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Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors1. Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2V617F (JAK2VF) mutation, which increases JAK–STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease1,2. Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2VF selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1β reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1β or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk. Accelerated atherosclerosis in a mouse model of clonal haematopoiesis is prevented by genetic interruption of AIM2 inflammasome activation or by inhibition of interleukin-1β.

Dendrobine has been reported to reduce blood lipid levels and apoptosis. The present study was designed to observe the effect of dendrobine in a model of ERS using vascular endothelial cells and to reveal the biological mechanisms and pathways responsible for the therapeutic effects of dendrobine on AS. Human umbilical vein endothelial cells (HUVECs) were pre-treated with various concentrations of dendrobine, followed by treatment with tunicamycin (TM) for the establishment of the cell models of ERS. The proliferation and apoptosis of HUVECs were detected by bromodeoxyuridine staining and flow cytometry, respectively. The target binding association was verified through dual luciferase reporter assay. It was found that TM treatment resulted in a low expression of miR-381-3p. Dendrobine treatment not only promoted the proliferation, but also inhibited the apoptosis of HUVECs induced by TM. The reduced expression of 78-kDa glucose-regulated protein, inositol-requiring enzyme 1, caspase-4, C/EBP homologous protein and caspase-3 was also observed following treatment with dendrobine. Dendrobine reduced the apoptosis of endothelial cells in the model of ERS by increasing miR-381-3p expression, and partially restored the cell proliferation level. This effect was significantly reduced after the expression of miR-381-3p was blocked. On the whole, the present study demonstrated that dendrobine upregulated miR-381-3p expression to inhibit apoptosis induced by ERS in HUVECs and this process was found to be mediated by caspase-4. The findings of the present study may provide new insight into the causes of endothelial cell apoptosis during AS and reveal the potent therapeutic effects of dendrobine in AS., GRAPHICAL ABSTRACT

There is the notion that infection with a virulent intestinal pathogen induces generally stronger mucosal adaptive immunity than the exposure to an avirulent strain. Whether the associated mucosal inflammation is important or redundant for effective induction of immunity is, however, still unclear. Here we use a model of auxotrophic Salmonella infection in germ-free mice to show that live bacterial virulence factor-driven immunogenicity can be uncoupled from inflammatory pathogenicity. Although live auxotrophic Salmonella no longer causes inflammation, its mucosal virulence factors remain the main drivers of protective mucosal immunity; virulence factor-deficient, like killed, bacteria show reduced efficacy. Assessing the involvement of innate pathogen sensing mechanisms, we show MYD88/TRIF, Caspase-1/Caspase-11 inflammasome, and NOD1/NOD2 nodosome signaling to be individually redundant. In colonized animals we show that microbiota metabolite cross-feeding may recover intestinal luminal colonization but not pathogenicity. Consequent immunoglobulin A immunity and microbial niche competition synergistically protect against Salmonella wild-type infection., Nature Communications, 11 (1), ISSN:2041-1723

Background Myocardial reperfusion injury is often accompanied by cell death and inflammatory reactions. Recently, pyroptosis is gradually recognized as pivotal role in cardiovascular disease. However, little is known about the regulatory role of beclin1 in the control of caspase-4 activation and pyroptosis. The present study confirmed whether beclin1 regulates caspase-4 mediated pyroptosis and thereby protects Human Cardiac microvascular endothelial cells (HCMECs) against injury. Methods TTC and Evan's blue dye, western blot, immunofluorescence and immunohistochemistry staining were performed in wild mice and transgenic mice with overexpression of beclin 1(BECN1-Tg). CMECs were transfected with a beclin1 lentivirus. The cell cytotoxicity was analyzed by LDH-Cytotoxicity Assay Kit. The protein levels of autophagy protein (Beclin1, p62 and LC3II/LC3I) and caspase-4/GSDMD pathway were determined by western blot. Autophagic vacuoles in cells were monitored with RFP-GFP-LC3 using fluorescence microscope. Results I/R caused caspase-4 activity and gasdermin D expression increase in vivo and in vitro. Overexpression of beclin1 in heart tissue and CMECs suppressed the caspase-4 activity and decreased the levels of gasdermin D; meanwhile beclin1 overexpression also reduced IL-1β levels, promoted autophagy (p62 expression was inhibited while LC3II expression was increased) in the heart and CMECs. Interestingly, beclin1 overexpression increased animal survival and attenuated myocardial infarct size (45 ± 6.13 vs 22 ± 4.37), no-reflow area (39 ± 5.22 vs 16 ± 2.54) post-myocardial ischemia reperfusion. Conclusions Induction of beclin-1 signaling can be a potential therapeutic target in myocardial reperfusion-induced microvascular injury.

Innate immune signaling pathways comprise multiple proteins that promote inflammation. This multistep means of information transfer suggests that complexity is a prerequisite for pathway design. Here, we test this hypothesis by studying caspases that regulate inflammasome-dependent inflammation. Several caspases differ in their ability to recognize bacterial lipopolysaccharide (LPS) and cleave interleukin-1β (IL-1β). No caspase is known to contain both activities, yet distinct caspases with complementary activities bookend an LPS-induced pathway to IL-1β cleavage. Using caspase-1/4 hybrid proteins present in canines as a guide, we identified molecular determinants of IL-1β cleavage specificity within human and murine caspase-1. This knowledge enabled the redesign of human caspase-4 to operate as a one-protein signaling pathway, which intrinsically links LPS detection to IL-1β cleavage and release, independent of inflammasomes. We identified caspase-4 homologs in multiple carnivorans that display the activities of redesigned human caspase-4. These findings illustrate natural signaling pathway diversity and highlight how multistep innate immune pathways can be condensed into a single protein.

Streptococcus pneumoniae (S. pneumoniae) causes severe pulmonary diseases, leading to high morbidity and mortality. It has been reported that inflammasomes such as NLR family pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2) play an important role in the host defense against S. pneumoniae infection. However, the role of NLRP6 in vivo and in vitro against S. pneumoniae remains unclear. Therefore, we investigated the role of NLRP6 in regulating the S. pneumoniae-induced inflammatory signaling pathway in vitro and the role of NLRP6 in the host defense against S. pneumoniae in vivo by using NLRP6−/− mice. The results showed that the NLRP6 inflammasome regulated the maturation and secretion of IL-1β, but it did not affect the induction of IL-1β transcription in S. pneumoniae-infected macrophages. Furthermore, the activation of caspase-1, caspase-11, and gasdermin D (GSDMD) as well as the oligomerization of apoptosis-associated speck-like protein (ASC) were also mediated by NLRP6 in S. pneumoniae-infected macrophages. However, the activation of NLRP6 reduced the expression of NF-κB and ERK signaling pathways in S. pneumoniae-infected macrophages. In vivo study showed that NLRP6−/− mice had a higher survival rate, lower number of bacteria, and milder inflammatory response in the lung compared with wild-type (WT) mice during S. pneumoniae infection, indicating that NLRP6 plays a negative role in the host defense against S. pneumoniae. Furthermore, increased bacterial clearance in NLRP6 deficient mice was modulated by the recruitment of macrophages and neutrophils. Our study provides a new insight on S. pneumoniae-induced activation of NLRP6 and suggests that blocking NLRP6 could be considered as a potential therapeutic strategy to treat S. pneumoniae infection.

P2X7 receptor activation induces the release of different cellular proteins, such as CD14, a glycosylphosphatidylinositol (GPI)-anchored protein to the plasma membrane important for LPS signaling via TLR4. Circulating CD14 has been found at elevated levels in sepsis, but the exact mechanism of CD14 release in sepsis has not been established. Here, we show for first time that P2X7 receptor induces the release of CD14 in extracellular vesicles, resulting in a net reduction in macrophage plasma membrane CD14 that functionally affects LPS, but not monophosphoryl lipid A, pro-inflammatory cytokine production. Also, we found that during a murine model of sepsis, P2X7 receptor activity is important for maintaining elevated levels of CD14 in biological fluids and a decrease in its activity results in higher bacterial load and exacerbated organ damage, ultimately leading to premature deaths. Our data reveal that P2X7 is a key receptor for helping to clear sepsis because it maintains elevated concentrations of circulating CD14 during infection., eLife digest When the immune system detects an infection, it often launches an inflammatory response to fight off the disease. This defense mechanism is activated by a cascade of signaling molecules that can aggravate inflammation, causing it to damage the body’s own tissues and organs. This life-threatening reaction is referred to as sepsis, and kills around 11 million people each year. New approaches are therefore needed to help alleviate the damage caused by this condition. The inflammatory response is often triggered by proteins called receptors, which sit on the surface of immune cells. When these receptors are activated, they induce cells to secrete proteins that travel around the body and activate immune cells that can eliminate the infection. In 2016, a group of researchers showed that a receptor called P2X7 stimulates the release of a signaling molecule called CD14. Patients with sepsis often have elevated amounts of CD14 in their bloodstream. Yet, it remained unclear what causes this rise in CD14 and what role this molecule plays in the development of sepsis. Now, Alarcón-Vila et al. – including some of the researchers involved in the 2016 study – have investigated the role of P2X7 in mice undergoing sepsis. This was done by puncturing the mice’s intestines, causing bacteria to leak out and initiate an over-active immune response. Alarcón-Vila et al. found that mice lacking the P2X7 receptor had less CD14 and struggled to eliminate the bacterial infection from their system. This increase in bacteria caused excessive damage to the mice’s organs, ultimately leading to premature death. These findings suggest that P2X7 plays an important role in preventing the onset of sepsis by helping maintain high levels of CD14 following infection. This result could help to identify new therapies that reduce the mortality rates of septic infections.

Sepsis is a life-threating multi-organ disease induced by host innate immunity to pathogen-derived endotoxins including lipopolysaccharide (LPS). Direct sensing of LPS by caspase-11 activates inflammasomes and causes lethal sepsis in mice. Inhibition of caspase-11 inflammasomes is important for the prevention of LPS-induced septic shock; however, whether a caspase-11 inflammasome-specific suppressive mechanism exists is unclear. Here we show that deficiency of GABARAP autophagy-related proteins results in over-activation of caspase-11 inflammasomes but not of canonical inflammasomes. Gate-16−/−Gabarap−/− macrophages exhibited elevated guanylate binding protein 2 (GBP2)-dependent caspase-11 activation and inflammatory responses. Deficiency of GABARAPs resulted in formation of GBP2-containing aggregates that promote IL-1β production. High mortality after low dose LPS challenge in Gate-16−/−Gabarap−/− mice primed with poly(I:C) or polymicrobial sepsis was ameliorated by compound GBP2 deficiency. These results reveal a critical function of Gate-16 and Gabarap to suppress GBP2-dependent caspase-11-induced inflammation and septic shock.

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.

Innate immune cells, epithelial cells, and many other cell types are capable of detecting infection or tissue injury, thus mounting regulated immune response. Inflammasomes are highly sophisticated and effective orchestrators of innate immunity. These oligomerized multiprotein complexes are at the center of various innate immune pathways, including modulation of the cytoskeleton, production and maturation of cytokines, and control of bacterial growth and cell death. Inflammasome assembly often results in caspase‐1 activation, which is an inflammatory caspase that is involved in pyroptotic cell death and release of inflammatory cytokines in response to pathogen patterns and endogenous danger stimuli. However, the nature of stimuli and inflammasome components are diverse. Caspase‐1 activation mediated release of mature IL‐1β and IL‐18 in response to canonical stimuli initiated by NOD‐like receptor (NLR), and apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC). On the other hand, caspase‐11 delineates a non‐canonical inflammasome that promotes pyroptotic cell death and non‐pyroptotic functions in response to non‐canonical stimuli. Caspase‐11 in mice and its homologues in humans (caspase‐4/5) belong to caspase‐1 family of cysteine proteases, and play a role in inflammation. Knockout mice provided new genetic tools to study inflammatory caspases and revealed the role of caspase‐11 in mediating septic shock in response to lethal doses of lipopolysaccharide (LPS). Recognition of LPS mediates caspase‐11 activation, which promotes a myriad of downstream effects that include pyroptotic and non‐pyroptotic effector functions. Therefore, the physiological functions of caspase‐11 are much broader than its previously established roles in apoptosis and cytokine maturation. Inflammation induced by exogenous or endogenous agents can be detrimental and, if excessive, can result in organ and tissue damage. Consequently, the existence of sophisticated mechanisms that tightly regulate the specificity and sensitivity of inflammasome pathways provides a fine‐tuning balance between adequate immune response and minimal tissue damage. In this review, we summarize effector functions of caspase‐11.

International audience; Cell death is ascribed as an essential biological process that is fundamental for the development of an organism along with its survival. The procedure comprises of apoptosis and pyroptosis. Pyroptosis is a programmed procedure for cell death which is inflammatory in nature and this pathway gets activated via human caspase-4, human caspase-11 and human caspase-5. The activation of this process leads to release of pro-inflammatory mediators including cytokines, alarmins, IL-18 and IL-1β. The pro-inflammatory mediators released via interaction of intracellular kinases direct the development of Rheumatoid arthritis. Rheumatoid arthritis is characterized as disorder/disease that is auto-immune and chronic in nature. It involves erosions in marginal bone along with articular cartilage which is responsible for joint destruction. The cytokine along with its complex network is responsible for inflammation. The process of pyroptosis is linked with the destruction of plasma membrane, that releases these mediators and excessive release of these mediators is linked with rheumatoid arthritis.

SUMMARY Either caspase-1 or caspase-11 can cleave gasdermin D to cause pyroptosis, eliminating intracellular replication niches. We previously showed that macrophages detect Burkholderia thailandensis via NLRC4, triggering the release of interleukin (IL)-18 and driving an essential interferon (IFN)-γ response that primes caspase-11. We now identify the IFN-γ-producing cells as a mixture of natural killer (NK) and T cells. Although both caspase-1 and caspase-11 can cleave gasdermin D in macrophages and neutrophils, we find that NLRC4-activated caspase-1 triggers pyroptosis in macrophages, but this pathway does not trigger pyroptosis in neutrophils. In contrast, caspase-11 triggers pyroptosis in both macrophages and neutrophils. This translates to an absolute requirement for caspase-11 in neutrophils during B. thailandensis infection in mice. We present an example of inflammasome sensors causing diverging outcomes in different cell types. Thus, cell fates are dictated not simply by the pathogen or inflammasome, but also by how the cell is wired to respond to detection events., Graphical Abstract, In Brief Kovacs et al. demonstrate that natural killer and T cells produce IFN-γ to prime caspase-11 during Burkholderia thailandensis infection. They demonstrate that in neutrophils, caspase-1 and caspase-11 activation lead to gasdermin D cleavage, but only caspase-11 activation leads to pyroptosis that is necessary for clearance of this cytosol-invasive pathogen in vivo.

The human non-canonical inflammasome controls caspase-4 activation and gasdermin-D-dependent pyroptosis in response to cytosolic bacterial lipopolysaccharide (LPS). Since LPS binds and oligomerizes caspase-4, the pathway is thought to proceed without dedicated LPS sensors or an activation platform. Here we report that interferon-induced guanylate-binding proteins (GBPs) are required for non-canonical inflammasome activation by cytosolic Salmonella or upon cytosolic delivery of LPS. GBP1 associates with the surface of cytosolic Salmonella seconds after bacterial escape from their vacuole, initiating the recruitment of GBP2-4 to assemble a GBP coat. The GBP coat then promotes the recruitment of caspase-4 to the bacterial surface and caspase activation, in absence of bacteriolysis. Mechanistically, GBP1 binds LPS with high affinity through electrostatic interactions. Our findings indicate that in human epithelial cells GBP1 acts as a cytosolic LPS sensor and assembles a platform for caspase-4 recruitment and activation at LPS-containing membranes as the first step of non-canonical inflammasome signaling., Detection of LPS derived from Gram-negative bacteria by innate immune receptors is a critical step in the host response. Here Santos and colleagues show human GBP1 binds to LPS resulting in non-canonical inflammasome activation.

Mesenchymal stem cells (MSCs) have been used in cell-based therapies for a variety of disorders. Some factors such as inflammatory mediators in the diseased area might damage the survival of MSCs and affect their efficacy. Pyroptosis is a form of programmed necrosis as a response for immune cells to cytosolic pathogenic stimuli. Whether MSCs develop pyroptosis under pathological stimulation, its underlying mechanism and biological significance are still unclear. Here, we found that LPS, flagellin, dsDNA, nigericin (NIG), or LPS combined with nigericin (LPS/NIG) could not induce pyroptosis in adipose-tissue-derived mesenchymal stem cells (ASCs). However, when applied the culture media collected from LPS/NIG-induced pyroptotic bone marrow-derived macrophages (BMDMs) to incubate ASCs, ASCs developed pyroptosis. Inhibition of caspases or deletion of Caspase-1/11 in ASCs did not affect the pyroptotic macrophage media-triggered ASC pyroptosis while ablation of Caspase-1/11 abolished BMDM pyroptosis induced by LPS/NIG. Media collected from LPS/NIG stimulated Gsdmd−/− or Caspase-1/11−/− BMDMs could not induce pyroptosis of ASCs. In addition, RNA-seq analysis showed that interferon (IFN)-stimulated genes were upregulated in pyroptotic ASCs. Adding IFNβ could boost LPS/NIG stimulated BMDM media-induced ASC pyroptosis. Surprisingly, the pyroptotic ASCs had a lower bactericidal ability to P. Aeruginosa. Taken together, induction of ASC pyroptosis requires gasdermin D or caspase-1/11-dependent mediators and IFNβ from pyroptotic macrophages.

OBJECTIVE: To study the protective effect of isoflurane preconditioning on hepatic ischemia-reperfusion (I/R) injury mediated by the noncanonical pyroptosis pathway. METHODS: Thirty C57BL/6 mice were randomly divided into sham-operated group, isoflurane group and I/R group, and in the latter two groups, hepatic I/R injury was induced by clamping the portal vein for 30 min. In isoflurane group, the mice were pretreated with 1.4% isoflurane 30 min before the surgery. The protective effect of isoflurane preconditioning against hepatic I/R injury was evaluated by assessing the pathological score of HE staining of the liver tissue and serum ALT and AST levels. Serum IL-1β and IL-18 levels and the protein expression of GSDMS were detected by ELISA and Western blotting to evaluate the inhibitory effect of isoflurane preconditioning on pyroptosis. Western blotting and immunofluroescence were used to detect the protein expression of caspase-11 in the liver tissues to evaluate the inhibitory effect of isoflurane preconditioning on noncanonical pyroptosis pathway. RESULTS: The Suzuki's score of the liver tissue was significantly higher in I/R group than in the sham group (P < 0.05), while the score in the isoflurane group was significantly lower than that in the I/R group (P < 0.05). Serum ALT and AST levels significantly increased in the sham group (P < 0.05), and were significantly lower in isoflurane group than in I/R group (P < 0.05). The serum levels of IL-1β and IL-18 were significantly higher in I/R group than in sham group (P < 0.05), and were significantly lower in isoflurane group than in I/R group (P < 0.05). The expression of GSDMD in the I/R group was significantly higher than that in sham group, and was significantly lower in isoflurane group than in I/R group (P < 0.05). The hepatic expression of caspase-11 was significantly higher in I/R group than in sham group (P < 0.05), and was significantly lower in isoflurane group than in I/R group (P < 0.05). CONCLUSION: Isoflurane preconditioning has protective effect against hepatic I/R injury, which is related to the inhibition of the noncanonical pyroptosis pathway.

Programmed cell death plays crucial roles in organismal development and host defense. Recent studies have highlighted mechanistic overlaps and extensive, multifaceted crosstalk between pyroptosis, apoptosis, and necroptosis, three programmed cell death pathways traditionally considered autonomous. The growing body of evidence, in conjunction with the identification of molecules controlling the concomitant activation of all three pathways by pathological triggers, has led to the development of the concept of PANoptosis. During PANoptosis, inflammatory cell death occurs through the collective activation of pyroptosis, apoptosis, and necroptosis, which can circumvent pathogen-mediated inhibition of individual death pathways. Many of the molecular details of this emerging pathway are unclear. Here, we describe the activation of PANoptosis by bacterial and viral triggers and report protein interactions that reveal the formation of a PANoptosome complex. Infection of macrophages with influenza A virus, vesicular stomatitis virus, Listeria monocytogenes, or Salmonella enterica serovar Typhimurium resulted in robust cell death and the hallmarks of PANoptosis activation. Combined deletion of the PANoptotic components caspase-1 (CASP1), CASP11, receptor-interacting serine/threonine-protein kinase 3 (RIPK3), and CASP8 largely protected macrophages from cell death induced by these pathogens, while deletion of individual components provided reduced or no protection. Further, molecules from the pyroptotic, apoptotic, and necroptotic cell death pathways interacted to form a single molecular complex that we have termed the PANoptosome. Overall, our study identifies pathogens capable of activating PANoptosis and the formation of a PANoptosome complex.

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 liver is the main organ responsible for bacterial and endotoxin clearance. Pyroptosis is a form of proinflammatory programmed cell death activated by caspase-1/11 and gasdermin D (GadD). Pyroptosis protects the host against bacterial infection, however, overactive pyroptosis can lead to organ injury. Glutamine (GLN) is a specific amino acid with anti-inflammatory and immunomodulatory properties. This study investigated the effects of GLN pretreatment on liver pyroptosis in a mouse model of polymicrobial sepsis. Mice were assigned to sham, sepsis control (Sepsis-C), and sepsis GLN (Sepsis-G) groups. The sham and Sepsis-C groups were fed the AIN-93G diet. The Sepsis-G group was provided with identical diet components except that part of the casein was replaced by GLN. After feeding the respective diets for 2 weeks, a cecal ligation and puncture (CLP) procedure was performed in the sepsis groups. An antibiotic was administered after CLP. Mice were sacrificed at either 24 or 72 h after CLP. The results showed that sepsis resulted in upregulated liver caspase-1/11 expression. Compared to the Sepsis-C group, the Sepsis-G group had higher liver caspase-11 and NLRP3 gene expressions at 24 h and lower active caspase-1/11 and cleaved GadD protein levels at 72 h after sepsis. Additionally, liver inflammatory cytokine gene expressions had decreased by 72 h post-CLP. The findings suggest that prophylactic administration of GLN initially upregulated liver pyroptosis to eradicate pathogens, yet the process of pyroptosis was suppressed in the late phase of sepsis. This may have beneficially attenuated liver inflammation and injury in an antibiotic-treated septic condition.

Inflammasomes are intracellular signaling complexes that are assembled in response to a variety of pathogenic or physiologic stimuli to initiate inflammatory responses. Ubiquitously present LPS in Gram-negative bacteria induces NLRP3 inflammasome activation that requires caspase-11. We have recently demonstrated that IFN regulatory factor (IRF) 8 was dispensable for caspase-11–mediated NLRP3 inflammasome activation during LPS transfection; however, its role in Gram-negative bacteria–mediated NLRP3 inflammasome activation remains unknown. In this study, we found that IRF8 promotes NLRP3 inflammasome activation in murine bone marrow–derived macrophages (BMDMs) infected with Gram-negative bacteria such as Citrobacter rodentium, Escherichia coli, or Pseudomonas aeruginosa mutant strain ΔpopB. Moreover, BMDMs deficient in IRF8 showed substantially reduced caspase-11 activation and gasdermin D cleavage, which are required for NLRP3 inflammasome activation. Mechanistically, IRF8-mediated phosphorylation of IRF3 was required for Ifnb transcription, which in turn triggered the caspase-11–dependent NLRP3 inflammasome activation in the infected BMDMs. Overall, our findings suggest that IRF8 promotes NLRP3 inflammasome activation during infection with Gram-negative bacteria.

In this study, we used LS174T cells as a model to investigate the protective effects of icariin and phosphorylated icariin on LPS-induced goblet cell dysfunction. Our results indicated that icariin and phosphorylated icariin increased the cell viability and decreased lactate dehydrogenase activity in LPS-treated LS174T cells. Icariin and phosphorylated icariin attenuated LPS-induced changes in mucin 2 synthesis and secretion. Besides, Icariin and phosphorylated icariin reduced the levels of ROS, MDA, and H2O2 and increased the activity of SOD, GPx, CAT, and T-AOC in LPS-treated LS174T cells. Moreover, the levels of IL-1β, IL-6, IL-8, and TNF-α were reduced in the Icariin and phosphorylated icariin group. Furthermore, Icariin and phosphorylated icariin decreased gene abundance or enzyme activity of Bip, XBP1, GRP78, CHOP, caspase-3, and caspase-4 in LPS-treated LS174T cells. Our data suggest that Icariin and phosphorylated icariin effectively attenuate LPS-induced intestinal goblet cell function damage through regulating oxidative stress, inflammation, apoptosis, and mucin expression.

BACKGROUND: Human and mouse studies implicate the inflammasome in the pathogenesis of inflammatory bowel diseases, though the effects in mice are variable. The noncanonical inflammasome activator caspase-11 (Casp11) reportedly attenuates acute dextran sodium sulfate (DSS) colitis in mice. However, the effects of Casp11 on chronic experimental colitis and factors that influence the impact of Casp11 on acute DSS colitis are unknown. METHODS: We studied the role of Casp11 in Il10(-/-) mice and acute and chronic DSS colitis mouse models. We quantified colonic Casp11 mRNA using quantative polymerase chain reaction and colitis using weight loss, blinded histological scoring, IL-12/23p40 secretion by colonic explants, and fecal lipocalin-2. We determined fecal microbial composition using 16S amplicon sequencing. RESULTS: We detected increased colonic Casp11 mRNA in Il10(-/-) mice with chronic colitis, but not in mice with DSS colitis. The presence of Casp11 did not alter the severity of chronic colitis in DSS-treated or Il10(-/-) mice. Contrary to prior reports, we initially observed that Casp11 exacerbates acute DSS colitis. Subsequent experiments in the same animal facility revealed no effect of Casp11 on acute DSS colitis. There were pronounced stochastic changes in the fecal microbiome over this time. The majority of bacterial taxa that changed over time in wild-type vs Casp11(-/-) mice belong to the Clostridiales. CONCLUSIONS: Casp11 does not impact chronic experimental colitis, and its effects on acute DSS colitis vary with environmental factors including the microbiota, particularly Clostridiales. Stochastic drifts in intestinal microbiota composition, even in mice in the same housing facility, should be considered when interpreting studies of acute DSS colitis models.

Summary: The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1β secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1β production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPS-induced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3-deficient THP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis. : Shiga toxin 2 is a major virulence factor of enterohemorrhagic E. coli. Platnich et al. show that Shiga toxin 2 and co-transported lipopolysaccharide activate caspase-4, gasdermin D, and mitochondrial ROS upstream of the NLRP3 inflammasome in macrophages. Inflammasome activation may play a role in the pathogenesis of enterohemorrhagic E. coli infection and disease. Keywords: Shiga toxin, inflammasome, NLRP3, caspase-4, gasdermin D, enterohemorrhagic Escherichia coli, macrophages

Commercially available saponins are extracted from Quillaja saponaria barks, being Quil A® the most widely used. Nanoparticulate immunostimulating complexes (ISCOMs or ISCOMATRIX) formulated with these, are able to stimulate strong humoral and cellular immune responses. Recently, we formulated novel ISCOMs replacing QuilA® by QB-90 (IQB-90), a Quillaja brasiliensis leaf-extracted saponin fraction, and reported that IQB-90 improved antigen uptake, and induced systemic and mucosal antibody production, and T-cell responses. However, its mechanism of action remains unclear. In this study we provide a deeper insight into the immune stimulatory properties of QB-90 and ISCOMATRIX-like based on this fraction (IMXQB-90). We show herein that, when used as a viral vaccine adjuvant, QB-90 promotes an “immunocompetent environment”. In addition, QB-90 and IMXQB-90 induce immune-cells recruitment at draining-lymph nodes and spleen. Subsequently, we prove that QB-90 or IMXQB-90 stimulated dendritic cells secret IL-1β by mechanisms involving Caspase-1/11 and MyD88 pathways, implying canonical inflammasome activation. Finally, both formulations induce a change in the expression of cytokines and chemokines coding genes, many of which are up-regulated. Findings reported here provide important insights into the molecular and cellular mechanisms underlying the adjuvant activity of Q. brasiliensis leaf-saponins and its respective nanoparticles.

The avian origin influenza A virus (IAV) H7N9 has caused a considerable number of human infections associated with high rates of death since its emergence in 2013. As a vital component of the host innate immune system, the nucleotide-binding domain leucine-rich repeat containing receptor, pyrin domain containing 3 (NLRP3) inflammasome plays a critical role against H1N1 viral infection. However, the function of NLRP3 inflammasome in host immunological responses to the lethal H7N9 virus is still obscure. Here, we demonstrated that mice deficient for NLRP3 inflammasome components, including NLRP3, caspase-1, and Apoptosis-associated speck-like protein containing a CARD (ASC), were less susceptible to H7N9 viral challenge than wild type (WT) controls. Inflammasome deficiency in these animals led to significantly milder mortality and less pulmonary inflammation compared with WT mice. Furthermore, IL-1 receptor deficient mice also exhibited a higher survival rate than WT controls. Thus, our study reveals that the NLRP3 inflammasome is deleterious for the host during H7N9 infection in mice, which is due to an overwhelming inflammatory response via caspase-1 activation and associated IL-1 signal. Therefore, fine-tuning the activity of NLRP3 inflammasome or IL-1 signaling may be beneficial for the host to control H7N9 associated lethal pathogenesis.

Summary Inflammasomes are multimeric protein complexes that initiate inflammatory cascades. Their activation is a hallmark of many infectious or inflammatory diseases. Their composition and activity are specified by proinflammatory stimuli. For example, the NLRP3 inflammasome is activated in response to cell damage and K + efflux, whereas the AIM2 inflammasome is activated in response to cytosolic DNA. We used Legionella pneumophila , an intracellular bacterial pathogen that activates multiple inflammasomes, to elucidate the molecular mechanisms regulating inflammasome activation during infection. Upon infection, the AIM2 inflammasome engaged caspase-1 to induce pore formation in the cell membrane, which then caused K + -efflux-mediated activation of NLRP3. Thus, the AIM2 inflammasome amplifies signals of infection, triggering noncanonical activation of NLRP3. During infection, AIM2 and caspase-11 induced membrane damage, which was sufficient and essential for activating the NLRP3 inflammasome. Our data reveal that different inflammasomes regulate one another's activity to ensure an effective immune response to infection.

Gout is characterized by attacks of arthritis with hyperuricemia and monosodium urate (MSU) crystal-induced inflammation within joints. Innate immune responses are the primary drivers for tissue destruction and inflammation in gout. MSU crystals engage the Nlrp3 inflammasome, leading to the activation of caspase-1 and production of IL-1β and IL-18 within gout-affected joints, promoting the influx of neutrophils and monocytes. Here, we show that caspase-11 -/- mice and their derived macrophages produce significantly reduced levels of gout-specific cytokines including IL-1β, TNFα, IL-6, and KC, while others like IFNγ and IL-12p70 are not altered. IL-1β induces the expression of caspase-11 in an IL-1 receptor-dependent manner in macrophages contributing to the priming of macrophages during sterile inflammation. The absence of caspase-11 reduced the ability of macrophages and neutrophils to migrate in response to exogenously injected KC in vivo. Notably, in vitro, caspase-11 -/- neutrophils displayed random migration in response to a KC gradient when compared to their WT counterparts. This phenotype was associated with altered cofilin phosphorylation. Unlike their wild-type counterparts, caspase-11 -/- neutrophils also failed to produce neutrophil extracellular traps (NETs) when treated with MSU. Together, this is the first report demonstrating that caspase-11 promotes neutrophil directional trafficking and function in an acute model of gout. Caspase-11 also governs the production of inflammasome-dependent and -independent cytokines from macrophages. Our results offer new, previously unrecognized functions for caspase-11 in macrophages and neutrophils that may apply to other neutrophil-mediated disease conditions besides gout.

International audience; Caspase‐4, the cytosolic LPS sensor, and gasdermin D, its downstream effector, constitute the non‐canonical inflammasome, which drives inflammatory responses during Gram‐negative bacterial infections. It remains unclear whether other proteins regulate cytosolic LPS sensing, particularly in human cells. Here, we conduct a genome‐wide CRISPR/Cas9 screen in a human monocyte cell line to identify genes controlling cytosolic LPS‐mediated pyroptosis. We find that the transcription factor, IRF2, is required for pyroptosis following cytosolic LPS delivery and functions by directly regulating caspase‐4 levels in human monocytes and iPSC‐derived monocytes. CASP4, GSDMD, and IRF2 are the only genes identified with high significance in this screen highlighting the simplicity of the non‐canonical inflammasome. Upon IFN‐γ priming, IRF1 induction compensates IRF2 deficiency, leading to robust caspase‐4 expression. Deficiency in IRF2 results in dampened inflammasome responses upon infection with Gram‐negative bacteria. This study emphasizes the central role of IRF family members as specific regulators of the non‐canonical inflammasome.

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.

Gasdermin D (GSDMD) cleavage by caspase-1 or caspase-11 inflammasomes triggers pyroptosis, a lytic form of cell death protective against intracellular bacteria. In this study, we examine the role of GSDMD in a mouse model of melioidosis. Gsdmd−/− mice were more susceptible than wild-type mice to intranasal infection with Burkholderia thailandensis. Production of IL-18, but not IL-1β, was decreased in Gsdmd−/− infected mice. Despite lower IL-18, IFN-γ was produced in similar amounts in wild-type and Gsdmd−/− mice. In vitro, secretion of both IL-1β and IL-18 by macrophages or dendritic cells infected with B. thailandensis was dependent on GSDMD. Surprisingly, wild-type or GSDMD-deficient neutrophils secreted similar amounts of IL-1β, suggesting these cells may be the source of the GSDMD-independent IL-1β detected in vivo. Recombinant GSDMD was able to directly kill B. thailandensis in vitro upon processing by active caspase-1. Moreover, bacteria harvested from wild-type, but not Gsdmd−/−, macrophages were more susceptible to the microbicidal effect of hydrogen peroxide or human β-defensin-3. Finally, we provide evidence that pyroptosis of in vitro infected macrophages is directly microbicidal. Taken together, these results indicate that the protective action of GSDMD in melioidosis is primarily due to induction of pyroptosis and direct killing of bacteria rather than production of cytokines.

peer-reviewed Inflammation of the post-partum uterus is a normal physiological event, crucial for tissue involution and repair. However, in the bovine, some cows fail to resolve this inflammation, resulting in endometritis, which compromises fertility. Earlier work has identified upregulated expression of the potent inflammatory cytokine IL-1β early post-partum, in cows which subsequently develop endometritis. As a result, targeting IL-1β expression holds potential as a novel treatment for this disease, yet the regulatory mechanisms contributing to IL-1β expression in the bovine endometrium remain unknown. To investigate this, endometrial tissue samples were obtained 7 and 21 days post-partum (DPP) from cows that were diagnosed with endometritis at 21 DPP and cows that experienced a physiological level of inflammation throughout involution. IL-1β was measured by qPCR, ELISA, and immunohistochemistry. Seven DPP, endometrial IL-1β protein levels were significantly higher in animals that proceeded to develop endometritis at 21 DPP. IL-1β production could be detected in luminal and glandular epithelium, in underlying stromal fibroblasts as well as infiltrating immune cells. To investigate the mechanisms regulating IL-1β expression, primary endometrial epithelial cells, stromal fibroblasts and PBMCs were stimulated with LPS and the inflammasome activator nigericin. Stromal fibroblast cells were particularly potent producers of IL-1β. Basolateral LPS stimulation of polarized epithelial cells induced IL1B mRNA and a previously undescribed IL-1β protein isoform, with preferential protein secretion into the apical compartment. Key inflammasome components [nod-like receptor protein 3 (NLRP3), nima-related kinase-7 (NEK7), apoptosis speck like protein containing a CARD (ASC), and gasdermin-D] were expressed by endometrial cells following stimulation. Endometrial cell stimulation in the presence of NLRP3 receptor (MCC950) and pan-caspase (Z-VAD-FMK) inhibitors blocked IL-1β production, demonstrating its dependence on the NLRP3 inflammasome and on caspase activity. Furthermore, caspase-4 specific siRNA prevented IL-1β production, confirming that inflammasome activation in endometrial cells is caspase-4 but not caspase-1 dependent, as shown in other species. Identifying the tissue- and species-specificity of inflammasome assembly and activation has critical relevance for our understanding of inflammation and suggests new therapeutic targets to enhance the resolution of inflammatory pathologies including endometritis in cattle.

International audience; Inflammatory caspases, activated within the inflammasome, are responsible for the maturation and secretion of IL-1beta/IL-18. Although their expression in psoriasis was shown several years ago, little is known about the role of inflammatory caspases in the context of psoriasis. Here, we confirmed that caspases 1, 4, and 5 are activated in lesional skin from psoriasis patients. We showed in three psoriasis-like models that inflammatory caspases are activated, and accordingly, caspase 1/11 invalidation or pharmacological inhibition by Ac-YVAD-CMK (i.e., Ac-Tyr-Val-Ala-Asp-chloromethylketone) injection induced a decrease in ear thickness, erythema, scaling, inflammatory cytokine expression, and immune cell infiltration in mice. We observed that keratinocytes were primed to secrete IL-1beta when cultured in conditions mimicking psoriasis. Generation of chimeric mice by bone marrow transplantation was carried out to decipher the respective contribution of keratinocytes and/or immune cells in the activation of inflammatory caspases during psoriasis-like inflammatory response. Our data showed that the presence of caspase 1/11 in the immune system is sufficient for a fully inflammatory response, whereas the absence of caspase 1/11 in keratinocytes/fibroblasts had no impact. In summary, our study indicates that inflammatory caspases activated in immune cells are implicated in psoriasis pathogenesis.

Vibrio cholerae is a Gram-negative enteropathogen causing potentially life-threatening cholera disease outbreaks, for which the World Health Organization currently registers 2-4 million cases and similar to 100.000 cholera-associated deaths annually worldwide. Genomic Vibrio cholerae research revealed that the strains causing this ongoing cholera pandemic are members of the El Tor biotype, which fully replaced the Classical biotype that caused former cholera pandemics. While both of these biotypes express the characteristic Cholera Toxin (CT), the El Tor biotype additionally expresses the accessory toxins hemolysin (hlyA) and multifunctional auto-processing repeat-in-toxin (MARTX). Previous studies demonstrated that the Classical biotype of Vibrio cholerae triggers caspase-11-dependent non-canonical inflammasome activation in macrophages following CT-mediated cytosolic delivery of LPS. In contrast to the Classical biotype, we here show that El Tor Vibrio cholerae induces IL-1 beta maturation and secretion in a caspase-11- and CT-independent manner. Instead, we show that El Tor Vibrio cholerae engages the canonical Nlrp3 inflammasome for IL-1 beta secretion through its accessory hlyA toxin. We further reveal the capacity of this enteropathogen to engage the canonical Pyrin inflammasome as an accessory mechanism for IL-1 beta secretion in conditions when the pro-inflammatory hlyA-Nlrp3 axis is blocked. Thus, we show that the V. cholerae El Tor biotype does not trigger caspase-11 activation, but instead triggers parallel Nlrp3- and Pyrin-dependent pathways toward canonical inflammasome activation to induce IL-1 beta-mediated inflammatory responses. These findings further unravel the complex inflammasome activating mechanisms that can be triggered when macrophages face the full arsenal of El Tor Vibrio cholerae toxins, and as such increase our understanding of host-pathogen interactions in the context of the Vibrio cholerae biotype associated with the ongoing cholera pandemic.

Lipopolysaccharides (LPS) are a major component of the outer membrane of Gram-negative bacteria and are pathogen-associated molecular patterns recognized by the TLR4/MD2 complex that induces an inflammatory response. Recently, the cytosolic receptors caspase-4/-5/-11 that bind LPS inside the cell and trigger inflammasome activation or pyroptosis, have been identified. Despite the important roles of caspase-4 in human immune responses, few studies have investigated its biochemical characteristics and interactions with LPS. Since caspase-4 (C258A) purified from an Escherichia coli host forms aggregates, monomeric proteins including full-length caspase-4, caspase-4 (C258A), and the CARD domain of caspase-4 have been purified from the insect cell system. Here, we report the overexpression and purification of monomeric caspase-4 (C258A) and CARD domain from E. coli and demonstrate that purified caspase-4 (C258A) and CARD domain bind large LPS micelles and disaggregate them to small complexes. As the molar ratio of caspase-4 to LPS increases, the size of the caspase-4/LPS complex decreases. Our results present a new function of caspase-4 and set the stage for structural and biochemical studies, and drug discovery targeting LPS/caspase-4 interactions by establishing a facile purification method to obtain large quantities of purified caspase-4 (C258A) and the CARD domain.

Innate immune response against Brucella abortus involves activation of Toll-like receptors (TLRs) and NOD-like receptors (NLRs). Among the NLRs involved in the recognition of B. abortus are NLRP3 and AIM2. Here, we demonstrate that B. abortus triggers non-canonical inflammasome activation dependent on caspase-11 and gasdermin-D (GSDMD). Additionally, we identify that Brucella-LPS is the ligand for caspase-11 activation. Interestingly, we determine that B. abortus is able to trigger pyroptosis leading to pore formation and cell death, and this process is dependent on caspase-11 and GSDMD but independently of caspase-1 protease activity and NLRP3. Mice lacking either caspase-11 or GSDMD were significantly more susceptible to infection with B. abortus than caspase-1 knockout or wild-type animals. Additionally, guanylate-binding proteins (GBPs) present in mouse chromosome 3 participate in the recognition of LPS by caspase-11 contributing to non-canonical inflammasome activation as observed by the response of Gbpchr3-/- BMDMs to bacterial stimulation. We further determined by siRNA knockdown that among the GBPs contained in mouse chromosome 3, GBP5 is the most important for Brucella LPS to be recognized by caspase-11 triggering IL-1β secretion and LDH release. Additionally, we observed a reduction in neutrophil, dendritic cell and macrophage influx in spleens of Casp11-/- and Gsdmd-/- compared to wild-type mice, indicating that caspase-11 and GSDMD are implicated in the recruitment and activation of immune cells during Brucella infection. Finally, depletion of neutrophils renders wild-type mice more susceptible to Brucella infection. Taken together, these data suggest that caspase-11/GSDMD-dependent pyroptosis triggered by B. abortus is important to infection restriction in vivo and contributes to immune cell recruitment and activation., Author summary Brucella abortus is the causative agent of brucellosis, a zoonotic disease that affects both humans and cattle. In humans, it is characterized by undulant fever and chronic symptoms as arthritis, endocarditis, and meningitis, while in cattle it causes abortion and infertility. Due to its difficult diagnosis and treatment, it leads to severe economic losses and human suffering. Recently, a novel non-canonical inflammasome pathway was described that involves sensing of bacterial LPS by an intracellular receptor termed caspase-11 and leads to pyroptosis and non-canonical NLRP3 inflammasome activation. Here, we show that B. abortus or its purified LPS is able to activate the non-canonical inflammasome. In this process, activated caspase-11 leads to GSDMD-dependent pyroptosis. Moreover, this pathway was dependent of IFN-induced GBP proteins, mainly GBP5. To analyze the role of caspase-1, caspase-11 and GSDMD in controlling B. abortus infection, we infected knockout (KO) mice for these molecules and we observed that caspase-11 and GSDMD KO animals were more susceptible to infection compared to wild-type animals. Casp11-/- and Gsdmd-/- animals also recruited less immune cells in mouse spleens compared to wild-type animals in response to B. abortus. Thus, caspase-11 and GSDMD are major components of the innate immune system to restrict B. abortus in vivo. This pathway of bacterial sensing by the host immune system is important to future development of drugs and vaccines that may contribute to the control of brucellosis worldwide.

BACKGROUND: Cardiac dysfunction is a common manifestation of sepsis and is associated with early increases in inflammation and decreases in myocardial protein synthesis. However, little is known regarding the molecular mechanisms regulating protein homeostasis during the recovery phase after the removal of the septic nidus. Therefore, the purpose of this study was to investigate diverse signal transduction pathways that regulate myocardial protein synthesis and degradation. METHODS: Adult male C57BL/6 mice were used to identify potential mechanisms mediating the acute (24 h) effect of cecal ligation and puncture (CLP) as well as long-term changes that manifest during the chronic (10 d) recovery phase. RESULTS: Acutely, sepsis decreased cardiac protein synthesis that was associated with reduced phosphorylation of S6K1/S6 but not 4E-BP1. Sepsis also decreased proteasome activity, although with no change in MuRF1 and atrogin-1 mRNA expression. Sepsis acutely increased apoptosis (increased caspase-3 and PARP cleavage), autophagosome formation (increased LC3B-II), and canonical inflammasome activity (increased NLRP3, TMS1, cleaved caspase-1). In contrast, during the recovery phase, independent of a difference in food consumption, global protein synthesis was increased, the early repression in proteasome activity was restored to basal levels, while stimulation of apoptosis, autophagosome formation and the canonical inflammasome pathway had abated. However, during recovery there was a selective stimulation of the non-canonical inflammasome pathway as evidenced by activation of caspase-11 with cleavage of Gasdermin D. CONCLUSION: These data demonstrate a temporally distinct homeostatic shift in the cardiac proteostatic response to acute infection and recovery.

SUMMARY Caspase-11 sensing of intracellular lipopolysaccharide (LPS) plays critical roles during infections and sepsis. However, the key cell types that sense intracellular LPS and their contributions to the host responses at the organismal level are not completely clear. Here, we show that macrophage/monocyte-specific caspase-11 plays a dominant role in mediating the pathological manifestations of endotoxemia, including gasdermin D (GSDMD) activation, interleukin (IL)-1β, IL-18, and damage-associated molecular pattern (DAMP) release, tissue damage, and death. Surprisingly, caspase-11 expression in CD11c+ cells and intestinal epithelial cells (IECs) plays minor detrimental roles in LPS shock. In contrast, caspase-11 expression in neutrophils is dispensable for LPS-induced lethality. Importantly, caspase-11 sensing of intracellular LPS in LyzM+ myeloid cells and MRP8+ neutrophils, but not CD11c+ cells and IECs, is necessary for bacterial clearance and host survival during intracellular bacterial infection. Thus, we reveal hierarchical cell-type-specific roles of caspase-11 that govern the host-protective and host-detrimental functions of the cytosolic LPS surveillance., Graphical abstract, In brief Kumari et al. reveal hierarchical cell-type-specific roles of caspase-11 that govern the host-protective and host-detrimental functions of the cytosolic LPS surveillance pathway during bacterial infections and sepsis, respectively.

Summary The activator and composition of the NLRP6 inflammasome remain poorly understood. We find that lipoteichoic acid (LTA), a molecule produced by Gram-positive bacteria, binds and activates NLRP6. In response to cytosolic LTA or infection with Listeria monocytogenes, NLRP6 recruited caspase-11 and caspase-1 via the adaptor ASC. NLRP6 activation by LTA induced processing of caspase-11, which promoted caspase-1 activation and interleukin-1β (IL-1β)/IL-18 maturation in macrophages. Nlrp6−/− and Casp11−/− mice were less susceptible to L. monocytogenes infection, which was associated with reduced pathogen loads and impaired IL-18 production. Administration of IL-18 to Nlrp6−/− or Casp11−/− mice restored the susceptibility of mutant mice to L. monocytogenes infection. These results reveal a previously unrecognized innate immunity pathway triggered by cytosolic LTA that is sensed by NLRP6 and exacerbates systemic Gram-positive pathogen infection via the production of IL-18.

The CANTOS trial (Canakinumab Antiinflammatory Thrombosis Outcome Study) showed that antagonism of interleukin (IL)-1β reduces coronary heart disease in patients with a previous myocardial infarction and evidence of systemic inflammation, indicating that pathways required for IL-1β secretion increase cardiovascular risk. IL-1β and IL-18 are produced via the NLRP3 inflammasome in myeloid cells in response to cholesterol accumulation, but mechanisms linking NLRP3 inflammasome activation to atherogenesis are unclear. The cholesterol transporters ATP binding cassette A1 and G1 (ABCA1/G1) mediate cholesterol efflux to high-density lipoprotein, and Abca1/g1 deficiency in myeloid cells leads to cholesterol accumulation.To interrogate mechanisms connecting inflammasome activation with atherogenesis, we used mice with myeloid Abca1/g1 deficiency and concomitant deficiency of the inflammasome components Nlrp3 or Caspase-1/11. Bone marrow from these mice was transplanted into LdlrMyeloid Abca1/g1 deficiency increased plasma IL-18 levels in LdlrCholesterol accumulation in myeloid cells activates the NLRP3 inflammasome, which enhances neutrophil accumulation and neutrophil extracellular trap formation in atherosclerotic plaques. Patients with Tangier disease, who have increased myeloid cholesterol content, showed markers of inflammasome activation, suggesting human relevance.

Caspase-11 is a key upstream modulator for activation of inflammatory response under pathological conditions. In this study, we investigated the roles of caspase-11 in the maturation of interleukin-1β (IL-1β) and development of renal interstitial fibrosis in vivo and in vitro. Mice were subjected to unilateral ureteral obstruction (UUO). The mice were treated with either caspase-11 inhibitor wedelolactone (Wed, 30 mg/kg/day, ig) for 7 days or caspase-11 siRNA (10 nmol/20 g body weight per day, iv) for 14 days. The mice were euthanized on day 14, their renal tissue and blood sample were collected. We found that the obstructed kidney had significantly higher caspase-11 levels and obvious tubular injury and interstitial fibrosis. Treatment with Wed or caspase-11 siRNA significantly mitigated renal fibrosis in UUO mice, evidenced by the improved histological changes. Furthermore, caspase-11 inhibition significantly blunted caspase-1 activation, IL-1β maturation, transforming growth factor-β (TGF-β), fibronectin, and collagen I expressions in the obstructed kidney. Renal tubular epithelial NRK-52E cells were treated in vitro with angiotensin (Ang, 1 μmol/L), which stimulated caspase-11 activation and IL-1β maturation. Treatment with IL-1β (20 ng/ml) significantly increased the expression of TGF-β, fibronectin, and collagen I in the cells. Ang II-induced expression of TGF-β, fibronectin, and collagen I were suppressed by caspase-11 siRNA or Wed. Finally, we revealed using co-immunoprecipitation that caspase-11 was able to interact with caspase-1 in NRK-52E cells. These results suggest that caspase-11 is involved in UUO-induced renal fibrosis. Elevation of caspase-11 in the obstructed kidney promotes renal fibrosis by stimulating caspase-1 activation and IL-1β maturation.

Gasdermin B (GSDMB) has been reported to be associated with immune diseases in humans, but the detailed molecular mechanisms remain unsolved. The N-terminus of GSDMB by itself, unlike other gasdermin family proteins, does not induce cell death. Here, we show that GSDMB is highly expressed in the leukocytes of septic shock patients, which is associated with increased release of the gasdermin D (GSDMD) N-terminus. GSDMB expression and the accumulation of the N-terminal fragment of GSDMD are induced by the activation of the non-canonical pyroptosis pathway in a human monocyte cell line. The downregulation of GSDMB alleviates the cleavage of GSDMD and cell death. Consistently, the overexpression of GSDMB promotes GSDMD cleavage, accompanied by increased LDH release. We further found that GSDMB promotes caspase-4 activity, which is required for the cleavage of GSDMD in non-canonical pyroptosis, by directly binding to the CARD domain of caspase-4. Our study reveals a GSDMB-mediated novel regulatory mechanism for non-canonical pyroptosis and suggests a potential new strategy for the treatment of inflammatory diseases.

Stearoyl lysophosphatidylcholine (LPC) exerts protective effect during endotoxemia and in experimental sepsis, but the underlying mechanism is unclear. Here, we demonstrated that stearoyl LPC could block caspase-11-mediated macrophage pyroptosis. In vitro, stearoyl LPC significantly decreased caspase-11 activation and pyroptosis induced by lipopolysaccharide (LPS) plus cholera toxin subunit B independent of the receptor G2A. Stearoyl LPC did not affect LPS uptake by mouse peritoneal macrophages but did significantly inhibit the interaction between LPS and caspase-11. Moreover, stearoyl LPC treatment conferred significant protection against lethal endotoxemia and significantly reduced the release of IL-1α and IL-1β. These findings identify stearoyl LPC as an inhibitor of LPS-mediated caspase-11 activation. This mechanism could explain the protective action of stearoyl LPC in experimental sepsis and endotoxemia.

Caspases exert critical functions in diverse cell death pathways, including apoptosis and pyroptosis, but some caspases also have roles in the processing of cytokines into their functional forms during inflammation. The roles of many caspases have been unravelled by the generation of knockout mice, but still very little is known about the overlapping functions of caspases as only a few studies report on double or triple caspase knockout mice. For example, the functions of caspase-12 in cell death and inflammation, on its own or overlapping with the functions of caspase-1 and caspase-11, are only poorly understood. Therefore, we generated a novel mutant mouse strain lacking all three inflammatory caspases, caspases-1, -11 and -12. Analysis under steady state conditions showed no obvious differences between caspase-1/11/12(−/−) and wildtype (WT) mice. Since caspases-1 and -11 are involved in endotoxic shock, we analysed the response of caspase-1/11/12(−/−) mice to high-dose LPS injection. Interestingly, we could not detect any differences in responses between caspase-1/11/12(−/−) mice vs. caspase-1/11 double knockout mice. Furthermore, cell lines generated from caspase-1/11/12(−/−) mice showed no differences in their apoptotic or necroptotic responses to a diverse set of cytotoxic drugs in vitro when compared to WT cells. Importantly, these drugs also included ER stress-inducing agents, such as thapsigargin and tunicamycin, a form of cell death for which a critical pro-apoptotic function of caspase-12 has previously been reported. Additionally, we found no differences between caspase-1/11/12(−/−) and WT mice in their in vivo responses to the ER stress-inducing agent, tunicamycin. Collectively, these findings reveal that caspase-12 does not have readily recognisable overlapping roles with caspases-1 and -11 in the inflammatory response induced by LPS and in necroptosis and apoptosis induced by diverse cytotoxic agents, including the ones that elicit ER stress.

Summary The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-β-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.