The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- (also known as Rbck1-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3-/-Casp8-/-Hoil-1-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.

Department of Medical Biology The University of Melbourne Melbourne Victoria Australia

Institute of General Pathology Università Cattolica del Sacro Cuore Rome Italy

Laboratory of Adaptive Immunity Institute of Molecular Genetics Czech Academy of Sciences Prague Czech Republic

Pattern Recognition Receptor Discovery Performance Unit Immuno Inflammation Therapeutic Area GlaxoSmithKline Collegeville PA USA

The Walter and Eliza Hall Institute of Medical Research Parkville Victoria Australia

UCL Cancer Institute University College London London UK

UCL Great Ormond Street Institute of Child Health London UK

University of Texas Health Science Center San Antonio TX USA

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Shimizu Y, Taraborrelli L, Walczak H. Linear ubiquitination in immunity. Immunological reviews. 2015;266:190–207. doi: 10.1111/imr.12309. PubMed DOI PMC

Sasaki Y, et al. Defective immune responses in mice lacking LUBAC-mediated linear ubiquitination in B cells. EMBO J. 2013;32:2463–2476. doi: 10.1038/emboj.2013.184. PubMed DOI PMC

Emmerich CH, et al. Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains. Proc Natl Acad Sci U S A. 2013;110:15247–15252. doi: 10.1073/pnas.1314715110. PubMed DOI PMC

Berger SB, et al. Cutting Edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice. J Immunol. 2014;192:5476–5480. doi: 10.4049/jimmunol.1400499. PubMed DOI PMC

Gerlach B, et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature. 2011;471:591–596. doi: 10.1038/nature09816. PubMed DOI

Kumari S, et al. Sharpin prevents skin inflammation by inhibiting TNFR1-induced keratinocyte apoptosis. eLife. 2014;3 doi: 10.7554/eLife.03422. PubMed DOI PMC

Rickard JA, et al. TNFR1-dependent cell death drives inflammation in Sharpin-deficient mice. eLife. 2014;3 doi: 10.7554/eLife.03464. PubMed DOI PMC

Peltzer N, et al. HOIP deficiency causes embryonic lethality by aberrant TNFR1-mediated endothelial cell death. Cell Rep. 2014;9:153–165. doi: 10.1016/j.celrep.2014.08.066. PubMed DOI

Boisson B, et al. Immunodeficiency, autoinflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency. Nat Immunol. 2012;13:1178–1186. doi: 10.1038/ni.2457. PubMed DOI PMC

Boisson B, et al. Human HOIP and LUBAC deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia. J Exp Med. 2015;212:939–951. doi: 10.1084/jem.20141130. PubMed DOI PMC

Tokunaga F, et al. Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation. Nat Cell Biol. 2009 doi: 10.1038/ncb1821. PubMed DOI

Tokunaga F, et al. Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation. Nat Cell Biol. 2009;11:123–132. doi: 10.1038/ncb1821. PubMed DOI

Stieglitz B, Morris-Davies AC, Koliopoulos MG, Christodoulou E, Rittinger K. LUBAC synthesizes linear ubiquitin chains via a thioester intermediate. EMBO Rep. 2012;13:840–846. doi: 10.1038/embor.2012.105. PubMed DOI PMC

Sato Y, et al. Specific recognition of linear ubiquitin chains by the Npl4 zinc finger (NZF) domain of the HOIL-1L subunit of the linear ubiquitin chain assembly complex. Proc Natl Acad Sci U S A. 2011;108:20520–20525. doi: 10.1073/pnas.1109088108. PubMed DOI PMC

Kaiser WJ, et al. RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature. 2011;471:368–372. doi: 10.1038/nature09857. PubMed DOI PMC

Oberst A, et al. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature. 2011;471:363–367. doi: 10.1038/nature09852. PubMed DOI PMC

Alvarez-Diaz S, et al. The Pseudokinase MLKL and the Kinase RIPK3 Have Distinct Roles in Autoimmune Disease Caused by Loss of Death-Receptor-Induced Apoptosis. Immunity. 2016 doi: 10.1016/j.immuni.2016.07.016. PubMed DOI PMC

Clapes T, Lefkopoulos S, Trompouki E. Stress and Non-Stress Roles of Inflammatory Signals during HSC Emergence and Maintenance. Front Immunol. 2016;7:487. doi: 10.3389/fimmu.2016.00487. PubMed DOI PMC

Pietras EM. Inflammation: a key regulator of hematopoietic stem cell fate in health and disease. Blood. 2017;130:1693–1698. doi: 10.1182/blood-2017-06-780882. PubMed DOI PMC

Harris PA, et al. Discovery of a First-in-Class Receptor Interacting Protein 1 (RIP1) Kinase Specific Clinical Candidate (GSK2982772) for the Treatment of Inflammatory Diseases. Journal of Medicinal Chemistry. 2017;60:1247–1261. doi: 10.1021/acs.jmedchem.6b01751. PubMed DOI

Roderick JE, et al. Hematopoietic RIPK1 deficiency results in bone marrow failure caused by apoptosis and RIPK3-mediated necroptosis. Proc Natl Acad Sci U S A. 2014;111:14436–14441. doi: 10.1073/pnas.1409389111. PubMed DOI PMC

Rickard JA, et al. RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis. Cell. 2014;157:1175–1188. doi: 10.1016/j.cell.2014.04.019. PubMed DOI

Dillon CP, et al. Survival function of the FADD-CASPASE-8-cFLIP(L) complex. Cell Rep. 2012;1:401–407. doi: 10.1016/j.celrep.2012.03.010. PubMed DOI PMC

Peltzer N, Darding M, Walczak H. Holding RIPK1 on the Ubiquitin Leash in TNFR1 Signaling. Trends Cell Biol. 2016;26:445–461. doi: 10.1016/j.tcb.2016.01.006. PubMed DOI

Gustafsson E, Brakebusch C, Hietanen K, Fassler R. Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice. Journal of cell science. 2001;114:671–676. PubMed

Murphy JM, et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity. 2013;39:443–453. doi: 10.1016/j.immuni.2013.06.018. PubMed DOI

Newton K, Sun X, Dixit VM. Kinase RIP3 is dispensable for normal NF-kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4. Molecular and cellular biology. 2004;24:1464–1469. PubMed PMC

Salmena L, et al. Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. Genes & development. 2003;17:883–895. doi: 10.1101/gad.1063703. PubMed DOI PMC

Kelliher MA, et al. The death domain kinase RIP mediates the TNF-induced NF-kappaB signal. Immunity. 1998;8:297–303. PubMed

Haas TL, et al. Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction. Mol Cell. 2009;36:831–844. doi: 10.1016/j.molcel.2009.10.013. PubMed DOI

TBK1 and IKKε prevent TNF-induced cell death by RIPK1 phosphorylation