Interleukin-1β (IL-1β) is a potent inflammatory cytokine that is usually cleaved and activated by inflammasome-associated caspase-1. To determine whether IL-1β activation is regulated by inhibitor of ...apoptosis (IAP) proteins, we treated macrophages with an IAP-antagonist “Smac mimetic” compound or genetically deleted the genes that encode the three IAP family members cIAP1, cIAP2, and XIAP. After Toll-like receptor priming, IAP inhibition triggered cleavage of IL-1β that was mediated not only by the NLRP3-caspase-1 inflammasome, but also by caspase-8 in a caspase-1-independent manner. In the absence of IAPs, rapid and full generation of active IL-1β by the NLRP3-caspase-1 inflammasome, or by caspase-8, required the kinase RIP3 and reactive oxygen species production. These results demonstrate that activation of the cell death-inducing ripoptosome platform and RIP3 can generate bioactive IL-1β and implicate them as additional targets for the treatment of pathological IL-1-driven inflammatory responses.
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► IAP inhibition induces NLRP3 inflammasome-dependent and -independent IL-1 activation ► Genetic deletion of the three IAPs (cIAP1, cIAP2, XIAP) activates IL-1 ► Inflammasome-independent IL-1 maturation is mediated by caspase-8 cleavage ► RIP3 signaling, and not cell death, activates IL-1
RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive ...inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3-caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1β inflammatory responses independent of MLKL and necroptotic cell death.
The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14, when expressed in tumors, causes cachexia and that ...antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor, rather than host, is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia, thereby extending lifespan and improving quality of life for cancer patients.
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•Tumors expressing Fn14 cause cachexia in mice•Fn14 antibodies extend lifespan by inhibiting tumor-induced cachexia•Fn14- and TWEAK-deficient mice succumb to cancer cachexia•Tumor Fn14 signaling, rather than host, is responsible for inducing cachexia
Antibodies against the TWEAK receptor Fn14 prevent tumor-induced cachexia and extend lifespan by inhibiting weight loss and inflammation, although having only moderate effects on tumor growth.
Interferon (IFN) induction of IFN‐stimulated genes (ISGs) creates a formidable protective antiviral state. However, loss of appropriate control mechanisms can result in constitutive pathogenic ISG ...upregulation. Here, we used genome‐scale loss‐of‐function screening to establish genes critical for IFN‐induced transcription, identifying all expected members of the JAK‐STAT signaling pathway and a previously unappreciated epigenetic reader, bromodomain‐containing protein 9 (BRD9), the defining subunit of non‐canonical BAF (ncBAF) chromatin‐remodeling complexes. Genetic knockout or small‐molecule‐mediated degradation of BRD9 limits IFN‐induced expression of a subset of ISGs in multiple cell types and prevents IFN from exerting full antiviral activity against several RNA and DNA viruses, including influenza virus, human immunodeficiency virus (HIV1), and herpes simplex virus (HSV1). Mechanistically, BRD9 acts at the level of transcription, and its IFN‐triggered proximal association with the ISG transcriptional activator, STAT2, suggests a functional localization at selected ISG promoters. Furthermore, BRD9 relies on its intact acetyl‐binding bromodomain and unique ncBAF scaffolding interaction with GLTSCR1/1L to promote IFN action. Given its druggability, BRD9 is an attractive target for dampening ISG expression under certain autoinflammatory conditions.
Synopsis
Interferon signaling induces a potent antiviral state in cells. This study uses genome‐scale CRISPR/Cas9 screening as a basis to identify and characterize a new role for BRD9 in cellular interferon‐stimulated gene expression and antiviral activity.
The non‐canonical chromatin remodeling complex component BRD9 is required for interferon‐stimulated gene expression.
BRD9 function relies on its acetyl‐binding bromodomain and unique DUF3512 scaffolding domain.
Depletion of BRD9 limits the antiviral action of interferon against several RNA and DNA viruses.
Small molecule degraders of BRD9 may permit dampening of pathogenic autoinflammatory gene expression.
Interferon signaling induces a potent antiviral state in cells. This study uses genome‐scale CRISPR/Cas9 screening as a basis to identify and characterize a new role for BRD9 in cellular interferon‐stimulated gene expression and antiviral activity.
Necroptosis is a form of regulated necrosis that results in cell death and content release after plasma membrane permeabilization. However, little is known about the molecular events responsible for ...the disruption of the plasma membrane. Here, we find that early increase in cytosolic calcium in TNF-induced necroptosis is mediated by treatment with a Smac mimetic via the TNF/RIP1/TAK1 survival pathway. This does not require the activation of the necrosome and is dispensable for necroptosis. Necroptosis induced by the activation of TLR3/4 pathways does not trigger early calcium flux. We also demonstrate that necroptotic plasma membrane rupture is mediated by osmotic forces and membrane pores around 4 nm in diameter. This late permeabilization step represents a hallmark in necroptosis execution that is cell and treatment independent and requires the RIP1/RIP3/MLKL core. In support of this, treatment with osmoprotectants reduces cell damage in an in vivo necroptosis model of ischemia-reperfusion injury.
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•Early calcium signaling in TSZ necroptosis correlates with cellular levels of cIAP1/2•Calcium flux is induced by a Smac mimetic and is dispensable for necroptosis execution•Pores ∼4 nm in diameter mediate final plasma membrane disruption in necroptosis
Ros et al. find that calcium flux induced by a Smac mimetic is dispensable for necroptosis execution. The authors identify the formation of calcium-independent nanopores at the plasma membrane and find that this is important for necroptosis execution.
Birinapant is a smac-mimetic (SM) in clinical trials for treating cancer. SM antagonize inhibitor of apoptosis (IAP) proteins and simultaneously induce tumor necrosis factor (TNF) secretion to render ...cancers sensitive to TNF-induced killing. To enhance SM efficacy, we screened kinase inhibitors for their ability to increase TNF production of SM-treated cells. We showed that p38 inhibitors increased TNF induced by SM. Unexpectedly, even though p38 is required for Toll-like receptors to induce TNF, loss of p38 or its downstream kinase MK2 increased induction of TNF by SM. Hence, we show that the p38/MK2 axis can inhibit or promote TNF production, depending on the stimulus. Importantly, clinical p38 inhibitors overcame resistance of primary acute myeloid leukemia to birinapant.
•Genetic loss or chemical inhibition of p38 or MK2 increases SM-induced TNF•p38 and MK2 inhibit SM-induced phosphorylation of JNK and ERK•The combination of SM and p38 inhibitor is well tolerated in vivo•p38 and MK2 inhibitors potentiate SM killing of AML in vivo
Lalaoui et al. show that inhibition of p38 or its downstream kinase MK2, in contrast to reducing Toll-like receptor-mediated tumor necrosis factor (TNF) production, increases TNF production upon smac-mimetic (SM) treatment and enhances the anti-tumor efficacy of SM.
Loss of inhibitor of apoptosis proteins (IAPs), particularly cIAP1, can promote production of tumor necrosis factor (TNF) and sensitize cancer cell lines to TNF-induced necroptosis by promoting ...formation of a death-inducing signaling complex containing receptor-interacting serine/threonine-protein kinase (RIPK) 1 and 3. To define the role of IAPs in myelopoiesis, we generated a mouse with cIAP1, cIAP2, and XIAP deleted in the myeloid lineage. Loss of cIAPs and XIAP in the myeloid lineage caused overproduction of many proinflammatory cytokines, resulting in granulocytosis and severe sterile inflammation. In vitro differentiation of macrophages from bone marrow in the absence of cIAPs and XIAP led to detectable levels of TNF and resulted in reduced numbers of mature macrophages. The cytokine production and consequent cell death caused by IAP depletion was attenuated by loss or inhibition of TNF or TNF receptor 1. The loss of RIPK1 or RIPK3, but not the RIPK3 substrate mixed lineage kinase domain-like protein, attenuated TNF secretion and thereby prevented apoptotic cell death and not necrosis. Our results demonstrate that cIAPs and XIAP together restrain RIPK1- and RIPK3-dependent cytokine production in myeloid cells to critically regulate myeloid homeostasis.
•cIAPs and XIAP negatively regulate cytokine production, including TNF to disrupt myeloid lineage differentiation.•IAPs prevent RIPK1 and RIPK3 activity to limit cytokine production prior to cell death.
A role for cellular inhibitors of apoptosis (IAPs cIAPs) in preventing CD95 death has been suspected but not previously explained mechanistically. In this study, we find that the loss of cIAPs leads ...to a dramatic sensitization to CD95 ligand (CD95L) killing. Surprisingly, this form of cell death can only be blocked by a combination of RIP1 (receptor-interacting protein 1) kinase and caspase inhibitors. Consistently, we detect a large increase in RIP1 levels in the CD95 death-inducing signaling complex (DISC) and in a secondary cytoplasmic complex (complex II) in the presence of IAP antagonists and loss of RIP1-protected cells from CD95L/IAP antagonist-induced death. Cells resistant to CD95L/IAP antagonist treatment could be sensitized by short hairpin RNA-mediated knockdown of cellular FLICE-inhibitory protein (cFLIP). However, only cFLIPL and not cFLIPS interfered with RIP1 recruitment to the DISC and complex II and protected cells from death. These results demonstrate a fundamental role for RIP1 in CD95 signaling and provide support for a physiological role of caspase-independent death receptor-mediated cell death.
Background and Aims Tumor necrosis factor (TNF) is a cytokine that promotes inflammation and contributes to pathogenesis of inflammatory bowel diseases. Unlike other cells and tissues, intestinal ...epithelial cells undergo rapid cell death upon exposure to TNF, by unclear mechanisms. We investigated the roles of inhibitor of apoptosis proteins (IAPs) in the regulation of TNF-induced cell death in the intestinal epithelium of mice and intestinal organoids. Methods RNA from cell lines and tissues was analyzed by quantitative polymerase chain reaction, protein levels were analyzed by immunoblot assays. BIRC2 (also called cIAP1) was expressed upon induction from lentiviral vectors in young adult mouse colon (YAMC) cells. YAMC cells, the mouse colon carcinoma cell line MC38, the mouse macrophage cell line RAW 264.7, or mouse and human organoids were incubated with second mitochondrial activator of caspases (Smac)-mimetic compound LCL161 or recombinant TNF-like weak inducer of apoptosis (TNFSF12) along with TNF, and cell death was quantified. C57BL/6 mice with disruption of Xiap, Birc2 (encodes cIAP1), Birc3 (encodes cIAP2), Tnfrsf1a , or Tnfrsf1b ( Tnfrsf1a and b encode TNF receptors) were injected with TNF or saline (control); liver and intestinal tissues were collected and analyzed for apoptosis induction by cleaved caspase 3 immunohistochemistry. We also measured levels of TNF and alanine aminotransferase in serum from mice. Results YAMC cells, and mouse and human intestinal organoids, died rapidly in response to TNF. YAMC and intestinal crypts expressed lower levels of XIAP, cIAP1, cIAP2, and cFLIP than liver tissue. Smac-mimetics reduced levels of cIAP1 and XIAP in MC38 and YAMC cells, and Smac-mimetics and TNF-related weak inducer of apoptosis increased TNF-induced cell death in YAMC cells and organoids—most likely by sequestering and degrading cIAP1. Injection of TNF greatly increased levels of cell death in intestinal tissue of cIAP1-null mice, compared with wild-type C57BL/6 mice, cIAP2-null mice, or XIAP-null mice. Excessive TNF-induced cell death in the intestinal epithelium was mediated TNF receptor 1. Conclusions In a study of mouse and human cell lines, organoids, and tissues, we found cIAP1 to be required for regulation of TNF-induced intestinal epithelial cell death and survival. These findings have important implications for the pathogenesis of TNF-mediated enteropathies and chronic inflammatory diseases of the intestine.
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