The interplay of IKK, NF- κb and RIPK1 signaling in the regulation of cell death, tissue homeostasis and inflammation

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1 DOI: /imr INVITED REVIEW The interplay of IKK, NF- κb and RIPK1 signaling in the regulation of cell death, tissue homeostasis and inflammation Vangelis Kondylis 1,2,3 Snehlata Kumari 1,2,3 Katerina Vlantis 1,2,3 Manolis Pasparakis 1,2,3 1 Institute for Genetics, University of Cologne, Cologne, Germany 2 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany 3 Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Correspondence Manolis Pasparakis, CECAD Research Center, Institute for Genetics, University of Cologne, Cologne, Germany. pasparakis@uni-koeln.de Funding information ERC, Grant/Award Number: ; DFG, Grant/Award Number: SFB670, SFB829, SFB1218 and SPP1656; European Commission, Grant/Award Number: ; Deutsche Krebshilfe, Grant/Award Number: ; Helmholtz Alliance Preclinical Comprehensive Cancer Center; Worldwide Cancer Research, Grant/Award Number: Summary Regulated cell death pathways have important functions in host defense and tissue homeostasis. Studies in genetic mouse models provided evidence that cell death could cause inflammation in different tissues. Inhibition of RIPK3- MLKL- dependent necroptosis by FADD and caspase- 8 was identified as a key mechanism preventing inflammation in epithelial barriers. Moreover, the interplay between IKK/NF- κb and RIPK1 signaling was recognized as a critical determinant of tissue homeostasis and inflammation. NEMO was shown to regulate RIPK1 kinase activity- mediated apoptosis by NFκB- dependent and independent functions, which are critical for averting chronic tissue injury and inflammation in the intestine and the liver. In addition, RIPK1 was shown to exhibit kinase activity- independent functions that are essential for preventing cell death, maintaining tissue architecture and inhibiting inflammation. In the intestine, RIPK1 acts as a scaffold to prevent epithelial cell apoptosis and preserve tissue integrity. In the skin, RIPK1 functions via its RHIM to counteract ZBP1/DAI- dependent activation of RIPK3- MLKL- dependent necroptosis and inflammation. Collectively, these studies provided evidence that the regulation of cell death signaling plays an important role in the maintenance of tissue homeostasis, and suggested that cell death could be causally involved in the pathogenesis of inflammatory diseases. KEYWORDS apoptosis, IKK/NF-κB signaling, inflammation, necroptosis, RIPK1 1 INTRODUCTION Inflammation is a primarily beneficial reaction that is important for host defense and wound healing. However, excessive and/or prolonged inflammatory responses contribute to the pathogenesis of severe diseases, therefore immune responses need to be stringently regulated to ensure efficient host defense and prevent tissue damage and disease. Cell death has been long recognized as an important mechanism in host defense particularly against viral infections. 1 The presence of dead cells was often documented as a prominent pathological feature of inflamed tissues, but until recently cell death was This article is part of a series of reviews covering Molecular Mechanisms of Cell Death appearing in Volume 277 of Immunological Reviews. considered a secondary consequence of the immune response. 2 More recent studies provided evidence that cell death could also act as a potent trigger of inflammation, suggesting that cell death could contribute to the pathogenesis of inflammatory diseases. 3 5 Signaling pathways controlling immune responses but also cell survival and cell death have critical functions in the regulation of tissue homeostasis and inflammation. The nuclear factor kappa B (NF- κb) pathway controls immune responses and cell survival by regulating the expression of proinflammatory but also pro- survival genes. 6 NFκB transcription factors form dimers that are kept inactive in resting cells by association with proteins of the inhibitor of NF- κb (IκB) family. Upon activation by a variety of stimuli, IκB proteins are phosphorylated by the IκB kinase (IKK) complex, composed of the IKK1/IKKα Immunological Reviews. 2017;277: wileyonlinelibrary.com/journal/imr 2017 John Wiley & Sons A/S. 113 Published by John Wiley & Sons Ltd

2 114 KONDYLIS et al. and IKK2/IKKβ catalytic subunits and the NF- κb essential modulator (NEMO) regulatory subunit. IKK- mediated phosphorylation targets IκB proteins for K48 ubiquitination and proteasomal degradation, which allows NF- κb dimers to accumulate in the nucleus where they induce the expression of target genes. 6 Another key regulator of inflammation, cell survival and cell death is receptor interacting protein kinase 1 (RIPK1). RIPK1 exhibits kinase activity- dependent functions that trigger cell death, but it also exerts kinase- independent scaffold functions regulating inflammatory signaling and cell survival. 4,7,8 NF- κb and RIPK1 regulate signaling downstream of many receptors involved in inflammation. The tumor necrosis factor receptor 1 (TNFR1) signaling cascade is the best characterized pathway engaging IKK/NF- κb and RIPK1 to regulate inflammation and cell death. TNF is a potent immunoregulatory cytokine with an important role in host defense but is also a key pathogenic factor in many chronic inflammatory diseases, such as inflammatory bowel disease (IBD), including Crohn s disease and ulcerative colitis, rheumatoid arthritis and psoriasis. 9,10 While the role of TNF as a pathogenic cytokine in inflammatory diseases is well established through numerous clinical studies, the mechanisms by which TNF induces pathogenic inflammatory responses remain poorly understood. TNF signals primarily via TNFR1, a ubiquitously expressed cell surface receptor. Binding of the trimeric TNF ligand to TNFR1 induces receptor trimerization triggering the formation of a receptor proximal signaling complex, often described as complex I (Figure 1). TNFR1 associated death domain protein (TRADD) and RIPK1 are rapidly recruited to the cytoplasmic tail of the activated TNFR1 via death domain homotypic interactions. Studies in knockout cells showed that TRADD and RIPK1 bind independently to TNFR1. 11,12 TRADD recruits TNF receptor associated factor 2 (TRAF2) via its TRAF- interacting domain, which in turn brings the E3 ubiquitin ligases cellular inhibitors of apoptosis 1 and 2 (ciap1/2) into the complex. 11,12 ciap1/2 ubiquitinate RIPK1 and other components of the TNFR1 complex primarily with K63- linked ubiquitin chains. 13,14 ciap1/2- dependent ubiquitination of complex I proteins facilitates the recruitment of the linear ubiquitin assembly complex (LUBAC), composed of HOIP, HOIL- 1 and Sharpin, which adds M1- linked ubiquitin chains on RIPK1 and other components. 13,14 These ubiquitination events provide a signal for the recruitment of two distinct protein complexes that are essential for the activation of downstream signaling. 13,14 The IKK complex is recruited to complex I via the binding of the NEMO UBAN domain to linear ubiquitin chains. In addition, the TAK1/TAB2/3 complex is recruited via the binding of TAB2/3 to K63 ubiquitin chains. When the IKK and TAK1/TAB complexes are brought into close proximity, TAK1 phosphorylates IKK2 triggering its autophosphorylation that is required for full activation. 15 While normally TNF does not induce cell death in most cell types, inhibition of NF- κb sensitizes cells to TNF- induced apoptosis. Treatment of cells with inhibitors of transcription (e.g. actinomycin D) or translation (e.g. cycloheximide) also sensitizes cells to TNF- induced cell death, suggesting that NF- κb- dependent gene transcription prevents TNF- induced apoptosis. More recently, treatment of cells with SMAC- mimetic drugs that cause the rapid degradation of ciap1/2 were also shown to sensitize cells to TNF- induced cell death. 4,13 Currently, TNFR1 is believed to induce apoptosis via two distinct protein complexes (Figure 1): complex IIa depends on TRADD to recruit FIGURE 1 The TNFR1 signaling cascade. TNF binding to TNFR1 induces a receptor proximal signaling complex (complex I)-mediated activation of the IKK/NF- κb signaling pathway. In cells with compromised pro- survival signaling, TNFR1 can induce apoptosis by TRADDdependent (complex IIa) or RIPK1- dependent (complex IIb) activation of FADD- caspase- 8, or necroptosis by the RIPK1- RIPK3- MKLK complex (necrosome)

3 KONDYLIS et al. 115 FADD and caspase- 8 resulting in apoptosis in response to NF- κb inhibition or treatment with cycloheximide; complex IIb depends on RIPK1 for the recruitment of FADD and caspase- 8 and the induction of apoptosis in response to treatment with SMAC mimetics. 13,16 18 Activation of RIPK1 catalytic activity and likely its autophosphorylation is required for complex IIb- mediated apoptosis. When caspase- 8 is inhibited, TNFR1 can kill cells by necroptosis, a more recently described type of regulated necrotic cell death that is induced by RIPK3 and its substrate, the pseudokinase mixed lineage kinase like (MLKL) Activated RIPK1 recruits RIPK3 via RIP homotypic interaction motif (RHIM)- dependent interaction, resulting in the activation and autophosphorylation of RIPK3 in a complex often referred to as necrosome. 3,4,13,14,23 Phosphorylated RIPK3 recruits and phosphorylates MLKL, which acts as the downstream executer of necroptosis. 19 Although the mechanism by which MLKL kills the cell remains poorly understood, phosphorylation by RIPK3 induces the localization of MLKL into the plasma membrane, where it has been suggested to form pores that likely contribute to cell death. 24 The interplay between IKK/NF- κb and RIPK1 signaling has emerged as a critical regulator of cell survival and cell death that contributes to tissue homeostasis and inflammation. Much of the recent progress in this field has been driven by in vivo studies in genetic mouse models that provided novel insights on the physiological and pathological functions of these pathways. In this review, we aim to discuss primarily our own work using mouse models to study the role of IKK/NF- κb and RIPK1 signaling in the regulation cell death, inflammation and tissue homeostasis. We will also attempt to put these findings into context with relation to the current state of the art in the field and outline some of the remaining open questions. 2 CROSS- TALK BETWEEN IKK, NF- κb AND RIPK1 SIGNALING IN TISSUE HOMEOSTASIS 2.1 IKK/NF- κb signaling maintains intestinal homeostasis by inhibiting RIPK1- mediated apoptosis of intestinal epithelial cells The intestinal epithelium provides a physical and immunological barrier that is critical for the maintenance of healthy immune homeostasis in the gut. 25,26 In addition to forming a mechanical barrier between mucosal immune cells and the luminal contents, intestinal epithelial cells (IEC) actively communicate with both intestinal bacteria and host immune cells and regulate their interactions. Many receptors sensing bacterial products but also cytokine receptors activate the IKK/ NF- κb pathway, which controls the expression of immunomodulatory cytokines and chemokines but also genes regulating cell survival. 6 Therefore, IKK/NF- κb signaling in IECs was expected to play an important role in the regulation of intestinal homeostasis and inflammation. In order to study the role of IKK/NF- κb signaling in the intestinal epithelium, several years ago we generated mice lacking NEMO in IECs (NEMO IEC-KO ) and found that these animals developed spontaneously severe colon inflammation. 27 This finding was counterintuitive considering the potent pro- inflammatory properties of NF- κb, as inhibition of NF- κb was expected to prevent inflammation. Starting from the observation that the intestinal epithelium of NEMO IEC-KO mice contained increased numbers of dying cells, we reasoned that NEMO deficiency could trigger intestinal inflammation by sensitizing IECs to death. Increased death of IECs could compromise the integrity of the intestinal barrier and allow luminal bacteria to invade the mucosa eliciting inflammation. Indeed, when NEMO IEC-KO mice were raised under germ- free conditions they did not develop colitis, providing experimental evidence that the presence of the microbiota was essential for the development of intestinal inflammation. 28 In addition, MyD88 deficiency also prevented the development of colitis in NEMO IEC-KO mice, suggesting that bacteria cause inflammation by triggering TLR signaling. 27 The development of colitis in NEMO IEC-KO mice also depended on TNF as TNFR1 deficiency inhibited inflammation. 27 TNF is a key pathogenic factor in intestinal inflammation and TNF neutralizing antibodies are highly effective in the treatment of IBD, including Crohn s disease and Ulcerative colitis. 10 However, the cellular targets of TNF signaling that are important for the pathogenesis of IBD remain elusive. IECspecific knockout of TNFR1 prevented the development of colitis in NEMO IEC-KO mice, showing that in this model TNF drives intestinal inflammation by activating TNFR1 signaling in IECs. 28 In addition to preventing inflammation, IEC- specific TNFR1 deficiency suppressed IEC death, suggesting that TNFR1- induced death of NEMO- deficient IECs could trigger colitis by disrupting the intestinal barrier. Considering the established role of NF- κb in inhibiting TNF- induced apoptosis, we reasoned that NEMO prevented IEC death by activating pro- survival NFκB signaling. Surprisingly, complete inhibition of NF- κb- dependent gene expression in IECs, achieved by combined knockout of all three NF- κb subunits capable to activate gene transcription (RelA, c- Rel, RelB), did not cause colitis, indicating that NEMO maintains colonic epithelial integrity and prevents inflammation by exerting NF- κbindependent functions that are important for IEC survival. 28 To address the role of IEC death in triggering intestinal inflammation in this model, we assessed the role of apoptosis and necroptosis in NEMO IEC-KO mice. While immunostaining with antibodies recognizing cleaved caspase- 3 readily detected apoptotic IECs in the colonic epithelium of NEMO IEC-KO mice, in the absence of reliable reagents detecting necroptotic cell death in mouse tissues, we could not exclude that necroptosis could also be involved. Therefore, we employed genetic studies in an attempt to dissect the potential contribution of apoptosis and necroptosis to the phenotype. Indeed, IEC- specific FADD knockout combined with systemic RIPK3 deficiency fully prevented colitis development in NEMO IEC-KO mice, providing experimental evidence that IEC death, by apoptosis or/and necroptosis, causes the disease. RIPK3 deficiency alone resulted in partial protection from colitis as only 30% of NEMO IEC-KO Ripk3 / mice exhibited a similar degree of inflammation as NEMO IEC-KO mice. In the remaining animals, disease severity was ameliorated indicating an involvement of RIPK3 in NEMO IEC-KO colitis pathogenesis, though it is currently not clear whether this is due to any of the described apoptosis-, necroptosis- or immune- related functions of RIPK3. 4,29 Genetic studies in NEMO IEC-KO Mlkl / mice would be required to specifically address the

4 116 KONDYLIS et al. potential contribution of IEC necroptosis to the intestinal pathology of NEMO IEC KO mice. NEMO IEC-KO FADD IEC-KO mice developed colitis due to the important role of FADD in preventing RIPK3- mediated necroptosis (see below), which did not allow addressing directly the role of FADD- dependent apoptosis in NEMO IEC-KO mice. We then examined the role of RIPK1, which is a critical regulator of TNFR1- induced apoptosis and necroptosis upstream of FADD- caspase- 8 and RIPK3- MLKL. 4,7,8 Importantly, inhibition of RIPK1 kinase activity by crossing NEMO IEC-KO mice with Ripk1 D138N/D138N knock- in mice expressing kinase inactive RIPK1 from the endogenous Ripk1 locus prevented IEC death and the development of colitis. 28 Collectively, these studies showed that TNFR1- mediated and RIPK1 kinase activity- dependent death of NEMO- deficient IECs causes colitis in NEMO IEC-KO mice. NEMO- deficient IECs appear to die primarily by apoptosis but the potential contribution of necroptosis in NEMO IEC-KO mice remains to be addressed (Figure 2). In contrast to the severe inflammation in the colon, the small intestine of NEMO IEC-KO animals did not exhibit overt signs of inflammation but showed changes in the tissue architecture characterized by the presence of apoptotic IECs in the crypt area and reduced numbers of Paneth cells, 28 specialized secretory cells located at the crypt base that produce anti- microbial factors and contribute to the formation of the stem cell niche. 26 Lack of RIPK1 kinase activity or combined knockout of FADD and RIPK3 restored Paneth cell numbers in NEMO IEC-KO mice, showing that IEC death causes the small intestinal pathology in these animals. 28 RIPK3 deficiency did not have any effect in preventing or ameliorating enterocyte death and loss of Paneth cells in the small intestine of NEMO IEC-KO mice, arguing against a role of necroptosis. As opposed to the colon, IEC death and lack of Paneth cells in the small intestine were not prevented by the absence of epithelial TNFR1, Fas and TRAILR, but also by MyD88 deficiency or the lack of the intestinal microbiota. 28 Therefore, RIPK1 kinase activity- mediated IEC apoptosis drives small intestinal pathology in NEMO IEC-KO mice, but the upstream pathway(s) that induce RIPK1 activation in the small intestine remain elusive (Figure 2). In contrast to the colon where epithelial NF- κb deficiency did not phenocopy NEMO deficiency, NF- κb IEC-KO mice also lacked Paneth cells and showed increased IEC death in the ileum, albeit with reduced severity compared to NEMO IEC-KO animals. 28 Moreover, consistent with earlier findings that RelA IEC-KO mice showed increased IEC apoptosis and reduced expression of anti- microbial peptides, 30 we also found that mice with IEC- specific RelA knockout showed increased numbers of apoptotic cells in the small intestinal crypts and had reduced numbers of Paneth cells. 28 Notably, inhibition of RIPK1 kinase activity by crossing with Ripk1 D138N/D138N mice suppressed IEC apoptosis and prevented Paneth cell loss in RelA IEC-KO mice. 28 Therefore, RIPK1 kinase activity mediated apoptosis causes death of IECs and loss of Paneth cells in both NEMO IEC-KO and NF- κb IEC-KO mice, showing that NF- κb- dependent mechanisms control the survival of IECs in the small intestine (Figure 2). FIGURE 2 IKK/NF- κb signaling regulates RIPK1-mediated apoptosis and inflammation. A, NEMO expression in epithelial cells is essential for the maintenance of immune homeostasis and the prevention of inflammation in the colon. NEMO deficiency causes TNF- mediated, RIPK1- dependent colonocyte apoptosis, resulting in disruption of the intestinal barrier and bacteria- induced colon inflammation. NEMO prevents RIPK1- dependent apoptosis in colonic epithelial cells primarily by NF- κb- independent functions likely acting directly at the level of RIPK1, but NF- κb- dependent expression of pro- survival genes may also be involved. B, NEMO prevents epithelial cell apoptosis and Paneth cell loss in the small intestine by activating NF- κb- dependent mechanisms. Deficiency in NEMO or NF- κb causes RIPK1- dependent epithelial cell apoptosis and loss of Paneth cells independently of death receptors, the microbiota and TLR signaling. The upstream mediators driving RIPK1 activation in the ileum remain unknown. C, NEMO prevents chronic liver disease and hepatocellular carcinoma by NF- κb- dependent and -independent functions. NEMO deficiency triggers RIPK1- dependent hepatocyte apoptosis resulting in compensatory hepatocyte proliferation, chronic hepatitis and the development of liver cancer. The upstream mechanisms inducing RIPK1 kinase activity- mediated apoptosis of NEMO- deficient hepatocytes are independent of death receptors and remain unknown

5 KONDYLIS et al. 117 These results provided experimental evidence that IKK/NF- κb signaling maintains homeostasis and prevents inflammation in the intestine by inhibiting RIPK1 kinase activity- dependent death of IECs, however the mechanisms by which IKK/NF- κb signaling restrains epithelial cell death in the colon and small intestine seem to be different. In the colon, NEMO inhibits RIPK1 kinase activity- mediated death of IECs in an NF- κb- independent manner. Earlier studies in cell lines suggested that NEMO prevents RIPK1 from engaging in the formation of death- inducing signaling complexes with FADD and caspase ,32 In addition, a more recent study suggested that IκB kinases inhibit TNFinduced cell death by directly phosphorylating and inhibiting RIPK1 kinase activity independently of downstream NF- κb activation. 33 These studies would be consistent with an NF- κb- independent function of IKKs to restrict RIPK1- mediated apoptosis in the colon, although the role of IKK- mediated RIPK1 phosphorylation in the regulation of TNFinduced cell death remains to be studied in well characterized genetic and physiologically relevant models. Our finding that combined IECspecific knockout of IKK1 and IKK2 caused colitis while single knockouts did not, 27 are consistent with a redundant role of the two kinases in restraining RIPK1 activation. In contrast to the colon, in the small intestine RIPK1 is inhibited by NF- κb- dependent mechanisms that remain elusive at present. 28 This role of IKK subunits and of NF- κb transcription factors in preventing RIPK1 kinase activity- dependent cell death appears to be effective in a broader setting outside the small intestine, as lack of RIPK1 kinase activity also prevented embryonic lethality of Nemo -/ and Rela / animals The interplay between IKK/NF- κb and RIPK1 in liver homeostasis The liver is an organ with phenomenal capacity to regenerate. However, under physiological conditions, differentiated hepatocytes show a very low turnover rate ( days in mice/rats), compared to intestinal epithelial or epidermal cells, and rest in G 0 phase. Liver regeneration is only induced upon extensive tissue loss or injury, most commonly caused by alcohol, drug or metabolic toxicity, viral or other microbial infections, and surgical resection. 34 Hepatocellular death is a key cellular event observed in liver pathologies leading to liver inflammation and fibrosis in order to promote organ regeneration and preserve functionality. Yet, liver regeneration in the context of chronic liver damage and persistent inflammation predisposes to hepatocarcinogenesis. Therefore, the regulation of hepatocyte survival is critical for the maintenance of liver homeostasis and the prevention of liver disease and cancer. 35,36 We have studied the role of IKK/NF- κb signaling in the liver by generating mice lacking key pathway components in liver parenchymal cells (LPC). Mice with LPC- specific deletion of NEMO (NEMO LPC-KO ) developed a spontaneous liver pathology recapitulating many features of chronic liver diseases, including early liver damage, activation of a regenerative response, hepatitis, macromolecular steatosis and fibrosis. 37 All NEMO LPC-KO mice developed dysplastic nodules in their livers, with a fraction of them progressing to hepatocellular carcinomas (HCC) at the age of 1 year. 37 Hepatocyte apoptosis is a prominent and early event of the spontaneous liver pathology developing in NEMO LPC-KO mice. This led to the hypothesis that NEMO deficiency causes chronic liver disease and cancer by sensitizing hepatocytes to apoptosis, which subsequently triggers compensatory hepatocyte proliferation that in the context of chronic liver inflammation culminates in hepatocarcinogenesis. 37,38 To experimentally validate this hypothesis, we employed mice lacking both NEMO and FADD in LPCs. Indeed, FADD deficiency in NEMO LPC-KO FADD LPC-KO mice strongly inhibited hepatocyte apoptosis and prevented the development of liver tumors. 37,39 Consistently, an independent study showed that LPC- specific knockout of caspase- 8 also protected NEMO LPC-KO mice from hepatocyte apoptosis and the development of liver cancer. 40 These findings suggested that hepatocyte apoptosis induced by death receptors drives the liver pathology in NEMO LPC-KO mice. Surprisingly NEMO LPC-KO mice with hepatocyte- specific deficiency of TNFR1, Fas or TRAILR, individually or combined, were not protected from the liver pathology, suggesting that these death receptors are not essential drivers of hepatocyte apoptosis in this model. 39 In light of an earlier study reporting that systemic TNFR1 deficiency largely ameliorated liver damage and tumorigenesis, 41 we also generated and analyzed NEMO LPC-KO Tnfr1 / mice but found that systemic lack of TNFR1 did not prevent liver damage and only had a mild effect in slowing down tumor progression. 42 Therefore, TNFR1 is not required for hepatocyte apoptosis but TNFR1 signaling in cells other than LPCs appears to contribute to liver tumor progression in NEMO LPC-KO mice. Given its key role in activating canonical NF- κb signaling, we reasoned that NEMO deficiency might sensitize hepatocytes to apoptosis by inhibiting NF- κb- mediated pro- survival gene expression. Though an earlier study showed that mice with hepatocyte- specific knockout of RelA did not develop spontaneous pathology, 43 this could be due to functional redundancy between NF- κb subunits in the liver. To address the role of NF- κb- dependent gene transcription in the liver, we generated mice lacking all three NF- κb subunits capable of gene transactivation (RelA, c- Rel and RelB) in LPCs. Although these triple NF- κb LPC-KO (RelA/RelB/c-Rel LPC-KO ) mice were highly susceptible to endotoxic shock- induced liver damage, they did not develop the spontaneous severe liver pathology of NEMO LPC-KO mice and also did not show any signs of dysplasia in the liver. 42 These genetic studies provided unequivocal experimental evidence that NF- κb- dependent gene transcription is not essential for hepatocyte survival and normal liver homeostasis under steady state conditions, and suggested that NEMO exerts additional, NF- κb- independent, functions that are important to prevent hepatocyte apoptosis, chronic liver disease and development of HCC (Figure 2). To genetically dissect the pathway driving spontaneous hepatocyte apoptosis in NEMO LPC-KO mice, we addressed the function of RIPK1. Crossing the NEMO LPC-KO mice with Ripk1 D138N/D138N mice expressing kinase inactive RIPK1 strongly inhibited hepatocyte apoptosis, chronic liver disease and liver tumorigenesis, showing that RIPK1 kinase activity is required for the death of NEMO- deficient hepatocytes. 42 Although RIPK1 kinase activity promotes both apoptosis and necroptosis, the finding that FADD or caspase- 8 knockout could also prevent chronic liver disease and liver tumorigenesis in NEMO LPC-KO

6 118 KONDYLIS et al. mice suggested that in these mice RIPK1 kinase activity drives exclusively apoptosis. This is supported by the finding that RIPK3 deficiency did not prevent or ameliorate chronic liver disease and hepatocarcinogenesis in NEMO LPC-KO mice. 42 The very low expression of RIPK3 in hepatocytes compared to other cells is the most likely reason why hepatocytes are generally resistant to necroptosis. 44 In contrast to the lack of its kinase activity, ablation of RIPK1 protein expression in LPCs failed to protect NEMO LPC KO mice from chronic liver damage and hepatocarcinogenesis, indicating that RIPK1 exhibits complex kinase- dependent and - independent functions in regulating survival of NEMO- deficient hepatocytes. We reasoned that in the absence of RIPK1, NEMO- deficient hepatocytes could undergo apoptosis induced by TRADD- dependent mechanisms and used mice lacking TRADD in LPCs to validate this hypothesis. While TRADD knockout in LPCs did not protect NEMO LPC-KO mice from liver damage and HCC development, combined deficiency of TRADD and RIPK1 fully prevented hepatocyte apoptosis, chronic liver disease and hepatocarcinogenesis in NEMO LPC-KO mice. 42 These results are consistent with a model where RIPK1 kinase activity drives the formation of a FADD- caspase- 8 containing, apoptosis- inducing signaling complex in NEMO-deficient hepatocytes. In the absence of RIPK1 scaffolding functions, TRADD can induce hepatocyte apoptosis and drive the liver pathology in NEMO LPC-KO mice. These findings are intriguing and raise several questions. Considering that TNFR1, Fas and TRAILR, the three death receptors best studied for their role in inducing cell death, are not required for the spontaneous hepatocyte apoptosis in NEMO LPC-KO mice, the upstream receptors driving FADD- caspase- 8- dependent cell death in the NEMO- deficient hepatocytes remain unknown. Death receptors 3 and 6 (DR3 and DR6) can also induce FADD- caspase- 8- dependent apoptosis 45 and could potentially contribute to hepatocyte death in NEMO LPC-KO mice. DR3 was recently reported to induce necroptosis in addition to apoptosis, but since it is mainly expressed in lymphocytes and innate immune cells this receptor is unlikely to play an important role in hepatocytes. 46 The function of DR6 remains poorly understood 47 and its role as potential trigger of apoptosis in NEMO- deficient hepatocytes needs to be experimentally addressed. To formally exclude a role of death receptors and possible functional redundancies between them, additional studies will be needed ideally in mice lacking all death receptors, an admittedly challenging task considering that this would need the generation of mice carrying six homozygous mutations in addition to the Cre transgene. Another possibility would be that the spontaneous apoptosis of NEMO- deficient hepatocytes is not triggered by upstream (death) receptors but by the unprompted formation of intracellular death- inducing signaling complexes due to the destabilization of pro- survival proteins, such as cflip, ciap1/2 and TRAF2, as previously described upon induction of genotoxic stress. 48,49 Recent work in mice showing that combined ablation of RIPK1 and TRAF2 in LPCs triggered spontaneous hepatocyte apoptosis, cholestasis, and chronic hepatitis, leading eventually to liver tumorigenesis provided additional support that destabilization of pro- survival signaling complexes could be critically involved in triggering hepatocyte death. 50 Notably, deficiency of RIPK1 or TRAF2 alone were not sufficient to trigger spontaneous hepatocyte apoptosis in vivo suggesting that these proteins exhibit redundant or independent pro- survival functions that need to be simultaneously impaired to trigger spontaneous liver damage. Another important question relates to the NF- κb- independent mechanism(s) by which NEMO prevents RIPK1 activation and apoptosis. Biochemical data in primary hepatocytes showed that NEMO deficiency triggers the formation of a death- inducing signaling complex containing RIPK1, FADD and caspase Notably, the formation of this complex depends on RIPK1 kinase activity, suggesting that the presence of NEMO restrains RIPK1 activation and the formation of the apoptosis- inducing protein complex. 42 As discussed above, IKK1/ IKK2 were reported to directly phosphorylate RIPK1 and this phosphorylation was suggested to inhibit RIPK1- dependent apoptosis. 33 Therefore, one could envisage that NEMO inhibits RIPK1 by facilitating its phosphorylation by IKK1 and IKK2. However, a recent study suggested that RIPK1 phosphorylation in the liver is inhibited by LPCspecific knockout of both IKK1 and IKK2 but not by lack of NEMO, 51 arguing against a function of NEMO in controlling IKK- dependent RIPK1 phosphorylation. In this case, how IKK1/IKK2 are recruited in the vicinity of RIPK1 and whether they directly target RIPK1 or facilitate its phosphorylation by other kinases remains to be clarified. The capacity of NEMO to bind linear ubiquitin chains generated by LUBAC is likely related to its pro- survival function(s) in hepatocytes, as mice with hepatocyte- specific knockout of HOIP developed a spontaneous liver pathology resembling that of NEMO LPC-KO mice, manifesting with hepatocyte apoptosis, chronic liver disease and liver tumorigenesis. 52 While inhibition of NF- κb- dependent gene transcription was not sufficient to phenocopy NEMO deficiency in the liver, it is likely that inhibition of canonical NF- κb synergizes with NF- κb- independent NEMO functions to cause the severe liver disease in NEMO LPC-KO mice. This is supported by our results showing that expression of a constitutively active form of IKK2 (IKK2ca), which does not require NEMO to induce NF- κb activation, rescued the death of NEMOdeficient hepatocytes. 42 Importantly, RelA deficiency largely abolished the protective effect of IKK2ca expression in the liver of NEMO LPC-KO mice, demonstrating that constitutive IKK2 activity prevented hepatocyte apoptosis mainly by inducing RelA- dependent gene transcription. 42 This raised the question of which NF- κb target genes mediate the protective IKK2ca effect. An obvious candidate would be cflip L, a classical NF- κb- regulated gene, 53 whose protein levels are significantly downregulated in the liver of NEMO LPC-KO mice. 42 However, cflip L mrna expression was not affected in NEMO- or NF- κb- deficient hepatocytes or when IKK2ca was overexpressed. 42 In contrast, mrna expression of other NF- κb responsive genes encoding proteins with anti- apoptotic and anti- oxidant functions, such as ciap1/2, Tnfaip3/A20 and SOD2, were significantly upregulated in IKK2ca- expressing hepatocytes, suggesting that these factors could contribute to the prevention of apoptosis in NEMO- deficient hepatocytes. 42 Collectively, these results indicate that NEMO functions at two different levels to prevent hepatocyte apoptosis: It directly inhibits the formation of the death- inducing signaling complex by acting on

7 KONDYLIS et al. 119 RIPK1 and at the same time promotes the activation of canonical NFκB- mediated transcription of pro- survival genes (Figure 2). The presence of these two distinct checkpoints preventing hepatocyte death could explain why neither complete inhibition of NF- κb- dependent gene transcription nor RIPK1 deficiency in LPCs could cause considerable spontaneous liver pathology. This model also predicts that combined deficiency in RIPK1 and NF- κb in LPCs could sensitize hepatocytes to spontaneous apoptosis and cause chronic liver disease and cancer similarly to NEMO deficiency. LPC- specific knockout of each of the two IκB kinases did not cause spontaneous pathology, however combined lack of both IKK1 and IKK2 in LPCs caused severe cholestatic liver disease, a phenotype different from the liver pathology of NEMO LPC-KO mice. 54 Concurrent ablation of NEMO and IKK1 in LPCs caused similar cholestatic liver disease, suggesting that IKK1 regulates additional pro- survival functions that when combined with NEMO or IKK2 deficiency cause biliary epithelial cell death. 54 It was initially suggested that IKK1 knockout could cause the cholestatic liver disease by inhibiting non- canonical NF- κb activation, but NEMO LPC-KO RelB LPC-KO mice did not develop similar pathology arguing against a role of the alternative NF- κb pathway. 42 Ablation of RIPK1 was recently reported to prevent biliary damage and cholestatic liver disease in mice with LPC- specific knockout of IKK1 and IKK2, suggesting that the additional function of IKK1 could be related to the modulation of RIPK1 activity and is compatible with the proposed redundant function of IKK1 and IKK2 in phosphorylating RIPK1. 33,51 However, it should be noted that this IKK1 function appears to biliary epithelial cell- specific as RIPK1 deficiency did not prevent hepatocyte apoptosis in IKK1/IKK2- deficient hepatocytes. 51 Therefore, additional studies will be required to assess the cell type specificity and the potential physiological significance of these phosphorylation events on RIPK1 for liver homeostasis using suitable in vivo genetic mouse models of chronic liver damage. 2.3 Epithelial IKK- NF- κb signaling in skin homeostasis Consistent with its function in the intestinal epithelium and the liver, NEMO expression in keratinocytes has an important role for the maintenance of healthy skin homeostasis. The first indication that NEMO is important for skin homeostasis was the finding that NEMO mutations cause Incontinentia Pigmenti (IP), a human genetic disease defined by male embryonic lethality and the development of characteristic skin lesions among other symptoms in heterozygous females. 55 NEMO deficiency caused embryonic lethality in male mice, while heterozygous females developed inflammatory skin lesions resembling those of IP patients. 56,57 Epidermal keratinocytespecific NEMO deficiency caused inflammatory skin lesions both in males and in heterozygous females, demonstrating that inflammation was caused by NEMO knockout in keratinocytes. 58 Interestingly, the inflammatory skin lesions in both human and mouse NEMO heterozygous females develop early after birth but subsequently subside and disappear during adulthood. The mosaic presence of wild type and NEMO- deficient keratinocytes due to random X- chromosome inactivation was proposed as a key mechanism contributing to the transient development of the skin pathology in human IP patients and in heterozygous NEMO knockout mice. 56 In particular, the transient nature of the skin lesions during adulthood was attributed to the death of NEMO- deficient keratinocytes and their replacement by wild type cells. Indeed, large number of dying keratinocytes were detected in early skin lesions of IP patients and heterozygous NEMO knockout female mice suggesting that cell death was a key aspect of the phenotype TNFR1 knockout prevented the early postnatal development of skin lesions in mice homozygous or heterozygous for NEMO knockout in keratinocytes, however these mice developed inflammatory skin lesions later in life between 4 and 6 months of age suggesting that TNF has an important role early on but TNFindependent pathways drive skin lesion development in adults. 58 Based on these results, it is likely that TNF- induced killing of NEMOdeficient keratinocytes is a key aspect of the skin pathology in both mice and human IP patients. Particularly considering the important role of RIPK1 kinase activity- dependent apoptosis in driving the spontaneous pathologies in mice lacking NEMO in intestinal epithelial cells or in LPC, it is likely that RIPK1- dependent keratinocyte apoptosis contributes to the skin pathology caused by NEMO deficiency. However, the role of TNF- induced cell death and particularly the specific contribution of apoptosis and necroptosis in the development of inflammatory skin lesions in mice lacking NEMO in keratinocytes has not been experimentally addressed. In contrast to the intestinal epithelium and the liver, where knockout of IKK2 did not cause spontaneous pathology, mice with epidermal keratinocyte- specific IKK2 deficiency (IKK2 E-KO ) developed severe inflammatory skin lesions shortly after birth resulting in death before postnatal day The development of skin lesions in these IKK2 E-KO mice depends on TNFR1 signaling in keratinocytes, as epidermis- specific knockout of TNFR1 rescued the phenotype. 61 IKK2- deficient keratinocytes expressed increased amounts of IL- 24 both in vivo and in response to TNF stimulation in vitro, suggesting that IL- 24 overexpression could be implicated in the development of skin inflammation in IKK2 E-KO mice. Indeed, knockout of IL- 22R1, which is essential for IL- 22 and IL- 24 signaling, 62 strongly delayed and ameliorated skin lesion development in IKK2 E-KO mice. 61 Considering that IL- 22 deficiency did not have any effect in the skin phenotype of IKK2 E-KO mice, these results provided functional evidence that IL- 24 contributes to skin inflammation in this model. 61 Interestingly, human psoriatic epidermis showed increased expression of IL- 24, and human wild type primary keratinocytes treated with a highly specific IKK2 inhibitor also produced increased amounts of IL- 24 in response to TNF stimulation, suggesting that regulation of IL- 24 expression by IKK2 could contribute to the pathogenesis of skin inflammation in psoriasis. 61 Furthermore, although keratinocyte- specific ablation of RelA did not cause spontaneous skin inflammation, combined knockout of RelA and crel in the epidermis caused psoriasis- like inflammatory skin lesions In contrast to the presence of large numbers of dying keratinocytes in the epidermis of NEMO E-KO mice, keratinocyte death does not seem to be a prominent feature of the

8 120 KONDYLIS et al. IKK2- deficient or RelA- crel- deficient epidermis during the early stages of lesion development, raising the question whether death of keratinocytes plays a role in the pathogenesis of skin inflammation caused by inhibition of IKK2 or NF- κb signaling. Future studies will be needed to address the role of TNF- induced cell death, apoptotic or necroptotic, in these models. Linear ubiquitination of RIPK1 and other TNFR1 signaling components by LUBAC contributes to activation of NF- κb and pro- survival signaling. 14 Mutations disrupting the expression of Sharpin, one of the LUBAC components, causes the severe chronic proliferative dermatitis (cpdm) phenotype in mice, 67 suggesting that linear ubiquitination regulates skin immune homeostasis. Genetic studies showed that skin lesion development in Sharpin cpdm/cpdm mice depends on TNF signaling via TNFR1 in epidermal keratinocytes The presence of large numbers of apoptotic keratinocytes in the epidermis of Sharpin cpdm/ cpdm mice suggested that TNFR1- induced keratinocyte death could be important in driving the skin pathology. Indeed, combined inhibition of FADD- caspase- 8-dependent apoptosis and RIPK3- MLKL-dependent necroptosis fully prevented the development of skin lesions in these animals. 68,69 RIPK3 or MLKL deficiency alone had only a mild effect in delaying the skin lesions, showing that skin inflammation in Sharpin cpdm/cpdm mice is caused primarily by TNFR1- mediated keratinocyte apoptosis. 68,69 Notably, inhibition of RIPK1 kinase activity by crossing Sharpin cpdm/cpdm with knock- in mice expressing kinase inactive RIPK1 also prevented keratinocyte death and skin lesion development, demonstrating that Sharpin- dependent LUBAC signaling maintains skin homeostasis by preventing RIPK1 kinase- dependent keratinocyte apoptosis FADD AND CASPASE- 8 MAINTAIN TISSUE HOMEOSTASIS BY PREVENTING NECROPTOSIS 3.1 FADD and caspase- 8 inhibit epithelial cell necroptosis and prevent inflammation in the intestine The adapter protein FADD recruits and activates caspase- 8 and is essential for death receptor- induced apoptosis. 45 To study the role of FADD- caspase- 8- mediated apoptosis in the intestine, we generated mice with IEC- specific knockout of FADD (FADD IEC-KO ). Surprisingly, FADD IEC-KO mice developed spontaneous gut inflammation affecting both the colon and the small intestine, characterized by ulcerating lesions, increased infiltration of immune cells and expression of inflammatory cytokines, as well as loss of Paneth cells. 72 Immunohistological analysis of intestinal sections from FADD IEC-KO mice revealed the presence of large numbers of dead IECs that did not stain with antibodies recognizing cleaved caspase- 3, indicating FADD- deficient epithelial cells underwent non- apoptotic cell death that could provide the trigger for the development of intestinal inflammation. The discovery that inhibition of caspase- 8 sensitized cells to RIPK3- dependent necroptosis provided clues for a possible mechanism that could cause the intestinal pathology in FADD IEC-KO mice. Indeed, RIPK3 deficiency prevented IEC death, loss of Paneth cells and intestinal inflammation in FADD IEC-KO mice. 72 In addition, inhibition of CYLD deubiquitinase activity by expression of a catalytically- deficient truncated CYLD specifically in IECs also largely prevented IEC death, Paneth cell loss and intestinal inflammation in FADD IEC-KO mice. 72 These results provided experimental evidence that FADD deficiency caused intestinal FIGURE 3 FADD and caspase- 8 inhibit RIPK3- mediated necroptosis and inflammation. A, FADD expression in epithelial cells is essential to prevent the development of colitis. FADD deficiency causes TNFR1- mediated RIPK3- dependent necroptosis resulting in disruption of the intestinal barrier and bacteria-driven inflammation. Necroptosis of epithelial cells may also contribute to colitis presumably by the release of DAMPs from necroptotic cells. B, FADD and caspase- 8 prevent loss of Paneth cells and the development of ileitis. FADD or caspase- 8 deficiency causes TNFR1-, microbiota- and TLR- independent, RIPK3- mediated epithelial cell necroptosis resulting in Paneth cell loss and ileitis. The upstream receptors and pathways triggering necroptosis in the small intestine remain unknown. C, FADD and caspase- 8 are essential for the maintenance of skin homeostasis. FADD or caspase- 8 deficiency cause RIPK3- dependent keratinocyte necroptosis triggering skin inflammation likely via the release of DAMPs by necroptotic keratinocytes. TNFR1- dependent but also TNFR1- independent mechanisms drive keratinocyte necroptosis and skin inflammation in mice with keratinocyte- restricted FADD or caspase- 8 knockout

9 KONDYLIS et al. 121 inflammation by sensitizing IECs to RIPK3- dependent necroptosis that was dependent also on CYLD catalytic activity (Figure 3). FADD IEC-KO mice did not develop colitis when raised under germ- free conditions showing that the presence of the microbiota was required for triggering the intestinal inflammation in this model. Moreover, systemic MyD88 deficiency also prevented colitis development in FADD IEC-KO mice, suggesting that intestinal bacteria cause intestinal inflammation by activating MyD88- dependent TLR signaling. 72 Furthermore, TNF deficiency strongly ameliorated the development of colon inflammation in FADD IEC-KO mice showing that TNF plays an important role but also TNF- independent mechanisms contribute to the pathology. 72 In contrast to their critical involvement in driving the colonic inflammatory pathology, lack of the microbiota but also MyD88 or TNF knockout did not significantly alleviate ileitis and loss of Paneth cells in FADD IEC-KO mice suggesting that different upstream pathways trigger necroptosis of FADD- deficient IECs in the small and large intestine. 72 Finally, genetic loss of RIPK1 kinase activity strongly inhibited but did not fully prevent IEC death, Paneth cell loss and the development of colitis in FADD IEC-KO mice, 73 indicating that RIPK1- dependent but also RIPK1- independent mechanisms drive RIPK3- induced IEC necroptosis in these mice (Figure 3). Mice with IEC- specific knockout of caspase- 8 also developed ileitis with lack of Paneth cells but did not develop spontaneous colitis, although they were highly sensitive to chemically- induced experimental colon inflammation. 74 In a subsequent study, it was shown that ileitis and loss of Paneth cells in Casp8 IEC-KO mice were dependent on RIPK3 but persisted in the absence of TNFR1, similarly to FADD IEC-KO mice. 75 Moreover, acute systemic deletion of caspase- 8 in adult mice caused intestinal epithelial cell death and inflammation that were inhibited in the absence of RIPK3. 76 Together, these studies showed that FADD and caspase- 8 inhibit RIPK3- dependent death of IECs to maintain intestinal homeostasis and prevent inflammation (Figure 3). Although the effect of MLKL deficiency has not been studied in mice with IECspecific loss of FADD or caspase- 8, Fadd / Mlkl / or caspase-8 / Mlkl / mice were not reported to develop intestinal pathology, 77 further supporting that FADD or caspase- 8 deficiency cause intestinal pathology by sensitizing IECs to RIPK3- MLKL- dependent necroptosis. 3.2 FADD and caspase- 8 inhibit keratinocyte necroptosis and prevent skin inflammation Genetic mouse models provided evidence that FADD and caspase- 8 have important functions in keratinocytes that are essential for the maintenance of skin homeostasis. Two independent studies showed that keratinocyte- specific knockout of caspase- 8 (Casp8 E-KO ) caused severe skin inflammation in mice but proposed different mechanisms. 78,79 Lee et al. 79 proposed that loss of caspase- 8 causes the p38 MAPKmediated production of IL- 1α from keratinocytes triggering inflammation. Kovalenko et al. showed that knockout of IL- 1α and IL- 1β but also pharmacological inhibition of p38 could not prevent skin inflammation caused by loss of caspase- 8 in keratinocytes. Instead, they suggested that caspase- 8 deficiency caused enhanced activation of RIG- I, presumably by endogenous activators generated during keratinocyte differentiation, triggering IRF3- dependent inflammation. 78 In a subsequent study, Weinlich et al. 76 could not confirm a role of the RIG- I pathway in this model, as they found that MAVS knockout did not prevent skin inflammation in mice with inducible caspase- 8 knockout. Mice with epidermal keratinocyte- specific knockout of FADD (FADD E-KO ) also developed severe skin inflammation similarly to the Casp8 E-KO mice. 80 RIPK3 deficiency fully prevented skin lesion development in FADD E-KO animals, providing experimental evidence that RIPK3- dependent necroptosis of FADD- deficient keratinocytes causes skin inflammation. 80 Similarly, RIPK3 knockout also prevented the skin lesions caused by inducible caspase- 8 knockout, suggesting that necroptosis of caspase- 8- deficient keratinocytes causes skin inflammation also in this model. 76 However, in light of the rapidly expanding functions of RIPK3 that appear not to depend on necroptosis, 29 rescue of inflammation by RIPK3 deficiency cannot be considered anymore as a proof that necroptosis is involved. Additional studies in MLKL- deficient mice will be required to formally demonstrate that keratinocyte necroptosis drives the inflammatory skin disease caused by FADD or caspase- 8 knockout in keratinocytes. However, the absence of inflammatory skin lesions in Fadd / Mlkl / or Casp8 / Mlkl / mice 77 very strongly suggests that RIPK3- MLKL- dependent necroptosis of FADD- or caspase- 8- deficient keratinocytes is the key event triggering skin inflammation. TNF seems to be an important but not exclusive driver of skin inflammation in both FADD E-KO and Casp8 E-KO mice, as TNF or TNFR1 deficiency delayed and ameliorated but could not fully prevent the development of skin lesions in these animals. 78,80 Epidermis- specific Fas deficiency did not affect skin lesion development in FADD E-KO mice, showing that Fas is not involved in this model. 80 However, potential redundant functions of TNFR, Fas, TRAIL and other death receptors in driving necroptosis of FADD- or caspase- 8- deficient keratinocytes cannot be excluded. Finally, the development of skin lesions in FADD E-KO mice was also considerably delayed by the lack of CYLD catalytic activity in keratinocytes, suggesting that CYLD contributes to the induction of necroptosis in FADD- deficient keratinocytes. 80 Collectively, these studies showed that FADD and caspase- 8 prevent RIPK3- dependent keratinocyte necroptosis downstream of TNFR1 and other receptors, and this function is essential for the maintenance of skin homeostasis and the prevention of inflammation (Figure 3). 4 KINASE- INDEPENDENT RIPK1 FUNCTIONS MAINTAIN TISSUE HOMEOSTASIS BY INHIBITING APOPTOSIS AND NECROPTOSIS 4.1 RIPK1 inhibits intestinal epithelial cell apoptosis While lack of RIPK1 kinase activity did not cause spontaneous pathology, mice lacking RIPK1 in IECs (RIPK1 IEC-KO ) developed severe intestinal disease manifesting with apoptotic death of IECs in the colon and small intestine, villous atrophy, reduced body weight, and early death within the first month of age. 73,81 Apoptosis of RIPK1- deficient IECs and early lethality of RIPK1 IEC-KO mice was partially dependent