NLRP3 Inflammasome Components Research Articles

Repurposing the KCa3.1 Blocker Senicapoc for Ischemic Stroke

Senicapoc, a small molecule inhibitor of the calcium-activated potassium channel KCa3.1, was safe and well-tolerated in clinical trials for sickle cell anemia. We previously reported proof-of-concept data suggesting that both pharmacological inhibition and genetic deletion of KCa3.1 reduces infarction and improves neurologic recovery in rodents by attenuating neuroinflammation. Here we evaluated the potential of repurposing senicapoc for ischemic stroke. In cultured microglia, senicapoc inhibited KCa3.1 currents with an IC50 of 7 nM, reduced Ca2+ signaling induced by the purinergic agonist ATP, suppressed expression of pro-inflammatory cytokines and enzymes (iNOS and COX-2), and prevented induction of the inflammasome component NLRP3. When transient middle cerebral artery occlusion (tMCAO, 60 min) was induced in male C57BL/6 J mice, twice daily administration of senicapoc at 10 and 40 mg/kg starting 12 h after reperfusion dose-dependently reduced infarct area determined by T2-weighted magnetic resonance imaging (MRI) and improved neurological deficit on day 8. Ultra-high-performance liquid chromatography/mass spectrometry analysis of total and free brain concentrations demonstrated sufficient KCa3.1 target engagement. Senicapoc treatment significantly reduced microglia/macrophage and T cell infiltration and activation and attenuated neuronal death. A different treatment paradigm with senicapoc started at 3 h and MRI on day 3 and day 8 revealed that senicapoc reduces secondary infarct growth and suppresses expression of inflammation markers, including T cell cytokines in the brain. Lastly, we demonstrated that senicapoc does not impair the proteolytic activity of tissue plasminogen activator (tPA) in vitro. We suggest that senicapoc could be repurposed as an adjunctive immunocytoprotective agent for combination with reperfusion therapy for ischemic stroke.

Altered Lnc-EGFR, SNHG1, and LincRNA-Cox2 Profiles in Patients with Relapsing-Remitting Multiple Sclerosis: Impact on Disease Activity and Progression.

Relapsing-remitting multiple sclerosis (RRMS) is the most prevalent MS subtype. Ample evidence has indicated that long noncoding RNAs (lncRNAs) are crucial players in autoimmune and inflammatory disorders. This study investigated the expression of lnc-EGFR, SNHG1, and lincRNA-Cox2 in RRMS patients during active relapses and in remission. Additionally, the expression of FOXP3, a master transcription factor for regulatory T cells, and NLRP3-inflammasome-related genes were determined. Relationships between these parameters and MS activity and annualized relapse rate (ARR) were also evaluated. The study included 100 Egyptian participants: 70 RRMS patients (35 during relapse and 35 in remission) and 30 healthy controls. RRMS patients showed significant downregulation of lnc-EGFR and FOXP3 and dramatic upregulation of SNHG1, lincRNA-Cox2, NLRP3, ASC, and caspase-1 compared to controls. Lower serum TGF-β1 and elevated IL-1β levels were observed in RRMS patients. Notably, patients during relapses displayed more significant alterations than those in remission. Lnc-EGFR was positively correlated with FOXP3 and TGF-β1 and negatively correlated with ARR, SNHG1, lincRNA-Cox2, and NLRP3 inflammasome components. Meanwhile, SNHG1 and lincRNA-Cox2 were positively correlated with ARR, NLRP3, ASC, caspase-1, and IL-1β. Excellent diagnostic performance for lnc-EGFR, FOXP3, and TGF-β1 was demonstrated, while all biomarkers exhibited strong prognostic potential for predicting relapses. Finally, the differential expression of lnc-EGFR, SNHG1, and lincRNA-Cox2 in RRMS patients, especially during relapses, suggests their involvement in RRMS pathogenesis and activity. Correlation between their expression and ARR implies relationships to disease progression. Our findings also highlight their promising roles as biomarkers for RRMS.

Taurodeoxycholate, a GPCR19 agonist, ameliorates atopic dermatitis in Balb/c mice.

Keratinocytes are pivotal cells in the pathogenesis of atopic dermatitis (AD) as much as Th2 cells. In this sense, regulation of pro-inflammatory features of keratinocytes might be useful for AD patients. P2X7R-mediated activation of NLRP3 inflammasome (N3I) in keratinocytes and myeloid cells plays crucial roles in AD. Nonetheless, inhibition of P2X7R has not been feasible because of polymorphisms and ubiquitous expression of P2X7R. Here, we report that GPCR19 colocalizes with P2X7R, and a GPCR19 agonist (taurodeoxycholate [TDCA]) inhibits the activation of P2X7R. Noncistronically, TDCA inhibits NF-kB activation via the adenylate cyclase-PKA pathway and BzATP-mediated Ca++ mobilization. Cistronically, TDCA suppresses the expression of P2X7R and N3I components in keratinocytes. NLRP3 oligomerization and the production of mature IL-1β and IL-18 was suppressed by TDCA treatment in keratinocytes. Topical TDCA treatment ameliorates proinflammatory features of AD in mice induced by DNCB, MC903, or oxazolone. Taken together, a GPCR19 agonist such as TDCA might inhibit P2X7R-mediated N3I activation of keratinocytes, which is crucial for the pathogenesis of AD.

FAM69C functions as a kinase for eIF2α and promotes stress granule assembly

Stress granules are dynamic cytoplasmic ribonucleoprotein granules that assemble in response to cellular stress. Aberrant formation of stress granules has been linked to neurodegenerative diseases. However, the molecular mechanisms underlying the initiation of stress granules remain elusive. Here we report that the brain-enriched protein kinase FAM69C promotes stress granule assembly through phosphorylation of eukaryotic translation initiation factor 2 (eIF2α). FAM69C physically interacts with eIF2α and functions as a stress-specific kinase for eIF2α, leading to stress-induced protein translation arrest and stress granule assembly. Primary microglia derived from Fam69c knockout mice exhibit aberrant stress granule assembly in response to oxidative stress and ATP. Defective stress granule assembly in microglia correlates with the formation of ASC specks and NLRP3 inflammasome activation, whereas induction of stress granule precludes inflammasome formation. Consistently, increased NLRP3 levels, caspase-1 cleavage and Il18 expression corroborate microglia-associated neuroinflammation in aged Fam69c knockout mice. Our study demonstrates that FAM69C is critical for stress granule assembly and suggests its role in the regulation of microglia function.

NLRP3 inflammasome activation By 17β-estradiol is a potential therapeutic target in hepatocellular carcinoma treatment

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it mostly arises as a consequence of persistent chronic inflammation. Recently, NLRP3 inflammasome has caught the attention of many research groups due to its involvement in different types of cancer. However, its direct role in HCC remains elusive. Our study aimed to evaluate the role of NLRP3 inflammasome and pyroptosis in HCC and to clarify the potential mechanism by which 17β-estradiol (E2) can be used as a protective factor against HCC. NLRP3, caspase-1 (CASP1) as well as gasdermin-D (GSDMD) mRNA expression levels were assessed in human HCC tissues and adjacent non-cancerous liver tissues. Also, HepG2 HCC cells were cultured and treated with E2, followed by detection of the mRNA levels of these three genes. Our results revealed that NLRP3, CASP1, and GSDMD mRNA expressions were significantly lower in HCC tissues than in controls, and this under-expression was closely correlated with advanced HCC stages and grades. In contrast, HepG2 HCC cells displayed significantly higher expression levels of NLRP3 inflammasome components and GSDMD in the two E2-treated groups compared to the untreated group. Also, NLRP3, CASP1, and GSDMD mRNA expression levels were positively correlated with each other. This study confirmed that lack of NLRP3 inflammasome is involved in HCC progression and 17β-estradiol-induced activation of NLRP3 inflammasome may be effective in HCC treatment as it inhibited tumor cell growth and proliferation by triggering CASP1-dependent pyroptosis in HCC cells.

In Silico Structural and Functional Analyses of NLRP3 Inflammasomes to Provide Insights for Treating Neurodegenerative Diseases.

Inflammasomes are cytoplasmic intracellular multiprotein complexes that control the innate immune system's activation of inflammation in response to derived chemicals. Recent advancements increased our molecular knowledge of activation of NLRP3 inflammasomes. Although several studies have been done to investigate the role of inflammasomes in innate immunity and other diseases, structural, functional, and evolutionary investigations are needed to further understand the clinical consequences of NLRP3 gene. The purpose of this study is to investigate the structural and functional impact of the NLRP3 protein by using a computational analysis to uncover putative protein sites involved in the stabilization of the protein-ligand complexes with inhibitors. This will allow for a deeper understanding of the molecular mechanism underlying these interactions. It was found that human NLRP3 gene coexpresses with PYCARD, NLRC4, CASP1, MAVS, and CTSB based on observed coexpression of homologs in other species. The NACHT, LRR, and PYD domain-containing protein 3 is a key player in innate immunity and inflammation as the sensor subunit of the NLRP3 inflammasome. The inflammasome polymeric complex, consisting of NLRP3, PYCARD, and CASP1, is formed in response to pathogens and other damage-associated signals (and possibly CASP4 and CASP5). Comprehensive structural and functional analyses of NLRP3 inflammasome components offer a fresh approach to the development of new treatments for a wide variety of human disorders.

The Correlation between Increased Expressions of NLRP3 Inflammasome Components in Peripheral Blood Mono-Nuclear Cells and Plaque Vulnerability in Human Carotid Atherosclerosis.

We aimed to determine whether NLRP3 inflammasomes in peripheral blood mononuclear cells (PBMC) were associated with carotid plaque instability in carotid atherosclerosis patients. Consecutive 38 carotid atherosclerosis with vulnerable plaques, 22 carotid atherosclerosis with stable plaques, and 40 healthy subjects with no carotid or coronary artery stenosis were enrolled. They were referred to the Second Hospital of Dalian Medical University from 2013-2019. Carotid plaques were evaluated by modified plaque vulnerability risk score (MPVRS) and pathological assessment. The mRNA and protein expression of NLRP3 inflammasome components in PBMC were determined by quantitative real time PCR and Western blot analysis or ELISA. When consecutive study subjects undergoing carotid endarterectomy were divided into stable (≤4) and unstable (>4) plaque groups according to the MPVRS, the unstable plaque group had significantly raised mRNA and protein expression of NLRP3 inflammasome components in PBMC as compared with the stable plaque group and healthy subject group. Furthermore, subjects with higher NLRP3 protein expression in PBMC had greater incidence of cerebrovascular events. Increased NLRP3 inflammasome components in PBMC is associated with instability of human carotid atherosclerotic plaques, suggesting NLRP3 inflammasome as a potential biomarker for monitoring carotid plaque instability.

Luteolin-rich fraction from Perilla frutescens seed meal inhibits spike glycoprotein S1 of SARS-CoV-2-induced NLRP3 inflammasome lung cell inflammation via regulation of JAK1/STAT3 pathway: A potential anti-inflammatory compound against inflammation-induced long-COVID.

The multi-systemic inflammation as a result of COVID-19 can persevere long after the initial symptoms of the illness have subsided. These effects are referred to as Long-COVID. Our research focused on the contribution of the Spike protein S1 subunit of SARS-CoV-2 (Spike S1) on the lung inflammation mediated by NLRP3 inflammasome machinery and the cytokine releases, interleukin 6 (IL-6), IL-1beta, and IL-18, in lung epithelial cells. This study has attempted to identify the naturally- occurring agents that act against inflammation-related long-COVID. The seed meal of Perilla frutescens (P. frutescens), which contains two major dietary polyphenols (rosmarinic acid and luteolin), has been reported to exhibit anti-inflammation activities. Therefore, we have established the ethyl acetate fraction of P. frutescens seed meal (PFEA) and determined its anti-inflammatory effects on Spike S1 exposure in A549 lung cells. PFEA was established using solvent-partitioned extraction. Rosmarinic acid (Ra) and luteolin (Lu) in PFEA were identified using the HPLC technique. The inhibitory effects of PFEA and its active compounds against Spike S1-induced inflammatory response in A549 cells were determined by RT-PCR and ELISA. The mechanistic study of anti-inflammatory properties of PFEA and Lu were determined using western blot technique. PFEA was found to contain Ra (388.70 ± 11.12 mg/g extract) and Lu (248.82 ± 12.34 mg/g extract) as its major polyphenols. Accordingly, A549 lung cells were pre-treated with PFEA (12.5-100 μg/mL) and its two major compounds (2.5-20 μg/mL) prior to the Spike S1 exposure at 100 ng/mL. PFEA dose-dependently exhibited anti-inflammatory properties upon Spike S1-exposed A549 cells through IL-6, IL-1β, IL-18, and NLRP3 gene suppressions, as well as IL-6, IL-1β, and IL-18 cytokine releases with statistical significance (p < 0.05). Importantly, Lu possesses superior anti-inflammatory properties when compared with Ra (p < 0.01). Mechanistically, PFEA and Lu effectively attenuated a Spike S1-induced inflammatory response through downregulation of the JAK1/STAT3-inflammasome-dependent inflammatory pathway as evidenced by the downregulation of NLRP3, ASC, and cleaved-caspase-1 of the NLRP3 inflammasome components and by modulating the phosphorylation of JAK1 and STAT3 proteins (p < 0.05). The findings suggested that luteolin and PFEA can modulate the signaling cascades that regulate Spike S1-induced lung inflammation during the incidence of Long-COVID. Consequently, luteolin and P. frutescens may be introduced as potential candidates in the preventive therapeutic strategy for inflammation-related post-acute sequelae of COVID-19.

Increased expression of NLRP3 inflammasome components in granulosa cells and follicular fluid interleukin(IL)-1beta and IL-18 levels in fresh IVF/ICSI cycles in women with endometriosis.

The inflammasomes are a family of recently described multi-protein cytoplasmic sensors that orchestrate the inflammatory response and participate in a variety of inflammatory conditions. We hypothesized that the activation of pyrin domain‑containing protein 3 (NLRP3) inflammasome by granulosa cells (hGCs) may be activated in women with endometriosis and influence oocyte maturation and IVF outcomes. We performed a cross-sectional study to investigate the NLRP3 inflammasome status in follicular fluid (FF) and in hGCs from 44 women undergoing controlled ovarian stimulation for IVF/ICSI. Study subjects were divided into two groups according to the infertility etiology: group with tubal or male factor (control, n = 22) vs. group with endometriosis (n = 22). The FF IL-1beta and IL-18 levels in the endometriosis group were significantly higher than those in the non-endometriosis group, i.e., 5010pg/mL and 2738pg/mL, respectively (p < 0.05). No correlation was found between clinical pregnancy and live birth rate and analyzed inflammasome component levels (p > 0.05). In addition, the hGCs from endometriosis women demonstrated high expression of NLRP3 inflammasome at both protein and mRNA levels. Higher expression of inflammasome components within the ovary compartment may result from the exaggerated inflammatory state associated with endometriosis and thus impact the fertility of these women.

IL-37 alleviates Coxsackievirus B3-induced viral myocarditis via inhibiting NLRP3 inflammasome-mediated pyroptosis

Our study aims to verify the potential effects and underlying mechanisms of IL-37 in Coxsackievirus B3 (CVB3)-induced viral myocarditis (VMC). VMC model was established by intraperitoneal injection of CVB3 into 6-week-old male balb-c mice on day 0. Each mouse of the IL-37-control group and IL-37-VMC CVB3 groups was intraperitoneally injected with IL-37 on day 4 and day 7. The cardiac function was evaluated by transthoracic echocardiography including LVEF, LVFE, IVSs and IVSd. Myocardial injury was measured by Elisa for serum cTnI. The inflammation infiltration and fibrosis were evaluated by hematoxylin and eosin (HE) staining and Masson staining. The expression levels of NLRP3 inflammasome components in pyroptosis were determined by western blot, Elisa, and immunofluorescent analysis. We also detected the expression of IL-37-IL-1R8 in PBMCs by immunofluorescence after injection with CVB3 and IL-37. Compared with the VMC group, mice received CVB3 and IL-37 have improved cardiac function, reduced inflammation infiltration and fibrosis, and with lower expression of cTnI, IL-1β, IL-18 and NLRP3 inflammasome component. IL-37 weakened the upregulation of GSDMD and phosphorylation of NF-κB p65 induced by CVB3. Exogenous addition of IL-37 with CVB3 further increases the production of IL-37-IL-1R8 -IL-18RA complex in vitro. Our findings indicate that IL-37 alleviates CVB3-induced VMC, which may be produced by inhibiting NLRP3 inflammasome-mediated pyroptosis, NF-κB signaling pathway, and IL-37-IL-1R8 -IL-18RA complex.

Stromal Cell-Activated Bone Marrow Neutrophils Provide BAFF in Newly Diagnosed and Treated Multiple Myeloma

Background: In many solid cancers, increased numbers of intra-tumoral neutrophils are associated with poor prognosis. Interestingly, and in contrast to peripheral tissues, the bone marrow (BM) is unique in its continuous occupation by high numbers of neutrophils, making these cells likely candidates to influence the tumor microenvironment in BM malignancies such as multiple myeloma (MM). Accordingly, a high neutrophil/T-cell ratio is associated with inferior progression-free survival in MM. Recently, we identified inflammatory mesenchymal stromal cells (iMSC) in the MM BM that transcribe myeloid modulation factors, including chemokines that bind the neutrophil-expressed receptors CXCR1 and 2. This led us to hypothesize that neutrophil-stromal cell crosstalk in MM might lead to neutrophil alterations and by virtue of their abundance, the creation of a pro-tumor MM environment. Results: To determine whether BM neutrophils of MM patients differed from those of controls, we first assessed phenotypic alterations by flow cytometry. Compared to non-cancer controls, mature neutrophils from the MM BM had an activated phenotype, evidenced by increased numbers of CD62L negative cells (37% vs. 5%, p = 0.03, N = 5 newly diagnosed MM, 3 controls), switching of CD11b to its activated isoform (74% vs. 17%, p = 0.03), and upregulation of CXCR1 (mean fluorescent intensity [MFI] 7574 vs. 1831, p = 0.03) and CXCR2 (MFI 2284 vs. 813, p = 0.03). To assess the extent of activation, we performed single cell RNA sequencing of the entire BM granulocytic lineage (N = 6 MM, 4 controls). This confirmed the activation status of mature neutrophils in MM, and suggested an association with inflammation, as mature neutrophils of patients with MM were characterized by increased transcription of inflammatory mediators such as CXCL8, OSM and SLPI, inflammasome components IL1B, NLRP3 and CASP1, and various toll-like receptors (TLRs). One of the most prominent genes differentially expressed in mature neutrophils from MM patients was TNFSF13B, which encodes the MM cell survival factor BAFF (Figure 1). A transcriptomic comparison of TNFSF13B levels across hematopoietic and non-hematopoietic cells from the BM revealed that transcription of this gene was highest in mature neutrophils found in the MM BM. Combined with the fact that mature neutrophils are the most abundant hematological cells in the MM BM (37.7 ± 9% of all nucleated cells, N = 3), this finding suggests that neutrophils are a major source of BAFF in MM. As iMSC transcribe many factors with the potential to modulate neutrophils, we hypothesized that neutrophilic activation was mediated by iMSC. Coculture with IL1β-activated iMSC-like cells, but not non-inflammatory MSCs, led to activation of naïve neutrophils, as evidenced by the acquisition of a surface protein expression profile almost identical to that of BM neutrophils from MM patients. Moreover, RNA-sequencing of neutrophils cocultured with iMSC revealed increased transcription of inflammatory genes as observed in MM BM neutrophils (Figure 2). Importantly, interactions between naïve neutrophils and iMSC resulted in release of BAFF protein by neutrophils (29pg/mL vs. 15pg/mL on MSC, p = 0.008), suggesting that stromal cell-neutrophil interactions can drive a pro-MM environment by activation of local neutrophils. Finally, we assessed the durability of neutrophilic activation in patients undergoing first-line treatment. Single cell RNA sequencing of BM neutrophils from patients after ASCT and consolidation treatment revealed persistent neutrophil activation including high transcription of TNFSF13B. This was supported by increased levels of BAFF protein in BM plasma of MM patients post-consolidation compared to controls (3541 pg/mL vs. 411 pg/mL, p <0.0001, N = 52 MM and 18 controls). To assess if neutrophil activation and increased BAFF levels persist long-term, we determined levels of BAFF in BM supernatants of patients 1 year after consolidation therapy that did (N = 33) or did not (N = 19) receive daratumumab maintenance. Intriguingly, even after 1 year of maintenance therapy, and even in patients that were still MRD negative, protein levels of BAFF remained elevated (1096 pg/mL, p <0.0001). Conclusion: The continued presence of activated and BAFF-producing BM neutrophils during treatment creates a MM-supportive environment with the potential to impact malignant plasma cell survival and disease recurrence. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 15059: Effects of Cigarette Smoke on Inflammation and Atrial Fibrillation

Introduction: Cigarette smoking is a major risk factor for occurrence and progression of several cardiovascular diseases. Smoking is associated with an increased risk of Atrial Fibrillation (AF). AF is an extremely common cardiac rhythm disorder that is associated with substantial morbidity and mortality. One of the main contributors for the initiation, progression and persistence of AF is inflammation. Cigarette smoke (CS) is known to increase inflammation. CS is known to increase pro-inflammatory cytokines and AF progression is associated with increased levels of these pro-inflammatory cytokines. Therefore, it is of great clinical importance to elucidate the underlying mechanisms contributing to smoking induced inflammation. The amplification of the pro-inflammatory cytokines is mediated by inflammasome and recently, the direct involvement of the NLRP3 (NACHT, leucine-rich repeat, and pyrin domain containing protein 3), inflammasome and AF has been demonstrated in patients with AF. The central hypothesis to be tested is that patients with AF and exposed to CS will demonstrate a heightened inflammation through the activation of NLRP3 inflammasome compared to patients in normal sinus rhythm (NSR) and non-smoking AF patients. Methods: Human atrial tissues and blood were obtained from patients undergoing coronary artery bypass graft surgery at UC Davis Medical Center in accordance with the approved UC Davis IRB protocol. An integrated approach using molecular biology, flow cytometry, and electrophysiology was used to test the components of NLRP3 inflammasome, inflammatory cytokines and structural remodeling in smoking and non-smoking AF and NSR patients using blood and human atrial tissue. Results: Our results demonstrate a significant increase in protein expression of component of the NLRP3 inflammasome including NLRP3, ASC and Caspase-1 in smoking AF and NSR patients compared to non-smoking patients. There was a significant increase in inflammatory cytokines activated by the NLRP3 inflammasome, IL-18 and IL-1β in in smoking AF and NSR patients compared to non-smoking patients. Conclusions: Our study not only provides important mechanistic insights into the roles of inflammasome in AF patients exposed to CS

Identification of key proteins of cytopathic biotype bovine viral diarrhoea virus involved in activating NF-κB pathway in BVDV-induced inflammatory response

ABSTRACT Bovine viral diarrhoea virus (BVDV) is the etiologic agent of bovine viral diarrhea-mucosal disease, one of the most important viral diseases in cattle, with inflammatory diarrhea, enteritis, and mucosa necrosis as the major clinical manifestations. NF-κB is an important transcription complex that regulates the expression of genes involved in inflammation and immune responses. NLRP3 inflammasome plays a key role in the development of inflammatory diseases. However, whether the activation of NF-κB is crucial for BVDV infection-induced inflammatory responses remains unclear. The results of our present study showed that BVDV infection significantly activated the NF-κB pathway and promoted the expression of NLRP3 inflammasome components (NLRP3, ASC, pro-caspase 1) as well inflammatory cytokine pro-IL-1β in BVDV-infected bovine cells, resulting in the cleavage of pro-caspase 1 and pro-IL-1β into active form caspase 1 and IL-1β. However, the levels of the NLRP3 inflammasome components and inflammatory cytokines were obviously inhibited, as well the cleavage of pro-caspase 1 and pro-IL-1β in the pre-treated bovine cells with NF-κB-specific inhibitors after BVDV infection. Further, cytopathic biotype BVDV (cpBVDV) Erns and NS5A proteins with their key functional domains contributed to BVDV-induced inflammatory responses via activating the NF-κB pathway were confirmed experimentally. Especially, the NS5A can promote cholesterol synthesis and accelerate its augmentation, further activating the NF-κB signalling pathway. Conclusively, our data elucidate that the activation of NF-κB signaling pathway plays a crucial role in cpBVDV infection-induced inflammatory responses.

Microglial NLRP3 Inflammasome Induces Excitatory Synaptic Loss Through IL-1β-Enriched Microvesicle Release: Implications for Sepsis-Associated Encephalopathy.

Acute cerebral dysfunction is a pathological state common in severe infections and a pivotal determinant of long-term cognitive outcomes. Current evidence indicates that a loss of synaptic contacts orchestrated by microglial activation is central in sepsis-associated encephalopathy. However, the upstream signals that lead to microglial activation and the mechanism involved in microglial-mediated synapse dysfunction in sepsis are poorly understood. This study investigated the involvement of the NLRP3 inflammasome in microglial activation and synaptic loss related to sepsis. We demonstrated that septic insult using the cecal ligation and puncture (CLP) model induced the expression of NLRP3 inflammasome components in the brain, such as NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1, and IL-1β. Immunostaining techniques revealed increased expression of the NLRP3 inflammasome in microglial cells in the hippocampus of septic mice. Meanwhile, an in vitro model of primary microglia stimulated with LPS exhibited an increase in mitochondrial reactive oxygen species (ROS) production, NLRP3 complex recruitment, and IL-1β release. Pharmacological inhibition of NLRP3, caspase-1, and mitochondrial ROS all decreased IL-1β secretion by microglial cells. Furthermore, we found that microglial NLRP3 activation is the main pathway for IL-1β-enriched microvesicle (MV) release, which is caspase-1-dependent. MV released from LPS-activated microglia induced neurite suppression and excitatory synaptic loss in neuronal cultures. Moreover, microglial caspase-1 inhibition prevented neurite damage and attenuated synaptic deficits induced by the activated microglial MV. These results suggest that microglial NLRP3 inflammasome activation is the mechanism of IL-1β-enriched MV release and potentially synaptic impairment in sepsis.

Birefringent crystals deposition and inflammasome expression in human atheroma plaques by levels of uricemia

To verify the monosodium urate (MSU) crystal deposition in artery walls following a structure assessment and to assess NLRP3 inflammasome expression in human atheroma plaques by levels of uricemia. Patients with peripheral arterial disease who were candidates for amputation were recruited and classified as normouricemic or hyperuricemic. During surgery, an artery segment from the amputated limb was sampled, divided and fixed separately by cryo-embedding, 100% ethanol or Glyo-fixx. Samples were assessed by compensated polarized-light microscopy to identify MSU crystals on the artery walls. Afterwards, macrophages, neutrophils and NLRP3 inflammasome components at the plaque were categorized by immunostaining and compared between normouricemics and hyperuricemics. Thirty artery samples from 27 patients were studied; 10 (37.0%) participants were hyperuricemic. Birefringent needle-shaped crystals were found in three samples (10.0%), all processed by frozen sectioning. Other methods showed no crystals. No accompanying inflammatory process was noted, and the presence of crystals was equally distributed across ranges of uricemia, making it unlikely they were MSU crystals. Regarding immunostaining, 28 artery samples were available for analysis, with similar infiltration of macrophages and neutrophils. NLRP3 and gasdermin-D expression were significantly greater in hyperuricemics compared to normouricemics (P=0.044 and P=0.017, respectively). ASC content was numerically larger in hyperuricemics as well, while caspase-1 and IL-1beta expression were similar between groups. The presence of MSU crystals on artery walls was not confirmed. Hyperuricemia was associated with greater NLRP3 and gasdermin-D expression on human atheroma plaques in patients with peripheral artery disease.

Loganin Prevents Hepatic Steatosis by Blocking NLRP3 Inflammasome Activation.

Activation of the NLRP3 inflammasome is a necessary process to induce fibrosis in nonalcoholic fatty liver disease (NAFLD). Nonalcoholic steatohepatitis (NASH) is a kind of NAFLD that encompasses the spectrum of liver disease. It is characterized by inflammation and ballooning of hepatocytes during steatosis. We tested whether inhibiting the NLRP3 inflammasome could prevent the development and pathology of NASH. We identified loganin as an inhibitor of the NLRP3 inflammasome and investigated whether in vivo administration of loganin prevented NASH symptoms using a methionine-choline deficient (MCD) diet model in mice. We found that loganin inhibited the NLRP3 inflammasome activation triggered by ATP or nigericin, as shown by suppression of the production of interleukin (IL)-1β and caspase-1 (p10) in mouse primary macrophages. The speck formation of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) was blocked by loganin, showing that the assembly of the NLRP3 inflammasome complex was impaired by loganin. Administration of loganin reduced the clinical signs of NASH in mice fed the MCD diet, including hepatic inflammation, fat accumulation, and fibrosis. In addition, loganin reduced the expression of NLRP3 inflammasome components in the liver. Our findings indicate that loganin alleviates the inflammatory symptoms associated with NASH, presumably by inhibiting NLRP3 inflammasome activation. In summary, these findings imply that loganin may be a novel nutritional and therapeutic treatment for NASH-related inflammation.

Formation and activity of NLRP3 inflammasome and histopathological changes in the lung of corpses with COVID-19.

COVID-19 is a contagious disease that attacks many organs but the lungs are the main organs affected. The inflammasome activation results in the exacerbation of inflammatory response in infectious disease. The aim of this study is to investigate the formation and activity of the NLRP3 inflammasome complex and the histopathological changes caused by the coronavirus in the lung of deceased persons with COVID-19. In total, 10 corpses; 5 corpses with no history of any infectious diseases and COVID-19 and 5 corpses with the cause of death of COVID-19 were included in this study. Lung tissue samples were harvested during autopsy under safe conditions. Fresh tissues in each group were used to measure the genes expression and proteins level of NLRP3, ASC, Caspase-1, IL-1β, IL-6 and TNF-α and a routine hematoxylin and eosin staining was performed for histological assessment. Data are represented as the means ± SD. Statistical significance difference was accepted at a p-value less than 5%. The NLRP3, ASC, Caspase-1, IL-1β, IL-6 and TNF-α genes expression and proteins level were elevated in the lung of the COVID-19 group in comparison with the control group. Histological findings presented the increasing number of polymorphonuclear leukocytes, macrophages and also pulmonary fibrosis in the lungs of corpses with the cause of death of COVID-19. High expression of NLRP3 inflammasome components and its relation with the pathophysiology of the coronavirus-infected lung suggested that targeting the NLRP3 inflammasome could be helpful in achieving a more effective treatment in patients with COVID-19.

NLRP3-inflammasome activation in male reproductive system diseases.

The nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome, a multiprotein complex belonging to the innate immune system, plays a key role in the chronic inflammatory response, through the production of proinflammatory cytokines, IL-1β and IL-18, which can elicit their effects through receptor activation, both locally and systemically. Furthermore, it has been demonstrated the interaction of NLRP3 inflammasome components with redox signaling, endoplasmic reticulum stress, and mitochondrial function. A growing literature reported the involvement of NLRP3 platform dysregulation in the pathophysiology of different chronic diseases so it has been proposed that the inhibition of NLRP3 inflammasome could represent a new potential therapeutic target in the management of autoimmune and chronic inflammatory diseases, including cancer. In addition, it has been demonstrated that Sars-CoV2 preferentially activates NLRP3 inflammasome, strongly contributing to the hyperinflammatory state responsible for COVID-19. Recently, in vitro and animal models of both infectious and non-infectious male genital tract diseases affecting fertility, demonstrated the activation of the innate immune system, leading to increased levels of pro-inflammatory cytokines, as well as apoptosis and pyroptosis and that it was likely mediated by activation of the NLRP3 inflammasome. The objective of this review was to analyze the evidence on the role and the mechanisms by which NLRP3-inflammasome pathway activation may exert detrimental effects on the male reproductive system. Furthermore, although the literature data are still discordant, this review also highlighted the possible connection between SARS-CoV-2 infection/NLRP3 activation/oxidative stress and male infertility.

Macrophages in gouty inflammation

Gout disorder is likely to be the most common cause of inflammatory arthritis. Hyperuricemia leads to formation of poorly soluble crystalline urate salts in tissues and joints, thus being etiological factor of the gout. The review summarizes modern views on the mechanisms of autoinflammation in gout disorder and it provides an outlook for the new treatment approaches. Inflammation in gout is related to phagocytosis of the urate crystals and the macrophagic NLRP3 inflammosome activation. This process consists of preliminary stage and proinflammatory cytokine generation phases. During the preliminary phase, soluble and crystalline urate salts initiate mRNA transcription for IL-1 and other proinflammatory cytokines precursors in macrophages, synthesis of NLRP3 inflammosome components, and formation of immune memory. Enhanced transcription of IL-1 and other cytokine precursors is implemented via PRAS49-AKT-mTOR signaling, IL- 1ra anti-inflammantory factor and TLR-MyD88-IRAK-NF-B pathway; the enhanced synthesis of NLRP3 inflammasome components provided via the TLR-NF-B pathway. The immune memory develops due to the epigenetic modifications, associated with (de-) acetylation and (de-) methylation of histones and DNA. During the next phase, the effect of soluble and crystalline urate salts upon macrophages promotes NLRP3 inflammosome activation, due to the following events: 1. К+, Cl- and Са2+ ionic currents shift; 2. lysosomic and mitochondrial damage, leading to the cathepsin B release and enhancement of reactive oxygen production, respectively; 3. NLRP3 relocation between the endoplasmic reticulum, Golgi complex and cytosol; 4. alteration of the NLRP3 structure due to auxiliary proteins attachment, phosphorylation, ubiquitination and acetylation. The NLRP3 inflammosome activity results into increased caspase 1 production which, in turn, produces IL-1 and pyroptotic pores proteins. The pyroptotic pores allow IL-1 passage, which further amplifies inflammation. During the pyroptosis, soluble and crystalline urate salts release from the cells, further increasing inflammation and tissue damage. Understanding the mechanisms of gout-associated inflammation helps us to formulate promising approaches to development of novel treatments. Macrophages are the key cells to crystal-induced inflammation development. Thus, new biothechnologies based on macrophage engineering may appear to be prospective in gout treatment. In this review, we have analyzed the prospects of M3 phenotype macrophages (AB-M3) usage in therapy, unlike M1 and M2 phenotypes, is able to produce anti-inflammatory cytokines in response to the inflammatory factors and, therefore, to inhibit crystal-induced inflammation. Meanwhile, unlike the anti-IL-1 medications, e.g., canakinumab, targeting a single end product of the gout inflammation (IL-1), AB-M3 macrophages inhibit not only IL-1 production, but also a group of other inflammatory cytokines.

Evidence for Monocyte Reprogramming in a Long-Term Postsepsis Study.

OBJECTIVES:This study sought to identify monocyte alterations from septic patients after hospital discharge by evaluating gene expression of inflammatory mediators and monocyte polarization markers. It was hypothesized that sepsis reprograms the inflammatory state of monocytes, causing effects that persist after hospital discharge and influencing patient outcomes.DESIGN:The gene expression patterns of inflammatory receptors, M1 and M2 macrophage polarization markers, NLRP3 inflammasome components, and pro- and anti-inflammatory cytokines in monocytes were assessed.PATIENTS:Thirty-four patients from the University of São Paulo Hospital, during the acute sepsis phase (phase A), immediately after ICU discharge (phase B), and 3 months (phase C), 6 months (phase D), 1 year (phase E), and 3 years (phase F) after discharge, were included. Patients that died during phases A and B were grouped separately, and the remaining patients were collectively termed the survivor group.MEASUREMENTS AND MAIN RESULTS:The gene expression of toll-like receptor (TLR)2 and TLR4 (inflammatory receptors), NLRP3, NFκB1, adaptor molecule apoptosis-associated speck-like protein containing a CARD, caspase 1, caspase 11, and caspase 12 (NLRP3 inflammasome components), interleukin-1α, interleukin-1β, interleukin-18, and high-mobility group box 1 protein (proinflammatory cytokines), interleukin-10 (anti-inflammatory cytokine), C-X-C motif chemokine ligand 10, C-X-C motif chemokine ligand 11, and interleukin-12p35 (M1 inflammatory polarization markers), and C-C motif chemokine ligand 14, C-C motif chemokine ligand 22, transforming growth factor-beta (TGF-β), SR-B1, and peroxisome proliferator-activated receptor γ (M2 anti-inflammatory polarization and tissue repair markers) was upregulated in monocytes from phase A until phase E compared with the control group.CONCLUSIONS:Sepsis reprograms the inflammatory state of monocytes, probably contributing to postsepsis syndrome development and mortality.