FOR DOCTORAL DEGREE ( Ph . D . ) By SSV

Tuberculosis infection remains a global health problem and the emergence of multidrugresistant TB (MDR-TB) adds further challenges in the battle to stop the spread of infection. Macrophages are the primary host cells to be infected with Mycobacterium tuberculosis (Mtb), and are therefore key cells in the initial control of TB infection. However, to survive and persist in activated macrophages, Mtb has developed strategies to counteract antimicrobial immunity. This thesis work aimed to explore macrophage responses in Mtbinfected cells, the nature of innate effector mechanisms and how these can be enhanced. For this purpose, we used an in vitro macrophage infection model as well as an organotypic lung tissue model system. The thesis work is based on four papers, Study I and III, involved studies on the effects of the immunomodulatory compounds, vitamin D3 (VitD3) and phenylbutyrate (PBA), on the induction of antimicrobial peptides, primarily human cathelicidin LL-37, and the ability to kill intracellular Mtb. In Study I, we observed that Mtb can down-regulate the expression of LL-37 in infected macrophages, but this effect was effectively counteracted by treatment with VitD3 and/or PBA. PBA and/or VitD3 prevented intracellular Mtb growth via induction of LL-37 as well as activation of autophagy in Mtb-infected macrophages. In Study III, we further demonstrated that PBA+VitD3 enhanced intracellular Mtb killing of both MDR-TB and drug-susceptible strains and this effect was dependent on LL-37. In Study IV, we explored the role of VitD3 in polarization of macrophages in comparison to activation with conventional stimuli such as GM-CSF (polarization of inflammatory M1 macrophages) or MCSF (polarization of anti-inflammatory M2 macrophages). The findings from Study IV, suggested that polarization with VitD3 enhanced the ability of Mtb-infected macrophages to control intracellular Mtb growth in comparison to M1 and M2 subsets. Enhanced Mtb growth control was associated with elevated levels of pro-inflammatory cytokines and LL-37, but reduced Mtb-induced expression of the immunosuppressive enzyme IDO (indoleamine 2,3-dioxygenase). Study II, aimed to investigate the expression and function of matrix metalloproteinases (MMPs) in early TB granuloma formation using the organotypic lung tissue model that enabled studies on macrophage-Mtb interactions in a more physiological environment in tissue. In this study, we found that pre-treatment of macrophages with the global MMP inhibitor, marimastat, resulted in effective inhibition of TB granuloma formation, which was associated with reduced Mtb growth detected in the lung tissue model. Thus, reduced degradation of extracellular matrix proteins, could prevent Mtb multiplication and spread of the infection. The main conclusion from this thesis work is that immunomodulatory compounds with the ability to boost or block innate effector mechanisms in Mtb-infected macrophages may be used as adjunct host-directed therapies that could support standard anti-TB drugs to enhance clinical recovery from TB. LIST OF SCIENTIFIC PAPERS I. Rokeya Sultana Rekha, SSV Jagadeeswara Rao Muvva, Min Wan, Rubhana Raqib, Peter Bergman, Susanna Brighenti, Gudmuundur H Gudmundsson and Birgitta Agerberth. Phenylbutyrate induces LL-37dependent autophagy and intracellular killing of Mycobacterium tuberculosis in human macrophages. Autophagy. 2015 Sep; 11(9): 1688– 1699. II. Venkata R.Parasa, Jagadeeswara Rao Muvva, Jeronimo F. Rose, Clara Braian, Susanna Brighenti and Maria Lerm. Inhibition of Tissue Matrix Metalloproteinases Interferes with Mycobacterium tuberculosis-Induced Granuloma Formation and Reduces Bacterial Load in a Human Lung Tissue Model. Frontiers in Microbiology. 2017; 8: 2370. III. Jagadeeswara Rao Muvva, Sultan Ahmed, Rokeya Sultana Rekha, Thomas schon, Ramona Groenheit, Peter Bergman, Susanna Brighenti and Birgitta Agerberth. Vitamin Dand Phenylbutyrate-mediated growth inhibition of multidrug-resistant Mycobacterium tuberculosis in human macrophages is dependent on the antimicrobial peptide LL-37. Manuscript. IV. Jagadeeswara Rao Muvva, Venkata Ramanarao Parasa, Maria Lerm, Mattias Svensson and Susanna Brighenti.Polarization of human macrophages with vitamin D promotes control of Mycobacterium tuberculosis infection. Manuscript. ADDITIONAL PUBLICATIONS Min Wan, Xiao Tang, Rokeya Sultana Rekha, S. S. V. Jagadeeswara Rao Muvva, Susanna Brighenti, Birgitta Agerberth and Jesper Z. Haeggstrom. Prostaglandin E2 suppresses hCAP18/LL-37 expression in human macrophages via EP2/EP4: implications for treatment of Mycobacterium tuberculosis infection. FASEB Journal. 2018 May; 32(5):2827-2840. Tadepally Lakshmikanth Frank Heuts, S. S. V. Jagadeeswara Rao Muvva, Robert P. A. Wallin, Anna-Karin Persson, Cyril Fauriat, Steven E. Applequist, Hans-Gustaf Ljunggren, Petter Hoglund, Klas Karre, Mattias Svensson and Julius G. Juarez. In vivo engineering of mobilized stem cell grafts with the immunomodulatory drug FTY720 for allogeneic transplantation. European Journal of Immunology. 2016 July; 46(7):1758-69. CONTENTS 1 Background on TB infection . 1 1.1 Pathogenesis of TB infection . 1 1.2 The TB granuloma . 2 1.3 Macrophage polarization in TB infection . 4 1.4 Role of matrix metalloproteinases in the TB granuloma . 6 1.5 Immune evasion mechanisms of MTB . 7 1.6 Antimicrobial effector mechanisms in TB infection . 7 1.6. 1 NO and ROS production: . 8 1.6. 2 Antimicrobial peptides: . 8 1.6. 3 Autophagy: . 9 1.6. 4 Macrophage extracellular traps (MET’s): . 10 1.6. 5 Cytokines: . 11 1.7 Negative regulators of protective TB immunity . 12 1.8 Drug resistance in TB . 13 1.9 Immunomodulatory compounds-Host-directed therapies in TB . 15 1.9. 1 Vitamin D: . 16 1.9. 2 Phenylbutyrate: . 18 2 Aims, materials and Methods . 19 2.1 Aims . 19 2.2 Background on disease models of TB infection . 20 2.3 Materials and methods . 20 2.3.1 Cell cultures: . 22 2.3.2 Bacterial cultures: . 22 2.3. 3 Human organotypic lung tissue model: . 23 2.3. 4 Inhibitors: . 23 2.3. 5 Knockdown/silencing of CAMP: . 23 2.3. 6 Quantitative PCR: . 23 2.3. 7 ELISA and western blot: . 24 2.3. 8 Microscopy: . 24 2.3. 9 Flow cytometry: . 25 2.3.10 Bacterial growth: . 25 2.4 Statistical analyses . 26 2.5 Ethical considerations . 26 3 Results and discussion . 27 3.1 STUDY I . 27 3.1.1 Background . 27 3.1.2 Mtb-mediated down-regulation of LL-37 expression in human primary macrophages can be counteracted by PBA+VitD3 . 28 3.1.3 Activation of autophagy in Mtb-infected macrophages by PBA and VitD3 treatment is dependent on LL-37 . 29 3.1.4 LL-37 activates macrophage autophagy in an autocrine or paracrine manner via the P2X7 receptor . 30 3.2 STUDY III . 31 3.2.1 Background . 31 3.2.2 PBA+VitD3 reduced intracellular growth of MDR-TB as well as drug-susceptible Mtb strains and possessed additive effects together with RIF and INH .