Tumor Necrosis Factor- (TNF) and Toll-like receptor (TLR) signaling play key roles in coordinating immune responses, by driving the transcriptional activation of pro-inflammatory genes. However, it has long been recognized that they can also trigger apoptotic cell death. More recently, it has been shown that these signals can also induce another form of programmed cell death, called necroptosis. While the discovery of necroptosis has generated considerable interest, the physiological role of this alternate cell death program remains elusive. In particular, necroptosis is blocked by the pro-apoptotic protease caspase-8, so most studies rely on genetic ablation or chemical inhibition of caspase-8 to trigger necroptosis. This raises a question: when does TNF or TLR-mediated necroptosis occur under physiological conditions? We have shown that caspase-8 must act in concert with its paralog FLIP to block necroptosis, and FLIP is potently up-regulated by TNF and TLR transcriptional signaling. Many types of infection and stress lead to inhibition of inflammatory signaling or general inhibition of protein synthesis. We therefore propose that the absence of FLIP-rather than inhibition of caspase-8-provides a general mechanism for cellular sensitization to necroptosis. We further hypothesize that necroptosis is itself inflammatory, because cells dying by necroptosis release damage- associated signaling molecules that activate immune cells. To address this possibility, we will focus on three specific questions: 1) How is the pro-necroptotic kinase RIPK3 activated, and how is this activation suppressed by caspase-8/FLIP? We have created a system in which multiple steps of RIPK3 can be controlled. We will use this system to test the hypothesis that RIPK3 activation requires phosphorylation- dependent assembly and propagation of a RIPK3 oligomer, and that caspase-8/FLIP directly blocks this process. 2) How is suppression of caspase-8/FLIP relieved to allow necroptosis under physiological conditions? We hypothesize that inhibitors of NF-kB signaling, or of general protein translation, sensitize cells to necroptosis by preventing FLIP expression. We will test this model in multiple cell types using pathologically relevant models of bacterial and viral infection, as well as ER stress. We will also consider how FLIP levels are controlled at both mRNA and protein levels. 3) How does the immune system respond to necroptotic vs. apoptotic cell death? We hypothesize that the mechanism by which a cell dies is important, because necroptosis releases inflammatory molecules that are contained or eliminated during apoptosis. To test this idea, we have created a system that allows us to trigger apoptosis or necroptosis using a non-toxic drug. We will use this system to analyze innate and adaptive immune responses to cell death. Together, the work proposed here seeks to understand the causes and consequences of necroptosis in vivo, and to thereby allow rational design of therapies that manipulate this process in infection, autoimmune disease, and cancer.

Public Health Relevance

Cell death occurs in response to many types of stress; how the immune system responds to dying cells is therefore crucial in determining the outcome of infection, limiting tumor progression, and preventing harmful autoimmune disease. This project explores how cells 'decide' between inflammatory and non-inflammatory cell death, and the consequences of this decision for subsequent immune responses.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI108685-03
Application #
8986155
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Singleton, Kentner L
Project Start
2014-01-01
Project End
2018-12-31
Budget Start
2016-01-01
Budget End
2016-12-31
Support Year
3
Fiscal Year
2016
Total Cost
$498,196
Indirect Cost
$201,356
Name
University of Washington
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Brault, Michelle; Olsen, Tayla M; Martinez, Jennifer et al. (2018) Intracellular Nucleic Acid Sensing Triggers Necroptosis through Synergistic Type I IFN and TNF Signaling. J Immunol 200:2748-2756
Ando, Kiyohiro; Parsons, Melissa J; Shah, Richa B et al. (2017) NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus. J Cell Biol 216:1795-1810
Orozco, Susana; Oberst, Andrew (2017) RIPK3 in cell death and inflammation: the good, the bad, and the ugly. Immunol Rev 277:102-112
Daniels, Brian P; Snyder, Annelise G; Olsen, Tayla M et al. (2017) RIPK3 Restricts Viral Pathogenesis via Cell Death-Independent Neuroinflammation. Cell 169:301-313.e11
Giampazolias, Evangelos; Zunino, Barbara; Dhayade, Sandeep et al. (2017) Mitochondrial permeabilization engages NF-?B-dependent anti-tumour activity under caspase deficiency. Nat Cell Biol 19:1116-1129
Kolb, Joseph P; Oguin 3rd, Thomas H; Oberst, Andrew et al. (2017) Programmed Cell Death and Inflammation: Winter Is Coming. Trends Immunol 38:705-718
Gutierrez, Kimberley D; Davis, Michael A; Daniels, Brian P et al. (2017) MLKL Activation Triggers NLRP3-Mediated Processing and Release of IL-1? Independently of Gasdermin-D. J Immunol 198:2156-2164
Nogusa, Shoko; Thapa, Roshan J; Dillon, Christopher P et al. (2016) RIPK3 Activates Parallel Pathways of MLKL-Driven Necroptosis and FADD-Mediated Apoptosis to Protect against Influenza A Virus. Cell Host Microbe 20:13-24
Oberst, Andrew (2016) Death in the fast lane: what's next for necroptosis? FEBS J 283:2616-25
Philip, Naomi H; DeLaney, Alexandra; Peterson, Lance W et al. (2016) Activity of Uncleaved Caspase-8 Controls Anti-bacterial Immune Defense and TLR-Induced Cytokine Production Independent of Cell Death. PLoS Pathog 12:e1005910

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