Leptin signalling regulates transcriptional differences in granulosa cells from genetically obese mice but not the activation of NLRP3 inflammasome.

Obesity is associated with increased ovarian inflammation and the establishment of leptin resistance. We presently investigated the role of impaired leptin signalling on transcriptional regulation in granulosa cells (GCs) collected from genetically obese mice. Furthermore, we characterised the association between ovarian leptin signalling, the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and macrophage infiltration in obese mice. After phenotype characterisation, ovaries were collected from distinct group of animals for protein and mRNA expression analysis: (i) mice subjected to a diet-induced obesity (DIO) protocol, where one group was fed a high-fat diet (HFD) and another a standard chow diet (CD) for durations of 4 or 16 weeks; (ii) mice genetically deficient in the long isoform of the leptin receptor (ObRb; db/db); (iii) mice genetically deficient in leptin (ob/ob); and (iv) mice rendered pharmacologically hyperleptinemic (LEPT). Next, GCs from antral follicles isolated from db/db and ob/ob mice were subjected to transcriptome analysis. Transcriptional analysis revealed opposing profiles in genes associated with steroidogenesis and prostaglandin action between the genetic models, despite the similarities in body weight. Furthermore, we observed no changes in the mRNA and protein levels of NLRP3 inflammasome components in the ovaries of db/db mice or in markers of M1 and M2 macrophage infiltration. This contrasted with the downregulation of NLRP3 inflammasome components and M1 markers in ob/ob and 16-wk HFD-fed mice. We concluded that leptin signalling regulates NLRP3 inflammasome activation and the expression of M1 markers in the ovaries of obese mice in an ObRb-dependent and ObRb-independent manner. Furthermore, we found no changes in the expression of leptin signalling and NLRP3 inflammasome genes in GCs from db/db and ob/ob mice, which was associated with no effects on macrophage infiltration genes, despite the dysregulation of genes associated with steroidogenesis in homozygous obese db/db. Our results suggest that: (i) the crosstalk between leptin signalling, NLRP3 inflammasome and macrophage infiltration takes place in ovarian components other than the GC compartment; and (ii) transcriptional changes in GCs from homozygous obese ob/ob mice suggest structural rearrangement and organisation, whereas in db/db mice the impairment in steroidogenesis and secretory activity.

The heavy subunit of ferritin stimulates NLRP3 inflammasomes in hepatic stellate cells through ICAM-1 to drive hepatic inflammation.

Serum ferritin concentrations increase during hepatic inflammation and correlate with the severity of chronic liver disease. Here, we report a molecular mechanism whereby the heavy subunit of ferritin (FTH) contributes to hepatic inflammation. We found that FTH induced activation of the NLRP3 inflammasome and secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) in primary rat hepatic stellate cells (HSCs) through intercellular adhesion molecule-1 (ICAM-1). FTH-ICAM-1 stimulated the expression of Il1b, NLRP3 inflammasome activation, and the processing and secretion of IL-1ß in a manner that depended on plasma membrane remodeling, clathrin-mediated endocytosis, and lysosomal destabilization. FTH-ICAM-1 signaling at early endosomes stimulated Il1b expression, implying that this endosomal signaling primed inflammasome activation in HSCs. In contrast, lysosomal destabilization was required for FTH-induced IL-1ß secretion, suggesting that lysosomal damage activated inflammasomes. FTH induced IL-1ß production in liver slices from wild-type mice but not in those from Icam1-/- or Nlrp3-/- mice. Thus, FTH signals through its receptor ICAM-1 on HSCs to activate the NLRP3 inflammasome. We speculate that this pathway contributes to hepatic inflammation, a key process that stimulates hepatic fibrogenesis associated with chronic liver disease.

Inflammasome-Related Genetic Polymorphisms as Severity Biomarkers of COVID-19.

The most critical forms of coronavirus disease 2019 (COVID-19) are associated with excessive activation of the inflammasome. Despite the COVID-19 impact on public health, we still do not fully understand the mechanisms by which the inflammatory response influences disease prognosis. Accordingly, we aimed to elucidate the role of polymorphisms in the key genes of the formation and signaling of the inflammasome as biomarkers of COVID-19 severity. For this purpose, a large and well-defined cohort of 377 COVID-19 patients with mild (n = 72), moderate (n = 84), severe (n = 100), and critical (n = 121) infections were included. A total of 24 polymorphisms located in inflammasome-related genes (NLRP3, NLRC4, NLRP1, CARD8, CASP1, IL1B, IL18, NFKB1, ATG16L1, and MIF) were genotyped in all of the patients and in the 192 healthy controls (HCs) (who were without COVID-19 at the time of and before the study) by RT-qPCR. Our results showed that patients with mild, moderate, severe, and critical COVID-19 presented similar allelic and genotypic distribution in all the variants studied. No statistically significant differences in the haplotypic distribution of NLRP3, NLRC4, NLRP1, CARD8, CASP1, IL1B, and ATG16L1 were observed between COVID-19 patients, who were stratified by disease severity. Each stratified group of patients presented a similar genetic distribution to the HCs. In conclusion, our results suggest that the inflammasome polymorphisms studied are not associated with the worsening of COVID-19.

L-AP Alleviates Liver Injury in Septic Mice by Inhibiting Macrophage Activation via Suppressing NF-κB and NLRP3 Inflammasome/Caspase-1 Signal Pathways.

Liver injury and progressive liver failure are severe life-threatening complications in sepsis, further worsening the disease and leading to death. Macrophages and their mediated inflammatory cytokine storm are critical regulators in the occurrence and progression of liver injury in sepsis, for which effective treatments are still lacking. l-Ascorbic acid 6-palmitate (L-AP), a food additive, can inhibit neuroinflammation by modulating the phenotype of the microglia, but its pharmacological action in septic liver damage has not been fully explored. We aimed to investigate L-AP's antisepticemia action and the possible pharmacological mechanisms in attenuating septic liver damage by modulating macrophage function. We observed that L-AP treatment significantly increased survival in cecal ligation and puncture-induced WT mice and attenuated hepatic inflammatory injury, including the histopathology of the liver tissues, hepatocyte apoptosis, and the liver enzyme levels in plasma, which were comparable to NLRP3-deficiency in septic mice. L-AP supplementation significantly attenuated the excessive inflammatory response in hepatic tissues of septic mice in vivo and in cultured macrophages challenged by both LPS and ATP in vitro, by reducing the levels of NLRP3, pro-IL-1ß, and pro-IL-18 mRNA expression, as well as the levels of proteins for p-I-κB-α, p-NF-κB-p65, NLRP3, cleaved-caspase-1, IL-1ß, and IL-18. Additionally, it impaired the inflammasome ASC spot activation and reduced the inflammatory factor contents, including IL-1ß and IL-18 in plasma/cultured superannuants. It also prevented the infiltration/migration of macrophages and their M1-like inflammatory polarization while improving their M2-like polarization. Overall, our findings revealed that L-AP protected against sepsis by reducing macrophage activation and inflammatory cytokine production by suppressing their activation in NF-κB and NLRP3 inflammasome signal pathways in septic liver.

Functional diversity of NLRP3 gain-of-function mutants associated with CAPS autoinflammation.

NLRP3-associated autoinflammatory disease is a heterogenous group of monogenic conditions caused by NLRP3 gain-of-function mutations. The poor functional characterization of most NLRP3 variants hinders diagnosis despite efficient anti-IL-1 treatments. Additionally, while NLRP3 is controlled by priming and activation signals, gain-of-functions have only been investigated in response to priming. Here, we characterize 34 NLRP3 variants in vitro, evaluating their activity upon induction, priming, and/or activation signals, and their sensitivity to four inhibitors. We highlight the functional diversity of the gain-of-function mutants and describe four groups based on the signals governing their activation, correlating partly with the symptom severity. We identify a new group of NLRP3 mutants responding to the activation signal without priming, associated with frequent misdiagnoses. Our results identify key NLRP3 residues controlling inflammasome activity and sensitivity to inhibitors, and antagonistic mechanisms with broader efficacy for therapeutic strategies. They provide new insights into NLRP3 activation, an explanatory mechanism for NLRP3-AID heterogeneity, and original tools for NLRP3-AID diagnosis and drug development.

Increased NLRP1 mRNA and Protein Expression Suggests Inflammasome Activation in the Dorsolateral Prefrontal and Medial Orbitofrontal Cortex in Schizophrenia.

Schizophrenia is a complex mental condition, with key symptoms marked for diagnosis including delusions, hallucinations, disorganized thinking, reduced emotional expression, and social dysfunction. In the context of major developmental hypotheses of schizophrenia, notably those concerning maternal immune activation and neuroinflammation, we studied NLRP1 expression and content in the postmortem brain tissue of 10 schizophrenia and 10 control subjects. In the medial orbitofrontal cortex (Brodmann's area 11/12) and dorsolateral prefrontal cortex (area 46) from both hemispheres of six schizophrenia subjects, the NLRP1 mRNA expression was significantly higher than in six control brains (p < 0.05). As the expression difference was highest for the medial orbitofrontal cortex in the right hemisphere, we assessed NLRP1-immunoreactive pyramidal neurons in layers III, V, and VI in the medial orbitofrontal cortex in the right hemisphere of seven schizophrenia and five control brains. Compared to controls, we quantified a significantly higher number of NLRP1-positive pyramidal neurons in the schizophrenia brains (p < 0.01), suggesting NLRP1 inflammasome activation in schizophrenia subjects. Layer III pyramidal neuron dysfunction aligns with working memory deficits, while impairments of pyramidal neurons in layers V and VI likely disrupt predictive processing. We propose NLRP1 inflammasome as a potential biomarker and therapeutic target in schizophrenia.

Identification of circular RNA hsa_circ_0034621 as a novel biomarker for carotid atherosclerosis and the potential function as a regulator of NLRP3 inflammasome.

BACKGROUND AND AIMS: NLRP3 inflammasome plays a key role in vascular inflammation and atherosclerosis. Circular RNAs (circRNAs) are involved in disease development by regulating gene expression, and have emerged as promising novel disease biomarkers. This study aimed to identify the NLRP3 inflammasome-associated circRNA biomarkers of carotid atherosclerosis. METHODS: Based on the differential expression profiles of circRNAs in patients with carotid artery plaque (CAP) and healthy controls, hsa_circ_0043621, hsa_circ_0051995, and hsa_circ_0123388 were screened and validated using real-time quantitative polymerase chain reaction (RT-qPCR). Potential circRNA-miRNA-mRNA interactions were explored using a luciferase assay. The biological roles of the validated circRNAs were investigated in human umbilical vein endothelial cells (HUVECs) using Western blotting, transwell, and CCK-8 assays. Clinical significance was assessed using receiver operating characteristic (ROC) curves and logistic regression analysis. RESULTS: The expression levels of all candidate circRNAs were significantly higher in patients with CAP than in controls (p<0.05), which was consistent with the results of the microarray analysis. Overexpression of hsa_circ_0043621 significantly increased the expression of NLRP3, induced migration of HUVECs, and inhibited cell proliferation. hsa_circ_0043621 demonstrated reasonable diagnostic accuracy for CAP detection and increased intima-media thickness (IMT). hsa_circ_0043621 upregulation was an independent predictor of an increased risk of CAP and increased IMT. CONCLUSIONS: hsa_circ_0043621 is a valuable circulating biomarker of carotid atherosclerosis and may contribute to its pathogenesis by regulating the NLRP3 inflammasome.

Gasdermin D promotes influenza virus-induced mortality through neutrophil amplification of inflammation.

Influenza virus activates cellular inflammasome pathways, which can be both beneficial and detrimental to infection outcomes. Here, we investigate the function of the inflammasome-activated, pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice (Gsdmd-/-) significantly attenuates influenza virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected Gsdmd-/- mice exhibit decreased inflammatory gene signatures shown by lung transcriptomics. Among these, diminished neutrophil gene activation signatures are corroborated by decreased detection of neutrophil elastase and myeloperoxidase in KO mouse lungs. Indeed, directly infected neutrophils are observed in vivo and infection of neutrophils in vitro induces release of DNA and tissue-damaging enzymes that is largely dependent on GSDMD. Neutrophil depletion in infected WT mice recapitulates the reductions in mortality, lung inflammation, and lung dysfunction observed in Gsdmd-/- animals, while depletion does not have additive protective effects in Gsdmd-/- mice. These findings implicate a function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a therapeutic avenue for treating severe influenza.

SARS-CoV-2 virus-like particle variants alpha and delta mimic the native viruses in their differential inflammasome activating potential.

The emerging SARS-CoV-2 variants are evolving to evade human immunity and differ in their pathogenicity. While evasion of the variants from adaptive immunity is widely investigated, there is a paucity of knowledge about their interactions with innate immunity. Inflammasome assembly is one of the most potent mechanisms of the early innate response to viruses, but when it is inappropriate, it can perpetuate tissue damage. In this study, we focused on the capacity of SARS-CoV-2 Alpha and Delta variants to activate the NLRP3 inflammasome. We compared the macrophage activation, particularly the inflammasome formation, using Alpha- and Delta-spike virus-like particles (VLPs). We found that VLPs of both variants activated the inflammasome even without a priming step. Delta-spike VLPs had a significantly stronger effect on triggering pyroptosis and inflammasome assembly in THP-1 macrophages than did Alfa-spike VLPs. Cells treated with Delta VLPs showed greater cleavage of caspase-1 and IL-1ß release. Furthermore, Delta VLPs induced stronger cytokine secretion from macrophages and caused essential impairment of mitochondrial respiration in comparison to Alpha VLPs. Additionally, infection of primary human monocyte-derived macrophages with the SARS-CoV-2 variants confirmed the observations in VLPs. Collectively, we revealed that SARS-CoV-2 Delta had a greater impact on the inflammasome activation, cell death and mitochondrial respiration in macrophages than did the Alpha variant. Importantly, the differential response to the SARS-CoV-2 variants can influence the efficacy of therapies targeting the host's innate immunity.

Folic Acid Rescues Dopaminergic Neurons in MPTP-Induced Mice by Inhibiting the NLRP3 Inflammasome and Ameliorating Mitochondrial Impairment.

Parkinson's disease (PD) is marked by the degeneration of dopaminergic neurons of the substantia nigra (SN), with neuroinflammation and mitochondrial dysfunction being key contributors. The neuroprotective potential of folic acid (FA) in the dopaminergic system of PD was assessed in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model. MPTP (20 mg/kg of body weight) was administered to C57BL/6J mice to simulate PD symptoms followed by FA treatment (5 mg/kg of body weight). Behavioral tests, pole, rotarod, and open-field tests, evaluated motor function, while immunohistochemistry, ELISA, RT-qPCR, and Western blotting quantified neuroinflammation, oxidative stress markers, and mitochondrial function. FA supplementation considerably improved motor performance, reduced homocysteine levels and mitigated oxidative damage in the SN. The FA-attenuated activation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome lessened glial cell activity and reduced neuroinflammation. At the molecular level, FA reduced DNA damage, downregulated phosphorylated p53, and induced the expression of peroxisome proliferator-activated receptor α coactivator 1α (PGC-1α), enhancing mitochondrial function. Therefore, FA exerts neuroprotection in MPTP-induced PD by inhibiting neuroinflammation via NLRP3 inflammasome suppression and promoting mitochondrial integrity through the p53-PGC-1α pathway. Notable limitations of our study include its reliance on a single animal model and the incompletely elucidated mechanisms underlying the impact of FA on mitochondrial dynamics. Future investigations will explore the clinical utility of FA and its molecular mechanisms, further advancing it as a potential therapeutic for managing and delaying the progression of PD.

Guanylate-binding protein-5 is involved in inflammasome activation by bacterial DNA but only the cooperation of multiple GBPs accounts for control of Brucella abortus infection.

Introduction: Guanylate-binding proteins (GBPs) are produced in response to pro-inflammatory signals, mainly interferons. The most studied cluster of GBPs in mice is on chromosome 3. It comprises the genes for GBP1-to-3, GBP5 and GBP7. In humans, all GBPs are present in a single cluster on chromosome 1. Brucella abortus is a Gram-negative bacterium known to cause brucellosis, a debilitating disease that affects both humans and animals. Our group demonstrated previously that GBPs present on murine chromosome 3 (GBPchr3) is important to disrupt Brucella-containing vacuole and GBP5 itself is important to Brucella intracellular LPS recognition. In this work, we investigated further the role of GBPs during B. abortus infection. Methods and results: We observed that all GBPs from murine chromosome 3 are significantly upregulated in response to B. abortus infection in mouse bone marrow-derived macrophages. Of note, GBP5 presents the highest expression level in all time points evaluated. However, only GBPchr3-/- cells presented increased bacterial burden compared to wild-type macrophages. Brucella DNA is an important Pathogen-Associated Molecular Pattern that could be available for inflammasome activation after BCV disruption mediated by GBPs. In this regard, we observed reduced IL-1ß production in the absence of GBP2 or GBP5, as well as in GBPchr3-/- murine macrophages. Similar result was showed by THP-1 macrophages with downregulation of GBP2 and GBP5 mediated by siRNA. Furthermore, significant reduction on caspase-1 p20 levels, LDH release and Gasdermin-D conversion into its mature form (p30 N-terminal subunit) was observed only in GBPchr3-/- macrophages. In an in vivo perspective, we found that GBPchr3-/- mice had increased B. abortus burden and higher number of granulomas per area of liver tissue, indicating increased disease severity. Discussion/conclusion: Altogether, these results demonstrate that although GBP5 presents a high expression pattern and is involved in inflammasome activation by bacterial DNA in macrophages, the cooperation of multiple GBPs from murine chromosome 3 is necessary for full control of Brucella abortus infection.

P2RX7 gene variants associate with altered inflammasome assembly and reduced pyroptosis in chronic nonbacterial osteomyelitis (CNO).

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1ß and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.

Knockdown of NR4A1 alleviates doxorubicin-induced cardiotoxicity through inhibiting the activation of the NLRP3 inflammasome.

Doxorubicin (DOX) is a widely used antitumor drug, but its clinical applicability is hampered by the unfortunate side effect of DOX-induced cardiotoxicity (DIC). In our current study, we retrieved three high-throughput sequencing datasets related to DIC from the Gene Expression Omnibus (GEO) datasets. We conducted differential analysis using R (DESeq2) to pinpoint differentially expressed genes (DEGs, and identified 11 genes that were consistently altered in both the control and DOX-treated groups. Notably, our Random Forest analysis of these three GEO datasets highlighted the significance of nuclear receptor subfamily 4 group A member 1 (NR4A1) in the context of DIC. The DOX-induced mouse model and cell model were used for the in vivo and in vitro studies to reveal the role of NR4A1 in DIC. We found that silencing NR4A1 by adeno-associated virus serotype 9 (AAV9) contained shRNA in vivo alleviated the DOX-induced cardiac dysfunction, cardiomyocyte injury and fibrosis. Mechanistically, we found NR4A1 silencing was able to inhibit DOX-induced the cleavage of NLRP3, IL-1ß and GSDMD in vivo. Further in vitro studies have shown that inhibition of NR4A1 suppressed DOX-induced cytotoxicity and oxidative stress through the same molecular mechanism. We prove that NR4A1 plays a critical role in DOX-induced cardiotoxicity by inducing pyroptosis via activation of the NLRP3 inflammasome, and it might be a promising therapeutic target for DIC.

A mouse model to distinguish NLRP6-mediated inflammasome-dependent and -independent functions.

The NOD-like receptor (NLR) family pyrin domain containing 6 (NLRP6) serves as a sensor for microbial dsRNA or lipoteichoic acid (LTA) in intestinal epithelial cells (IECs), and initiating multiple pathways including inflammasome pathway and type I interferon (IFN) pathway, or regulating nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. NLRP6 can exert its function in both inflammasome-dependent and inflammasome-independent manners. However, there is no tool to distinguish the contribution of individual NLRP6-mediated pathway to the physiology and pathology in vivo. Here, we validated that Arg39 and Trp50 residues in the pyrin domain (PYD) of murine NLRP6 are required for ASC recruitment and inflammasome activation, but are not important for the RNA binding and PYD-independent NLRP6 oligomerization. We further generated the Nlrp6R39E&W50E mutant mice, which showed reduced inflammasome activation in either steady state intestine or during viral infection. However, the type I IFN production in cells or intestine tissue from Nlrp6R39E&W50E mutant mice remain normal. Interestingly, NLRP6-mediated inflammasome activation or the IFN-I production seems to play distinct roles in the defense responses against different types of RNA viruses. Our work generated a useful tool to study the inflammasome-dependent role of NLRP6 in vivo, which might help to understand the complexity of multiple pathways mediated by NLRP6 in response to the complicated and dynamic environmental cues in the intestine.

Pathogen recognition pathway gene variants and inflammasome sensors gene expression in tuberculosis patients under treatment.

BACKGROUND: Several epidemiological studies have suggested that genetic variations in encoding pattern recognition receptors (PRRs) genes such as Toll Like Receptors (TLRs) and their signaling products, may influence the susceptibility, severity and outcome of tuberculosis (TB). After sensing a pathogen, the cell responds producing an inflammatory response, to restrain the pathogen's successful course of infection. Herein we assessed single nucleotide polymorphisms (SNP) and gene expression from pathogen recognition and inflammasome pathways in Brazilian TB patients. METHODS AND RESULTS: For genetic association analysis we included MYD88 and TLR4, PRRs sensing proteins. Allele distribution for MYD88 rs6853 (A > G) and TLR4 rs7873784 (C > G) presented conserved among the tested samples with statistically differential distribution in TB patients versus controls. However, when testing according to sample ethnicity (African or Caucasian-derived individuals) we identified that the rs6853 G/G genotype was associated with a lower susceptibility to TB in Caucasian population. Meanwhile, the rs7873784 G/G genotype was associated with a higher TB susceptibility in Afro-descendant ethnicity individuals. We also aimed to verify MYD88 and the inflammasome genes NLRP1 and NLRC4 expression in order to connect to active TB and/or clinical aspects. CONCLUSIONS: We identified that inflammasome gene expression in TB patients under treatment display a similar pattern as in healthy controls, indicating that TB treatment impairs NLRP1 inflammasome activation.

The molecular mechanism of MiR-26a-5p regulates autophagy and activates NLRP3 inflammasome to mediate cardiomyocyte hypertrophy.

OBJECTIVE: Many studies have found that miR-26a-5p plays an essential role in the progression of pathological cardiac hypertrophy, however, there is still no evidence on whether miR-26a-5p is related to the activation of autophagy and NLRP3 inflammasome. And the mechanism of miR-26a-5p and NLRP3 inflammasome aggravating pathological cardiac hypertrophy remain unclear. METHODS: Cardiomyocytes were treated with 200µM PE to induce cardiac hypertrophy and intervened with 10mM NLRP3 inhibitor INF39. In addition, we also used the MiR-26a-5p mimic and inhibitor to transfect PE-induced cardiac hypertrophy. RT-qPCR and western blotting were used to detect the expressions of miR-26a-5p, NLRP3, ASC and Caspase-1 in each group, and we used α-SMA immunofluorescence to detect the change of cardiomyocyte area. The expression levels of autophagy proteins LC3, beclin-1 and p62 were detected by western blotting. Finally, we induced the SD rat cardiac hypertrophy model through aortic constriction (TAC) surgery. In the experimental group, rats were intervened with MiR-26a-5p mimic, MiR-26a-5p inhibitor, autophagy inhibitor 3-MA, and autophagy activator Rapamycin. RESULTS: In cell experiments, we observed that the expression of miR-26a-5p was associated with cardiomyocyte hypertrophy and increased surface area. Furthermore, miR-26a-5p facilitated autophagy and activated the NLRP3 inflammasome pathway, which caused changes in the expression of genes and proteins including LC3, beclin-1, p62, ACS, NLRP3, and Caspase-1. We discovered similar outcomes in the TAC rat model, where miR-26a-5p expression corresponded with cardiomyocyte enlargement and fibrosis in the cardiac interstitial and perivascular regions. In conclusion, miR-26a-5p has the potential to regulate autophagy and activate the NLRP3 inflammasome, contributing to the development of cardiomyocyte hypertrophy. CONCLUSION: Our study found a relationship between the expression of miR-26a-5p and cardiomyocyte hypertrophy. The mechanism behind this relationship appears to involve the activation of the NLRP3 inflammasome pathway, which is caused by miR-26a-5p promoting autophagy. Targeting the expression of miR-26a-5p, as well as inhibiting the activation of autophagy and the NLRP3 inflammasome pathway, could offer additional treatments for pathological cardiac hypertrophy.

The contributions of deleterious rare alleles in NLRP12 and inflammasome-related genes to polymyalgia rheumatica.

Polymyalgia rheumatica (PMR) is a chronic inflammatory disease characterized by arthralgia and myalgia of the shoulder and hip girdles, and fever. PMR is linked to autoimmune diseases and autoinflammatory disorders. Exome sequencing has revealed the roles of rare variants in some diseases. Causative genes for monogenic autoinflammatory disorders might be candidate genes for the selective exome analysis of PMR. We investigated rare variants in the coding and boundary regions of candidate genes for PMR. Exome sequencing was performed to analyze deleterious rare variants in candidate genes, and the frequencies of the deleterious rare alleles in PMR were compared with those of Japanese population controls. Deleterious rare alleles in the NLRL12 gene were associated with PMR (P = 0.0069, Pc = 0.0415, odds ratio [OR] 4.49, 95% confidence interval [CI] 1.79-11.27). A multigene analysis demonstrated the deleterious rare allele frequency of the candidate genes for autoinflammatory disorders was also increased in PMR (P = 0.0016, OR 3.69, 95%CI 1.81-7.54). The deleterious rare allele frequencies of the candidate genes including NLRP12 were increased in PMR patients, showing links to autoinflammatory disorders in the pathogenesis of PMR.

VEXAS syndrome is characterized by inflammasome activation and monocyte dysregulation.

Acquired mutations in the UBA1 gene were recently identified in patients with severe adult-onset auto-inflammatory syndrome called VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic). However, the precise physiological and clinical impact of these mutations remains poorly defined. Here we study a unique prospective cohort of VEXAS patients. We show that monocytes from VEXAS are quantitatively and qualitatively impaired and display features of exhaustion with aberrant expression of chemokine receptors. In peripheral blood from VEXAS patients, we identify an increase in circulating levels of many proinflammatory cytokines, including IL-1ß and IL-18 which reflect inflammasome activation and markers of myeloid cells dysregulation. Gene expression analysis of whole blood confirms these findings and also reveals a significant enrichment of TNF-α and NFκB signaling pathways that can mediate cell death and inflammation. This study suggests that the control of the nflammasome activation and inflammatory cell death could be therapeutic targets in VEXAS syndrome.

METTL14 contributes to acute lung injury by stabilizing NLRP3 expression in an IGF2BP2-dependent manner.

Acute lung injury (ALI) as well as its more severe form, acute respiratory distress syndrome (ARDS), frequently leads to an uncontrolled inflammatory response. N6-methyladenosine (m6A) modification was associated with the progression of several inflammatory diseases. However, the role of methyltransferase-like 14 (METTL14)-mediated m6A methylation in ALI/ARDS remains unclear. Here, we reported an increase in overall expression levels of m6A and METTL14 in circulating monocyte-derived macrophages recruited to the lung following ALI, which is correlated with the severity of lung injury. We further demonstrated the critical function of METTL14 in activating NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome in vitro and in mouse models of ALI/ARDS, and validated NLRP3 as the downstream target of METTL14 by the m6A RNA immunoprecipitation (MeRIP) and RIP assays. Mechanistically, METTL14-methylated NLRP3 transcripts were subsequently recognized by insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), an m6A reader, which stabilized NLRP3 mRNA. Furthermore, we observed that IGF2BP2 knockdown diminished LPS-induced ALI in mice by downregulating NLRP3 expression. In summation, our study revealed that the molecular mechanism underlying the pathogenesis of ALI/ARDS involves METTL14-mediated activation of NLRP3 inflammasome in an IGF2BP2 dependent manner, thereby demonstrating the potential of METTL14 and IGF2BP2 as promising biomarkers and therapeutic targets for ALI/ARDS treatment.

Palmitic acid activates NLRP3 inflammasome through NF-κB and AMPK-mitophagy-ROS pathways to induce IL-1ß production in large yellow croaker (Larimichthys crocea).

Studies on marine fish showed that vegetable oils substituted for excessive fish oil increased interleukin-1ß (IL-1ß) production. However, whether the nucleotide-binding oligomerization domain, leucine-rich repeat-containing family, pyrin domain-containing-3 (NLRP3) inflammasome has a substantial role in fatty acid-induced IL-1ß production in fish remains unclear. The associated specific mechanism is also unknown. In this study, nlrp3, caspase-1 and apoptosis-associated speck-like protein containing a CARD (asc) were successfully cloned, and NLRP3 inflammasome consisted of NLRP3, caspase-1 and ASC in large yellow croaker. Primary hepatocytes of fish incubated with palmitic acid (PA) exhibited the highest expression of pro-inflammatory genes (il-1ß and tnfα) and NLRP3 inflammasome related genes (nlrp3, caspase-1 and asc), caspase-1 activity and IL-1ß production among different treatments. Furthermore, PA-induced NLRP3 inflammasome activation was confirmed to require two signals: the first signal was that PA promoted the NF-κB (P65) protein into the nucleus, and NF-κB increased NLRP3 promoter activity and nlrp3 transcription. The second signal was that PA inhibited AMPK phosphorylation and decreased mitophagy by inhibiting the expression of PINK and parkin proteins, thereby damaging the mitochondria that could not be effectively cleared. Mitochondrial damage generated excessive amounts of reactive oxygen species, which activated the NLRP3 inflammasome and then induced caspase-1 activity and IL-1ß production. Therefore, excessive dietary PA activated NLRP3 inflammasome through NF-κB and AMPK-mitophagy-ROS pathways to induce IL-1ß production, thereby leading to inflammation in fish.