Die Regulation des Zelltods ist ein kritischer Schritt in der Embryonalentwicklung, der Gewebehomöostase und bei der Abwehr eindringender Krankheitserreger. Hierbei spielt Caspase-8 (Casp8) eine entscheidende Rolle, indem das Enzym extrinsische Apoptose induziert. Außerdem hemmt Casp8 Nekroptose und sichert so die embryonale Entwicklung. Wir zeigen hier, dass eine weitere wichtige Funktion der enzymatische Aktivität von Casp8 die Hemmung der Pyroptose ist. Um die spezifische Rolle der enzymatischen Aktivität von Casp8 zu untersuchen, haben wir eine Mauslinie etabliert, welche eine Punktmutation in der Substratbindetasche von Casp8 (Mutation des katalytischen Cysteins 362 zu Serin) aufweist. Diese Mutation führt zum Verlust der enzymatischer Aktivität von Casp8 C362S. Die Expression von katalytisch inaktiver Casp8 C362S resultiert in eine embryonale Letalität bei E10,5 aufgrund von kardiovaskulären Defekten, ähnlich zu Casp8-/-Mäusen. Die Blockade von Nekroptose durch zusätzliche Deletion von MLKL verhinderte den kardiovaskulären Phänotyp, verursachte jedoch unerwartet die perinatale Letalität von Casp8 C362S/C362S Mäusen. Dies deutet darauf hin, dass der Verlust der enzymatischen Aktivität von Casp8 die perinatale Entwicklung durch zusätzliche, Nekroptose-unabhängigen Funktionen beeinträchtigt. Der spezifische Verlust der katalytischen Aktivität von Casp8 in Darmepithelzellen (IECs) führte zu einer Darmentzündung ähnlich zu Casp8 IEC-KO Mäusen. Eine zusätzliche Deletion von MLKL verschlimmerte die Entzündung des Darms und verursachte ein vorzeitiges Versterben von Casp8 C362S/IEC-/Mlkl-/-Mäusen durch die Induktion des pyroptischen Zelltods. In diesem Zusammenhang konnten Prozesse, welche charakteristisch für Pyroptose sind, wie die Bildung von ASC-Specks, die Aktivierung von Casp1 sowie die Produktion von IL-1β beobachtet werden. Unsere Zellkulturanalysen zeigen, dass katalytisch inaktive Casp8 mit ASC kolokalisiert, sowie ASC-Nukleation und Casp1-Aktivierung induziert. Dementsprechend verhinderte die Deletion von ASC oder Casp1 die Entzündung des Darms und den vorzeitigen Tod von Casp8 C362S/C362S /Mlkl-/-Mäusen. Diese Ergebnisse zeigen eine noch unbekannte und unerwartete Rolle für Casp8 als Proteingerüst eines Signalkomplexes, welcher unter Gegebenheiten bei denen Apoptose und Nekroptose blockiert sind, gebildet wird.

Z-DNA and Z-RNA are left-handed double helix nucleic acid structures with poorly understood biological function 1 – 3 . Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM-1) is a nucleic acid sensor containing two Zα domains that bind Z-DNA 4 , 5 and Z-RNA 6 – 8 . ZBP1 mediates host-defence against certain viruses 6 , 7 , 9 – 14 by sensing viral nucleic acids 6 , 7 , 10 . RIPK1 deficiency or mutation of its RIP homotypic interaction motif (RHIM) triggers ZBP1-dependent necroptosis and inflammation in mice 15 , 16 , however, the mechanisms inducing ZBP1 activation in the absence of viral infection remain elusive. Here we show that Zα-dependent sensing of endogenous ligands induces ZBP1-mediated perinatal lethality in mice expressing RIPK1 with mutated RHIM ( Ripk1 mR/mR ) and skin inflammation in mice with epidermis-specific RIPK1 deficiency (RIPK1 E-KO ), as well as colitis in mice with intestinal epithelial-specific FADD deficiency (FADD IEC-KO ). Consistently, functional Zα domains were required for ZBP1-induced necroptosis in fibroblasts that express RIPK1 with mutated RHIM or were treated with caspase inhibitors. Moreover, inhibition of nuclear export triggered Zα-dependent activation of RIPK3 in the nucleus resulting in cell death, suggesting that ZBP1 may recognise Z-form nucleic acids (Z-NA) in the nucleus. We found that ZBP1 constitutively bound cellular double stranded RNA (dsRNA) in a Zα-dependent manner. Furthermore, endogenous retroelement (ERE)-derived complementary reads were detected in epidermal RNA, suggesting that ERE-derived dsRNA may act as Zα domain ligand triggering ZBP1 activation. Collectively, our results provide evidence that sensing of endogenous Z-NA by ZBP1 triggers RIPK3-dependent necroptosis and inflammation, which could underlie the development of chronic inflammatory conditions particularly in patients with mutations in the RIPK1 and CASPASE-8 genes 17 – 20 .

SummaryIntestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor κB (NF-κB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-κB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.

Pathways controlling intestinal epithelial cell (IEC) death regulate gut immune homeostasis and contribute to the pathogenesis of inflammatory bowel diseases. Here, we show that caspase-8 and its adapter FADD act in IECs to regulate intestinal inflammation downstream of Z-DNA binding protein 1 (ZBP1)-and tumor necrosis factor receptor-1 (TNFR1)-mediated receptor interacting protein kinase 1 (RIPK1) and RIPK3 signaling. Mice with IEC-specific FADD or caspase-8 deficiency developed colitis dependent on mixed lineage kinase-like (MLKL)-mediated epithelial cell necroptosis. However, MLKL deficiency fully prevented ileitis caused by epithelial caspase-8 ablation, but only partially ameliorated ileitis in mice lacking FADD in IECs. Our genetic studies revealed that caspase-8 and gasdermin-D (GSDMD) were both required for the development of MLKL-independent ileitis in mice with epithelial FADD deficiency. Therefore, FADD prevents intestinal inflammation downstream of ZBP1 and TNFR1 by inhibiting both MLKL-induced necroptosis and caspase-8-GSDMD-dependent pyroptosis-like death of epithelial cells.

Dysregulated autophagy and ER stress are involved in the etiology of human IBD. Aden et al. show that loss of ATG16L1 function renders intestinal epithelial cells vulnerable to IL-22–induced ER stress and necroptosis via STING signaling, which induces ileal inflammation in vivo.

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Necroptosis and pyroptosis are inflammatory forms of regulated necrotic cell death as opposed to apoptosis that is generally considered immunologically silent. Recent studies revealed unexpected links in the pathways regulating and executing cell death in response to activation of signalling cascades inducing apoptosis, necroptosis and pyroptosis. Emerging evidence suggests that RIPK1 and caspase-8 control the crosstalk between apoptosis, necroptosis and pyroptosis and determine the type of cell death induced in response to activation of cell death signalling.

Receptor interacting protein kinase 1 (RIPK1) regulates cell death and inflammatory responses downstream of TNFR1 and other receptors, and has been implicated in the pathogenesis of inflammatory and degenerative diseases. RIPK1 kinase activity induces apoptosis and necroptosis, however the mechanisms and phosphorylation events regulating RIPK1dependent cell death signaling remain poorly understood. Here we show that RIPK1 autophosphorylation at serine 166 plays a critical role for the activation of RIPK1 kinase-dependent apoptosis and necroptosis. Moreover, we show that S166 phosphorylation is required for RIPK1 kinase-dependent pathogenesis of inflammatory pathologies in vivo in four relevant mouse models. Mechanistically, we provide evidence that trans autophosphorylation at S166 modulates RIPK1 kinase activation but is not by itself sufficient to induce cell death. These results show that S166 autophosphorylation licenses RIPK1 kinase activity to induce downstream cell death signaling and inflammation, suggesting that S166 phosphorylation can serve as a reliable biomarker for RIPK1 kinase-dependent pathologies.