Immunity against Moraxella catarrhalis requires guanylate-binding proteins and caspase-11-NLRP3 inflammasomes.

Moraxella catarrhalis is an important human respiratory pathogen and a major causative agent of otitis media and chronic obstructive pulmonary disease. Toll-like receptors contribute to, but cannot fully account for, the complexity of the immune response seen in M. catarrhalis infection. Using primary mouse bone marrow-derived macrophages to examine the host response to M. catarrhalis infection, our global transcriptomic and targeted cytokine analyses revealed activation of immune signalling pathways by both membrane-bound and cytosolic pattern-recognition receptors. We show that M. catarrhalis and its outer membrane vesicles or lipooligosaccharide (LOS) can activate the cytosolic innate immune sensor caspase-4/11, gasdermin-D-dependent pyroptosis, and the NLRP3 inflammasome in human and mouse macrophages. This pathway is initiated by type I interferon signalling and guanylate-binding proteins (GBPs). We also show that inflammasomes and GBPs, particularly GBP2, are required for the host defence against M. catarrhalis in mice. Overall, our results reveal an essential role for the interferon-inflammasome axis in cytosolic recognition and immunity against M. catarrhalis, providing new molecular targets that may be used to mitigate pathological inflammation triggered by this pathogen.

NLRP3 is its own gatekeeper: a group hug of NLRP3 monomers controls inflammation.

A recent study by Hochheiser et al. describes the cryo-electron microscopy (cryoEM) structure of an autoinhibited nucleotide-binding domain-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3 (NLRP3) decamer that assembles via LRR interactions and is further stabilized by the small-molecule NLRP3-specific inhibitor CRID3 binding into a cleft within the NACHT domain. The study provides a springboard for the development of novel NLRP3-based therapies.

Inhibition of NLRP3 inflammasome by MCC950 under hypoxia alleviates photoreceptor apoptosis via inducing autophagy in Müller glia.

NLRP3 inflammasome activation has emerged as a critical initiator of inflammatory response in ischemic retinopathy. Here, we identified the effect of a potent, selective NLRP3 inhibitor, MCC950, on autophagy and apoptosis under hypoxia. Neonatal mice were exposed to hyperoxia for 5 days to establish oxygen-induced retinopathy (OIR) model. Intravitreal injection of MCC950 was given, and then autophagy and apoptosis markers were assessed. Retinal autophagy, apoptosis, and related pathways were evaluated by western blot, immunofluorescent labeling, transmission electron microscopy, and TUNEL assay. Autophagic activity in Müller glia after NLRP3 inflammasome inhibition, together with its influence on photoreceptor death, was studied using western blot, immunofluorescence staining, mRFP-GFP-LC3 adenovirus transfection, cell viability, proliferation, and apoptosis assays. Results showed that activation of NLRP3 inflammasome in Müller glia was detected in OIR model. MCC950 could improve impaired retinal autophagic flux and attenuate retinal apoptosis while it regulated the retinal AMPK/mTOR/ULK-1 pathway. Suppressed autophagy and depressed proliferation capacity resulting from hypoxia was promoted after MCC950 treatment in Müller glia. Inhibition of AMPK and ULK-1 pathway significantly interfered with the MCC950-induced autophagy activity, indicating MCC950 positively modulated autophagy through AMPK/mTOR/ULK-1 pathway in Müller cells. Furthermore, blockage of autophagy in Müller glia significantly induced apoptosis in the cocultured 661W photoreceptor cells, whereas MCC950 markedly preserved the density of photoreceptor cells. These findings substantiated the therapeutic potential of MCC950 against impaired autophagy and subsequent apoptosis under hypoxia. Such protective effect might involve the modulation of AMPK/mTOR/ULK-1 pathway. Targeting NLRP3 inflammasome in Müller glia could be beneficial for photoreceptor survival under hypoxic conditions.

Prevention of the foreign body response to implantable medical devices by inflammasome inhibition.

SignificanceImplantable electronic medical devices (IEMDs) are used for some clinical applications, representing an exciting prospect for the transformative treatment of intractable conditions such Parkinson's disease, deafness, and paralysis. The use of IEMDs is limited at the moment because, over time, a foreign body reaction (FBR) develops at the device-neural interface such that ultimately the IEMD fails and needs to be removed. Here, we show that macrophage nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activity drives the FBR in a nerve injury model yet integration of an NLRP3 inhibitor into the device prevents FBR while allowing full healing of damaged neural tissue to occur.

MCC950 reduces autophagy and improves cognitive function by inhibiting NLRP3-dependent neuroinflammation in a rat model of Alzheimer's disease.

Alzheimer's disease (AD) is the seventh most common cause of mortality and one of the major causes of disability and vulnerability in the elderly. AD is characterized by gradual cognitive deterioration, the buildup of misfolded amyloid beta (Aß) peptide, and the generation of neurofibrillary tangles. Despite enormous scientific progress, there is no effective cure for AD. Thus, exploring new treatment options to stop AD or at least slow down its progress is important. In this study, we investigated the potential therapeutic effects of MCC950 on NLRP3-mediated inflammasome-driven inflammation and autophagy in AD. Rats treated with streptozotocin (STZ) exhibited simultaneous activation of the NLRP3 inflammasome and autophagy, as confirmed by Western blot, immunofluorescence, and co-immunoprecipitation analyses. MCC950, a specific NLRP3 inhibitor, was intraperitoneally administered (50 mg/kg body weight) to rats with AD-like symptoms induced by intracerebroventricular STZ injections (3 mg/kg body weight). MCC950 effectively suppressed STZ-induced cognitive impairment and anxiety by inhibiting NLRP3-dependent neuroinflammation. Moreover, our findings indicate that MCC950 exerts neuroprotective effects by attenuating autophagy in neuronal cells. The inhibiting effects of MCC950 on inflammasome activation and autophagy were reproduced in vitro, provding further mechansistic insights into MCC950 therapeutic action. Our findings suggest that MCC950 impedes the progression of AD and may also improve cognitive function through the mitigation of autophagy and NLRP3 inflammasome inhibition.

Blockage of the NLRP3 inflammasome by MCC950 inhibits migration and invasion in adenomyosis.

RESEARCH QUESTION: Does the NOD-like receptor protein 3 (NLRP3) inflammasome have an effect in adenomyosis? DESIGN: Fresh-frozen endometrial tissues and paraffin specimens were obtained from endometrial tissues from patients with adenomyosis and controls. Western blot, quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were applied to assess expression of the NLRP3 inflammasome components. Primary eutopic endometrial stromal cells were isolated from the uteri of patients with adenomyosis. After NLRP3 was knocked down using small interfering RNA, proliferation, invasion and epithelial-mesenchymal transition (EMT) were evaluated using EdU, CCK8, transwell assays and western blot. Importantly, a mouse model of adenomyosis was established to evaluate the effects of the NLRP3 inhibitor MCC950 on the formation of adenomyosis. RESULTS: Expression of the NLRP3 inflammasome components was elevated in the ectopic or eutopic endometrium of patients with adenomyosis. NLRP3 knockdown inhibited migration, invasion and EMT in endometrial cells and primary endometrial cells (P < 0.0001). MCC950, which blocks the NLRP3 inflammasome, reduced migration and invasion of endometrial cells (P < 0.01) and primary endometrial cells (P < 0.0001) considerably. Importantly, in the mouse model of adenomyosis, MCC950 had a mitigating effect on the severity of adenomyosis (P < 0.01). CONCLUSIONS: NLRP3 was found to enhance migration, invasion and EMT of human endometrial cells in adenomyosis. Notably, the NLRP3 inhibitor MCC950 reduced migration and invasion of endometrial cells effectively. Furthermore, in the mouse model of adenomyosis, MCC950 exhibited a therapeutic effect by alleviating the severity of adenomyosis.

The bioenergetics of traumatic brain injury and its long-term impact for brain plasticity and function.

Mitochondria provide the energy to keep cells alive and functioning and they have the capacity to influence highly complex molecular events. Mitochondria are essential to maintain cellular energy homeostasis that determines the course of neurological disorders, including traumatic brain injury (TBI). Various aspects of mitochondria metabolism such as autophagy can have long-term consequences for brain function and plasticity. In turn, mitochondria bioenergetics can impinge on molecular events associated with epigenetic modifications of DNA, which can extend cellular memory for a long time. Mitochondrial dysfunction leads to pathological manifestations such as oxidative stress, inflammation, and calcium imbalance that threaten brain plasticity and function. Hence, targeting mitochondrial function may have great potential to lessen the outcomes of TBI.

MCC950 as a promising candidate for blocking NLRP3 inflammasome activation: A review of preclinical research and future directions.

The NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome is a key component of the innate immune system that triggers inflammation and pyroptosis and contributes to the development of several diseases. Therefore, blocking the activation of the NLRP3 inflammasome has therapeutic potential for the treatment of these diseases. MCC950, a selective small molecule inhibitor, has emerged as a promising candidate for blocking NLRP3 inflammasome activation. Ongoing research is focused on elucidating the specific targets of MCC950 as well as assessfing its metabolism and safety profile. This review discusses the diseases that have been studied in relation to MCC950, with a focus on stroke, Alzheimer's disease, liver injury, atherosclerosis, diabetes mellitus, and sepsis, using bibliometric analysis. It then summarizes the potential pharmacological targets of MCC950 and discusses its toxicity. Furthermore, it traces the progression from preclinical to clinical research for the treatment of these diseases. Overall, this review provides a solid foundation for the clinical therapeutic potential of MCC950 and offers insights for future research and therapeutic approaches.

MCC950 Ameliorates Diabetic Muscle Atrophy in Mice by Inhibition of Pyroptosis and Its Synergistic Effect with Aerobic Exercise.

Diabetic muscle atrophy is an inflammation-related complication of type-2 diabetes mellitus (T2DM). Even though regular exercise prevents further deterioration of atrophic status, there is no effective mediator available for treatment and the underlying cellular mechanisms are less explored. In this study, we investigated the therapeutic potential of MCC950, a specific, small-molecule inhibitor of NLRP3, to treat pyroptosis and diabetic muscle atrophy in mice. Furthermore, we used MCC950 to intervene in the protective effects of aerobic exercise against muscle atrophy in diabetic mice. Blood and gastrocnemius muscle (GAS) samples were collected after 12 weeks of intervention and the atrophic state was assessed. We initially corroborated a diabetic muscle atrophy phenotype in db/db mice (D) by comparison with control m/m mice (W) by examining parameters such as fasting blood glucose (D vs. W: 24.47 ± 0.45 mmol L-1 vs. 4.26 ± 0.6 mmol L-1, p < 0.05), grip strength (D vs. W: 166.87 ± 15.19 g vs. 191.76 ± 14.13 g, p < 0.05), exercise time (D vs. W: 1082.38 ± 104.67 s vs. 1716 ± 168.55 s, p < 0.05) and exercise speed to exhaustion (D vs. W: 24.25 ± 2.12 m min-1 vs. 34.75 ± 2.66 m min-1, p < 0.05), GAS wet weight (D vs. W: 0.07 ± 0.01 g vs. 0.13 ± 0.01 g, p < 0.05), the ratio of GAS wet weight to body weight (D vs. W: 0.18 ± 0.01% vs. 0.54 ± 0.02%, p < 0.05), and muscle fiber cross-sectional area (FCSA) (D vs. W: 1875 ± 368.19 µm2 vs. 2747.83 ± 406.44 µm2, p < 0.05). We found that both MCC950 (10 mg kg-1) treatment and exercise improved the atrophic parameters that had deteriorated in the db/db mice, inhibited serum inflammatory markers and significantly attenuated pyroptosis in atrophic GAS. In addition, a combined MCC950 treatment with exercise (DEI) exhibited a further improvement in glucose uptake capacity and muscle performance. This combined treatment also improved the FCSA of GAS muscle indicated by Laminin immunofluorescence compared to the group with the inhibitor treatment alone (DI) (DEI vs. DI: 2597 ± 310.97 vs. 1974.67 ± 326.15 µm2, p < 0.05) or exercise only (DE) (DEI vs. DE: 2597 ± 310.97 vs. 2006.33 ± 263.468 µm2, p < 0.05). Intriguingly, the combination of MCC950 treatment and exercise significantly reduced NLRP3-mediated inflammatory factors such as cleaved-Caspase-1, GSDMD-N and prevented apoptosis and pyroptosis in atrophic GAS. These findings for the first time demonstrate that targeting NLRP3-mediated pyroptosis with MCC950 improves diabetic muscle homeostasis and muscle function. We also report that inhibiting pyroptosis by MCC950 can enhance the beneficial effects of aerobic exercise on diabetic muscle atrophy. Since T2DM and muscle atrophy are age-related diseases, the young mice used in the current study do not seem to fully reflect the characteristics of diabetic muscle atrophy. Considering the fragile nature of db/db mice and for the complete implementation of the exercise intervention, we used relatively young db/db mice and the atrophic state in the mice was thoroughly confirmed. Taken together, the current study comprehensively investigated the therapeutic effect of NLRP3-mediated pyroptosis inhibited by MCC950 on diabetic muscle mass, strength and exercise performance, as well as the synergistic effects of MCC950 and exercise intervention, therefore providing a novel strategy for the treatment of the disease.

Inflammasome inhibition protects dopaminergic neurons from α-synuclein pathology in a model of progressive Parkinson's disease.

Neuroinflammation has been suggested as a pathogenetic mechanism contributing to Parkinson's disease (PD). However, anti-inflammatory treatment strategies have not yet been established as a therapeutic option for PD patients. We have used a human α-synuclein mouse model of progressive PD to examine the anti-inflammatory and neuroprotective effects of inflammasome inhibition on dopaminergic (DA) neurons in the substantia nigra (SN). As the NLRP3 (NOD-, LRR- and pyrin domain-containing 3)-inflammasome is a core interface for both adaptive and innate inflammation and is also highly druggable, we investigated the implications of its inhibition. Repeat administration of MCC950, an inhibitor of NLRP3, in a PD model with ongoing pathology reduced CD4+ and CD8+ T cell infiltration into the SN. Furthermore, the anti-inflammasome treatment mitigated microglial activation and modified the aggregation of α-synuclein protein in DA neurons. MCC950-treated mice showed significantly less neurodegeneration of DA neurons and a reduction in PD-related motor behavior. In summary, early inflammasome inhibition can reduce neuroinflammation and prevent DA cell death in an α-synuclein mouse model for progressive PD.

Synthesis and Evaluation of [11C]MCC950 for Imaging NLRP3-Mediated Inflammation in Atherosclerosis.

Overexpression of the NLRP3 inflammasome has been attributed to the progressive worsening of a multitude of cardiovascular inflammatory diseases such as myocardial infarction, pulmonary arterial hypertension, and atherosclerosis. The recently discovered potent and selective NLRP3 inhibitor MCC950 has shown promise in hindering disease progression, but NLRP3-selective cardiovascular positron emission tomography (PET) imaging has not yet been demonstrated. We synthesized [11C]MCC950 with no-carrier-added [11C]CO2 fixation chemistry using an iminophosphorane precursor (RCY 45 ± 4%, >99% RCP, 27 ± 2 GBq/µmol, 23 ± 3 min, n = 6) and determined its distribution both in vivo and ex vivo in C57BL/6 and atherogenic ApoE-/- mice. Small animal PET imaging was performed in both strains following intravenous administration via the lateral tail vein and revealed considerable uptake in the liver that stabilized by 20 min (7-8.5 SUV), coincident with secondary renal excretion. Plasma metabolite analysis uncovered excellent in vivo stability of [11C]MCC950 (94% intact). Ex vivo autoradiography performed on excised aortas revealed heterogeneous uptake in atherosclerotic plaques of ApoE-/- mice in comparison to C57BL/6 controls (48 ± 17 %ID/m2 vs 18 ± 8 %ID/m2, p = 0.002, n = 4-5). Treatment of ApoE-/- mice with nonradioactive MCC950 (5 mg/kg, iv) 10 min prior to radiotracer administration increased uptake in the intestine (5.3 ± 1.8 %ID/g vs 11.0 ± 3.7 %ID/g, p = 0.04, n = 4-6) and in aortic lesions (48 ± 17 %ID/m2 vs 104 ± 15 %ID/m2, p = 0.0002, n = 5) by 108% and 117%, respectively, without significantly increasing plasma free fraction (fp, 1.3 ± 0.4% vs 1.7 ± 0.8%, n = 2). These results suggest that [11C]MCC950 uptake demonstrates specific binding and may prove useful for in vivo NLRP3 imaging in atherosclerosis.

The Selective NLRP3-inflammasome inhibitor MCC950 Mitigates Post-resuscitation Myocardial Dysfunction and Improves Survival in a Rat Model of Cardiac Arrest and Resuscitation.

PURPOSE: To investigate the effects of the selective NLRP3 inflammasome inhibitor MCC950 on post-resuscitation myocardial function and survival in a rat model of cardiopulmonary resuscitation (CPR). METHODS: Thirty-six Sprague Dawley rats were randomized into three groups: (1) MCC950, (2) control, and (3) sham. Each group consisted of a 6 h non-survival subgroup (n = 6) and a 48 h survival subgroup (n = 6). Ventricular fibrillation (VF) was induced and untreated for 6 min. CPR was initiated and continued for 8 min. Resuscitation was attempted with a 4 J defibrillation. MCC950 (10 mg/kg) or vehicle was administered via intraperitoneal injection immediately after the return of spontaneous circulation (ROSC). Myocardial function and sublingual microcirculation were measured after ROSC in the non-survival subgroups. Plasma levels of interleukin Iß (IL-1ß) and cardiac troponin I (cTnI) were measured at baseline and 6 h in the non-survival subgroups. Heart tissue was harvested to measure the NLRP3 inflammasome constituents, including NLRP3, apoptosis-associated speck-like protein (ASC), Caspase-1, and IL-1ß. Survival duration and neurologic deficit score (NDS) were recorded and evaluated among survival groups. RESULTS: Post-resuscitation myocardial function and sublingual microcirculation were improved in MCC950 compared with control (p < 0.05). IL-1ß and cTnI were decreased in MCC950 compared to control (p < 0.01). The MCC950 treated groups showed significantly reduced ASC, caspase-1, and IL-1ß compared with the control group (p < 0.05). Survival at 48 h after ROSC was greater in MCC950 (p < 0.05) with improved NDS (p < 0.05). CONCLUSION: Administration of MCC950 following ROSC mitigates post-resuscitation myocardial dysfunction and improves survival.

Macrophage-NLRP3 Activation Promotes Right Ventricle Failure in Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) often results in death from right ventricular failure (RVF). NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3)-macrophage activation may promote RVF in PAH. Objectives: Evaluating the contribution of the NLRP3 inflammasome in RV macrophages to PAH RVF. Methods: Rats with decompensated RV hypertrophy (monocrotaline [MCT] and Sugen-5416 hypoxia [SuHx]) were compared with compensated RV hypertrophy rats (pulmonary artery banding). Echocardiography and right heart catheterization were performed. Macrophages, atrial natriuretic peptides, and fibrosis were evaluated by microscopy or flow cytometry. NLRP3 inflammasome activation and cardiotoxicity were confirmed by immunoblot and in vitro strategies. MCT rats were treated with SC-144 (a GP130 antagonist) or MCC950 (an NLRP3 inhibitor). Macrophage-NLRP3 activity was evaluated in patients with PAH RVF. Measurements and Main Results: Macrophages, fibrosis, and atrial natriuretic peptides were increased in MCT and SuHx RVs but not in left ventricles or pulmonary artery banding rats. Although MCT RV macrophages were inflammatory, lung macrophages were antiinflammatory. CCR2+ macrophages (monocyte-derived) were increased in MCT and SuHx RVs and highly expressed NLRP3. The macrophage-NLRP3 pathway was upregulated in patients with PAH with decompensated RVs. Cultured MCT monocytes showed NLRP3 activation, and in coculture experiments resulted in cardiomyocyte mitochondrial damage, which MCC950 prevented. In vivo, MCC950 reduced NLRP3 activation and regressed pulmonary vascular disease and RVF. SC-144 reduced RV macrophages and NLRP3 content, prevented STAT3 (signal transducer and activator of transcription 3) activation, and improved RV function without regressing pulmonary vascular disease. Conclusions: NLRP3-macrophage activation occurs in the decompensated RV in preclinical PAH models and patients with PAH. Inhibiting GP130 or NLRP3 signaling improves RV function. The concept that PAH RVF results from RV inflammation rather than solely from elevated RV afterload suggests a new therapeutic paradigm.

Adenine model of chronic renal failure in rats to determine whether MCC950, an NLRP3 inflammasome inhibitor, is a renopreventive.

BACKGROUND: Chronic renal failure (CRF) is defined by a significant decline in renal function that results in decreased salt filtration and inhibition of tubular reabsorption, which ultimately causes volume enlargement. This study evaluated the potential renopreventive effects of the NLRP3 inflammasome inhibitor MCC950 in adenine-induced CRF in rats due to conflicting evidence on the effects of MCC950 on the kidney. METHODS: Since the majority of the kidney tubular abnormalities identified in people with chronic renal disease are comparable to those caused by adding 0.75 percent of adenine powder to a rat's diet each day for four weeks, this method has received broad approval as a model for evaluating kidney damage. Throughout the test, blood pressure was checked weekly and at the beginning. Additionally, oxidative stress factors, urine sample examination, histological modifications, and immunohistochemical adjustments of caspase-3 and interleukin-1 beta (IL-1) levels in renal tissues were carried out. RESULTS: Results revealed that MCC950, an inhibitor of the NLRP3 inflammasome, had a renopreventive effect, which was demonstrated by a reduction in blood pressure readings and an improvement in urine, serum, and renal tissue indicators that indicate organ damage. This was also demonstrated by the decrease in neutrophil gelatinase-associated lipocalin tubular expression (NGAL). The NLRP3 inflammasome inhibitor MCC950 was found to significantly alleviate the worsening renal cellular alterations evidenced by increased expression of caspase-3 and IL-1, according to immunohistochemical tests. CONCLUSION: The NLRP3 inflammasome inhibitor MCC950 demonstrated renopreventive effects in the CRF rat model, suggesting that it might be used as a treatment strategy to stop the progression of CRF.

NLRP3 inflammasome activation increases brain oxidative stress after transient global cerebral ischemia in rats.

Pupurpose of the study: Oxidative stress has been reported to be an important mechanism for brain damage following ischemic stroke. Recently, the involvement of cytosolic receptors capable of forming protein complexes called inflammasomes has been demonstrated to perpetuate oxidative stress. Herein, we report the effect of NLRP3 inhibition with MCC950 on brain oxidative stress in an animal model of transient global cerebral ischemia.Materials and methods: Male Wistar rats received an intracerebroventricularly (icv) injection of MCC950 (140 ng/kg) or saline and were subjected to sham procedure or ischemia/reperfusion (I/R). Twenty-four hours after I/R, myeloperoxidase (MPO) activity, nitrite/nitrate (N/N) concentration, lipid peroxidation, protein carbonyls formation, superoxide dismutase (SOD) and catalase (CAT) activity were determined in the prefrontal cortex, hippocampus, cortex, cerebellum and striatum. Results: After I/R, MPO activity increased in the prefrontal cortex, hippocampus, cortex and cerebellum and N/N concentration elevated in the prefrontal cortex, hippocampus and cortex, while MCC950 decreased this level except in hippocampus. After I/R, lipid peroxidation enhanced in the prefrontal cortex and cerebellum and increased the oxidative protein damage in both structures and hippocampus. MCC950 decreased lipid peroxidation in the prefrontal cortex and decreased protein oxidative damage in all brain structures except in the striatum. SOD activity decreased in the cortex after I/R and MCC950 reestablished these levels. CAT activity decreased in the prefrontal cortex, hippocampus and cerebellum after I/R and MCC950 reestablished these levels in the prefrontal cortex.Conclusion: Our data provide novel demonstration that inhibiting NLRP3 activation with MCC950 reduces brain oxidative damage after cerebral I/R in rats.

Intraperitoneal Injection of MCC950 Inhibits the Progression of Myopia in Form-Deprivation Myopic Mice.

Myopia, one of the most prevalent ocular diseases worldwide, is projected to affect nearly half of the global population by 2050. The main cause of myopia in most patients is axial myopia, which primarily occurs due to the elongation of the eyeball, driven by changes in the extracellular matrix (ECM) of scleral cells. Previous studies have shown that NLRP3, an important inflammatory mediator, plays a critical role in regulating the expression of MMP-2 in the sclera. This, in turn, leads to a decrease in the expression of Collagen-1, a major component of the scleral ECM, triggering the remodeling of the scleral ECM. This study aimed to investigate the effect of MCC950, an inhibitor of NLRP3, on the progression of myopia using a mouse form-deprivation myopia (FDM) model. The FDM mouse model was constructed by subjecting three-week-old C57BL/6J mice to form-deprivation. The mice were divided into experimental (n = 10/group; FDM2M, FDM4M, FDM2W, and FDM4W) and control groups (n = 5/group). The experimental groups were further categorized based on the duration of form deprivation (2 and 4 weeks, labeled as 2 and 4, respectively) and the type of injection received (MCC950 or saline, labeled as M and W, respectively). MCC950 was injected at a concentration of 50 mg/mL, with a dose of 10 mg per kilogram of body weight. Meanwhile, the saline group received the same volume of saline. Refraction and axial length measurements were performed for each eye. The expression levels of NLRP3, caspase-1, IL-1ß, IL-18, MMP-2, and Collagen-1 in the sclera were assessed using immunohistochemistry and Western blotting. The intraperitoneal injection of MCC950 did not significantly affect refraction or axial length in normal mice (p > 0.05). However, in FDM mice, MCC950 attenuated the elongation of the axial length and resulted in a smaller shift towards myopia compared to the saline group (FDM4M vs. FDM4W, p = 0.03 and p < 0.05, respectively). MCC950 decreased MMP-2 expression (p < 0.05) but increased Collagen-1 expression (p < 0.05) in the experimental eyes when compared to the saline group. Within the MCC950 group, the expression of MMP-2 was increased in the experimental eyes at 4 weeks (p < 0.05), while that of Collagen-1 was decreased (p < 0.05), which is consistent with changes in refractive error. Immunohistochemical analysis yielded similar results (p < 0.05). MCC950 also reduced the expression levels of NLRP3 (p = 0.03), caspase-1 (p < 0.05), IL-1ß (p < 0.05), and IL-18 (p < 0.05) in the experimental eyes compared to the saline group. Within the MCC950 group, the expression levels of NLRP3 and caspase-1 were comparable between the experimental and control eyes (p > 0.05), whereas IL-18 expression was higher in experimental eyes (p < 0.05). IL-1ß expression was higher in the experimental eyes only at week 4 (p < 0.05). The intraperitoneal injection of MCC950 can inhibit the progression of myopia in FDM mice, possibly by regulating collagen remodeling in the sclera through the NLRP3-MMP-2 signaling pathway. Therefore, MCC950 holds promise as a potential therapeutic agent for controlling the progression of myopia.

Hyperglycemia Aggravates Periodontitis via Autophagy Impairment and ROS-Inflammasome-Mediated Macrophage Pyroptosis.

Macrophage pyroptosis drives the secretion of IL-1ß, which has been recently reported to be a featured salivary biomarker for discriminating periodontitis in the presence of diabetes. This study aimed to explore whether macrophage pyroptosis plays a role in the development of diabetes mellitus-periodontitis, as well as potential therapeutic strategies. By establishing a model of experimental diabetes mellitus-periodontitis in rats, we found that IL-1ß and gasdermin D were highly expressed, leading to aggravated destruction of periodontal tissue. MCC950, a potent and selective molecule inhibitor of the NLRP3 inflammasome, effectively inhibited macrophage pyroptosis and attenuated alveolar bone losses in diabetes mellitus-periodontitis. Consistently, in vitro, high glucose could induce macrophage pyroptosis and thus promoted IL-1ß production in macrophages stimulated by lipopolysaccharide. In addition, autophagy blockade by high glucose via the mTOR-ULK1 pathway led to severe oxidative stress response in macrophages stimulated by lipopolysaccharide. Activation of autophagy by rapamycin, clearance of mitochondrial ROS by mitoTEMPO, and inhibition of inflammasome by MCC950 could significantly reduce macrophage pyroptosis and IL-1ß secretion. Our study demonstrates that hyperglycemia promotes IL-1ß production and pyroptosis in macrophages suffered by periodontal microbial stimuli. Modulation of autophagy activity and specific targeting of the ROS-inflammasome pathway may offer promising therapeutic strategies to alleviate diabetes mellitus-periodontitis.

NLRP3 inflammasome up-regulates major histocompatibility complex class I expression and promotes inflammatory infiltration in polymyositis.

OBJECTIVE: This study was designed to investigate the role of the nucleotide-binding-domain -and leucine-rich repeat -containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome in the pathogenesis of polymyositis (PM). METHODS: Immunochemistry was performed to analyze the NLRP3, caspase-1 and interleukin-1 beta (IL-1ß) expression in the muscle tissue of PM patients. Rat model of PM and C2C12 cell were used to investigate the potential role of NLRP3 inflammasome in PM. RESULTS: The percentage of CD 68+ macrophages, and the expression levels of NLRP3, caspase-1 and IL-1ß in the muscle tissue were elevated in 27 PM patients. LPS/ATP treatment resulted in activation of NLRP3 inflammasome and secretion of IL-1ß as well as interferons (IFNs) and monocyte chemotactic protein-1 (MCP-1) in the Raw 264.7 macrophages. Meanwhile, LPS/ATP challenged activation of NLRP3 inflammasome induced overexpression of major histocompatibility complex class I (MHC-I), a key molecular of PM in the co-cultured C2C12 cells. The effect was decreased by treatment of NLRP3 inflammasome inhibitor MCC950 or siRNA of NLRP3 inflammasome. These findings suggested certain levels of IL-1ß rather than IFNs up-regulated MHC-I expression in C2C12 cells. IL-1ß blockade using neutralizing IL-1ß monoclonal antibody or siRNA of IL-1ß suppressed MHC-I overexpression. In vivo, NLRP3 inflammasome inhibition by MCC950 reduced the expression of NLRP3, IL-1ß and MHC-I in the muscle tissue of PM modal rats. Also, it attenuated the intensity of muscle inflammation as well as the CRP, CK, and LDH levels in the serum. CONCLUSION: NLRP3/caspase-1/IL-1ß axis may play an important role in the development of PM. Inhibition of NLRP3 activation may hold promise in the treatment of PM.

MCC950 ameliorates the dementia symptom at the early age of line M83 mouse and reduces hippocampal α-synuclein accumulation.

Dementia with Lewy bodies (DLB) is the second most common type of neurodegenerative dementia after Alzheimer's disease (AD). Neuroinflammation plays an important role in neurodegenerative diseases. It is urgent to unravel the pathogenesis of DLB and find potential therapeutic drugs. Here, we investigated the pharmacological effects of the NLRP3 inflammasome inhibitor MCC950 in A53T α-synuclein transgenic line M83 mice aged 4 months. The behavioral tests including Y-maze, Barnes maze, nest building and Rotarod showed that MCC950 significantly improved the cognitive dysfunction symptom without affecting the motor coordination after consecutive intragastric administration every day for 5 weeks. Furthermore, immunostaining or immunoblotting experiments on the hippocampal tissue were performed, and the results suggested that MCC950 not only inhibited the expression of NLRP3, and suppressed the activation of astrocytes and microglia, but also promoted the mTOR-mediated autophagy pathway to reduce human α-synuclein accumulation. Our findings further demonstrate that line M83 mice may be used as an animal model for DLB research, and can provide preclinical evidences for the development of MCC950 as a promising therapeutic drug.

Paeonol protects against acute pancreatitis by inhibiting M1 macrophage polarization via the NLRP3 inflammasomes pathway.

The excessive inflammatory response mediated by macrophage is one of the key factors for the progress of acute pancreatitis (AP). Paeonol (Pae) was demonstrated to exert multiple anti-inflammatory effects. However, the role of Pae on AP is not clear. In the present study, we aimed to investigate the protective effect and mechanism of Pae on AP in vivo and vitro. In the caerulein-induced mild acute pancreatitis (MAP) model, we found that Pae administration reduced serum levels of amylase, lipase, IL-1ß and IL-6 and alleviated the histopathological manifestations of pancreatic tissue in a dose-dependent manner. And Pae decrease the ROS generated, restore mitochondrial membrane potential (ΔΨm), inhibit M1 macrophage polarization and NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs) in vitro. In addition, specific NLRP3 inhibitor MCC950 eliminated the protective effect of Pae on AP induced by caerulein in mice. Correspondingly, the inhibitory effect of Pae on ROS generated and M1 polarization was not observed in BMDMs with MCC950 in vitro. Taken together, our datas for the first time confirmed the protective effects of Pae on AP via the NLRP3 inflammasomes Pathway.