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 #
3R01AI108685-01S1
Application #
8910840
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Leitner, Wolfgang W
Project Start
2014-01-01
Project End
2018-12-31
Budget Start
2014-08-15
Budget End
2014-12-31
Support Year
1
Fiscal Year
2014
Total Cost
$19,998
Indirect Cost
$8,505
Name
University of Washington
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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
Lopez, Jonathan; Bessou, Margaux; Riley, Joel S et al. (2016) Mito-priming as a method to engineer Bcl-2 addiction. Nat Commun 7:10538
Oberst, Andrew (2016) Death in the fast lane: what's next for necroptosis? FEBS J 283:2616-25
Rodriguez, D A; Weinlich, R; Brown, S et al. (2016) Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis. Cell Death Differ 23:76-88
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
Ichim, Gabriel; Lopez, Jonathan; Ahmed, Shafiq U et al. (2015) Limited mitochondrial permeabilization causes DNA damage and genomic instability in the absence of cell death. Mol Cell 57:860-72
Yatim, Nader; Jusforgues-Saklani, Hélène; Orozco, Susana et al. (2015) RIPK1 and NF-κB signaling in dying cells determines cross-priming of CD8⁺ T cells. Science 350:328-34
Haller, Martina; Hock, Andreas K; Giampazolias, Evangelos et al. (2014) Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity. Autophagy 10:2269-78
Orozco, S; Yatim, N; Werner, M R et al. (2014) RIPK1 both positively and negatively regulates RIPK3 oligomerization and necroptosis. Cell Death Differ 21:1511-21
Philip, Naomi H; Dillon, Christopher P; Snyder, Annelise G et al. (2014) Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF-κB and MAPK signaling. Proc Natl Acad Sci U S A 111:7385-90