NLRP3 Activation Research Articles

Effect of fructose on anthracycline and HER-2 blocking agent mediated cardiotoxicity through NLRP3 and MyD88 mediated pathways: New roles of sweeteners in cardio-oncology.

e24020 Background: Combinatorial therapy based on anthracyclines and HER-2 blocking agents increases the risk of cardiomyopathy in women with breast cancer. Fructose is a monosaccharide associated with high risk of diabetes, non-alcoholic fatty liver disease, atherosclerosis and visceral obesity. A high fructose diet is a potential cardiometabolic risk factor and may increase cardiac risk in women with breast cancer treated with cardiotoxic drugs. In this study, we highlighted the role of sweeteners fructose in the worsening of cardiotoxicity induced by combinatorial therapy anthracycline-HER2-blocking agents. Methods: Human cardiomyocytes were pre-exposed with doxorubicin combined to trastuzumab (at 300 and 1000 nM) for 48 and 72h alone or combined to fructose at 0.25, 1 and 2.5 mM. After the incubation period, we performed the following tests: determination of cell viability, through mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of peroxisome proliferator-activated receptor-α; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6, CXCL-12). Results: High fructose increases significantly cell mortality of cardiomyocytes exposed to doxorubicin and trastuzumab through the activation of NLRP-3 and Myd-88 expression. Intracellular Ca++ levels were also increased of 4 times compared to non-fructose exposed cardiomyocytes; MDA and 4-HNA levels were increased of 48,3 and 51,2 % compared to non-fructose exposed cells (p<0.001). Fructose increases IL-1, IL-6 and CXCL-12 expression in cardiomyocytes in a concentration dependent manner, compared to doxorubicin-trastuzumab group (p<0.05). Conclusions: Data of the present study indicate that fructose induces a pro-inflammatory phenotype in human cardiac cells exposed to anticancer cardiotoxic drugs, providing even more data on the harmful role of fructose as a key player of cardiotoxic phenomena.

Combined Insults of a MASH Diet and Alcohol Binges Activate Intercellular Communication and Neutrophil Recruitment via the NLRP3-IL-1β Axis in the Liver.

Binge drinking in obese patients positively correlates with accelerated liver damage and liver-related death. However, the underlying mechanism and the effect of alcohol use on the progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) remain unexplored. Here, we show that short-term feeding of a metabolic-dysfunction-associated steatohepatitis (MASH) diet plus daily acute alcohol binges for three days induce liver injury and activation of the NLRP3 inflammasome. We identify that a MASH diet plus acute alcohol binges promote liver inflammation via increased infiltration of monocyte-derived macrophages, neutrophil recruitment, and NET release in the liver. Our results suggest that both monocyte-derived macrophages and neutrophils are activated via NLRP3, while the administration of MCC950, an NLRP3 inhibitor, dampens these effects.In this study, we reveal important intercellular communication between hepatocytes and neutrophils. We discover that the MASH diet plus alcohol induces IL-1β via NLRP3 activation and that IL-1β acts on hepatocytes and promotes the production of CXCL1 and LCN2. In turn, the increase in these neutrophils recruits chemokines and causes further infiltration and activation of neutrophils in the liver. In vivo administration of the NLRP3 inhibitor, MCC950, improves the early phase of MetALD by preventing liver damage, steatosis, inflammation, and immune cells recruitment.

Inflammasome functional activities in B lymphocytes.

Studies in animal models and human subjects have shown that, in addition to their implication in innate immunity, inflammasomes also can play a role in adaptive immunity. However, the contribution of the nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3) inflammasome pathway to adaptive immunity remains incompletely explored. Here, we show that NLRP3 plays an important role in different facets of B cell functions, including proliferation, antibody production, and secretion of inflammatory and anti-inflammatory cytokines. When exposed to B cell receptor engagement, Toll-like receptor activation, stimulation in conditions that mimic T cell-dependent responses, or NLRP3 activation, B cells manifest disparate responses and produce different cytokine patterns critical for modulating innate and adaptive immunity, indicating that the cytokines produced serve a critical link between the early innate immune response and the delayed adaptive immunity. Importantly, genetic ablation of nlrp3 reduced the inflammasome-mediated functions of B cells. We propose that, in the absence of other cell types, the potential of B lymphocytes to respond to NLRP3 engagement enables them to initiate inflammatory cascades through recruitment of other cell subsets, such as macrophages and neutrophils. Since NLRP3 activation of B cells is not followed by pyroptosis, even in the presence of a basal caspase-1 activity, this pathway acts as a bridge that optimizes interactions between the innate and adoptive branches of the immune response.

#984 Macrophage BRCC3 ameliorates sepsis-induced kidney injury by mediating renal NLRP3 inflammation

Abstract Background and Aims Sepsis-induced kidney injury (S-AKI) is an inflammatory disease characterized by high mortality rates. Macrophages are key inflammatory cells in the pathogenesis of S-AKI, and a rationale to target macrophage-endothelial cell interactions has been elucidated. Some study reported that BRCC3, a deubiquitylating enzyme, which is a critical regulator of NLRP3 activity in vitro experiments. However, the role of Macrophage BRCC3 role in S-AKI remains unclear. Method The S-AKI model was established by injecting lipopolysaccharide (LPS) into wild-type (WT) and macrophage-specific BRCC3 knockdown (KO-Mφ) mice. Transfecting RAW264.7 macrophages with siBRCC3 and establishing the co-culture system of RAW264.7 macrophages and murine tubular epithelial cells (mTECs) in Transwell. Results We found that macrophage BRCC3 deficiency ameliorates S-AKI. Compared with WT-S-AKI mice, the macrophage infiltration was decreased, the serum creatinine, BUN and the renal histologic injury scoring was decreased, as well as the expression of NLRP3 and Pro-Caspase-1 was clearly attenuated in KO-Mφ S-AKI mice. However, the serum IL-1β, IL-6, and TNF-α changes had on different between S-AKI groups. In vitro study, knockdown of BRCC3 in macrophages decreased NLRP3 inflammasome activation and cell apoptosis in mTECs. Mechanistically, BRCC3 knockdown promoted K63-linked ubiquitination of NLRP3 in macrophages. NLRP3 overexpression in mTEC abolished the renal protective effects of macrophages BRCC3 knockoff in S-AKI mice. Conclusion We showed that the macrophage BRCC3 as a key regulator in the activation of NLRP3 inflammasome by ubiquitination pathway in S-AKI. Our study unveils that macrophage BRCC3 has the potential to be the novel therapeutic target for S-AKI treatment.

Ganlu formula ethyl acetate extract (GLEE) blocked the development of experimental arthritis by inhibiting NLRP3 activation and reducing M1 type macrophage polarization

Ethnopharmacological relevanceThe Tibetan medicine Ganlu Formula, as a classic prescription, is widely used across the Qinghai-Tibet Plateau area of China, which has a significant effect on relieving the course of rheumatoid arthritis (RA). However, the active compounds and underlying mechanisms of Ganlu Formula in RA treatment remain largely unexplored. Aim of the studyThis study aimed to elucidate the active substances and potential mechanisms of the ethyl acetate extract of Ganlu Formula ethyl acetate extract (GLEE) in the treatment of RA. Materials and methodsUltra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was utilized to analyze and identify the chemical constituents within GLEE. Discovery Studio molecular virtual docking technology was utilized to dock the interaction of GLEE with inflammation-related pathway proteins. The GLEE gene library was obtained by transcriptome sequencing. Collagen-induced arthritic（CIA) rats were utilized to assess the antiarthritic efficacy of GLEE. Micro-CT imaging was employed to visualize the rat paw, and ultrasound imaging revealed knee joint effusion. Evaluation of synovial tissue pathological changes was conducted through hematoxylin-eosin staining and saffranine solid green staining, while immunohistochemical staining was employed to assess NLRP3 expression along with inflammatory markers. Immunofluorescence staining was utilized to identify M1 macrophages. ResultsMetabolomic analysis via UPLC-Q-TOF-MS identified 28 potentially bioactive compounds in GLEE, which interacted with the active sites of key proteins such as NLRP3, NF-κB, and STAT3 through hydrogen bonds, C–H bonds, and electrostatic attractions. In vitro analyses demonstrated that GLEE significantly attenuated NLRP3 inflammasome activation and inhibited the polarization of bone marrow-derived macrophages (BMDMs) towards the M1 phenotype. In vivo, GLEE not only prevented bone mineral density (BMD) loss but also reduced ankle swelling in CIA rats. Furthermore, it decreased the expression of the NLRP3 inflammasome and curtailed the release of inflammatory mediators within the knee joint. ConclusionGLEE effectively mitigated inflammatory responses in both blood and knee synovial membranes of CIA rats, potentially through the down-regulation of the NLRP3/Caspase-1/IL-1β signaling pathway and reduction in M1 macrophage polarization.

BRD4 promotes gouty arthritis through MDM2-mediated PPARγ degradation and pyroptosis

BackgroundGouty arthritis (GA) is characterized by monosodium urate (MSU) crystal accumulation that instigates NLRP3-mediated pyroptosis; however, the underlying regulatory mechanisms have yet to be fully elucidated. The present research endeavors to elucidate the regulatory mechanisms underpinning this MSU-induced pyroptotic cascade in GA.MethodsJ774 cells were exposed to lipopolysaccharide and MSU crystals to establish in vitro GA models, whereas C57BL/6 J male mice received MSU crystal injections to mimic in vivo GA conditions. Gene and protein expression levels were evaluated using real-time quantitative PCR, Western blotting, and immunohistochemical assays. Inflammatory markers were quantified via enzyme-linked immunosorbent assays. Pyroptosis was evaluated using immunofluorescence staining for caspase-1 and flow cytometry with caspase-1/propidium iodide staining. The interaction between MDM2 and PPARγ was analyzed through co-immunoprecipitation assays, whereas the interaction between BRD4 and the MDM2 promoter was examined using chromatin immunoprecipitation and dual-luciferase reporter assays. Mouse joint tissues were histopathologically evaluated using hematoxylin and eosin staining.ResultsIn GA, PPARγ was downregulated, whereas its overexpression mitigated NLRP3 inflammasome activation and pyroptosis. MDM2, which was upregulated in GA, destabilized PPARγ through the ubiquitin–proteasome degradation pathway, whereas its silencing attenuated NLRP3 activation by elevating PPARγ levels. Concurrently, BRD4 was elevated in GA and exacerbated NLRP3 activation and pyroptosis by transcriptionally upregulating MDM2, thereby promoting PPARγ degradation. In vivo experiments showed that BRD4 silencing ameliorated GA through this MDM2–PPARγ–pyroptosis axis.ConclusionBRD4 promotes inflammation and pyroptosis in GA through MDM2-mediated PPARγ degradation, underscoring the therapeutic potential of targeting this pathway in GA management.

Cigarette smoke impairs the hematopoietic supportive property of mesenchymal stem cells via the production of reactive oxygen species and NLRP3 activation

BackgroundMesenchymal stem cells (MSCs) play important roles in tissue homeostasis by providing a supportive microenvironmental niche for the hematopoietic system. Cigarette smoking induces systemic abnormalities, including an impeded recovery process after hematopoietic stem cell transplantation. However, the role of cigarette smoking-mediated alterations in MSC niche function have not been investigated.MethodsIn the present study, we investigated whether exposure to cigarette smoking extract (CSE) disrupts the hematopoietic niche function of MSCs, and pathways impacted. To investigate the effects on bone marrow (BM)-derived MSCs and support of hematopoietic stem and progenitor cells (HSPCs), mice were repeatedly infused with the CSE named 3R4F, and hematopoietic stem and progenitor cells (HSPCs) supporting function was determined. The impact of 3R4F on MSCs at cellular level were screened by bulk-RNA sequencing and subsequently validated through qRT-PCR. Specific inhibitors were treated to verify the ROS or NLRP3-specific effects, and the cells were then transplanted into the animal model or subjected to coculture with HSPCs.ResultsBoth direct ex vivo and systemic in vivo MSC exposure to 3R4F resulted in impaired engraftment in a humanized mouse model. Furthermore, transcriptomic profile analysis showed significantly upregulated signaling pathways related to reactive oxygen species (ROS), inflammation, and aging in 3R4F-treated MSCs. Notably, ingenuity pathway analysis revealed the activation of NLRP3 inflammasome signaling pathway in 3R4F-treated MSCs, and pretreatment with the NLRP3 inhibitor MCC950 rescued the HSPC-supporting ability of 3R4F-treated MSCs.ConclusionIn conclusion, these findings indicate that exposure to CSE reduces HSPCs supportive function of MSCs by inducing robust ROS production and subsequent NLRP3 activation.

Positive Effect of 6-Gingerol on Functional Plasticity of Microglia in a rat Model of LPS-induced Depression.

Neuroinflammation has emerged as a crucial factor in the development of depression. Despite the well-known anti-inflammatory properties of 6-gingerol, its potential impact on depression remains poorly understood. This study aimed to investigate the antidepressant effects of 6-gingerol by suppressing microglial activation. In vivo experiments were conducted to evaluate the effect of 6-gingerol on lipopolysaccharide (LPS)-induced behavioral changes and neuroinflammation in rat models. In vitro studies were performed to examine the neuroprotective properties of 6-gingerol against LPS-induced microglial activation. Furthermore, a co-culture system of microglia and neurons was established to assess the influence of 6-gingerol on the expression of synaptic-related proteins, namely synaptophysin (SYP) and postsynaptic density protein 95 (PSD95), which are influenced by microglial activation. In the in vivo experiments, administration of 6-gingerol effectively alleviated LPS-induced depressive behavior in rats. Moreover, it markedly suppressed the activation of rat prefrontal cortex (PFC) microglia induced by LPS and the activation of the NF-κB/NLRP3 inflammatory pathway, while also reducing the levels of inflammatory cytokines IL-1β and IL-18. In the in vitro experiments, 6-gingerol mitigated nuclear translocation of NF-κB p65, NLRP3 activation, and maturation of IL-1β and IL-18, all of which were induced by LPS. Furthermore, in the co-culture system of microglia and neurons, 6-gingerol effectively restored the decreased expression of SYP and PSD95. The findings of this study demonstrate the neuroprotective effects of 6-gingerol in the context of LPS-induced depression-like behavior. These effects are attributed to the inhibition of microglial hyperactivation through the suppression of the NF-κB/NLRP3 inflammatory pathway.

Molecular regulation of NLRP3 inflammasome activation during parasitic infection.

Parasitic diseases are a serious global health concern, causing many common and severe infections, including Chagas disease, leishmaniasis, and schistosomiasis. The NLRP3 inflammasome belongs to the NLR (nucleotide-binding domain leucine-rich-repeat-containing proteins) family, which are cytosolic proteins playing key roles in the detection of pathogens. NLRP3 inflammasomes are activated in immune responses to Plasmodium, Leishmania, Toxoplasma gondii, Entamoeba histolytica, Trypanosoma cruzi, and other parasites. The role of NLRP3 is not fully understood, but it is a crucial component of the innate immune response to parasitic infections and its functions as a sensor triggering the inflammatory response to the invasive parasites. However, while this response can limit the parasites' growth, it can also result in potentially catastrophic host pathology. This makes it essential to understand how NLRP3 interacts with parasites to initiate the inflammatory response. Plasmodium hemozoin, Leishmania glycoconjugate lipophosphoglycan (LPG) and E. histolytica Gal/GalNAc lectin can stimulate NLRP3 activation, while the dense granule protein 9 (GRA9) of T. gondii has been shown to suppress it. Several other parasitic products also have diverse effects on NLRP3 activation. Understanding the mechanism of NLRP3 interaction with these products will help to develop advanced therapeutic approaches to treat parasitic diseases. This review summarizes current knowledge of the NLRP3 inflammasome's action on the immune response to parasitic infections and aims to determine the mechanisms through which parasitic molecules either activate or inhibit its action.

Extracellular histones promote TWIK2-dependent potassium efflux and associated NLRP3 activation in alveolar macrophages during sepsis-induced lung injury.

Sepsis-induced acute lung injury (ALI) is a complex and life-threatening condition lacking specific and efficient clinical treatments. Extracellular histones, identified as a novel type of damage-associated molecular patterns, have been implicated in the inflammatory process of ALI. However, further elucidation is needed regarding the precise mechanism through which extracellular histones induce inflammation. The aim of this study was to investigate whether extracellular histones can activate NLRP3 inflammasome-mediated inflammation in alveolar macrophages (AMs) by affecting TWIK2-dependent potassium efflux. We conducted experiments using cecal ligation and puncture (CLP) C57BL/6 mice and extracellular histone-stimulated LPS-primed MH-Scells. The results demonstrated a significant increase in the levels of extracellular histones in the plasma and bronchoalveolar lavage fluid (BALF) of CLP mice. Furthermore, neutralizing extracellular histone mitigated lung injury and inflammation in CLP-induced ALI mice. In vitro studies confirmed that extracellular histones upregulated the expression of NLRP3 inflammasome activation-related proteins in MH-S cells, and this effect was dependent on increased potassium efflux mediated by the TWIK2 channel on the plasma membrane. Moreover, extracellular histones directly triggered a substantial influx of calcium, leading to increased Rab11 activity and facilitating the trafficking and location of TWIK2 to the plasma membrane. These findings underscore the critical role of extracellular histone-induced upregulation of TWIK2 expression on the plasma membrane of alveolar macrophages (AMs). This upregulation leads to potassium efflux and subsequent activation of the NLRP3 inflammasome, ultimately exacerbating lung inflammation and injury during sepsis.

Modified Danzhi XiaoyaoSan inhibits neuroinflammation via regulating TRIM31/NLRP3 inflammasome in the treatment of CUMS depression

The NLRP3 inflammasome is critically involved in the development of depression. The E3 ubiquitin ligase TRIM31 negatively regulates this process by promoting the degradation of NLRP3 through the ubiquitin-proteasome pathway. Modified Danzhi Xiaoyaosan (MDZXYS) has shown good therapeutic effect in both preclinical and clinical depression treatments, yet the underlying mechanisms of its antidepressant effects are not fully understood. In the present study, we aimed to explore the antidepressant mechanisms of MDZXYS, focusing on NLRP3 activation and ubiquitin-mediated degradation. We employed rats with depression induced by chronic unpredictable mild stress (CUMS) and conducted various behavioral tests, including the sucrose preference, forced swimming, and open field tests. Neuronal damage in CUMS-treated rats was assessed using Nissl staining. We measured proinflammatory cytokine levels using ELISA kits and analyzed NLRP3/TRIM31 protein expression via Western blotting and immunofluorescence staining. Our results disclosed that MDZXYS reversed CUMS-induced depression-like behaviors in rats, reduced proinflammatory cytokine levels (IL-1β), and ameliorated neuronal damage in the prefrontal cortex. Additionally, CUMS activated the NLRP3 inflammasome in the prefrontal cortex and upregulated the protein expression of TRIM31. After MDZXYS administration, the expression of NLRP3 inflammasome-associated proteins was reduced, while the expression level of TRIM31 was further increased. Through co-localized immunofluorescence staining, we observed a significant elevation in the co-localization expression of NLRP3 and TRIM31 in the prefrontal cortex of the MDZXYS group. These findings suggest that inhibiting NLRP3 inflammasome-mediated neuroinflammation by modulating the TRIM31signaling pathway may underlie the antidepressant effects of MDZXYS, and further support targeting NLRP3 as a novel approach for the prevention and treatment of depression.

Sulforaphane decreases oxidative stress and inhibits NLRP3 inflammasome activation in a mouse model of ulcerative colitis

Excessive oxidative stress and NLRP3 inflammasome activation are considered the main drivers of inflammatory bowel disease (IBD), and inhibition of inflammasomes ameliorates clinical symptoms and morphological manifestations of IBD. Herein, we examined the roles of NLRP3 activation in IBD and modulation of NLRP3 by sulforaphane (SFN), a compound with multiple pharmacological activities that is extracted from cruciferous plants. To simulate human IBD, we established a mouse colitis model by administering dextran sodium sulfate in the drinking water. SFN (25, 50 mg·kg−1·d−1, ig) or the positive control sulfasalazine (500 mg/kg, ig) was administered to colitis-affected mice for 7 days. Model mice displayed pathological alterations in colon tissue as well as classic symptoms of colitis beyond substantial tissue inflammation. Expression of NLRP3, ASC, and caspase-1 was significantly elevated in the colonic epithelium. The expression of NLRP3 inflammasomes led to activation of downstream proteins and increases in the cytokines IL-18 and IL-1β. SFN administration either fully or partially reversed these changes, thus restoring IL-18 and IL-1β, substantially inhibiting NLRP3 activation, and decreasing inflammation. SFN alleviated the inflammation induced by LPS and NLRP3 agonists in RAW264.7 cells by decreasing the levels of reactive oxygen species. In summary, our results revealed the pathological roles of oxidative stress and NLRP3 in colitis, and indicated that SFN might serve as a natural NLRP3 inhibitor, thereby providing a new strategy for alternative colitis treatment.

Coarse particulate matter (PM10) induce an inflammatory response through the NLRP3 activation

IntroductionPM exposure can induce inflammatory and oxidative responses; however, differences in these adverse effects have been reported depending on the chemical composition and size. Moreover, inflammatory mechanisms such as NLRP3 activation by PM10 have yet to be explored.ObjectiveTo assess the impact of PM10 on cell cytotoxicity and the inflammatory response through in vitro and in vivo models.MethodologyPeripheral blood mononuclear cells (PBMCs) from healthy donors were exposed to PM10. Cytotoxicity was determined using the LDH assay; the expression of inflammasome components and the production of pro-inflammatory cytokines were quantified through qPCR and ELISA, respectively; and the formation of ASC complexes was examined using confocal microscopy. For in vivo analysis, male C57BL6 mice were intranasally challenged with PM10 and bronchoalveolar lavage fluid was collected to determine cell counts and quantification of pro-inflammatory cytokines by ELISA. RNA was extracted from lung tissue, and the gene expression of inflammatory mediators was quantified.ResultsPM10 exposure induced significant cytotoxicity at concentrations over 100 µg/mL. Moreover, PM10 enhances the gene expression and release of pro-inflammatory cytokines in PBMCs, particularly IL-1β; and induces the formation of ASC complexes in a dose-dependent manner. In vivo, PM10 exposure led to cell recruitment to the lungs, which was characterized by a significant increase in polymorphonuclear cells compared to control animals. Furthermore, PM10 induces the expression of several inflammatory response-related genes, such as NLRP3, IL-1β and IL-18, within lung tissue.ConclusionBriefly, PM10 exposure reduced the viability of primary cells and triggered an inflammatory response, involving NLRP3 inflammasome activation and the subsequent production of IL-1β. Moreover, PM10 induces the recruitment of cells to the lung and the expression of multiple cytokines; this phenomenon could contribute to epithelial damage and, thus to the development and exacerbation of respiratory diseases such as viral infections.

NLRP3 Cys126 palmitoylation by ZDHHC7 Promotes Inflammasome Activation

Abstract Macrophages could response to pathogen and damage-associated molecular patterns through NLRP3-mediated inflammasome. Hyperactivation of NLRP3 is associated with human chronic inflammatory diseases. Therefore, accurate regulation of NLRP3 activation is important. Recent works show that NLRP3 is localized on trans-Golgi network (TGN), which is crucial for NLRP3 activation and consequently ASC recruitment. However, what regulates NLRP3 TGN localization is unknown. Here, we demonstrated that NLRP3 is palmitoylated on Cys126 by palmitoyl-acyltransferase ZDHHC7 in macrophages, which promotes resting NLRP3 localization to TGN and activated NLRP3 localization on dispersed TGN. As a result, ZDHHC7-mediated NLRP3 Cys126 palmitoylation is critical for recruitment and oligomerization of ASC after NLRP3 activation. Perturbating NLRP3 palmitoylation by ZDHHC7 knockout, pharmaceutically inhibition, or modification site mutation, will diminish NLRP3 activation and consequential Caspase-1/GSDMD cleavage, and cytokines secretion in macrophages. Furthermore, NLRP3 palmitoylation on Cys126 is vital for inflammasome activation in vivo, including endotoxic shock and peritonitis mouse models. Notably, this palmitoylation-regulated NLRP3 activation also contributes to multiple sclerosis development in mice. Therefore, our study provides new insight into NLRP3 regulation and suggests targeting ZDHHC7 or NLRP3 Cys126 could be a potential therapeutic strategy to treat human NLRP3-related disorders.

Abstract 3039: TOMM40 Knockdown In Macrophages Inhibits Oxidized LDL-induced NLRP3 Activation And Promotes Reverse Cholesterol Transport

Atherogenesis has been shown to be promoted by activation of the NLRP3 inflammasome, a cytosolic innate immune sensor activated by a broad range of factors, including oxidized LDL (oxLDL). Our group has shown that hepatocyte knockdown of Translocase of Outer Mitochondrial Membrane 40 ( TOMM40), which encodes a key component of the mitochondrial importer TOM complex, results in increased levels of oxysterols that activate the transcription factors LXRα and β. As LXR agonists have been shown to inhibit NLRP3 activation, we tested whether TOMM40 knockdown reduces NLRP3 activation in THP-1 monocyte-derived macrophages. On stimulating these cells with both the NLRP3 activator Nigericin and oxLDL following TOMM40 knockdown by siRNA, we found that pro-inflammatory cytokine release and NLRP3 activation were significantly inhibited (Two-way ANOVA, p<0.0001, n=6). We further showed that TOMM40 knockdown resulted in significant LXRβ upregulation (unpaired t-test, p=0.007, n=3), and reduced oxLDL-induced upregulation of both NLRP3 and CASP1 , which encodes a protein recruited in NLRP3 activation (One-way ANOVA, p=0.0007, n=3). Moreover, pre-treatment with an LXR antagonist, GSK2033, abrogated TOMM40 knockdown-induced NLRP3 inhibition, supporting our hypothesis that this inhibition is likely LXR dependent. Since LXRs are known to promote cholesterol efflux from macrophages, we also examined the effect of TOMM40 knockdown on expression of the cholesterol transporter genes, ABCA1, ABCG1, and SR-B1 , and found that ABCG1 and SR-B1 were significantly upregulated (unpaired t-test, p=0.02 and p=0.003 respectively, n=3). Taken together, these findings identify mechanisms by which TOMM40 knockdown exerts anti-inflammatory effects and promotes reverse cholesterol transport. While TOMM40 has been studied largely in relation to APOE and neurodegenerative disease due to its genomic location, we identify a novel potential impact of TOMM40 on atherogenesis.

Therapeutic potential of VBY-825 in MSU crystal-induced NLRP3 inflammasome activation

Abstract In gout patients, MSU crystals formed in the joints cause inflammation, and the activation of the NLRP3 inflammasome is believed to mediate these responses. Cathepsins play an important role in crystal-induced NLRP3 activation that results in subsequent speck formation, caspase-1 activation, IL-1β secretion, and pyroptosis. In this study, we investigated if cathepsins can also influence MSU-induced NLRP3 activation in a gout mouse model. VBY-825 is a reversible pan-cathepsin inhibitor, and we found that VBY-825 significantly suppressed IL-1β secretion and LDH levels from peritoneal naïve macrophages upon MSU stimulation in vito. In a mouse peritonitis model, we found that VBY-825 suppressed MSU-induced inflammation when compared to the control treatment. Interestingly, the inflammation demonstrated by histology and MPO activity in the joints was also reduced by VBY-825 treatment in MSU-induced arthritis. These findings suggest that cathepsins play a critical role in MSU-induced inflammation and could potentially be a target for gout treatment.

Loss of myeloid-KLF2 mediates LPS-induced mortality in neonatal pups by NLRP3 inflammasome signaling and altered neutrophil aging

Abstract Preterm neonates have disrupted myeloid cell activation and aging. KLF2 inhibits myeloid cell activation by repressing NF-kB, which controls NLRP3 transcription. In our myeloid-KLF2 deleted (MKO) murine model, postnatal day (P) 4 pups have higher LPS-induced mortality than controls but 100% survival in both groups after LPS at P12. P4 and P12 reflect preterm and term physiology, respectively. We aimed to to determine the role of neutrophil-NLRP3 and neutrophil aging in LPS-induced mortality in P4 MKO pups. Serum IL-1β was measured 4h post-LPS injection. BM neutrophils were treated with LPS and nigericin for NLRP3 protein expression in lysate and IL-1b levels in the supernatant. Daily MCC950 injections (P1-P4) were used to inhibit NLRP3 before LPS injection on P4. BM neutrophils were treated with LPS ex vivo and flow-sorted to examine CXCR4/CD62L expression. MKO pups have increased serum IL-1β, neutrophil-NLRP3 expression, and IL-1b release, P4>P12. In vivo NLRP3 inhibition rescued survival in P4 MKO and controls. P4 MKO have reduced BM and higher circulating neutrophils. MKO BM neutrophils are more aged (CD62Llo), but CXCR4lo, affecting aged neutrophil homing to the BM. Myeloid-KLF2 loss increases LPS mortality via NLRP3 activation in P4 pups. It accelerates neutrophil aging, but leads to persistence of aged neutrophils in the circulation by reducing CXCR4 expression. Future mechanistic studies will focus on KLF2's regulation of NLRP3 and CXCR4 expression at P4 and P12.

Abstract 108: Inflammasome Activation Via BRCC3-mediated NLRP3 Deubiquitylation Promotes Atherosclerosis In Tet2 Clonal Hematopoiesis

Clonal hematopoiesis (CH) has emerged as an independent risk factor for atherosclerotic cardiovascular disease (CVD) with activation of macrophage inflammasomes as a potential underlying mechanism. The NLRP3 inflammasome has a key role in promoting atherosclerosis in mouse models of Tet2 CH, while inhibition of the inflammasome product IL-1β appeared to particularly benefit patients with TET2 CH in CANTOS. TET2 is an epigenetic modifier that decreases promoter methylation. However, the mechanisms underlying macrophage NLRP3 inflammasome activation in TET2 deficiency and potential links with epigenetic modifications are poorly understood. We used cholesterol-loaded TET2 deficient murine and embryonic stem cell derived isogenic human macrophages to evaluate mechanisms of NLRP3 inflammasome activation in vitro and hypercholesterolemic Ldlr -/- mice modeling TET2 CH to assess the role of NLRP3 inflammasome activation in atherosclerosis. Tet2 deficiency acted synergistically with cholesterol loading in cell culture and with hypercholesterolemia in vivo to increase JNK1 phosphorylation and NLRP3 inflammasome activation. The mechanism of JNK activation in TET2 deficiency was increased promoter methylation and decreased expression of the JNK-inactivating dual specificity phosphatase, Dusp10 . Active Tet1-deadCas9 targeted editing of Dusp10 promoter methylation abolished cholesterol-induced inflammasome activation in Tet2 -deficient macrophages. Increased JNK1 signaling led to NLRP3 deubiquitylation and activation by the deubiquitinase BRCC3. Accelerated atherosclerosis and NETosis in Tet2 CH mice were reversed by holomycin, a BRCC3 deubiquitinase inhibitor, and also by hematopoietic deficiency of Abro1 , an essential scaffolding protein in the BRCC3-containing cytosolic complex. Hypercholesterolemia and TET2 deficiency converge on a common pathway of NLRP3 inflammasome activation mediated by JNK1 activation and BRCC3-mediated NLRP3 deubiquitylation with potential therapeutic implications for the prevention of CVD in TET2 CH.

Antioxidant and anti-Inflammatory effects of maltol on inflammasome activation in macrophages and mice

Abstract Maltol (3-hydroxy-2-methyl-4-pyrone) is used widely as a food and cosmetic supplement, and it has antioxidant and anti-inflammatory activities. Inflammasome causes the maturation and secretion of interleukin (IL)-1β and -18 through activation of caspase-1 (Casp1), which contributes to various inflammatory diseases. This study examined the effects of maltol on inflammasome activation in macrophages and mice. Lipopolysaccharide (LPS)-primed macrophages were treated with a trigger of NLRP3, NLRC4, AIM2, or non-canonical (NC) inflammasomes in the presence of maltol. The secretion of IL-1β and IL-18, and cleavage of Casp1 were analyzed as indices of inflammasome activation. Mice were injected with LPS and an NLRP3 trigger with or without maltol, and the peritoneal IL-1β secretions were observed. The effects of maltol on reactive oxygen species (ROS) production and Casp1 activity were analyzed to determine the mechanism. Maltol inhibited the activation of NLRP3 and NC inflammasomes, but it did not alter the other inflammasomes. Maltol also attenuated IL-1β secretion resulting from the inflammasome activation in mice. The anti-inflammatory mechanism of maltol was revealed by the inhibition of ROS production and Casp1 activity. Maltol is suggested as a candidate of anti-inflammasome molecules.

Role of NLRP3 Inflammasomes in Monocyte and Microglial Recruitments in Choroidal Neovascularization.

Although the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice and immunostaining techniques to confirm localization of NLRP3 inflammasomes in the laser-induced CNV (LCNV) lesions. Confocal microscopy was used to image and quantify LCNV volumes. MCC950 was used as NLRP3 inhibitor. ELISA and quantitative RT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1β protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that red fluorescent protein (RFP)-positive monocyte-derived macrophages and GFP-positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP-positive macrophages, Cx3cr1GFP-positive microglia, and other cells, resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice showed significantly increased lesion size compared with age-matched controls. Inhibition of NLRP3 resulted in decreased IL-1β mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1β.