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A hallmark ofEntamoeba histolytica(Eh) invasion in the gut is acute inflammation dominated by the secretion of pro-inflammatory cytokines TNF-α and IL-1β. This is initiated whenEhin contact with macrophages in the lamina propria activates caspase-1 by recruiting the NLRP3 inflammasome complex in a Gal-lectin andEhCP-A5-dependent manner resulting in the maturation and secretion of IL-1β and IL-18. Here, we interrogated the requirements and mechanisms forEh-induced caspase-4/1 activation in the cleavage of gasdermin D (GSDMD) to regulate bioactive IL-1β release in the absence of cell death in human macrophages. Unlike caspase-1, caspase-4 activation occurred as early as 10 min that was dependent onEhGal-lectin andEhCP-A5 binding to macrophages. By utilizing CRISPR-Cas9 gene editedCASP4/1,NLRP3 KOand ASC-def cells, caspase-4 activation was found to be independent of the canonical NLRP3 inflammasomes. In CRISPR-Cas9 gene editedCASP1macrophages, caspase-4 activation was significantly up regulated that enhanced the enzymatic cleavage of GSDMD at the same cleavage site as caspase-1 to induce GSDMD pore formation and sustained bioactive IL-1β secretion.Eh-induced IL-1β secretion was independent of pyroptosis as revealed by pharmacological blockade of GSDMD pore formation and in CRISPR-Cas9 gene editedGSDMD KOmacrophages. This was in marked contrast to the potent positive control, lipopolysaccharide + Nigericin that induced high expression of predominantly caspase-1 that efficiently cleaved GSDMD with high IL-1β secretion/release associated with massive cell pyroptosis. These results reveal thatEhtriggered “hyperactivated macrophages” allowed caspase-4 dependent cleavage of GSDMD and IL-1β secretion to occur in the absence of pyroptosis that may play an important role in disease pathogenesis.

The NLRP3 inflammasome has emerged as a central immune regulator that senses virulence factors expressed by microbial pathogens for triggering inflammation. Inflammation can be harmful and therefore this response must be tightly controlled. The mechanisms by which immune cells, such as macrophages, discriminate benign from pathogenic microbes to control the NLRP3 inflammasome remain poorly defined. Here we used live cell imaging coupled with a compendium of diverse clinical isolates to define how macrophages respond and activate NLRP3 when faced with the human yeast commensal and pathogen Candida albicans. We show that metabolic competition by C. albicans, rather than virulence traits such as hyphal formation, activates NLRP3 in macrophages. Inflammasome activation is triggered by glucose starvation in macrophages, which occurs when fungal load increases sufficiently to outcompete macrophages for glucose. Consistently, reducing Candida’s ability to compete for glucose and increasing glucose availability for macrophages tames inflammatory responses. We define the mechanistic requirements for glucose starvation-dependent inflammasome activation by Candida and show that it leads to inflammatory cytokine production, but it does not trigger pyroptotic macrophage death. Pyroptosis occurs only with some Candida isolates and only under specific experimental conditions, whereas inflammasome activation by glucose starvation is broadly relevant. In conclusion, macrophages use their metabolic status, specifically glucose metabolism, to sense fungal metabolic activity and activate NLRP3 when microbial load increases. Therefore, a major consequence of Candida-induced glucose starvation in macrophages is activation of inflammatory responses, with implications for understanding how metabolism modulates inflammation in fungal infections., Author summary Activation of the immune regulator NLRP3 inflammasome by microbial pathogens has been shown to play both protective and destructive roles in infection, underscoring the importance of tight control over NLRP3-driven inflammation to ensure host health. A key microbe recognised by NLRP3 is the human yeast commensal and pathogen Candida albicans, which is responsible for mucosal and invasive infections. We demonstrate that innate immune cells sense their metabolic status to trigger NLRP3 activation only when microbial numbers have reached dangerous levels. This regulation is a consequence of metabolic competition between C. albicans and macrophages for an essential nutrient–glucose. The NLRP3 inflammasome is activated when increased fungal load in the infection microenvironment drives down glucose levels, thereby causing glucose starvation in macrophages. Restoring glucose homeostasis in macrophages reduced NLRP3 activation and production of the proinflammatory cytokine IL-1β, suggesting that metabolism regulates NLRP3 inflammasome activity in fungal infections.

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., Author summary Intestinal epithelial cells (IECs) are located at the interface between the gut lumen and the mucosal immune system and form the first layer of defense against the invasive enteric pathogen Salmonella enterica serovar Typhimurium. To prevent Salmonella, and other pathogens from establishing a foothold in the gut, the host mobilizes the inflammasome to selectively eject infected/compromised IECs from the epithelial layer into the intestinal lumen. This involves the activation of the inflammatory caspases; caspase-1 and -11. The individual contributions of each caspase to intestinal host defense, as well as the importance of IEC-intrinsic inflammasomes have not been previously defined, due to the lack of Casp1−/− mice as well as appropriate IEC-intrinsic defense models. Here, we determined that both caspases contribute to controlling Salmonella pathogen burdens and IEC shedding in the mouse intestine. Caspase-1 appears to play a larger role at baseline since caspase-11 expression must be first induced through proinflammatory signalling. Our data also highlights that IEC-intrinsic caspase activation is sufficient for infection-induced cell shedding and that the intestinal epithelium is a key site for inflammasome-mediated immune defense.

The granulomatous lesion resulting from infection with the fungus Paracoccidioides brasiliensis is characterized by a compact aggregate of mature cells, surrounded by a fibroblast- and collagen-rich content. Granuloma formation requires signaling elicited by inflammatory molecules such as members of the interleukin-1 family. Two members of this family have been thoroughly studied, namely IL-1α and IL-1β. In this study, we addressed the mechanisms underlying IL-1α secretion and its functional role on the host resistance to fungal infection. We found that, the expression of caspase-11 triggered by P. brasiliensis infection of macrophages depends on IFN-β production, because its inhibition reduced procaspase-11 levels. Curiously, caspase-11 deficiency did not impair IL-1β production, however caspase-11 was required for a rapid pore-mediated cell lysis. The plasma membrane rupture facilitated the release of IL-1α, which was necessary to induce NO production and restrict fungal replication. Furthermore, P. brasiliensis-infected macrophages required IL-1α to produce optimal levels of IL-6, a major component of Th17 lymphocyte differentiation. Indeed, IL-1α deficiency accounted for a significant reduction of Th17 lymphocytes in lungs of infected mice, correlating with diminished neutrophil infiltration in the lungs. Strikingly, we identified that IL-1α directly reprograms the transcriptional profile of Th17-committed lymphocytes, increasing cellular proliferation, as for boosting IL-17 production by these cells. Beyond neutrophil chemotaxis in vivo, IL-17 also amplified IL-1α production by infected macrophages in vitro, endorsing a critical amplification loop of the inflammatory response. Therefore, our data suggest that the IFN-β/caspase-11/IL-1α pathway shapes a protective antifungal Th17 immunity, revealing a molecular mechanism underlying the cross-talk between innate and adaptive immunity., Author summary After infectious insults, the release of intracellular molecules from dying cells, such as IL-1α, provokes a local inflammation as an alert of tissue damage. In this study, we investigated the steps necessary for IL-1α-mediated inflammation and its implications after fungal infection. We identified a molecular mechanism in which IL-1α plays a pivotal role on the control of Paracoccidioides brasiliensis infection. We observed that after fungal recognition, macrophages produce IFN-β, a cytokine that promotes the expression of procaspase-11. This enzyme is then activated to trigger a rapid pore-mediated cell lysis, leading to the passive leakage of the cytosolic IL-1α. Once extracellularly, IL-1α functions via paracrine signaling on surrounding cells to enhance the inflammatory response against the pathogen. IL-1α coordinates nitric oxide and IL-6 production by macrophages upon infection, but it also acts directly on CD4+IL-17+ T lymphocytes by reprograming their transcriptional profile and potentiating IL-17 production. While NO has intrinsic fungicidal properties and IL-6 drives Th17 cell differentiation, IL-17 recruits neutrophils into lungs of infected mice. Furthermore, macrophages synthesize more IL-1α in response to an IL-17-rich milieu. Therefore, IL-1α initiates a sustained and self-perpetuating inflammatory loop that is required for host resistance to P. brasiliensis infection.

Innate recognition of invading intracellular pathogens is essential for regulating robust and rapid CD4+ T cell effector function, which is critical for host-mediated immunity. The intracellular apicomplexan parasite, Toxoplasma gondii, is capable of infecting almost any nucleated cell of warm-blooded animals, including humans, and establishing tissue cysts that persist throughout the lifetime of the host. Recognition of T. gondii by TLRs is essential for robust IL-12 and IFN-γ production, two major cytokines involved in host resistance to the parasite. In the murine model of infection, robust IL-12 and IFN-γ production have been largely attributed to T. gondii profilin recognition by the TLR11 and TLR12 heterodimer complex, resulting in Myd88-dependent IL-12 production. However, TLR11 or TLR12 deficiency failed to recapitulate the acute susceptibility to T. gondii infection seen in Myd88-/- mice. T. gondii triggers inflammasome activation in a caspase-1-dependent manner resulting in cytokine release; however, it remains undetermined if parasite-mediated inflammasome activation impacts IFN-γ production and host resistance to the parasite. Using mice which lack different inflammasome components, we observed that the inflammasome played a limited role in host resistance when TLR11 remained functional. Strikingly, in the absence of TLR11, caspase-1 and -11 played a significant role for robust CD4+ TH1-derived IFN-γ responses and host survival. Moreover, we demonstrated that in the absence of TLR11, production of the caspase-1-dependent cytokine IL-18 was sufficient and necessary for CD4+ T cell-derived IFN-γ responses. Mechanistically, we established that T. gondii-mediated activation of the inflammasome and IL-18 were critical to maintain robust CD4+ TH1 IFN-γ responses during parasite infection in the absence of TLR11., Author summary It is currently estimated that one third of the world’s population is seropositive for the parasite Toxoplasma gondii and this parasite can lead to serious illness and death in immunocompromised patients, and is one of the leading causes of foodborne-related deaths in the United States. Host immunity against the parasite has largely been attributed to recognition of the parasite-derived protein, profilin, by the innate Toll-like receptors (TLRs), TLR11 and TLR12. T. gondii also triggers inflammasome activation in a caspase-1-dependent manner resulting in cytokine release. However, how these innate recognition systems regulate TH1 immunity and host resistance remains largely unknown. Therefore, using genetically modified mice, we investigated TLR11-dependent and -independent host immunity against the parasite. Our research establishes that in the absence of TLR11, inflammasome activation and subsequent production of the inflammasome-dependent molecule, IL-18 are critical for host immunity to the parasite. These data provide novel mechanistic insight into how TLR and inflammasomes cooperate in regulation of TH1 immunity and host protection.

Infection with Burkholderia pseudomallei or B. thailandensis triggers activation of the NLRP3 and NLRC4 inflammasomes leading to release of IL-1β and IL-18 and death of infected macrophages by pyroptosis, respectively. The non-canonical inflammasome composed of caspase-11 is also activated by these bacteria and provides protection through induction of pyroptosis. The recent generation of bona fide caspase-1-deficient mice allowed us to reexamine in a mouse model of pneumonic melioidosis the role of caspase-1 independently of caspase-11 (that was also absent in previously generated Casp1-/- mice). Mice lacking either caspase-1 or caspase-11 were significantly more susceptible than wild type mice to intranasal infection with B. thailandensis. Absence of caspase-1 completely abolished production of IL-1β and IL-18 as well as pyroptosis of infected macrophages. In contrast, in mice lacking caspase-11 IL-1β and IL-18 were produced at normal level and macrophages pyroptosis was only marginally affected. Adoptive transfer of bone marrow indicated that caspase-11 exerted its protective action both in myeloid cells and in radio-resistant cell types. B. thailandensis was shown to readily infect mouse lung epithelial cells triggering pyroptosis in a caspase-11-dependent way in vitro and in vivo. Importantly, we show that lung epithelial cells do not express inflammasomes components or caspase-1 suggesting that this cell type relies exclusively on caspase-11 for undergoing cell death in response to bacterial infection. Finally, we show that IL-18’s protective action in melioidosis was completely dependent on its ability to induce IFNγ production. In turn, protection conferred by IFNγ against melioidosis was dependent on generation of ROS through the NADPH oxidase but independent of induction of caspase-11. Altogether, our results identify two non-redundant protective roles for caspase-1 and caspase-11 in melioidosis: Caspase-1 primarily controls pyroptosis of infected macrophages and production of IL-18. In contrast, caspase-11 mediates pyroptosis of infected lung epithelial cells., Author summary Burkholderia pseudomallei is a bacterium that infect macrophages and other cell types and causes a diseases called melioidosis. Inflammasomes are multiprotein complexes that control activation of the proteases caspase-1 and caspase-11 resulting in production of the inflammatory mediators IL-1β and IL-18 and death of infected cells. Mice deficient of caspase-1 or caspase-11 are more susceptible to infection with B. pseudomallei or the closely related B. thailandensis. Here we show that absence of caspase-1 completely abolished production of IL-1β and IL-18 as well as death of macrophages infected with B. thailandensis. In contrast, in the highly susceptible caspase-11-deficient mice, IL-1β and IL-18 production and macrophages death were not significantly affected. Rather, absence of caspase-11 abolished death of infected lung epithelial cells. Taken together, our results show that caspase-1 and caspase-11 have non-redundant protective roles in melioidosis: Caspase-1 primarily controls cell death of infected macrophages and production of IL-18. In contrast, caspase-11 mediates cell death of infected lung epithelial cells.

Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains undiscovered. Here, we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cell lines. Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion and gut inflammation in mice, which was associated with restricting bacterial colonization in vivo. Together, our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses.

Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in inflammatory responses. The inflammasome activates caspase-1 to process and secrete IL-1β. However, the mechanisms governing IL-1α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1α and IL-1β. Caspase-11 activation in response to Gram-negative bacteria requires Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon production. Whether additional bacterial signals trigger caspase-11 activation is unknown. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, even in the absence of flagellin, these secretion systems induce inflammasome activation and the release of IL-1α and IL-1β, but the inflammasome pathways that mediate this response are unclear. We observe rapid IL-1α and IL-1β release and cell death in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis. Unlike IL-1β, IL-1α secretion does not require caspase-1. Instead, caspase-11 activation is required for both IL-1α secretion and cell death in response to the activity of these secretion systems. Interestingly, whereas caspase-11 promotes IL-1β release in response to the type IV secretion system through the NLRP3/ASC inflammasome, caspase-11-dependent release of IL-1α is independent of both the NAIP5/NLRC4 and NLRP3/ASC inflammasomes as well as TRIF and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila. Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response important for host defense., Author Summary The inflammasome, a multiprotein complex, is critical for host defense against bacterial infection. The inflammasome activates the host protease caspase-1 to process and secrete IL-1β. Another caspase, caspase-11, can cause cell death and IL-1α release. The bacterial signals that trigger caspase-11 activation are poorly understood. A common feature of many bacterial pathogens is the ability to inject virulence factors and other bacterial molecules into the host cell cytosol by means of a variety of virulence-associated secretion systems. These secretion systems can introduce bacterial flagellin into the host cytosol, which leads to caspase-1 activation and cell death. However, many bacteria lack or down-regulate flagellin yet still activate the inflammasome. Here, we show that the type IV secretion system of Legionella pneumophila and the type III secretion system of Yersinia pseudotuberculosis rapidly trigger caspase-11 activation in a flagellin-independent manner. Caspase-11 activation mediates two separate inflammasome pathways: one leading to IL-1β processing and secretion, and one leading to cell death and IL-1α release. Furthermore, we find these caspase-11-regulated cytokines are critical for neutrophil recruitment to the site of infection and clearance of non-flagellated Legionella in vivo. Overall, our findings show that virulent bacteria activate a rapid caspase-11-dependent immune response that plays a critical role in host defense.