Search

Notch1 activation mediated by γ-secretase is critical for angiogenesis. GeneCards database predicted that Caspase-4 (CASP4, with murine ortholog CASP11) interacts with presenilin-1, the catalytic core of γ-secretase. Therefore, we investigated the role of CASP4/11 in angiogenesis.In vivo, we studied the role of Casp11 in several angiogenesis mouse models using Casp11 wild-type and knockout mice. In vitro, we detected the effects of CASP4 on endothelial functions and Notch signalling by depleting or overexpressing CASP4 in human umbilical vein endothelial cells (HUVECs). The functional domain responsible for the binding of CASP4 and presenilin-1 was detected by mutagenesis and co-immunoprecipitation.Casp11 deficiency impaired adult angiogenesis in ischaemic hindlimbs, melanoma xenografts and Matrigel plugs, but not the developmental angiogenesis of retina. Bone marrow transplantation revealed that the pro-angiogenic effect depended on CASP11 derived from non-haematopoietic cells. CASP4 expression was induced by inflammatory factors and CASP4 knockdown decreased cell viability, proliferation, migration and tube formation in HUVECs. Mechanistically, CASP4/11 deficiency increased Notch1 activation in vivo and in vitro, while CASP4 overexpression repressed Notch1 signalling in HUVECs. Moreover, CASP4 knockdown increased γ-secretase activity. The γ-Secretase inhibitor DAPT restored the effects of CASP4 siRNA on Notch1 activation and angiogenesis in HUVECs. Notably, the catalytic activity of CASP4/11 was dispensable. CASP4 directly interacted with presenilin-1 through the caspase recruitment domain (CARD).These findings reveal a critical role of CASP4/11 in adult angiogenesis and make this molecule a promising therapeutic target for angiogenesis-related diseases in the future.

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

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.

Ischemia/reperfusion (I/R) injury is a major cause of acute kidney injury (AKI) in clinic. The activation of NLRP3 inflammasome is associated with inflammation and renal injury in I/R-induced AKI. In the current study we explored the molecular and cellular mechanisms for NLRP3 inflammasome activation following renal I/R. Mice were subjected to I/R renal injury by clamping bilateral renal pedicles. We showed that I/R injury markedly increased caspase-11 expression and the cleavage of pannexin 1 (panx1) in the kidneys accompanied by NLRP3 inflammasome activation evidenced by the activation of caspase-1 and interlukin-1β (IL-1β) maturation. In Casp-11(−/−) mice, I/R-induced panx1 cleavage, NLRP3 inflammasome activation as well as renal functional deterioration and tubular morphological changes were significantly attenuated. In cultured primary tubular cells (PTCs) and NRK-52E cells, hypoxia/reoxygenation (H/R) markedly increased caspase-11 expression, NLRP3 inflammasome activation, IL-1β maturation and panx1 cleavage. Knockdown of caspase-11 attenuated all those changes; similar effects were observed in PTCs isolated from Casp-11(−/−) mice. In NRK-52E cells, overexpression of caspase-11 promoted panx1 cleavage; pretreatment with panx1 inhibitor carbenoxolone or knockdown of panx1 significantly attenuated H/R-induced intracellular ATP reduction, extracellular ATP elevation and NLRP3 inflammasome activation without apparent influence on H/R-induced caspase-11 increase; pretreatment with P2X7 receptor inhibitor AZD9056 also attenuated NLRP3 inflammasome activation. The above results demonstrate that the cleavage of panx1 by upregulated caspase-11 is involved in facilitating ATP release and then NLRP3 inflammasome activation in I/R-induced AKI. This study provides new insight into the molecular mechanism of NLRP3 inflammasome activation in AKI.

Pyroptosis has been reported to contribute to the traumatic brain injury (TBI) process. Ac-FLTD-CMK is a newly synthesized pyroptosis inhibitor. However, whether Ac-FLTD-CMK inhibits pyroptosis and plays a neuroprotective role after TBI is unknown. The present study aimed to determine the effects of Ac-FLTD-CMK on TBI in a mouse model. Male C57BL/6 mice were randomly divided into sham, TBI + vehicle, and TBI + Ac-FLTD-CMK groups. TBI was induced using a weight-drop apparatus. Intraventricular injection of Ac-FLTD-CMK was performed 30 min after TBI. Caspase-1, caspase-11, gasdermin-D (GSDMD), and caspase-3 expression in the peri-contusional cortex were assessed by western blotting. Interleukin-1β (IL-1β) and interleukin-18 (IL-18) expression in the peri-contusional cortex were measured using ELISA. Behavioral experiments, brain water content, Evans blue extravasation, lactate dehydrogenase (LDH) release, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining were also performed. The results showed that Ac-FLTD-CMK administration significantly downregulated caspase-1 p20, caspase-11 p20, GSDMD N-terminal, IL-1β, and IL-18 expression; reduced LDH release; alleviated neuronal death; attenuated brain edema and blood-brain barrier damage; and improved neurobehavioral function. These findings indicate that Ac-FLTD-CMK treatment suppresses pyroptosis and protects mice against TBI.

Severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARS–CoV-2 infections and that CASP4 expression correlates with severity of SARS–CoV-2 infection in humans. SARS–CoV-2–infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARS–CoV-2–infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1β, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARS–CoV-2–induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

Enteric neuronal degeneration, as seen in inflammatory bowel disease, obesity, and diabetes, can lead to gastrointestinal dysmotility. Pyroptosis is a novel form of programmed cell death but little is known about its role in enteric neuronal degeneration. We observed higher levels of cleaved caspase-1, a marker of pyroptosis, in myenteric ganglia of overweight and obese human subjects compared with normal-weight subjects. Western diet–fed (WD-fed) mice exhibited increased myenteric neuronal pyroptosis, delayed colonic transit, and impaired electric field stimulation–induced colonic relaxation responses. WD increased TLR4 expression and cleaved caspase-1 in myenteric nitrergic neurons. Overactivation of nitrergic neuronal NF-κB signaling resulted in increased pyroptosis and delayed colonic motility. In caspase-11–deficient mice, WD did not induce nitrergic myenteric neuronal pyroptosis and colonic dysmotility. To understand the contributions of saturated fatty acids and bacterial products to the steps leading to enteric neurodegeneration, we performed in vitro experiments using mouse enteric neurons. Palmitate and lipopolysaccharide (LPS) increased nitrergic, but not cholinergic, enteric neuronal pyroptosis. LPS gained entry to the cytosol in the presence of palmitate, activating caspase-11 and gasdermin D, leading to pyroptosis. These results support a role of the caspase-11–mediated pyroptotic pathway in WD-induced myenteric nitrergic neuronal degeneration and colonic dysmotility, providing important therapeutic targets for enteric neuropathy.

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: Endothelial dysfunction enhances vascular inflammation, which initiates pulmonary arterial hypertension (PAH) pathogenesis, further induces vascular remodeling and right ventricular failure. Activation of inflammatory caspases is an important initial event at the onset of pyroptosis. Studies have shown that caspase-1–mediated pyroptosis has played a crucial role in the pathogenesis of PAH. However, the role of caspase-11, another inflammatory caspase, remains to be elucidated. Therefore, the purpose of this study was to clarify the role of caspase-11 in the development of PAH and its mechanism on endothelial cell function. Methods: The role of caspase-11 in the progression of PAH and vascular remodeling was assessed in vivo. In vitro, the effect of caspase-4 silencing on the human pulmonary arterial endothelial cells pyroptosis was determined. Results: We confirmed that caspase-11 and its human homolog caspase-4 were activated in PAH animal models and TNF (tumor necrosis factor)-α–induced human pulmonary arterial endothelial cells. Caspase-11 −/− relieved right ventricular systolic pressure, right ventricle hypertrophy, and vascular remodeling in Sugen-5416 combined with chronic hypoxia mice model. Meanwhile, pharmacological inhibition of caspase-11 with wedelolactone exhibited alleviated development of PAH on the monocrotaline-induced rat model. Moreover, knockdown of caspase-4 repressed the onset of TNF-α–induced pyroptosis in human pulmonary arterial endothelial cells and inhibited the activation of pyroptosis effector GSDMD (gasdermin D) and GSDME (gasdermin E). Conclusions: These observations identified the critical role of caspase-4/11 in the pyroptosis pathway to modulate pulmonary vascular dysfunction and accelerate the progression of PAH. Our findings provide a potential diagnostic and therapeutic target in PAH.

Cytoplasmic recognition of microbial lipopolysaccharides (LPS) in human cells is elicited by the caspase-4 and caspase-5 noncanonical inflammasomes, which induce a form of inflammatory cell death termed pyroptosis. Here we show that LPS-mediated activation of caspase-4 also induces a stress response promoting cellular senescence, which is dependent on the caspase-4 substrate gasdermin-D and the tumor suppressor p53. Furthermore, we found that the caspase-4 noncanonical inflammasome is induced and assembled in response to oncogenic RAS signaling during oncogene-induced senescence (OIS). Moreover, targeting caspase-4 expression in OIS showed its critical role in the senescence-associated secretory phenotype and the cell cycle arrest induced in cellular senescence. Finally, we observed that caspase-4 induction occurs in vivo in mouse models of tumor suppression and ageing. Altogether, we are showing that cellular senescence is induced by cytoplasmic LPS recognition by the noncanonical inflammasome and that this pathway is conserved in the cellular response to oncogenic stress., This work was funded by Cancer Research UK (CRUK) (C47559/A16243 Training & Career Development Board - Career Development Fellowship), the University of Edinburgh Chancellor’s Fellowship R42576 MRC, and the Ministry of Science and Innovation of the Government of Spain (Proyecto PID2020-117860GB-I00 financiado por MCIN/ AEI /10.13039/501100011033). J.C.A. was supported by CRUK, the University of Edinburgh and is supported by the Spanish National Research Council (CSIC). P.H., I.F.D and N.T. were funded by the University of Edinburgh. A.Q. was funded by CRUK. J.F.P and A.B.L. are funded by NIH grants: 1R01AG068048-01; P01 AG062413; 1UG3 CA268103-01. J.B. was funded by BBSRC (BB/K017314/1). V.S-B is supported by funding from the University of Edinburgh and Medical Research Council (MC_UU_00009/2). F.R.M is funded by a Wellcome Trust Clinical Research Fellowship through the Edinburgh Clinical Academic Track (ECAT) (203913/Z/16/Z). M.M. was supported by CRUK Edinburgh Centre Award (C157/A25140). V.G.B. is funded by CRUK (C157/A24837) and the University of Edinburgh.

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.

Exposure to high dose radiation causes life-threatening acute and delayed effects. Defining the mechanisms of lethal radiation-induced acute toxicity of gastrointestinal and hematopoietic tissues are critical steps to identify drug targets to mitigate and protect against the Acute Radiation Syndrome (ARS). For example, one rational strategy would be to design pharmaceuticals that block cell death pathways to preserve tissue integrity in radiation sensitive organ systems including the gastrointestinal tract and hematopoietic compartment. A previous study reported that the inflammasome pathway, which mediates inflammatory cell death through pyroptosis, promotes the acute radiation syndrome (ARS) (1). However, we show that mice lacking the inflammatory executioner caspases, caspase-1 and caspase-11, are not protected from ARS when compared directly to littermates expressing caspase-1 and caspase-11. These results suggest that alternative pathways will need to be targeted by drugs that successfully mitigate and protect against the ARS.

Summary Zoonotic pathogens, such as COVID-19, reside in animal hosts before jumping species to infect humans. The Carnivora, like mink, carry many zoonoses, yet how diversity in host immune genes across species affect pathogen carriage is poorly understood. Here, we describe a progressive evolutionary downregulation of pathogen-sensing inflammasome pathways in Carnivora. This includes the loss of nucleotide-oligomerization domain leucine-rich repeat receptors (NLRs), acquisition of a unique caspase-1/-4 effector fusion protein that processes gasdermin D pore formation without inducing rapid lytic cell death, and the formation of a caspase-8 containing inflammasome that inefficiently processes interleukin-1β. Inflammasomes regulate gut immunity, but the carnivorous diet has antimicrobial properties that could compensate for the loss of these immune pathways. We speculate that the consequences of systemic inflammasome downregulation, however, can impair host sensing of specific pathogens such that they can reside undetected in the Carnivora., Graphical abstract, Highlights • Carnivorans lack key NLRs and express a unique caspase-1/-4 hybrid protein • This protein is defective in mediating activation of common inflammasome pathways • What little activity occurs is driven by caspase-8, rather than caspase-1/-4, Species of the order Carnivora have evolutionarily acquired the expression of a unique caspase-1/-4 hybrid protein. Digby et al. show that this protein is a poor mediator of NLRP3- and caspase-4-dependent inflammasome activation. This downregulation in inflammasome pathways could impair pathogen detection and facilitate transmission of zoonotic infections.

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.

Chemotherapy resistance is one of the most common causes of death among patients with ovarian cancer, and identifying novel antitumor agents is a priority. Here, we report that the novel molecule 2-(anaphthoyl)ethyltrimethylammonium iodide (α-NETA) induces epithelial ovarian cancer (EOC) cell pyroptosis through the gesdermin-d (GSDMD)/caspase-4 pathway. Furthermore, Cell Counting Kit-8 fluorescence-activated cell sorting analysis showed that α-NETA treatment led to cell death in different ovarian cancer cell lines, including Ho8910, Ho8910PM, and A2780. Morphologic examination by electron microscopy indicated that cells treated with α-NETA produced multiple microbubbles, typical of cells undergoing pyroptosis. α-NETA also significantly increased expression of pyroptosis-associated molecules including caspase-4 and GSDMD in EOC cells. Knockdown of either caspase-4 or GSDMD in ovarian cancer cells strongly interfered with α-NETA cell-killing activity, indicating that α-NETA acts through the pyroptosis pathway. In vivo, α-NETA treatment dramatically decreased the size of EOC tumors in mice. Our findings suggest that α-NETA represents a potential new antitumor molecule or lead compound for EOC chemotherapy.-Qiao, L., Wu, X., Zhang, J., Liu, L., Sui, X., Zhang, R., Liu, W., Shen, F., Sun, Y., Xi, X. α-NETA induces pyroptosis of epithelial ovarian cancer cells through the GSDMD/caspase-4 pathway.

Ferritin is the major iron storage molecule of vertebrates, which can be detected in serum under numerous conditions, including inflammatory, neurodegenerative, and malignant diseases. Given this character, serum ferritin is frequently used as a biomarker in clinical settings. How the ferritin secreted to the serum has attracted much attention. Although some studies have found ferritin was mediated via the endoplasmic reticulum (ER)-Golgi secretion pathway or secretory lysosomes trafficking pathway under normal conditions, the secretion pathway of ferritin under pathological conditions, especially in sepsis is not very clear. In this report, we adopt a murine sepsis model to study the secretion pathway of ferritin in sepsis. We demonstrated caspase-11-GSDMD pathway and associated pyroptosis are required for secretion of ferritin in vitro and in vivo in sepsis. Moreover, our work connects pyroptosis to serum ferritin secretion and suggests a passive release process of ferritin, enhancing our understanding of the mechanism of ferritin secretion.

Background/Aims: Acute kidney injury (AKI) is a serious complication of sepsis and has a high morbidity and mortality rate. Caspase-11 induces pyroptosis, a form of programmed cell death that plays a critical role in endotoxic shock, but its role in tubular epithelial cell death and whether it contributes to sepsis-associated AKI remains unknown. Methods: The caspase-11–/– mouse received an intraperitoneal injection of lipopolysaccharide (LPS, 40 mg/kg body weight). Caspase-11–/– renal tubular epithelial cells (RTECs) form C57BL caspase-11–/– mice were treated with LPS in vitro. The IL-1β ELISA kit and Scr assay kit were used to measure the level of interleukin-1β and serum creatinine. Annexin V-FITC assay and TUNEL staining assay were used to detect the cell death in different groups in vitro and in vivo. Western blot was performed to analyze the protein expression of caspase-11 and Gsdmdc1. Results: LPS-induced sepsis results in lytic death of RTECs, accompanied by increased expression of the pyroptosis-related proteins caspase-11 and Gsdmd. However, the increase in pyroptosis-related protein expression induced by LPS was attenuated with caspase-11 knockout, both in vitro and in vivo. Furthermore, when challenged with lethal doses of systemic LPS, pathologic abnormalities in renal structure, increased serum and kidney interleukin-1β, increased serum creatinine, and animal death were observed in wild-type mice but prevented in caspase-11–/– mice. Conclusions: Caspase-11-induced pyroptosis of RTECs is a key event during septic AKI, and targeting of caspase-11 in RTECs may serve as a novel therapeutic target in septic AKI.

The Gram-negative bacterium Porphyromonas gingivalis is strongly associated with periodontitis. We previously demonstrated that P2X7 receptor activation by extracellular ATP (eATP) triggers elimination of intracellular pathogens, such as Leishmania amazonensis, Toxoplasma gondii and Chlamydia trachomatis. We also showed that eATP-induced IL-1β secretion via the P2X7 receptor is impaired by P. gingivalis fimbriae. Furthermore, enhanced P2X7 receptor expression was detected in the maxilla of P. gingivalis-orally infected mice as well as in human periodontitis patients. Here, we examined the effect of P2X7-, caspase-1/11- and IL-1 receptor-mediated responses during P. gingivalis infection. P2X7 receptor played a large role in controlling P. gingivalis infection and P. gingivalis-induced recruitment of inflammatory cells, especially neutrophils. In addition, IL-1β secretion was detected at different time points only when P2X7 receptor was expressed and in the presence of eATP treatment ex vivo. Activation of P2X7 receptor and IL-1 receptor by eATP and IL-1β, respectively, promoted P. gingivalis elimination in macrophages. Interestingly, eATP-induced P. gingivalis killing was inhibited by the IL-1 receptor antagonist (IL-1RA), consistent with autocrine activation of the IL-1 receptor for P. gingivalis elimination. In vivo, caspase-1/11 and IL-1 receptor were also required for bacterial clearance, leukocyte recruitment and IL-1β production after P. gingivalis infection. Our data demonstrate that the P2X7-IL-1 receptor axis activation is required for effective innate immune responses against P. gingivalis infection.

Rationale: Pyroptosis is a morphologically and mechanistically distinct form of cell death and is characterized by GSDMD (gasdermin D) or GSDME (gasdermin E)-mediated necrosis with excessive inflammatory factor release. Cardiomyocyte necrosis and inflammation play key roles in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury. However, whether cardiomyocytes undergo pyroptosis and the underlying mechanism in myocardial I/R injury remain unclear. Objective: We aimed to investigate the role of pyroptosis in myocardial I/R injury. Methods and Results: In vivo and in vitro experiments were used to investigate pyroptosis of cardiomyocyte and the associated mechanisms during I/R injury. Wild-type, Myh6-Cre, and cardiomyocyte-specific GSDMD-deficient male mice were subjected to I/R. Human peripheral blood samples were collected from patients with acute ST-segment–elevation myocardial infarction or control patients at 0, 1, and 24 hours after percutaneous coronary intervention in our department. The serum levels of GSDMD were measured by ELISA. Hypoxia/reoxygenation induced cardiomyocyte pyroptosis and the release of mature IL (interleukin)-18 but not IL-1β, which mechanistically resulted from GSDMD cleavage by caspase-11 in cardiomyocytes. Furthermore, GSDMD gene deletion blocked hypoxia/reoxygenation-induced cardiomyocyte pyroptosis and IL-18 release. GSDMD and its pyroptosis-inducing N-terminal fragment were upregulated in myocardial tissues after I/R injury. Immunofluorescence analysis showed that GSDMD was mainly localized in cardiomyocytes. GSDMD deficiency in cardiomyocytes significantly reduced the I/R-induced myocardial infarct size. Moreover, increased GSDMD serum levels were detected in patients exhibiting I/R injury 1 hour after percutaneous coronary intervention for ST-segment–elevation myocardial infarction. Conclusions: Our results show that GSDMD-mediated cardiomyocyte pyroptosis is a key event during myocardial I/R injury and that the caspase-11/GSDMD pathway may be essential to this process. Additionally, GSDMD inhibition significantly reduces cardiomyocyte pyroptosis and I/R-induced myocardial injury.

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.

Asthma is an inflammatory lung disorder characterized by mucus hypersecretion, cellular infiltration, and bronchial hyper-responsiveness. House dust mites (HDM) are the most prevalent cause of allergic sensitization. Canonical and noncanonical inflammasomes are multiprotein complexes that assemble in response to pathogen or danger-associated molecular patterns (PAMPs or DAMPs). Murine caspase-11 engages the noncanonical inflammasome. We addressed the role of caspase-11 in mediating host responses to HDM and subsequent allergic inflammation using caspase-11-/- mice, which lack caspase-11 while express caspase-1. We found that HDM induce caspase-11 expression in vitro. The presence of IL-4 and IL-13 promote caspase-11 expression. Additionally, caspase-11-/- macrophages show reduced release of IL-6, IL-12, and KC, and express lower levels of costimulatory molecules (e.g., CD40, CD86 and MHCII) in response to HDM stimulation. Notably, HDM sensitization of caspase-11-/- mice resulted in similar levels of IgE responses and hypothermia in response to nasal HDM challenge compared to WT. However, analysis of cell numbers and cytokines in bronchiolar alveolar lavage fluid (BALF) and histopathology of representative lung segments demonstrate altered inflammatory responses and reduced neutrophilia in the airways of the caspase-11-/- mice. These findings indicate that caspase-11 regulates airway inflammation in response to HDM exposure.

Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1β) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia. Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia.

Inflammatory caspase-dependent cytosolic lipopolysaccharide (LPS) sensing is a critical arm of host defense against bacteria. How pathogens overcome this pathway to establish infections is largely unknown. Enterohemorrhagic Escherichia coli (EHEC) is a clinically important human pathogen causing hemorrhagic colitis and hemolytic uremic syndrome. We found that a bacteriophage-encoded virulence factor of EHEC, Shiga toxin (Stx), suppresses caspase-11-mediated activation of the cytosolic LPS sensing pathway. Stx was essential and sufficient to inhibit pyroptosis and interleukin-1 (IL-1) responses elicited specifically by cytosolic LPS. The catalytic activity of Stx was necessary for suppression of inflammasome responses. Stx impairment of inflammasome responses to cytosolic LPS occurs at the level of gasdermin D activation. Stx also suppresses inflammasome responses in vivo after LPS challenge and bacterial infection. Overall, this study assigns a previously undescribed inflammasome-subversive function to a well-known bacterial toxin, Stx, and reveals a new phage protein-based pathogen blockade of cytosolic immune surveillance.

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.

Peptidylarginine deiminases (PADs) are a family of calcium-dependent enzymes that are involved in a variety of human disorders, including cancer and autoimmune diseases. Although targeting PAD4 has shown no benefit in sepsis, the role of PAD2 remains unknown. Here, we report that PAD2 is engaged in sepsis and sepsis-induced acute lung injury in both human patients and mice. Pad2–/– or selective inhibition of PAD2 by a small molecule inhibitor increased survival and improved overall outcomes in mouse models of sepsis. Pad2 deficiency decreased neutrophil extracellular trap (NET) formation. Importantly, Pad2 deficiency inhibited Caspase-11–dependent pyroptosis in vivo and in vitro. Suppression of PAD2 expression reduced inflammation and increased macrophage bactericidal activity. In contrast to Pad2–/–, Pad4 deficiency enhanced activation of Caspase-11–dependent pyroptosis in BM-derived macrophages and displayed no survival improvement in a mouse sepsis model. Collectively, our findings highlight the potential of PAD2 as an indicative marker and therapeutic target for sepsis., Peptidylarginine deiminases 2 (PAD2) regulates neutrophil extracellular trap (NET) formation in sepsis and sepsis-induced acute lung injury.

This study aimed to investigate the mechanism of type I interferon (IFN) in aggravating sepsis in bacterial infection, focusing on the roles of Caspase-11 (Casp11) and Gasdermin D (Gsdmd) in this process. Type I interferons, including IFNα and IFNβ, were used to treat peritoneal macrophage harvested from wild-type or IFNα/βR1 knockout (KO) mice, of which the levels of Casp11 and Gsdmd were monitored using real-time polymerase chain reaction (RT-PCR) and Western blot, the exposure to phosphatidylserine was monitored by flow cytometry, and tissue factor (TF) activation was assessed by RT-PCR and TF chromogenic assay. Endotoxemia in wild-type mice led to upregulation of Casp11 and Gsdmd in myeloid cells, which in contrast was attenuated in IFNα/βR1 KO mice. IFNα or IFNβ treatment led to dose-dependent upregulation of Casp11 and Gsdmd in peritoneal macrophages harvested from wild-type mice, but induced negligible changes in IFNα/βR1 KO mice. Type I IFN promoted phosphatidylserine exposure in peritoneal macrophage from wild-type mice but not IFNα/βR1 KO mice. Type I IFN induced insignificant changes of TF expression levels in both wild-type mice and IFNα/βR1 KO mice, but the TF activity was markedly increased in wild-type mice after type I IFN treatment. Our data suggested that the upregulation of Casp11 and Gsdmd in myeloid cells and macrophages induced by endotoxemia was reliant on the expression of IFNα/βR1. IFNα or IFNβ treatment efficiently upregulated Casp11 and Gsdmd, phosphatidylserine exposure, and TF activity of macrophages. Therefore, type I IFN could aggravate sepsis through upregulating Casp11 and Gsdmd.

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.

The gut microbiota affects tissue physiology, metabolism, and function of both the immune and nervous systems. We found that intrinsic enteric-associated neurons (iEANs) in mice are functionally adapted to the intestinal segment they occupy; ileal and colonic neurons are more responsive to microbial colonization than duodenal neurons. Specifically, a microbially responsive subset of viscerofugal CART+ neurons, enriched in the ileum and colon, modulated feeding and glucose metabolism. These CART+ neurons send axons to the prevertebral ganglia and are polysynaptically connected to the liver and pancreas. Microbiota depletion led to NLRP6- and caspase 11–dependent loss of CART+ neurons and impaired glucose regulation. Hence, iEAN subsets appear to be capable of regulating blood glucose levels independently from the central nervous system.

Summary Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections., Graphical Abstract, Highlights • Mice lacking pyroptosis and apoptosis cannot control Salmonella infection • Macrophages lacking pyroptosis and apoptosis resist Salmonella-induced killing • Caspase-1 kills Salmonella-infected cells by activating GSDMD, BID, or other caspases • Caspase-1 and -8 act as cell death executioners when all cell death effectors are lost, The clearance of intracellular pathogens requires the killing of infected cells, but it remains unclear why host cells have so many different means of inducing programmed cell death. Doerflinger et al. demonstrate that interconnectivity between pyroptosis and apoptosis involving flexible deployment of caspases ensures control of Salmonella infection in mice.

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.

Background We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. Methods C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. Results CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. Conclusion Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.

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.

Inflammation associated with gram-negative bacterial infections is often instigated by the bacterial cell wall component lipopolysaccharide (LPS). LPS-induced inflammation and resulting life-threatening sepsis are mediated by the two distinct LPS receptors TLR4 and caspase-11 (caspase-4/-5 in humans). Whereas the regulation of TLR4 activation by extracellular and phago-endosomal LPS has been studied in great detail, auxiliary host factors that specifically modulate recognition of cytosolic LPS by caspase-11 are largely unknown. This study identifies autophagy-related and dynamin-related membrane remodeling proteins belonging to the family of Immunity-related GTPases M clade (IRGM) as negative regulators of caspase-11 activation in macrophages. Phagocytes lacking expression of mouse isoform Irgm2 aberrantly activate caspase-11-dependent inflammatory responses when exposed to extracellular LPS, bacterial outer membrane vesicles, or gram-negative bacteria. Consequently, Irgm2-deficient mice display increased susceptibility to caspase-11-mediated septic shock in vivo. This Irgm2 phenotype is partly reversed by the simultaneous genetic deletion of the two additional Irgm paralogs Irgm1 and Irgm3, indicating that dysregulated Irgm isoform expression disrupts intracellular LPS processing pathways that limit LPS availability for caspase-11 activation.

SummaryInflammasomes and interferons constitute two critical arms of innate immunity. Most facultative bacterial pathogens that inhabit the host cell cytosol avoid activating inflammasomes and are often resistant to killing by type I interferon (IFN-I). We report that the human pathogenRickettsia parkeri,an obligate intracellular pathogen that resides in the cytosol, is sensitive to IFN-I. The mechanism of IFN-I-dependent restriction requires the transcription factor IRF5, which upregulates anti-rickettsial factors including guanylate-binding proteins and iNOS. However,R. parkericurtails cGAS-dependent IFN-I production by causing caspase-11-dependent pyroptosis.In vivo, inflammasome activation antagonizes IFN-I production, enhancingR. parkeriabundance in the spleen. Mice lacking either IFN-I or IFN-γ signaling are resistant to infection, but mice lacking both rapidly succumb, revealing that both interferons are required to controlR. parkeri. This study illuminates how an obligate cytosolic pathogen exploits the intrinsic trade-off between cell death and cytokine production to escape killing by innate immunity.HighlightsRickettsiakilled by GBPs activates caspase-11 and GSDMD, promoting pyroptosisRickettsiaexploits pyroptosis to avoid cGAS-dependent type I interferonIRF5, GBPs, and iNOS contribute to controllingR. parkeriinfectionIfnar-/-Ifngr-/-mice succumb to infection, uncovering a mouse model to studyR. parkeri

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.

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.

Activation of the inflammasome is involved in the progression of retinal degenerative diseases, in particular, in the pathogenesis of Age-Related Macular Degeneration (AMD), with NLRP3 activation the focus of many investigations. In this study, we used genetic and pharmacological approaches to explore the role of the inflammasome in a mouse model of retinal degeneration. We identify that Casp1/11−/− mice have better-preserved retinal function, reduced inflammation and increased photoreceptor survivability. While Nlrp3−/− mice display some level of preservation of retinal function compared to controls, pharmacological inhibition of NLRP3 did not protect against photoreceptor cell death. Further, Aim2−/−, Nlrc4−/−, Asc−/−, and Casp11−/− mice show no substantial retinal protection. We propose that CASP-1-associated photoreceptor cell death occurs largely independently of NLRP3 and other established inflammasome sensor proteins, or that inhibition of a single sensor is not sufficient to repress the inflammatory cascade. Therapeutic targeting of CASP-1 may offer a more promising avenue to delay the progression of retinal degenerations.

Obesity is a chronic disease with rising worldwide prevalence and largely associated with several other comorbidities, such as cancer, non-alcoholic fatty liver disease (NAFLD), and metabolic syndrome. Hepatic steatosis, a hallmark of NAFLD, is strongly correlated with obesity and has been correlated with changes in the gut microbiota, which can promote its development through the production of short-chain fatty acids (SCFAs) that regulate insulin resistance, bile acid, choline metabolism, and inflammation. Recent studies have suggested a controversial role for the inflammasome/caspase-1 in the development of obesity and non-alcoholic steatohepatitis (NASH). Here, we evaluated the role of inflammasome NLRP3 and caspases 1/11 in the establishment of obesity and hepatic steatosis in diet-induced obese mice, correlating them with the global lipid profile of the liver and gut microbiota diversity. After feeding wild-type, caspases 1/11, and NLRP3 knockout mice with a standard fat diet (SFD) or a high-fat diet (HFD), we found that the caspases 1/11 knockout mice, but not NLRP3 knockout mice, were more susceptible to HFD-induced obesity, and developed enhanced hepatic steatosis even under SFD conditions. Lipidomics analysis of the liver, assessed by MALDI-MS analysis, revealed that the HFD triggered a significant change in global lipid profile in the liver of WT mice compared to those fed an SFD, and this profile was modified by the lack of caspases 1/11 and NLRP3. The absence of caspases 1/11 was also correlated with an increased presence of triacylglycerol in the liver. Gut microbial diversity analysis, using 16S rRNA gene sequencing, showed that there was also an increase of Proteobacteria and a higher Firmicutes/Bacteroidetes ratio in the gut of caspases 1/11 knockout mice fed an HFD. Overall, mice without caspases 1/11 harbored gut bacterial phyla involved with weight gain, obesity, and hepatic steatosis. Taken together, our data suggest an important role for caspases 1/11 in the lipid composition of the liver and in the modulation of the gut microbial community composition. Our results further suggest that HFD-induced obesity and the absence of caspases 1/11 may regulate both lipid metabolism and gut microbial diversity, and therefore may be associated with NAFLD and obesity.

Activated caspase-1 and caspase-11 induce inflammatory cell death in a process termed pyroptosis. Here we show that Prostaglandin E2 (PGE2) inhibits caspase-11-dependent pyroptosis in murine and human macrophages. PGE2 suppreses caspase-11 expression in murine and human macrophages and in the airways of mice with allergic inflammation. Remarkably, caspase-11-deficient mice are strongly resistant to developing experimental allergic airway inflammation, where PGE2 is known to be protective. Expression of caspase-11 is elevated in the lung of wild type mice with allergic airway inflammation. Blocking PGE2 production with indomethacin enhances, whereas the prostaglandin E1 analog misoprostol inhibits lung caspase-11 expression. Finally, alveolar macrophages from asthma patients exhibit increased expression of caspase-4, a human homologue of caspase-11. Our findings identify PGE2 as a negative regulator of caspase-11-driven pyroptosis and implicate caspase-4/11 as a critical contributor to allergic airway inflammation, with implications for pathophysiology of asthma., Caspase 11 activation involves transcriptional upregulation and proteolytic cleavage. Here the authors show that prostaglandin E2 prevents caspase-11-mediated pyroptosis, blocking caspase-11 mRNA and protein upregulation in macrophages and in vivo, and that mice lacking caspase-11 are strongly protected from allergic airway inflammation.

Peptic ulcers are caused by the interaction between bacterial and host factors. This study demonstrates enhanced expression of caspase-4 in peptic ulcer patient biopsies, indicating that pyroptosis and noncanonical inflammasome activity may be processes involved in peptic ulcer disease. We show that primary murine macrophages infected with Helicobacter pylori upregulate caspase-11 (the ortholog of human caspase-4), activate caspase-1, and secrete IL-1β. We demonstrate that misoprostol (a stable PGE1 analogue) decreased IL-1β secretion and delayed lethality in vivo in a murine peritonitis model. PGE2 was shown to inhibit caspase-11–driven pyroptosis and IL-1β secretion in macrophages. Overall, we provide evidence for a pathological role of caspase-4/11 in peptic ulcer disease and propose that targeting caspase-4 or inhibiting pyroptosis may have therapeutic potential in the management of peptic ulcers.

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.

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.

What We Already Know about This Topic What This Article Tells Us That Is New Background Pyroptosis, a type of proinflammatory programmed cell death, drives cytokine storm. Caspase-11–dependent macrophage pyroptosis contributes to mortality during sepsis. Sphingosine-1-phosphate receptor 2 (S1PR2) signaling can amplify interleukin-1β secretion in endotoxin-induced inflammation. Here, we hypothesized that S1PR2 signaling increases caspase-11–dependent macrophage pyroptosis and worsens Gram-negative sepsis outcome. Methods A Gram-negative sepsis model was induced through intraperitoneal injection of Escherichia coli. Primary peritoneal macrophages isolated from wild-type, S1pr2-deficient (S1pr2-/-), or nucleotide-binding oligomerization domain-like receptor protein-3–deficient mice were treated with E. coli. Caspase-11 activation, macrophage pyroptosis, and Ras homolog gene family, member A-guanosine triphosphate levels were assessed in those cells. Additionally, monocyte caspase-4 (an analog of caspase-11) expression and its correlation with S1PR2 expression were determined in patients with Gram-negative sepsis (n = 11). Results Genetic deficiency of S1PR2 significantly improved survival rate (2/10 [20%] in wild-type vs. 7/10 [70%] in S1pr2-/-, P = 0.004) and decreased peritoneal macrophage pyroptosis (pyroptosis rate: 35 ± 3% in wild-type vs. 10 ± 3% in S1pr2-/-, P < 0.001). Decreased caspase-11 activation in S1PR2 deficiency cells contributed to the reduced macrophage pyroptosis. In addition, RhoA inhibitor abrogated the amplified caspase-11 activation in wild-type or S1PR2-overexpressing cells. In patients with Gram-negative sepsis, caspase-4 increased significantly in monocytes compared to nonseptic controls and was positively correlated with S1PR2 (r = 0.636, P = 0.035). Conclusions S1PR2 deficiency decreased macrophage pyroptosis and improved survival in E. coli sepsis. These beneficial effects were attributed to the decreased caspase-11 activation of S1PR2-deficient macrophages. S1PR2 and caspase-11 may be promising new targets for treatment of sepsis.

Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells in the intestinal tract and cause a flu-like diarrheal illness. Innate immunity is key to limiting the expansion of parasitic stages early in infection. One mechanism in which it does this is through the generation of early cytokines, such as IL-18. The processing and secretion of mature IL-18 (and IL-1β) is mediated by caspase-1 which is activated within an inflammasome following the engagement of inflammasome-initiating sensors. We examined how the absence of caspase-1 and caspase-11, the adapter protein Asc, and other inflammasome components affects susceptibility to cryptosporidial infection by these and other key cytokines in the gut. We found that Casp-11−/−Casp-1−/− knockout mice have increased susceptibility to Cryptosporidium parvum infection as demonstrated by the 35-fold higher oocyst production (at peak infection) compared to wild-type mice. Susceptibility correlated with a lack of IL-18 in caspase-1 and caspase1/11 knockout mice, whereas IL-18 is significantly elevated in wildtype mice. IL-1β was not generated in any significant amount following infection nor was any increased susceptibility observed in IL-1β knockout mice. We also show that the adapter protein Asc is important to susceptibility, and that the caspase-1 canonical inflammasome signaling pathway is the dominant pathway in C. parvum resistance.

The innate immune system acts as the first line of defense against infection. One key component of the innate immune response to gram-negative bacterial infections is inflammasome activation. The caspase-11 (CASP11)-nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is activated by cytosolic lipopolysaccharide, a gram-negative bacterial cell wall component, to trigger pyroptosis and host defense during infection. Although several cellular signaling pathways have been shown to regulate CASP11-NLRP3 inflammasome activation in response to lipopolysaccharide, the upstream molecules regulating CASP11 activation during infection with live pathogens remain unclear. Here, we report that the understudied caspase-6 (CASP6) contributes to the activation of the CASP11-NLRP3 inflammasome in response to infections with gram-negative bacteria. Using in vitro cellular systems with bone marrow-derived macrophages and 293T cells, we found that CASP6 can directly process CASP11 by cleaving at Asp59 and Asp285, the CASP11 auto-cleavage sites, which could contribute to the activation of CASP11 during gram-negative bacterial infection. Thus, the loss of CASP6 led to impaired CASP11-NLRP3 inflammasome activation in response to gram-negative bacteria. These results demonstrate that CASP6 potentiates activation of the CASP11-NLRP3 inflammasome to produce inflammatory cytokines during gram-negative bacterial infections.