Elucidation of the cell signaling pathways mediating innate immunity and host-pathogen interactions

The ability to generate a robust immune response is integral to organismal homeostasis. Cells of the innate immune system are considered the first responders of immunity, and are therefore responsible for sensing both pathogens and endogenous danger signals and initiating a protective inflammatory response. To appropriately sense pathogens and danger signals, cells have developed intricate mechanisms for transducing signals from the extracellular environment into the cell. The integration of these signals is complex, resulting from crosstalk between many signaling pathways, but is critical to generating a coordinated biological response. In additional to the specialized mechanisms of innate immune cells to respond to antigens, these cells (like most) have evolved a complex set of adaptive mechanisms that maintain homeostasis during cell stress. Activation of innate immunity via pathogen invasion or the presence of danger signals can be considered an especially intense form of cell stress, thereby implicating these homeostatic pathways as components of the innate immune response. The work presented in this thesis relates to the molecular mechanisms by which cells of the innate immune system integrate signals from the microenvironment to produce a coordinated biological response. The aim was to elucidate the mechanisms by which innate macrophages transduce extracellular signals to activate important effector pathways, and to describe crosstalk between cell signaling pathways that mediate adaptive responses to cell stress. Finally, we looked to extend our understanding to pathophysiological settings, and investigated the mechanisms by which pathogens that cause cell stress generate an aberrant inflammatory response. In doing so, we described novel components of these signaling pathways, which may be exploited in designing novel therapeutics. In paper I, Gαi2 was identified as a critical signaling molecule in macrophage phenotype determination, functioning to transduce signals from the microenvironment to fine tune macrophage propensity towards an M1 inflammatory or M2 anti-inflammatory phenotype. In paper II, the immune receptor CD38 was shown activate the master transcriptional regulation of the autophagic/lysosome machinery, TFEB. We further identified the large kinase LRRK2 as essential in signal transduction downstream of CD38. In paper III, we described adaptive crosstalk between TFEB, an essential component of the cell stress response, and the typically proliferative WNT signaling pathway. Finally, in paper IV we describe how the SARS-Coronavirus open reading frame-3a causes multimodal necrotic death by activating multiple cell stress and innate immune pathways, resulting in aberrant inflammation. In summary, the work presented in this thesis extends our current understanding of the molecular mechanisms mediating the integration of signals in innate immune cells. We have identified several novel signaling mechanisms, which could lay the foundation for the development of targeted therapeutics. LIST OF SCIENTIFIC PAPERS I. Vural A*, Nabar NR*#, Hwang IY, Sohn S, Park C, Karlsson MCI, Blumer JB, Kehrl JH#. Gαi2 signaling regulates inflammasome priming and cytokine production by biasing macrophage phenotype determination. J Immunol, 2018, 202(5): 15101520. II. Nabar NR#, Shi CS, Hwang IY, Karlsson MCI, Kehrl JH#. Identification of a functional CD38-LRRK2-TFEB signaling pathway in immune cells. In Manuscript. III. Xiao X, Nabar NR#, Shi CS, Yue Y, Zhao W, Wang M, Kehrl JH#. Transcription Factor EB limits Wnt/β-catenin signaling by directly binding β-catenin and promoting its degradation. In Revision. IV. Yue Y, Nabar NR#, Shi CS, Kamenyeva O, Xiao X, Hwang IY, Wang M, Kehrl JH#. SARS-Coronavirus Open Reading Frame-3a drives multimodal necrotic cell death. Cell Death Dis, 2018, 9(9): 904. * Equal Contribution # Co-corresponding authors PUBLICATIONS NOT INCLUDED IN THE THESIS I. Nabar NR, Shi CS, Kehrl JH. Autophagy accompanies inflammasome activation moderating inflammation by eliminating active inflammasomes. In Hayat MA, Autophagy: Cancer, Other Patholgies, Inflammation, Immunity, Infection, and Aging, 2017, Vol 12 pp 343-357. II. Nabar NR, Shi CS, Kehrl JH. Signaling by the Toll-like Receptors induces autophagy through modification of Beclin 1: Molecular Mechanism. In Hayat MA, Immunology: Immunotoxicology, Immunopathology, and Immunotherapy, 2017, Vol 1 pp 75-84. III. Nabar NR#, Kehrl JH#. The Transcription Factor EB links cellular stress to the immune response. Yale J Biol Med, 2017, 90(2): 301-315. IV. Harris J, Lang T, Thomas JPW, Sukkar MB, Nabar NR, Kehrl JH. Autophagy and Inflammasomes. Mol Immunol, 2017, 86: 10-15. V. Nabar NR#, Kehrl JH#. Inflammasome inhibition links IRGM to innate immunity. Mol Cell, 2019 73(3): 391-392. VI. Shi CS, Nabar NR#, Huang NN, Kehrl JH#. SARS-CoV ORF8b triggers intracellular stress pathways and activates NRLP3 inflammasomes. Submitted. # Co-corresponding authors CONTENTS