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.

Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

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.

Posttranslational Modification as a Critical Determinant of Cytoplasmic Innate Immune Recognition.

Cell surface innate immune receptors can directly detect a variety of extracellular pathogens to which cytoplasmic innate immune sensors are rarely exposed. Instead, within the cytoplasm, the environment is rife with cellular machinery and signaling pathways that are indirectly perturbed by pathogenic microbes to activate intracellular sensors, such as pyrin, NLRP1, NLRP3, or NLRC4. Therefore, subtle changes in key intracellular processes such as phosphorylation, ubiquitination, and other pathways leading to posttranslational protein modification are key determinants of innate immune recognition in the cytoplasm. This concept is critical to establish the "guard hypothesis" whereby otherwise homeostatic pathways that keep innate immune sensors at bay are released in response to alterations in their posttranslational modification status. Originally identified in plants, evidence that a similar guardlike mechanism exists in humans has recently been identified, whereby a mutation that prevents phosphorylation of the innate immune sensor pyrin triggers a dominantly inherited autoinflammatory disease. It is also noteworthy that even when a cytoplasmic innate immune sensor has a direct ligand, such as bacterial peptidoglycan (NOD1 or NOD2), RNA (RIG-I or MDA5), or DNA (cGAS or IFI16), it can still be influenced by posttranslational modification to dramatically alter its response. Therefore, due to their existence in the cytoplasmic milieu, posttranslational modification is a key determinant of intracellular innate immune receptor functionality.

Tying the knot between cytokine and toll-like receptor signaling in gastrointestinal tract cancers.

Inflammation-associated malignancies of the gastrointestinal tract (GI), including those of the stomach and colon, collectively rank as the highest cause of cancer-related deaths worldwide. It has been well documented that the deregulated activation of the archetypal pro-inflammatory and oncogenic transcription factors nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription (STAT)3 is a common feature of GI cancers that invariably correlates with poor prognosis. Signal transducer and activator of transcription 3 and NF-κB are key downstream signal transducers of the interleukin (IL)-6 cytokine and toll-like receptor (TLR) families, respectively, and until recently, the potential involvement of these two families in the pathogenesis of cancer has been investigated in isolation. However, there is now emerging evidence of the complex interplay between the IL-6 cytokine and TLR families in GI tract cancers, with a surprising twist in the identification of a non-immune role for specific TLR family members. In this review, we discuss the molecular mechanisms associated with cross-talk between the IL-6 cytokine family/STAT3 signaling network and the TLR family/NF-κB signaling network, and we address the potential benefit of their therapeutic targeting in gastric and colorectal cancers.

STAT3 and STAT1 mediate IL-11-dependent and inflammation-associated gastric tumorigenesis in gp130 receptor mutant mice.

Deregulated activation of STAT3 is frequently associated with many human hematological and epithelial malignancies, including gastric cancer. While exaggerated STAT3 signaling facilitates an antiapoptotic, proangiogenic, and proproliferative environment for neoplastic cells, the molecular mechanisms leading to STAT3 hyperactivation remain poorly understood. Using the gp130(Y757F/Y757F) mouse model of gastric cancer, which carries a mutated gp130 cytokine receptor signaling subunit that cannot bind the negative regulator of cytokine signaling SOCS3 and is characterized by hyperactivation of the signaling molecules STAT1 and STAT3, we have provided genetic evidence that IL-11 promotes chronic gastric inflammation and associated tumorigenesis. Expression of IL-11 was increased in gastric tumors in gp130(Y757F/Y757F) mice, when compared with unaffected gastric tissue in wild-type mice, while gp130(Y757F/Y757F) mice lacking the IL-11 ligand-binding receptor subunit (IL-11Ralpha) showed normal gastric STAT3 activation and IL-11 expression and failed to develop gastric tumors. Furthermore, reducing STAT3 activity in gp130(Y757F/Y757F) mice, either genetically or by therapeutic administration of STAT3 antisense oligonucleotides, normalized gastric IL-11 expression and alleviated gastric tumor burden. Surprisingly, the genetic reduction of STAT1 expression also reduced gastric tumorigenesis in gp130(Y757F/Y757F) mice and coincided with reduced gastric inflammation and IL-11 expression. Collectively, our data have identified IL-11 as a crucial cytokine promoting chronic gastric inflammation and associated tumorigenesis mediated by excessive activation of STAT3 and STAT1.

Targeting RIP Kinases in Chronic Inflammatory Disease.

Chronic inflammatory disorders are characterised by aberrant and exaggerated inflammatory immune cell responses. Modes of extrinsic cell death, apoptosis and necroptosis, have now been shown to be potent drivers of deleterious inflammation, and mutations in core repressors of these pathways underlie many autoinflammatory disorders. The receptor-interacting protein (RIP) kinases, RIPK1 and RIPK3, are integral players in extrinsic cell death signalling by regulating the production of pro-inflammatory cytokines, such as tumour necrosis factor (TNF), and coordinating the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, which underpin pathological inflammation in numerous chronic inflammatory disorders. In this review, we firstly give an overview of the inflammatory cell death pathways regulated by RIPK1 and RIPK3. We then discuss how dysregulated signalling along these pathways can contribute to chronic inflammatory disorders of the joints, skin, and gastrointestinal tract, and discuss the emerging evidence for targeting these RIP kinases in the clinic.

Inflammasome Adaptor ASC Suppresses Apoptosis of Gastric Cancer Cells by an IL18-Mediated Inflammation-Independent Mechanism.

Inflammasomes are key regulators of innate immunity in chronic inflammatory disorders and autoimmune diseases, but their role in inflammation-associated tumorigenesis remains ill-defined. Here we reveal a protumorigenic role in gastric cancer for the key inflammasome adaptor apoptosis-related speck-like protein containing a CARD (ASC) and its effector cytokine IL18. Genetic ablation of ASC in the gp130F/F spontaneous mouse model of intestinal-type gastric cancer suppressed tumorigenesis by augmenting caspase-8-like apoptosis in the gastric epithelium, independently from effects on myeloid cells and mucosal inflammation. This phenotype was characterized by reduced activation of caspase-1 and NF-κB activation and reduced expression of mature IL18, but not IL1ß, in gastric tumors. Genetic ablation of IL18 in the same model also suppressed gastric tumorigenesis, whereas blockade of IL1ß and IL1α activity upon genetic ablation of the IL1 receptor had no effect. The specific protumorigenic role for IL18 was associated with high IL18 gene expression in the gastric tumor epithelium compared with IL1ß, which was preferentially expressed in immune cells. Supporting an epithelial-specific role for IL18, we found it to be highly secreted from human gastric cancer cell lines. Moreover, IL18 blockade either by a neutralizing anti-IL18 antibody or by CRISPR/Cas9-driven deletion of ASC augmented apoptosis in human gastric cancer cells. In clinical specimens of human gastric cancer tumors, we observed a significant positive correlation between elevated mature IL18 protein and ASC mRNA levels. Collectively, our findings reveal the ASC/IL18 signaling axis as a candidate therapeutic target in gastric cancer.Significance: Inflammasome activation that elevates IL18 helps drive gastric cancer by protecting cancer cells against apoptosis, with potential implications for new therapeutic strategies in this setting. Cancer Res; 78(5); 1293-307. ©2017 AACR.

NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease.

Anti-microbial signaling pathways are normally triggered by innate immune receptors when detecting pathogenic microbes to provide protective immunity. Here we show that the inflammasome sensor Nlrp1 aggravates DSS-induced experimental mouse colitis by limiting beneficial, butyrate-producing Clostridiales in the gut. The colitis-protective effects of Nlrp1 deficiency are thus reversed by vancomycin treatment, but recapitulated with butyrate supplementation in wild-type mice. Moreover, an activating mutation in Nlrp1a increases IL-18 and IFNγ production, and decreases colonic butyrate to exacerbate colitis. We also show that, in patients with ulcerative colitis, increased NLRP1 in inflamed regions of the colon is associated with increased IFN-γ. In this context, NLRP1, IL-18 or IFN-γ expression negatively correlates with the abundance of Clostridiales in human rectal mucosal biopsies. Our data identify the NLRP1 inflammasome to be a key negative regulator of protective, butyrate-producing commensals, which therefore promotes inflammatory bowel disease.

Myeloid-derived miR-223 regulates intestinal inflammation via repression of the NLRP3 inflammasome.

MicroRNA (miRNA)-mediated RNA interference regulates many immune processes, but how miRNA circuits orchestrate aberrant intestinal inflammation during inflammatory bowel disease (IBD) is poorly defined. Here, we report that miR-223 limits intestinal inflammation by constraining the nlrp3 inflammasome. miR-223 was increased in intestinal biopsies from patients with active IBD and in preclinical models of intestinal inflammation. miR-223-/y mice presented with exacerbated myeloid-driven experimental colitis with heightened clinical, histopathological, and cytokine readouts. Mechanistically, enhanced NLRP3 inflammasome expression with elevated IL-1ß was a predominant feature during the initiation of colitis with miR-223 deficiency. Depletion of CCR2+ inflammatory monocytes and pharmacologic blockade of IL-1ß or NLRP3 abrogated this phenotype. Generation of a novel mouse line, with deletion of the miR-223 binding site in the NLRP3 3' untranslated region, phenocopied the characteristics of miR-223-/y mice. Finally, nanoparticle-mediated overexpression of miR-223 attenuated experimental colitis, NLRP3 levels, and IL-1ß release. Collectively, our data reveal a previously unappreciated role for miR-223 in regulating the innate immune response during intestinal inflammation.

Aberrant actin depolymerization triggers the pyrin inflammasome and autoinflammatory disease that is dependent on IL-18, not IL-1ß.

Gain-of-function mutations that activate the innate immune system can cause systemic autoinflammatory diseases associated with increased IL-1ß production. This cytokine is activated identically to IL-18 by an intracellular protein complex known as the inflammasome; however, IL-18 has not yet been specifically implicated in the pathogenesis of hereditary autoinflammatory disorders. We have now identified an autoinflammatory disease in mice driven by IL-18, but not IL-1ß, resulting from an inactivating mutation of the actin-depolymerizing cofactor Wdr1. This perturbation of actin polymerization leads to systemic autoinflammation that is reduced when IL-18 is deleted but not when IL-1 signaling is removed. Remarkably, inflammasome activation in mature macrophages is unaltered, but IL-18 production from monocytes is greatly exaggerated, and depletion of monocytes in vivo prevents the disease. Small-molecule inhibition of actin polymerization can remove potential danger signals from the system and prevents monocyte IL-18 production. Finally, we show that the inflammasome sensor of actin dynamics in this system requires caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain, and the innate immune receptor pyrin. Previously, perturbation of actin polymerization by pathogens was shown to activate the pyrin inflammasome, so our data now extend this guard hypothesis to host-regulated actin-dependent processes and autoinflammatory disease.

STAT3-driven upregulation of TLR2 promotes gastric tumorigenesis independent of tumor inflammation.

Gastric cancer (GC) is associated with chronic inflammation; however, the molecular mechanisms promoting tumorigenesis remain ill defined. Using a GC mouse model driven by hyperactivation of the signal transducer and activator of transcription (STAT)3 oncogene, we show that STAT3 directly upregulates the epithelial expression of the inflammatory mediator Toll-like receptor (TLR)2 in gastric tumors. Genetic and therapeutic targeting of TLR2 inhibited gastric tumorigenesis, but not inflammation, characterized by reduced proliferation and increased apoptosis of the gastric epithelium. Increased STAT3 pathway activation and TLR2 expression were also associated with poor GC patient survival. Collectively, our data reveal an unexpected role for TLR2 in the oncogenic function of STAT3 that may represent a therapeutic target in GC.