Developmental and Viral Control of Programmed Necrosis 公开

Abstract

Apoptosis and necroptosis are complementary cell death pathways controlled by common signaling adaptors, kinases and proteases. Among the key players, caspase-8 stands out because this protease mediates tumor necrosis factor (TNF) family death receptor-induced apoptosis. TNF family receptors also control cytokine activation via NF-kB activation. Recently, caspase 8 has been shown to suppress programmed necrosis mediated via receptor-interacting protein (RIP)1 and RIP3. Either apoptosis or necrosis can follow death receptor-activation, dependent on the level of caspase 8 activity. In this dissertation, I describe my work on establishing role of RIP3-dependent necrosis in host defense and the essential function caspase 8 plays in suppressing RIP3 during mammalian development.

Viral pathogenesis relies upon modulation of cell death pathways that contribute to host defense by eliminating infected cells. Infection by murine cytomegalovirus induces RIP3-dependent necrosis. Virus-induced programmed necrosis is death receptor- and TLR-independent but is dependent on the pathogen sensor protein DNA-dependent activator of interferon regulatory factors (DAI) which forms an antiviral pro-necrotic complex upon binding to RIP3. The viral inhibitor of RIP activation (vIRA, encoded by the viral M45 gene) suppresses programmed necrosis by disrupting DAI-RIP3 interactions. Importantly, the attenuation of vIRA-deficient virus in wild type mice is normalized in RIP3^-/- or DAI^-/- mice. Thus, vIRA function validates necrosis as central to the elimination of infected cells in host defense.

Germ line disruption of caspase 8 leads to embryonic lethality in mice. Conditional elimination of caspase 8 has revealed an essential role of this enzyme in a range of tissues and cell types. To determine whether caspase 8 naturally holds RIP3-dependent death pathways in check, I generated embryos lacking both caspase 8 and RIP3. Remarkably, these Casp8^-/-Rip3^-/- double mutant mice were viable, matured into fertile adults and appear immunocompetent although they develop lymphadenopathy marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr). Thus, capsase 8 contributes to homeostatic control in the adult immune system; however, RIP3 and caspase 8 are both completely dispensable for mammalian development.

Table of Contents

Abstract........................................................................................................................... iii

Acknowledgements......................................................................................................... v

Table of Contents ........................................................................................................... vi

List of Figures and Tables ............................................................................................. viii

Chapter 1: Introduction ....................................................................................................... 1

Figures 1.1-1.4 and Table 1.1 ............................................................................... 11

Chapter 2: Pathogen subversion of RIP3-dependent necrosis

A. Introduction....................................................................................................... 17

B. Results............................................................................................................... 21

C. Discussion.......................................................................................................... 32

D. Conclusions........................................................................................................ 39

E. Materials and Methods..................................................................................... 40

F. Figures 2.1-2.5 and 2.S1-S3................................................................................ 46

Chapter 3: RIP3 mediates the embryonic lethality of caspase-8-deficient mice

A. Introduction and Summary.................................................................................. 61

B. Results and Discussion....................................................................................... 62

C. Materials and Methods..................................................................................... 70

D. Figures 3.1-3.4 and 3.S1-3.S5............................................................................ 75

Chapter 4: DAI-RIP3 complex mediates virus-induced programmed necrosis

A. Introduction....................................................................................................... 88

B. Results............................................................................................................... 92

C. Discussion........................................................................................................ 100

D. Materials and Methods................................................................................... 105

E. Figures 4.1-4.4 and 4.S1-4.S4.......................................................................... 108

Chapter 5: Toll-like Receptor 3 induces RIP1-independent necrosis via a TRIF-RIP3 complex

A. Introduction...................................................................................................... 119

B. Results............................................................................................................. 123

C. Discussion........................................................................................................ 129

D. Materials and Methods................................................................................... 134

E. Figures 5.1-5.6................................................................................................. 136

Chapter 6: General Discussion and Future Possibilities ..................................................... 144

Figures 6.1 1.1 .................................................................................................... 154

References...................................................................................................................... 155

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Virology
关键词	apoptosis host defense programmed necrosis
Committee Chair / Thesis Advisor	Mocarski, Edward S, Emory University
Committee Members	Weiss, David S, Emory University Boise, Lawrence, Emory University Speck, Sam, Emory University Caspary, Tamara, Emory University

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