NLRP3 Inflammasome Activation Research Articles

Cell-based in vitro hemoassay for evaluation of NLRP3-inflammasome activity

Currently, gouty arthritis is considered as a polygenic multifactorial autoinflammatory disease caused by activation of the NOD (nucleotide-binding domain) -like protein receptor 3 inflammasome NLRP3. The two cytokines IL-1β and IL-18 are considered important biomarkers of NLRP3 inflammasome activation. However, usually the concentration of IL-1β in donor sera is extremely low and found to be at the limit of detection level (1-3 pg/ml), while the concentration of circulating cytokine IL-18 in the sera of individual donors varies greatly. This results in difficulty using these biomarkers in the diagnosis of autoinflammatory diseases. We hypothesized that the patient’s blood cells which were sensitized in vivo to the presence of specific factors characteristic of autoinflammatory diseases, in particular, gouty arthritis, would produce increased amounts of the inflammasome-regulated cytokines compared to blood cells obtained from healthy donors. A comparison of the IL-18 cytokine in healthy donors and patients with gouty arthritis was carried out using 2 methods: a) by traditional analysis of the level of serum circulated IL-18 and b) by using a cell-based Hemoassay in vitro developed at the research institute “Biotech” SamGMU. The comparative analysis demonstrated the advantages of using an in vitro cell-based Hemoassay to assess the IL-18 cytokine status of patients. Serum IL-18 values varied widely and showed no significant difference between donors and patients with gouty arthritis. Using the developed cell-based Hemoassay in vitro, significant quantitative differences in the production of the inflammatory cytokine IL-18 produced by blood cells of potentially healthy donors and patients with gouty arthritis were detected. Blood cells of individual patients, sensitized in vivo with specific factors characteristic of gouty arthritis, produce increased concentrations of IL-18 in the cell growth medium in vitro compared to cells from healthy donors. Thus, the in vitro cell-based Hemoassay can be used for a more accurate assessment of the cytokine status of patients.

Bardoxolone methyl alleviates acute liver injury in mice by inhibiting NLRP3 inflammasome activation

To investigate the inhibitory effect of bardoxolone methyl (CDDO-Me) on activation of NLRP3 inflammasome and its mechanism for alleviating acute liver injury (ALI). Mouse bone marrow-derived macrophages (BMDM) and THP-1 cells were pre-treated with CDDO-Me followed by treatment with Nigericin, ATP, MSU, intracellular LPS transfection for activation of NLRP3 inflammasomes, or poly A: T for activation of AIM2 inflammasomes. The levels of caspase-1 and IL-1β in the cell culture supernatant was determined with Western blotting and ELISA to assess the inhibitory effect of CDDO-Me on NLRP3 inflammasomes and its specificity. In the animal experiment, male C57BL/6J mouse models of acetaminophen-induced ALI were treated with low-dose (20 mg/kg) and high-dose (40 mg/kg) CDDO-Me, and the changes in serum levels of IL-1β, TNF- α, AST and ALT were measured by ELISA and liver tissue pathology was observed using HE staining. In mouse BMDM and THP-1 cells, CDDO-Me dose-dependently inhibited the activation of NLRP3 inflammasomes without significantly affecting the secretion of non-inflammasome-related inflammatory factors IL-6 and TNF-α or AIM2 inflammasome activation. In the mouse models of ALI, CDDO-Me treatment at both the low and high doses significantly reduced serum levels of IL-1β, AST and ALT, ameliorated histological changes and reduced inflammatory cell infiltration in the liver tissue, and the effects exhibited a distinct dose dependence. CDDO-Me can specifically inhibit the activation of NLRP3 inflammasomes to alleviate acetaminophen-induced ALI in mice.

Multifunctional carbomer based ferulic acid hydrogel promotes wound healing in radiation-induced skin injury by inactivating NLRP3 inflammasome

BackgroundRadiation-induced skin injury is a significant adverse reaction to radiotherapy. However, there is a lack of effective prevention and treatment methods for this complication. Ferulic acid (FA) has been identified as an effective anti-radiation agent. Conventional administrations of FA limit the reaching of it on skin. We aimed to develop a novel FA hydrogel to facilitate the use of FA in radiation-induced skin injury.MethodsWe cross-linked carbomer 940, a commonly used adjuvant, with FA at concentrations of 5%, 10%, and 15%. Sweep source optical coherence tomography system, a novel skin structure evaluation method, was applied to investigate the influence of FA on radiation-induced skin injury. Calcein-AM/PI staining, CCK8 assay, hemolysis test and scratch test were performed to investigate the biocompatibility of FA hydrogel. The reducibility of DPPH and ABTS radicals by FA hydrogel was also performed. HE staining, Masson staining, laser Doppler blood flow monitor, and OCT imaging system are used to evaluate the degree of skin tissue damage. Potential differentially expressed genes were screened via transcriptome analysis.ResultsGood biocompatibility and in vitro antioxidant ability of the FA hydrogels were observed. 10% FA hydrogel presented a better mechanical stability than 5% and 15% FA hydrogel. All three concentrations of FA remarkably promoted the recovery of radiation-induced skin injury by reducing inflammation, oxidative conidiation, skin blood flow, and accelerating skin tissue reconstruction, collagen deposition. FA hydrogel greatly inhibiting the levels of NLRP3, caspase-1, IL-18, pro-IL-1β and IL-1β in vivo and vitro levels through restraining the activation of NLRP3 inflammasome. Transcriptome analysis indicated that FA might regulate wound healing via targeting immune response, inflammatory response, cell migration, angiogenesis, hypoxia response, and cell matrix adhesion.ConclusionsThese findings suggest that the novel FA hydrogel is a promising therapeutic method for the prevention and treatment of radiation-induced skin injury patients.Graphical

Corynoline alleviates hepatic ischemia-reperfusion injury by inhibiting NLRP3 inflammasome activation through enhancing Nrf2/HO-1 signaling.

Corynoline has displayed pharmacological effects in reducing oxidative stress and inflammatory responses in many disorders. However, its effects on hepatic ischemia-reperfusion (I/R) injury remain unclear. This study aimed to investigate the protective effects of corynoline against hepatic I/R injury and the underlying mechanisms. Rat models with hepatic I/R injury and BRL-3A cell models with hypoxia/reoxygenation (H/R) insult were constructed. Models were pretreated with corynoline and/or other inhibitors for functional and mechanistic examination. Corynoline pretreatment effectively mitigated hepatic I/R injury verified by reduced serum transaminase levels, improved histological damage scores, and decreased apoptosis rates. Additionally, corynoline pretreatment significantly inhibited I/R-triggered oxidative stress and inflammatory responses, as indicated by enhanced mitochondrial function, reduced levels of ROS and MDA, reduced neutrophil infiltration and suppressed proinflammatory cytokine release. In vitro experiments further showed that corynoline pretreatment increased cellular viability, decreased LDH activity, reduced cellular apoptosis, and inhibited oxidative stress and inflammatory injury in H/R-induced BRL-3A cells. Mechanistically, corynoline significantly increased Nrf2 nuclear translocation and expression levels of its target gene, HO-1. It also blocked NLRP3 inflammasome activation both in vivo and in vitro. Furthermore, pretreatment with Nrf2 inhibitor ML-385 counteracted the protective effect of corynoline on hepatic I/R injury. Ultimately, in vitro studies revealed that the NLRP3 activator nigericin could also nullified the protective effects of corynoline in BRL-3A cells, but had minimal impact on Nrf2 nuclear translocation. Corynoline can exert protective effects against hepatic I/R injury by inhibiting oxidative stress, inflammatory responses, and apoptosis. These effects may be associated with inhibiting ROS-induced NLRP3 inflammasome activation by enhancing Nrf2/HO-1 signaling. These data provide new understanding about the mechanism of corynoline action, suggesting it is a potential drug applied for the treatment and prevention of hepatic I/R injury.

Activation of osmo-sensitive LRRC8 anion channels in macrophages is important for micro-crystallin joint inflammation

Deposition of monosodium urate and calcium pyrophosphate (MSU and CPP) micro-crystals is responsible for painful and recurrent inflammation flares in gout and chondrocalcinosis. In these pathologies, the inflammatory reactions are due to the activation of macrophages responsible for releasing various cytokines including IL-1β. The maturation of IL-1β is mediated by the multiprotein NLRP3 inflammasome. Here, we find that activation of the NLRP3 inflammasome by crystals and concomitant production of IL-1β depend on cell volume regulation via activation of the osmo-sensitive LRRC8 anion channels. Both pharmacological inhibition and genetic silencing of LRRC8 abolish NLRP3 inflammasome activation by crystals in vitro and in mouse models of crystal-induced inflammation. Activation of LRRC8 upon MSU/CPP crystal exposure induces ATP release, P2Y receptor activation and intracellular calcium increase necessary for NLRP3 inflammasome activation and IL-1β maturation. We identify a function of the LRRC8 osmo-sensitive anion channels with pathophysiological relevance in the context of joint crystal-induced inflammation.

Diacetoxy-6-gingerdiol protects the extracellular matrix of nucleus pulposus cells and ameliorates intervertebral disc degeneration by inhibiting the IL-1β-mediated NLRP3 pathway

Intervertebral disc degeneration (IDD) is a common cause of low back pain, causing a huge emotional and economic burden on patients and society. Reduction of nucleus pulposus cells (NPC) and extracellular matrix (ECM) is the main feature of IDD, and NPC is the main source of ECM. Thermal apoptosis is a newly discovered form of cell death in recent years that differs significantly from apoptosis in terms of molecular mechanisms and cellular morphological changes. Diacetoxy-6-gingerdiol(D-6-G), a type of gingerol, has anti-inflammatory and antioxidant effects, but whether it has an inhibitory effect on cellular pyroptosis is not clear. Therefore, in the present study, we investigated the effect of D-6-G on the ECM of the nucleus pulposus oblongata under IL-1β treatment, as well as the mechanism of its effect on NLRP3 inflammasome and cellular focal death. In vitro cellular experiments demonstrated that D-6-G could bind to and inhibit the activity of NLRP3 inflammasome, and interestingly, D-6-G could also inhibit cellular pyroptosis and protect the nucleus pulposusry cellular microenvironment by activating the Nrf2/HO-1 axis. In conclusion, we found that D-6-G could inhibit NLRP3 inflammatory vesicle activity as well as cellular pyroptosis in NPCs and protect the ECM, suggesting the potential of D-6-G to delay IDD.

Helicobacter pylori CagA mediated mitophagy to attenuate the NLRP3 inflammasome activation and enhance the survival of infected cells

Helicobacter pylori (H. pylori) is one of the most common bacterial infections in the world, and its key virulence component CagA is the leading cause of gastric cancer. Mitophagy is a form of selective autophagy that eliminates damaged mitochondria and is essential for some viruses and bacteria to evade the immune system. However, the mechanisms by which CagA mediates H. pylori-induced mitophagy and NLRP3 inflammasome activation remain elusive. In this study, we reported that H. pylori primarily uses its CagA to induce mitochondrial oxidative damage, mitochondrial dysfunction, dynamic imbalance, and to block autophagic flux. Inhibition of mitophagy led to an increase in NLRP3 inflammasome activation and apoptosis and a decrease in the viability of H. pylori-infected cells. Our findings suggested that H. pylori induces mitochondrial dysfunction and mitophagy primarily via CagA. It reduces NLRP3 inflammasome activation to evade host immune surveillance and increases the survival and viability of infected cells, potentially leading to gastric cancer initiation and development. Our findings provide new insights into the pathogenesis of H. pylori-induced gastric cancer, and inhibition of mitophagy may be one of the novel techniques for the prevention and treatment of this disease.

Deciphering the mechanisms of the IL-6/CXCL1 and NOD-like receptor signaling pathways in otitis media with effusion in rodents

BackgroundOtitis media with effusion (OME) often leads to pediatric hearing loss and is influenced by innate and adaptive immune responses. Innate immunity serves as the non-specific first line of defense against OME. MethodsWe induced OME in rats using ovalbumin. We administered IL-6 monoclonal antibodies intranasally to inhibit IL-6, and we injected an NF-κB inhibitor intraperitoneally to explore the role of IL-6 in innate immunity and its interaction with the NOD-like receptor signaling pathway. We analyzed RNA-sequencing data with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways to assess signaling pathways involved in OME. We also utilized Western blot, quantitative real-time PCR, and immunohistochemistry on middle ear samples and used microscopy to identify immune cells in ear wash fluids. ResultsOur study suggests a pivotal role for IL-6 in the immune pathways of rats with OME via the regulation of CXCL1-mediated pathways. Increased levels of IL-6 and CXCL1 were observed in the middle ear tissues, and activation of the NLRP3 inflammasome in OME rats led to an immune response via NF-κB, thus promoting IL-6 and CXCL1 production, which was reduced by IL-6 antibody treatment. ConclusionsOur findings confirm that IL-6 and CXCL1 play significant roles in the innate immune response in OME in rodents, predominantly via the NOD-like receptor signaling pathway and NLRP3 inflammasome activation. This research sheds light on OME pathogenesis and its immune-related mechanisms.

Protective role of cytosolic prion protein against virus infection in prion-infected cells.

Cytosolic PrP has been detected in prion-infected cells and suggested to be involved in the neurotoxicity of prions. Here, we also detected cytosolic PrP in prion-infected cells. We further found that the nuclear translocation of NF-κB was disturbed in prion-infected cells and that the N-terminal potential nuclear translocation signal of PrP expressed in the cytosol disturbed the nuclear translocation of NF-κB. Thus, the N-terminal nuclear translocation signal of cytosolic PrP might play a role in prion neurotoxicity. Prion-like protein aggregates in other protein misfolding disorders, including Alzheimer's disease were reported to play a protective role against various environmental stimuli. We here showed that prion-infected cells were partially resistant to IAV/WSN infection due to the cytosolic PrP-mediated disturbance of the nuclear translocation of NF-κB, which consequently activated NLRP3 inflammasomes after IAV/WSN infection. It is thus possible that prions could also play a protective role in viral infections.

Enteric glial NLRP3 inflammasome contributes to gut mucosal barrier alterations in a mouse model of diet-induced obesity.

In the present study, we investigated the involvement of NLRP3 inflammasome in the intestinal epithelial barrier (IEB) changes associated with obesity, and its role in the interplay between enteric glia and intestinal epithelial cells (IECs). Wild-type C57BL/6J and NLRP3-KO (-/-) mice were fed with high-fat diet (HFD) or standard diet for 8 weeks. Colonic IEB integrity and inflammasome activation were assessed. Immunolocalization of colonic mucosal GFAP- and NLRP3-positive cells along with invitro coculture experiments with enteric glial cells (EGCs) and IECs allowed to investigate the potential link between altered IEB, enteric gliosis, and NLRP3 activation. HFD mice showed increased body weight, altered IEB integrity, increased GFAP-positive glial cells, and NLRP3 inflammasome hyperactivation. HFD-NLRP3-/- mice showed a lower increase in body weight, an improvement in IEB integrity and an absence of enteric gliosis. Coculture experiments showed that palmitate and lipopolysaccharide contribute to IEB damage and promote enteric gliosis with consequent hyperactivation of enteric glial NLRP3/caspase-1/IL-1β signaling. Enteric glial-derived IL-1β release exacerbates the IEB alterations. Such an effect was abrogated upon incubation with anakinra (IL-1β receptor antagonist) and with conditioned medium derived from silenced-NLRP3 glial cells. HFD intake elicits mucosal enteric gliotic processes characterized by a hyperactivation of NLRP3/caspase-1/IL-1β signaling pathway, that contributes to further exacerbate the disruption of intestinal mucosal barrier integrity. However, we cannot rule out the contribution of NLRP3 inflammasome activation from other cells, such as immune cells, in IEB alterations associated with obesity. Overall, our results suggest that enteric glial NLRP3 inflammasome might represent an interesting molecular target for the development of novel pharmacological approaches aimed at managing the enteric inflammation and intestinal mucosal dysfunctions associated with obesity.

Precise In Situ Delivery of a Photo-Enhanceable Inflammasome-Activating Nanovaccine Activates Anticancer Immunity.

A variety of state-of-the-art nanovaccines (NV) combined with immunotherapies have recently been developed to treat malignant tumors, showing promising results. However, immunosuppression in the tumor microenvironment (TME) restrains cytotoxic T-cell infiltration and limits the efficacy of immunotherapies in solid tumors. Therefore, tactics for enhancing antigen cross-presentation and reshaping the TME need to be explored to enhance the activity of NVs. Here, we developed photo-enhanceable inflammasome-activating NVs (PIN) to achieve precise in situ delivery of a tumor antigen and a hydrophobic small molecule activating the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3 inflammasome (NLRP3) pathway. Near-infrared light irradiation promoted PIN accumulation in tumor sites through photo-triggered charge reversal of the nanocarrier. Systematic PIN administration facilitated intratumoral NLRP3 inflammasome activation and antigen cross-presentation in antigen-presenting cells upon light irradiation at tumor sites. Furthermore, PIN treatment triggered immune responses by promoting the production of proinflammatory cytokines and activating antitumor immunity without significant systematic toxicity. Importantly, the PIN enhanced the efficacy of immune checkpoint blockade and supported the establishment of long-term immune memory in mouse models of melanoma and hepatocellular carcinoma. Collectively, this study reports a safe and efficient photoresponsive system for codelivery of antigens and immune modulators into tumor tissues, with promising therapeutic potential. Significance: The development of a photoresponsive nanovaccine with spatiotemporal controllability enables robust tumor microenvironment modulation and enhances the efficacy of immune checkpoint blockade, providing an effective immunotherapeutic strategy for cancer treatment. See related commentary by Zhen and Chen, p. 3709.

Retention of ES cell-derived 129S genome drives NLRP1 hypersensitivity and transcriptional deregulation in Nlrp3tm1Flv mice.

Immune response genes are highly polymorphic in humans and mice, with heterogeneity amongst loci driving strain-specific host defence responses. The inadvertent retention of polymorphic loci can introduce confounding phenotypes, leading to erroneous conclusions, and impeding scientific advancement. In this study, we employ a combination of RNAseq and variant calling analyses to identify a substantial region of 129S genome, including the highly polymorphic Nlrp1 locus, proximal to Nlrp3, in one of the most commonly used mouse models of NLRP3 deficiency (Nlrp3tm1Flv). We show that the presence of the Nlrp1129S locus leads to an increase in NLRP1B protein expression, and a sensitising of Nlrp3tm1Flv macrophages to NLRP1 inflammasome activation, independent of NLRP3 deficiency. Retention of 129S genome further leads to protein sequence differences and altered gene regulation across multiple cell types, including of the key tissue-resident macrophage marker, TIM4. Using alternative models of NLRP3 deficiency, including a previously undescribed conditional Nlrp3 allele enabling precise temporal and cell-type specific control over Nlrp3 deletion, we further show that NLRP3 contributes to Talabostat-driven IL-1β release. Our study also establishes a generic framework to identify functionally relevant SNPs and assess genomic contamination in transgenic mice using RNAseq data. This allows for unambiguous attribution of phenotypes to the target gene and advances the precision and reliability of research in the field of host defence responses.

Thrombin receptor PAR4 cross-activates the tyrosine kinase c-met in atrial cardiomyocytes.

Thrombin supports coagulation-independent inflammation via protease-activated receptors (PAR). PAR4 is specifically increased in obese human atria, correlating with NLRP3 inflammasome activation. PAR4-mediated NLRP3 inflammasome activation in atrial cardiomyocytes is not known, nor have signaling partners been identified. Thrombin transactivates the hepatocyte growth factor receptor in some cancer cells, so we examined PAR4/c-met cross-talk in atrial cardiomyocytes and its possible significance in obesity. Cardiomyocytes from right atrial appendages (RAA) of obese patients expressed more PAR1 and PAR4 compared to non-obese. In HL-1 atrial cardiomyocytes, thrombin induced caspase-1 auto-activation and IL-1β maturation; IL-1β secretion was evoked by PAR4-activating peptide (AP), but not PAR1-AP. PAR4-AP additionally increased phosphorylated CaMKII-Thr287, mTOR-Ser2481, and Akt-Ser473 while suppressing AMPK-Thr172 phosphorylation. Total kinase levels were largely unaltered. PAR4AP rapidly increased phosphorylated c-met in HL-1 cells and over time also transcriptionally upregulated c-met. The c-met inhibitor SGX-523 abrogated the effects of PAR4-AP on CaMKII/AKT/mTOR phosphorylation but did not affect PAR4-stimulated IL-1β production. Obese human RAA contained more IL-1β, phospho-c-met, and phospho-mTOR than non-obese RAA; CamKII phosphorylation was not modified. Atria from high-fat diet (HFD) versus chow-fed mice also contained more IL-1β, together with higher myeloperoxidase activity, Acta2 mRNA total and phosphorylated c-met; these increases were blunted in PAR4-/- HFD-fed mice. Thrombin cross-activates c-met via PAR4 in atrial cardiomyocytes. Transactivated c-met contributes partially to PAR4-mediated signaling, but NLRP3 inflammasome activation appears to be largely independent of c-met. Abundance of PAR4 and activated c-met increases with obesity, providing therapeutic targets for management of adiposity-driven AF.

Markers of extracellular matrix degradation and inflammasome activation are associated with carotid plaques in virally suppressed people with HIV in Botswana.

HIV is associated with increased risk of cardiovascular disease. We investigated soluble markers of extracellular matrix (ECM) remodeling and inflammation in relation to presence of carotid plaques in a well-characterized adult cross-sectional study of people with HIV (PWH) and matched people without HIV in Botswana. Using enzyme immunoassays we analyzed plasma ECM remodeling mediators including Galectin-3 (GAL-3), Cystatin B (CysB) and Growth/differentiation factor 15 (GDF-15) and the inflammatory marker IL-18 in 196 without HIV and 197 PWH of which 36 were ART-naïve. We found i) PWH had higher plasma levels of the ECM markers GAL-3 and CysB and the NLRP3 inflammasome activation marker IL-18, mainly in ART naïve participants, ii) PWH on ART had markedly higher GDF-15, associated with use of first generation nucleoside analogs; iii) high levels of CysB and IL-18 correlated with presence of carotid plaques. In PWH, high levels of CysB and IL-18 were associated with the presence of carotid plaques. For IL-18 this was observed in the study population as a whole, while the association for CysB was restricted to PWH.

The Association between the NLRP3 Inflammasome and Specific Long-Non Coding RNAs (lncRNAs) in Cancer; New Perspective and Summary of Recent Studies.

In response to cellular perturbations, a multimeric cytosolic protein complex known as the NLRP3 inflammasome assembles. As a result of this assembly, caspase-1 is activated, which encourages inflammatory cell death (pyroptosis) as well as the maturation and release of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18. Tumor etiology involves dysregulation of NLRP3 inflammasome activation, but because of conflicting results, its significance in the onset and spread of cancer is still up for debate. New research indicates that long-non coding RNAs (lncRNAs) play a crucial part in controlling the activity of the NLRP3 inflammasome in a number of disorders. Non-coding RNAs longer than 200 nucleotides are known as lncRNAs. Cancer development has been connected to its deregulation in the development of illnesses. Growing data suggests that controlling lncRNAs on the NLRP3 inflammasome plays a crucial role in the emergence and progression of cancer-related challenges. Here, we discuss over how lncRNAs control the NLRP3 inflammasome and how it functions in different types of cancer cells, offering new perspectives on creating new cancer treatment strategies in the future.

Abstract B050: Convergent signaling via C/EBPβ regulates MDSC-intrinsic NLRP3 inflammasomes to drive inflammatory CAF polarization

Abstract OBJECTIVE: Pivotal contributors to therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) are myeloid cell infiltration and signaling, and interleukin-1 (IL1)-mediated inflammatory polarization of cancer-associated fibroblasts (iCAF). We have shown that granulocytic myeloid- derived suppressor cell (gMDSC) promote iCAF polarization via NLRP3 inflammasome-derived IL-1β. Here, we sought to dissect the mechanisms regulating NLRP3 inflammasome induction in gMDSCs that drive iCAF polarization in PDAC. METHODS: Single-cell RNA sequencing (scRNA-seq) was performed in PKT mice treated with NLRP3i SB-NL02 vs vehicle. Single-Cell rEgulatory Network Inference and Clustering (SCENIC) analysis identified putative regulons binding upstream to Nlrp3 Transcription Start Site (TSS) in gMDSC subclusters in scRNA-seq data. CRISPR/cas9 silencing of Nlrp3 and Cebpb was performed in gMDSC-like J774M cells in vitro. These edited gMDSCs and J774M cells treated with/without SB-NL02 were co-cultured with Il6/Acta2 dual-reporter CAFs to examine iCAF polarization. RESULTS: PKT mice treated with NLRP3i SB-NL02 showed reduced tumor burden, gMDSC-intrinsic ASC oligomerization, and iCAF polarization in vivo. In silico analysis of intercellular single-cell communication showed striking attenuation in gMDSC-Nlrp3/Il1b:CAF-Il1r1 ligand-receptor crosstalk following NLRP3i treatment. To dissect mechanisms underlying NLRP3 regulation in gMDSCs that drive iCAF skewness, lineage trajectory inference revealed strong co-expression of Tlr4, Cxcr2, Nlrp3 and Il1b genes. Mechanistic studies in J774M gMDSCs showed activation of NLRP3 inflammasomes and IL-1βsecretion not only via canonical TLR4-MyD88 signaling, but also via CXCR2-mediated p38 MAPK induction. This was corroborated in vivo where ASC speck formation in intratumoral Ly6G+ gMDSCs was diminished in mice treated with CXCR2i AZD5069 and p38i peximetinib. To dissect how TLR4 and CXCR2-p38 signaling converge to regulate NLRP3, SCENIC analysis in single-cell gMDSCs transcriptomes revealed Cebpb as the top predicted regulon at the Nlrp3 TSS. In vitro studies using peximetinib and TLR4i (and combinations) revealed that p38 MAPK and TLR4-Myd88 signaling cooperatively regulate C/EBPβ, which in turn dictates Nlrp3 transcription. Cebpb silencing in J774M gMDSCs abolished Nlrp3 transcription, ASC assembly, and IL-1β secretion. ChIP-seq analysis showed strong occupancy of Cebpb on promoter regions of Nlrp3 upon inflammasome induction in gMDSCs. Finally, gMDSC-mediated CAF-Il6 induction in dual-reporter CAFs was significantly disrupted by co-culture with J774M-Cebpb KO cells, recapitulating the effect on iCAF mitigation demonstrated by co-cultures with J774M-Nlrp3 KO or gMDSCs pre-treated with SB-NL02. CONCLUSIONS: CXCR2-p38 and TLR4 signaling converge on C/EBPβ to regulate gMDSC-intrinsic NLRP3 inflammasomes, which drives iCAF polarization in PDAC. The role of C/EBPβ activation as a central node in imparting myeloid checkpoint function in gMDSCs warrants further exploration. Citation Format: Karthik Rajkumar, Andrew Adams, Haleh Amirian, Manan Patel, Erin Dickey, Harper Margaret Marsh, Siddharth Mehra, Nagaraj Nagathihalli, Nipun Merchant, Anna Bianchi, Jashodeep Datta. Convergent signaling via C/EBPβ regulates MDSC-intrinsic NLRP3 inflammasomes to drive inflammatory CAF polarization [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B050.

Human NLRP3 inflammasome activation leads to formation of condensate at the microtubule organizing center.

The NLRP3 inflammasome is a multi-protein molecular machine that mediates inflammatory responses in innate immunity. Its dysregulation has been linked to a large number of human diseases. Using cryogenic fluorescence-guided focused-ion-beam (cryo-FIB) milling and electron cryo-tomography (cryo-ET), we obtained 3-D images of the NLRP3 inflammasome in situ at various stages of its activation at macromolecular resolution. The cryo-tomograms unexpectedly reveal dense condensates of the human macrophage NLRP3 inflammasome that form within and around the microtubule organizing center (MTOC). We also find that following activation, the trans-Golgi network disperses and 50-nm NLRP3-associated vesicles appear which likely ferry NLRP3 to the MTOC. At later time points after activation, the electron-dense condensates progressively solidify and the cells undergo pyroptosis with widespread damaged mitochondria and autophagasomal structures.

Evaluation of the protective effects of chondroitin sulfate oligosaccharide against osteoarthritis via inactivation of NLRP3 inflammasome by in vivo and in vitro studies

Osteoarthritis (OA) is the most prevalent degenerative arthritis disease linked to aging, obesity, diet, and accumulation of octacalcium phosphate (OCP) crystals in joints. Current research has focused on inflammation and chondrocytes apoptosis as underlying OA mechanisms. Inflammatory cytokines like IL-1β activate matrix metalloproteinase-13 (MMP-13) and aggrecanase (the member of A Disintegrin and Metalloproteinase with Thrombospondin motifs family, ADAMTS), leading to cartilage matrix degradation. The NLRP3 inflammasome also contributes to OA pathogenesis by maturing IL-1β. Natural products like chondroitin sulfate oligosaccharides (oligo-CS) show promise in OA treatment by inhibiting inflammation. Our study evaluates the protective effects of oligo-CS against OA by targeting NLRP3 inflammation. Stimulating human SW1353 chondrocytes and human mononuclear macrophage THP-1 cells with OCP showed increased NLRP3 inflammation initiation, NF-κB pathway activation, and the production of inflammatory cytokines (IL-1β, IL-6) and the metabolic index (MMP-13, ADAMTS-5), leading to cartilage matrix degradation. However, oligo-CS treatment significantly reduced inflammation. In a 28-day in vivo study with C57BL/6 female mice, OCP was injected into their right knee and oligo-CS was orally administered. The OCP group exhibited significant joint space narrowing and chondrocyte loss, while the oligo-CS group maintained cartilage integrity. Oligo-CS groups also regulated gut microbiota composition to a healthier state. Taken together, our findings suggest that oligo-CS can be considered as a protective compound against OA.

Targeting the NLRP3 inflammasome in cochlear macrophages protects against hearing loss in chronic suppurative otitis media

The activation of the NLRP3 inflammasome has been linked to several inflammatory and autoinflammatory diseases. Despite cases of potential hearing improvement in immune-mediated diseases, direct evidence of the efficacy of targeting this mechanism in the inner ear is still lacking. Previously, we discovered that macrophages are associated with Sensorineural Hearing loss (SNHL) in Chronic Suppurative Otitis Media (CSOM), the leading cause of this permanent hearing loss in the developing world and incurring costs of $4 to $11 billion dollars in the United States. However, the underlying mechanism remained unknown. Here, we investigate how macrophages drive permanent hearing loss in CSOM. We first confirmed the occurrence of NLRP3 inflammasome activation in cochlear macrophages in CSOM. We then revealed that Outer Hair Cells (OHCs) were protected in CSOM by macrophage depletion and subsequently confirmed the same protection in the NLRP3 knockout condition. Furthermore, we showed that therapeutic inhibition of NLRP3 inflammasome activation and downstream inhibition of IL-1β protects OHCs in CSOM. Collectively, our data demonstrates that the main driver for hearing loss in CSOM is NLRP3 inflammasome activation in cochlear macrophages and this is therapeutically targetable, leading the way for the development of interventions to prevent the leading cause of permanent hearing loss and a costly disease in the developed world.

Protective effects of tetramethylpyrazine on myocardial ischemia/reperfusion injury involve NLRP3 inflammasome suppression by autophagy activation

Tetramethylpyrazine (TMP) belongs to the active ingredients of the traditional Chinese medicine Chuanxiong, which has a certain protective effect in myocardial ischemia–reperfusion (I/R) injury. It can improve postoperative cardiac function and alleviate ventricular remodeling in acute myocardial infarction patients. However, its specific protective mechanism is still unclear. In this study, a certain concentration of TMP was introduced into I/R mice or H9C2 cells after oxygen-glucose deprivation/reoxygenation (OGD/R) treatment to observe the effects of TMP on cardiomyocyte activity, cytotoxicity, apoptosis, autophagy, pyroptosis, and NLRP3 inflammasome activation. The results displayed that TMP intervention could reduce OGD/R and I/R-induced cardiomyocyte apoptosis, accelerate cellular activity and autophagy levels, and ameliorate myocardial tissue necrosis in I/R mice in a dose-dependent manner. Further, TMP prevented the formation of NLRP3 inflammasomes to suppress pyroptosis by increasing the level of cardiomyocyte autophagy after I/R and OGD/R modelling, the introduction of chloroquine to suppress autophagic activity in vivo and in vitro was further analyzed to confirm whether TMP inhibits NLRP3 inflammasome activation and pyroptosis by increasing autophagy, and we found the inhibitory effect of TMP on NLRP3 inflammasomes and its protective effect against myocardial injury were blocked when autophagy was inhibited with chloroquine. In conclusion, this experiment demonstrated that TMP unusually attenuated I/R injury in mice, and this protective effect was achieved by inhibiting the activation of NLRP3 inflammasomes through enhancing autophagic activity.