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Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses (1-3). Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response., Members of the death receptor family such as FAS and TNFR1 contain an intracellular death domain and require caspase-8 for the activation of effector caspases and consequent cell death by [...]

Innate immune signalling mediated by the nucleotide-binding and oligomerization domain (NOD) receptors for pathogen-associated constituents regulates the response to intracellular peptidoglycans present in Gram-negative and Gram-positive bacteria. Recently, Yeretssian et [...]

Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKy or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene ([Sharpin.sup.cpdm/cpdm]) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1β was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling., Members of the TNF receptor superfamily (TNFRSF) exert pleiotropic effects in immune and non-immune tissues (1) and their signalling is regulated by different types of ubiquitin chains (2-4). We and [...]

FAS (also called APO-1 and CD95) and its physiological ligand, FASL, regulate apoptosis of unwanted or dangerous cells, functioning as a guardian against autoimmunity and cancer development (1-4). Distinct cell [...]

Author(s): Vanessa S. Marsden [1]; Liam O'Connor [1, 2]; Lorraine A. O'Reilly [1]; John Silke [1]; Donald Metcalf [1]; Paul G. Ekert [1, 3]; David C. S. Huang [1]; Francesco [...]

Toxic epidermal necrolysis (TEN) is a fatal drug-induced skin reaction triggered by common medications and is an emerging public health issue 1-3 . Patients with TEN undergo severe and sudden epidermal detachment caused by keratinocyte cell death. Although molecular mechanisms that drive keratinocyte cell death have been proposed, the main drivers remain unknown, and there is no effective therapy for TEN 4-6 . Here, to systematically map molecular changes that are associated with TEN and identify potential druggable targets, we utilized deep visual proteomics, which provides single-cell-based, cell-type-resolution proteomics 7,8 . We analysed formalin-fixed, paraffin-embedded archived skin tissue biopsies of three types of cutaneous drug reactions with varying severity and quantified more than 5,000 proteins in keratinocytes and skin-infiltrating immune cells. This revealed a marked enrichment of type I and type II interferon signatures in the immune cell and keratinocyte compartment of patients with TEN, as well as phosphorylated STAT1 activation. Targeted inhibition with the pan-JAK inhibitor tofacitinib in vitro reduced keratinocyte-directed cytotoxicity. In vivo oral administration of tofacitinib, baricitinib or the JAK1-specific inhibitors abrocitinib or upadacitinib ameliorated clinical and histological disease severity in two distinct mouse models of TEN. Crucially, treatment with JAK inhibitors (JAKi) was safe and associated with rapid cutaneous re-epithelialization and recovery in seven patients with TEN. This study uncovers the JAK/STAT and interferon signalling pathways as key pathogenic drivers of TEN and demonstrates the potential of targeted JAKi as a curative therapy., (© 2024. The Author(s).)

Tumour immune evasion presents a significant challenge to the effectiveness of cancer immunotherapies. Recent advances in high-throughput screening techniques have uncovered that loss of antigen presentation and cytokine signalling pathways are central mechanisms by which tumours evade T cell immunity. To uncover additional vulnerabilities in tumour cells beyond the well-recognized antigen presentation pathway, we conducted a genome-wide CRISPR/Cas9 screen to identify genes that mediate resistance to chimeric-antigen receptor (CAR)-T cells, which function independently of classical antigen presentation. Our study revealed that loss of core-binding factor subunit beta (CBFβ) enhances tumour cell resistance to T cell killing, mediated through T cell-derived TNF. Mechanistically, RNA-sequencing and elemental analyses revealed that deletion of CBFβ disrupts numerous pathways including those involved in zinc homoeostasis. Moreover, we demonstrated that modulation of cellular zinc, achieved by supplementation or chelation, significantly altered tumour cell susceptibility to TNF by regulating the levels of inhibitor of apoptosis proteins. Consistent with this, treatment of tumour cells with a membrane-permeable zinc chelator had no impact on tumour cell viability alone, but significantly increased tumour cell lysis by CD8+ T cells in a TNF-dependent but perforin-independent manner. These results underscore the crucial role of intracellular zinc in regulating tumour cell susceptibility to T cell-mediated killing, revealing a novel vulnerability in tumour cells that might be exploited for the development of future cancer immunotherapeutics., (© 2024. The Author(s).)

Caspase-8 activity is required to inhibit necroptosis during embryogenesis in mice. In vitro studies have suggested that caspase-8 directly cleaves RIPK1, CYLD and the key necroptotic effector kinase RIPK3 to repress necroptosis. However, recent studies have shown that mice expressing uncleavable RIPK1 die during embryogenesis due to excessive apoptosis, while uncleavable CYLD mice are viable. Therefore, these results raise important questions about the role of RIPK3 cleavage. To evaluate the physiological significance of RIPK3 cleavage, we generated Ripk3 D333A/D333A mice harbouring a point mutation in the conserved caspase-8 cleavage site. These mice are viable, demonstrating that RIPK3 cleavage is not essential for blocking necroptosis during development. Furthermore, unlike RIPK1 cleavage-resistant cells, Ripk3 D333A/D333A cells were not significantly more sensitive to necroptotic stimuli. Instead, we found that the cleavage of RIPK3 by caspase-8 restricts NLRP3 inflammasome activation-dependent pyroptosis and IL-1β secretion when Inhibitors of APoptosis (IAP) are limited. These results demonstrate that caspase-8 does not inhibit necroptosis by directly cleaving RIPK3 and further underscore a role for RIPK3 in regulating the NLRP3 inflammasome., (© 2024. The Author(s).)

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

MLKL and RIPK3 are the core signaling proteins of the inflammatory cell death pathway, necroptosis, which is a known mediator and modifier of human disease. Necroptosis has been implicated in the progression of disease in almost every physiological system and recent reports suggest a role for necroptosis in aging. Here, we present the first comprehensive analysis of age-related histopathological and immunological phenotypes in a cohort of Mlkl -/- and Ripk3 -/- mice on a congenic C57BL/6 J genetic background. We show that genetic deletion of Mlkl in female mice interrupts immune system aging, specifically delaying the age-related reduction of circulating lymphocytes. -Seventeen-month-old Mlkl -/- female mice were also protected against age-related chronic sterile inflammation in connective tissue and skeletal muscle relative to wild-type littermate controls, exhibiting a reduced number of immune cell infiltrates in these sites and fewer regenerating myocytes. These observations implicate MLKL in age-related sterile inflammation, suggesting a possible application for long-term anti-necroptotic therapy in humans., (© 2023. The Author(s).)

Thymic epithelial cells (TECs) form a unique microenvironment that orchestrates T cell differentiation and immunological tolerance. Despite the importance of TECs for adaptive immunity, there is an incomplete understanding of the signalling networks that support their differentiation and survival. We report that the linear ubiquitin chain assembly complex (LUBAC) is essential for medullary TEC (mTEC) differentiation, cortical TEC survival and prevention of premature thymic atrophy. TEC-specific loss of LUBAC proteins, HOIL-1 or HOIP, severely impaired expansion of the thymic medulla and AIRE-expressing cells. Furthermore, HOIL-1-deficiency caused early thymic atrophy due to Caspase-8/MLKL-dependent apoptosis/necroptosis of cortical TECs. By contrast, deficiency in the LUBAC component, SHARPIN, caused relatively mild defects only in mTECs. These distinct roles for LUBAC components in TECs correlate with their function in linear ubiquitination, NFκB activation and cell survival. Thus, our findings reveal dual roles for LUBAC signaling in TEC differentiation and survival., (© 2021. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER + ) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER + breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER + cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage 1-7 . The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis 8 . Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1 D325A mutant mouse strain. Whereas Ripk1 -/- mice died postnatally from systemic inflammation, Ripk1 D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1 D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1 D325A/D325A and Ripk1 D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1 D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.

RIPK1 is an essential downstream component of many pattern recognition and death receptors. RIPK1 can promote the activation of caspase-8 induced apoptosis and RIPK3-MLKL-mediated necroptosis, however, during development RIPK1 limits both forms of cell death. Accordingly, Ripk1 -/- mice present with systemic cell death and consequent multi-organ inflammation, which is driven through the activation of both FADD-caspase-8 and RIPK3-MLKL signaling pathways causing perinatal lethality. TRADD is a death domain (DD) containing molecule that mediates signaling downstream of TNFR1 and the TLRs. Following the disassembly of the upstream receptor complexes either RIPK1 or TRADD can form a complex with FADD-caspase-8-cFLIP, via DD-DD interactions with FADD, facilitating the activation of caspase-8. We show that genetic deletion of Ripk1 licenses TRADD to complex with FADD-caspase-8 and activates caspase-8 during development. Deletion of Tradd provided no survival advantage to Ripk1 -/- animals and yet was sufficient to reduce the systemic cell death and inflammation, rescue the intestinal and thymic histopathologies, reduce cleaved caspases in most tissues and rescue the anemia observed in Ripk1 -/- neonates. Furthermore, deletion of Ripk3 is sufficient to rescue the neonatal lethality of Ripk1 -/- Tradd -/- animals and delays but does not completely prevent early mortality. Although Ripk3 deletion provides a significant survival advantage, Ripk1 -/- Tradd -/- Ripk3 -/- animals die between 22 and 49 days, are runty compared to littermate controls and present with splenomegaly. These findings reveal a new mechanism by which RIPK1 limits apoptosis through blocking TRADD recruitment to FADD and preventing aberrant activation of caspase-8.

The programmed cell death pathway, necroptosis, relies on the pseudokinase, Mixed Lineage Kinase domain-Like (MLKL), for cellular execution downstream of death receptor or Toll-like receptor ligation. Receptor-interacting protein kinase-3 (RIPK3)-mediated phosphorylation of MLKL's pseudokinase domain leads to MLKL switching from an inert to activated state, where exposure of the N-terminal four-helix bundle (4HB) 'executioner' domain leads to cell death. The precise molecular details of MLKL activation, including the stoichiometry of oligomer assemblies, mechanisms of membrane translocation and permeabilisation, remain a matter of debate. Here, we dissect the function of the two 'brace' helices that connect the 4HB to the pseudokinase domain of MLKL. In addition to establishing that the integrity of the second brace helix is crucial for the assembly of mouse MLKL homotrimers and cell death, we implicate the brace helices as a device to communicate pseudokinase domain phosphorylation event(s) to the N-terminal executioner 4HB domain. Using mouse:human MLKL chimeras, we defined the first brace helix and adjacent loop as key elements of the molecular switch mechanism that relay pseudokinase domain phosphorylation to the activation of the 4HB domain killing activity. In addition, our chimera data revealed the importance of the pseudokinase domain in conferring host specificity on MLKL killing function, where fusion of the mouse pseudokinase domain converted the human 4HB + brace from inactive to a constitutive killer of mouse fibroblasts. These findings illustrate that the brace helices play an active role in MLKL regulation, rather than simply acting as a tether between the 4HB and pseudokinase domains.

Mixed lineage kinase domain-like (MLKL)-dependent necroptosis is thought to be implicated in the death of mycobacteria-infected macrophages, reportedly allowing escape and dissemination of the microorganism. Given the consequent interest in developing inhibitors of necroptosis to treat Mycobacterium tuberculosis (Mtb) infection, we used human pharmacologic and murine genetic models to definitively establish the pathophysiological role of necroptosis in Mtb infection. We observed that Mtb infection of macrophages remodeled the intracellular signaling landscape by upregulating MLKL, TNFR1, and ZBP1, whilst downregulating cIAP1, thereby establishing a strong pro-necroptotic milieu. However, blocking necroptosis either by deleting Mlkl or inhibiting RIPK1 had no effect on the survival of infected human or murine macrophages. Consistent with this, MLKL-deficiency or treatment of humanized mice with the RIPK1 inhibitor Nec-1s did not impact on disease outcomes in vivo, with mice displaying lung histopathology and bacterial burdens indistinguishable from controls. Therefore, although the necroptotic pathway is primed by Mtb infection, macrophage necroptosis is ultimately restricted to mitigate disease pathogenesis. We identified cFLIP upregulation that may promote caspase 8-mediated degradation of CYLD, and other necrosome components, as a possible mechanism abrogating Mtb's capacity to coopt necroptotic signaling. Variability in the capacity of these mechanisms to interfere with necroptosis may influence disease severity and could explain the heterogeneity of Mtb infection and disease.

The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death 2-8 . In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype 9-11 . Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1 -/- (also known as Rbck1 -/- ) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3 -/- Casp8 -/- Hoil-1 -/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Smac-mimetics are emerging as promising anti-cancer agents and are being evaluated in clinical trials for a variety of malignancies. Smac-mimetics can induce TNF production from a subset of tumor cells and simultaneously sensitize them to TNF-induced apoptosis. However, TNF derived from other cellular sources, such as cytotoxic lymphocytes (CLs) within the tumor, may also contribute to the anti-tumor activity of SMs. Here, we show that CD8 + T cells and NK cells potently kill tumor cells in the presence of the SM, birinapant. Enhanced CL killing occurred through TNF secretion upon tumor antigen recognition or NK-activating receptor ligation. Importantly, the perforin/granzyme route to CL-mediated tumor cell killing was dispensable for the efficacy of birinapant, emphasizing the importance of the TNF-mediated apoptosis pathway. Time-lapse microscopy revealed that birinapant sensitized tumor cells to apoptosis as bystanders and to membrane-bound TNF delivered to tumor cells within the immunological synapse. Furthermore, PD-L1 expression on tumor cells suppressed antigen-driven TNF production by CD8 + T cells, which could be antagonized through PD-1 blockade. Importantly, the elevated levels of TNF produced upon PD-1 blockade further enhanced tumor cell killing when combined with birinapant. The combined anti-tumor activity of IAP antagonism and PD-1 blockade occurred independently of perforin-mediated tumor cell death. Taken together, we identify CL-derived TNF as a potent effector of birinapant mediated anti-tumor immunity and opportunity for combination therapy through co-inhibition of immune checkpoints.

The mechanisms that underpin the production of small molecules and cytokines that lead to inflammation or programmed cell death are intricately intertwined. So much so that some of the proteins that contribute to the transcriptional up regulation of cytokines can switch their role in the right circumstances to generate cell death-inducing complexes. This entwinement is reflected in the fact that inflammation helps an organism fight pathogens and that therefore pathogens are under an evolutionary pressure to interfere with this process. Cell death is therefore a defensive measure that may serve to deny pathogens a host cell, expose pathogens to the immune system and also provide additional inflammatory information to the host. Clearly such a system must be tightly regulated and ubiquitylation is a post-translational protein modification that is at the heart of this regulation. In this review, we discuss the regulatory ubiquitin events that dictate the formation and activation of death-inducing complexes containing RIPK1/FADD/caspase-8, and examine how these events collectively determine cell fate.

Peptido-mimetic inhibitor of apoptosis protein (IAP) antagonists (Smac mimetics (SMs)) can kill tumour cells by depleting endogenous IAPs and thereby inducing tumour necrosis factor (TNF) production. We found that interferon-γ (IFNγ) synergises with SMs to kill cancer cells independently of TNF- and other cell death receptor signalling pathways. Surprisingly, CRISPR/Cas9 HT29 cells doubly deficient for caspase-8 and the necroptotic pathway mediators RIPK3 or MLKL were still sensitive to IFNγ/SM-induced killing. Triple CRISPR/Cas9-knockout HT29 cells lacking caspase-10 in addition to caspase-8 and RIPK3 or MLKL were resistant to IFNγ/SM killing. Caspase-8 and RIPK1 deficiency was, however, sufficient to protect cells from IFNγ/SM-induced cell death, implying a role for RIPK1 in the activation of caspase-10. These data show that RIPK1 and caspase-10 mediate cell death in HT29 cells when caspase-8-mediated apoptosis and necroptosis are blocked and help to clarify how SMs operate as chemotherapeutic agents.

Cell death signalling pathways contribute to tissue homeostasis and provide innate protection from infection. Adaptor proteins such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3), TIR-domain-containing adapter-inducing interferon-β (TRIF) and Z-DNA-binding protein 1 (ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI) that contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM) play a key role in cell death and inflammatory signalling 1-3 . RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis 4,5 . Here, we report that the bacterial pathogen enteropathogenic Escherichia coli (EPEC) uses the type III secretion system (T3SS) effector EspL to degrade the RHIM-containing proteins RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This requires a previously unrecognized tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaves within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL leads to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of tumour necrosis factor (TNF), lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C))-induced necroptosis and inflammatory signalling. Furthermore, EPEC infection inhibits TNF-induced phosphorylation and plasma membrane localization of mixed lineage kinase domain-like pseudokinase (MLKL). In vivo, EspL cysteine protease activity contributes to persistent colonization of mice by the EPEC-like mouse pathogen Citrobacter rodentium. The activity of EspL defines a family of T3SS cysteine protease effectors found in a range of bacteria and reveals a mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signalling pathways.

Sirtuins can promote deacetylation of a wide range of substrates in diverse cellular compartments and regulate many cellular processes¹,². Recently Narayan et al., reported that SIRT2 was required for necroptosis based on their findings that SIRT2 inhibition, knock-down or knock-out prevented necroptosis. We sought to confirm and explore the role of SIRT2 in necroptosis and tested four different sources of the SIRT2 inhibitor AGK2, three independent siRNAs against SIRT2, and cells from two independently generated Sirt2−/− mouse strains, however we were unable to show that inhibiting or depleting SIRT2 protected cells from necroptosis. Furthermore, Sirt2−/− mice succumbed to TNF induced Systemic Inflammatory Response Syndrome (SIRS) more rapidly than wild type mice while Ripk3−/− mice were resistant. Our results therefore question the importance of SIRT2 in the necroptosis cell death pathway.