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Wild-Derived Allele of Tmem173 Potentiates an Alternate Signaling Response to Cytosolic DNA

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Wild-Derived Allele of Tmem173 Potentiates an Alternate Signaling Response to Cytosolic DNA Wild-derived allele of Tmem173 potentiates an alternate signaling response to cytosolic DNA. A dissertation submitted by Guy Surpris In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Immunology TUFTS UNIVERSITY Sackler School of Graduate Biomedical Sciences May 2016 Adviser: Alexander Poltorak ABSTRACT The cellular recognition of cytosolic DNA is critical for maintaining homeostasis and to signal warnings to prevent the spread of pathogens such as HSV1 or Listeria. Inborn mutations in the human population determine the susceptibility or ability to clear infection. Mouse models of infectious disease are an invaluable resource for the study of these mechanisms of disease progression. However, classical laboratory mouse strains do not always recapitulate the diversity in immune responses found in the human population. Wild derived mice are an excellent source of genomic and phenotype diversity in the lab. Herein, we report and characterize phenotype variations in the wild-derived mouse strain MOLF/Ei and classical lab mouse strain in interferon stimulated gene induction to cytosolic DNA species. Using forward genetic analysis, we identified multiple loci that confer attenuated IFNβ production in MOLF/Ei macrophages to pathogen derived cytosolic di-nucleotides. Fine mapping of a major locus of linkage revealed a novel polymorphic allele of Tmem173 (STING). The MOLF allele of Tmem173 produces a protein with multiple amino acid changes, and an internal 6 amino acid deletion. Most of these amino acid changes are confined to the understudied N-terminus. These polymorphisms in MOLF STING altogether confer a lack of induction of the IFNβ promoter in an overexpression assay that seems to be attributed to the most N-terminal proximal mutations. The loss-of-function of MOLF STING is inherited in macrophages of C57Bl/6 mice congenic for the MOLF Tmem173 allele. C57Bl/6 2 macrophages congenic for MOLF STING display a shifted interferon signal profile in response to different c-di-nucleotides when compared to wild-type C57Bl/6. Furthermore, expression of MOLF STING in STING-/- MEFs show attenuated trafficking of MOLF STING after c-di-nucleotide activation compared to B6 STING. Several, human alleles of STING confer a loss-of-function or gain-of-function phenotype that may dramatically affect immunity. The study of this novel allele in mice will aid in the mechanistic study the STING pathway that has recently garnered much attention as being central to the response to cytosolic DNA. 3 To know, is to know that you know nothing. That is the meaning of true knowledge. Socrates 4 Acknowledgements I would like to thank my family for loving and nurturing my curiosity and love for science. Thank you Sasha for bringing me into your lab. Thanks for sharing excitement for the work that we have done together. It was a blessing to have a mentor such as you who always encouraged, and believed in me. I have learned how to think original scientific ideas, and many life lessons. Thank you Irina for your friendship, and for your dependable support. I will not forget all the molecular biology techniques that I have learned. 5 DEDICATION I would like to dedicate this work to my family, and to the people whom I intend to serve. 6 TABLE OF CONTENTS ABSTRACT .......................................................................................................................... 2 Acknowledgements ............................................................................................................. 5 DEDICATION ...................................................................................................................... 6 TABLE OF CONTENTS ....................................................................................................... 7 LIST OF TABLES .............................................................................................................. 11 LIST OF FIGURES ............................................................................................................. 12 LIST OF ABBREVIATIONS ............................................................................................... 15 CHAPTER 1 - Introduction .............................................................................................. 17 Type I IFNs in Physiology and Pathogenesis ............................................................... 17 DNA Damage sensing and Type I Interferons ........................................................... 19 PRRs .............................................................................................................................. 22 DNA Sensors ................................................................................................................ 26 STING ............................................................................................................................ 29 SELF-DNA and the overwhelmed DDR leads to STING mediated autoimmunity ..... 38 Forward genetics is an unbiased approach of explaining gene’s function ............. 38 MOLF mice variability ................................................................................................. 41 CHAPTER 2 - Methods .................................................................................................... 44 Reagents .................................................................................................................... 44 7 Mice .......................................................................................................................... 44 DNA Sequencing ...................................................................................................... 44 SNP analysis ............................................................................................................. 45 Species alignment .................................................................................................... 45 Isolation of Primary Peritoneal Macrophages ...................................................... 46 Genotyping and Quantitative Trait Locus Mapping (QTL) .................................. 46 Plasmids .................................................................................................................... 47 Luciferase assay ........................................................................................................... 47 Overexpression assay ............................................................................................. 47 CDN stimulus dependent Luciferase assay ........................................................... 48 Western Blotting ....................................................................................................... 48 Listeria monocytogenes, HSV1, and Sendai infection ............................................. 49 ELISA ....................................................................................................................... 49 Quantitative PCR RNA analysis ............................................................................... 50 RNASeq workflow .................................................................................................... 50 Lentiviral transduction of STING -/- MEFs ............................................................. 51 Confocal imaging of MEFs ....................................................................................... 51 CHAPTER 3 - MOLF macrophages lack interferon responses to DNA viruses, Listeria, and cytosolic nucleotides. ............................................................................................... 53 MOLF macrophages lack IFNβ responses to DNA viruses ......................................... 55 CHAPTER 4 - Forward genetic screen reveals linkage to Tmem173 as candidate gene. ........................................................................................................................................... 66 8 N2 Panel responses to cytosolic nucleotides and pathogens ........................................ 67 CHAPTER 5 - Identification of polymorphisms in MOLF Tmem173. ........................... 79 CHAPTER 6 - MOLF STING is deficient in activating the IFNβ and NF-kB promoter elements in Luciferase assay. ............................................................................................ 92 CHAPTER 7 - Characterization of Interferon responses in Congenic mice reveals further defect of MOLF STING. ..................................................................................... 106 CHAPTER 8 - Mapping genes that interact with MOLF STING on the MOLF background to confer responsiveness. ........................................................................ 117 CHAPTER 9 - Localization of STING during signaling. ............................................... 126 CHAPTER 10 - RNA-seq and Pathway analysis of STING dependent responses in macrophages ................................................................................................................... 135 DISCUSSION ................................................................................................................... 147 Summary Findings ...................................................................................................... 148 STING dependent IL-6 production ............................................................................. 150 Complementation ........................................................................................................ 151 STING signalosomes .................................................................................................
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