Search

Clostridioides difficile (C. difficile), is a major cause of nosocomial diarrhea and colitis. C. difficile flagellin FliC contributes toxins to gut inflammation by interacting with the immune Toll-like receptor 5 (TLR5) to activate nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) signaling pathways. Flagella of intracellular pathogens can activate the NLR family CARD domain-containing protein 4 (NLRC4) inflammasome pathway. In this study, we assessed whether flagellin of the extracellular bacterium C. difficile internalizes into epithelial cells and activates the NLRC4 inflammasome. Confocal microscopy showed internalization of recombinant green fluorescent protein (GFP)-FliC into intestinal Caco-2/TC7 cell line. Full-length GFP-FliC activates NLRC4 in Caco-2/TC7 cells in contrast to truncated GFP-FliC lacking the C-terminal region recognized by the inflammasome. FliC induced cleavage of pro-caspase-1 into two subunits, p20 and p10 as well as gasdermin D (GSDMD), suggesting the caspase-1 and NLRC4 inflammasome activation. In addition, colocalization of GFP-FliC and pro-caspase-1 was observed, indicating the FliC-dependent NLRC4 inflammasome activation. Overexpression of the inflammasome-related interleukin (interleukin (IL)-1β, IL-18, and IL-33) encoding genes as well as increasing of the IL-18 synthesis was detected after cell stimulation. Inhibition of I-kappa-B kinase alpha (IKK-α) decreased the FliC-dependent inflammasome interleukin gene expression suggesting a role of the NF-κB pathway in regulating inflammasome. Altogether, these results suggest that FliC internalizes into the Caco-2/TC7 cells and activates the intracellular NLRC4 inflammasome thus contributing to the inflammatory process of C. difficile infection.

Ischemic stroke (also called cerebral ischemia) is one of the leading causes of death and severe disability worldwide. NLR inflammasomes play a crucial role in sensing cell damage in response to a harmful stimuli and modulating the inflammatory response, promoting the release of pro-inflammatory cytokines such as IL-18 and IL-1β following ischemic injury. Therefore, a neuroprotective effect is achieved by inhibiting the expression, assembly, and secretion of inflammasomes, thus limiting the extent of brain detriment and neurological sequelae. This review aims to illustrate the molecular characteristics, expression levels, and assembly of NLRP3 (nucleotide-binding oligomerization domain-like receptor [NLR] family pyrin-domain-containing 3) inflammasome, the most studied in the literature, in order to discover promising therapeutic implications. In addition, we provide some information regarding the contribution of NLRP1, NLRP2, and NLRC4 inflammasomes to ischemic stroke pathogenesis, highlighting potential therapeutic strategies that require further study.

Summary: Zoonotic pathogens, such as COVID-19, reside in animal hosts before jumping species to infect humans. The Carnivora, like mink, carry many zoonoses, yet how diversity in host immune genes across species affect pathogen carriage is poorly understood. Here, we describe a progressive evolutionary downregulation of pathogen-sensing inflammasome pathways in Carnivora. This includes the loss of nucleotide-oligomerization domain leucine-rich repeat receptors (NLRs), acquisition of a unique caspase-1/-4 effector fusion protein that processes gasdermin D pore formation without inducing rapid lytic cell death, and the formation of a caspase-8 containing inflammasome that inefficiently processes interleukin-1β. Inflammasomes regulate gut immunity, but the carnivorous diet has antimicrobial properties that could compensate for the loss of these immune pathways. We speculate that the consequences of systemic inflammasome downregulation, however, can impair host sensing of specific pathogens such that they can reside undetected in the Carnivora.

Salmonella spvC gene, encoding a phosphothreonine lyase on host mitogen-activated protein kinases, facilitates systemic infection of Salmonella while the precise mechanisms remain elusive. Autophagy and pyroptosis dependent on the activation of inflammasomes, as parts of innate immune response, contribute to host defense against Salmonella infection. Recently, we reported that spvC could inhibit pyroptosis. To explore the effect of spvC on autophagy and the relationship between its function in pyroptosis and autophagy, infection models of macrophages J774A.1 and epithelial HeLa cells co-cultured with Salmonella Typhimurium wild type, spvC deletion, site-directed mutant which lacks phosphothreonine lyase activity, or complemented strain were established. The levels of LC3 turnover and Beclin 1 of J774A.1 cells were determined by western blot. Confocal laser scanning microscopy was used to visualize the autophagic flux after being transfected with mRFP-GFP-LC3 plasmid in HeLa cells. Results showed that SpvC inhibited autophagosome formation through its phosphothreonine lyase activity. Additionally, analysis of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) and NLR with CARD domain-containing 4 (NLRC4) in J774A.1 cells indicated that spvC decreased the protein levels of NLRP3 and NLRC4, which were significantly changed by autophagy inhibitor Bafilomycin A1. Together, our observations reveal a novel mechanism of spvC in Salmonella pathogenesis and host inflammatory response via inhibiting autophagy and NLRP3 as well as NLRC4. These pathways and their subversion by diverse pathogen virulence determinants are expected to throw light on the design of anti-infective agents.

Fibrotic Interstitial lung diseases (ILDs) are complex disorders of variable clinical behaviour. The majority of them cause significant morbidity, whilst Idiopathic Pulmonary Fibrosis (IPF) is recognised as the most relentless. NLRP3, AIM2, and NLRC4 inflammasomes are multiprotein complexes driving IL-1β release; a proinflammatory and profibrotic cytokine. Several pathogenetic factors associated with IPF are identified as inflammasome activators, including increases in mtROS and bacterial burden. Mitochondrial oxidation and alterations in bacterial burden in IPF and other ILDs may lead to augmented inflammasome activity in airway macrophages (AMs). IPF (n=14), non-IPF-ILDs (n=12) patients and healthy subjects (n=12) were prospectively recruited and AMs were isolated from bronchoalveolar lavage. IL-1β release resulting from NLRP3, AIM2 and NLRC4 inflammasomes stimulation in AMs were determined and baseline levels of mitochondrial ROS and microbial burden were also measured. Our results showed that NLRP3 was more inducible in IPF and other ILDs compared to controls. Additionally, following AIM2 activation IL-1β release was significantly higher in IPF compared to controls, whereas similar trends were observed in Non-IPF-ILDs. NLRC4 activation was similar across groups. mtROS was significantly associated with heightened NLRP3 and AIM2 activation, and mitochondrial antioxidant treatment limited inflammasome activation. Importantly, microbial burden was linked to baseline IL-1β release and AIM2 and IL-18 relative expression independently of mtROS. In conclusion, the above findings suggested a link between the overactivation of NLRP3 and AIM2 inflammasomes, driven by mitochondrial oxidation, in the pathogenesis of lung fibrosis while changes in the microbiota may prime the inflammasome in the lungs.

BackgroundNOD-like receptor family CARD-containing 4 protein (NLRC4) is a cytosolic protein that forms an inflammasome in response to flagellin and type 3 secretion system (T3SS) proteins from invading Gram-negative bacteria. NLRC4 mutations have been recently identified in early-onset severe autoinflammatory disorders. In this study, we reported a novel mutation in NLRC4 in two Chinese patients, who manifested with recurrent urticaria and arthralgia.MethodsWe summarized the clinical data of the two patients. Gene mutations were identified by whole-exome sequencing (WES). Swiss-PdbViewer was used to predict the pathogenicity of the identified mutations. Cytokine levels and caspase-1 activation were detected in the patient PBMCs with lipopolysaccharide (LPS) stimulation. All previously published cases with NLRC4 mutations were reviewed.ResultsWe identified a missense heterozygous mutation (c.514G>A, p.Gly172Ser), which was located in the highly conserved residue of nucleotide-binding domain (NBD) of NLRC4. The mutation did not alter the expression of NLRC4 protein, but induced considerably much higher production of IL-1β and IL-6 in patient PBMCs than in healthy controls after LPS stimulation. Four NLRC4 inflammasomopathy phenotypes have been described, with severe inflammatory diseases including macrophage activation syndrome, enterocolitis and NOMID in patients with mutations in the NBD and HD1 domains, whereas a mild clinical phenotype was associated with two mutations in the WHD domain of NLRC4.ConclusionWe identified a novel mutation in the NBD domain, and the patients just presented with a mild inflammatory phenotype. Thus, our findings reinforce the diversity of NLRC4 mutations and expand the clinical spectrum of associated diseases.

Inflammasomes comprise a family of cytosolic multi-protein complexes that modulate the activation of cysteine-aspartate-specific protease 1 (caspase-1) and promote the maturation and secretion of interleukin (IL)-1β and IL-18, leading to an inflammatory response. Different types of inflammasomes are defined by their sensor protein which recognizes pathogenic ligands and then directs inflammasome assembly. Although the specific molecular mechanisms underlying the activation of most inflammasomes are still unclear, NLRC4 inflammasomes have emerged as multifaceted agents of the innate immune response, playing important roles in immune defense against a variety of pathogens. Other studies have also expanded the scope of NLRC4 inflammasomes to include a range of inherited human autoimmune diseases as well as proposed roles in cancer. In this review article, we provide an updated overview of NLRC4 inflammasomes, describing their composition, activation mechanisms and roles in both microbial infections and other disease conditions.

The effect of autoinflammatory diseases on severe acute respiratory syndrome coronavirus 2 infection remains unknown. We report a case of coronavirus disease 2019 (COVID-19) in a patient with autoinflammation with infantile enterocolitis with inflammatory flares due to a mutation in the inflammasome component NLRC4. This case highlights the role of immunosuppression in patients with autoinflammation with COVID-19. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is characterized by chronic airway infection, inflammation, and tissue damage that lead to progressive respiratory failure. NLRP3 and NLRC4 are cytoplasmic pattern recognition receptors that activate the inflammasome, initiating a caspase-1-mediated response. We hypothesized that gain-offunction inflammasome responses are associated with worse outcomes in children with CF. We genotyped nonsynonymous variants in NLRP3 and the NLRC4 pathway from individuals in the EPIC (Early Pseudomonas Infection Control) Observational Study cohort and tested for association with CF outcomes. We generated knockouts of NLRP3 and NLRC4 in human macrophage-like cells and rescued knockouts with wild-type or variant forms of NLRP3 and NLRC4. We identified a SNP in NLRP3, p.(Q705K), that was associated with a higher rate of P. aeruginosa colonization (N = 609; P = 0.01; hazard ratio, 2.3 [Cox model]) and worsened lung function over time as measured by forced expiratory volume in 1 second (N = 445; P = 0.001 [generalized estimating equation]). We identified a SNP in NLRC4, p.(A929S), that was associated with a lower rate of P. aeruginosa colonization as part of a composite of rare variants (N = 405; P = 0.045; hazard ratio, 0.68 [Cox model]) and that was individually associated with protection from lung function decline (P < 0.001 [generalized estimating equation]). Rescue of the NLRP3 knockout with the p.(Q705K) variant produced significantly more IL-1b in response to NLRP3 stimulation than rescue with the wild type (P = 0.020 [Student's t test]). We identified a subset of children with CF at higher risk of early lung disease progression. Knowledge of these genetic modifiers could guide therapies targeting inflammasome pathways. [ABSTRACT FROM AUTHOR]

Salmonella spvC gene, encoding a phosphothreonine lyase on host mitogen-activated protein kinases, facilitates systemic infection of Salmonella while the precise mechanisms remain elusive. Autophagy and pyroptosis dependent on the activation of inflammasomes, as parts of innate immune response, contribute to host defense against Salmonella infection. Recently, we reported that spvC could inhibit pyroptosis. To explore the effect of spvC on autophagy and the relationship between its function in pyroptosis and autophagy, infection models of macrophages J774A.1 and epithelial HeLa cells co-cultured with Salmonella Typhimurium wild type, spvC deletion, site-directed mutant which lacks phosphothreonine lyase activity, or complemented strain were established. The levels of LC3 turnover and Beclin 1 of J774A.1 cells were determined by western blot. Confocal laser scanning microscopy was used to visualize the autophagic flux after being transfected with mRFP-GFP-LC3 plasmid in HeLa cells. Results showed that SpvC inhibited autophagosome formation through its phosphothreonine lyase activity. Additionally, analysis of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) and NLR with CARD domain-containing 4 (NLRC4) in J774A.1 cells indicated that spvC decreased the protein levels of NLRP3 and NLRC4, which were significantly changed by autophagy inhibitor Bafilomycin A1. Together, our observations reveal a novel mechanism of spvC in Salmonella pathogenesis and host inflammatory response via inhibiting autophagy and NLRP3 as well as NLRC4. These pathways and their subversion by diverse pathogen virulence determinants are expected to throw light on the design of anti-infective agents. [ABSTRACT FROM AUTHOR]

Inflammasomes comprise a family of cytosolic multi-protein complexes that modulate the activation of cysteine-aspartate-specific protease 1 (caspase-1) and promote the maturation and secretion of interleukin (IL)-1β and IL-18, leading to an inflammatory response. Different types of inflammasomes are defined by their sensor protein which recognizes pathogenic ligands and then directs inflammasome assembly. Although the specific molecular mechanisms underlying the activation of most inflammasomes are still unclear, NLRC4 inflammasomes have emerged as multifaceted agents of the innate immune response, playing important roles in immune defense against a variety of pathogens. Other studies have also expanded the scope of NLRC4 inflammasomes to include a range of inherited human autoimmune diseases as well as proposed roles in cancer. In this review article, we provide an updated overview of NLRC4 inflammasomes, describing their composition, activation mechanisms and roles in both microbial infections and other disease conditions. [ABSTRACT FROM AUTHOR]

Background: NOD-like receptor family CARD-containing 4 protein (NLRC4) is a cytosolic protein that forms an inflammasome in response to flagellin and type 3 secretion system (T3SS) proteins from invading Gram-negative bacteria. NLRC4 mutations have been recently identified in early-onset severe autoinflammatory disorders. In this study, we reported a novel mutation in NLRC4 in two Chinese patients, who manifested with recurrent urticaria and arthralgia. Methods: We summarized the clinical data of the two patients. Gene mutations were identified by whole-exome sequencing (WES). Swiss-PdbViewer was used to predict the pathogenicity of the identified mutations. Cytokine levels and caspase-1 activation were detected in the patient PBMCs with lipopolysaccharide (LPS) stimulation. All previously published cases with NLRC4 mutations were reviewed. Results: We identified a missense heterozygous mutation (c.514G>A, p.Gly172Ser), which was located in the highly conserved residue of nucleotide-binding domain (NBD) of NLRC4. The mutation did not alter the expression of NLRC4 protein, but induced considerably much higher production of IL-1β and IL-6 in patient PBMCs than in healthy controls after LPS stimulation. Four NLRC4 inflammasomopathy phenotypes have been described, with severe inflammatory diseases including macrophage activation syndrome, enterocolitis and NOMID in patients with mutations in the NBD and HD1 domains, whereas a mild clinical phenotype was associated with two mutations in the WHD domain of NLRC4. Conclusion: We identified a novel mutation in the NBD domain, and the patients just presented with a mild inflammatory phenotype. Thus, our findings reinforce the diversity of NLRC4 mutations and expand the clinical spectrum of associated diseases. [ABSTRACT FROM AUTHOR]

Fibrotic Interstitial lung diseases (ILDs) are complex disorders of variable clinical behaviour. The majority of them cause significant morbidity, whilst Idiopathic Pulmonary Fibrosis (IPF) is recognised as the most relentless. NLRP3, AIM2, and NLRC4 inflammasomes are multiprotein complexes driving IL-1β release; a proinflammatory and profibrotic cytokine. Several pathogenetic factors associated with IPF are identified as inflammasome activators, including increases in mtROS and bacterial burden. Mitochondrial oxidation and alterations in bacterial burden in IPF and other ILDs may lead to augmented inflammasome activity in airway macrophages (AMs). IPF (n=14), non-IPF-ILDs (n=12) patients and healthy subjects (n=12) were prospectively recruited and AMs were isolated from bronchoalveolar lavage. IL-1β release resulting from NLRP3, AIM2 and NLRC4 inflammasomes stimulation in AMs were determined and baseline levels of mitochondrial ROS and microbial burden were also measured. Our results showed that NLRP3 was more inducible in IPF and other ILDs compared to controls. Additionally, following AIM2 activation IL-1β release was significantly higher in IPF compared to controls, whereas similar trends were observed in Non-IPF-ILDs. NLRC4 activation was similar across groups. mtROS was significantly associated with heightened NLRP3 and AIM2 activation, and mitochondrial antioxidant treatment limited inflammasome activation. Importantly, microbial burden was linked to baseline IL-1β release and AIM2 and IL-18 relative expression independently of mtROS. In conclusion, the above findings suggested a link between the overactivation of NLRP3 and AIM2 inflammasomes, driven by mitochondrial oxidation, in the pathogenesis of lung fibrosis while changes in the microbiota may prime the inflammasome in the lungs. [ABSTRACT FROM AUTHOR]

Inflammasomes are molecular machines that carry out inflammatory responses on challenges by pathogens and endogenous dangers. Dysregulation of inflammasome assembly and regulation is associated with numerous human diseases from autoimmunity to cancer. In recent years, significant advances have been made in understanding the mechanism of inflammasome signaling using structural approaches. Here, we review inflammasomes formed by the NLRP1, NLRP3, and NLRC4 sensors, which are well characterized structurally, and discuss the structural and functional diversity among them. [ABSTRACT FROM AUTHOR]

Innate inflammation is crucial for ischemic stroke development. NLRP6, a nucleotide-binding and oligomerization domain-like receptors (NLRs) family member, regulates innate inflammation. Whether NLRP6 regulates neurological damage and neuroinflammation during ischemic stroke remains unclear. We report that NLRP6 is abundantly expressed in microglia and significantly upregulated in the ischemic brain. The brain injury severity was alleviated in NLRP6-deficient mice after ischemic stroke, as evidenced by reduced cerebral infarct volume, decreased neurological deficit scores, improved histopathological morphological changes, ameliorated neuronal denaturation, and relief of sensorimotor dysfunction. In the co-culture OGD/R model, NLRP6 deficiency prevented neuronal death and attenuated microglial cell injury. NLRP6 deficiency blocked several NLRs inflammasomes' activation and abrogated inflammasome-related cytokine production by decreasing the expression of the common effector pro-caspase-1. NLRP6 deficiency reduced pro-caspase-1's protein level by inducing proteasomal degradation. These findings confirm the neuroprotective role of NLRP6 deficiency in ischemic stroke and its underlying regulation mechanism in neuroinflammation and provide a potential therapeutic target for ischemic stroke. Working model for NLRP6 deficiency blocking the activation of several NLRs inflammasomes in ischemic stroke. (A) NLR receptor proteins, such as NLRP6, NLRP3, and NLRC4, interact with ASC and further recruit pro-caspase-1, finally forming several inflammasomes. These inflammasomes produce the biologically active cytokines IL-1β and IL-18, leading to neuroinflammation in ischemic stroke. (B) NLRP6 deficiency mediates the proteasomal degradation of pro-caspase-1, resulting in the blockage of several NLRs inflammasome assemblies and alleviating neuroinflammation in ischemic stroke. [Display omitted] • NLRP6 deficiency exerted a neuroprotective role in ischemic stroke. • NLRP6 deficiency inhibited inflammasome-dependent inflammation in ischemic stroke. • NLRP6 deficiency blocked NLRs inflammasomes activation by decreasing pro-caspase-1. • NLRP6 deficiency reduced the pro-caspase-1 by inducing its proteasomal degradation. [ABSTRACT FROM AUTHOR]

• Spinal motoneurons express NLRP1, NLRP3, NLRC4 AIM2 and the inflammasome adaptor ASC. • Immunoreactivity of AIM2 was detected in all types of glial cells. • Glial type-specific immunolabelling was observed for NLRP1 and NLRC4. • Increased NLRC4 is associated with pathogenesis in SOD1(G93A) mice and ALS patients. Inflammasomes are key components of the innate immune system and activation of these multiprotein platforms is a crucial event in the etiopathology of amyotrophic lateral sclerosis (ALS). Inflammasomes consist of a pattern recognition receptor (PRR), the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and caspase 1. Exogenous or endogenous "danger signals" can trigger inflammasome assembly and promote maturation and release of pro-inflammatory cytokines, including interleukin 1β. Previous studies have demonstrated presence and activation of NLRP3 in spinal cord tissue from SOD1(G93A) mice and human sporadic ALS (sALS) patients. However, regulation and cell type-specific localization of other well-known PRRs has not yet been analysed in ALS. Here, we explored gene expression, protein concentration and cell type-specific localization of the NLRP1, NLRC4 and AIM2 inflammasomes in spinal cord samples from SOD1(G93A) mice and sALS patients. Transcription levels of NLRP1 and NLRC4, but not AIM2, were elevated in symptomatic SOD1(G93A) animals. Immunoblotting revealed elevated protein levels of NLRC4, which were significantly increased in sALS vs. control patients. Immunofluorescence studies revealed neuronal labelling of all investigated PRRs. Staining of AIM2 was detected in all types of glia, whereas glial type-specific labelling was observed for NLRP1 and NLRC4. Our findings revealed pathology-related and cell type-specific differences in the expression of subsets of PRRs. Besides NLRP3, NLRC4 appears to be linked more closely to ALS pathogenesis. [ABSTRACT FROM AUTHOR]

Objectives: Targeted inhibition of inflammatory response can reduce diabetic cerebral ischemia–reperfusion (I/R) injure. Pyroptosis is characterized by caspase-1 dependence and the release of a large number of pro-inflammatory factors. LncRNA-Fendrr is associated with a variety of diseases, but Fendrr has not been studied in diabetic cerebral I/R. NLR-family CARD-containing protein 4 (NLRC4) regulate the pyroptosis of microglia cells. This study was designed to investigate whether Fendrr is involved in the effects of diabetic cerebral I/R injury. Methods: The diabetic brain I/R model in mice was constructed. Mouse microglia cell line BV-2 cells were exposed to high glucose followed by hypoxia/reoxygenation (H/R). Fendrr and some pyroptosis-associated proteins were detected by qRT-PCR, western blot or ELISA. HE staining was used to detect pathological changes. Microglia pyroptosis was detected by TUNEL staining. RNA pull-down and RNA Immunoprecipitation were used to detect binding of Fendrr to HERC2 (E3 ubiquitin ligase), and CO-IP detected binding of HERC2 to NLRC4. The ubiquitination of NLRC4 was detected by ubiquitination experiments. Results: Fendrr was significantly increased in the diabetic cerebral I/R model, and NLRC4 inflammatory complex and pyroptosis mediated inflammatory factors were increased. NLRC4 and inflammatory cytokines associated with pyroptosis were decreased in the high glucose-treated hypoxia/reoxygenation (H/R)-induced microglia after Fendrr knockdown. Fendrr bound to HERC2 protein, and HERC2 bound to NLRC4. Meanwhile, Fendrr could inhibit the ubiquitination of NLRC4, HERC2 promoted the ubiquitination of NLRC4 protein. Moreover, the effect of Fendrr overexpression in the diabetic cerebral I/R model of microglia can be reversed by HERC2 overexpression. Conclusion: Fendrr can protect against the ubiquitination and degradation of NLRC4 protein through E3 ubiquitin ligase HERC2, thereby accelerating the pyroptosis of microglia. [ABSTRACT FROM AUTHOR]

BackgroundPseudomonas aeruginosa (PA) is one of the most common Gram-negative bacteria causing hospital-acquired pulmonary infection, with high drug resistance and mortality. Therefore, it is urgent to introduce new non-antibiotic treatment strategies. Mesenchymal stem cells (MSCs), as important members of the stem cell family, were demonstrated to alleviate pathological damage in acute lung injury. However, the potential mechanism how MSC alleviate acute lung infection caused by PA remains unclear.ObjectiveThe purpose of this study was to investigate the effects of Adipose-derived mesenchymal stem cells (ASCs) on acute pulmonary infections and the possible mechanisms how ASCs reduce pulmonary inflammation induced by PA.MethodsThe therapeutic and mechanistic effects of ASCs on PA pulmonary infection were evaluated respectively in a murine model as well as in an in vitro model stimulated by PA and co-cultured with ASCs.Results1. ASCs treatment significantly reduced the bacterial load, inflammation of lung tissue and histopathological damage by PA. 2. PA infection mainly activated Nod-like receptor containing a caspase activating and recruitment domain 4 (NLRC4) inflammasome in the lung of mice. ASCs attenuated acute lung infection in mice by inhibiting NLRC4 inflammasome activation. 3. NLRC4−/− mice showed a significant improvement in survival rate and lung bacterial load after PA infection. 4. ASCs mainly increased expression and secretion of STC‐1 in response to PA‐stimulated NLRC4 inflammasome activation.ConclusionsPA infection attenuated macrophage phagocytosis through activation of NLRC4 inflammasome in macrophages, which eventually led to pulmonary inflammatory damage in mouse; ASCs reduced the activation of NLRC4 inflammasome in macrophages induced by PA infection, thereby increasing the phagocytic ability of macrophages, and ultimately improving lung tissue damage in mouse; ASCs may inhibit NLRC4 inflammasome through the secretion of STC-1.

• Inflammasome inhibition improves neurologic outcome and infarct size. • Inflammasome inhibition reduces neuro-inflammation. • NLRP3 is the major contributor among the inflammasomes. • NLRP3 is mainly expressed within ischemic neurons. Cerebral ischemia induces a profound neuro-inflammatory response, but the underlying molecular mechanisms are poorly understood. Inflammasomes (NLRP1, NLRP3, NLRC4, AIM2) are intracellular multi-protein complexes which can induce sets of pro-inflammatory cyto- and chemokines, and thereby guide inflammation. We, here, assessed the functional role of NLRP3 in ischemia/reperfusion (I/R) injury in a mouse model of transient cerebral ischemia. Ischemic stroke was induced in C57Bl/6 mice by 60 min transient middle cerebral artery occlusion (tMCAO) and 3, 7 or 23 h of reperfusion, a paradigm of I/R injury. The expression patterns of inflammasomes in the ischemic hemispheres were evaluated by semiquantitative real-time PCR and Western Blot analysis accompanied by protein localization using immunocytochemistry. Finally, animals were treated with the inflammasome inhibitors Sulforaphane, Genipin, MCC950 or vehicle, directly before or upon recanalization after tMCAO. Stroke outcome was assessed, including infarct size and functional deficits, local inflammatory response, neuronal survival as well as blood–brain barrier function on day 1 after tMCAO. After tMCAO the relative gene expression levels of NLRP3 increased 20-30x within 1 day in the ischemic hemisphere which translated into an increased expression of NLRP3 in neurons. Accordingly, the gene expression levels of the NLRP3-modulator, Bruton's Tyrosine Kinase (BTK), and the NLRP3-inducible cytokine IL-1β significantly rose. Lesser or non-significant changes were seen for the other inflammasomes. Application of inflammasome inhibitors covering all inflammasomes or specifically NLRP3 significantly reduced infarct volumes when given before or after tMCAO and was accompanied by clear evidence for reduced activation of caspase 1. This stroke attenuating effect coincided with less immune cell infiltration in the ischemic hemisphere and preservation of the blood–brain barrier integrity. Our data show that induction of the NLRP3 inflammasome in neurons drives neuroinflammation in acute ischemic stroke. Early blockade of NLRP3 protects from I/R injury by mitigating inflammation and stabilizing the blood–brain barrier. [ABSTRACT FROM AUTHOR]

Background: Pseudomonas aeruginosa (PA) is one of the most common Gram-negative bacteria causing hospital-acquired pulmonary infection, with high drug resistance and mortality. Therefore, it is urgent to introduce new non-antibiotic treatment strategies. Mesenchymal stem cells (MSCs), as important members of the stem cell family, were demonstrated to alleviate pathological damage in acute lung injury. However, the potential mechanism how MSC alleviate acute lung infection caused by PA remains unclear. Objective: The purpose of this study was to investigate the effects of Adipose-derived mesenchymal stem cells (ASCs) on acute pulmonary infections and the possible mechanisms how ASCs reduce pulmonary inflammation induced by PA. Methods: The therapeutic and mechanistic effects of ASCs on PA pulmonary infection were evaluated respectively in a murine model as well as in an in vitro model stimulated by PA and co-cultured with ASCs. Results: 1. ASCs treatment significantly reduced the bacterial load, inflammation of lung tissue and histopathological damage by PA. 2. PA infection mainly activated Nod-like receptor containing a caspase activating and recruitment domain 4 (NLRC4) inflammasome in the lung of mice. ASCs attenuated acute lung infection in mice by inhibiting NLRC4 inflammasome activation. 3. NLRC4−/− mice showed a significant improvement in survival rate and lung bacterial load after PA infection. 4. ASCs mainly increased expression and secretion of STC‐1 in response to PA‐stimulated NLRC4 inflammasome activation. Conclusions: PA infection attenuated macrophage phagocytosis through activation of NLRC4 inflammasome in macrophages, which eventually led to pulmonary inflammatory damage in mouse; ASCs reduced the activation of NLRC4 inflammasome in macrophages induced by PA infection, thereby increasing the phagocytic ability of macrophages, and ultimately improving lung tissue damage in mouse; ASCs may inhibit NLRC4 inflammasome through the secretion of STC-1. [ABSTRACT FROM AUTHOR]

Subarachnoid hemorrhage is associated with high morbidity and mortality and lacks effective treatment. Pyroptosis is a crucial mechanism underlying early brain injury after subarachnoid hemorrhage. Previous studies have confirmed that tumor necrosis factor-stimulated gene-6 (TSG-6) can exert a neuroprotective effect by suppressing oxidative stress and apoptosis. However, no study to date has explored whether TSG-6 can alleviate pyroptosis in early brain injury after subarachnoid hemorrhage. In this study, a C57BL/6J mouse model of subarachnoid hemorrhage was established using the endovascular perforation method. Our results indicated that TSG-6 expression was predominantly detected in astrocytes, along with NLRC4 and gasdermin-D (GSDMD). The expression of NLRC4, GSDMD and its N-terminal domain (GSDMD-N), and cleaved caspase-1 was significantly enhanced after subarachnoid hemorrhage and accompanied by brain edema and neurological impairment. To explore how TSG-6 affects pyroptosis during early brain injury after subarachnoid hemorrhage, recombinant human TSG-6 or a siRNA targeting TSG-6 was injected into the cerebral ventricles. Exogenous TSG-6 administration downregulated the expression of NLRC4 and pyroptosis-associated proteins and alleviated brain edema and neurological deficits. Moreover, TSG-6 knockdown further increased the expression of NLRC4, which was accompanied by more severe astrocyte pyroptosis. In summary, our study revealed that TSG-6 provides neuroprotection against early brain injury after subarachnoid hemorrhage by suppressing NLRC4 inflammasome activation-induced astrocyte pyroptosis., Competing Interests: None

Bacteria-released components can modulate host innate immune response in the absence of direct host cell–bacteria interaction. In particular, bacteria-derived outer membrane vesicles (OMVs) were recently shown to activate host caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide. However, further precise understanding of innate immune-modulation by bacterial OMVs remains elusive. Here, we present evidence that flagellated bacteria-released OMVs can trigger NLRC4 canonical inflammasome activation via flagellin delivery to the cytoplasm of host cells. Salmonella typhimurium-derived OMVs caused a robust NLRC4-mediated caspase-1 activation and interleukin-1β secretion in macrophages in an endocytosis-dependent, but guanylate-binding protein-independent manner. Notably, OMV-associated flagellin is crucial for Salmonella OMV-induced inflammasome response. Flagellated Pseudomonas aeruginosa-released OMVs consistently promoted robust NLRC4 inflammasome activation, while non-flagellated Escherichia coli-released OMVs induced NLRC4-independent non-canonical inflammasome activation leading to NLRP3-mediated interleukin-1β secretion. Flagellin-deficient Salmonella OMVs caused a weak interleukin-1β production in a NLRP3-dependent manner. These findings indicate that Salmonella OMV triggers NLRC4 inflammasome activation via OMV-associated flagellin in addition to a mild induction of non-canonical inflammasome signaling via OMV-bound lipopolysaccharide. Intriguingly, flagellated Salmonella-derived OMVs induced more rapid inflammasome response than flagellin-deficient Salmonella OMV and non-flagellated Escherichia coli-derived OMVs. Supporting these in vitro results, Nlrc4-deficient mice showed significantly reduced interleukin-1β production after intraperitoneal challenge with Salmonella-released OMVs. Taken together, our results here propose that NLRC4 inflammasome machinery is a rapid sensor of bacterial OMV-bound flagellin as a host defense mechanism against bacterial pathogen infection.

The NLRC4 inflammasome assembles in response to detection of bacterial invasion, and NLRC4 activation leads to the production of IL-1β and IL-18 together with pyroptosis-mediated cell death. Missense activating mutations in NLRC4 cause autoinflammatory disorders whose symptoms are distinctly dependent on the site of mutation and other aspects of the genetic background. To determine the involvement of IL-1β and IL-18 in the inflammation induced by NLRC4 mutation, we depleted IL-1β, IL-18, or both cytokines in Nlrc4-transgenic mice in which mutant Nlrc4 is expressed under the MHC class II promoter (Nlrc4-H443P-Tg mice). The deletion of the Il1b or Il18 gene in Nlrc4-H443P-Tg mice reduced the neutrophil numbers in the spleen, and mice with deletion of both genes had an equivalent number of neutrophils compared to wild-type mice. Deletion of Il1b ameliorated but did not eliminate bone marrow hyperplasia, while mice deficient in Il18 showed no bone marrow hyperplasia. In contrast, tail bone deformity remained in the presence of Il18 deficiency, but Il1b deficiency completely abolished bone deformity. The decreased bone density in Nlrc4-H443P-Tg mice was counteracted by Il1b but not Il18 deficiency. Our results demonstrate the distinct effects of IL-1β and IL-18 on NLRC4-induced inflammation among tissues, which suggests that blockers for each cytokine should be utilized depending on the site of inflammation.

The interplay between M. tuberculosis (Mtb) and humans is multifactorial. The susceptibility/resistance profile and the establishment of clinical tuberculosis (TB) still remains elusive. The gain-of-function variant rs10754558 in the NLRP3 gene (found in 30% of the world population) confers protection against the development of TB, indicating a prominent role played by NLRP3 inflammasome against Mtb. Through genotype-guided assays and various Mtb strains (BCG, H37Rv, Beijing-1471, MP287/03), we demonstrate that Mtb strains activate inflammasome according to the NLRP3/IL-1ß or NLRC4/IL18 preferential axis. NLRP3 and NLRC4 genetic variants contribute to the presentation of TB. For the first time, we have shown that loss-of-function variants in NLRC4 significantly contribute to the development of extra-pulmonary TB. The analysis of inflammasome activation in a cohort of TB patients and their “household contacts” (CNT) revealed that plasma IL-1ß/IFN-α ratio lets us distinguish patients from Mtb-exposed-but-healthy individuals from an endemic region. Moreover, NLRP3 inflammasome seemed “exhausted” in TB patients compared to CNT, indicating a more efficient activation of inflammasome in resistant individuals. These findings suggest that inflammasome genetics as well as virulence-dependent level of inflammasome activation contribute to the onset of a susceptible/resistant profile among Mtb-exposed individuals.

Recent research has led to novel findings in inflammasome biology and genetics that altered the diagnosis and management of patients with autoinflammatory syndromes caused by NLRP3-, Pyrin-, NLRP1-, and NLRC4-inflammasomes and spurred the development of novel treatments. The use of next-generation sequencing in clinical practice allows for rapid diagnosis and the detection of somatic mutations that cause autoinflammatory diseases. Clinical differences in patients with NLRP3, pyrin, and NLRP1 inflammasomopathies, and the constitutive elevation of unbound free serum IL-18 that predisposes to the development of macrophage activation syndrome (MAS) in patients with gain-of function mutations in NLRC4 led to the screening and the characterization of novel diseases presenting with constitutively elevated serum IL-18 levels, and start to unravel the biology of “high IL-18 states” that translate into the use of biomarkers that improve diagnosis and monitoring of disease activity and investigations of treatments that target IL-18 and IFN-gamma which promise to improve the management and outcome of these conditions. Lastly, advances in structural modeling by cryo-electron microscopy (cryo-EM) of gasdermin, and of NLRP3- and NLRC4-inflammasome assembly, and the characterization of post-translational modifications (PTM) that regulate inflammasome activation, coupled with high-throughput screening (HTS) of libraries of inflammasome-inhibiting compounds, promise a new generation of treatments for patients with inflammasome-mediated diseases.

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

Introduction: Bacterial flagellin, as a pathogen-associated molecular pattern (PAMP), can activate both innate and adaptive immunity. Its unique structural characteristics endow an effective and flexible adjuvant activity, which allow the design of different types of vaccine strategies to prevent various diseases. This review will discuss recent progress in the mechanism of action of flagellin and its prospects for use as a vaccine adjuvant. Areas covered: Herein we summarize various types of information related to flagellin adjuvants from PubMed, including structures, signaling pathways, natural immunity, and extensive applications in vaccines, and it discusses the immunogenicity, safety, and efficacy of flagellin-adjuvanted vaccines in clinical trials. Expert commentary: It is widely accepted that as an adjuvant, flagellin can induce an enhanced antigen-specific immune response. Flagellin adjuvants will allow more effective flagellin-based vaccines to enter clinical trials. Furthermore, vaccine formulations containing PAMPs are crucial to exert the maximum potential of vaccine antigens. Therefore, combinations of flagellin-adjuvanted vaccines with other adjuvants that act in a synergistic manner, particularly TLR ligands, represent a promising method for tailoring targeted vaccines to meet specific requirements.

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether this innate sensing mechanism has any regulatory effect on the expression of stimulatory ligands by the pathogen. During infection with Salmonella and many other pathogens, flagellin is a major activator of NLRC4 inflammasome-mediated macrophage pyroptosis and pathogen eradication. Salmonella switches to a flagellin-low phenotype as infection progresses to avoid this mechanism of clearance by the host. However, the host cues that Salmonella perceives to undergo this switch remain unclear. Here, we report an unexpected role of the NLRC4 inflammasome in promoting expression of its microbial ligand, flagellin, and identify a role for type 1 IFN signaling in switching of Salmonella to a flagellin-low phenotype. Early in infection, activation of NLRC4 by flagellin initiates pyroptosis and concomitant release of lysophospholipidswhich in turn enhance expression of flagellin by Salmonella thereby amplifying its ability to elicit cell death. TRIF-dependent production of type 1 IFN, however, later represses NLRC4 and the lysophospholipid biosynthetic enzyme iPLA2, causing a decline in intracellular lysophospholipids that results in down-regulation of flagellin expression by Salmonella. These findings reveal a previously unrecognized immune-modulating regulatory cross-talk between endosomal TLR signaling and cytosolic NLR activation with significant implications for the establishment of infection with Salmonella. [ABSTRACT FROM AUTHOR]

Bacteria-released components can modulate host innate immune response in the absence of direct host cell–bacteria interaction. In particular, bacteria-derived outer membrane vesicles (OMVs) were recently shown to activate host caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide. However, further precise understanding of innate immune-modulation by bacterial OMVs remains elusive. Here, we present evidence that flagellated bacteria-released OMVs can trigger NLRC4 canonical inflammasome activation via flagellin delivery to the cytoplasm of host cells. Salmonella typhimurium -derived OMVs caused a robust NLRC4-mediated caspase-1 activation and interleukin-1β secretion in macrophages in an endocytosis-dependent, but guanylate-binding protein-independent manner. Notably, OMV-associated flagellin is crucial for Salmonella OMV-induced inflammasome response. Flagellated Pseudomonas aeruginosa -released OMVs consistently promoted robust NLRC4 inflammasome activation, while non-flagellated Escherichia coli -released OMVs induced NLRC4-independent non-canonical inflammasome activation leading to NLRP3-mediated interleukin-1β secretion. Flagellin-deficient Salmonella OMVs caused a weak interleukin-1β production in a NLRP3-dependent manner. These findings indicate that Salmonella OMV triggers NLRC4 inflammasome activation via OMV-associated flagellin in addition to a mild induction of non-canonical inflammasome signaling via OMV-bound lipopolysaccharide. Intriguingly, flagellated Salmonella -derived OMVs induced more rapid inflammasome response than flagellin-deficient Salmonella OMV and non-flagellated Escherichia coli -derived OMVs. Supporting these in vitro results, Nlrc4 -deficient mice showed significantly reduced interleukin-1β production after intraperitoneal challenge with Salmonella -released OMVs. Taken together, our results here propose that NLRC4 inflammasome machinery is a rapid sensor of bacterial OMV-bound flagellin as a host defense mechanism against bacterial pathogen infection. [ABSTRACT FROM AUTHOR]

The interplay between M. tuberculosis (Mtb) and humans is multifactorial. The susceptibility/resistance profile and the establishment of clinical tuberculosis (TB) still remains elusive. The gain-of-function variant rs10754558 in the NLRP3 gene (found in 30% of the world population) confers protection against the development of TB, indicating a prominent role played by NLRP3 inflammasome against Mtb. Through genotype-guided assays and various Mtb strains (BCG, H37Rv, Beijing-1471, MP287/03), we demonstrate that Mtb strains activate inflammasome according to the NLRP3/IL-1ß or NLRC4/IL18 preferential axis. NLRP3 and NLRC4 g enetic variants contribute to the presentation of TB. For the first time, we have shown that loss-of-function variants in NLRC4 significantly contribute to the development of extra-pulmonary TB. The analysis of inflammasome activation in a cohort of TB patients and their "household contacts" (CNT) revealed that plasma IL-1ß/IFN-α ratio lets us distinguish patients from Mtb-exposed-but-healthy individuals from an endemic region. Moreover, NLRP3 inflammasome seemed "exhausted" in TB patients compared to CNT, indicating a more efficient activation of inflammasome in resistant individuals. These findings suggest that inflammasome genetics as well as virulence-dependent level of inflammasome activation contribute to the onset of a susceptible/resistant profile among Mtb-exposed individuals. [ABSTRACT FROM AUTHOR]

Recent research has led to novel findings in inflammasome biology and genetics that altered the diagnosis and management of patients with autoinflammatory syndromes caused by NLRP3-, Pyrin-, NLRP1-, and NLRC4-inflammasomes and spurred the development of novel treatments. The use of next-generation sequencing in clinical practice allows for rapid diagnosis and the detection of somatic mutations that cause autoinflammatory diseases. Clinical differences in patients with NLRP3, pyrin, and NLRP1 inflammasomopathies, and the constitutive elevation of unbound free serum IL-18 that predisposes to the development of macrophage activation syndrome (MAS) in patients with gain-of function mutations in NLRC4 led to the screening and the characterization of novel diseases presenting with constitutively elevated serum IL-18 levels, and start to unravel the biology of "high IL-18 states" that translate into the use of biomarkers that improve diagnosis and monitoring of disease activity and investigations of treatments that target IL-18 and IFN-gamma which promise to improve the management and outcome of these conditions. Lastly, advances in structural modeling by cryo-electron microscopy (cryo-EM) of gasdermin, and of NLRP3- and NLRC4-inflammasome assembly, and the characterization of post-translational modifications (PTM) that regulate inflammasome activation, coupled with high-throughput screening (HTS) of libraries of inflammasome-inhibiting compounds, promise a new generation of treatments for patients with inflammasome-mediated diseases. [ABSTRACT FROM AUTHOR]

Post-hypoxic/ischemic neuroinflammation is selectively driven by sterile inflammation, which implies the interplay of brain-intrinsic immune cells with other neural cells and immigrated peripheral immune cells. The resultant inflammatory cascade evolves extra- and intracellular pathogen and danger-associated receptors. The latter interacts with multiprotein complexes termed inflammasomes. The NLRP3 inflammasome is one of the best-described inflammasomes. However, its impact on post-ischemic neuroinflammation and its role in neuroprotection after ischemic stroke are still under debate. Microglial cells are known to be the main source of neuroinflammation; hence, we depleted NLRP3 in BV-2 microglial cells using shRNA to investigate its role in IL-1β maturation and phagocytosis after hypoxia (oxygen-glucose-deprivation (OGD)). We also examined the expression profiles of other inflammasomes (NLRC4, AIM2, ASC) and caspase-1 activity after OGD. OGD triggered caspase-1 activity and increased IL-1β secretion in BV-2 cells with no alteration after NLRP3 depletion. The expression of the AIM2 inflammasome was significantly higher after OGD in NLRP3-depleted cells, whereas NLRC4 was unaltered in all groups. Interestingly, OGD induced a complete inactivation of phagocytic activity in wild-type cells, while in NLRP3-depleted BV-2, this inactivity was restored after hypoxia. Our findings indicate a minor role of NLRP3 in the inflammatory response after hypoxic/ischemic stimulus. However, NLRP3 seems to play a pivotal role in the regulation of post-ischemic phagocytosis. This might be a prerequisite for the putative neuroprotective effect. [ABSTRACT FROM AUTHOR]

• IL-1β plays a critical role in the pathogenesis of Alzheimer's disease. • Inflammasome complex pave the way for IL-1β production in Alzheimer's disease. • IL-1β and caspase-1 production are associated with NLRC4 inflammasome. • NLRC4 inflammasome was involved in neuroinflammation and memory impairment. Inflammatory response through interleukin-1β (IL-1β) plays a key role in the pathogenesis of Alzheimer's disease (AD). However, the molecular mechanism of pro-IL-1β processing in AD is not clearly defined. The current study was designed to investigate which of the inflammasome complexes are critical for IL-1β production in AD. An experimental model for Alzheimer like disease was induced in male Wistar rats and Morris Water Maze was used to evaluate the function of learning and memory. The expression of genes involved in inflammasome complex including NLRP1, NLRP3, NLRC4, AIM2, ASC, IL18, IL-1β and caspase-1 was determined via Real-time PCR. Hematoxylin and Eosin (H&E) staining and Immunohistochemistry (IHC) for CD45 was applied to assess inflammatory cells infiltration. Furthermore, caspase-1, IL-1β and phosphorylated tau (p-Tau) protein expressing cells were investigated in the lesion area using immunofluorescence staining technique. The behavioral study revealed that streptozotocin (STZ) injection significantly impaired learning and memory function. In addition, the infiltration of inflammatory cells was confirmed in the hippocampus region of STZ-treated animals. Furthermore, a significant increase in the expression level of NLRC4 inflammasome, ASC and IL-1β was identified in STZ-treated animals. In contrast, no significant difference was observed in other inflammasome components including NLRP1, NLRP3, AIM2, IL-18 and caspase-1 in STZ-treated group compared with the control group. Moreover, the number of caspase-1, IL-1β and p-Tau protein positive cells were remarkably increased in STZ-treated animals. Based on the obtained results, it can be concluded that increased production of IL-1β, caspase-1 and p-Tau through association with NLRC4 inflammasome may be involved in neuroinflammation and memory impairment in AD, which creates a new horizon in this regard. Hence, strategies targeting NLRC4 inflammasome could be beneficial for the treatment of AD. [ABSTRACT FROM AUTHOR]

Acidovorax avenae is a flagellated, pathogenic bacterium to various plant crops that has also been found in human patients with haematological malignancy, fever, and sepsis; however, the exact mechanism for infection in humans is not known. We hypothesized that the human innate immune system could be responsive to the purified flagellin isolated from A. avenae, named FLA-AA. We observed the secretion of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8 by treating FLA-AA to human dermal fibroblasts, as well as macrophages. This response was exclusively through TLR5, which was confirmed by using TLR5-overexpression cell line, 293/hTLR5, as well as TLR5-specific inhibitor, TH1020. We also observed the secretion of inflammatory cytokine, IL-1β, by the activation of NLRC4 with FLA-AA. Overall, our results provide a molecular basis for the inflammatory response caused by FLA-AA in cell-based assays.

Activation of the NLRC4 inflammasome is crucial for defense against bacterial species that have flagellin or the type III secretion system (T3SS). We have discovered the role of interferon regulatory factor 8 (IRF8) in mediating NLRC4 inflammasome activation. IRF8 is required for the transcription of genes encoding NAIPs, thereby enabling cellular detection of flagellin or T3SS proteins. In vivo, IRF8 is important for NLRC4 inflammasome–dependent cytokine production, bacterial clearance, and ultimately, host survival. By introducing IRF8 as a player in inflammasome regulation, our study provides a new perspective on that process.

BackgroundInflammatory response during urinary tract infection (UTI) is mediated by innate immune defense. Nod like receptors (NLRs) have been proposed to work simultaneously beside TLR pathways to mediate pro-inflammatory response and maintain tissue homeostasis. Some in vitro reports have showed the involvement of NLRP3 inflammasome during uropathogenic Escherichia coli (UPEC) mediated UTI. So we have sought to determine the status of various inflammasomes and their components in UPEC mediated UTI.MethodsA total of 186 females experiencing the first episode of UTI were recruited for the study and forty were found to be positive for UPEC (≥105 CFU/ml) in their urine (N = 40). Further, we analyzed the expression of NLRP3, NLRC4, NAIP, AIM2, ASC, CASPASE-4, and CASPASE-1 gene at mRNA and protein level in the blood of UPEC confirmed study subjects through real time qPCR and immunoblotting. Healthy females (N = 40) visiting the OPD for health checkups, family planning advice and subjects undergoing routine medical examinations, were recruited as healthy control subjects. Pro-inflammatory cytokines (IL-6, IL-8, IFN-γ, TNF-α and MCP-1) were measured in the plasma of patients and controls through ELISA. For investigation of the involvement of NLRC4 and NLRP3 inflammasome, in vitro studies were performed using co-immunoprecipitation and confocal microscopy.ResultsMost of the inflammatory regulators studied (i.e., NLRP3, NAIP, NLRC4, ASC, and CASPASE-1) were found to be up-regulated at both mRNA and protein levels in the UPEC infected UTI patients. Also, pro-inflammatory cytokines (IL-6, IL-8, IFN-γ, TNF-α, and MCP-1) were found to be up-regulated in the patients group. However, no significant difference was observed in the expression of AIM2 and CASPASE-4 genes at both mRNA and protein levels. Further, in vitro studies have shown the involvement of NLRC4 inflammasome in UPEC infected THP1 derived macrophages.ConclusionInvolvement of NLRP3 and NLRC4 inflammasomes in UPEC infected UTI is evident from our findings. This is the first report showing levels of inflammasome and its components in UTI patients suggesting a possible role during UPEC mediated UTI. We have also reported the involvement of NLRC4 inflammasome for the first time during UTI infection.

Introduction: NLRP3 inflammasome plays a key role in dendritic cells (DC) activation in response to vaccine adjuvants, however we previously showed that it is not properly activated in DC from HIV-infected patients (HIV-DC), explaining, at least in part, the poor response to immunization of these patients. Taking in account that several cytoplasmic receptors are able to activate inflammasome, and that bacterial components are considered as a novel and efficient adjuvant, we postulated that bacterial flagellin (FLG), a natural ligand of NAIP/NLRC4 inflammasome, could rescue the activation of the complex in HIV-DC.Objective: Demonstrate that FLG is able to activate monocyte-derived dendritic cells from HIV-infected individuals better than LPS, and to what extent the entity of inflammasome activation differs between DC from HIV-infected patients and healthy donors.Methods: Monocyte-derived dendritic cells from HIV-infected patients (HIV-DC) and healthy donors (HD-DC) were stimulated with FLG, and inflammasome as well as DC activation (phenotypic profile, cytokine production, autologous lymphocytes activation) were compared. Chemical and genetic inhibitors were used to depict the relative contribution of NLRC4 and NLRP3 in HIV/HD-DC response to FLG.Results: FLG properly activates HD-DC and HIV-DC. FLG induces higher inflammasome activation than LPS in HIV-DC. FLG acts through NLRC4 and NLRP3 in HD-DC, but at a lesser extent in HIV-DC due to intrinsic NLRP3 defect.Conclusions: FLG by-passes NLRP3 defect in HIV-DC, through the activation of NAIP/NLRC4 inflammasome, indicating possible future use of the bacterial component as an efficient adjuvant in immunocompromised individuals.

Sesamin (Ses) is a natural lignan abundantly present in sesame and sesame oil. Pyroptosis, a newly identified type of pro-inflammatory programmed necrosis, contributes to the development of non-alcoholic steatohepatitis (NASH) when hepatocyte pyroptosis is excessive. In this study, Ses treatment demonstrated an improvement in hepatic damage in mice with high-fat, high-cholesterol diet-induced NASH and palmitate (PA)-treated mouse primary hepatocytes. Notably, we discovered, for the first time, that Ses could alleviate hepatocyte pyroptosis both in vivo and in vitro . Furthermore, treatment with phorbol myristate acetate, a protein kinase Cδ (PKCδ) agonist, increased PKCδ phosphorylation and attenuated the protective effects of Ses against pyroptosis in PA-treated mouse primary hepatocytes. Mechanistically, Ses treatment alleviated hepatocyte pyroptosis in NASH, which was associated with the regulation of the PKCδ/nod-like receptor family CARD domain-containing protein 4/caspase-1 axis. This study introduces a novel concept and target, suggesting the potential use of functional factors in food to alleviate liver damage caused by NASH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Zhou, Zhang, Wang, Lv, Wang, Bai, Hua and Guo.)

Inflammasomes are intracellular, multiprotein supercomplexes that mediate a post-translational inflammatory response to both pathogen and endogenous danger signals. They consist of a sensor, the adapter ASC, and the protease caspase 1 and, following their activation, lead to cl1β, as well as lytic cell death. Due to this potent inflammatory capacity, understanding inflammasome biology is important in many pathological conditions. It is increasingly clear that inflammasomes are particularly relevant in macrophages, which express a diverse range of inflammasome sensors. In these two chapters, we detail methods to isolate and differentiate human macrophages, murine bone marrow-derived macrophages, and murine microglia and stimulate the inflammasomes known to be expressed in macrophages, including the AIM2, NLRP3, NLRC4, NLRP1, and non-canonical inflammasomes. Furthermore, we describe the methodology required to measure the various results of inflammasome activation including ASC speck formation, monitoring lytic cell death and cytokine release, as well as caspase-1 activation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The inflammasome is a cytosolic immune signaling complex that induces inflammation and pyroptosis. Inflammasome complexes respond to a variety of pathogens, as well as danger or homeostasis-altering signals; they can play critical roles in the development of autoinflammatory conditions and cancer. Studies have now provided additional insights into the activation mechanisms and regulation of established inflammasome complexes, including NLRP1b, NLRP3, NOD-like receptor family apoptosis inhibitory protein (NAIP)–NLRC4, absent in melanoma (AIM)2, caspase-11, and pyrin. New activators and regulators of emerging NLRP6 and NLRP9b inflammasome complexes have also been described. We highlight the latest progress in our understanding of the molecular mechanisms governing inflammasome activation and pyroptosis, including the discovery of the pore-forming protein gasdermin D (GSDMD). We also discuss the importance of inflammasome activators and regulators in health and disease. The inflammasome family is expanding and now includes the canonical AIM2, NAIP-NLRC4, NLRP1, NLRP3, NLRP6, NLRP9b, and pyrin inflammasomes and the noncanonical caspase-11 inflammasome. Inflammasome sensors detect a growing constellation of pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), and homeostasis-altering molecular processes (HAMPs). Activation of human caspase-1, -4, and -5, or mouse caspase-1 and -11, induces cleavage of the pore-forming protein GSDMD, mediating pyroptosis. Assembly and signaling of the inflammasome often requires transcriptional and translational regulation, and spatial localization of inflammasome components at the site of organelles, such as the Golgi and mitochondria. Targeting inflammasome components has potential therapeutic value in the treatment of certain infections, cancers, and inflammatory conditions. [ABSTRACT FROM AUTHOR]

NLRC4 [nucleotide-binding domain and leucine-rich repeat (NLR) family, caspase recruitment domain (CARD) containing 4] is an innate immune receptor, which, upon detection of certain pathogens or internal distress signals, initiates caspase-1–mediated interleukin-1β maturation and an inflammatory response. A gain-of-function mutation, H443P in NLRC4, causes familial cold autoinflammatory syndrome (FCAS) characterized by cold-induced hyperactivation of caspase-1, enhanced interleukin-1β maturation, and inflammation. Although the H443P mutant shows constitutive activity, the mechanism involved in hyperactivation of caspase-1 by NLRC4-H443P upon exposure of cells to lower temperature is not known. Here, we show that heat shock cognate protein 70 (HSC70) complexes with NLRC4 and negatively regulates caspase-1 activation by NLRC4-H443P in human cells. Compared with NLRC4, the structurally altered NLRC4-H443P shows enhanced interaction with HSC70. Nucleotide binding- and leucine-rich repeat domains of NLRC4, but not its CARD, can engage in complex formation with HSC70. Knockdown of HSC70 enhances apoptosis-associated speck-like protein containing a CARD (ASC)-speck formation and caspase-1 activation by NLRC4-H443P. Exposure to subnormal temperature results in reduced interaction of NLRC4-H443P with HSC70, and an increase in its ability to form ASC specks and activate caspase-1. Unlike the NLRC4-H443P mutant, another constitutively active mutant (NLRC4-V341A) associatedwith autoinflammatory diseases, but not FCAS, showed neither enhanced interaction with HSC70 nor an increase in inflammasome formation upon exposure to subnormal temperature. Our results identify HSC70 as a negative regulator of caspase-1 activation by the temperature-sensitive NLRC4-H443P mutant. We also show that low-temperature–induced hyperactivation of caspase-1 by NLRC4-H443P is due to loss of inhibition by HSC70. [ABSTRACT FROM AUTHOR]

Background: Inflammatory response during urinary tract infection (UTI) is mediated by innate immune defense. Nod like receptors (NLRs) have been proposed to work simultaneously beside TLR pathways to mediate pro-inflammatory response and maintain tissue homeostasis. Some in vitro reports have showed the involvement of NLRP3 inflammasome during uropathogenic Escherichia coli (UPEC) mediated UTI. So we have sought to determine the status of various inflammasomes and their components in UPEC mediated UTI. Methods: A total of 186 females experiencing the first episode of UTI were recruited for the study and forty were found to be positive for UPEC (≥105 CFU/ml) in their urine (N = 40). Further, we analyzed the expression of NLRP3 , NLRC4 , NAIP , AIM2 , ASC , CASPASE-4 , and CASPASE-1 gene at mRNA and protein level in the blood of UPEC confirmed study subjects through real time qPCR and immunoblotting. Healthy females (N = 40) visiting the OPD for health checkups, family planning advice and subjects undergoing routine medical examinations, were recruited as healthy control subjects. Pro-inflammatory cytokines (IL-6, IL-8, IFN-γ, TNF-α and MCP-1) were measured in the plasma of patients and controls through ELISA. For investigation of the involvement of NLRC4 and NLRP3 inflammasome, in vitro studies were performed using co-immunoprecipitation and confocal microscopy. Results: Most of the inflammatory regulators studied (i.e., NLRP3, NAIP, NLRC4, ASC , and CASPASE-1) were found to be up-regulated at both mRNA and protein levels in the UPEC infected UTI patients. Also, pro-inflammatory cytokines (IL-6, IL-8, IFN-γ, TNF-α, and MCP-1) were found to be up-regulated in the patients group. However, no significant difference was observed in the expression of AIM2 and CASPASE-4 genes at both mRNA and protein levels. Further, in vitro studies have shown the involvement of NLRC4 inflammasome in UPEC infected THP1 derived macrophages. Conclusion: Involvement of NLRP3 and NLRC4 inflammasomes in UPEC infected UTI is evident from our findings. This is the first report showing levels of inflammasome and its components in UTI patients suggesting a possible role during UPEC mediated UTI. We have also reported the involvement of NLRC4 inflammasome for the first time during UTI infection. [ABSTRACT FROM AUTHOR]

This study was aimed to determine Gastrodin (GAS) and its underlying signaling pathway involved in suppression of inflammasome specifically in reactive astrocytes that are featured prominently in different neurological conditions or diseases including cerebral ischemia. For this purpose, TNA2 astrocytes in cultures were exposed to oxygen–glucose–deprivation (OGD) mimicking hypoxic cerebral ischemia. Separately, TNA2 cells were pretreated with GAS prior to OGD exposure. Additionally, Stattic, an inhibitor of STAT3 signaling pathway, was used to ascertain its involvement in regulating inflammasome in astrocytes exposed to OGD. In parallel to the above, adult rats subjected to middle cerebral artery occlusion (MCAO) with or without GAS pretreatment were sacrificed at different time points to determine the effects of GAS on astrocyte inflammasome. TNA2 astrocytes in different treatments as well as reactive astrocytes in MCAO were processed for immunofluorescence labeling and Western blot analysis for various protein markers. In the latter, protein expression levels of p-STAT3, NLRP3, and NLRC4 were markedly increased in TNA2 astrocytes exposed to OGD. Remarkably, the expression levels of these biomarkers were significantly suppressed by GAS. Of note, GAS especially at dose 20 μM inhibited NLRP3 and NLRC4 expression levels most substantially. Moreover, GAS inhibited the downstream proteins caspase-1 and IL-18. Concomitantly, GAS significantly suppressed the expression of STAT3 and NF-κB signaling pathway. It is noteworthy that Stattic at dose 100 μM inhibited STAT3 pathway and NF-κB activation in TNA2 astrocytes, an effect that was shared by GAS. In MCAO, GAS was found to effectively attenuate p-STAT3 immunofluorescence intensity in reactive astrocytes. Arising from the above, it is concluded that GAS is anti-inflammatory as it effectively suppresses inflammasome in OGD-stimulated astrocytes as well as in reactive astrocytes in MCAO via STAT3 and NF-κB signaling expression coupled with decreased expression of caspase-1 and IL-18. [ABSTRACT FROM AUTHOR]

Introduction: NLRP3 inflammasome plays a key role in dendritic cells (DC) activation in response to vaccine adjuvants, however we previously showed that it is not properly activated in DC from HIV-infected patients (HIV-DC), explaining, at least in part, the poor response to immunization of these patients. Taking in account that several cytoplasmic receptors are able to activate inflammasome, and that bacterial components are considered as a novel and efficient adjuvant, we postulated that bacterial flagellin (FLG), a natural ligand of NAIP/NLRC4 inflammasome, could rescue the activation of the complex in HIV-DC. Objective: Demonstrate that FLG is able to activate monocyte-derived dendritic cells from HIV-infected individuals better than LPS, and to what extent the entity of inflammasome activation differs between DC from HIV-infected patients and healthy donors. Methods: Monocyte-derived dendritic cells from HIV-infected patients (HIV-DC) and healthy donors (HD-DC) were stimulated with FLG, and inflammasome as well as DC activation (phenotypic profile, cytokine production, autologous lymphocytes activation) were compared. Chemical and genetic inhibitors were used to depict the relative contribution of NLRC4 and NLRP3 in HIV/HD-DC response to FLG. Results: FLG properly activates HD-DC and HIV-DC. FLG induces higher inflammasome activation than LPS in HIV-DC. FLG acts through NLRC4 and NLRP3 in HD-DC, but at a lesser extent in HIV-DC due to intrinsic NLRP3 defect. Conclusions: FLG by-passes NLRP3 defect in HIV-DC, through the activation of NAIP/NLRC4 inflammasome, indicating possible future use of the bacterial component as an efficient adjuvant in immunocompromised individuals. [ABSTRACT FROM AUTHOR]

Highlights • Low serum level of IL-18 represents a protective factor against MS development. • Genetic variants associated to constitutive inflammasome activation contribute to MS severity. • Loss-of-function NLRC4 SNP was associated with better treatment response in MS patients. • MS monocytes presented high expression of inflammasome genes and produced more IL-1ß than controls. Abstract Background Multiple sclerosis (MS) is a neurodegenerative disease of central nervous system (CNS) with autoimmune and inflammatory characteristics, and a still uncertain pathogenesis. Early events as well as evolution of MS are heterogeneous (three main clinical forms) and multifactorial. Genome-wide association studies indicate that MS pathogenesis shares features with both autoimmune and inflammatory diseases. Innate immunity has been recently proved to be an important factor in MS. Genetic variants in inflammasome components have been associated with both autoimmune and neurodegenerative diseases, letting us hypothesize that inflammasome, and related cytokines IL-1ß and IL-18, could represent important contributors in MS pathogenesis, and eventually explain, at least in part, the heterogeneity observed in MS patients. Aim To evaluate the contribution of inflammasome in MS, in term of (a) genetic effect on development, severity and/or prognosis, and (b) complex activation in peripheral blood as a measure of systemic inflammation. Methods Functional genetic variants in inflammasome components were analyzed in a cohort of MS patients, by the use of allele-specific assays and qPCR. Multivariate analysis was performed based on clinical form (recurrent remittent/RR, primary progressive/PP or secondary progressive/SP), severity index (EDSS) and progression index (PI), response to IFN-ß treatment. Peripheral blood monocytes (PBM) of patients were examined for inflammasome activation and expression profile. Results and discussion Variants associated with low serum levels of IL-18 were significantly less frequent in MS patients than in controls, suggesting a protective role of diminished IL-18-mediate inflammation in MS development. On the other hands, gain-of-function variants in NLRP3 (Q705K) and IL1B (-511 C >T) associated with severity and progression of MS, suggesting that a constitutive activation of NLRP3 inflammasome could represent a risk factor for MS clinical presentation. Accordingly, -511C >T SNP resulted more frequent in progressive forms than in RR MS, reinforcing the idea that increased inflammasome activation characterized bad prognosis of MS. Altogether these findings corroborate previous data about the harmful role of NLRP3 inflammasome in experimental autoimmune encephalitis (EAE). Moreover, we reported for the first time the beneficial effect of NLRC4 rs479333 G >C variant in MS progression and in the response to IFN-ß treatment. This intronic polymorphism have been previously associated to decreased NLRC4 transcription and low IL-18 serum level, indicated once more that less activation of inflammasome and IL-18 production are beneficial for MS patients. PBM analysis showed that MS cells express higher level of inflammasome genes than HD ones, and are more prone to respond to a classical NLRP3 stimulus than HD. [ABSTRACT FROM AUTHOR]

Inflammasomes are signaling hubs that activate inflammatory caspases to drive cytokine maturation and cell lysis. Inflammasome activation by Salmonella Typhimurium infection or Salmonella‐derived molecules is extensively studied in murine myeloid cells. Salmonella‐induced inflammasome signaling in human innate immune cells, is however, poorly characterized. Here, we show that Salmonella mutation to inactivate the Salmonella pathogenicity island‐2 type III secretion system (SPI2 T3SS) potentiates S. Typhimurium‐induced inflammasome responses from primary human macrophages, resulting in strong IL‐1β production and macrophage death. Inactivation of the SPI1 T3SS diminished human macrophage responses to WT and ΔSPI2 Salmonella. Salmonella ΔSPI2 elicited a mixed inflammasome response from human myeloid cells, in which NLR family CARD‐domain containing protein 4 (NLRC4) and NLR family PYRIN‐domain containing protein 3 (NLRP3) perform somewhat redundant functions in generating IL‐1β and inducing pyroptosis. Our data suggest that Salmonella employs the SPI2 T3SS to subvert SPI1‐induced NLRP3 and NLRC4 inflammasome responses in human primary macrophages, in a species‐specific immune evasion mechanism. The Gram‐negative bacterium Salmonella Typhimurium suppresses human macrophage inflammasome responses via the Salmonella Pathogenicity Island 2. [ABSTRACT FROM AUTHOR]

Highlights • Inflammasomes play a role in inflammatory responses and cell death. • Functional roles for inflammasomes in microglia are thus far unknown. • Microglial cells produce and release inflammasome components following ischemia. • NLRC4 inflammasome promotes apoptotic and pyroptotic microglial death. • Inhibition of caspase-1 protects against apoptotic and pyroptotic microglial death. Abstract Stroke is the second leading cause of death in the world and a major cause of long-term disability. Recent evidence has provided insight into a newly described inflammatory mechanism that contributes to neuronal and glial cell death, and impaired neurological outcome following ischemic stroke – a form of sterile inflammation involving innate immune complexes termed inflammasomes. It has been established that inflammasome activation following ischemic stroke contributes to neuronal cell death, but little is known about inflammasome function and cell death in activated microglial cells following cerebral ischemia. Microglia are considered the resident immune cells that function as the primary immune defense in the brain. This study has comprehensively investigated the expression and activation of NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in isolates of microglial cells subjected to simulated ischemic conditions and in the brain following ischemic stroke. Immunoblot analysis from culture media indicated microglial cells release inflammasome components and inflammasome activation-dependent pro-inflammatory cytokines following ischemic conditions. In addition, a functional role for NLRC4 inflammasomes was determined using siRNA knockdown of NLRC4 and pharmacological inhibitors of caspase-1 and -8 to target apoptotic and pyroptotic cell death in BV2 microglial cells under ischemic conditions. In summary, the present study provides evidence that the NLRC4 inflammasome complex mediates the inflammatory response, as well as apoptotic and pyroptotic cell death in microglial cells under in vitro and in vivo ischemic conditions. [ABSTRACT FROM AUTHOR]

Infection is a dynamic biological process underpinned by a complex interplay between the pathogen and the host. Microbes from all domains of life, including bacteria, viruses, fungi, and protozoan parasites, have the capacity to cause infection. Infection is sensed by the host, which often leads to activation of the inflammasome, a cytosolic macromolecular signaling platform that mediates the release of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18 and cleavage of the pore-forming protein gasdermin D, leading to pyroptosis. Host-mediated sensing of the infection occurs when pathogens inject or carry pathogen-associated molecular patterns (PAMPs) into the cytoplasm or induce damage that causes cytosolic liberation of danger-associated molecular patterns (DAMPs) in the host cell. Recognition of PAMPs and DAMPs by inflammasome sensors, including NLRP1, NLRP3, NLRC4, NAIP, AIM2, and Pyrin, initiates a cascade of events that culminate in inflammation and cell death. However, pathogens can deploy virulence factors capable of minimizing or evading host detection. This review presents a comprehensive overview of the mechanisms of microbe-induced activation of the inflammasome and the functional consequences of inflammasome activation in infectious diseases. We also explore the microbial strategies used in the evasion of inflammasome sensing at the host-microbe interaction interface. [ABSTRACT FROM AUTHOR]