Tuberculosis Transcriptomics: Host Protection and ImmuneTown Evasion Mechanisms Cape of Mumin OZTURK Thesis submitted to the University of Cape Town in fulfillment of the University degree Doctor of Philosophy Cytokines and Disease Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Cape Town Component and Institute of Infectious Diseases and Molecular Medicine (IDM), Division of Immunology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa. The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………………………………………………….4 ABSTRACT……………………………………………………………………………………….5 LIST OF PUBLICATIONS………………………………………………………………………..6 LIST OF ABBREVIATIONS……………………………………………………………………..7 CHAPTER I LITERATURE REVIEW…………………………………………………………...9 1.1 HISTORY of TUBERCULOSIS………………………………………………………9 1.2 TUBERCULOSIS: CURRENT SITUATION and INSIGHTS……………………...15 1.3 TUBERCULOSIS IMMUNOPATHOLOGY……………………………………….16 1.4 MTB AND MACROPHAGE INTERPLAY………………………………………...21 CHAPTER II TRANSCRIPTOMICS OF MTB INFECTED MACROPHAGES………………29 2.1 ROLE OF FUNCTIONAL GENOMICS TO UNDERSTAND IMMUNE RESPONSES ……………………………………………………………………………29 2.2 CAP ANALYSIS GENE EXPRESSION (CAGE) SEQUENCING………………..30 2.3 EXPERIMENTAL DESIGN and REFINEMENT of CAGE DATA……………….36 2.4 TRANSCRIPTIONAL CHANGES INDUCED by HN878 INFECTION………….41 2.5 DISCUSSION……………………………………………………………………….52 CHAPTER III BATF2: AN IMMUNE EVASION GENE IN VIVO…………………………...55 BATF2 CAUSES INFLAMMATION IN MICE AND IS A PREDICTIVE HUMAN MARKER FOR TUBERCULOSIS DISEASE PROGRESSION……………………….58 ABSTRACT……………………………………………………………………...59 INTRODUCTION………………………………………………………….....…59 RESULTS………………………………………………………………………..60 DISCUSSION……………………………………………………………………64 METHODS………………………………………………………………………66 CHAPTER IV PRKCD: A HOST PROTECTIVE GENE IN VIVO……………………………79 PROTEIN KINASE C-DELTA (PKCD), A MARKER OF INFLAMMATION AND TUBERCULOSIS DISEASE PROGRESSION IN HUMANS, IS IMPORTANT FOR OPTIMAL MACROPHAGE KILLING EFFECTOR FUNCTIONS AND SURVIVAL IN MICE…………………………………………………………………………………82 2 ABSTRACT……………………………………………………………………...83 INTRODUCTION……………………………………………………………….83 RESULTS………………………………………………………………………..84 DISCUSSION……………………………………………………………………90 METHODS………………………………………………………………………94 FIGURE LEGENDS……………………………………………………………..97 CHAPTER V CONCLUSIONS………………………………………………………………..113 REFERENCES…………………………………………………………………………………114 3 ACKNOWLEDGEMENTS I would like to thank my supervisors Prof. Frank Brombacher and Dr Reto Guler for giving me the chance of participating in an intriguing and fruitful study. They have been exceptional mentors, providing intellectual support at every step. I am grateful to Dr Reto Guler for his insightful advices, encouragement of independent thinking and finding time for discussions. I also want to thank Dr. Suraj Parihar, who taught me literally the basics of experimental immunology and microbiology. I want to thank as well my friends and colleagues in Brombacher group for their support. Thank you for the laughs, the frustration and the successes. Science is not easy, but it doesn’t matter when you’re working on it alongside friends rather than colleagues. Furthermore, I am indebted to my supportive partner Kerry for her endurance over 4 years. Last but not least, I would like to express my gratitude to my mother and father, who have always supported me throughout my scientific career. 4 ABSTRACT Mycobacterium tuberculosis (Mtb) is the leading cause of death from an infectious disease. The success of the pathogen lies in its ability to subvert hostile intracellular macrophage environment. We performed genome-wide transcriptional deep sequencing on total RNA in murine bone marrow-derived macrophages (BMDM) infected with hypervirulent Beijing strain (HN878) in an extensive time kinetic manner using single molecule sequencer and cap analysis gene expression (CAGE) technique. CAGE analysis revealed nearly 36000 unique RNA transcripts with approximately 16000 are not unannotated to a specific gene. This thesis addressed global changes in RNA expression levels in macrophages infected with Mtb in a time kinetic manner to pinpoint novel host protection and immune evasion genes and elucidate the role of these genes in vitro macrophage assays and in vivo knockout mouse studies. The data in this thesis showed that basic leucine zipper transcription factor 2 (Batf2) was an important factor that regulates inflammatory responses in Mtb infection. Deletion of Batf2 led to the survival of mice with reduced lung inflammation and histopathology due to reduced recruitment of inflammatory macrophages. We also showed that Batf2 was highly expressed in peripheral blood from adolescents who progressed from infection to tuberculosis disease and a predictive human biomarker for tuberculosis disease. In contrast to Batf2, we showed that Protein Kinase C-delta (PKC-δ) deficient mice are highly susceptible to tuberculosis and human lung proteomics dataset revealed that PKC-δ was highly upregulated in the necrotic and cavitory regions of human granulomas in multi-drug resistant subjects. PKC-δ deficient mice had a significant reduction in alveolar macrophages and dendritic cells, reduced accumulation of lipid bodies and serum fatty acids. In vitro experiments showed that PKCδ was required for optimal killing effector functions which were independent of phagosome maturation and autophagy in primary murine macrophages. Our studies suggested that these novel genes play a role in the immune response to Mtb and should be studied more thoroughly to evaluate their potential in possible TB interventions. 5 List of Publications In addition to the work presented in this thesis, the author has made significant contributions in following publications. 1) IRNdb: the database of immunologically relevant non-coding RNAs. Elena Denisenko, Daniel Ho, Ousman Tamgue, Mumin Ozturk, Harukazu Suzuki, Frank Brombacher, Reto Guler, Sebastian Schmeier. Database 2016, baw138 2) Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics. Sugata Roy, Sebastian Schmeier, Erik Arner, Tanvir Alam, Suraj P Parihar, Mumin Ozturk, Ousman Tamgue, Hideya Kawaji, Michiel JL de Hoon, Masayoshi Itoh, Timo Lassmann, Piero Carninci, Yoshihide Hayashizaki, Alistair RR Forrest, Vladimir B Bajic, Reto Guler, Frank Brombacher, Harukazu Suzuki, Fantom Consortium. Nucleic acids research 2015, 43-14: 6969-6982 3) Batf2/Irf1 induces inflammatory responses in classically activated macrophages, lipopolysaccharides, and mycobacterial infection. Sugata Roy, Reto Guler, Suraj P Parihar, Sebastian Schmeier, Bogumil Kaczkowski, Hajime Nishimura, Jay W Shin, Yutaka Negishi, Mumin Ozturk, Ramona Hurdayal, Atsutaka Kubosaki, Yasumasa Kimura, Michiel JL de Hoon, Yoshihide Hayashizaki, Frank Brombacher, Harukazu Suzuki. The Journal of Immunology 2015,194-12: 6035-6044 4) IL-4Rα-Dependent Alternative Activation of Macrophages Is Not Decisive for Mycobacterium tuberculosis Pathology and Bacterial Burden in Mice. Reto Guler, Suraj P Parihar, Suzana Savvi, Erin Logan, Anita Schwegmann, Sugata Roy, Natalie E Nieuwenhuizen, Mumin Ozturk, Sebastian Schmeier, Harukazu Suzuki, Frank Brombacher. PloS one 2015, 310: e012107 6 List of Abbreviations ARG1 Arginase 1 ATG5 Autophagy protein 5 ATP Adenosine triphosphate BC Before Christ BCG Bacillus Calmette Guérin CCL Chemokine (C-C motif) ligand CD Cluster of Differentiation C/EBP CCAAT-enhancer-binding proteins cGAS cGMP- AMP Synthase CLIP class II-associated Ii peptide CIITA MHC II transcriptional transactivator CXCR C-X-C chemokine receptor DAP Diaminopimelic acid DC-SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin DNA Deoxyribonucleic Acid ESX Early secretory antigenic target (ESAT6) protein family secretion G-CSF Granulocyte colony stimulating factor GTP Guanosine 5′-triphosphate HIV Human Immunodeficiency Virus HLA-DR Human Leukocyte Antigen – antigen D Related IDO Indoleamine-pyrrole 2,3-dioxygenase IFNγ Interferon γ IL- Interleukin iNKT invariant Natural Killer T cells iNOS inducible nitric oxide synthase IRF3 Interferon regulatory factor 3 IRG Interferon Response Genes LTBI Latent Tuberculosis Infection LYSM Lysozyme M MARCO Macrophage Receptor with Collagenous Structure MBL Mannose-binding lectin 7 MCL Macrophage C-type lectin MCP-1 Monocyte chemoattractant protein-1 MHC Major Histocompatibility complex MMP Matrix Metalloproteinase MTB Mycobacterium tuberculosis MYD88 Myeloid differentiation primary response gene 88 NDK Nucleoside diphosphate kinase NFκB Nuclear factor κB NOD Nucleotide-binding oligomerization domain-containing protein NOX2 Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase PAS Para-aminosalicylic acid PCR Polymerase Chain Reaction PGE2 Prostaglandin E2 pH Potential of Hydrogen PhD Doctor of Philosophy PHOX phagocyte oxidase PI3P Phosphatidylinositol 3-phosphate PMN Polymorphonuclear leukocytes PPAR Peroxisome proliferator-activated receptor PtpA Protein tyrosine phosphatase A RAC1 Ras-related C3 botulinum toxin substrate 1 ROS Reactive Oxygen Species SapM Secreted acid phosphatase M SP-A Surfactant protein A STING Stimulator