Caspase-1 Activation Research Articles

Abstract 15308: NLRP3 Activation and Calcium-Dependent Contractile Function in Rat Right Ventricle Cardiomyocytes (RVCMs) Are Sexually Dimorphic and Controlled by 17β-Estradiol-via Estrogen Receptor-α

Introduction: NLRP3 inflammasome activation promotes right ventricle (RV) contractile dysfunction in pulmonary hypertension (PH). However, the role of NLRP3 signaling in RVCMs has not been studied. 17β-estradiol (E2) improves RVCM function in PH. Hypothesis: E2, via estrogen receptor-α (ERα), prevents RVCM NLRP3 inflammasome activation and contractile dysfunction in experimental PH. Methods: RV failure in male and female Sprague Dawley rats were induced with monocrotaline (MCT). Co-localization of NLRP3 and its partner ASC was assessed. RVCMs isolated from healthy male or female wildtype (WT) or ERα loss of function mutant (ERα mut ) rats were treated with NLRP3 activators lipopolysaccharide (LPS 1 μg/mL, 4 h) and ATP (2 mM, 10 min) ± E2 (1 nM, 24 h) or NLRP3 inhibitor MCC950 (1 μM, 30 min). RVCM contractility and Ca 2+ levels were evaluated via IONOPTIX system. Protein levels of NLRP3 and its targets were assessed. ERα binding to the NLRP3 promoter was assessed by immunoprecipitation. RNA-seq was performed in RV tissues from male and intact or ovariectomized female rats with RV failure. Results: RNA-seq demonstrated that transcripts involved in NLRP3 activation are E2-regulated. RVCMs from male, but not female, MCT rats showed increased NLRP3 and ASC co-localization/activation (p<0.05). In addition, male MCT rats RVs exhibited increased activation of NLRP3 downstream mediators caspase-1 and IL-1β (p<0.05). Cultured male RVCMs treated with LPS+ATP demonstrated more pronounced NLRP3 activation and decreased contractility and Ca 2+ levels than female RVCMs (p<0.05). NLRP3 activation in RVCMs from male rats resulted in reduced contractility; this was abolished with NLRP3 inhibition (p<0.05). E2 treatment in male RVCMs reduced NLRP3 and ASC interaction and increased contractility Ca 2+ levels in male WT but not in ERα mut RVCMs (p<0.05). IP demonstrated ERα binding to the NLRP3 promoter. Conclusion: NLRP3 signaling in RVCMs from MCT-PH rats is sexually dimorphic, with more pronounced activation and contractile dysfunction in males. E2 prevents RVCM NLRP3 activation and contractile dysfunction in an ERα-dependent manner, likely mediated by ERα binding to the NLRP3 promoter. NLRP3 activation may be therapeutically targetable in PH patients of either sex.

Pseudomonas aeruginosa-Derived DnaJ Induces the Expression of IL-1β by Engaging the Interplay of p38 and ERK Signaling Pathways in Macrophages.

As members of pathogen-associated molecular patterns, bacterial heat shock proteins (HSPs) are widely recognized for their role in initiating innate immune responses. This study aimed to examine the impact of DnaJ, a homolog of HSP40 derived from Pseudomonas aeruginosa (P. aeruginosa), on the regulation of IL-1β expression in macrophages. We demonstrated that DnaJ modulates macrophages to secrete IL-1β by activating NF-κB and MAPK signaling pathways. Specifically, ERK was identified as a positive mediator for IL-1β expression, while p38 acted as a negative mediator. These results suggest that the reciprocal actions of these two crucial MAPKs play a vital role in controlling IL-1β expression. Additionally, the reciprocal actions of MAPKs were found to regulate the activation of inflammasome-related molecules, including vimentin, NLRP3, caspase-1, and GSDMD. Furthermore, our investigation explored the involvement of CD91/CD40 in ERK signaling-mediated IL-1β production from DnaJ-treated macrophages. These findings emphasize the importance of understanding the signaling mechanisms underlying IL-1β induction and suggest the potential utility of DnaJ as an adjuvant for stimulating inflammasome activation.

Mechanisms of Inflammatory Caspase Activation in Sickle Cell Disease

Background: Sickle cell disease (SCD) is an inherited red blood cell disorder that impacts millions of people worldwide. Patients with SCD experience chronic hemolysis and have elevated serum levels of free heme. Free heme activates a variety of inflammatory pathways that contribute to the serious clinical complications of SCD. One of the inflammatory pathways activated by heme is the inflammatory caspase pathway. Our lab previously showed that heme exposure in macrophages leads to the activation of inflammatory caspase proteins caspase-1 and caspase-4. Activation of these proteins by heme leads to IL-1β release and pyroptosis, a form of inflammatory cell death. Patients with SCD have increased circulating IL-1β levels, indicating this pathway is active in patients. In mouse models, inflammatory caspases are required for hemolysis-induced neutrophil recruitment and lethality, indicating these proteins are central to hemolysis-induced inflammation. Despite the key role these proteins play following hemolysis, it remains unknown how heme activates inflammatory caspases. Interestingly, both caspase-1 and caspase-4 are required for heme-induced IL-1β release while caspase-4 alone is required for heme-induced pyroptosis. In fact, pyroptosis likely is required for IL-1β release from the cell. Based on this data, we hypothesized that caspase-4 is the apical step in this pathway and a key druggable target for controlling unchecked inflammation in SCD. Results: We first sought to determine if caspase-4 activation is upstream of caspase-1 activation. To assess this, we used an imaging-based caspase activation reporter assay. Using this system, we found that siRNA knock-down of caspase-4 in primary human macrophages significantly inhibited heme-induced caspase-1 activation (63.8% vs 26.3%, n =4, p = 0.015). This result shows that caspase-4 is upstream of heme-induced caspase-1 activation and confirmed caspase-4 activation is the initial step in this pathway. Next, we sought to determine how caspase-4 activation occurs. Other known activators of caspase-4 induce activation by directly binding to caspase-4 and inducing self-oligomerization. To determine if heme acts in a similar manner, we used a published heme-binding prediction algorithm called HeMoQuest. We found multiple predicted heme-binding sites in caspase-4. The dissociation constants for these binding sites are less than 1 µM, suggesting strong binding affinity. Importantly, there are no predicted heme binding sites in caspase-1, indicating caspase-4 is the only direct heme receptor in this pathway. To validate these binding predictions, we performed molecular docking experiments and confirmed there are multiple heme-binding sites in caspase-4. Specifically, we found two heme-binding regions in the p10 catalytic subunit of caspase-4 comprising 2-4 essential residues. We next confirmed that heme directly interacts with caspase-4 in vitro. Using a pull-down assay with heme conjugated to agarose beads, we observed heme bound to purified caspase-4 and to casapase-4 in macrophage cell-free extracts. Heme did not interact with proteins structurally related to caspase-4 including caspase-1, caspase-7, and caspase-9 and caspase-4 was not pulled down by control agarose. This confirms the specificity of the heme-caspase-4 interaction. To determine if heme binding to caspase-4 results in activation, we performed native PAGE analysis of purified caspase-4 after exposure to heme. Heme induced formation of a caspase-4 oligomer around 200 kDa in size, four-fold larger than the inactive caspase-4 monomer. Other compounds known to induce IL-1β release from cells, like microbial toxin nigericin, did not induce caspase-4 oligomerization, demonstrating the specificity of heme's effect on caspase-4. Conclusions: We have determined that caspase-4 is a direct intracellular heme receptor and that heme exposure leads to caspase-4 activation, which in turn facilitates caspase-1 activation, IL-1β release and cell death. Our future work will elucidate the functional effects of heme binding to caspase-4 in mouse models of sickle cell disease. Caspase-4 is an attractive therapeutic target as it is required for both functional outputs of inflammatory caspase activation, IL-1β release and pyroptosis. Inhibiting heme-caspase-4 interactions would provide a specific way to block the harmful effects of this pathway in patients.

Mitochondria-Mediated (MAVS) Innate Immune Signaling Drives Inflammation-Induced Bone Marrow Failure

Bone marrow failure syndromes (BMFS) are life threatening diseases, characterized by impaired fitness of hematopoietic stem cells (HSC) and ineffective hematopoiesis causing the absence of one or more hematopoietic lineages in the peripheral blood. Substantial clinical data indicate that hyperactivity of inflammatory cytokines, including TNFα, IL-6, and transforming growth factor-β (TGFβ), directly contribute to BMF. Inflammatory challenges result in irreversible HSC functional decline, including decreased repopulation potential and myeloid-bias differentiation. We recently showed that the combination of inflammatory challenges, including polyinosinc:polycytidilic acid (pIC - a double stranded RNA [dsRNA]) plus high TGFβ causes chronic pancytopenia, anemia, expanded hematopoietic stem and progenitor cell pools, and enhanced bone marrow dysplasia, mimicking a myelodysplastic syndrome (MDS) that developed 3-4 months after pIC challenge (Javier et.al. Haematologica 2022). However, the mechanisms behind this association and/or causality are still ill-defined. Here, we show that mitochondrial antiviral signaling (MAVS) protein can mediate the negative effect of pIC and TGFβ on HSC functions and BMF/ MDS development. Differential gene expression analyses in HSCs suggested that pIC plus TGFβ altered expression of nuclear-encoded mitochondrial genes. Mitochondria serve as platform for the activation of MAVS and downstream innate immune signaling in response to dsRNA. We thus examined the role of mitochondria and MAVS in pIC plus TGFβ-induced BMF/MDS. We show that overexpression TGFβ and pIC together cause alterations in mitochondrial proteins and membrane potential as well as enhanced caspase1 activity, but not the necroptotic signals p-RIP1 and p-RIP3, in HSCs. pIC exposure caused an increase in MAVS expression, and a redistribution into large aggregates that localize onto mitochondria in HSCs, indicative of MAVS activation (two days after pIC exposure in vivo). The MAVS aggregates resolved into smaller and dispersed clusters by 3 months after pIC exposure in control mice. In contrast, in HSCs from TGFβ overexpressing mice, MAVS aggregates persisted 3 months after pIC exposure. To determine the impact of augmented MAVS in dsRNA+TGFβ-driven BMF, we used MAVS deficient mice crossed with TGFβ overexpressing mice. We found that absence of MAVS protein restored normal white blood cell count, including neutrophils and lymphocytes, restored normal red blood cell but not platelet counts in TGFβ overexpressing mice after pIC challenge, compared to controls. Remarkably, MAVS deficiency partially rescued the dysplastic features of bone marrow myeloid cells in TGFβ overexpressing mice after pIC challenge. MAVS-deficiency also reduced the increased caspase1 activity and lowers mitochondria membrane potential in HSCs of these mice. In serial competitive transplant studies, MAVS-deficiency conferred HSC repopulation advantage. These findings suggest that pIC+TGFβ overexpression mediated BMF/MDS phenotype depends on chronic activation of MAVS. Because MAVS aggregation depends on mitochondrial fusion, we used genetic loss of the mitochondrial fission regulator Drp1. Drp1-deficiency caused severe mitochondrial aggregation in HSCs; this was associated with sustained MAVS aggregation. In response to pIC, Drp1-deficient mice exhibited increased caspase-1 activity, increased mitochondrial membrane potential and increase in HSC numbers 6 months after pIC challenges. Drp1 loss resulted in anemia and mild pancytopenia. Interestingly, Drp1-deficient bone marrow myeloid progenitors exhibited severe dysplastic features after pIC challenged. These findings suggest that chronic mitochondrial and MAVS aggregation are sufficient to drive BMF/MDS phenotype. Finally, we investigated the levels of epigenetic markers (acetyl H3K27 and methyl H3 Lys4) in HSCs after pIC stress. H4K4me3 levels were decreased in HSCs long-term after pIC challenges [6 months], this was further decreased in Drp1-deficient HSCs. In conclusion, these findings indicate that non-genetic factors contribute to the onset of BMF/MDS. Mitochondria and MAVS protein together mediate an innate immune mechanism that drives BMF/MDS. This study opens the possibility to target mitochondria-related proteins as a therapeutic strategy to restore ineffective hematopoiesis due to chronic inflammation.

JAK2-V617F Promotes up-Regulation of Pro-Inflammatory Cytokines Including IL-1 upon Adhesion-Induced Activation of β1/β2 Integrins

Introduction In JAK2-V617F (JAK2-VF) mutated cells, over-activation of β1/β2 integrin inside-out signaling has been described (Edelmann-Stephan et al., 2017, JCI). Integrins play a fundamental role in cell interaction and cell invasion during inflammatory processes. In patients suffering from Myeloproliferative Neoplasms (MPNs), highly elevated pro-inflammatory cytokine levels including IL-1β play an important role in pathophysiology. Importantly, Rai et al. (2023, BioRχ) have shown that IL-1β promotes initiation of MPN. In this study, we investigated a potential link between integrin over-activation and elevated pro-inflammatory cytokines including IL-1α and IL-1β in JAK2-VF induced disease. Methods We employed (1) murine myeloid progenitor 32D cells co-transfected with EPO-R and JAK2-WT or JAK2-VF, (2) granulocytes and hematopoietic progenitor cell populations from total bone marrow of Vav1-Cre x JAK2 +/+ and Vav1-Cre x JAK2 VF/+ mice (described by Mullally et al. 2010), and (3) human granulocytes isolated from PB of JAK2-VF positive patients and healthy volunteers. Cells were allowed to adhere on immobilized VCAM-1/ICAM-1 or on BSA (control) and subsequently subjected to further analysis including (1) qPCR analysis and ELISA of a panel of inflammatory cytokines (IL-1β, IL-1α, TNF-α, IL-6, CXCL10), (2) Western-blotting monitoring integrin outside-in signaling pathways, (3) FLICA assays assessing adhesion-induced active caspase-1, (4) RNAseq of mouse and human granulocytes (3 JAK2-VF patients, 2 controls). Results In the 32D cell model, adhesion on mVCAM-1/mICAM-1 strongly induced mRNA expression of IL-1α, IL1-β, IL-6, TNF-α and CXCL10. Interestingly, cytokine RNA induction was significantly higher in 32D JAK2-VF cells as compared to the 32D JAK2-WT cells (Figure 1). By applying a panel of integrin signaling inhibitors we could show an essential involvement of pivotal signaling nodes such as PI3K, Syk, FAK and NFκB, respectively. Next, cytokine proteins were investigated by ELISA. Adhesion on mVCAM-1/mICAM-1 increased intracellular IL-1α and IL-1β cytokine levels. Again, this effect was strongly amplified by the JAK2-VF mutation (Figure 2). Importantly, both cytokines were found in their mature form, indicating intact protein processing. RNAseq of bone marrow (BM) derived mouse granulocytes (including immature pro-and pre-neutrophils) also showed significant upregulation of a number of pro-inflammatory genes including Nlrp3, Il1a, Il1b, Tnf, Cxcl2 and Cxcl10 upon adhesion on mVCAM-1/mICAM-1. However, no significant differences between WT and VF cells were observed. In contrast, RNAseq of VCAM-1/ICAM-1 stimulated human JAK2-VF positive PB granulocytes (which represent terminally differentiated cells) revealed no cytokine up-regulation. Based on this finding, we hypothesize that the adhesion-induced cytokine phenotype is restricted to more immature cell populations as observed in the 32D cell model. Therefore, we are currently investigating (by qPCR and intracellular IL-1 immunophenotyping) hematopoietic progenitor cell populations from Vav1-Cre x JAK2 +/+ and Vav1-Cre x JAK2 VF/+ mice. Since active IL-1β requires processing by the inflammasome, FLICA assays for active caspase-1 were performed using mouse hematopoietic progenitor cells. Importantly, CD41+ cells and megakaryocyte progenitors (MKP) showed an increased FLICA signal upon adhesion on mVCAM-1/mICAM-1. This indicates that caspase 1 is activated upon adhesion in distinct progenitor cell populations. Finally, to support our hypothesis that JAK2-VF augments induction of pro-inflammatory cytokines upon adhesion to VCAM-1/ICAM-1, Vav1-Cre x JAK2 VF/+ mice (n=8) were treated in vivo with anti-β1/β2-integrin antibodies for 1 week. As expected we found a strong reduction of IL-1α concentration in serum of antibody treated mice (528.7 ± 82.2 pg/ml) as compared to PBS-treated control mice (1302.3 ± 370.4 pg/ml, p = 0.0541) Conclusion Here, we show a functional link between elevated cytokine levels and integrin activation in JAK2-VF induced disease. Adhesion to VCAM-1/ICAM-1 promoted the expression of a number of pro-inflammatory cytokines which are known to play important roles in initiation, clinical symptoms and prognosis of MPNs. Further studies aimed at precisely defining the primary cell populations involved in the mouse MPN disease models as well as in humans are currently ongoing.

Mechanism of Microglial Cell Activation in the Benzophenone-3 Exposure Model.

Benzophenone-3 (BP-3) is the most commonly used UV filter in cosmetics, which is absorbed through the skin and crosses the blood-brain barrier. This compound increases extracellular glutamate concentrations, lipid peroxidation, the number of microglia cells and induces process of apoptosis. The aim of this study was to determine the effect of BP-3 on the activation and polarization of microglial cells in the frontal cortex and hippocampus of adult male rats exposed to BP-3 prenatally and then for two weeks in adulthood. It has been found, that exposure to BP-3 reduced the expression of the marker of the M2 phenotype of glial cells in both examined brain structures. An increase in the CD86/CD206 microglial phenotype ratio, expression of transcription factor NFκB and activity of caspase-1 were observed only in the frontal cortex, whereas BP-3 increased the level of glucocorticoid receptors in the hippocampus. The in vitro study conducted in the primary culture of rat frontal cortical microglia cells showed that BP-3 increased the LPS-stimulated release of pro-inflammatory cytokines IL-1α, IL-1β, TNFα, but in cultures without LPS there was decreased IL-1α, IL-6 and TNFα production, while the IL-18 and IP-10 was elevated. The obtained results indicate that differences in the level of immunoactivation between the frontal cortex and the hippocampus may result from the action of this compound on glucocorticoid receptors. In turn, changes in cytokine production in microglial cells indicate that BP-3 aggravates the LPS-induced immunoactivation.

Inflammasome-triggered IL-18 controls skin inflammation in the progression of Buruli ulcer

Buruli ulcer is an emerging chronic infectious skin disease caused by Mycobacterium ulcerans. Mycolactone, an exotoxin produced by the bacterium, is the only identified virulence factor so far, but the functions of this toxin and the mechanisms of disease progression remain unclear. By interfering Sec61 translocon, mycolactone inhibits the Sec61-dependent co-translational translocation of newly synthesized proteins, such as induced cytokines and immune cell receptors, into the endoplasmic reticulum. However, in regard to IL-1β, which is secreted by a Sec61-independent mechanism, mycolactone has been shown to induce IL-1β secretion via activation of inflammasomes. In this study, we clarified that cytokine induction, including that of IL-1β, in infected macrophages was suppressed by mycolactone produced by M. ulcerans subsp. shinshuense, despite the activation of caspase-1 through the inflammasome activation triggered in a manner independent of mycolactone. Intriguingly, mycolactone suppressed the expression of proIL-1β as well as TNF-α at the transcriptional level, suggesting that mycolactone of M. ulcerans subsp. shinshuense may exert additional inhibitory effect on proIL-1β expression. Remarkably, constitutively produced IL-18 was cleaved and mature IL-18 was actually released from macrophages infected with the causative mycobacterium. IL-18-deficient mice infected subcutaneously with M. ulcerans exhibited exacerbated skin inflammation during the course of disease progression. On the other hand, IL-1β controls bacterial multiplication in skin tissues. These results provide information regarding the mechanisms and functions of the induced cytokines in the pathology of Buruli ulcer.

Anti-inflammatory activity of calmodulin-lysine N-methyltransferase through suppressing the caspase-11 non-canonical inflammasome

Calmodulin (CaM)-lysine N-methyltransferase (CAMKMT) is a novel methyltransferase that catalyzes lysine trimethylation in CaM. However, its specific roles in inflammatory responses and diseases remain unclear. In this study, we investigated the effects of CAMKMT on caspase-11 non-canonical inflammasomes. CAMKMT expression levels were significantly decreased during inflammatory responses activated by caspase-11 non-canonical inflammasome in macrophages. Moreover, CaM lysine trimethylation was markedly inhibited, but no change was observed in CaM expression during these inflammatory responses in macrophages. Activation of the CaM downstream effectors, CaM-dependent proteinkinase kinase 2 and CaM-dependent proteinkinase type IV, was also inhibited during inflammatory responses activated by caspase-11 non-canonical inflammasome in macrophages. Notably, forced expression of CAMKMT restrained caspase-11 non-canonical inflammasome activation via inhibiting proteolytic activation of caspase-11 and gasdermin D (GSDMD), which in turn suppressed pyroptosis and the release of interleukin (IL)-1β and IL-18 in macrophages. Finally, an in vivo study revealed that CAMKMT ameliorated lipopolysaccharide (LPS)-stimulated acute lethal sepsis in mice by increasing the survival rate and reducing the serum levels of IL-1 β. These findings suggest CAMKMT as a novel methyltransferase that plays an anti-inflammatory role through restraining caspase-11 non-canonical inflammasome in macrophages.

SHP2 potentiates anti-PD-1 effectiveness through intervening cell pyroptosis resistance in triple-negative breast cancer

Triple negative breast cancer (TNBC) presents a formidable challenge due to the lack of effective treatment modalities. Immunotherapy stands as a promising therapeutic approach; however, the emergence of drug resistance mechanisms within tumor cells, particularly those targeting apoptosis and pyroptosis, has hampered its clinical efficacy. SHP2 is intricately involved in diverse physiological processes, including immune cell proliferation, infiltration, and tumor progression. Nevertheless, the precise contribution of SHP2 to tumor cell pyroptosis resistance remains inadequately understood. Herein, we demonstrate that SHP2 inhibition hampers the proliferative, migratory, and invasive capabilities of TNBC, accompanied by noticeable alterations in cellular membrane architecture. Mechanistically, we provide evidence that SHP2 depletion triggers the activation of Caspase-1 and GSDMD, resulting in GSDMD-dependent release of LDH, IL-1β, and IL-18. Furthermore, computational analyses and co-localization investigations substantiate the hypothesis that SHP2 may hinder pyroptosis through direct binding to JNK, thereby impeding JNK phosphorylation. Our cellular experiments further corroborate these findings by demonstrating that JNK inhibition rescues pyroptosis induced by SHP2 knockdown. Strikingly, in vivo experiments validate the suppressive impact of SHP2 knockdown on tumor progression via enhanced JNK phosphorylation. Additionally, SHP2 knockdown augments tumor sensitivity to anti-PD-1 therapy, thus reinforcing the pro-pyroptotic effects and inhibiting tumor growth. In summary, our findings elucidate the mechanism by which SHP2 governs TNBC pyroptosis, underscoring the potential of SHP2 inhibition to suppress cell pyroptosis resistance and its utility as an adjunctive agent for tumor immunotherapy.

Cordyceps militaris Solid Medium Extract Alleviates Lipoteichoic Acid-Induced MH-S Inflammation by Inhibiting TLR2/NF-κB/NLRP3 Pathways.

The aim of this study was to investigate the inhibitory effects of Cordyceps militaris solid medium extract (CME) and cordycepin (COR) on LTA-induced inflammation in MH-S cells and their mechanisms of action. In this study, the establishment of an LTA-induced MH-S inflammation model was determined, the CCK-8 method was used to determine the safe concentration range for a drug for COR and CME, the optimal concentration of COR and CME to exert anti-inflammatory effects was further selected, and the expression of inflammatory factors of TNF-α, IL-1β, IL-18, and IL-6 was detected using ELISA. The relative expression of TNF-α, IL-1β, IL-18, IL-6, IL-10, TLR2 and MyD88 mRNA was detected using RT-PCR, and the IL-1β, IL-18, TLR2, MyD88, NF-κB p-p65, NLRP3, pro-caspase-1, Caspase-1 and ASC protein expression in the cells were detected using Western blot; immunofluorescence assay detected the expression of Caspase-1 in MH-S cells. The results revealed that both CME and COR inhibited the levels of IL-1β, IL-18, IL-6, and TNF-α in the supernatants of LTA-induced MH-S cells and the mRNA expression levels of IL-1β, IL-18, IL-6, TNF-α, TLR2 and MyD88, down-regulated the LTA-induced IL-1β, IL-18, TLR2 in MH-S cells, MyD88, NF-κB p-p65/p65, NLRP3, ASC, pro-caspase-1, and caspase-1 protein expression levels, and inhibited LTA-induced caspase-1 activation in MH-S cells. In conclusion, CME can play a therapeutic role in LTA-induced inflammation in MH-S cells via TLR2/NF-κB/NLRP3, and may serve as a potential drug for bacterial pneumonia caused by Gram-positive bacteria.

Novel insights into IL-37: an anti-inflammatory cytokine with emerging roles in anti-cancer process.

Interleukin-37 (IL-37) is a newly discovered member of IL-1 family. The cytokine was proved to have extensive protective effects in infectious diseases, allergic diseases, metabolic diseases, autoimmune diseases and tumors since its discovery. IL-37 was mainly produced by immune and some non-immune cells in response to inflammatory stimulus. The IL-37 precursors can convert into the mature forms after caspase-1 cleavage and activation intracellularly, and then bind to Smad-3 and transfer to the nucleus to inhibit the production and functions of proinflammatory cytokines; extracellularly, IL-37 binds to cell surface receptors to form IL-37/IL-18Rα/IL-1R8 complex to exert immunosuppressive function via inhibiting/activating multiple signal pathways. In addition, IL-37 can attenuate the pro-inflammatory effect of IL-18 through directly or forming an IL-37/IL-18BP/IL-18Rβ complex. Therefore, IL-37 has the ability to suppress innate and acquired immunity of the host, and effectively control inflammatory stimulation, which was considered as a new hallmark of cancer. Specifically, it is concluded that IL-37 can inhibit the growth and migration of tumor cells, prohibit angiogenesis and mediate the immunoregulation in tumor microenvironment, so as to exert effective anti-tumor effects. Importantly, latest studies also showed that IL-37 may be a novel therapeutic target for cancer monitoring. In this review, we summarize the immunoregulation roles and mechanisms of IL-37 in anti-tumor process, and discuss its progress so far and potential as tumor immunotherapy.

Interrogating direct NLRP3 engagement and functional inflammasome inhibition using cellular assays

As a key regulator of the innate immune system, the NLRP3 inflammasome responds to a variety of environmental insults through activation of caspase-1 and release of the proinflammatory cytokines IL-1β and IL-18. Aberrant NLRP3 inflammasome function is implicated in numerous inflammatory diseases, spurring drug discovery efforts at NLRP3 as a therapeutic target. A diverse array of small molecules is undergoing preclinical/clinical evaluation with a reported mode of action involving direct modulation of the NLRP3 pathway. However, for a subset of these ligands the functional link between live-cell target engagement and pathway inhibition has yet to be fully established. Herein we present a cohort of mechanistic assays to both query direct NLRP3 engagement in cells, and functionally interrogate different nodes of NLRP3 pathway activity. This system enabled the stratification of potency for five confirmed NLRP3 inhibitors, and identification of two reported NLRP3 inhibitors that failed to demonstrate direct pathway antagonism.

Diallyl disulfide attenuates pyroptosis via NLRP3/Caspase-1/IL-1β signaling pathway to exert a protective effect on hypoxic-ischemic brain damage in neonatal rats

Hypoxic-ischemic encephalopathy (HIE) is a perinatal brain disease caused by hypoxia in neonates. It is one of the leading causes of neonatal death in the perinatal period, as well as disability beyond the neonatal period. Due to the lack of a unified and comprehensive treatment strategy for HIE, research into its pathogenesis is essential. Diallyl disulfide (DADS) is an allicin extract, with detoxifying, antibacterial, and cardiovascular disease protective effects. This study aimed to determine whether DADS can alleviate HIE induced brain damage in rats and oxygen-glucose deprivation (OGD)-induced pyroptosis in PC12 cells, as well as whether it can inhibit pyroptosis via the NLRP3/Caspase-1/IL-1β signaling pathway. In vivo, DADS significantly reduced the cerebral infarction volume, alleviated inflammatory reaction, reduced astrocyte activation, promoted tissue structure recovery, improved pyroptosis caused by HIE and improved the prognosis following HI injury. In vitro findings indicated that DADS increased cell activity, decreased LDH activity and reduced the expression of pyroptosis-related proteins, including IL-1β, IL-18, and certain inflammatory factors in PC12 cells caused by OGD. Mechanistically, DADS inhibited pyroptosis and protected against HIE via the NLRP3/Caspase-1/IL-1β pathway. The specific inhibitor of caspase-1, VX-765, inhibited caspase-1 activation, and IL-1β expression was determined. Additionally, the overexpression of NLRP3 reversed the protective effect of allicin against OGD-induced pyroptosis. In conclusion, these findings demonstrated that DADS inhibits the NLRP3/Caspase-1/IL-1β signaling pathway and decreases HI brain damage.

Discovery and characterisation of quinazolines and 8-Azaquinazolines as NLRP3 agonists with oral bioavailability in mice

The NLRP3 inflammasome is a multiprotein complex that plays a critical role in activating the immune system in response to danger signals. Small molecule agonists of NLRP3 may offer clinical benefits in cancer immunology either as a monotherapy or in combination with checkpoint blockade, where it is hypothesised that their application can help to initiate an antitumor immune response. In this study, we report the discovery of quinazolines and 8-azaquinazolines as NLRP3 agonists and their chemical optimization to afford compounds with oral bioavailability in mice. We confirm that these compounds engage the NLRP3 inflammasome by verifying their dependence upon lipopolysaccharide (LPS) priming for cytokine release and the activation of Caspase-1. We further demonstrate pathway engagement through loss of activity in an NLRP3-knockout THP1 cell line. Based on their pharmacokinetic profile and biological activity, these compounds represent valuable tools to evaluate the therapeutic potential of NLRP3 activation in a pre-clinical setting.

Giardia VSPAS7 protein attenuates Giardia intestinalis-induced host macrophage pyroptosis

BackgroundThe unicellular protozoan parasite Giardia intestinalis, which primarily infects humans and animals such as cattle and sheep, is having a major negative impact on public health. Giardia is able to evade the recognition and elimination of the host immune system because of the trophozoite surface and extracellular vesicles (EVs) covered by variant-specific surface proteins (VSPs). As key proteins for immune evasion, whether VSPs can regulate Giardia-induced pyroptosis and promote Giardia evasion of host immune responses has not been reported.MethodsTo examine the role of Giardia VSPAS7 on Giardia-induced activation of the signaling pathway, secretion of pro-inflammatory cytokines, pyroptosis and the mechanism involved, we constructed the pcDNA3.1-vspas7 expression plasmid and transfected this plasmid into mouse macrophages. Key proteins for pyroptosis, IL-1β secretion and LDH release were detected in pcDNA3.1-vspas7-transfected wild-type (WT) cells and NLRP3-deficient cells by western blot, ELISA and LDH assays, respectively. The interactions of Giardia VSPAS7 and mouse NLRP3 were examined using immunofluorescence assays (IFA), co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays.ResultsVSPAS7 could decrease the levels of phosphorylated-p65 (P-p65), P-IκBα and P-ERK caused by Giardia and reduce the production levels of Giardia-induced pro-inflammatory cytokine IL-6, IL-12 p40 and TNF-α. The results showed that VSPAS7 inhibited Giardia-mediated activation of NF-κB, ERK/MAPK signaling and secretion of pro-inflammatory cytokines. Furthermore, VSPAS7 suppressed Giardia-induced macrophage pyroptosis by reducing GSDMD cleavage, caspase-1 activation, IL-1β secretion and LDH release. We further found that VSPAS7 could interact with mouse NLRP3 directly, and in NLRP3-deficient cells the suppression of Giardia-induced macrophage pyroptosis by VSPAS7 was significantly attenuated.ConclusionsOverall, VSPAS7 could inhibit Giardia-induced activation of signaling pathways and pyroptosis in host macrophages, allowing Giardia evasion of host immune responses. Studies on Giardia VSP-mediated immune evasion provide an important theoretical basis for in-depth studies on Giardia pathogenicity.Graphical abstract

Genetic mechanisms of bacteria recognition by cattle innate immunity receptors

The research identified a list of molecules involved in the mechanisms of innate immunity in cattle and the recognition of bacterial pathogens. The current list of molecular receptors has expanded to include TLR receptors and the recently defined NOD-like receptors (NLRs): NOD, NALP, NAIP, and IPAF. TLR molecules are designed to transmit a ligand-binding signal on the cell surface or endosome and activate specific molecules of bacterial origin in the cytosol, such as peptidoglycans, RNA, toxins and flagellins. The obtained data on the molecular structure of TLR and NLR receptors indicate their anti-inflammatory role, mediated by signals through nuclear transcription factor κB and activation of caspase-1 in the inflammasome. It has been shown that the role of immunosensors of extracellular and intracellular perception of bacteria in regulating inflammation is synergistic. Mutations in TLR and NOD receptors are associated with autoimmune inflammatory syndromes. This review examines the body's ways of recognising intracellular pathogens, describes the problem of their mimicry from the animal immune system, and the molecular mechanisms of such interactions. Variants of molecular interactions of innate immune receptors with peptidoglycans, bacterial DNA and toxins, cell wall compartments, and bacterial flagellin receptors are also considered. This study aimed to analyse the current understanding of the genetic and molecular structure of the immune response to bacterial environmental factors and the mechanisms and characteristics of the reaction of the animal body.

Ubiquitin-specific peptidase 25 exacerbated osteoarthritis progression through facilitating TXNIP ubiquitination and NLRP3 inflammasome activation

Several members of the ubiquitin-specific proteases (USPs) family have been revealed to regulate the progression of osteoarthritis (OA). The current study aimed to investigate the role and the underlying mechanism of USP25 in IL-1β-induced chondrocytes and OA rat model. It was discovered that IL-1β stimulation upregulated USP25, increased ROS level, and suppressed cell viability in rat chondrocytes. Besides, USP25 knockdown alleviated IL-1β-induced injury by decreasing ROS level, attenuating pyroptosis, and downregulating the expression of IL-18, NLRP3, GSDMD-N, active caspase-1, MMP-3, and MMP-13. Furthermore, we discovered that USP25 affected the IL-1β-induced injury in chondrocytes in a ROS-dependent manner. Moreover, USP25 was revealed to interact with TXNIP, and USP25 knockdown increased the ubiquitination of TXNIP. The pro-OA effect of USP25 abundance could be overturned by TXNIP suppression in IL-1β-induced chondrocytes. Finally, in vivo experiment results showed that USP25 inhibition alleviated cartilage destruction in OA rats. In conclusion, we demonstrated that USP25 stimulated the overproduction of ROS to activate the NLRP3 inflammasome via regulating TXNIP, resulting in increased pyroptosis and inflammation in OA.

The Shigella flexneri virulence factor apyrase is released inside eukaryotic cells to hijack host cell fate.

Intestinal epithelial cells represent the first line of defense from invading enteric pathogens. During the course of infection, pro-inflammatory programmed cell death is an effective way to eliminate invading microbes and to create a localized inflammatory environment. On the other hand, pathogens evolved countless strategies to overcome cell death and to keep the host alive ensuring their spread. It was previously shown that Shigella flexneri apyrase interacts with OmpA to contribute to a proper polar exposition of IcsA, which mediates actin-based motility. However, apyrase is also an ATP-diphosphohydrolase whose catalytic activity function has not been elucidated yet. Herein, we demonstrated that apyrase contributes to the manipulation of host cell fate by S. flexneri since it is released within the host cell cytoplasm during infection to degrade intracellular ATP. Thus, apyrase contributes to prevent caspase-1 activation, thereby downregulating the activation of pyroptosis in infected cells. Overall, apyrase is involved in the modulation of host cell survival and dampens the inflammatory response.IMPORTANCEIn this paper, we demonstrated that apyrase is released within the host cell cytoplasm during infection to target the intracellular ATP pool. By degrading intracellular ATP, apyrase contributes to prevent caspases activation, thereby inhibiting the activation of pyroptosis in infected cells. Our results show, for the first time, that apyrase is involved in the modulation of host cell survival, thereby aiding this pathogen to dampen the inflammatory response. This work adds a further piece to the puzzle of Shigella pathogenesis. Due to its increased spread worldwide, prevention and controlling strategies are urgently needed. Overall, this study highlighted apyrase as a suitable target for an anti-virulence therapy to tackle this pathogen.

Activation of AIM2 by hepatitis B virus results in antiviral immunity that suppresses hepatitis C virus during coinfection.

Clinical data suggest that Hepatitis C virus (HCV) levels are generally lower in Hepatitis B virus (HBV) co-infected patients, but the mechanism is unknown. Here, we show that HBV, but not HCV, activated absent in melanoma-2. This in turn results in inflammasome-mediated cleavage of pro-IL-18, leading to an innate immune activation cascade that results in increased interferon-γ, suppressing both viruses.

Total alkaloids of Sophora alopecuroides alleviate atherosclerosis by regulating macrophage pyroptosis induced by reactive oxygen species-NLRP3 signaling axis

Cardiovascular and cerebrovascular diseases are common diseases. This study aimed to investigate the role of total alkaloids of Sophora alopecuroides (TASA) in atherosclerosis and exploring the underlying mechanism. Mice were given normal diet (ND), high-fat diet (HFD), and TASA plus ND or HFD. The mice were further administered with NLRP3 shRNA and shControl. After treatment, Hematoxylin and Eosin and Oil Red O staining assessed histological changes. CD31/caspase-1 double staining was detected by fluorescent staining and pyroptosis expression assessed by CD31/TUNEL double staining. Western blot results determined caspase-1 expression in HFD mice. TASA treatment suppressed plaque formation in HFD-fed ApoE-/- mice but insignificantly altered the lesion area in ND-fed ApoE-/- mice. Caspase-1 content and TUNEL positive cells were significantly elevated in mice fed with HFD or ND, whilst advent of TASA further enhanced the activation of caspase-1 in HFD mice. TASA treatment reduced activity of ROS, inhibited NLRP3 inflammasome and regulated macrophage pyroptosis. Inhibition of NLRP3 decreased the lesion area of atherosclerosis and lipid deposition. TASA inhibited the expression of ROS to reduce macrophages necrosis and secretion of inflammatory mediators, thereby alleviating inflammation, delaying plaque formation and stabilizing plaques in atherosclerosis.