A 360° view of the inflammasome: Mechanisms of activation, cell death, and diseases.

Inflammasomes are critical sentinels of the innate immune system that respond to threats to the host through recognition of distinct molecules, known as pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), or disruptions of cellular homeostasis, referred to as homeostasis-altering molecular processes (HAMPs) or effector-triggered immunity (ETI). Several distinct proteins nucleate inflammasomes, including NLRP1, CARD8, NLRP3, NLRP6, NLRC4/NAIP, AIM2, pyrin, and caspases-4/-5/-11. This diverse array of sensors strengthens the inflammasome response through redundancy and plasticity. Here, we present an overview of these pathways, outlining the mechanisms of inflammasome formation, subcellular regulation, and pyroptosis, and discuss the wide-reaching effects of inflammasomes in human disease.

NLRP1 restricts porcine deltacoronavirus infection via IL-11 inhibiting the phosphorylation of the ERK signaling pathway.

Continuously emerging highly pathogenic coronaviruses remain a major threat to human and animal health. Porcine deltacoronavirus (PDCoV) is a newly emerging enterotropic swine coronavirus that causes large-scale outbreaks of severe diarrhea disease in piglets. Unlike other porcine coronaviruses, PDCoV has a wide range of species tissue tropism, including primary human cells, which poses a significant risk of cross-species transmission. Nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 1 (NLRP1) has a key role in linking host innate immunity to microbes and the regulation of inflammatory pathways. We now report a role for NLRP1 in the control of PDCoV infection. Overexpression of NLRP1 remarkably suppressed PDCoV infection, whereas knockout of NLRP1 led to a significant increase in PDCoV replication. A mechanistic study revealed that NLRP1 suppressed PDCoV replication in cells by upregulating IL-11 expression, which in turn inhibited the phosphorylation of the ERK signaling pathway. Furthermore, the ERK phosphorylation inhibitor U0126 effectively hindered PDCoV replication in pigs. Together, our results demonstrated that NLRP1 exerted an anti-PDCoV effect by IL-11-mediated inhibition of the phosphorylation of the ERK signaling pathway, providing a novel antiviral signal axis of NLRP1-IL-11-ERK. This study expands our understanding of the regulatory network of NLRP1 in the host defense against virus infection and provides a new insight into the treatment of coronaviruses and the development of corresponding drugs.IMPORTANCECoronavirus, which mainly infects gastrointestinal and respiratory epithelial cells in vivo, poses a huge threat to both humans and animals. Although porcine deltacoronavirus (PDCoV) is known to primarily cause fatal diarrhea in piglets, reports detected in plasma samples from Haitian children emphasize the potential risk of animal-to-human spillover. Finding effective therapeutics against coronaviruses is crucial for controlling viral infection. Nucleotide-binding oligomerization-like receptor (NLR) family pyrin domain-containing 1 (NLRP1), a key regulatory factor in the innate immune system, is highly expressed in epithelial cells and associated with the pathogenesis of viruses. We demonstrate here that NLRP1 inhibits the infection of the intestinal coronavirus PDCoV through IL-11-mediated phosphorylation inhibition of the ERK signaling pathway. Furthermore, the ERK phosphorylation inhibitor can control the infection of PDCoV in pigs. Our study emphasizes the importance of NLRP1 as an immune regulatory factor and may open up new avenues for the treatment of coronavirus infection.

The Yersinia Type III Secretion System as a Tool for Studying Cytosolic Innate Immune Surveillance.

Microbial pathogens have evolved complex mechanisms to interface with host cells in order to evade host defenses and replicate. However, mammalian innate immune receptors detect the presence of molecules unique to the microbial world or sense the activity of virulence factors, activating antimicrobial and inflammatory pathways. We focus on how studies of the major virulence factor of one group of microbial pathogens, the type III secretion system (T3SS) of human pathogenic Yersinia, have shed light on these important innate immune responses. Yersinia are largely extracellular pathogens, yet they insert T3SS cargo into target host cells that modulate the activity of cytosolic innate immune receptors. This review covers both the host pathways that detect the Yersinia T3SS and the effector proteins used by Yersinia to manipulate innate immune signaling.

A type VI secretion system in Burkholderia species cenocepacia and orbicola triggers distinct macrophage death pathways independent of the pyrin inflammasome.

The Burkholderia cepacia complex contains opportunistic pathogens that cause chronic infections and inflammation in the lungs of people with cystic fibrosis. Two closely related species within this complex are Burkholderia cenocepacia and the recently classified Burkholderia orbicola. B. cenocepacia and B. orbicola encode a type VI secretion system and the effector TecA, which is detected by the pyrin/caspase-1 inflammasome, and triggers macrophage inflammatory death. We previously showed that the pyrin inflammasome was dispensable for lung inflammation in mice infected with B. orbicola AU1054, indicating this species activates an alternative pathway of macrophage inflammatory death. Notably, B. cenocepacia strains J2315 and K56-2 can damage macrophage phagosomes, and K56-2 triggers activation of the caspase-11 inflammasome, which detects cytosolic lipopolysaccharide. Here, we investigated inflammatory cell death in pyrin- (Mefv-/-) or caspase-1/caspase-11- (Casp1/11-/-) deficient mouse macrophages infected with B. cenocepacia J2315 or K56-2 or B. orbicola AU1054 or PC184. Macrophage inflammatory death was measured by cleavage of gasdermin D protein, the release of cytokines IL-1α and IL-1ß, and plasma membrane rupture. We found that J2315 and K56-2 are detected by the caspase-11 inflammasome in Mefv-/- macrophages, resulting in IL-1ß release. By contrast, inflammasome activation was not detected in Mefv-/- macrophages infected with AU1054 or PC184. Instead, AU1054 triggered an alternative macrophage inflammatory death pathway that required TecA and resulted in plasma membrane rupture and IL-1α release. Structural modeling of TecA orthologs in B. cenocepacia and B. orbicola suggested that amino acid changes in the latter may underlie its ability to trigger a non-inflammasome macrophage death pathway.

NLRP3 Inflammasome Activation Through Heart-Brain Interaction Initiates Cardiac Inflammation and Hypertrophy During Pressure Overload.

BACKGROUND: Mechanical stress on the heart, such as high blood pressure, initiates inflammation and causes hypertrophic heart disease. However, the regulatory mechanism of inflammation and its role in the stressed heart remain unclear. IL-1ß (interleukin-1ß) is a proinflammatory cytokine that causes cardiac hypertrophy and heart failure. Here, we show that neural signals activate the NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3) inflammasome for IL-1ß production to induce adaptive hypertrophy in the stressed heart. METHODS: C57BL/6 mice, knockout mouse strains for NLRP3 and P2RX7 (P2X purinoceptor 7), and adrenergic neuron-specific knockout mice for SLC17A9, a secretory vesicle protein responsible for the storage and release of ATP, were used for analysis. Pressure overload was induced by transverse aortic constriction. Various animal models were used, including pharmacological treatment with apyrase, lipopolysaccharide, 2'(3')-O-(4-benzoylbenzoyl)-ATP, MCC950, anti-IL-1ß antibodies, clonidine, pseudoephedrine, isoproterenol, and bisoprolol, left stellate ganglionectomy, and ablation of cardiac afferent nerves with capsaicin. Cardiac function and morphology, gene expression, myocardial IL-1ß and caspase-1 activity, and extracellular ATP level were assessed. In vitro experiments were performed using primary cardiomyocytes and fibroblasts from rat neonates and human microvascular endothelial cell line. Cell surface area and proliferation were assessed. RESULTS: Genetic disruption of NLRP3 resulted in significant loss of IL-1ß production, cardiac hypertrophy, and contractile function during pressure overload. A bone marrow transplantation experiment revealed an essential role of NLRP3 in cardiac nonimmune cells in myocardial IL-1ß production and cardiac phenotype. Pharmacological depletion of extracellular ATP or genetic disruption of the P2X7 receptor suppressed myocardial NLRP3 inflammasome activity during pressure overload, indicating an important role of ATP/P2X7 axis in cardiac inflammation and hypertrophy. Extracellular ATP induced hypertrophic changes of cardiac cells in an NLRP3- and IL-1ß-dependent manner in vitro. Manipulation of the sympathetic nervous system suggested sympathetic efferent nerves as the main source of extracellular ATP. Depletion of ATP release from sympathetic efferent nerves, ablation of cardiac afferent nerves, or a lipophilic ß-blocker reduced cardiac extracellular ATP level, and inhibited NLRP3 inflammasome activation, IL-1ß production, and adaptive cardiac hypertrophy during pressure overload. CONCLUSIONS: Cardiac inflammation and hypertrophy are regulated by heart-brain interaction. Controlling neural signals might be important for the treatment of hypertensive heart disease.

Pyrin Inflammasome Activation Defines Colchicine-Responsive SURF Patients from FMF and Other Recurrent Fevers.

Syndrome of undifferentiated recurrent fever (SURF) is characterized by recurrent fevers, a lack of confirmed molecular diagnosis, and a complete or partial response to colchicine. Despite the clinical similarities to familial Mediterranean fever (FMF), the underlying inflammatory mechanisms of SURF are not yet understood. We here analyzed the in vitro activation of the pyrin inflammasome in a cohort of SURF patients compared to FMF and PFAPA patients. Peripheral blood mononuclear cells (PBMC) were collected from SURF (both colchicine-treated and untreated), FMF, PFAPA patients, and healthy donors. PBMC were stimulated ex vivo with Clostridium difficile toxin A (TcdA) and a PKC inhibitor (UCN-01), in the presence or absence of colchicine. The assembly of the pyrin inflammasome was evaluated by measuring the presence of apoptosis-associated Speck-like protein containing caspase recruitment domain (ASC) specks in monocytes using flow cytometry. IL-1ß secretion was quantified using an ELISA assay. No differences in TcdA-induced activation of pyrin inflammasome were observed among FMF, PFAPA, and healthy donors. Untreated SURF patients showed a reduced response to TcdA, which was normalized after colchicine treatment. In contrast to FMF, SURF patients, similar to PFAPA patients and healthy donors, did not exhibit pyrin inflammasome activation in response to UCN-01-mediated pyrin dephosphorylation. These data demonstrate that in vitro functional analysis of pyrin inflammasome activation can differentiate SURF from FMF and PFAPA patients, suggesting the involvement of the pyrin inflammasome in the pathophysiology of SURF.

A novel Nlrp3 knock-in mouse model with hyperactive inflammasome in development of lethal inflammation.

NOD-like receptor family, pyrin domain-containing 3 (NLRP3) is a central protein contributing to human inflammatory disorders, including cryopyrin-associated periodic syndrome and sepsis. However, the molecular mechanisms and functions of NLRP3 activation in various diseases remain unknown. Here, we generated gain-of-function knock-in mice associated with Muckle-Wells syndromes using the Cre-LoxP system allowing for the constitutive T346M mutation of NLRP3 to be globally expressed in all cells under the control of tamoxifen. The mice were treated with tamoxifen for 4 days before determining their genotype by PCR and sequence analysis. In vitro, we found that bone marrow-derived macrophage from homozygous T346M mutation mice displayed a robust ability to produce IL-1ß in response to lipopolysaccharide exposure. Moreover, ASC specks and oligomerization were observed in the homozygous mutant bone marrow-derived macrophages in the presence of lipopolysaccharides alone. Mechanistically, K+ and Ca2+ depletion and mitochondrial depolarization contribute to the hyperactivation of mutant NLRP3. In vivo, homozygous mice carrying the T346M mutation exhibit weight loss and mild inflammation in the resting state. In the lipopolysaccharide-mediated sepsis model, homozygous mutant mice exhibited higher mortality and increased serum circulating cytokine levels, accompanied by serious liver injury. Furthermore, an increase in myeloid cells in the spleen has been suggested to be a risk factor for inducing sepsis sensitivity. Altogether, we describe a cryopyrin-associated syndrome animal model with the T346M mutation of NLRP3 and suggest that the hyperactivated inflammasome aggregated by the mutant NLRP3 lowers the inflammatory response threshold both in vitro and in vivo.

Helicobacter pylori secretary Proteins-Induced oxidative stress and its role in NLRP3 inflammasome activation.

Helicobacter pylori-associated stomach infection is a leading cause of gastric ulcer and related cancer. H. pylori modulates the functions of infiltrated immune cells to survive the killing by reactive oxygen and nitrogen species (ROS and RNS) produced by these cells. Uncontrolled immune responses further produce excess ROS and RNS which lead to mucosal damage. The persistent oxidative stress is a major cause of gastric cancer. H. pylori regulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), nitric oxide synthase 2 (NOS2), and polyamines to control ROS and RNS release through lesser-known mechanisms. ROS and RNS produced by these pathways differentiate macrophages and T cells from protective to inflammatory phenotype. Pathogens-associated molecular patterns (PAMPs) induced ROS activates nuclear oligomerization domain (NOD), leucine rich repeats (LRR) and pyrin domain-containing protein 3 (NLRP3) inflammasome for the release of pro-inflammatory cytokines. This study evaluates the role of H. pylori secreted concentrated proteins (HPSCP) related oxidative stress role in NLRP3 inflammasome activation and macrophage differentiation. To perceive the role of ROS/RNS, THP-1 and AGS cells were treated with 10 µM diphenyleneiodonium (DPI), 50 µM salicyl hydroxamic acid (SHX), 5 µM Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), which are specific inhibitors of NADPH oxidase (NOX), Myeloperoxidase (MPO), and mitochondrial oxidative phosphorylation respectively. Cells were also treated with 10 µM of NOS2 inhibitor l-NMMA and 10 µM of N-acetyl cysteine (NAC), a free radical scavenger·H2O2 (100 µM) treated and untreated cells were used as positive controls and negative control respectively. The expression of gp91phox (NOX2), NOS2, NLRP3, CD86 and CD163 was analyzed through fluorescent microscopy. THP-1 macrophages growth was unaffected whereas the gastric epithelial AGS cells proliferated in response to higher concentration of HPSCP. ROS and myeloperoxidase (MPO) level increased in THP-1 cells and nitric oxide (NO) and lipid peroxidation significantly decreased in AGS cells. gp91phox expression was unchanged, whereas NOS2 and NLRP3 downregulated in response to HPSCP, but increased after inhibition of NO, ROS and MPO in THP-1 cells. HPSCP upregulated the expression of M1 and M2 macrophage markers, CD86 and CD163 respectively, which was decreased after the inhibition of ROS. This study concludes that there are multiple pathways which are generating ROS during H. pylori infection which further regulates other cellular processes. NO is closely associated with MPO and inhibition of NLRP3 inflammasome. The low levels of NO and MPO regulates gastrointestinal tract homeostasis and overcomes the inflammatory response of NLRP3. The ROS also plays crucial role in macrophage polarization hence alter the immune responses duing H. pylori pathogenesis.

Pyrin variant E148Q potentiates inflammasome activation and the effect of pathogenic mutations in cis.

OBJECTIVE: The p.E148Q variant in pyrin is present in different populations at a frequency of up to 29%, and has been associated with diseases, including vasculitis and FMF. The pathogenicity of p.E148Q in FMF is unclear, even when observed in cis or in trans to a single, typically recessive, pathogenic mutation. We performed functional validation to determine whether p.E148Q increases the ability of pyrin to form an active inflammasome complex in cell lines. METHODS: We interrogated the Australian Autoinflammatory Disease RegistrY (AADRY) to find candidate inheritance patterns for the p.E148Q variant in pyrin. Different pyrin variant combinations were tested in HEK293T cells stably expressing the adaptor protein apoptosis-associated speck-like (ASC), which were analysed by flow cytometry to visualize inflammasome formation, with and without stimulation by Clostridioides difficile toxin B (TcdB). Inflammasome-dependent cytokine secretion was also quantified by ELISA of supernatants from THP-1 cells transduced with lentiviral expression vectors. RESULTS: In AADRY, we observed the p.E148Q allele in individuals with autoinflammatory diseases alone or in conjunction with other pyrin variants. Two FMF families harboured the allele p.E148Q-M694I in cis with dominant heritability. In vitro, p.E148Q pyrin could spontaneously potentiate inflammasome formation, with increased IL-1ß and IL-18 secretion. p.E148Q in cis to classical FMF mutations provided significant potentiation of inflammasome formation. CONCLUSION: The p.E148Q variant in pyrin potentiates inflammasome activation in vitro. In cis, this effect is additive to known pathogenic FMF mutations. In some families, this increased effect could explain why FMF segregates as an apparently dominant disease.

Old paradigms and new concepts in familial Mediterranean fever (FMF): an update 2023.

Familial Mediterranean fever (FMF) is a hereditary autoinflammatory disease characterized by recurrent attacks of fever and polyserositis. Its first description as a new entity was published by Siegal in 1945. Colchicine has been the treatment of choice for this disease since 1972. Significant progress has been made over the years in understanding FMF's clinical features, diagnosis, mode of inheritance, pathogenesis and therapeutic approach. However, many old paradigms related to FMF have proven inaccurate, leading to the emergence of new concepts that provide more precise insights. The term 'FMF' is no longer appropriate as the disease is found beyond the Mediterranean basin. The concept of diagnosis based only upon clinical ground proved to be wrong. The paradigm that MEFV mutations in FMF lead to loss of function of the encoded peptide pyrin turned out to be a gain of function mutation. Finally, the concept that as a genetic disease FMF should be treated for life was found to be inaccurate for the subpopulation of the heterozygote patients. Thus, the breakthroughs of identifying the gene associated with the disease (MEFV) and the deciphering of its pathogenesis revolutionized our old paradigms and replaced them with new and more precise insights.

Pregnancy outcomes in patients with familial Mediterranean fever: systematic review and meta-analysis.

OBJECTIVE: The relationship between FMF and pregnancy outcomes remains unclear. This systematic review and meta-analysis aimed to clarify this association. METHODS: Electronic databases-PubMed, Web of Science, Cochrane, and EMBASE-were searched on 20 December 2022, using specific search terms. Case-control, cohort, and randomized clinical trial studies comparing patients with FMF and healthy controls were considered eligible. We excluded systematic reviews, meta-analyses, case series with fewer than five cases, republished articles without new findings on pregnancy outcomes, studies targeting paternal FMF, and those not published in English. The results were summarized in the form of odds ratios (ORs) and 95% CIs, using a random-effects model. This study was registered in the University hospital Medical Information Network Clinical Trials Registry (Japan) as UMIN000049827. RESULTS: The initial electronic search identified 611 records, of which 9 were included in this meta-analysis (177 735 pregnancies, 1242 with FMF, and 176 493 healthy controls). FMF was significantly associated with increased odds of preterm deliveries (OR, 1.67; 95% CI, 1.05-2.67; I2 = 22%) and insignificantly associated with increased odds of fetal growth restriction (OR, 1.45; 95% CI, 0.90-2.34; I2 = 0%) and hypertensive disorders during pregnancy (OR, 1.28; 95% CI, 0.87-1.87; I2 = 0%). CONCLUSION: FMF was significantly associated with preterm delivery and insignificantly associated with fetal growth restriction and hypertensive disorders. All of the included studies were observational studies. Treatment characteristics were not fully collected from the articles, and further analysis of treatments for FMF in pregnancy is still warranted.

Pre-electroacupuncture Ameliorates Cerebral Ischemia-reperfusion Injury by Inhibiting Microglial RhoA/pyrin/GSDMD Signaling Pathway.

Cerebral ischemia reperfusion injury is a severe neurological impairment that occurs after blood flow reconstruction in stroke, and microglia cell pyroptosis is one of its important mechanisms. Electroacupuncture has been shown to be effective in mitigating and alleviating cerebral ischemia reperfusion injury by inhibiting neuroinflammation, reducing cellular pyroptosis, and improving neurological function. In this experiment, we divided the rats into three groups, including the sham operation (Sham) group, the middle cerebral artery occlusion/reperfusion (MCAO/R) group, and the pre-electroacupuncture (EAC) group. Pre-electroacupuncture group was stimulated with electroacupuncture of a certain intensity on the Baihui (GV 20) and Dazhui (GV 14) of the rat once a day from the 7th day to the 1st day before the MCAO/R operation. The extent of cerebral infarction was detected by TTC staining. A modified Zea-Longa five-point scale scoring system was used to determine neurologic function in MCAO rats. The number of neurons and morphological changes were accessed by Nissl staining and HE staining. The cellular damage was detected by TUNEL staining. In addition, the expression levels of RhoA, pyrin, GSDMD, Caspase1, cleaved-Caspase1, Iba-1, CD206, and ROCK2 were examined by western blotting and immunofluorescence. The results found that pre-electroacupuncture significantly attenuated neurological impairment and cerebral infarction compared to the post-MCAO/R rats. In addition, pre-electroacupuncture therapy promoted polarization of microglia to the neuroprotective (M2) phenotype. In addition, pre-electroacupuncture inhibited microglia pyroptosis by inhibiting RhoA/pyrin/GSDMD signaling pathway, thereby reducing neuronal injury and increasing neuronal survival in the MCAO/R rats. Taken together, these results demonstrated that pre-acupuncture could attenuate cerebral ischemia-reperfusion injury by inhibiting microglial pyroptosis. Therefore, pre-electroacupuncture might be a potential preventive strategy for ischemic stroke patients.

NLRP3 activation in neutrophils induces lethal autoinflammation, liver inflammation, and fibrosis.

Sterile inflammation is a central element in liver diseases. The immune response following injurious stimuli involves hepatic infiltration of neutrophils and monocytes. Neutrophils are major effectors of liver inflammation, rapidly recruited to sites of inflammation, and can augment the recruitment of other leukocytes. The NLRP3 inflammasome has been increasingly implicated in severe liver inflammation, fibrosis, and cell death. In this study, the role of NLRP3 activation in neutrophils during liver inflammation and fibrosis was investigated. Mouse models with neutrophil-specific expression of mutant NLRP3 were developed. Mutant mice develop severe liver inflammation and lethal autoinflammation phenocopying mice with a systemic expression of mutant NLRP3. NLRP3 activation in neutrophils leads to a pro-inflammatory cytokine and chemokine profile in the liver, infiltration by neutrophils and macrophages, and an increase in cell death. Furthermore, mutant mice develop liver fibrosis associated with increased expression of pro-fibrogenic genes. Taken together, the present work demonstrates how neutrophils, driven by the NLRP3 inflammasome, coordinate other inflammatory myeloid cells in the liver, and propagate the inflammatory response in the context of inflammation-driven fibrosis.

Circulating perturbation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is associated to cardiac remodeling and NLRP3 inflammasome in cardiovascular patients with insulin resistance risk.

Lipidome perturbation occurring during meta-inflammation is associated to left ventricle (LV) remodeling though the activation of the NLRP3 inflammasome, a key regulator of chronic inflammation in obesity-related disorders. Little is known about phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as DAMP-induced NLRP3 inflammasome. Our study is aimed to evaluate if a systemic reduction of PC/PE molar ratio can affect NLRP3 plasma levels in cardiovascular disease (CVD) patients with insulin resistance (IR) risk. Forty patients from IRCCS Policlinico San Donato were enrolled, and their blood samples were drawn before heart surgery. LV geometry measurements were evaluated by echocardiography and clinical data associated to IR risk were collected. PC and PE were quantified by ESI-MS/MS. Circulating NLRP3 was quantified by an ELISA assay. Our results have shown that CVD patients with IR risk presented systemic lipid impairment of PC and PE species and their ratio in plasma was inversely associated to NLRP3 levels. Interestingly, CVD patients with IR risk presented LV changes directly associated to increased levels of NLRP3 and a decrease in PC/PE ratio in plasma, highlighting the systemic effect of meta-inflammation in cardiac response. In summary, PC and PE can be considered bioactive mediators associated to both the NLRP3 and LV changes in CVD patients with IR risk.

Tanshinone IIA Alleviates Early Brain Injury after Subarachnoid Hemorrhage in Rats by Inhibiting the Activation of NF-κB/NLRP3 Inflammasome.

The abnormal activation of the nuclear factor-kappa B (NF-κB)/nod-like receptor family-pyrin domain-containing 3 (NLRP3) signaling pathway is closely related to early brain injury after subarachnoid hemorrhage (SAH). Targeting the NLRP3-inflammasome has been considered an efficient therapy for the local inflammatory response after SAH. Tanshinone IIA (Tan IIA), a major component extracted from Salvia miltiorrhiza, has been reported to have anti-inflammatory effects. The aim of this study was to investigate the effect and mechanism of Tan IIA on early brain injury after SAH. In vivo SAH injury was established by endovascular perforation technique in Sprague-Dawley rats. Limb-placement test and corner turning test were used to measure the behavior. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, hematoxylin-eosin (H&E) staining, and immunofluorescence were used to evaluate the nerve damage. Real-time RT quantitative PCR (RT-qPCR) was used to quantify the levels of inflammatory factors. Western blot was performed for the activation of the NF-κB/NLRP3 pathway. An in vitro SAH model was used to validate the conclusion. We found that the neurobehavioral impairment and cerebral edema in SAH model rats given Tan IIA were alleviated. Further study demonstrated that Tan IIA could inhibit SAH-secondary neuronal apoptosis around hematoma and alleviate brain injury. Tan IIA down-regulated the expression of interleukin-6 (IL)-6, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor (TNF)-α, and inhibited the activation of NF-κB. And the overexpression of pro-inflammatory factors NLRP3, IL-1ß, and IL-18 induced after SAH was also reversed by Tan IIA. In conclusions, Tan IIA could inhibit the NF-κB/NLRP3 inflammasome activation to protect and ameliorate SAH-followed early brain injury, and may be a preventive and therapeutic strategy against SAH.

Identification of Azalamellarin N as a Pyroptosis Inhibitor.

Pyroptosis is a form of regulated cell death that promotes inflammation; it attracts much attention because its dysregulation leads to various inflammatory diseases. To help explore the precise mechanisms by which pyroptosis is regulated, in this study, we searched for chemical compounds that inhibit pyroptosis. From our original compound library, we identified azalamellarin N (AZL-N), a hexacyclic pyrrole alkaloid, as an inhibitor of pyroptosis induced by R837 (also called imiquimod), which is an agonist of the intracellular multiprotein complex nucleotide-binding and oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. However, whereas the effect of AZL-N on R837-induced pyroptosis was relatively weak, AZL-N strongly inhibited pyroptosis induced by extracellular ATP or nigericin, which are different types of NLRP3 inflammasome agonists. This was in contrast with the results that MCC950, a well-established NLRP3 inhibitor, consistently inhibited pyroptosis irrespective of the type of stimulus. We also found that AZL-N inhibited activation of caspase-1 and apoptosis-associated speck-like proteins containing a caspase activation and recruitment domain (ASC), which are components of the NLRP3 inflammasome. Analysis of the structure-activity relationship revealed that a lactam ring of AZL-N, which has been shown to contribute to the strong binding of AZL-N to its known target protein kinases, is required for its inhibitory effects on pyroptosis. These results suggest that AZL-N inhibits pyroptosis by targeting molecule(s), which may be protein kinase(s), that act upstream of NLRP3 inflammasome activation, rather than by directly targeting the components of the NLRP3 inflammasome. Further identification and analysis of target molecule(s) of AZL-N will shed light on the regulatory mechanisms of pyroptosis, particularly those depending on proinflammatory stimuli.

Endothelial Dysfunction in Obesity and Therapeutic Targets.

Parallel to the increasing prevalence of obesity in the world, the mortality from cardiovascular disease has also increased. Low-grade chronic inflammation in obesity disrupts vascular homeostasis, and the dysregulation of adipocyte-derived endocrine and paracrine effects contributes to endothelial dysfunction. Besides the adipose tissue inflammation, decreased nitric oxide (NO)-bioavailability, insulin resistance (IR), and oxidized low-density lipoproteins (oxLDLs) are the main factors contributing to endothelial dysfunction in obesity and the development of cardiorenal metabolic syndrome. While normal healthy perivascular adipose tissue (PVAT) ensures the dilation of blood vessels, obesity-associated PVAT leads to a change in the profile of the released adipo-cytokines, resulting in a decreased vasorelaxing effect. Higher stiffness parameter ß, increased oxidative stress, upregulation of pro-inflammatory cytokines, and nicotinamide adenine dinucleotide phosphate (NADP) oxidase in PVAT turn the macrophages into pro-atherogenic phenotypes by oxLDL-induced adipocyte-derived exosome-macrophage crosstalk and contribute to the endothelial dysfunction. In clinical practice, carotid ultrasound, higher leptin levels correlate with irisin over-secretion by human visceral and subcutaneous adipose tissues, and remnant cholesterol (RC) levels predict atherosclerotic disease in obesity. As a novel therapeutic strategy for cardiovascular protection, liraglutide improves vascular dysfunction by modulating a cyclic adenosine monophosphate (cAMP)-independent protein kinase A (PKA)-AMP-activated protein kinase (AMPK) pathway in PVAT in obese individuals. Because the renin-angiotensin-aldosterone system (RAAS) activity, hyperinsulinemia, and the resultant IR play key roles in the progression of cardiovascular disease in obesity, RAAS-targeted therapies contribute to improving endothelial dysfunction. By contrast, arginase reciprocally inhibits NO formation and promotes oxidative stress. Thus, targeting arginase activity as a key mediator in endothelial dysfunction has therapeutic potential in obesity-related vascular comorbidities. Obesity-related endothelial dysfunction plays a pivotal role in the progression of type 2 diabetes (T2D). The peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone (thiazolidinedione), is a popular drug for treating diabetes; however, it leads to increased cardiovascular risk. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin (EMPA) significantly improves endothelial dysfunction and mortality occurring through redox-dependent mechanisms. Although endothelial dysfunction and oxidative stress are alleviated by either metformin or EMPA, currently used drugs to treat obesity-related diabetes neither possess the same anti-inflammatory potential nor simultaneously target endothelial cell dysfunction and obesity equally. While therapeutic interventions with glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide or bariatric surgery reverse regenerative cell exhaustion, support vascular repair mechanisms, and improve cardiometabolic risk in individuals with T2D and obesity, the GLP-1 analog exendin-4 attenuates endothelial endoplasmic reticulum stress.

Hypoxia-inducible factor 1α activates the NLRP3 inflammasome to regulate epithelial differentiation in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is an upper airway inflammation disease associated with hypoxia-mediated inflammation. The effect of hypoxia-inducible factor 1α (HIF-1α) on NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation in the pathogenesis of sinonasal mucosa is unclear. OBJECTIVE: We investigated the effect and mechanism of HIF-1α on NLRP3 inflammasome activation in the primary human nasal epithelial cells (hNECs). METHODS: We measured the expression levels of HIF-1α and the NLRP3 inflammasome in nasal biopsy samples and hNECs derived from negative controls (healthy) and patients with CRS with and without nasal polyps, then further analyzed the specific mechanism of HIF-1α regulation of the NLRP3 inflammasome and its effect on hNEC differentiation. RESULTS: Increased mRNA and protein expression levels of HIF-1α and the NLRP3 inflammasome were found in all CRS biopsy samples. HIF-1α enhanced expression of phosphorylated NLRP3 (S295) in both HEK293T cells and hNECs; it also promoted recruitment of caspase-1 and apoptotic speck-like protein containing caspase recruitment domain (aka ASC) by NLRP3. HIF-1α also improved NLRP3's stability by preventing NLRP3 degradation caused by hypoxia-mediated inflammation. In addition, HIF-1α could also increase expression of Mucin5AC and decrease expression of α-tubulin by promoting activation of the NLRP3 inflammasome in hNECs. In addition, HIF-1α could also directly promote P63 expression in hNECs. CONCLUSION: HIF-1α could potentially induce cilia loss and enhance the proliferation of goblet cells, possibly mediated by the regulation of NLRP3 phosphorylation in CRS inflammation.

Complex regulation of alarmins S100A8/A9 and secretion via gasdermin D pores exacerbates autoinflammation in familial Mediterranean fever.

BACKGROUND: Familial Mediterranean fever (FMF), caused by mutations in the pyrin-encoding MEFV gene, is characterized by uncontrolled caspase-1 activation and IL-1ß secretion. A similar mechanism drives inflammation in cryopyrin-associated periodic fever syndrome (CAPS) caused by mutations in NLRP3. CAPS and FMF, however, result in largely different clinical manifestations, pointing to additional, autoinflammatory pathways involved in FMF. Another hallmark of FMF is extraordinarily high expression of S100A8 and S100A9. These alarmins are ligands of Toll-like receptor 4 and amplifiers of inflammation. However, the relevance of this inflammatory pathway for the pathogenesis of FMF is unknown. OBJECTIVE: This study investigated whether mutations in pyrin result in specific secretion of S100A8/A9 alarmins through gasdermin D pores' amplifying FMF pathology. METHODS: S100A8/A9 levels in FMF patients were quantified by enzyme-linked immunosorbent assay. In vitro models with knockout cell lines and specific protein inhibitors were used to unravel the S100A8/A9 secretion mechanism. The impact of S100A8/A9 to the pathophysiology of FMF was analyzed with FMF (MEFVV726A/V726A) and S100A9-/- mouse models. Pyrin-S100A8/A9 interaction was investigated by coimmunoprecipitation, immunofluorescence, and enzyme-linked immunosorbent assay studies. RESULTS: The S100A8/A9 complexes directly interacted with pyrin. Knocking out pyrin, caspase-1, or gasdermin D inhibited the secretion of these S100 alarmins. Inflammatory S100A8/A9 dimers were inactivated by tetramer formation. Blocking this inactivation by targeted S100A9 deletion in a murine FMF model demonstrated the relevance of this novel autoinflammatory pathway in FMF. CONCLUSION: This is the first proof that members of the S100 alarmin family are released in a pyrin/caspase-1/gasdermin D-dependent pathway and directly drive autoinflammation in vivo.

NLRP3 Negative Regulation Mechanisms in the Resting State and Its Implications for Therapeutic Development.

The NACHT-, leucine-rich-repeat-, and pyrin domain-containing protein 3 (NLRP3) is a critical intracellular sensor of the innate immune system that detects various pathogen- and danger-associated molecular patterns, leading to the assembly of the NLRP3 inflammasome and release of interleukin (IL) 1ß and IL-18. However, the abnormal activation of the NLRP3 inflammasome has been implicated in the pathogenesis of autoinflammatory diseases such as cryopyrin-associated autoinflammatory syndromes (CAPS) and common diseases such as Alzheimer's disease and asthma. Recent studies have revealed that pyrin functions as an indirect sensor, similar to the plant guard system, and is regulated by binding to inhibitory 14-3-3 proteins. Upon activation, pyrin transitions to its active form. NLRP3 is predicted to follow a similar regulatory mechanism and maintain its inactive form in the cage model, as it also acts as an indirect sensor. Additionally, newly developed NLRP3 inhibitors have been found to inhibit NLRP3 activity by stabilizing its inactive form. Most studies and reviews on NLRP3 have focused on the activation of the NLRP3 inflammasome. This review highlights the molecular mechanisms that regulate NLRP3 in its resting state, and discusses how targeting this inhibitory mechanism can lead to novel therapeutic strategies for NLRP3-related diseases.