Necroptosis at a glance

Necroptotic cell death is marked by plasma membrane rupture and leakage of damage-associated molecular patterns (DAMPs), such as the high mobility group box 1 protein (HMGB1) and ATP, which activate innate immune receptors and promote immune cell infiltration (Galluzzi et al., 2018). These inflammatory effects facilitate the host response to pathogens but can also lead to tissue damage and pathologies. Necroptosis is mediated by adaptors that contain the receptor-interacting protein kinase homotypic interaction motif (RHIM), a tetrapeptide core flanked by hydrophobic residues, which forms amyloid fibrils to facilitate cell death signaling (Li et al., 2012; Mompeán et al., 2018; Wu et al., 2021). In mammals, there are four RHIM-containing adaptors: receptor-interacting serine/threonine-protein kinases 1 and 3 (RIPK1 and RIPK3, respectively), TIR domain-containing adaptor-inducing interferon-β (TRIF, also known as TICAM1), and Z-DNA binding protein 1 (ZBP1) (see poster). RIPK1 and RIPK3 are activated by death receptors (DRs) belonging to the TNF receptor superfamily, which include TNFR1 (also known as TNFRSF1A), Fas (also known as CD95 or APO-1), TRAIL-R1 (also known as DR4 or TNFRSF10A) and TRAIL-R2 (also known as DR5 or TNFRSF10B). In mouse cells, RIPK1 plays a pivotal role in DR-induced necroptosis but is dispensable for necroptosis induced by the microbial immune receptors Toll-like receptor 3 (TLR3) and Toll-like receptor 4 (TLR4), and viruses (Nailwal and Chan, 2019). Moreover, both type I and type II interferons (IFNs) can induce transcription of the double-stranded RNA (dsRNA)-binding protein kinase R (PKR, also known as EIFAK2), which then interacts with RIPK1 and RIPK3 to stimulate necroptosis (Thapa et al., 2013). IFNs also promote necroptosis through STAT1-dependent expression of ZBP1 and mixed lineage kinase domain-like (MLKL) (Yang et al., 2020; Knuth et al., 2019; Ingram et al., 2019) (see poster). As RIPK1 and RIPK3 are both cleavage substrates of caspase 8, necroptosis is optimally induced when caspase 8 is inhibited. Here, we provide an overview of the machinery that regulates necroptosis and its biological functions in health and disease.

Most of the studies on necroptosis signaling center on the TNFR1 pathway. Upon stimulation with TNF, TNFR1 forms a membrane-associated complex termed complex I, which recruits TNFR1-associated death domain protein (TRADD) and RIPK1 through homotypic death domain interactions. Other factors recruited to complex I include TNFR-associated factor 2 (TRAF2), the E3 ubiquitin ligases cellular inhibitor of apoptosis 1 and 2 (cIAP1 and cIAP2, also known as BIRC2 and BIRC3, respectively), transforming growth factor-β-activated kinase (TAK1, also known as MAP3K7), the TAK1-binding adaptors TAB1 and TAB2, the linear ubiquitin chain assembly complex (LUBAC), and the inhibitor of nuclear factor (NF)-κB kinase (IKK) complex (see poster). cIAP-1 and -2 and LUBAC mediate K63-linked and M1-linked RIPK1 ubiquitylation, respectively, to facilitate the recruitment of TAK1 and the IKK complex components NF-κB essential modulator (NEMO, also known as IKBKG), IKKα (also known as CHUK) and IKKβ (also known as IKBKB) (Zhang et al., 2019; Dziedzic et al., 2018). TAK1 stimulates IKK-mediated phosphorylation and proteasomal degradation of IκBα (also known as NFKBIA) to relieve the inhibition on NF-κB, thereby facilitating NF-κB nuclear translocation and gene transcription (see poster). NF-κB switches on expression of pro-survival factors such as cellular FLICE-like inhibitor protein (cFLIP, also known as CFLAR), and cIAP-1 and -2. Thus, complex I is primarily a pro-survival signaling platform (Witt and Vucic, 2017).

A second, more long-lasting complex termed complex II is formed after dissolution of the plasma membrane-bound complex I (Micheau and Tschopp, 2003). The ubiquitin scaffold of RIPK1 restricts formation of complex II, since inhibitor of apoptosis protein (IAP) antagonists that cause cIAP-1 and -2 auto-ubiquitylation and proteasomal degradation, thereby promoting RIPK1 deubiquitylation, greatly enhance formation of complex II (Varfolomeev et al., 2007; Vince et al., 2007; Wang et al., 2008). The deubiquitylation enzyme cylindromatosis (CYLD) and its adaptor SPATA2 promote complex II formation by facilitating deubiquitylation of RIPK1 (Moquin et al., 2013; Wei et al., 2017; Elliott et al., 2016; Kupka et al., 2016).

Complex II is a 2 MDa macromolecular complex that contains RIPK1, the apoptosis adaptor FADD and caspase 8 (Feoktistova et al., 2011; Tenev et al., 2011) (see poster). Binding of the long form of cFLIP (cFLIP_L) to complex II promotes caspase 8 oligomerization and activity, whereas cFLIP short form (cFLIP_S) inhibits caspase 8 through blockade of caspase 8 chain elongation (Muendlein et al., 2020; Shindo et al., 2016). Active caspase 8 cleaves downstream substrates, such as caspase 3, to induce apoptosis. The apoptosis-inducing complex II is also referred to as complex IIa, or ripoptosome, to distinguish it from the necroptosis-inducing complex IIb, which is also known as the necrosome (see below). In addition to apoptosis substrates, active caspase 8 also cleaves the key necroptosis adaptors RIPK1, RIPK3 and CYLD (Chan et al., 2003; Zhang et al., 2009; O'Donnell et al., 2011). Biochemical and genetic evidence suggests that RIPK1 cleavage has a far more significant role in limiting necroptosis than that of RIPK3 or CYLD (Newton et al., 2019; Lalaoui et al., 2020; Tao et al., 2020). Hence, caspase 8 inhibition is a priming signal for necroptosis.

Inhibition of caspase 8 preserves the integrity of RIPK1, promoting recruitment of RIPK3 to complex IIb. Structural studies indicate that the RHIMs of RIPK1 and RIPK3 mediate assembly of an amyloid-like structure that promotes the recruitment and phosphorylation of the effector molecule MLKL at positions T357 and S358 (for human MLKL) at the C-terminal pseudokinase domain (PD) (Wang et al., 2014a). Interestingly, a recent report shows that human RIPK3 binds to MLKL in quiescent cells to maintain it in an inactive state (Meng et al., 2021). Whether this counter-regulation is conserved in other species is unknown at present. The phosphorylation of MLKL changes its conformation to expose the four-helical bundle (4HB) at the N terminus, leading to oligomerization and membrane targeting (Su et al., 2014; Sethi et al., 2022). Interestingly, MLKL binds to inositol phosphates, which causes conformational change to promote MLKL oligomerization and plasma membrane targeting (Dovey et al., 2018; McNamara et al., 2019) (see poster). Although direct evidence that MLKL forms membrane-penetrating pores is still lacking, these results nonetheless strongly support MLKL as the effector molecule that triggers membrane rupture and the release of DAMPs in necroptosis.

In contrast to TNFR1, DRs such as Fas and TRAIL (TNFSF10) receptors directly recruit FADD and caspase 8 into the death-inducing signaling complex (DISC) at the plasma membrane. Caspase 8 inhibition, complex IIb formation and the RIPK1–RIPK3–MLKL axis are involved in DR-induced necroptosis, similarly to the TNFRI pathway (Holler et al., 2000) (see poster). Therefore, DR-induced necroptosis mechanistically resembles that of TNFR1-induced necroptosis.

In mouse cells, RIPK1 is dispensable for necroptosis induced by the pattern recognition receptors (PRRs) TLR3 and TLR4 (He et al., 2011; Kaiser et al., 2013). TLR4 is activated by lipopolysaccharide (LPS) from Gram-negative bacteria and can signal through the adaptors MyD88 or TRIF. By contrast, TLR3 recognizes dsRNA from viruses and signals exclusively through TRIF (Brubaker et al., 2015). Under conditions of caspase 8 inhibition, TRIF binds RIPK3 through RHIM–RHIM interactions to promote downstream activation of MLKL and necroptosis (He et al., 2011; Kaiser et al., 2013).

ZBP1, a cytosolic sensor that recognizes left-handed Z-form nucleic acids through two Zα domains at the N terminus (Balachandran and Mocarski, 2021; Takaoka et al., 2007), is another RHIM-containing adaptor that can trigger RIPK3- and MLKL-dependent necroptosis in a RIPK1-independent manner. ZBP1 can be activated by viral nucleic acids (Sridharan et al., 2017; Kesavardhana and Kanneganti, 2020; Koehler et al., 2021; Zhang et al., 2020a), endogenous retrovirus elements (EREs) (Wang et al., 2020a; Jiao et al., 2020) or mitochondrial DNA (Szczesny et al., 2018). Z-form nucleic acid binding triggers ZBP1 interaction with RIPK3 through their respective RHIMs. Although ZBP1 contains two RHIMs in the C terminus, only the more N-terminal RHIM has a dominant role in RIPK3 binding (Yang et al., 2020). Under normal physiological conditions, ZBP1–RIPK3-mediated necroptosis is quelled by RIPK1 in a RHIM-dependent manner (Lin et al., 2016; Newton et al., 2016). This inhibition is relieved in response to Z-form RNA generated during viral infections or EREs, when endogenous RIPK1 is inactivated (Zhang et al., 2020a; Sridharan et al., 2017; Koehler et al., 2021).

RIPK1 is phosphorylated at multiple sites, as has been revealed by mass spectrometry studies (Jaco et al., 2017; Degterev et al., 2008; Krishnan et al., 2015). Activated RIPK1 is marked by autophosphorylation at S166, which licenses RIPK1 to fully unleash its apoptotic and necroptotic activity (Laurien et al., 2020). This death-inducing activity of RIPK1 is restricted by several upstream kinases. For instance, TAK1 and the p38 substrate MAPKAP kinase-2 (MK2, also known as MAPKAPK2) phosphorylate RIPK1 at S321 and S336 to inhibit its death-inducing activity (Jaco et al., 2017; Geng et al., 2017; Menon et al., 2017; Dondelinger et al., 2017). Moreover, TAK1 stimulates IKKα and IKKβ, which in turn phosphorylate RIPK1 at S25 to limit RIPK1-induced cell death (Dondelinger et al., 2019). TANK-binding kinase 1 (TBK1) and IKKε (IKBKE) also inhibit RIPK1-induced cell death by phosphorylation of RIPK1 at T189 (Lafont et al., 2018; Xu et al., 2018; Taft et al., 2021). Furthermore, the autophagy-initiating kinase ULK1 phosphorylates RIPK1 at S357 to restrain complex IIb formation (Wu et al., 2020b) (see poster). The regulation of RIPK1 activity by multiple kinases is consistent with the crucial functions of RIPK1 in cell death, inflammation and organismal fitness.

In mouse cells, RIPK3 phosphorylation at T231 and S232 (S227 in human RIPK3) is believed to occur through autophosphorylation (Chen et al., 2013; Sun et al., 2012), since this modification is lost in cells expressing kinase-inactive RIPK3 (Newton et al., 2014). However, some reports suggest that casein kinase 1 (CK1) is responsible for this phosphorylation (Hanna-Addams et al., 2020; Lee et al., 2019b). The protein phosphatase 1B (PPM1B) has been reported to reverse this phosphorylation (Chen et al., 2013). Interestingly, CK1G2 (also known as CSNK1G2) appears to inhibit RIPK3 and necroptosis through physical sequestration rather than direct phosphorylation (Li et al., 2020a). Once RIPK3 is activated, it recruits and phosphorylates the downstream effector MLKL at S345 (T357 and S358 in human MLKL) in the C-terminal PD (Sun et al., 2012; Chen et al., 2013). Comparison of the structures of full length MLKL and phosphomimetic mutants of the PD indicates that MLKL phosphorylation causes a conformational change that induces PD dimerization (Murphy et al., 2013; Zhang et al., 2021; Petrie et al., 2018). Furthermore, mutations that disrupt this interaction block necroptosis (Zhang et al., 2021). Functional studies using synthetic monobodies that bind to distinct domains of human MLKL further corroborate the importance of PD dimerization as a critical step in MLKL activation (Garnish et al., 2021). Thus, MLKL activation is a multi-step process that involves MLKL phosphorylation, PD dimerization, oligomerization of the N-terminal 4HB, and membrane insertion (Hildebrand et al., 2014; Su et al., 2014). In addition, the tyrosine kinases TYRO3, AXL and MER (also known as MERTK; referred to collectively as TAM kinases) have been reported to phosphorylate MLKL at Y376 to promote MLKL oligomerization (Najafov et al., 2019). Paradoxically, human RIPK3 appears to bind to and maintain MLKL in an inactive state prior to necroptosis induction (Meng et al., 2021). In support of a potential role of human RIPK3 in restricting MLKL activity, RIPK3-mediated inhibitory phosphorylation of MLKL at S82 (S83 in human MLKL), S158 and S228 have recently been reported in mouse (Tanzer et al., 2015; Zhu et al., 2022) (see poster). More work is needed to validate whether these regulatory mechanisms are conserved across species.

Mass spectrometry experiments have revealed that RIPK1 is ubiquitylated at multiple lysine residues (de Almagro et al., 2017; Li et al., 2020b). Mutation of mouse RIPK1 at K376 (which corresponds to K377 in human RIPK1), the target of ubiquitylation by cIAPs, leads to exaggerated RIPK1-driven apoptosis and necroptosis (Tang et al., 2019; Kist et al., 2021). This explains why IAP antagonists potently sensitize RIPK1-mediated apoptosis and necroptosis. The NF-κB target A20 (also known as TNFAIP3) exhibits E3 ubiquitin ligase and ubiquitin hydrolase activities. Early work suggests that A20 removes K63-linked ubiquitin chains on RIPK1 and replaces them with K48-linked ubiquitin chains to promote proteasomal degradation of RIPK1 (Wertz et al., 2004). More recent work suggests that A20 can also protect cells by stabilizing the ubiquitin scaffold on complex I (Priem et al., 2019). Furthermore, A20 dampens K63-linked RIPK3 ubiquitylation at K5, an event that promotes necrosome assembly (Onizawa et al., 2015).

LUBAC, which consists of the catalytic subunit heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1, also known as RBCK1), HOIL-1-interacting protein (HOIP, also known as RNF31) and the regulatory subunit shank-associated RH domain-interacting protein (SHARPIN), is the E3 ubiquitin ligase that regulates RIPK1 linear ubiquitylation (Witt and Vucic, 2017). Disruption of LUBAC activity often leads to exaggerated RIPK1 activity and necroptosis (Peltzer et al., 2018; Taraborrelli et al., 2018), indicating that M1-linked ubiquitylation also restricts RIPK1 activity and necroptosis (de Almagro et al., 2017). Conversely, the deubiquitylases CYLD and OTULIN hydrolyze the ubiquitin chains on RIPK1 to facilitate formation of the death-inducing complex IIb and necroptosis (Keusekotten et al., 2013; Moquin et al., 2013). Other E3 ubiquitin ligases that have been shown to inhibit necroptosis include Mind bomb-2 (MIB2), which ubiquitylates RIPK1 at K377 and K634 (Feltham et al., 2018), and carboxy terminus of Hsp70-interacting protein (CHIP, also known as STUB1), which ubiquitylates RIPK1 at K571, K604 and K627 (Seo et al., 2016). However, their roles in necroptosis will require further experimental validation.

The E3 ubiquitin ligase Pellino 1 (PELI1) has been reported to promote K48-linked ubiquitylation and degradation of RIPK3 (Choi et al., 2018). However, another study has reported that PELI1 stimulates K63-linked ubiquitylation of RIPK1 at K115 to induce necrosome assembly (Wang et al., 2017). Knock-in mice expressing a K115R mutant form of RIPK1 develop normally with no overt signs of deregulated RIPK1 function (Kist et al., 2021), indicating that this ubiquitylation is not critical for necroptosis. Other E3 ubiquitin ligases that have been reported to stimulate K48-linked ubiquitylation and proteasomal degradation of RIPK3 include tripartite motif containing protein 25 (TRIM25), which ubiquitylates RIPK3 at K501 (Mei et al., 2021); CHIP, which ubiquitylates RIPK3 at K55 and K363 (Seo et al., 2016); and Parkin (PRKN), which ubiquitylates RIPK3 at K197, K302 and K364 (Lee et al., 2019a). The ubiquitin-specific peptidase 22 (USP22) removes ubiquitin chains on RIPK3 at K42, K351 and K518 to promote necrosome assembly (Roedig et al., 2021). MLKL ubiquitylation at multiple sites has also been detected (Garcia et al., 2021; Liu et al., 2021a) (see poster). While it is not clear how all the individual modifications affect MLKL function, K63-linked ubiquitylation at K219 might enhance the necroptotic activity of MLKL (Garcia et al., 2021). In addition, a recent report suggests that RIPK3 is O-GlcNAcylated at T467 (Li et al., 2019). The proximity of this position to the RHIM means that RIPK3 O-GlcNAcylation interferes with necroptosis and, therefore, inhibition of RIPK3 O-GlcNAcylation greatly increases inflammatory gene expression and necroptosis in LPS-treated macrophages (Li et al., 2019). The functional relevance of these ubiquitin marks and O-GlcNAcylation in necroptosis will require further validation.

The importance of necroptosis in host defense against pathogen infection was first demonstrated in vaccinia virus (VACV) infection (Chan et al., 2003). VACV encodes the serpin Spi2 (also known as B13R), which inhibits caspase 8 to sensitize infected cells to TNF-induced and RIPK1–RIPK3-dependent necroptosis (Cho et al., 2009). Mice deficient in RIPK3 or RIPK1 kinase activity fail to control viral replication and succumb to the infection (Cho et al., 2009; Polykratis et al., 2014). Conversely, the interferon inhibitor E3L from VACV prevents ZBP1 activation by binding to and sequestering its ligand Z-form nucleic acid (Koehler et al., 2017). In the absence of this E3L function, Z-form nucleic acid generated during VACV infection activates ZBP1 and RIPK3-dependent necroptosis (Koehler et al., 2021; Koehler et al., 2017). Thus, VACV activates two parallel necroptosis responses: one that is triggered by TNF and RIPK1 and a second pathway that is stimulated by viral nucleic acid and ZBP1.

In contrast to VACV, the evolutionarily related orthopoxvirus cowpox virus (CPXV) disrupts necroptosis signaling through the viral inducer of RIPK3 degradation (vIRD), which promotes K48-linked ubiquitylation and proteasomal degradation of RIPK3 (Liu et al., 2021b). Thus, both RIPK1- and ZBP1-induced necroptosis are impaired. Importantly, functional vIRD that targets RIPK3 is present in most of the naturally found orthopoxviruses. In contrast to orthopoxviruses, avipoxviruses do not express vIRD but encode a class of inactive viral MLKL xenologs (vMLKLs) that bind to RIPK3 to inhibit host MLKL activation (Petrie et al., 2019) (see poster). These findings strongly implicate necroptosis inhibition as a critical mechanism for poxviruses to maintain their fitness.

The M45 gene from the betaherpesvirus murine cytomegalovirus (MCMV) encodes a RHIM-containing viral inhibitor of RIP activation (vIRA), which sequesters RIPK3 and ZBP1 in a RHIM-dependent manner (Upton et al., 2012; Pham et al., 2019; Sridharan et al., 2017). Mutant MCMV that lacks a functional RHIM in vIRA triggers premature ZBP1- and RIPK3-induced necroptosis and abortive viral replication (Upton et al., 2012). Human cytomegalovirus (HCMV) also inhibits TNF-induced necroptosis; however, the underlying mechanism is yet to be defined (Omoto et al., 2015).

The alphaherpesviruses herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2) encode the RHIM-containing necroptosis inhibitors ICP6 and ICP10, respectively, both of which also inhibit caspase 8 (Wang et al., 2014b; Yu et al., 2016; Huang et al., 2015; Guo et al., 2018; Mocarski et al., 2015). The capsid protein ORF20 from varicella zoster virus (VZV) also contains a RHIM that can inhibit ZBP1 and necroptosis (Steain et al., 2020). Surprisingly, while ICP6 and ICP10 inhibit necroptosis in human cells, they activate necroptosis in mouse cells (Yu et al., 2016), suggesting that necroptosis might contribute to host species restriction of herpesviruses. Moreover, RHIM inhibitors exert different effects in different species, further highlighting interspecies differences of the necroptosis machinery (Chen et al., 2013; Tanzer et al., 2016).

In addition to viruses, necroptosis inhibition has also recently been described for bacterial pathogens. The type III secretion system of certain enteropathogenic Escherichia coli (EPEC) contains EspL, an atypical cysteine protease that cleaves the four mammalian RHIM-containing adaptors (Pearson et al., 2017). Cleavage of the RHIM adaptors near the RHIM domain inactivates their functions, thereby preventing necroptosis. In studies of mouse infection with Citrobacter rodentium, EspL has been shown to facilitate persistent colonization by the bacteria (Pearson et al., 2017). These results highlight the expanding role of necroptosis in host defense against pathogens.

Necroptosis contributes to a variety of human diseases by driving acute and chronic inflammation of multiple tissues and organs. Elevated RIPK3 and MLKL phosphorylation has been detected in the airway of chronic obstructive pulmonary disease (COPD) patients (Lu et al., 2021; Chen et al., 2021). In agreement with these results, deletion of Ripk3 or Mlkl inhibits airway inflammation in mouse models of cigarette smoke-induced COPD (Lu et al., 2021; Wang et al., 2020c). In the liver, increased expression of active RIPK3 and MLKL, and increased hepatocyte cell death have been detected in non-alcoholic fatty liver disease (NAFLD) patients (Gautheron et al., 2014; Afonso et al., 2015), and Ripk3 deficiency has been found to protect mice against NAFLD (Afonso et al., 2015; Wu et al., 2020a). Since hepatocytes do not express RIPK3 at steady state (The Human Protein Atlas; https://www.proteinatlas.org/), the effects of RIPK3 in chronic liver disease might be attributable to immune cells. A role for RIPK3 in atherosclerosis has also been reported (Karunakaran et al., 2016). Early reports suggest that pharmacological inhibition of RIPK1 or Ripk3 deficiency delays disease onset in the mutant superoxide dismutase 1 (SOD1^G93A) mouse model of amyotrophic lateral sclerosis (ALS) and multiple sclerosis (Ito et al., 2016; Ofengeim et al., 2015). However, this observation was not replicated in two subsequent studies using the same ALS mouse model (Dermentzaki et al., 2019; Wang et al., 2020b). More work is needed to clearly define the role of necroptosis in neurodegeneration.

In addition to chronic inflammation, necroptosis is also involved in acute injury caused by ischemia–reperfusion injury (IRI). For instance, Ripk3 deficiency protects mice from IRI in the heart (Zhang et al., 2016; Lichý et al., 2019; Horvath et al., 2021), kidney (Linkermann et al., 2013) and the brain (Vieira et al., 2014; Zhang et al., 2020b). The role of necroptosis in acute liver injury is more controversial, with data both supporting and refuting a role for RIPK3 in acetaminophen-induced liver injury (Ramachandran et al., 2013; Li et al., 2014; Dara et al., 2015).

Selective loss of RIPK3 protein expression is widely observed in cancers of different tissue origins (Moriwaki et al., 2015; He et al., 2009). Indeed, low or complete loss of RIPK3 expression results in a poor prognosis for cancer survival (Lim et al., 2021). Although the mechanism is not fully defined, the oncogenes BRAF and AXL have been shown to promote selective loss of RIPK3 expression in cancer (Najafov et al., 2018). Moreover, RIPK3 gene promoter methylation can also lead to loss of RIPK3 protein expression in cancer (Koo et al., 2015; Tan et al., 2021; Najafov et al., 2019). The tendency for cancer to evolve ways to avoid necroptosis suggests that reinvigorating necroptosis is an attractive strategy in tumor immunotherapy (Rucker and Chan, 2022) (see poster).

Emerging evidence strongly supports a role for necroptosis in host defense against viral and bacterial pathogens. However, the potent immunogenic activity of necroptosis can be a double-edged sword and cause disease pathologies. The significant role of necroptosis in many inflammatory diseases has led to growing interest in targeting the pathway for therapy (Jensen et al., 2020; Harris et al., 2017; Weisel et al., 2017). Currently, RIPK1 inhibitors are being tested for safety and efficacy in human diseases ranging from ulcerative colitis, rheumatoid arthritis and Alzheimer's disease (Degterev et al., 2019). In preclinical mouse models, necroptotic cell immunization has shown promise as a prophylactic agent to ward off subsequent tumor challenge (Snyder et al., 2019; Aaes et al., 2020). The future is bright, but successful delivery of targeted necroptosis therapy will require further investigation of the physiological functions and interplay of necroptosis signal adaptors in immune and parenchymal cells.

We thank members of the Chan Lab for discussion and comments on the manuscript.