An outline of necrosome triggers

Necroptosis was initially identified as a backup cell death program when apoptosis is blocked. However, it is now recognized as a cellular defense mechanism against infections and is presumed to be a detrimental factor in several pathologies driven by cell death. Necroptosis is a prototypic form of regulated necrosis that depends on activation of the necrosome, which is a protein complex in which receptor interacting protein kinase (RIPK) 3 is activated. The RIP homotypic interaction motif (RHIM) is the core domain that regulates activation of the necrosome. To date, three RHIM-containing proteins have been reported to activate the kinase activity of RIPK3 within the necrosome: RIPK1, Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF), and DNA-dependent activator of interferon regulatory factors (DAI). Here, we review and discuss commonalities and differences of the increasing number of activators of the necrosome. Since the discovery that activation of mixed lineage kinase domain-like (MLKL) by RIPK3 kinase activity is crucial in necroptosis, interest has increased in monitoring and therapeutically targeting their activation. The availability of new phospho-specific antibodies, pharmacologic inhibitors, and transgenic models will allow us to further document the role of necroptosis in degenerative, inflammatory and infectious diseases.

Introduction Rudolf Virchow (1821-1902, founder of the cell theory (Omnis cellula e cellula) and cellular pathology, referred to tissue injury as ''parenchymatous inflammation'' and introduced the idea that tissue injury is caused by pathological changes within the cells. In 1858, he introduced the notion of cell death as a potential basis for pathology, with 'necrobiosis' being a physiological process of spontaneous wearing out of living parts from the body and 'necrosis' an accidental process. The term 'necrosis' comes from the Greek word 'nekros,' which means 'dead body. ' Virchow's necrobiosis-necrosis dichotomy resembles to some extent the current apoptosis-necrosis classification [1]. Together with cellular and molecular insights into inflammation came a shift in our understanding of the molecular interplay between cell death and inflammation at the site of tissue injury. This emerging field of research is crucial for understanding organismal homeostasis and how its processes contribute to a growing list of inflammatory and degenerative pathologies. Although cell death during inflammation was initially considered a manifestation of tissue damage, it was later recognized as a mechanism for eliminating pathogens and regulating inflammation by exposing or releasing molecular patterns that attract and alter the functions of other cells [2]. More recently, it became clear that phagocytosis of apoptotic cells can also initiate anti-inflammatory and tissue-regenerative responses [3,4]. The current notion that not only apoptotic but also necrotic cell death is molecularly controlled by defined signaling mechanisms has increased the interest in studying regulated necrosis in the context of development, homeostasis and inflammation. Although RIPK3 knockout mice and MLKL knockout mice do not show an overt phenotypic abnormality under non-challenged conditions [5][6][7][8], it became clear that RIPK3-mediated necroptosis is a highly controlled cell death program that is executed when negative regulators such as caspase-8, IAPs or even RIPK1 are absent [9][10][11][12][13][14][15][16][17]. This suggests that the default mode during development and homeostasis is strong inhibition of the necroptosis pathway. Regulated necrosis, like passive necrosis due to physico-chemical insult, is caused by loss of plasma membrane integrity leading to cellular rounding followed by swelling (oncosis). When the immune system recognizes cellular content exposed or released due to loss of membrane integrity, it initiates an inflammatory response. Regulated necrosis can be classified into several cell death modalities, such as necroptosis, parthanatos, ferroptosis, cyclophilin D-dependent necrosis, (n)etosis and pyroptosis [18]. Each type of regulated necrosis has particular biochemical features, yet it is not clear whether the common morphological features of these forms of cell death share or converge on common pathways. Other forms of cellular death are being identified, such as entosis [19], autosis [20,21] and autoschizis [22].
Necroptosis, the best-characterized form of regulated necrosis, is mediated by the concerted action of receptor interacting protein kinase (RIPK) 3 and mixed lineage kinase domain-like (MLKL). In this review, we provide a snapshot of the activation of RIPK3 within the necrosome, typically by the three RHIM-containing proteins RIPK1, Toll/IL-1 receptor domain-containing adaptor inducing IFN-b (TRIF) and DNA-dependent activator of interferon regulatory factors (DAI). We briefly discuss the pluripotent roles of RIPK1 and RIPK3 in gene regulation and cell death induction.

RIPK1-dependent necroptosis
The mechanism of RIPK1-dependent necroptosis has been discovered mostly from studying tumor necrosis factor (TNF) signaling under conditions favoring cell death. This is because the major function of TNFR1, like that of DR3 (TRAMP/APO-3), is to induce pro-survival and pro-inflammatory genes, in contrast to some other death receptor family members such as CD95 (FAS/APO-1), TRAILR1 (DR4) and TRAILR2 (APO-2/TRICK/DR5/ KILLER). The following model is currently proposed for TNFR1 signaling: sensing of trimeric TNF by TNFR1 induces the assembly of a primary receptor-bound complex that triggers activation of signaling pathways leading to gene induction [23][24][25]. Subsequently, assembly of a secondary TNFR1-unbound cytosolic complex induces cell death. For FAS and TRAILR1/2, the opposite situation is observed [26]. This sequential signaling provides a backup response by the secondary cytosolic complex in case the default pathway activated by the receptor-associated complex fails to resolve the infectious or inflammatory condition. Typically, pathogens and (epi)genetic factors can interfere with gene activation or cell death induction. Thus, this sequential signaling probably evolved as a host defense against pathogens or conditions that might perturb either pathway.
In this review, we will briefly describe cell death signaling downstream of TNFR1, with a focus on necrosome activation. A cytosolic cell death-inducing complex is formed upon stimulation of TNFR1 only in conditions that sensitize to cell death, for example when cellular inhibitor of apoptosis proteins (c-IAPs) are absent (IAP-antagonist treatment), TAK1 or translation is inhibited, or RIPK1 is deubiquitinated [18]. This cytosolic complex (often referred to as complex II), which is composed of at least RIPK1, the death-fold-containing proteins Fas-associated protein via death domain (FADD), CASP8, and cFLIP ( Fig. 1), induces either apoptosis or necroptosis. The formation and/or activity of complex II is tightly regulated by inhibitor jB kinases (IKKs) through mechanisms that are either dependent or independent of NF-jB [27].

DAI-dependent necroptosis
In addition to RIPK1 and TRIF, a third RHIM-containing protein, DAI, has been reported to activate the necrosome (Fig. 1). The DAI pathway is typically activated in response to DNA viruses and leads to inhibition of viral replication [72]. Like TLR signaling, the intracellular DNA sensor activates the NF-jB and IRF3 pathways to promote the synthesis of cytokines and interferons, which is dependent on RHIM-mediated recruitment of RIPK1 [73]. In addition, in response to DNA viruses, DAI induces necroptosis through RHIM-mediated activation of RIPK3 in the noncanonical necrosome [74]. As a virus-encoded countermeasure, the murine cytomegalovirus (CMV) M45-encoded viral inhibitor of RIP activation (vIRA) acts as a RHIM competitor and blocks necroptosis, which explains the virus's successful replication in the host. The potency of this cell autonomous host defense pathway is demonstrated by the remarkable attenuation of M45-deficient viruses in mice. Importantly, as in RIPK3-deficient mice, mCMV lacking M45 has the same pathogenesis in DAI-deficient mice, consistent with the notion of the existence of a DAI-RIPK3 complex as the natural target of M45 [74]. M45 encodes a ribonucleotide reductase (RNR) lacking enzymatic activity. Interestingly, many RNRs from herpesviruses also encode a RHIM [75]. This suggests that viral inhibitors that target the RIPKs via the RHIM represent a common viral evasion strategy.

The Janus faces of RIPK1
The 'two faces' of RIPK1 refers to its dual role. It has a cell death inhibitory role that is shown by the massive cell death observed in RIPK1-deficient models, whereas its necroptosis-inducing capacity is executed by its kinase activity. In the absence of RIPK1, massive apoptosis is observed in cells [34,76], in postnatal death knockout mice [77], and in intestinal specific knockout mice [12,13]. It was initially thought that this was due to the role of RIPK1 in mediating NF-jB activation, which results in the expression of survival genes such as Flip L [77]. In this respect, cFLIP L -CASP8 heterodimers have partial enzymatic activity, leading to incomplete cleavage of CASP8 [78,79], and this consequently prevents apoptosis. Nevertheless, it is thought that CASP8 has some local activity within complex II resulting in cleavage of RIPKs and CYLD [80], which may contribute to the anti-necroptotic role of CASP8. However, mounting evidence questions the necessity or uniqueness of the role of NF-jB activation in controlling cell death. For example, NF-jB is still activated in response to TNF stimulation in the absence of RIPK1 in cultured MEF cells [81] and in intestinal organoids [13]. TAB2-deficient mice have a functional NF-jB pathway, yet they die from massive liver apoptosis like mice deficient in p65, IKKb, TAK1 or NEMO [82]. Moreover, the rescue of mutant RIPK1 kinase-dead knockin mice from TNF-induced shock [10,11,83,84] and from the lethal TNF-induced inflammation in Sharpin mutant mice [83] also calls into question the dominance of NF-jB activation (that occurs in a RIPK1 kinase independent way). This is underscored by the recent finding that IKKa and IKKb control RIPK1-mediated cell death independently of NF-jB activation [27]. The dual role of RIPK1 in controlling cell death is also illustrated by the perinatal death of RIPK1 knockout mice due to the aberrant activation of caspase-8 and RIPK3; mice lacking all three enzymes survived to adulthood [10,14,85]. Indeed, in addition to its anti-apoptotic role, RIPK1 also prevents RIPK3-driven necroptosis promoted by IFN and the TLR-adapter TRIF [14]. Since RIPK1 is reported to be essential for RIPK3 activation and subsequent necroptosis induction by TNF, the identification of settings in which RIPK1 actively suppresses RIPK3 was surprising. Moreover, conditional depletion of RIPK1 leads to apoptosis in the intestine and necroptosis in the skin [12,13]. This dynamic interplay and interdependence of these complex II components confers a crucial host-defense function to limit pathogen spread, especially when any one of these processes is disrupted [72,86]. This may explain why this complex interrelationship exists and why ablation of specific elements (including RIPK1, FADD, caspase-8 and cFLIP) push the system to lethality [87]. In line with this reasoning, the tissues most affected by disruption of these gene products (intestine, lung, skin, endothelium, hematopoietic cells) represent crucial barriers to infection that are constantly engaged by pathogens [88]. Depending on the tissue, cell type and developmental stage, RIPK1 can certainly either activate or inhibit cell death.

Concluding remarks
There has been a revival of interest in the close interconnection between cell death and inflammation originally recognized by Virchow. This interconnection is emphasized by some recent findings that classical cell death inducers such as caspase-8 and RIPK3 seem to act also upstream of inflammasome activation in a cell autonomous way [93][94][95][96][97][98]. However, the precise mechanisms of this interaction are unclear. RIPK1 as well as RIPK3 and other cytosolic TNFR complex II components have been implicated in regulating cell death and inflammation, though if Alleviates ischemic brain injury [47] Is cardioprotective [175] Improves outcome after controlled cortical impact [176] Alleviates retinal ischemia reperfusion injury [177] Alleviates spinal cord injury pathology in rats [178] Alleviates injury in subtotal nephrectomised rats [179] Nec-1s Nec-1s treatment reduces disease severity in experimental autoimmune encephalitis model [180] NSA Nec-1 or NSA block death in models of both sporadic and familial ALS (ex vivo) [181] Dabrafenib Targets RIPK3 and alleviates acetaminophen-induced liver injury [182] Upregulated RIPK3 expression potentiates MLKL phosphorylation-mediated programmed necrosis in toxic epidermal necrolysis [183] Nec-1, SanglifehrinA,  Protects from renal ischemia reperfusion injury [99] Nec-1, cyclosporine A, 3-AB Protects from remote lung injury after receiving ischemic renal allografts in rats [101]  Multiple sclerosis Increased levels of RIPK1, RIPK3 and necrosome formation in lesions [180] Breast cancer Weak RIPK3 expression [158] Melanoma Melanoma cell lines lack RIPK3 expression, whereas primary melanocytes strongly express RIPK3 [186] HIV Dysfunctional HIV-specific CD8? T cell proliferation is associated with increased caspase-8 activity and mediated by necroptosis [187] Leukemia RIPK3 is downregulated [188] these functions could be uncoupled is not clear. In addition, the potential signal transduction interplay between parenchymal cell necrosis and some forms of necrosis that occur in immune cells, such as pyroptosis and netosis, remains unknown. Considering the central role of RHIM domains in controlling the cell death induced by several stimuli, small molecules that disrupt RHIM signaling might also be therapeutically useful. The number of genetic (Table 3) and pharmacological studies (Table 4) demonstrating an important role for RIPK1, RIPK3 or MLKL in murine experimental disease models is still increasing, highlighting the therapeutic potential of these necrosome members. In addition, the expression and activation of RIPK1, RIPK3 and MLKL is being increasingly explored in biopsies of patients with particular pathologies driven by cell death and inflammation ( Table 5). The availability of new phospho-specific antibodies, pharmacologic inhibitors and transgenic models will allow us to document further the role of necroptosis in degenerative, inflammatory and infectious diseases. It is noteworthy that therapeutic targeting of only necroptosis might be insufficient in some complex pathologies, as exemplified by the additive protective effect of targeting different types of regulated necrosis [99][100][101]. This observed redundancy of necrosome proteins and interplay between different modalities of necrotic cell death in vivo is an intriguing topic for further research and will generate further insight into how the targeting of these molecules in some cases looks very effective.