Cutting Edge: RIPK1 Kinase Inactive Mice Are Viable and Protected from TNF-Induced Necroptosis In Vivo

This information is current as Apostolos Polykratis, Nicole Hermance, Matija Zelic, of October 1, 2021. Justine Roderick, Chun Kim, Trieu-My Van, Thomas H. Lee, Francis K. M. Chan, Manolis Pasparakis and Michelle A. Kelliher J Immunol 2014; 193:1539-1543; Prepublished online 11 July 2014; doi: 10.4049/jimmunol.1400590 Downloaded from http://www.jimmunol.org/content/193/4/1539

Supplementary http://www.jimmunol.org/content/suppl/2014/07/11/jimmunol.140059 http://www.jimmunol.org/ Material 0.DCSupplemental References This article cites 17 articles, 5 of which you can access for free at: http://www.jimmunol.org/content/193/4/1539.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology

Cutting Edge: RIPK1 Kinase Inactive Mice Are Viable and Protected from TNF-Induced Necroptosis In Vivo x x x Apostolos Polykratis,*,†,‡,1 Nicole Hermance, ,1 Matija Zelic, ,1 Justine Roderick, ,†,‡ ,†,‡ { ‖ Chun Kim,* Trieu-My Van,* Thomas H. Lee,x Francis K. M. Chan, Manolis Pasparakis,*,†,‡,2 and Michelle A. Kelliher ,2 The serine/threonine kinase RIPK1 is recruited to called “necroptosis.” TNF binding to TNFR1 leads to the TNFR1 to mediate proinflammatory signaling and to formation of an intracellular complex that includes RIPK1, regulate TNF-induced cell . A RIPK1 deficiency TRADD, TRAF2, cIAP1/2, and the components of the linear results in perinatal lethality, impaired NFkB and ubiquitin chain assembly complex (complex I) (1). RIPK1 is MAPK signaling, and sensitivity to TNF-induced apo- modified by the addition of K63-linked and linear ubiquitin ptosis. Chemical inhibitor and in vitro–reconstitution chains, resulting in the recruitment of NEMO/IKKg and the Downloaded from studies suggested that RIPK1 displays distinct kinase TAK1/TAB2/TAB3 complex to mediate IKK activation (1). activity–dependent and –independent functions. To Deubiquitination of RIPK1 by CYLD or A20 results in for- determine the contribution of RIPK1 kinase to in- mation of a cytosolic complex, containing RIPK1, FADD, flammation in vivo, we generated knock-in mice en- cFLIP, and 8 (complex IIb) (2). Caspase 8 then dogenously expressing catalytically inactive RIPK1 cleaves and inactivates RIPK1, RIPK3, and CYLD and 2 2 stimulates (3–5). In the absence of caspase 8 or http://www.jimmunol.org/ D138N. Unlike Ripk1 / mice, which die shortly after D138N/D138N when apoptosis is inhibited, RIPK1 interacts with RIPK3 to birth, Ripk1 mice are viable. Cells expressing induce necroptosis via the recruitment of MLKL (2). RIPK1 D138N are resistant to TNF- and polyinosinic- Necroptosis is known to contribute to several types of polycytidylic acid–induced necroptosis in vitro, and pathological injury and can be triggered by TNF family Ripk1D138N/D138N mice are protected from TNF-induced D138N/D138N members (TNF, Fas, and TRAIL), TLRs (TLR3 and TLR4), shock in vivo. Moreover, Ripk1 mice fail to or the DNA sensor (DAI) (2). Necroptosis is thought to control vaccinia virus replication in vivo. This study depend on auto- and transphosphorylation of RIPK1 and provides genetic evidence that the kinase activity of RIPK3, resulting in the RIPK3-mediated recruitment of RIPK1 is not required for survival but is essential for MLKL (6). Although there is ample genetic evidence linking by guest on October 1, 2021 TNF-, TRIF-, and viral-initiated necroptosis. The RIPK3 to necroptosis that occurs during development, in- Journal of Immunology, 2014, 193: 1539–1543. flammation, and viral infection (6), genetic evidence impli- cating RIPK1 in necroptosis in vivo is limited (7). IPK1 is the founding member of a serine-threonine Materials and Methods kinase family that transduces inflammatory and cell Mice death signals following death receptor ligation, acti- R Ripk1D138N/D138N knock-in mice were generated by mutating the conserved vation of pattern recognition receptors, and DNA damage. aspartate (D) at position 138 to asparagine (D138N). The Ripk1D138N con- RIPK1 is the core component of TNF-induced signaling struct was introduced into Bruce 4 embryonic stem cells derived from C57BL/6 mice. Mice were maintained at the specific pathogen–free animal complexes mediating NFkB and MAPK activation, apoptosis, facilities of the University of Massachusetts Medical School and the Institute and an alternative form of caspase-independent for Genetics at the University of Cologne. All animal procedures were con-

*Institute for Genetics, University of Cologne, 50674 Cologne, Germany; †Cologne F.K.M.C. provided B13R-deficient virus and advice on design and interpretation of Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of viral experiments. N.H., M.Z., J.R., A.P., C.K., and T.-M.V. performed the experi- Cologne, 50931 Cologne, Germany; ‡Centre for Molecular Medicine, University of Cologne, ments. M.A.K. and M.P. coordinated the project and wrote the manuscript. x 50931 Cologne, Germany; Department of Cancer Biology, University of Massachusetts { ‖ Address correspondence and reprint requests to Dr. Michelle A. Kelliher or Dr. Manolis Medical School, Worcester, MA 01605; Genentech, San Francisco, CA 94080; and De- Pasparakis, Department of Cancer Biology, University of Massachusetts Medical School, partment of Pathology, University of Massachusetts Medical School, Worcester, MA 01605 Lazare Research Building, 364 Plantation Street, Worcester, MA 01605 (M.A.K.) or 1A.P., N.H., and M.Z. contributed equally to this work. Institute for Genetics, Centre for Molecular Medicine, and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, 2M.P. and M.A.K. shared last authorship. D-50931 Cologne, Germany (M.P.). E-mail addresses: michelle.kelliher@umassmed. Received for publication March 7, 2014. Accepted for publication June 18, 2014. edu (M.A.K.) or [email protected] (M.P.) This work was supported by National Institutes of Health/National Institute of The online version of this article contains supplemental material. Allergy and Infectious Diseases Grant AI075118 (to M.A.K.). M.P. acknowledges Abbreviations used in this article: BMDM, bone marrow–derived ; m, funding from the European Research Council (2012-ADG_20120314), the German mouse; MEF, mouse embryonic fibroblast; Nec-1, necrostatin-1; poly (I:C), Research Foundation (SFB670, SFB829, SPP1656), the European Commission polyinosinic-polycytidylic acid; VV, vaccinia virus. (FP7 Grants 223404 [Masterswitch] and 223151 [InflaCare]), and the Deutsche Krebshilfe (Grant 110302). Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 T.H.L. and M.A.K. designed and generated the targeting construct and A.P. performed the gene targeting in embryonic stem cells and generated the RIPK1 knock-in mice. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400590 1540 CUTTING EDGE: RIPK1 KINASE REQUIRED FOR NECROPTOSIS

ducted in accordance with national and institutional guidelines. Sex- and age- matched mice were used in all experiments. In vitro kinase assay and immunoblotting Mouse embryonic fibroblasts (MEFs) were left untreated or treated with mouse (m)TNF-a (50 ng/ml), RIPK1 was immunoprecipitated, and the kinase assay was performed in the presence or absence of Necrostatin-1 (Nec- 1; 30 mM) in kinase buffer containing 10 mCi g-[32P]-ATP. The samples were separated by SDS-PAGE and visualized by autoradiography. Wild-type or Ripk1D138N/D138N MEFs or bone marrow–derived (BMDMs) were left untreated or treated with 10 ng/ml mTNF, 25 mg/ml polyinosinic- polycytidylic acid [poly (I:C)], or 100 ng/ml LPS for the indicated time periods, and signaling was examined as described previously (8). Necroptosis assays 2 2 2 2 Wild-type, Ripk1D138N/D138N, Ripk1 / , and Ripk3 / MEFs were treated with 10 mg/ml cycloheximide, 20 mM zVAD-fmk, 10 mM Smac mimetic, or 30 mm Nec-1 and then treated with 10 ng/ml mTNF. Wild-type or Ripk1D138N/D138N BMDMs were treated with 20 mM zVAD-fmk or 30 mM Nec-1 and then with 50 mg/ml poly(I:C), and cell survival was determined by neutral red assay.

TNF-induced shock experiments Downloaded from 2 2 Age- and sex-matched wild-type, Ripk1D138N/D138N and Ripk3 / mice were injected with 9 mg mTNF only or with zVAD-fmk (16.7 mg/kg). Body temperature and survival were determined. Vaccinia virus infections D138N/D138N 2/2

Ten-week-old wild-type, Ripk1 and Ripk3 mice were infected http://www.jimmunol.org/ with 2 3 106 PFU Western Reserve or B13R-deficient strain of vaccinia virus (VV) via the i.p. route. Tissue extracts were harvested, and titers were de- termined by plaque assays on Vero cells. Results and Discussion To reveal the in vivo physiologic role(s) of the serine/threonine kinase activity of RIPK1, we generated knock-in mice expressing a kinase-inactive mutant RIPK1 from the endog- enous Ripk1 locus. The mutation introduced resulted in the by guest on October 1, 2021 replacement of the conserved aspartate (D) at position 138 within the activation loop of the RIPK1 kinase domain with asparagine (Ripk1D138N) (Fig. 1A). To determine whether the D138N mutation alters the kinase activity of RIPK1, we immunoprecipitated RIPK1 from Ripk1D138N/D138N and wild-type MEFs that were left untreated or stimulated with TNF and performed an in vitro kinase assay in the presence of g-[32P]-ATP. RIPK1 autophosphorylation was detected in both unstimulated and TNF-treated wild-type cells, which was inhibited by the RIPK1 inhibitor Nec-1 (Fig. 1B). Al- though an equivalent amount of the RIPK1 D138N protein was immunoprecipitated, no RIPK1 autophosphorylation was observed, even in the presence of TNF (Fig. 1B). Moreover, the D138N mutation appeared to have no detectable effects on RIPK1 expression levels (Fig. 1C). RIPK1 is recruited to TNFR1 to mediate the activation of NFkB, p38 MAPK, JNK, and ERK. RIPK1 is also recruited to the TLR3/4 adapter TRIF and contributes to TRIF- mediated NFkB activation and necroptosis (2, 9). A role for the kinase activity of RIPK1 in TNF-induced ERK activation FIGURE 1. Generation of kinase-inactive Ripk1 allele. (A) Schematic diagram has been suggested (10). Therefore, we examined TNF- and of the mouse Ripk1 locus and the Ripk1 D138N allele. (B) The D138N mu- TRIF-dependent signaling in MEFs and primary BMDMs. tation impairs RIPK1 kinase activity. Wild-type and Ripk1D138N/D138N MEFs were left untreated or stimulated with mTNF and an in vitro kinase assay per- formed in the presence or absence of Nec-1. The amount of RIPK1 in each required for TNF-induced NFkB, JNK, p38 MAPK, or ERK activation. immunoprecipitation was determined by immunoblotting with a RIPK1 Ab. (C) Wild-type and Ripk1D138N/D138N macrophages were left untreated or treated RIPK1 protein levels in Ripk1D138N/D138N mice. RIPK1 protein levels were ex- with TNF for the time periods indicated, and protein lysates were probed aminedbyimmunoblottinglysatesfromMEFsandmacrophagesisolatedfrom with phospho-specific Abs for JNK, p38 MAPK, and ERK. Lysates also were wild-type and Ripk1D138N/D138N mice. (D) The kinase activity of RIPK1 is not probed with Abs to total IkBa, JNK1/2, p38a, ERK, and b-actin. The Journal of Immunology 1541

Cells expressing RIPK1 D138N showed normal activation of these signaling pathways in response to TNF, poly (I:C), and LPS (Fig. 1D, Supplemental Fig. 2). These data are consistent with studies using Nec-1 to inhibit RIPK1 (11, 12) and with 2 2 our prior studies using Ripk1 / MEFs reconstituted with a kinase-inactive form of RIPK1 (8).

The kinase activity of RIPK1 is not essential for survival or for TNF- or TRIF-dependent activation of NFkB or MAPK signaling Unlike mice with a complete RIPK1 deficiency, which die at/ around the time of birth (13), Ripk1D138N/D138N mice are born at the expected Mendelian ratios and show no gross or histological abnormalities (Supplemental Fig. 1). A complete RIPK1 deficiency results in perinatal death, with evidence of cell death in the thymus, lymph nodes, and s.c. tissue, which is accompanied by an inflammatory response characterized by granulocyte and macrophage infiltration (13). To determine

whether loss of RIPK1 kinase activity causes similar pathology Downloaded from in vivo, we examined tissues from Ripk1D138N/D138N mice for evidence of cell death and inflammation. Histopathological examination and cleaved caspase 3 staining of tissues from Ripk1D138N/D138N mice revealed no evidence of cell death or inflammation (Supplemental Fig. 1). These results demonstrate that the kinase activity of RIPK1 is not required for mouse http://www.jimmunol.org/ FIGURE 2. Ripk1D138N/D138N MEFs and BMDMs are protected from TNF- survival. D138N/D138N 2/2 and poly (I:C)–induced necroptosis. (A) Wild-type, Ripk1 , Ripk1 , 2 2 and Ripk3 / MEFs were treated with zVAD-fmk, Nec-1, or cycloheximide prior D138N/D138N 2 2 Ripk1 MEFs and macrophages are resistant to TNF- and to treatment with mTNF. (B)Wild-type,Ripk1D138N/D138N,andRipk1 / MEFs poly (I:C)–induced necroptosis were treated with TNF and/or Smac mimetic and cell viability was determined. C D138N/D138N Necroptosis can be initiated by TNF or pattern recognition ( ) Wild-type or Ripk1 BMDMs were treated with zVAD-fmk and/ receptors (2). To investigate necroptotic death in response to or Nec-1 prior to treatment with poly (I:C).Cellviabilitywasanalyzedbyneutral 2 2 2 2 red assay, and the percentage of survival is shown. ***p , 0.0001. TNF, wild-type, Ripk1D138N/D138N, Ripk1 / ,orRipk3 /

MEFs were treated with cycloheximide, the pan caspase in- by guest on October 1, 2021 proved essential for TNF- and poly (I:C)–induced necroptosis hibitor zVAD-fmk, and/or Nec-1 prior to the addition of in vitro. mTNF. Ripk1D138N/D138N MEFs were protected from TNF/ zVAD-induced necroptosis, but they exhibited similar levels Ripk1D138N/D138N mice are protected from TNF-induced of TNF-induced apoptosis as wild-type cells (Fig. 2A). As hypothermia expected, a RIPK1 deficiency sensitized cells to TNF-induced TNF administration to mice mimics the histopathological and apoptosis, and a RIPK1 or RIPK3 deficiency rendered cells pathophysiologic changes associated with septic shock. We resistant to TNF-induced necroptosis (Fig. 2A). The kinase challenged Ripk1D138N/D138N mice, as well as wild-type and 2 2 activity also was implicated in TNF-induced apoptosis that Ripk3 / mice, with 9 mg mTNF, a dose determined previ- was induced under conditions of cIAP depletion (14). Con- ously to induce death in 100% of treated wild-type mice. In 2 2 sistent with these studies, we found that RIPK1 and its kinase contrast to wild-type controls, Ripk1D138N/D138N and Ripk3 / activity are required for apoptosis induced by TNF and Smac mice were protected from hypothermia, and all of the ani- mimetic treatment (Fig. 2B). mals survived the TNF challenge (Fig. 3). Thus, expression of The TLR3/4-specific adapter TRIF recruits RIPK1 via the a kinase-inactive form of RIPK1 was sufficient to provide rip homotypic interaction motif to mediate NFkB activation; protection from TNF-induced hypothermia and mortality in when apoptosis is inhibited, it is thought to recruit RIPK1 the presence or absence of caspase inhibition (Fig. 3). Collec- and RIPK3 to mediate necroptosis (6, 9). However, TRIF- tively, these genetic studies reveal that the mortality induced by dependent necroptosis can occur in the absence of RIPK1 and TNF in this shock model reflects RIPK1- and RIPK3-mediated may be mediated by the direct recruitment of RIPK3 to TRIF necroptotic death in vivo. and TLR3/4 (15). To determine whether the kinase activity of Necroptosis has emerged as an important host response to RIPK1 mediates TRIF-dependent necroptosis, BMDMs from viral infection. VV-infected cells become susceptible to nec- D138N/D138N wild-type or Ripk1 mice were left untreated or roptosis because the virus encodes the caspase inhibitor B13R, treatedwithzVAD-fmkand/orNec-1for1hpriortostimu- which inhibits caspase 8 and consequently stimulates RIPK3- lation with poly (I:C). Poly (I:C) and zVAD-fmk treatment of dependent necroptosis (16). We found that Ripk1D138N/D138N wild-type BMDMs induced cell death that was prevented by mice were unable to control VV replication, resulting in Nec-1 pretreatment (Fig. 2C). We found that Ripk1D138N/D138N significant increases in viral titers in the spleen and liver of BMDMs were protected from poly (I:C)–induced necroptosis infected mice (Fig. 4A, 4B). Consistent with the viral loads 2 2 (Fig. 2C). Thus, an absence of RIPK1 kinase activity had no in these animals, and as observed in Ripk3 / mice (16), detectable effect on TNF-induced apoptosis (TNF/Cx), but it Ripk1D138N/D138N mice exhibit reduced liver inflammation 1542 CUTTING EDGE: RIPK1 KINASE REQUIRED FOR NECROPTOSIS

FIGURE 3. Ripk1D138N/D138N mice are protected from TNF-induced shock. Body temperatures and survival of wild- 2 2 type, Ripk1D138N/D138N,andRipk3 / mice injected with mTNF (A and B) (p , 0.0001) or mTNF and zVAD-fmk (C and D)(p , 0.0001).

compared with infected wild-type mice (Fig. 4D, 4E). In nase activity of RIPK1 does not contribute to proinflammatory Downloaded from contrast, infection of wild-type and Ripk1D138N/D138N mice signaling induced by TNF or TLR3/4 ligands (Supplemental with the B13R-deficient virus, which lacks the caspase 8 in- Fig. 2). Apoptosis induced by TNF and cycloheximide treat- hibitor, yielded similar viral titers in both strains (Fig. 4C). ment was unaffected in Ripk1D138N/D138N MEFs, whereas the These data demonstrate that the host defense to VV infection kinase activity was required for apoptosis induced by TNF and depends on necroptosis mediated by RIPK3 and the kinase Smac mimetic treatment (Fig. 2). Thus, the proinflammatory activity of RIPK1. and prosurvival functions of RIPK1 are kinase independent, http://www.jimmunol.org/ Our in vivo results show that the kinase activity of RIPK1 is whereas its prodeath functions remain kinase dependent. not required for mouse survival, but it has an essential role in In conclusion, our in vivo studies in Ripk1D138N/D138N mice TNF-induced necroptosis. In contrast to in vitro studies in demonstrate that the kinase is not responsible for the pro- which necroptosis is observed in the absence of RIPK1 (11, 15, survival function(s) of RIPK1, but it is indispensable for 17), we find that MEFs and primary BMDMs that express TNF-induced necroptosis in vivo. Our data also provide ge- RIPK1 D138N are resistant to TNF- and TRIF-induced netic evidence to implicate RIPK1 in viral-initiated nec- necroptosis. Consistent with published studies (8, 12), the ki- roptosis. Our findings support the development of more by guest on October 1, 2021

FIGURE 4. The kinase activity of RIPK1 is required for protection against VV infection in vivo. VV titers in the spleens (A)andlivers(B) of wild-type, Ripk1D138N/D138N, 2 2 and Ripk3 / mice. (C) B13R-deficient viral titers of wild-type and Ripk1D138N/D138N mice (p =0.24).(D) Histological examination of liver inflammation stained with 2 2 H&E. Arrows represent areas of liver inflammation. (E) Number of focal inflammatory regions for infected wild-type, Ripk1D138N/D138N,andRipk3 / mice *p , 0.05, **p , 0.01, ***p , 0.0001. The Journal of Immunology 1543 stable, potent, and selective RIPK1 inhibitors to control the 8. Lee, T. H., J. Shank, N. Cusson, and M. A. Kelliher. 2004. The kinase activity of Rip1 is not required for tumor factor-alpha-induced IkappaB kinase or p38 systemic inflammation associated with chronic infection, MAP kinase activation or for the ubiquitination of Rip1 by Traf2. J. Biol. Chem. sepsis, or other types of tissue injury. 279: 33185–33191. 9. Meylan, E., K. Burns, K. Hofmann, V. Blancheteau, F. Martinon, M. Kelliher, and J. Tschopp. 2004. RIP1 is an essential mediator of Toll-like receptor 3-induced NF- kappa B activation. Nat. Immunol. 5: 503–507. Disclosures 10. Devin, A., Y. Lin, and Z. G. Liu. 2003. The role of the death-domain kinase RIP in The authors have no financial conflicts of interest. tumour-necrosis-factor-induced activation of mitogen-activated protein kinases. EMBO Rep. 4: 623–627. 11. Cho, Y., T. McQuade, H. Zhang, J. Zhang, and F. K. Chan. 2011. RIP1- dependent and independent effects of necrostatin-1 in necrosis and T cell activa- References tion. PLoS ONE 6: e23209. 1. Chen, Z. J. 2012. Ubiquitination in signaling to and activation of IKK. Immunol. 12. Degterev, A., J. Hitomi, M. Germscheid, I. L. Ch’en, O. Korkina, X. Teng, Rev. 246: 95–106. D. Abbott, G. D. Cuny, C. Yuan, G. Wagner, et al. 2008. Identification of RIP1 2. Vanden Berghe, T., A. Linkermann, S. Jouan-Lanhouet, H. Walczak, and kinase as a specific cellular target of necrostatins. Nat. Chem. Biol. 4: 313–321. P. Vandenabeele. 2014. Regulated necrosis: the expanding network of non- 13. Kelliher, M. A., S. Grimm, Y. Ishida, F. Kuo, B. Z. Stanger, and P. Leder. 1998. apoptotic cell death pathways. Nat. Rev. Mol. Cell Biol. 15: 135–147. The death domain kinase RIP mediates the TNF-induced NF-kappaB signal. Im- 3. Feng, S., Y. Yang, Y. Mei, L. Ma, D. E. Zhu, N. Hoti, M. Castanares, and M. Wu. munity 8: 297–303. 2007. Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by 14. Wang, L., F. Du, and X. Wang. 2008. TNF-alpha induces two distinct caspase-8 removal of kinase domain. Cell. Signal. 19: 2056–2067. activation pathways. Cell 133: 693–703. 4. O’Donnell, M. A., E. Perez-Jimenez, A. Oberst, A. Ng, R. Massoumi, R. Xavier, 15. Kaiser, W. J., H. Sridharan, C. Huang, P. Mandal, J. W. Upton, P. J. Gough, D. R. Green, and A. T. Ting. 2011. Caspase 8 inhibits programmed necrosis by C. A. Sehon, R. W. Marquis, J. Bertin, and E. S. Mocarski. 2013. Toll-like receptor processing CYLD. Nat. Cell Biol. 13: 1437–1442. 3-mediated necrosis via TRIF, RIP3, and MLKL. J. Biol. Chem. 288: 31268–31279. 5. Lin, Y., A. Devin, Y. Rodriguez, and Z. G. Liu. 1999. Cleavage of the death domain 16. Cho, Y. S., S. Challa, D. Moquin, R. Genga, T. D. Ray, M. Guildford, and kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev. 13: 2514–2526. F. K. Chan. 2009. Phosphorylation-driven assembly of the RIP1-RIP3 complex 6. Moriwaki, K., and F. K. Chan. 2013. RIP3: a molecular switch for necrosis and regulates programmed necrosis and virus-induced inflammation. Cell 137: 1112– Downloaded from inflammation. Genes Dev. 27: 1640–1649. 1123. 7. Zhang, H., X. Zhou, T. McQuade, J. Li, F. K. Chan, and J. Zhang. 2011. Func- 17. Moujalled, D. M., W. D. Cook, T. Okamoto, J. Murphy, K. E. Lawlor, J. E. Vince, tional complementation between FADD and RIP1 in embryos and lymphocytes. and D. L. Vaux. 2013. TNF can activate RIPK3 and cause programmed necrosis in Nature 471: 373–376. the absence of RIPK1. Cell Death Dis. 4: e465. http://www.jimmunol.org/ by guest on October 1, 2021