NAIP-NLRC4 Inflammasomes Coordinate Intestinal Epithelial Cell Expulsion with Eicosanoid and IL-18 Release via Activation of Caspase-1 and -8.

Intestinal epithelial cells (IECs) form a critical barrier against pathogen invasion. By generation of mice in which inflammasome expression is restricted to IECs, we describe a coordinated epithelium-intrinsic inflammasome response in vivo. This response was sufficient to protect against Salmonella tissue invasion and involved a previously reported IEC expulsion that was coordinated with lipid mediator and cytokine production and lytic IEC death. Excessive inflammasome activation in IECs was sufficient to result in diarrhea and pathology. Experiments with IEC organoids demonstrated that IEC expulsion did not require other cell types. IEC expulsion was accompanied by a major actin rearrangement in neighboring cells that maintained epithelium integrity but did not absolutely require Caspase-1 or Gasdermin D. Analysis of Casp1-/-Casp8-/- mice revealed a functional Caspase-8 inflammasome in vivo. Thus, a coordinated IEC-intrinsic, Caspase-1 and -8 inflammasome response plays a key role in intestinal immune defense and pathology.

Human NAIP/NLRC4 and NLRP3 inflammasomes detect Salmonella type III secretion system activities to restrict intracellular bacterial replication.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that uses two distinct type III secretion systems (T3SSs), termed Salmonella pathogenicity island (SPI)-1 and SPI-2, to deliver virulence factors into the host cell. The SPI-1 T3SS enables Salmonella to invade host cells, while the SPI-2 T3SS facilitates Salmonella's intracellular survival. In mice, a family of cytosolic immune sensors, including NAIP1, NAIP2, and NAIP5/6, recognizes the SPI-1 T3SS needle, inner rod, and flagellin proteins, respectively. Ligand recognition triggers assembly of the NAIP/NLRC4 inflammasome, which mediates caspase-1 activation, IL-1 family cytokine secretion, and pyroptosis of infected cells. In contrast to mice, humans encode a single NAIP that broadly recognizes all three ligands. The role of NAIP/NLRC4 or other inflammasomes during Salmonella infection of human macrophages is unclear. We find that although the NAIP/NLRC4 inflammasome is essential for detecting T3SS ligands in human macrophages, it is partially required for responses to infection, as Salmonella also activated the NLRP3 and CASP4/5 inflammasomes. Importantly, we demonstrate that combinatorial NAIP/NLRC4 and NLRP3 inflammasome activation restricts Salmonella replication in human macrophages. In contrast to SPI-1, the SPI-2 T3SS inner rod is not sensed by human or murine NAIPs, which is thought to allow Salmonella to evade host recognition and replicate intracellularly. Intriguingly, we find that human NAIP detects the SPI-2 T3SS needle protein. Critically, in the absence of both flagellin and the SPI-1 T3SS, the NAIP/NLRC4 inflammasome still controlled intracellular Salmonella burden. These findings reveal that recognition of Salmonella SPI-1 and SPI-2 T3SSs and engagement of both the NAIP/NLRC4 and NLRP3 inflammasomes control Salmonella infection in human macrophages.

Salmonella enterica Serovar Typhimurium Induces NAIP/NLRC4- and NLRP3/ASC-Independent, Caspase-4-Dependent Inflammasome Activation in Human Intestinal Epithelial Cells.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes diseases ranging from gastroenteritis to systemic infection and sepsis. Salmonella uses type III secretion systems (T3SS) to inject effectors into host cells. While these effectors are necessary for bacterial invasion and intracellular survival, intracellular delivery of T3SS products also enables detection of translocated Salmonella ligands by cytosolic immune sensors. Some of these sensors form multimeric complexes called inflammasomes, which activate caspases that lead to interleukin-1 (IL-1) family cytokine release and pyroptosis. In particular, the Salmonella T3SS needle, inner rod, and flagellin proteins activate the NAIP/NLRC4 inflammasome in murine intestinal epithelial cells (IECs), which leads to restriction of bacterial replication and extrusion of infected IECs into the intestinal lumen, thereby preventing systemic dissemination of Salmonella. While these processes are quite well studied in mice, the role of the NAIP/NLRC4 inflammasome in human IECs remains unknown. Unexpectedly, we found the NAIP/NLRC4 inflammasome is dispensable for early inflammasome responses to Salmonella in both human IEC lines and enteroids. Additionally, NLRP3 and the adaptor protein ASC are not required for inflammasome activation in Caco-2 cells. Instead, we observed a necessity for caspase-4 and gasdermin D pore-forming activity in mediating inflammasome responses to Salmonella in Caco-2 cells. These findings suggest that unlike murine IECs, human IECs do not rely on NAIP/NLRC4 or NLRP3/ASC inflammasomes and instead primarily use caspase-4 to mediate inflammasome responses to Salmonella pathogenicity island 1 (SPI-1)-expressing Salmonella.

Intestinal restriction of Salmonella Typhimurium requires caspase-1 and caspase-11 epithelial intrinsic inflammasomes.

We investigated the role of the inflammasome effector caspases-1 and -11 during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Salmonella burdens were significantly greater in the intestines of caspase-1/11 deficient (Casp1/11-/-), Casp1-/- and Casp11-/- mice, as compared to wildtype mice. To determine if this reflected IEC-intrinsic inflammasomes, enteroid monolayers were derived and infected with Salmonella. Casp11-/- and wildtype monolayers responded similarly, whereas Casp1-/- and Casp1/11-/- monolayers carried significantly increased intracellular burdens, concomitant with marked decreases in IEC shedding and death. Pretreatment with IFN-γ to mimic inflammation increased caspase-11 levels and IEC death, and reduced Salmonella burdens in Casp1-/- monolayers, while high intracellular burdens and limited cell shedding persisted in Casp1/11-/- monolayers. Thus caspase-1 regulates inflammasome responses in IECs at baseline, while proinflammatory activation of IECs reveals a compensatory role for caspase-11. These results demonstrate the importance of IEC-intrinsic canonical and non-canonical inflammasomes in host defense against Salmonella.

Gasdermin-D and Caspase-7 are the key Caspase-1/8 substrates downstream of the NAIP5/NLRC4 inflammasome required for restriction of Legionella pneumophila.

Inflammasomes are cytosolic multi-protein complexes that detect infection or cellular damage and activate the Caspase-1 (CASP1) protease. The NAIP5/NLRC4 inflammasome detects bacterial flagellin and is essential for resistance to the flagellated intracellular bacterium Legionella pneumophila. The effectors required downstream of NAIP5/NLRC4 to restrict bacterial replication remain unclear. Upon NAIP5/NLRC4 activation, CASP1 cleaves and activates the pore-forming protein Gasdermin-D (GSDMD) and the effector caspase-7 (CASP7). However, Casp1-/- (and Casp1/11-/-) mice are only partially susceptible to L. pneumophila and do not phenocopy Nlrc4-/-mice, because NAIP5/NLRC4 also activates CASP8 for restriction of L. pneumophila infection. Here we show that CASP8 promotes the activation of CASP7 and that Casp7/1/11-/- and Casp8/1/11-/- mice recapitulate the full susceptibility of Nlrc4-/- mice. Gsdmd-/- mice exhibit only mild susceptibility to L. pneumophila, but Gsdmd-/-Casp7-/- mice are as susceptible as the Nlrc4-/- mice. These results demonstrate that GSDMD and CASP7 are the key substrates downstream of NAIP5/NLRC4/CASP1/8 required for resistance to L. pneumophila.

Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells.

Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.

Intestinal Epithelial Cell Tyrosine Kinase 2 Transduces IL-22 Signals To Protect from Acute Colitis.

In the intestinal tract, IL-22 activates STAT3 to promote intestinal epithelial cell (IEC) homeostasis and tissue healing. The mechanism has remained obscure, but we demonstrate that IL-22 acts via tyrosine kinase 2 (Tyk2), a member of the Jak family. Using a mouse model for colitis, we show that Tyk2 deficiency is associated with an altered composition of the gut microbiota and exacerbates inflammatory bowel disease. Colitic Tyk2(-/-) mice have less p-STAT3 in colon tissue and their IECs proliferate less efficiently. Tyk2-deficient primary IECs show reduced p-STAT3 in response to IL-22 stimulation, and expression of IL-22-STAT3 target genes is reduced in IECs from healthy and colitic Tyk2(-/-) mice. Experiments with conditional Tyk2(-/-) mice reveal that IEC-specific depletion of Tyk2 aggravates colitis. Disease symptoms can be alleviated by administering high doses of rIL-22-Fc, indicating that Tyk2 deficiency can be rescued via the IL-22 receptor complex. The pivotal function of Tyk2 in IL-22-dependent colitis was confirmed in Citrobacter rodentium-induced disease. Thus, Tyk2 protects against acute colitis in part by amplifying inflammation-induced epithelial IL-22 signaling to STAT3.

Host-compound foraging by intestinal microbiota revealed by single-cell stable isotope probing.

The animal and human intestinal mucosa secretes an assortment of compounds to establish a physical barrier between the host tissue and intestinal contents, a separation that is vital for health. Some pathogenic microorganisms as well as members of the commensal intestinal microbiota have been shown to be able to break down these secreted compounds. Our understanding of host-compound degradation by the commensal microbiota has been limited to knowledge about simplified model systems because of the difficulty in studying the complex intestinal ecosystem in vivo. In this study, we introduce an approach that overcomes previous technical limitations and allows us to observe which microbial cells in the intestine use host-derived compounds. We added stable isotope-labeled threonine i.v. to mice and combined fluorescence in situ hybridization with high-resolution secondary ion mass spectrometry imaging to characterize utilization of host proteins by individual bacterial cells. We show that two bacterial species, Bacteroides acidifaciens and Akkermansia muciniphila, are important host-protein foragers in vivo. Using gnotobiotic mice we show that microbiota composition determines the magnitude and pattern of foraging by these organisms, demonstrating that a complex microbiota is necessary in order for this niche to be fully exploited. These results underscore the importance of in vivo studies of intestinal microbiota, and the approach presented in this study will be a powerful tool to address many other key questions in animal and human microbiome research.

Type I interferons have opposing effects during the emergence and recovery phases of colitis.

The contribution of the innate immune system to inflammatory bowel disease (IBD) is under intensive investigation. Research in animal models has demonstrated that type I interferons (IFN-Is) protect from IBD. In contrast, studies of patients with IBD have produced conflicting results concerning the therapeutic potential of IFN-Is. Here, we present data suggesting that IFN-Is play dual roles as regulators of intestinal inflammation in dextran sodium sulfate (DSS)-treated C57BL/6 mice. Though IFN-Is reduced acute intestinal damage and the abundance of colitis-associated intestinal bacteria caused by treatment with a high dose of DSS, they also inhibited the resolution of inflammation after DSS treatment. IFN-Is played an anti-inflammatory role by suppressing the release of IL-1ß from the colon MHC class II(+) cells. Consistently, IL-1 receptor blockade reduced the severity of inflammation in IFN-I receptor-deficient mice and myeloid cell-restricted ablation of the IFN-I receptor was detrimental. The proinflammatory role of IFN-Is during recovery from DSS treatment was caused by IFN-I-dependent cell apoptosis as well as an increase in chemokine production and infiltrating inflammatory monocytes and neutrophils. Thus, IFN-Is play opposing roles in specific phases of intestinal injury and inflammation, which may be important for guiding treatment strategies in patients.

Spatiotemporal proteomic profiling of cellular responses to NLRP3 agonists.

Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 3 (NLRP3) is an innate immune sensor that forms an inflammasome in response to various cellular stressors. Gain-of-function mutations in NLRP3 cause autoinflammatory diseases and NLRP3 signalling itself exacerbates the pathogenesis of many other human diseases. Despite considerable therapeutic interest, the primary drivers of NLRP3 activation remain controversial due to the diverse array of signals that are integrated through NLRP3. Here, we mapped subcellular proteome changes to lysosomes, mitochondrion, EEA1-positive endosomes, and Golgi caused by the NLRP3 inflammasome agonists nigericin and CL097. We identified several common disruptions to retrograde trafficking pathways, including COPI and Shiga toxin-related transport, in line with recent studies. We further characterized mouse NLRP3 trafficking throughout its activation using temporal proximity proteomics, which supports a recent model of NLRP3 recruitment to endosomes during inflammasome activation. Collectively, these findings provide additional granularity to our understanding of the molecular events driving NLRP3 activation and serve as a valuable resource for cell biological research. We have made our proteomics data accessible through an open-access Shiny browser to facilitate future research within the community, available at: https://harperlab.connect.hms.harvard.edu/inflame/. We will display anonymous peer review for this manuscript on pubpub.org (https://harperlab.pubpub.org/pub/nlrp3/) rather than a traditional journal. Moreover, we invite community feedback on the pubpub version of this manuscript, and we will address criticisms accordingly.

Interferon regulatory factor 6 (IRF6) determines intestinal epithelial cell development and immunity.

Intestinal epithelial cell (IEC) responses to interferon (IFN) favor antiviral defense with minimal cytotoxicity, but IEC-specific factors that regulate these responses remain poorly understood. Interferon regulatory factors (IRFs) are a family of nine related transcription factors, and IRF6 is preferentially expressed by epithelial cells, but its roles in IEC immunity are unknown. In this study, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) screens found that Irf6 deficiency enhanced IFN-stimulated antiviral responses in transformed mouse IECs but not macrophages. Furthermore, knockout (KO) of Irf6 in IEC organoids resulted in profound changes to homeostasis and immunity gene expression. Irf6 KO organoids grew more slowly, and single-cell ribonucleic acid sequencing indicated reduced expression of genes in epithelial differentiation and immunity pathways. IFN-stimulated gene expression was also significantly different in Irf6 KO organoids, with increased expression of stress and apoptosis-associated genes. Functionally, the transcriptional changes in Irf6 KO organoids were associated with increased cytotoxicity upon IFN treatment or inflammasome activation. These data indicate a previously unappreciated role for IRF6 in IEC biology, including regulation of epithelial development and moderation of innate immune responses to minimize cytotoxicity and maintain barrier function.

Galanin is a modulator of eccrine sweat gland secretion.

The neuropeptide galanin has been ascribed different roles in modulating physiological functions in the skin. The present study examined the function of galanin in eccrine sweat gland physiology. We demonstrated secretion of galanin by sweat glands in vivo by radioimmunoassay of human sweat (20-192 fmol galanin/ml). Furthermore, human sweat glands expressed galanin receptors GalR2 and GalR3. Using chamber short-circuit current (Isc) measurements showed that application of galanin to human NCL-SG3 cells led to a significant increase in Isc, which was inhibited by the presence of chloride channel blockers and in chloride-free Krebs solution. Additionally, application of SNAP 37889, a non-peptidergic selective antagonist of GalR3, abolished the effect of galanin on Isc. In summary, our results show that galanin can regulate transepithelial chloride ion transport and fluid secretion by stimulating GalR3 in NCL-SG3 cells and demonstrate a possible important extraneural function of galanin in sweat gland physiology.

Inducing Pyroptosis with FlaTox, RodTox, or NeedleTox.

Targeted activation of the NAIP-NLRC4 inflammasome has proven very useful in the study of pyroptosis. FlaTox and derivative LFn-NAIP-ligand cytosolic delivery systems offer a unique opportunity to interrogate both ligand recognition and downstream effects of the NAIP-NLRC4 inflammasome pathway. Here we describe how to stimulate the NAIP-NLRC4 inflammasome in vitro and in vivo. We describe experimental setup and specific considerations for treatment of macrophages in vitro and in vivo injections using a murine model of systemic inflammasome activation. The in vitro readouts of inflammasome activation propidium iodide uptake and lactate dehydrogenase (LDH) release as well as the in vivo readouts of hematocrit and body temperature measurement are described.

Epithelial Pyroptosis in Host Defense.

Pyroptosis is a lytic form of cell death that is executed by a family of pore-forming proteins called gasdermins (GSDMs). GSDMs are activated upon proteolysis by host proteases including the proinflammatory caspases downstream of inflammasome activation. In myeloid cells, GSDM pore formation serves two primary functions in host defense: the selective release of processed cytokines to initiate inflammatory responses, and cell death, which eliminates a replicative niche of the pathogen. Barrier epithelia also undergo pyroptosis. However, unique mechanisms are required for the removal of pyroptotic epithelial cells to maintain epithelial barrier integrity. In the following review, we discuss the role of epithelial inflammasomes and pyroptosis in host defense against pathogens. We use the well-established role of inflammasomes in intestinal epithelia to highlight principles of epithelial pyroptosis in host defense of barrier tissues, and discuss how these principles might be shared or distinctive across other epithelial sites.

Innate immune sensing by epithelial barriers.

Epithelial cells in barrier tissues perform a critical immune function by detecting, restricting, and often directly eliminating extrinsic pathogens. Membrane-bound and cytosolic pattern recognition receptors in epithelial cells bind to diverse ligands, detecting pathogen components and behaviors and stimulating cell-autonomous immunity. In addition to directly acting as first-responders to pathogens, epithelial cells detect commensal-derived and diet-derived products to promote homeostasis. Recent advances have clarified the array of molecular sensors expressed by epithelial cells, and how epithelial cells responses are wired to promote homeostatic balance while simultaneously allowing elimination of pathogens. These new studies emphatically position epithelial cells as central to an effective innate immune response.

Inflammasome activation leads to cDC1-independent cross-priming of CD8 T cells by epithelial cell-derived antigen.

The innate immune system detects pathogens and initiates adaptive immune responses. Inflammasomes are central components of the innate immune system, but whether inflammasomes provide sufficient signals to activate adaptive immunity is unclear. In intestinal epithelial cells (IECs), inflammasomes activate a lytic form of cell death called pyroptosis, leading to epithelial cell expulsion and the release of cytokines. Here, we employed a genetic system to show that simultaneous antigen expression and inflammasome activation specifically in IECs is sufficient to activate CD8+ T cells. By genetic elimination of direct T cell priming by IECs, we found that IEC-derived antigens were cross-presented to CD8+ T cells. However, cross-presentation of IEC-derived antigen to CD8+ T cells only partially depended on IEC pyroptosis. In the absence of inflammasome activation, cross-priming of CD8+ T cells required Batf3+ dendritic cells (conventional type one dendritic cells [cDC1]), whereas cross-priming in the presence of inflammasome activation required a Zbtb46+ but Batf3-independent cDC population. These data suggest the existence of parallel inflammasome-dependent and inflammasome-independent pathways for cross-presentation of IEC-derived antigens.

Lytic Cell Death in Specific Microglial Subsets Is Required for Preventing Atypical Behavior in Mice.

Microglial cells are known to contribute to brain development and behaviors, but the mechanisms behind such functions are not fully understood. Here, we show that mice deficient in inflammasome regulators, including caspase-1 (Casp1), NLR family pyrin domain containing 3 (Nlrp3), IL-1 receptor (Il-1r), and gasdermin D (Gsdmd), exhibit behavior abnormalities characterized by hyperactivity and low anxiety levels. Furthermore, we found that expression of Casp1 in CX3CR1+ myeloid cells, which includes microglia, is required for preventing these abnormal behaviors. Through tissue clearing and 3D imaging, we discovered that small numbers of Cx3cr1-GFP+ fetal microglial cells formed clusters and underwent lytic cell death in the primitive thalamus and striatum between embryonic day (E)12.5 and E14.5. This lytic cell death was diminished in Casp1-deficient mice. Further analysis of the microglial clusters showed the presence of Pax6+ neural progenitor cells (NPCs); thus, we hypothesized that microglial lytic cell death is important for proper neuronal development. Indeed, increased numbers of neurons were observed in the thalamic subset in adult Casp1-/- brains. Finally, injection of drug inhibitors of NLRP3 and CASP1 into wild-type (WT) pregnant mice from E12.5 to E14.5, the period when lytic cell death was detected, was sufficient to induce atypical behaviors in offspring. Taken together, our data suggests that the inflammasome cascade in microglia is important for regulating neuronal development and normal behaviors, and that genetic or pharmacological inhibition of this pathway can induce atypical behaviors in mice.

PGD2 and CRTH2 counteract Type 2 cytokine-elicited intestinal epithelial responses during helminth infection.

Type 2 inflammation is associated with epithelial cell responses, including goblet cell hyperplasia, that promote worm expulsion during intestinal helminth infection. How these epithelial responses are regulated remains incompletely understood. Here, we show that mice deficient in the prostaglandin D2 (PGD2) receptor CRTH2 and mice with CRTH2 deficiency only in nonhematopoietic cells exhibited enhanced worm clearance and intestinal goblet cell hyperplasia following infection with the helminth Nippostrongylus brasiliensis. Small intestinal stem, goblet, and tuft cells expressed CRTH2. CRTH2-deficient small intestinal organoids showed enhanced budding and terminal differentiation to the goblet cell lineage. During helminth infection or in organoids, PGD2 and CRTH2 down-regulated intestinal epithelial Il13ra1 expression and reversed Type 2 cytokine-mediated suppression of epithelial cell proliferation and promotion of goblet cell accumulation. These data show that the PGD2-CRTH2 pathway negatively regulates the Type 2 cytokine-driven epithelial program, revealing a mechanism that can temper the highly inflammatory effects of the anti-helminth response.

The NAIP/NLRC4 inflammasome in infection and pathology.

In this review we give an overview of the NAIP/NLRC4 activation mechanism as well as the described roles of this inflammasome, with a focus on in vivo infection and pathology. After ligand recognition by NAIP sensor proteins the NAIP/NLRC4 inflammasome forms through oligomerization with the NLRC4 adaptor to activate Caspase-1. The activating ligands are intracellular bacterial flagellin or type-3 secretion system components, delivered by pathogens. In vivo experiments indicate a role in macrophages during lung, spleen and liver infection and systemic sepsis like conditions, as well as in intestinal epithelial cells. Upon NAIP/NLRC4 activation in the intestine, epithelial cell extrusion is triggered in addition to the canonical inflammasome outcomes of cytokine cleavage and pyroptosis. Human patients with auto-activating mutations in NLRC4 present with an autoinflammatory syndrome including enterocolitis. Although one of the better understood inflammasomes in terms of mechanism, tissue specific functions of NAIP/NLRC4 are only beginning to be understood.

NLRC4 inflammasome activation is NLRP3- and phosphorylation-independent during infection and does not protect from melanoma.

The NAIP/NLRC4 inflammasome is a cytosolic sensor of bacteria that activates caspase-1 and initiates potent immune responses. Structural, biochemical, and genetic data demonstrate that NAIP proteins are receptors for bacterial ligands, while NLRC4 is a downstream adaptor that multimerizes with NAIPs to form an inflammasome. NLRC4 has also been proposed to suppress tumor growth, though the underlying mechanism is unknown. Further, NLRC4 is phosphorylated on serine 533, which was suggested to be critical for its function. In the absence of S533 phosphorylation, it was proposed that another inflammasome protein, NLRP3, can induce NLRC4 activation. We generated a new Nlrc4-deficient mouse line and mice with S533D phosphomimetic or S533A nonphosphorylatable NLRC4. Using these models in vivo and in vitro, we fail to observe a requirement for phosphorylation in NLRC4 inflammasome function. Furthermore, we find no role for NLRP3 in NLRC4 function, or for NLRC4 in a model of melanoma. These results clarify our understanding of the mechanism and biological functions of NAIP/NLRC4 activation.