Caspase-11 mediated inflammasome activation in macrophages by systemic infection of A. actinomycetemcomitans exacerbates arthritis.

Clinical studies have shown that Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is associated with aggressive periodontitis and can potentially trigger or exacerbate rheumatoid arthritis (RA). However, the mechanism is poorly understood. Here, we show that systemic infection with A. actinomycetemcomitans triggers the progression of arthritis in mice anti-collagen antibody-induced arthritis (CAIA) model following IL-1ß secretion and cell infiltration in paws in a manner that is dependent on caspase-11-mediated inflammasome activation in macrophages. The administration of polymyxin B (PMB), chloroquine, and anti-CD11b antibody suppressed inflammasome activation in macrophages and arthritis in mice, suggesting that the recognition of lipopolysaccharide (LPS) in the cytosol after bacterial degradation by lysosomes and invasion via CD11b are needed to trigger arthritis following inflammasome activation in macrophages. These data reveal that the inhibition of caspase-11-mediated inflammasome activation potentiates aggravation of RA induced by infection with A. actinomycetemcomitans. This work highlights how RA can be progressed by inflammasome activation as a result of periodontitis-associated bacterial infection and discusses the mechanism of inflammasome activation in response to infection with A. actinomycetemcomitans.

Interleukin-17F suppressed colon cancer by enhancing caspase 4 mediated pyroptosis of endothelial cells.

The combination of anti-angiogenic treatment and immunotherapy presents a promising strategy against colon cancer. Interleukin-17F (IL-17F) emerges as a critical immune cell cytokine expressed in colonic epithelial cells, demonstrating potential in inhibiting angiogenesis. In order to clarify the roles of IL-17F in the colon cancer microenvironment and elucidate its mechanism, we established a mouse colon carcinoma cell line CT26 overexpressing IL-17F and transplanted it subcutaneously into syngeneic BALB/c mice. We also analyzed induced colon tumor in IL-17F knockout and wild type mice. Our results demonstrated that IL-17F could suppress colon tumor growth in vivo with inhibited angiogenesis and enhanced recruitment of cysteine-cysteine motif chemokine receptor 6 (CCR6) positive immune cells. Additionally, IL-17F suppressed the tube formation, cell growth and migration of endothelial cells EOMA in vitro. Comprehensive bioinformatics analysis of transcriptome profiles between EOMA cells and those treated with three different concentrations of IL-17F identified 109 differentially expressed genes. Notably, a potential new target, Caspase 4, showed increased expressions after IL-17F treatment in endothelial cells. Further molecular validation revealed a novel downstream signaling for IL-17F: IL-17F enhanced Caspase 4/GSDMD signaling of endothelial cells, CT26 cells and CT26 transplanted tumors, while IL-17F knockout colon tumors exhibited decreased Caspase 4/GSDMD signaling. The heightened expression of the GSDMD N-terminus, coupled with increased cellular propidium iodide (PI) uptake and lactate dehydrogenase (LDH) release, revealed that IL-17F promoted pyroptosis of endothelial cells. Altogether, IL-17F could modulate the colon tumor microenvironment with inhibited angiogenesis, underscoring its potential as a therapeutic target for colon cancer.

Plexin C1 influences immune response to intracellular LPS and survival in murine sepsis.

BACKGROUND: Intracellular sensing of lipopolysaccharide (LPS) is essential for the immune response against gram-negative bacteria and results in activation of caspase-11 and pyroptotic cell death with fatal consequences in sepsis. We found the neuronal guidance receptor plexin C1 (PLXNC1) influences the intracellular response to LPS. METHODS: We employed a murine model of sepsis via cecal ligation and binding (CLP), using PLXNC1-/- mice and littermate controls, and additionally transfected murine bone-marrow-derived macrophages (BMDMs) from both genotypes with LPS to achieve activation of the noncanonical inflammasome ex vivo. Additionally, we transfected the PLXNC1 ligand SL4c-d in vivo and ex vivo to examine its effect on intracellular LPS response. RESULTS: We found the neuronal guidance receptor PLXNC1 dampens the intracellular response to LPS by interacting with adenylate cyclase 4 (ADCY4) and protein kinase A activity, which in turn diminishes caspase-11 expression. The absence of PLXNC1 results in excessive inflammation marked by increased cytokine release, increased secondary organ injury and reduced sepsis survival in a murine sepsis model induced by CLP. Notably, administration of SL4c-d-peptide ligand of PLXNC1-reduces the inflammatory response during CLP-induced sepsis and improves survival. CONCLUSIONS: These results elucidate a previously unknown mechanism for PLXNC1 suppressing excessive noncanonical inflammasome activity and offer a new potential target for treatment of sepsis with its detrimental effects.

Caspase-11 signaling promotes damage to hippocampal CA3 to enhance cognitive dysfunction in infection.

BACKGROUND: Cognitive dysfunction caused by infection frequently emerges as a complication in sepsis survivor patients. However, a comprehensive understanding of its pathogenesis remains elusive. METHODS: In our in vivo experiments, an animal model of endotoxemia was employed, utilizing the Novel Object Recognition Test and Morris Water Maze Test to assess cognitive function. Various techniques, including immunofluorescent staining, Western blotting, blood‒brain barrier permeability assessment, Limulus Amebocyte Lysate (LAL) assay, and Proximity-ligation assay, were employed to identify brain pathological injury and neuroinflammation. To discern the role of Caspase-11 (Casp11) in hematopoietic or non-hematopoietic cells in endotoxemia-induced cognitive decline, bone marrow chimeras were generated through bone marrow transplantation (BMT) using wild-type (WT) and Casp11-deficient mice. In vitro studies involved treating BV2 cells with E. coli-derived outer membrane vesicles to mimic in vivo conditions. RESULTS: Our findings indicate that the deficiency of Casp11-GSDMD signaling pathways reverses infection-induced cognitive dysfunction. Moreover, cognitive dysfunction can be ameliorated by blocking the IL-1 effect. Mechanistically, the absence of Casp11 signaling significantly mitigated blood‒brain barrier leakage, microglial activation, and synaptic damage in the hippocampal CA3 region, ultimately leading to improved cognitive function. CONCLUSION: This study unveils the crucial contribution of Casp11 and GSDMD to cognitive impairments and spatial memory loss in a murine sepsis model. Targeting Casp11 signaling emerges as a promising strategy for preventing or treating cognitive dysfunction in patients with severe infections.

GSDMB involvement in the pathogenesis of abdominal aortic aneurysm through regulation of macrophage non-canonical pyroptosis.

Abdominal aortic aneurysm (AAA) is a dangerous condition affecting the aorta. Macrophage pyroptosis, phenotypic transformation, and apoptosis of aortic smooth muscle cells (ASMCs) are pivotal mechanisms in AAA pathogenesis. This study explores how Gasdermin B (GSDMB) regulates macrophage non-canonical pyroptosis and its impact on the phenotypic transformation and apoptosis of ASMCs, thereby unveiling the role of GSDMB in AAA pathogenesis. Immunofluorescence analysis was used to assess the expression levels and localization of GSDMB, cysteinyl aspartate-specific protease-4 (Caspase-4), and N-terminal of cleaved GSDMD (N-GSDMD) in AAA tissues. A cell model that mimics macrophage non-canonical pyroptosis was established by treating THP-1 cells with lipopolysaccharide (LPS). THP-1 cells with reduced or increased GSDMB were generated using small interfering RNA (siRNA) or plasmids. Co-culture experiments involving THP-1 cells and HASMCs were conducted to explore the impact of GSDMB on HASMCs. The mitochondrial reactive oxygen species (mtROS) scavenger Mito-TEMPO lowered mtROS levels in THP-1 cells. Our findings revealed that GSDMB was significantly upregulated in AAA macrophages, which was accompanied by robust non-canonical pyroptosis. THP-1 cells showed non-canonical pyroptosis in response to LPS, which was accompanied by an increase in GSDMB. Further research demonstrated that altering GSDMB, either by knockdown or overexpression, can affect macrophage non-canonical pyroptosis as well as the phenotypic transformation and apoptosis of HASMCs. LPS-induced non-canonical pyroptosis in THP-1 cells was associated with an increase in mtROS, whereas Mito-TEMPO effectively decreased non-canonical pyroptosis and the expression of GSDMB. These findings suggest that GSDMB plays a role in AAA macrophage non-canonical pyroptosis, which influences the phenotypic transformation and apoptosis of HASMCs. The mtROS-Dynamin-Related Protein 1 (Drp1) axis is likely to regulate the GSDMB-mediated non-canonical pyroptosis.

Caspase-4/11 promotes hyperlipidemia and chronic kidney disease-accelerated vascular inflammation by enhancing trained immunity.

To determine whether hyperlipidemia and chronic kidney disease (CKD) have a synergy in accelerating vascular inflammation via trained immunity (TI), we performed aortic pathological analysis and RNA-Seq of high-fat diet-fed (HFD-fed) 5/6 nephrectomy CKD (HFD+CKD) mice. We made the following findings: (a) HFD+CKD increased aortic cytosolic LPS levels, caspase-11 (CASP11) activation, and 998 gene expressions of TI pathways in the aorta (first-tier TI mechanism); (b) CASP11-/- decreased aortic neointima hyperplasia, aortic recruitment of macrophages, and casp11-gasdermin D-mediated cytokine secretion; (c) CASP11-/- decreased N-terminal gasdermin D (N-GSDMD) membrane expression on aortic endothelial cells and aortic IL-1B levels; (d) LPS transfection into human aortic endothelial cells resulted in CASP4 (human)/CASP11 (mouse) activation and increased N-GSDMD membrane expression; and (e) IL-1B served as the second-tier mechanism underlying HFD+CKD-promoted TI. Taken together, hyperlipidemia and CKD accelerated vascular inflammation by promoting 2-tier trained immunity.

Caspase-4 in glioma indicates deterioration and unfavorable prognosis by affecting tumor cell proliferation and immune cell recruitment.

Gliomas are the most common malignant tumors of the central nervous system, accounting for approximately 80% of all malignant brain tumors. Accumulating evidence suggest that pyroptosis plays an essential role in the progression of cancer. Unfortunately, the effect of the pyroptosis-related factor caspase-4 (CASP4) on immunotherapy and drug therapy for tumors has not been comprehensively investigated. In this study, we systematically screened six hub genes by pooling differential pyroptosis-related genes in The Cancer Genome Atlas (TCGA) glioma data and the degree of centrality of index-related genes in the protein-protein interaction network. We performed functional and pathway enrichment analyses of the six hub genes to explore their biological functions and potential molecular mechanisms. We then investigated the importance of CASP4 using Kaplan-Meier survival analysis of glioma patients. TCGA and the Chinese Glioma Genome Atlas (CGGA) databases showed that reduced CASP4 expression leads to the potent clinical deterioration of glioma patients. Computational analysis of the effect of CASP4 on the infiltration level and recruitment of glioma immune cells revealed that CASP4 expression was closely associated with a series of tumor-suppressive immune checkpoint molecules, chemokines, and chemokine receptors. We also found that aberrant CASP4 expression correlated with chemotherapeutic drug sensitivity. Finally, analysis at the cellular and tissue levels indicated an increase in CASP4 expression in glioma, and that CASP4 inhibition significantly inhibited the proliferation of glioma cells. Thus, CASP4 is implicated as a new prognostic biomarker for gliomas with the potential to further guide immunotherapy and chemotherapy strategies for glioma patients.

TrxR1 is involved in the activation of Caspase-11 by regulating the oxidative-reductive status of Trx-1.

Sepsis is a common complication of infections that significantly impacts the survival of critically patients. Currently, effective pharmacological treatment strategies are lacking. Auranofin, known as an inhibitor of Thioredoxin reductase (TrxR), exhibits anti-inflammatory activity, but its role in sepsis is not well understood. Here, we demonstrate the significant inhibitory effect of Auranofin on sepsis in a cecal ligation and puncture (CLP) mouse model. In vitro, Auranofin inhibits pyroptosis triggered by Caspase-11 activation. Further investigations reveal that inhibiting TrxR1 suppresses macrophage pyroptosis induced by E. coli, while TrxR2 does not exhibit this effect. TrxR1, functioning as a reductase, regulates the oxidative-reductive status of Thioredoxin-1 (Trx-1). Mechanistically, the modulation of Trx-1's reductive activity by TrxR1 may be involved in Caspase-11 activation-induced pyroptosis. Additionally, inhibiting TrxR1 maintains Trx-1 in its oxidized state. The oxidized form of Trx-1 interacts with Caveolin-1 (CAV1), regulating outer membrane vesicle (OMV) internalization. In summary, our study suggests that inhibiting TrxR1 suppresses OMV internalization by maintaining the oxidized form of Trx-1, thereby restricting Caspase-11 activation and alleviating sepsis.

Caspase-4 promotes metastasis and interferon-γ-induced pyroptosis in lung adenocarcinoma.

Caspase-4 (CASP4) is a member of the inflammatory caspase subfamily and promotes inflammation. Here, we report that CASP4 in lung adenocarcinoma cells contributes to both tumor progression via angiogenesis and tumor hyperkinesis and tumor cell killing in response to high interferon (IFN)-γ levels. We observe that elevated CASP4 expression in the primary tumor is associated with cancer progression in patients with lung adenocarcinoma. Further, CASP4 knockout attenuates tumor angiogenesis and metastasis in subcutaneous tumor mouse models. CASP4 enhances the expression of genes associated with angiogenesis and cell migration in lung adenocarcinoma cell lines through nuclear factor kappa-light chain-enhancer of activated B cell signaling without stimulation by lipopolysaccharide or tumor necrosis factor. CASP4 is induced by endoplasmic reticulum stress or IFN-γ via signal transducer and activator of transcription 1. Most notably, lung adenocarcinoma cells with high CASP4 expression are more prone to IFN-γ-induced pyroptosis than those with low CASP4 expression. Our findings indicate that the CASP4 level in primary lung adenocarcinoma can predict metastasis and responsiveness to high-dose IFN-γ therapy due to cancer cell pyroptosis.

Inhibition of caspase-11 under inflammatory conditions suppresses chondrogenic differentiation.

Caspase-11 is the murine homologue of human caspases-4 and -5 and is involved in mediating the inflammatory response. However, its functions are often confused and misinterpreted with the more important and better described caspase-1. Therefore, this study focused exclusively on the specific roles of caspase-11, both in cartilage formation and in the inflammatory environment. The presence of caspase-11 during mouse limb development and in chondrogenic cell cultures was investigated by immunofluorescence detection. Subsequently, the function of caspase-11 was downregulated and the affected molecules investigated. The expression analysis applied for osteo/chondrogenesis associated factors and inflammatory cytokines. Simultaneously, morphological appearance of the micromass cultures was evaluated. The results revealed that caspase-11 is physiologically present during cartilage development, but its inhibition under physiological conditions has no significant effect on chondrogenic differentiation. However, in an inflammatory environment, inhibition and downregulation of caspase-11 leads to reduced differentiation of cartilage nodules. Additionally, reduced expression of several genes including Col2a1 and Sp7 and conversely increased expression of Mmp9 were observed. In the cytokine expression panel, a significant decrease was found in molecules that, along with the inflammatory function, may also be involved in cartilage differentiation. The findings bring new information about caspase-11 in chondrogenesis and show that its downregulation under inflammatory conditions reduces cartilage formation.

Characterization of the Apoptotic and Antimicrobial Activities of Two Initiator Caspases of Sea Cucumber Apostichopus japonicus.

Caspase (CASP) is a protease family that plays a vital role in apoptosis, development, and immune response. Herein, we reported the identification and characterization of two CASPs, AjCASPX1 and AjCASPX2, from the sea cucumber Apostichopus japonicus, an important aquaculture species. AjCASPX1/2 share similar domain organizations with the vertebrate initiator caspases CASP2/9, including the CARD domain and the p20/p10 subunits with conserved functional motifs. However, compared with human CASP2/9, AjCASPX1/2 possess unique structural features in the linker region between p20 and p10. AjCASPX1, but not AjCASPX2, induced marked apoptosis of human cells by activating CASP3/7. The recombinant proteins of AjCASPX2 and the CARD domain of AjCASPX2 were able to bind to a wide range of bacteria, as well as bacterial cell wall components, and inhibit bacterial growth. AjCASPX1, when expressed in Escherichia coli, was able to kill the host bacteria. Under normal conditions, AjCASPX1 and AjCASPX2 expressions were most abundant in sea cucumber muscle and coelomocytes, respectively. After bacterial infection, both AjCASPX1 and AjCASPX2 expressions were significantly upregulated in sea cucumber tissues and cells. Together, these results indicated that AjCASPX1 and AjCASPX2 were initiator caspases with antimicrobial activity and likely functioned in apoptosis and immune defense against pathogen infection.

Caspase-5: Structure, Pro-Inflammatory Activity and Evolution.

Caspase-5 is a protease that induces inflammation in response to lipopolysaccharide (LPS), a component of the cell envelope of Gram-negative bacteria. The expression level of the CASP5 gene is very low in the basal state, but strongly increases in the presence of LPS. Intracellular LPS binds to the caspase activation and recruitment domain (CARD) of caspase-5, leading to the formation of a non-canonical inflammasome. Subsequently, the catalytic domain of caspase-5 cleaves gasdermin D and thereby facilitates the formation of cell membrane pores through which pro-inflammatory cytokines of the interleukin-1 family are released. Caspase-4 is also able to form a non-canonical inflammasome upon binding to LPS, but its expression is less dependent on LPS than the expression of caspase-5. Caspase-4 and caspase-5 have evolved via the duplication of a single ancestral gene in a subclade of primates, including humans. Notably, the main biomedical model species, the mouse, has only one ortholog, namely caspase-11. Here, we review the structural features and the mechanisms of regulation that are important for the pro-inflammatory roles of caspase-5. We summarize the interspecies differences and the evolution of pro-inflammatory caspases in mammals and discuss the potential roles of caspase-5 in the defense against Gram-negative bacteria and in sepsis.

Brain endothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

African swine fever virus infection regulates pyroptosis by cleaving gasdermin A via active caspase-3 and caspase-4.

African swine fever, caused by the African swine fever virus (ASFV), is a viral hemorrhagic disease that affects domestic pigs and wild boars. ASFV infection causes extensive tissue damage, and the associated mechanism is poorly understood. Pyroptosis is characterized by the activation of inflammatory caspases and pore formation in the cellular plasma membrane, resulting in the release of inflammatory cytokines and cell damage. How ASFV infection regulates pyroptosis remains unclear. Here, using siRNA assay and overexpression methods, we report that ASFV infection regulated pyroptosis by cleaving the pyroptosis execution protein gasdermin A (GSDMA). ASFV infection activated caspase-3 and caspase-4, which specifically cleaved GSDMA at D75-P76 and D241-V242 to produce GSDMA into five fragments, including GSDMA-N1-75, GSDMA-N1-241, and GSDMA-N76-241 fragments at the N-terminal end of GSDMA. Only GSDMA-N1-241, which was produced in the late stage of ASFV infection, triggered pyroptosis and inhibited ASFV replication. The fragments, GSDMA-N1-75 and GSDMA-N76-241, lose the ability to induce pyroptosis. Overall ASFV infection differentially regulates pyroptosis by GSDMA in the indicated phase, which may be conducive to its own replication. Our findings reveal a novel molecular mechanism for the regulation of pyroptosis.

Adipose tissue IL-18 production is independent of caspase-1 and caspase-11.

BACKGROUND: Inflammation in adipose tissue, resulting from imbalanced caloric intake and energy expenditure, contributes to the metabolic dysregulation observed in obesity. The production of inflammatory cytokines, such as IL-1ß and IL-18, plays a key role in this process. While IL-1ß promotes insulin resistance and diabetes, IL-18 regulates energy expenditure and food intake. Previous studies have suggested that caspase-1, activated by the Nlrp3 inflammasome in response to lipid excess, mediates IL-1ß production, whereas activated by the Nlrp1b inflammasome in response to energy excess, mediates IL-18 production. However, this has not been formally tested. METHODS: Wild-type and caspase-1-deficient Balb/c mice, carrying the Nlrp1b1 allele, were fed with regular chow or a high-fat diet for twelve weeks. Food intake and mass gain were recorded weekly. At the end of the twelve weeks, glucose tolerance and insulin resistance were evaluated. Mature IL-18 protein levels and the inflammatory process in the adipose tissue were determined. Fasting lipid and cytokine levels were quantified in the sera of the different experimental groups. RESULTS: We found that IL-18 production in adipose tissue is independent of caspase-1 activity, regardless of the metabolic state, while Nlrp3-mediated IL-1ß production remains caspase-1 dependent. Additionally, caspase-1 null Balb/c mice did not develop metabolic abnormalities in response to energy excess from the high-fat diet. CONCLUSION: Our findings suggest that IL-18 production in the adipose tissue is independent of Nlrp3 inflammasome and caspase-1 activation, regardless of caloric food intake. In contrast, Nlrp3-mediated IL-1ß production is caspase-1 dependent. These results provide new insights into the mechanisms underlying cytokine production in the adipose tissue during both homeostatic conditions and metabolic stress, highlighting the distinct roles of caspase-1 and the Nlrp inflammasomes in regulating inflammatory responses.

A bacterial toxin co-opts caspase-3 to disable active gasdermin D and limit macrophage pyroptosis.

During infections, host cells are exposed to pathogen-associated molecular patterns (PAMPs) and virulence factors that stimulate multiple signaling pathways that interact additively, synergistically, or antagonistically. The net effect of such higher-order interactions is a vital determinant of the outcome of host-pathogen interactions. Here, we demonstrate one such complex interplay between bacterial exotoxin- and PAMP-induced innate immune pathways. We show that two caspases activated during enterohemorrhagic Escherichia coli (EHEC) infection by lipopolysaccharide (LPS) and Shiga toxin (Stx) interact in a functionally antagonistic manner; cytosolic LPS-activated caspase-11 cleaves full-length gasdermin D (GSDMD), generating an active pore-forming N-terminal fragment (NT-GSDMD); subsequently, caspase-3 activated by EHEC Stx cleaves the caspase-11-generated NT-GSDMD to render it nonfunctional, thereby inhibiting pyroptosis and interleukin-1ß maturation. Bacteria typically subvert inflammasomes by targeting upstream components such as NLR sensors or full-length GSDMD but not active NT-GSDMD. Thus, our findings uncover a distinct immune evasion strategy where a bacterial toxin disables active NT-GSDMD by co-opting caspase-3.

Fluoride induces pyroptosis via IL-17A-mediated caspase-1/11-dependent pathways and Bifidobacterium intervention in testis.

Fluoride, a ubiquitous environmental pollutant, poses a significant public health threat. Our previous study revealed a correlation between fluoride-induced testicular pyroptosis and male reproductive dysfunction. However, the underlying mechanism remains unclear. Wild-type and interleukin 17A knockout mice were exposed to sodium fluoride (100 mg/L) in deionized drinking water for 18 weeks. Bifidobacterium intervention (1 × 109 CFU/mL, 0.2 mL/day, administered via gavage) commenced in the 10th week. Sperm quality, testicular morphology, key pyroptosis markers, spermatogenesis key genes, IL-17A signaling pathway, and pyroptosis pathway related genes were determined. The results showed that fluoride reduced sperm quality, damaged testicular morphology, affected spermatogenesis, elevated IL-17A levels, and induced testicular pyroptosis. Bifidobacterium intervention alleviated adverse reproductive outcomes. Fluoride-activated testicular pyroptosis through both typical and atypical pathways, with IL-17A involvement. Bifidobacterium supplementation attenuated pyroptosis by downregulating IL-17A, inhibiting NLRP3 and PYRIN-mediated caspase-1 and caspase-11 dependent pathways in testis, thereby alleviating fluoride-induced male reproductive damage. In summary, this study uncovers the mechanism underlying fluorine-induced testicular pyroptosis and illustrates the novel protecting feature of Bifidobacterium against fluoride-induced harm to male reproduction, along with its potential regulatory mechanism. These results provide fresh perspectives on treating male reproductive dysfunction resulting from fluoride or other environmental toxins.

Transcription suppression of GABARAP mediated by lncRNA XIST-EZH2 interaction triggers caspase-11-dependent inflammatory injury in ulcerative colitis.

BACKGROUND: We have previously found that enhancer of zeste homolog 2 (EZH2) is correlated with inflammatory infiltration and mucosal cell injury in ulcerative colitis (UC). This study aims to analyze the role of X-inactive specific transcript (XIST), a possible interactive long non-coding RNA of EZH2, in UC and to explore the mechanisms. METHODS: C57BL/6N mice were treated with dextran sulfate sodium (DSS), and mouse colonic mucosal epithelial cells were treated with DSS and lipopolysaccharide (LPS) for UC modeling. The UC-related symptoms in mice, and the viability and apoptosis of mucosal epithelial cells were determined. Inflammatory injury in animal and cellular models were assessed through the levels of ACS, occludin, IL-1ß, IL-18, TNF-α, caspase-1, and caspase-11. Molecular interactions between XIST, EZH2, and GABA type A receptor-associated protein (GABARAP) were verified by immunoprecipitation assays, and their functions in inflammatory injury were determined by gain- or loss-of-function assays. RESULTS: XIST was highly expressed in DSS-treated mice and in DSS + LPS-treated mucosal epithelial cells. It recruited EZH2, which mediated gene silencing of GABARAP through H3K27me3 modification. Silencing of XIST alleviated body weight loss, colon shortening, and disease active index of mice and reduced inflammatory injuries in their colon tissues. Meanwhile, it reduced apoptosis and inflammation in mucosal epithelial cells. However, these alleviating effects were blocked by either EZH2 overexpression or GABARAP knockdown. Rescue experiments identified caspase-11 as a key effector mediating the inflammatory injury following GABARAP loss. CONCLUSION: This study suggests that the XIST-EZH2 interaction-mediated GABARAP inhibition activates caspase-11-dependent inflammatory injury in UC.

DNA hypomethylation promotes the expression of CASPASE-4 which exacerbates inflammation and amyloid-ß deposition in Alzheimer's disease.

Alzheimer's disease (AD) is the sixth leading cause of death in the USA. It is established that neuroinflammation contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the brain. For instance, microglia release pro-inflammatory products such as IL-1ß which is highly implicated in AD pathobiology. The mechanisms underlying the transition of microglia to proinflammatory promoters of AD remain largely unknown. To address this gap, we performed reduced representation bisulfite sequencing (RRBS) to profile global DNA methylation changes in human AD brains compared to no disease controls. We identified differential DNA methylation of CASPASE-4 (CASP4), which when expressed promotes the generation of IL-1ß and is predominantly expressed in immune cells. DNA upstream of the CASP4 transcription start site was hypomethylated in human AD brains, which was correlated with increased expression of CASP4. Furthermore, microglia from a mouse model of AD (5xFAD) express increased levels of CASP4 compared to wild-type (WT) mice. To study the role of CASP4 in AD, we developed a novel mouse model of AD lacking the mouse ortholog of CASP4 and CASP11, which is encoded by mouse Caspase-4 (5xFAD/Casp4-/-). The expression of CASP11 was associated with increased accumulation of pathologic protein aggregate amyloid-ß (Aß) and increased microglial production of IL-1ß in 5xFAD mice. Utilizing RNA-sequencing, we determined that CASP11 promotes unique transcriptomic phenotypes in 5xFAD mouse brains, including alterations of neuroinflammatory and chemokine signaling pathways. Notably, in vitro, CASP11 promoted generation of IL-1ß from macrophages in response to cytosolic Aß through cleavage of downstream effector Gasdermin D (GSDMD). Therefore, here we unravel the role for CASP11 and GSDMD in the generation of IL-1ß in response to Aß and the progression of pathologic inflammation in AD. Overall, our results demonstrate that overexpression of CASP4 due to differential DNA methylation in AD microglia contributes to the progression of AD pathobiology. Thus, we identify CASP4 as a potential target for immunotherapies for the treatment and prevention of AD.

Native architecture of a human GBP1 defense complex for cell-autonomous immunity to infection.

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo-electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our "open conformer" model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.