Neisseria meningitidis activates pyroptotic pathways in a mouse model of meningitis: role of a two-partner secretion system.

There is evidence that in infected cells in vitro the meningococcal HrpA/HrpB two-partner secretion system (TPS) mediates the exit of bacteria from the internalization vacuole and the docking of bacteria to the dynein motor resulting in the induction of pyroptosis. In this study we set out to study the role of the HrpA/HrpB TPS in establishing meningitis and activating pyroptotic pathways in an animal model of meningitis using a reference serogroup C meningococcal strain, 93/4286, and an isogenic hrpB knockout mutant, 93/4286ΩhrpB. Survival experiments confirmed the role of HrpA/HrpB TPS in the invasive meningococcal disease. In fact, the ability of the hrpB mutant to replicate in brain and spread systemically was impaired in mice infected with hrpB mutant. Furthermore, western blot analysis of brain samples during the infection demonstrated that: i. N. meningitidis activated canonical and non-canonical inflammasome pyroptosis pathways in the mouse brain; ii. the activation of caspase-11, caspase-1, and gasdermin-D was markedly reduced in the hrpB mutant; iii. the increase in the amount of IL-1ß and IL-18, which are an important end point of pyroptosis, occurs in the brains of mice infected with the wild-type strain 93/4286 and is strongly reduced in those infected with 93/4286ΩhrpB. In particular, the activation of caspase 11, which is triggered by cytosolic lipopolysaccharide, indicates that during meningococcal infection pyroptosis is induced by intracellular infection after the exit of the bacteria from the internalizing vacuole, a process that is hindered in the hrpB mutant. Overall, these results confirm, in an animal model, that the HrpA/HrpB TPS plays a role in the induction of pyroptosis and suggest a pivotal involvement of pyroptosis in invasive meningococcal disease, paving the way for the use of pyroptosis inhibitors in the adjuvant therapy of the disease.

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.

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.

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.

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 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.

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 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.

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.

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.

Role of Histiocyte-Derived frHMGB1 as a Facilitator in Noncanonical Pyroptosis of Monocytes/Macrophages in Lethal Sepsis.

In this study, we investigated the role of the noncanonical pyroptosis pathway in the progression of lethal sepsis. Our findings emphasize the significance of noncanonical pyroptosis in monocytes/macrophages for the survival of septic mice. We observed that inhibiting pyroptosis alone significantly improved the survival rate of septic mice and that the HMGB1 A box effectively suppressed this noncanonical pyroptosis, thereby enhancing the survival of septic mice. Additionally, our cell in vitro experiments unveiled that frHMGB1, originating from lipopolysaccharide-carrying histiocytes, entered macrophages via RAGE, resulting in the direct activation of caspase 11 and the induction of noncanonical pyroptosis. Notably, A box's competitive binding with lipopolysaccharide impeded its entry into the cell cytosol. These findings reveal potential therapeutic strategies for slowing the progression of lethal sepsis by modulating the noncanonical pyroptosis pathway.

Structural insights into cytokine cleavage by inflammatory caspase-4.

Inflammatory caspases are key enzymes in mammalian innate immunity that control the processing and release of interleukin-1 (IL-1)-family cytokines1,2. Despite the biological importance, the structural basis for inflammatory caspase-mediated cytokine processing has remained unclear. To date, catalytic cleavage of IL-1-family members, including pro-IL-1ß and pro-IL-18, has been attributed primarily to caspase-1 activities within canonical inflammasomes3. Here we demonstrate that the lipopolysaccharide receptor caspase-4 from humans and other mammalian species (except rodents) can cleave pro-IL-18 with an efficiency similar to pro-IL-1ß and pro-IL-18 cleavage by the prototypical IL-1-converting enzyme caspase-1. This ability of caspase-4 to cleave pro-IL-18, combined with its previously defined ability to cleave and activate the lytic pore-forming protein gasdermin D (GSDMD)4,5, enables human cells to bypass the need for canonical inflammasomes and caspase-1 for IL-18 release. The structure of the caspase-4-pro-IL-18 complex determined using cryogenic electron microscopy reveals that pro-lL-18 interacts with caspase-4 through two distinct interfaces: a protease exosite and an interface at the caspase-4 active site involving residues in the pro-domain of pro-IL-18, including the tetrapeptide caspase-recognition sequence6. The mechanisms revealed for cytokine substrate capture and cleavage differ from those observed for the caspase substrate GSDMD7,8. These findings provide a structural framework for the discussion of caspase activities in health and disease.

Caspase-4 dimerisation and D289 auto-processing elicit an interleukin-1ß-converting enzyme.

The noncanonical inflammasome is a signalling complex critical for cell defence against cytosolic Gram-negative bacteria. A key step in the human noncanonical inflammasome pathway involves unleashing the proteolytic activity of caspase-4 within this complex. Caspase-4 induces inflammatory responses by cleaving gasdermin-D (GSDMD) to initiate pyroptosis; however, the molecular mechanisms that activate caspase-4 and govern its capacity to cleave substrates remain poorly defined. Caspase-11, the murine counterpart of caspase-4, acquires protease activity within the noncanonical inflammasome by forming a dimer that self-cleaves at D285 to cleave GSDMD. These cleavage events trigger signalling via the NLRP3-ASC-caspase-1 axis, leading to downstream cleavage of the pro-IL-1ß cytokine precursor. Here, we show that caspase-4 first dimerises then self-cleaves at two sites-D270 and D289-in the interdomain linker to acquire full proteolytic activity, cleave GSDMD, and induce cell death. Surprisingly, caspase-4 dimerisation and self-cleavage at D289 generate a caspase-4 p34/p9 protease species that directly cleaves pro-IL-1ß, resulting in its maturation and secretion independently of the NLRP3 inflammasome in primary human myeloid and epithelial cells. Our study thus elucidates the key molecular events that underpin signalling by the caspase-4 inflammasome and identifies IL-1ß as a natural substrate of caspase-4.

The Protective Effects of Goitrin on LPS-Induced Septic Shock in C57BL/6J Mice via Caspase-11 Non-Canonical Inflammasome Inhibition.

Septic shock is defined as a subset of sepsis, which is associated with a considerably high mortality risk. The caspase-11 non-canonical inflammasome is sensed and activated by intracellular lipopolysaccharide (LPS) leading to pyroptosis, it plays a critical role in septic shock. However, there are few known drugs that can control caspase-11 non-canonical inflammasome activation. We report here that goitrin, an alkaloid from Radix Isatidis, shows protective effects in LPS-induced septic shock and significant inhibitory effect in caspase-11 non-canonical inflammasome pathway. Male C57BL/6J were injected intraperitoneally with LPS (20 mg/kg) to induce experimental septic shock. The results demonstrated that the survival rates of mice pretreated with goitrin or Toll-like receptor 4 (TLR4) inhibitor TKA-242 increased, and LPS-induced hypothermia and lung damage improved by inhibiting inflammatory response. Elucidating the detailed mechanism, we surprisingly found goitrin is really different from TAK-242, it independent of the TLR4 signal activation, but significantly inhibited the activation of caspase-11 non-canonical inflammasome, including cleaved caspase-11 and N-terminal fragment of gasdermin D (GSDMD-NT). Furthermore, with a nonlethal dose of the TLR3 agonist poly(I:C)-primed and subsequently challenged with LPS to induce caspase-11-mediated lethal septic shock, the efficacy of goitrin had been verified. Those results revealed the effect of goitrin in protective against LPS-induced septic shock via inhibiting caspase-11 non-canonical inflammasome, which provided a new therapeutic strategy for clinical treatment of septic shock.

Cigarette smoke promotes inflammasome-independent activation of caspase-1 and -4 leading to gasdermin D cleavage in human macrophages.

Mechanisms and consequences of gasdermin D (GSDMD) activation in cigarette smoke (CS)-associated inflammation and lung disease are unknown. GSDMD is a downstream effector of caspase-1, -8, and -4. Upon cleavage, GSDMD generates pores into cell membranes. Different degrees of GSDMD activation are associated with a range of physiological outputs ranging from cell hyperactivation to pyroptosis. We have previously reported that in human monocyte-derived macrophages CS extract (CSE) inhibits the NLRP3 inflammasome and shifts the response to lipopolysaccharide (LPS) towards the TLR4-TRIF axis leading to activation of caspase-8, which, in turn, activates caspase-1. In the present work, we investigated whether other ASC-dependent inflammasomes could be involved in caspase activation by CSE and whether caspase activation led to GSDMD cleavage and other downstream effects. Presented results demonstrate that CSE promoted ASC-independent activation of caspase-1 leading to GSDMD cleavage and increased cell permeability, in the absence of cell death. GSDMD cleavage was strongly enhanced upon stimulation with LPS+CSE, suggesting a synergistic effect between the two stimuli. Noteworthy, CSE promoted LPS internalization leading to caspase-4 activation, thus contributing to increased GSDMD cleavage. Caspase-dependent GSDMD cleavage was associated with mitochondrial superoxide generation. Increased cleaved GSDMD was found in lung macrophages of smokers compared to ex-smokers and non-smoking controls. Our findings revealed that ASC-independent activation of caspase-1, -4, and -8 and GSDMD cleavage upon exposure to CS may contribute to macrophage dysfunction and feed the chronic inflammation observed in the smokers' lung.

Mitochondrial calpain-5 truncates caspase-4 during endoplasmic reticulum stress.

Calpains are cysteine proteases activated in response to intracellular calcium signaling. Activated calpains regulate various cellular functions by degrading substrate molecules in a site-specific manner. Although most calpains are localized in the cytosol, we previously reported that calpain-5 exists in the mitochondria. The mitochondrial calpain-5 is activated during endoplasmic reticulum (ER) stress. However, the substrate of calpain-5, as well as the physiological significance of calpain-5 activation, has not yet been elucidated. In the present study, we treated HeLa cells with A23187, tunicamycin, or hydrogen peroxide to induce intracellular calcium increase, resulting in cell death. The cells treated with A23187 or tunicamycin exhibited the activation of calpain-5 and truncation of caspase-4. The truncation of caspase-4 was inhibited by the repression of calpain-5 expression with the appropriate siRNA. Additionally, both calpain-5 and caspase-4 were observed in the mitochondria. Our study is the first to demonstrate that the activation of mitochondrial calpain-5 triggers the truncation of caspase-4, suggesting that mitochondrial calpain-5 regulates the downstream pathway of caspase-4, including cell death and the inflammatory cascade. The results of the present study provide new insights into ER-stress-related diseases such as Alzheimer's disease and cancer. These perspectives allow us to propose new therapeutic strategies such as the development of inhibitors or activators of calpain-5, which may be useful in the development of treatment for ER-stress-related diseases.

A Fluorescence-Polarization-Based Lipopolysaccharide-Caspase-4 Interaction Assay for the Development of Inhibitors.

Recognition of intracellular lipopolysaccharide (LPS) by Caspase-4 (Casp-4) is critical for host defense against Gram-negative pathogens. LPS binds to the N-terminal caspase activation and recruitment domain (CARD) of procaspase-4, leading to auto-proteolytic activation followed by pro-inflammatory cytokine release and pyroptotic cell death. Aberrant hyper-activation of Casp-4 leads to amplification of the inflammatory response linked to sepsis. While the active site of a caspase has been targeted with peptide inhibitors, inhibition of LPS-Casp-4 interaction is an emerging strategy for the development of selective inhibitors with a new mode of action for treating infectious diseases and sepsis induced by LPS. In this study, a high-throughput screening (HTS) system based on fluorescence polarization (FP) was devised to identify inhibitors of the LPS and Casp-4 interaction. Using HTS and IC50 determination and subsequently showing inhibited Casp-4 activity, we demonstrated that the LPS-Casp-4 interaction is a druggable target for Casp-4 inhibition and possibly a non-canonical inflammatory pathway.

Vitamin B12 enhances the antitumor activity of 1,25-dihydroxyvitamin D3 via activation of caspases and targeting actin cytoskeleton.

BACKGROUND: 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is an effective anticancer agent, and when combined with other agents it shows superior activities. Vitamin B12 has been shown to contribute to increasing the effectiveness of anticancer drugs when used in combination. Thus, the current study aimed at investigating the anticancer potential of the combination of 1,25(OH)2D3 and vitamin B12. METHODS: MTT assay was used to determine the cytotoxic activity of combining 1,25(OH)2D3 and vitamin B12 against six different cancer cell lines and one normal cell line. The surviving fraction after clonogenic assay was measured, and the effects of 1,25(OH)2D3/B12 combination on the activity of different caspases, cell adhesion, actin cytoskeleton, cell morphology, and percentage of polarized cells were evaluated. RESULTS: Vitamin B12 did not cause cytotoxicity, however, it enhanced the cytotoxicity of 1,25(OH)2D3 against cancer cells. The cytotoxic effects of 1,25(OH)2D3 and its combination with vitamin B12 was not evident in the normal mammary MCF10A cell line indicating cancer cell-specificity. The cytotoxic effects of 1,25(OH)2D3/B12 combination occurred in a dose-dependent manner and was attributed to apoptosis induction which was mediated by caspase 4 and 8. Moreover, 1,25(OH)2D3/B12-treated cells showed enhanced inhibition of clonogenic tumor growth, reduced cell adhesion, reduced cell area, reduced percentage of cell polarization, and disorganized actin cytoskeleton resulting in reduced migratory phenotype when compared to cells treated with 1,25(OH)2D3 alone. CONCLUSION: 1,25(OH)2D3 and vitamin B12 exhibited synergistic anticancer effects against different cancer cell lines. The combination therapy of 1,25(OH)2D3 and vitamin B12 may provide a potential adjunctive treatment option for some cancer types.

The role of caspase-1, caspase-4 and NLRP3 in regulating the host cell response evoked by uropathogenic Escherichia coli.

The inflammasome-associated proteins caspase-1, caspase-4 and NLRP3 have been emphasised to be essential in the host cell response during urinary tract infection (UTI) by regulating IL-1ß release. Our aim was to investigate how the inflammasome-associated proteins regulate the cell response of bladder epithelial cells during infection with uropathogenic Escherichia coli (UPEC). Human bladder epithelial cells (5637) and CRISPR/Cas9 generated caspase-1, caspase-4 and NLRP3 knockdown cells were stimulated with the UPEC strain CFT073. Using Olink proteomics and real time RT-PCR, we showed that caspase-1, caspase-4 and NLRP3 are vital for the expression of many inflammatory genes and proteins from bladder epithelial cells. When investigating the effect of inflammasome-associated proteins on neutrophils, we found that conditioned medium from UPEC-infected caspase-4 knockdown cells significantly increased phagocytosis of CFT073 and significantly decreased ROS production from neutrophils. In contrast, conditioned medium from UPEC-infected NLRP3 knockdown cells significantly decreased the phagocytosis of CFT073 and significantly increased the ROS production from neutrophils. In conclusion, we showed that the inflammasome-associated proteins contribute to the host cell response during UPEC infection.