RIPK3 cleavage is dispensable for necroptosis inhibition but restricts NLRP3 inflammasome activation.

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 Ripk3D333A/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, Ripk3D333A/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-1ß 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.

Gasdermin and MLKL necrotic cell death effectors: Signaling and diseases.

Diverse inflammatory conditions, from infections to autoimmune disease, are often associated with cellular damage and death. Apoptotic cell death has evolved to minimize its inflammatory potential. By contrast, necrotic cell death via necroptosis and pyroptosis-driven by membrane-damaging MLKL and gasdermins, respectively-can both initiate and propagate inflammatory responses. In this review, we provide insights into the function and regulation of MLKL and gasdermin necrotic effector proteins and drivers of plasma membrane rupture. We evaluate genetic evidence that MLKL- and gasdermin-driven necrosis may either provide protection against, or contribute to, disease states in a context-dependent manner. These cumulative insights using gene-targeted mice underscore the necessity for future research examining pyroptotic and necroptotic cell death in human tissue, as a basis for developing specific necrotic inhibitors with the potential to benefit a spectrum of pathological conditions.

IFNγ: Priming for death.

TNF signaling does not result in cell death unless multiple inhibitory signals are overcome, which can be accomplished by simultaneous signaling through IFNγ. In this issue, Deng and colleagues (http://doi.org/10.1083/jcb.202305026) dissect the mechanisms by which IFNγ signaling combines with TNF to mediate cell death through caspase-8, discussed by James E. Vince.

CD45 limits early Natural Killer cell development.

The clinical development of Natural Killer (NK) cell-mediated immunotherapy marks a milestone in the development of new cancer therapies and has gained traction due to the intrinsic ability of the NK cell to target and kill tumor cells. To fully harness the tumor killing ability of NK cells, we need to improve NK cell persistence and to overcome suppression of NK cell activation in the tumor microenvironment. The trans-membrane, protein tyrosine phosphatase CD45, regulates NK cell homeostasis, with the genetic loss of CD45 in mice resulting in increased numbers of mature NK cells. This suggests that CD45-deficient NK cells might display enhanced persistence following adoptive transfer. However, we demonstrate here that adoptive transfer of CD45-deficiency did not enhance NK cell persistence in mice, and instead, the homeostatic disturbance of NK cells in CD45-deficient mice stemmed from a developmental defect in the progenitor population. The enhanced maturation within the CD45-deficient NK cell compartment was intrinsic to the NK cell lineage, and independent of the developmental defect. CD45 is not a conventional immune checkpoint candidate, as systemic loss is detrimental to T and B cell development, compromising the adaptive immune system. Nonetheless, this study suggests that inhibition of CD45 in progenitor or stem cell populations may improve the yield of in vitro generated NK cells for adoptive therapy.

Mind bomb 2 limits inflammatory dermatitis in Sharpin mutant mice independently of cell death.

Skin inflammation is a complex process implicated in various dermatological disorders. The chronic proliferative dermatitis (cpd) phenotype driven by the cpd mutation (cpdm) in the Sharpin gene is characterized by dermal inflammation and epidermal abnormalities. Tumour necrosis factor (TNF) and caspase-8-driven cell death causes the pathogenesis of Sharpincpdm mice; however, the role of mind bomb 2 (MIB2), a pro-survival E3 ubiquitin ligase involved in TNF signaling, in skin inflammation remains unknown. Here, we demonstrate that MIB2 antagonizes inflammatory dermatitis in the context of the cpd mutation. Surprisingly, the role of MIB2 in limiting skin inflammation is independent of its known pro-survival function and E3 ligase activity. Instead, MIB2 enhances the production of wound-healing molecules, granulocyte colony-stimulating factor, and Eotaxin, within the skin. This discovery advances our comprehension of inflammatory cytokines and chemokines associated with cpdm pathogenesis and highlights the significance of MIB2 in inflammatory skin disease that is independent of its ability to regulate TNF-induced cell death.

A1 is induced by pathogen ligands to limit myeloid cell death and NLRP3 inflammasome activation.

Programmed cell death pathways play an important role in innate immune responses to infection. Activation of intrinsic apoptosis promotes infected cell clearance; however, comparatively little is known about how this mode of cell death is regulated during infections and whether it can induce inflammation. Here, we identify that the pro-survival BCL-2 family member, A1, controls activation of the essential intrinsic apoptotic effectors BAX/BAK in macrophages and monocytes following bacterial lipopolysaccharide (LPS) sensing. We show that, due to its tight transcriptional and post-translational regulation, A1 acts as a molecular rheostat to regulate BAX/BAK-dependent apoptosis and the subsequent NLRP3 inflammasome-dependent and inflammasome-independent maturation of the inflammatory cytokine IL-1ß. Furthermore, induction of A1 expression in inflammatory monocytes limits cell death modalities and IL-1ß activation triggered by Neisseria gonorrhoeae-derived outer membrane vesicles (NOMVs). Consequently, A1-deficient mice exhibit heightened IL-1ß production in response to NOMV injection. These findings reveal that bacteria can induce A1 expression to delay myeloid cell death and inflammatory responses, which has implications for the development of host-directed antimicrobial therapeutics.

The role of caspase-8 in inflammatory signalling and pyroptotic cell death.

The programmed cell death machinery exhibits surprising flexibility, capable of crosstalk and non-apoptotic roles. Much of this complexity arises from the diverse functions of caspase-8, a cysteine-aspartic acid protease typically associated with activating caspase-3 and - 7 to induce apoptosis. However, recent research has revealed that caspase-8 also plays a role in regulating the lytic gasdermin cell death machinery, contributing to pyroptosis and immune responses in contexts such as infection, autoinflammation, and T-cell signalling. In mice, loss of caspase-8 results in embryonic lethality from unrestrained necroptotic killing, while in humans caspase-8 deficiency can lead to an autoimmune lymphoproliferative syndrome, immunodeficiency, inflammatory bowel disease or, when it can't cleave its substrate RIPK1, early onset periodic fevers. This review focuses on non-canonical caspase-8 signalling that drives immune responses, including its regulation of inflammatory gene transcription, activation within inflammasome complexes, and roles in pyroptotic cell death. Ultimately, a deeper understanding of caspase-8 function will aid in determining whether, and when, targeting caspase-8 pathways could be therapeutically beneficial in human diseases.

A Streamlined Method for Detecting Inflammasome-Induced ASC Oligomerization Using Chemical Crosslinking.

The apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) functions as the integral adaptor protein between inflammasome sensors such as NOD-like receptor protein 3 (NLRP3) and the inflammatory caspase, caspase-1. Inflammasome sensor triggering allows recruitment of ASC and the formation of long amyloid-like ASC oligomers that enable binding and proximity-induced activation of caspase-1. The detection of ASC oligomerization thus constitutes a highly specific and direct test for inflammasome complex formation and activation. Here, we describe a simplified and streamlined method for the detection of ASC oligomers via Western blotting, using the chemical crosslinking reagent disuccinimidyl suberate.

FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins.

Staphylococcus aureus causes severe infections such as pneumonia and sepsis depending on the pore-forming toxin Panton-Valentine leukocidin (PVL). PVL kills and induces inflammation in macrophages and other myeloid cells by interacting with the human cell surface receptor, complement 5a receptor 1 (C5aR1). C5aR1 expression is tighly regulated and may thus modulate PVL activity, although the mechanisms involved remain incompletely understood. Here, we used a genome-wide CRISPR/Cas9 screen and identified F-box protein 11 (FBXO11), an E3 ubiquitin ligase complex member, to promote PVL toxicity. Genetic deletion of FBXO11 reduced the expression of C5aR1 at the mRNA level, whereas ectopic expression of C5aR1 in FBXO11-/- macrophages, or priming with LPS, restored C5aR1 expression and thereby PVL toxicity. In addition to promoting PVL-mediated killing, FBXO11 dampens secretion of IL-1ß after NLRP3 activation in response to bacterial toxins by reducing mRNA levels in a BCL-6-dependent and BCL-6-independent manner. Overall, these findings highlight that FBXO11 regulates C5aR1 and IL-1ß expression and controls macrophage cell death and inflammation following PVL exposure.

Caspase-8-driven apoptotic and pyroptotic crosstalk causes cell death and IL-1ß release in X-linked inhibitor of apoptosis (XIAP) deficiency.

Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1ß maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1ß release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1ß maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency.

Epithelial-to-Mesenchymal Transition Supports Ovarian Carcinosarcoma Tumorigenesis and Confers Sensitivity to Microtubule Targeting with Eribulin.

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. SIGNIFICANCE: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

The escape of Candida albicans from macrophages is enabled by the fungal toxin candidalysin and two host cell death pathways.

The egress of Candida hyphae from macrophages facilitates immune evasion, but it also alerts macrophages to infection and triggers inflammation. To better define the mechanisms, here we develop an imaging assay to directly and dynamically quantify hyphal escape and correlate it to macrophage responses. The assay reveals that Candida escapes by using two pore-forming proteins to permeabilize macrophage membranes: the fungal toxin candidalysin and Nlrp3 inflammasome-activated Gasdermin D. Candidalysin plays a major role in escape, with Nlrp3 and Gasdermin D-dependent and -independent contributions. The inactivation of Nlrp3 does not reduce hyphal escape, and we identify ETosis via macrophage extracellular trap formation as an additional pathway facilitating hyphal escape. Suppressing hyphal escape does not reduce fungal loads, but it does reduce inflammatory activation. Our findings explain how Candida escapes from macrophages by using three strategies: permeabilizing macrophage membranes via candidalysin and engaging two host cell death pathways, Gasdermin D-mediated pyroptosis and ETosis.

Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death.

Pathogen recognition and TNF receptors signal via receptor interacting serine/threonine kinase-3 (RIPK3) to cause cell death, including MLKL-mediated necroptosis and caspase-8-dependent apoptosis. However, the post-translational control of RIPK3 is not fully understood. Using mass-spectrometry, we identified that RIPK3 is ubiquitylated on K469. The expression of mutant RIPK3 K469R demonstrated that RIPK3 ubiquitylation can limit both RIPK3-mediated apoptosis and necroptosis. The enhanced cell death of overexpressed RIPK3 K469R and activated endogenous RIPK3 correlated with an overall increase in RIPK3 ubiquitylation. Ripk3 K469R/K469R mice challenged with Salmonella displayed enhanced bacterial loads and reduced serum IFNγ. However, Ripk3 K469R/K469R macrophages and dermal fibroblasts were not sensitized to RIPK3-mediated apoptotic or necroptotic signaling suggesting that, in these cells, there is functional redundancy with alternate RIPK3 ubiquitin-modified sites. Consistent with this idea, the mutation of other ubiquitylated RIPK3 residues also increased RIPK3 hyper-ubiquitylation and cell death. Therefore, the targeted ubiquitylation of RIPK3 may act as either a brake or accelerator of RIPK3-dependent killing.

No longer married to inflammasome signaling: the diverse interacting pathways leading to pyroptotic cell death.

For over 15 years the lytic cell death termed pyroptosis was defined by its dependency on the inflammatory caspase, caspase-1, which, upon pathogen sensing, is activated by innate immune cytoplasmic protein complexes known as inflammasomes. However, this definition of pyroptosis changed when the pore-forming protein gasdermin D (GSDMD) was identified as the caspase-1 (and caspase-11) substrate required to mediate pyroptotic cell death. Consequently, pyroptosis has been redefined as a gasdermin-dependent cell death. Studies now show that, upon liberation of the N-terminal domain, five gasdermin family members, GSDMA, GSDMB, GSDMC, GSDMD and GSDME can all form plasma membrane pores to induce pyroptosis. Here, we review recent research into the diverse stimuli and cell death signaling pathways involved in the activation of gasdermins; death and toll-like receptor triggered caspase-8 activation of GSDMD or GSMDC, apoptotic caspase-3 activation of GSDME, perforin-granzyme A activation of GSDMB, and bacterial protease activation of GSDMA. We highlight findings that have begun to unravel the physiological situations and disease states that result from gasdermin signaling downstream of inflammasome activation, death receptor and mitochondrial apoptosis, and necroptosis. This new era in cell death research therefore holds significant promise in identifying how distinct, yet often networked, pyroptotic cell death pathways might be manipulated for therapeutic benefit to treat a range of malignant conditions associated with inflammation, infection and cancer.

CXCL11 expressing C57BL/6 mice have intact adaptive immune responses to viral infection.

The chemokine receptor CXCR3 is expressed on immune cells to co-ordinate lymphocyte activation and migration. CXCR3 binds three chemokine ligands, CXCL9, CXCL10 and CXCL11. These ligands display distinct expression patterns and ligand signaling biases; however, how each ligand functions individually and collaboratively is incompletely understood. CXCL9 and CXCL10 are considered pro-inflammatory chemokines during viral infection, while CXCL11 may induce a tolerizing state. The investigation of the individual role of CXCL11 in vivo has been hampered as C57BL/6 mice carry several mutations that result in a null allele. Here, CRISPR/Cas9 was used to correct these mutations on a C57BL/6 background. It was validated that CXCL11KI mice expressed CXCL11 protein in dendritic cells, spleen and lung. CXCL11KI mice were largely phenotypically indistinguishable from C57BL/6 mice, both at steady-state and during two models of viral infection. While CXCL11 expression did not modify acute antiviral responses, this study provides a new tool to understand the role of CXCL11 in other experimental settings.

Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway.

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.

The ubiquitylation of IL-1ß limits its cleavage by caspase-1 and targets it for proteasomal degradation.

Interleukin-1ß (IL-1ß) is activated by inflammasome-associated caspase-1 in rare autoinflammatory conditions and in a variety of other inflammatory diseases. Therefore, IL-1ß activity must be fine-tuned to enable anti-microbial responses whilst limiting collateral damage. Here, we show that precursor IL-1ß is rapidly turned over by the proteasome and this correlates with its decoration by K11-linked, K63-linked and K48-linked ubiquitin chains. The ubiquitylation of IL-1ß is not just a degradation signal triggered by inflammasome priming and activating stimuli, but also limits IL-1ß cleavage by caspase-1. IL-1ß K133 is modified by ubiquitin and forms a salt bridge with IL-1ß D129. Loss of IL-1ß K133 ubiquitylation, or disruption of the K133:D129 electrostatic interaction, stabilizes IL-1ß. Accordingly, Il1bK133R/K133R mice have increased levels of precursor IL-1ß upon inflammasome priming and increased production of bioactive IL-1ß, both in vitro and in response to LPS injection. These findings identify mechanisms that can limit IL-1ß activity and safeguard against damaging inflammation.

Deletion of IKK2 in haematopoietic cells of adult mice leads to elevated interleukin-6, neutrophilia and fatal gastrointestinal inflammation.

The IκB kinase complex, consisting of IKK1, IKK2 and the regulatory subunit NEMO, is required for NF-κB signalling following the activation of several cell surface receptors, such as members of the Tumour Necrosis Factor Receptor superfamily and the Interleukin-1 Receptor. This is critical for haematopoietic cell proliferation, differentiation, survival and immune responses. To determine the role of IKK in the regulation of haematopoiesis, we used the Rosa26Cre-ERT2 Cre/lox recombination system to achieve targeted, haematopoietic cell-restricted deletion of the genes for IKK1 or IKK2 in vivo. We found that the IKK complex plays a critical role in haematopoietic cell development and function. Deletion of IKK2, but not loss of IKK1, in haematopoietic cells led to an expansion of CD11b/Gr-1-positive myeloid cells (neutrophilia), severe anaemia and thrombocytosis, with reduced numbers of long-term haematopoietic stem cells (LT-HSCs), short-term haematopoietic stem cells (ST-HSCs) and multipotential progenitor cells (MPPs), increased circulating interleukin-6 (IL-6) and severe gastrointestinal inflammation. These findings identify distinct functions for the two IKK catalytic subunits, IKK1 and IKK2, in the haematopoietic system.

Specific NLRP3 Inhibition Protects Against Diabetes-Associated Atherosclerosis.

Low-grade persistent inflammation is a feature of diabetes-driven vascular complications, in particular activation of the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome to trigger the maturation and release of the inflammatory cytokine interleukin-1ß (IL-1ß). We investigated whether inhibiting the NLRP3 inflammasome, through the use of the specific small-molecule NLRP3 inhibitor MCC950, could reduce inflammation, improve vascular function, and protect against diabetes-associated atherosclerosis in the streptozotocin-induced diabetic apolipoprotein E-knockout mouse. Diabetes led to an approximately fourfold increase in atherosclerotic lesions throughout the aorta, which were significantly attenuated with MCC950 (P < 0.001). This reduction in lesions was associated with decreased monocyte-macrophage content, reduced necrotic core, attenuated inflammatory gene expression (IL-1ß, tumor necrosis factor-α, intracellular adhesion molecule 1, and MCP-1; P < 0.05), and reduced oxidative stress, while maintaining fibrous cap thickness. Additionally, vascular function was improved in diabetic vessels of mice treated with MCC950 (P < 0.05). In a range of cell lines (murine bone marrow-derived macrophages, human monocytic THP-1 cells, phorbol 12-myristate 13-acetate-differentiated human macrophages, and aortic smooth muscle cells from humans with diabetes), MCC950 significantly reduced IL-1ß and/or caspase-1 secretion and attenuated leukocyte-smooth muscle cell interactions under high glucose or lipopolysaccharide conditions. In summary, MCC950 reduces plaque development, promotes plaque stability, and improves vascular function, suggesting that targeting NLRP3-mediated inflammation is a novel therapeutic strategy to improve diabetes-associated vascular disease.