1 Figure 1-1 IL-12 / IFN-g feedback loop during M. tuberculosis infection 23 Figure 1-2 Human Toll-like receptors and ligands 24 Figure 1-3 Cell wall structure of mycobacteria 25 CHAPTER 2 Figure 1 T-cell cytokines differentially influence TLR2/1-induced expression of the antimicrobial peptides cathelicidin and DEFB4. 43 Figure 2 IFN-g and IL-4 upregulate vitamin D pathway genes in TLR2/1-activated monocytes 43 Figure 3 Effects of IL-4 on 1,25D3 responsiveness 44 Figure 4 IFN-g and IL-4 differentially regulate vitamin D metabolism 44 Figure 5 Regulation of 24-hydroxlase activity by IL-4 45 Figure S1 IL-4 and 1,25D3 similarly regulate vitamin D metabolism 48 Figure S2 mRNA expression levels of cytochrome p450 enzymes in TLR2/1 activated monocytes with or without IL-4 48 vi CHAPTER 3 Figure 3-1 Comparison of gene expression patterns induced by M. tuberculosis lipoprotein and tRNA 80 Figure 3-2 Network analysis of pathways and functions 81 Figure 3-3 Integrated model of central pathways and functions 82 Figure 3-4 Analysis of Th1 genes and validation in PBMC 83 Figure 3-5 Role of IL-18 and NK cells 84 Figure 3-6 Role for TLR8 85 Figure 3-7 Comparison of cytokine secretion by nucleic acid PAMP and synergy between TLR8 and TLR3 86 Figure 3-8 IFN-g expression in PBMC 87 TABLES CHAPTER 3 Table 1-1 Top significant differentially expressed genes 88 vii ACKNOWLEDGEMENTS I would like to thank my mentor, Robert Modlin, for giving me the opportunity to join his lab, for his constant encouragement, and for helping me find the story in my data. I would also like to thank my committee members, Linda Baum, Genhong Cheng and Steve Smale, for their guidance and insightful feedback. I am grateful to the past and present members of the Modlin Lab, especially Stephan Krutzik, who began this project, and Mirjam Schenk; for their help with experiments and discussions of results. I was fortunate to share this journey with my fellow ModLab PhDs – Susan, Manali and Angeline; grad school would have been nowhere near as fun without their friendship. Finally, I could not have succeeded without the love and support of my parents, Jim and Michele; my sister, Leah; and my fiancé, Yasser; who kept me going through the challenging times and who are a constant source of happiness in my life. Chapter 1 contains reprints of figures used with permission. Figure 1-1 is adapted with permission from Nature Publishing Group (License #3960000587574). Figure 1-2 is adapted with permission from Elsevier (License #3960431065745). Figure 1-3 is adapted with permission from Nature Publishing Group (License #3960421067287). Chapter 2 is a reprint of the full citation Edfeldt, K., Liu, P. T., Chun, R., Fabri, M., Schenk, M., Wheelwright, M., Keegan, C., Krutzik, S. R., Adams, J. S., Hewison, M., and Modlin, R. L. 2010. T-cell cytokines differentially control human monocyte viii antimicrobial responses by regulating vitamin D metabolism. Proc Natl Acad Sci U S A 107:22593-22598. Chapter 3 is a version of a manuscript in preparation for publication. Contributors include Stephan Krutzik, Mirjam Schenk, Jing Lu, Matteo Pellegrini, Barry Bloom, John Belisle, Sarah Fortune, Peter Dedon, and all work was performed with direction by Robert Modlin. ix VITA 2009 Bachelor of Science, Microbiology, Immunology and Molecular Genetics / Biology University of California, Los Angeles 2011-2012 Teaching Assistant Department of Microbiology, Immunology and Molecular Genetics University of California, Los Angeles 2009-2016 Graduate Student Researcher Department of Microbiology, Immunology and Molecular Genetics University of California, Los Angeles PUBLICATIONS Keegan, C., Krutzik, S., Schenk, M., Lu, J., Pellegrini, M., Bloom, B. R., Belisle, J. T., Fortune, S., Dedon, P., and Modlin, R. L. Mycobacterium tuberculosis tRNA triggers a gene network for instruction of Th1 cells. In preparation for submission. Edfeldt, K., P.T. Liu, R. Chun, M. Fabri, M. Schenk, M. Wheelwright, C. Keegan, S.R. Krutzik, J.S. Adams, M. Hewison, and R.L. Modlin. 2010. T-cell cytokines differentially control human monocyte antimicrobial responses by regulating vitamin D metabolism. Proc Natl Acad Sci U S A 107:22593-22598. McHardy, I., C. Keegan, J.H. Sim, W. Shi, and R. Lux. 2010. Transcriptional profiles of Treponema denticola in response to environmental conditions. PLoS One 5:e13655. x CHAPTER 1 Introduction Innate and adaptive immune response to Mycobacterium tuberculosis 1 Tuberculosis Mycobacterium tuberculosis, the etiologic agent of tuberculosis, has been a plague on humanity since ancient times. The genomes of ancestral strains of M. tuberculosis have been identified and sequenced from Neolithic human remains (Hershkovitz et al., 2008), and the disease was common in the 5th century BCE where Hippocrates reported, “Consumption was the most considerable of the diseases which then prevailed, and the only one which proved fatal to many persons” (Hippocrates and Adams, 1938). Currently, one third of the world’s population is infected, and tuberculosis remains one of the leading global infectious disease threats (WHO, 2015). The most common form of disease is pulmonary tuberculosis, an affliction of the lungs characterized by fever, chest pain, persistent cough, coughing up blood and weight loss. Extrapulmonary tuberculosis, disease occurring outside of the lungs, may take several forms including Pott’s disease (infection of the spine), scrofula (infection of the lymph nodes), and miliary tuberculosis (extensive disseminated infection) (Frith, 2014). For much of history, tuberculosis was thought to be an inherited condition. However, in 1882, Robert Koch presented definitive proof that tuberculosis was a transmissible bacterial infection. He demonstrated the presence of bacilli in infected tissues which he isolated and grew in culture, then used the cultured bacteria to infect guinea pigs and reconstitute disease. For this discovery, he was awarded the Nobel Prize in 1905. Still, there was no effective treatment for tuberculosis until the 1940’s 2 when treatment with the antibiotic streptomycin was found to be successful (Zumla et al., 2013), although antibiotic resistance swiftly developed. At this time, the treatment regimen for susceptible strains involves a six month course of a combination of antibiotics, isoniazid and rifampicin, which have toxic side effects. Premature discontinuation of treatment has fueled the rise of resistant