NLR Family Pyrin Domain Containing 3 Protein Research Articles

Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Attenuate LPS/Nigericin-Induced Inflammasome Activation by Inhibiting ASC Speck Formation in THP-1 Monocytes.

Inflammasomes are multi-protein complexes, which are formed in response to tissue injury, infections, and metabolic stress. However, aberrant inflammasome activation has been linked to several inflammatory diseases. Anthocyanins have been reported to attenuate NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, but the influence of grape/blueberry anthocyanins and especially their gut-derived metabolites on NLRP3 inflammasome activation in human monocytes remains unclear. Therefore, human leukemic monocytes (THP-1 cells, Tohoku Hospital Pediatrics-1 cells) were preincubated with different concentrations of grape/blueberry anthocyanins, homovanillyl alcohol, or 2,4,6-trihydroxybenzaldehyde (THBA) before the NLRP3 inflammasome was activated by lipopolysaccharide and/or nigericin. Apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, as well as ASC and NLRP3 protein expression, were determined using flow cytometry. Caspase-1 activity was measured in cultured cells, and pro-inflammatory cytokine secretion was determined using enzyme-linked immunosorbent assays. Anthocyanins and their metabolites had no effect on ASC or NLRP3 protein expression. However, THBA significantly inhibited ASC speck formation in primed and unprimed THP-1 monocytes, while caspase-1 activity was significantly declined by grape/blueberry anthocyanins. Furthermore, reduced inflammasome activation resulted in lower pro-inflammatory cytokine secretion. In conclusion, our results show for the first time that grape/blueberry anthocyanins and their gut-derived metabolites exert anti-inflammatory effects by attenuating NLRP3 inflammasome activation in THP-1 monocytes.

Er:YAG laser suppresses pro-inflammatory cytokines expression and inflammasome in human periodontal ligament fibroblasts with Porphyromonas gingivalis-lipopolysaccharide stimulation

Background/purposePeriodontitis is an inflammatory condition of the tooth-supporting structures triggered by the host's immune response towards the bacterial deposits around the teeth. It is well acknowledged that pro-inflammatory interleukin (IL)-6, IL-8, MCP-1 as well as the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, are the key modulators in the activation of this response. Erbium-doped yttrium-aluminium-garnet (Er:YAG) laser, a solid-state crystal laser have been commonly used in the treatment of periodontal diseases. However, little is understood about the molecular mechanism of the Er:YAG laser, especially in targeting the host immune response brought on by periodontal pathogens. Hence, the current study focused on the protective effects of Er:YAG laser on periodontitis in-vitro in terms of pro-inflammatory cytokines, chemokines and NLRP3 inflammasome expressions. Materials and methodsHuman periodontal ligament fibroblast (PDLFs) were first stimulated with lipopolysaccharides (LPS) from P. gingivalis (Pg-LPS) to simulate periodontitis. Cells were then irradiated with Er:YAG laser of ascending energy densities (3.6–6.3 J/cm2), followed by cell proliferation and wound healing assay. Next, the effects of Er:YAG laser on the expressions of IL-6, IL-8, MCP-1, NLRP3, and cleaved GSDMD were examined. ResultsPg-LPS was found to reduce cell's proliferation rate and wound healing ability in PDLFs and these were rescued by Er:YAG laser irradiation. In addition, LPS stimuli resulted in a marked upregulation in the secretion of IL-6, IL-8 and MCP-1 as well as the mRNA and protein expression of NLRP3 and cleaved-GSDMD protein whereas Er:YAG laser suppressed the elicited phenomena. ConclusionTo our knowledge, this is the first study to look into the laser's implication on the NLRP3 inflammasome in periodontitis models. Our study reveals a crucial role of Er:YAG laser in ameliorating periodontitis in-vitro through the modulation of IL-6, IL-8, MCP-1 and the NLRP3 inflammasome and highlights that the control of the NLRP3 inflammasome may become a potential approach for periodontitis.

Allicin protects against LPS-induced cardiomyocyte injury by activating Nrf2-HO-1 and inhibiting NLRP3 pathways.

Allicin is a bioactive compound with potent antioxidative activity and plays a protective effect in myocardial damage and fibrosis. The role and mechanism of Allicin in septic cardiomyopathy are unclear. In this study, we investigated the effects and underlying mechanisms of Allicin on lipopolysaccharide (LPS) induced injury in H9c2 cardiomyocytes. H9c2 cardiomyocyte cells were pretreated with Allicin (0, 25, 50, and 100 µM) for 2h, followed by incubation with LPS (10µg/mL) for 24h at 37°C. Cell viability (cell counting kit-8 [CCK-8]), apoptosis (TUNEL staining), oxidative stress (malondialdehyde [MDA] and superoxide dismutase [SOD]), and cytokines release (Interleukin beta [IL-β], Interleukin 6 [IL-6], and tumor necrosis factor-alpha [TNF-α]) were determined. The mRNA and protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NLR family pyrin domain containing 3 (NLRP3) signaling pathway molecules were quantified by real-time quantitative PCR (RT-qPCR) and western blot, respectively. Allicin had no effect on H9c2 cell viability but attenuated LPS-induced injury, with increased cell viability, reduction in inflammatory cytokines release, apoptosis, reduced MDA, and increased SOD (P < 0.05). Additionally, Allicin increased Nrf2 and cellular HO-1 expressions in LPS-treated H9c2 cells. Moreover, Allicin modulated the NLRP3 inflammasome, increased the cleaved caspase-1 (p10) protein, and attenuated the LPS-induced increase in NLRP3, pro-IL-1β, and IL-1β proteins. Silencing of Nrf2 by siRNA (siNrf2) significantly attenuated Allicin-induced increase in cell viability and HO-1 and decrease in NLRP3 protein in LPS-stimulated H9c2 cells. Allicin protects cardiomyocytes against LPS‑induced injury through activation of Nrf2/HO-1 and inhibition of NLRP3 signaling pathways.

Effects and clinical significance of NLRP3 inflammasome activated by IL-17A in CRSwNP

Objective: To investigate the effects and clinical significance of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activated by interleukin (IL)-17A in chronic rhinosinusitis with nasal polyps (CRSwNP). Methods: Patients underwent nasal endoscopic surgery in the Third Affiliated Hospital of Sun Yat-sen University from January 2020 to December 2021 were collected, including 28 CRSwNP (including 19 males and 9 females, aged 19 to 67 years), 22 chronic rhinosinusitis without nasal polyps (CRSsNP) and 22 controls. qRT-PCR was used to detect the expressions of IL-17A, NLRP3, IL-1β and IL-18 in the three groups, and their correlations were analyzed. The positions of IL-17A, NLRP3 and IL-18 in nasal polys were analyzed by immunofluorescence. Western Blotting and ELISA were employed to detect the expression of NLRP3, IL-1β and IL-18 in the human nasal epithelial cells after using IL-17A stimulation or IL-17A receptor inhibitor. Immunofluorescence was used to observe the NLRP3, IL-1β, and IL-18 protein expression after IL-17A stimulating human nasal epithelial cells, and after the use of IL-17A receptor inhibitor and NLRP3 inhibitor MCC950. The correlations between NLRP3, IL-1β, IL-18 and CT scores, nasal endoscopic scores, visual analogue scale (VAS) scores, and sino-nasal outcome test (SNOT) 22 scores of CRSwNP patients were analyzed. SPSS 20.0 software was used for statistical analysis. Results: The expressions of IL-17A, NLRP3, IL-1β and IL-18 in the tissues of CRSwNP patients were significantly higher than those in CRSsNP group(P=0.018,P<0.001,P=0.005, P=0.016) and the control group(all P<0.001). IL-17A was positively correlated with the expression of NLRP3, IL-1β, and IL-18(r ralue was 0.643,0.650,0.629,respectively, all P<0.05). IL-17A, NLRP3, and IL-18 were co-localized in the epithelial propria of polyp tissue. IL-17A stimulated the expressions of NLRP3, IL-1β, and IL-18 in human nasal epithelial cells. After the use of IL-17A receptor inhibitor, the expressions of NLRP3, IL-1β, and IL-18 were significantly down-regulated. After the use of NLRP3 inhibitor MCC950, IL-17A was significantly down-regulated to promote the expression of NLRP3, IL-1β, and IL-18. The expressions of NLRP3, IL-1β and IL-18 were positively correlated with CT, nasal endoscopy, VAS, and SNOT22 scores in patients with CRSwNP. Conclusions: IL-17A promotes the release of IL-1β and IL-18 by activating the NLRP3 inflammasome and aggravates the severity of the disease in CRSwNP.

Hepatitis E virus infection remodels the mature tRNAome in macrophages to orchestrate NLRP3 inflammasome response

Hepatitis E virus (HEV) infection has been shown to activate NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in macrophages, a key mechanism of causing pathological inflammation, but the mechanisms regulating this response remain poorly understood. Here, we report that the mature tRNAome dynamically responds to HEV infection in macrophages. This directs IL-1β expression, the hallmark of NLRP3 inflammasome activation, at mRNA and protein levels. Conversely, pharmacological inhibition of inflammasome activation abrogates HEV-provoked tRNAome remodeling, revealing a reciprocal interaction between the mature tRNAome and the NLRP3 inflammasome response. Remodeling the tRNAome results in improved decoding of codons directing leucine- and proline synthesis, which are the major amino acid constituents of IL-1β protein, whereas genetic or functional interference with tRNAome-mediated leucine decoding impairs inflammasome activation. Finally, we demonstrated that the mature tRNAome also actively responds to lipopolysaccharide (a key component of gram-negative bacteria)-triggered inflammasome activation, but the response dynamics and mode of actions are distinct from that induced by HEV infection. Our findings thus reveal the mature tRNAome as a previously unrecognized but essential mediator of host response to pathogens and represent a unique target for developing anti-inflammatory therapeutics.

Tanshinone IIA mediates protection from diabetes kidney disease by inhibiting oxidative stress induced pyroptosis

Ethnopharmacological relevanceSalvia miltiorrhiza is widely used traditional Chinese medicine in the treatment of diabetes kidney disease (DKD). Tanshinone IIA (Tan IIA) are one of the main components of the root of red-rooted Salvia miltiorrhiza Bunge. However, whether Tan IIA delay the progression of DKD and the underlying mechanisms are unknown. Aim of the studyClarify the mechanisms underlying the occurrence and progression of DKDs from a novel viewpoint and confirm the function and mechanism of Tan IIA. Materials and methodsWe experimented with models of DKD (db/db mice) and cultured human renal glomerular endothelial cells (HRGECs). We measured the biochemical indicators of mouse blood and urine to confirmed that Tan IIA exerted protective effects on the kidneys of db/db mice. Renal histopathology and immunohistochemical staining were used to determine the role of Tan IIA. High glucose-induced HRGECs pyroptosis based on the results of western blot, CCK-8 cell viability test, calcein/PI staining, ROS/superoxide anion generation and transmission electron microscope. We also confirmed that Tan IIA alleviated HRGEC pyroptosis through the same methods. The relationships between oxidative induction and regulation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation were investigated using western blot following the application of an NLRP3 inhibitor and oxidative stress inhibitor. ResultsTan IIA alleviated kidney injury and improved the levels of urine, blood indicators, the expression of NLRP3 and thioredoxin-interacting protein (Txnip) in db/db mice kidney. In vitro, high glucose inhibited HRGECs viability, increased ROS generation, enhanced the proportion of propidium iodide-stained cells. In addition, we discovered the expression of GSDMD-NT, NLRP3, cleaved IL-1β, cleaved caspase-1, and Txnip increased, but the expression of Trx1 decreased after treated by high glucose. These changes were partially ameliorated by Tan IIA. ConclusionHyperglycemia could induce pyroptosis in renal glomerular endothelial cells. However, Tan IIA could delay the progression of DKD by inhibiting pyroptosis by regulating the Txnip/NLRP3 inflammasome.

Apigenin Alleviated High-Fat-Diet-Induced Hepatic Pyroptosis by Mitophagy-ROS-CTSB-NLRP3 Pathway in Mice and AML12 Cells

Apigenin is considered the most-known natural flavonoid and is abundant in a wide variety of fruits and vegetables. A high fat diet (HFD) can induce liver injury and hepatocyte death in multiple ways. Pyroptosis is an innovative type of programmed cell death. Moreover, excessive pyroptosis of hepatocytes leads to liver injury. We used HFD to induce liver cell pyroptosis in C57BL/6J mice in this work. After gavage of apigenin, apigenin can significantly reduce the level of lactate dehydrogenase (LDH) in liver tissue ignited by HFD and reduce the levels of NLRP3 (NOD-like receptor family pyrin domain containing 3), the N-terminal domain of GSDMD (GSDMD-N), cleaved-caspase 1, cathepsin B (CTSB), interleukin-1β (IL-1β) and interleukin-18 (IL-18) protein expression and the colocalization of NLRP3 and CTSB and increase the level of lysosomal associated membrane protein-1 (LAMP-1) protein expression, thus alleviating cell pyroptosis. In a further in vitro mechanism study, we find that palmitic acid (PA) can induce pyroptosis in AML12 cells. After adding apigenin, apigenin can clear the damaged mitochondria through mitophagy and reduce the generation of intracellular reactive oxygen species (ROS), thus alleviating CTSB release caused by lysosomal membrane permeabilization (LMP), reducing the LDH release caused by PA and reducing the levels of NLRP3, GSDMD-N, cleaved-caspase 1, CTSB, IL-1β, and IL-18 protein expression. By adding the mitophagy inhibitor cyclosporin A (CsA), LC3-siRNA, the CTSB inhibitor CA-074 methyl ester (CA-074 Me), and the NLRP3 inhibitor MCC950, the aforementioned results were further confirmed. Therefore, our results show that HFD-fed and PA can damage mitochondria, promote the production of intracellular ROS, enhance the lysosomal membrane permeabilization (LMP), and cause the leakage of CTSB, thus activating the NLRP3 inflammatory body and inducing pyroptosis in C57BL/6J mice and AML12 cells, while apigenin alleviates this phenomenon through the mitophagy-ROS-CTSB-NLRP3 pathway.

Melatonin-Driven NLRP3 Inflammation Inhibition Via Regulation of NF-κB Nucleocytoplasmic Transport: Implications for Postoperative Cognitive Dysfunction.

The aseptic inflammatory response of the central nervous system is one of the important causes of neurodegenerative diseases in individuals and is also recognized in postoperative cognitive dysfunction (POCD). Inflammasome is thought to be closely related to brain homeostasis. However, there are few drugs targeting the inflammasome to suppress inflammation in clinical practice. Here, we showed that the neuroinflammatory response mediated by the NLRP3 (NLR family, pyrin domain containing 3) inflammasome was involved in the pathological process of POCD. Melatonin protected mice from nerve damage by inhibiting activation of the NLRP3-caspase-1-interleukin 1 beta (IL-β) pathway and thus reduced the secretion of IL-1β inflammatory factors in microglia. Further research found that melatonin has a potential binding effect with NLRP3 protein, and at the same time could reduce the phosphorylation of nuclear factor kappa-B (NF-κB) and inhibit its nuclear translocation. The underlying mechanism was that melatonin inhibited the expression of acetylation of histone H3 and melatonin attenuated the binding of NF-κb to the NLRP3 promoter region 1-200bp, where there are two potential binding target sites of NF-κb and NLRP3, namely the sequences 5'-GGGAACCCCC-3' and 5'-GGAAATCCA -3'. Therefore, we confirmed a novel mechanism of action of melatonin in the prevention and treatment of POCD.

Upregulation of HTRA1 mediated by the lncRNA NEAT1/miR-141-3p axis contributes to endometriosis development through activating NLRP3 inflammasome-mediated pyroptotic cell death and cellular inflammation

The present study identified a novel upstream long chain non-coding (lncRNA) NEAT1/miR-141-3p/HTRA1 axis that regulated the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome to modulate endometriosis (EM) development. Specifically, clinical data suggested that the expression of NLRP3 and apoptosis-associated speck-like protein containing CARD (ASC), the cleavage of caspase-1 and gasdermin D (GSDMD), and the production of inflammatory cytokines (interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-18) were all significantly increased in the ectopic endometrium (EE) tissues, compared to the normal endometrium (NE) tissues. Then, through analyzing the datasets from GEO database (GSE2339, GSE58178, and GSE7305) using the GEO2R bioinformatics tools, we verified that HtrA Serine Peptidase 1 (HTRA1) was especially enriched in the EE tissues compared to the NE tissues. To further confirm the biological functions of HTRA1, HTRA1 was overexpressed or downregulated in primary human endometrial stromal cells (hESCs) isolated from NE tissues or EE tissues, respectively. The results showed that upregulation of HTRA1 activated NLRP3 inflammasome-mediated pyroptotic cell death and cellular inflammation in NE-derived hESCs, whereas silencing of HTRA1 played an opposite role in EE-derived hESCs. In addition, the lncRNA NEAT1/miR-141-3p axis was screened as the upstream regulator of HTRA1. Mechanistically, lncRNA NEAT1 sponged miR-141-3p to positively regulate HTRA1 in a competing endogenous RNA (ceRNA) mechanisms-dependent manner. The recovery experiments in hESCs from NE and EE tissues confirmed that lncRNA NEAT1 overexpression promoted NLRP3 inflammasome-mediated pyroptotic cell death through regulating the miR-141-3p/HTRA1 axis. Taken together, this study firstly uncovered the underlying mechanisms by which a novel lncRNA NEAT1/miR-141-3p/HTRA1-NLRP3 pathway contributed to the development of EM, which provided novel diagnostic and therapeutic biomarkers for this disease.

The Effects of Curcumin on Inflammasome: Latest Update.

Curcumin, a traditional Chinese medicine extracted from natural plant rhizomes, has become a candidate drug for the treatment of different diseases due to its anti-inflammatory, anticancer, antioxidant, and antibacterial activities. Curcumin is generally beneficial to improve human health with anti-inflammatory and antioxidative properties as well as antitumor and immunoregulatory properties. Inflammasomes are NLR family, pyrin domain-containing 3 (NLRP3) proteins that are activated in response to a variety of stress signals and that promote the proteolytic conversion of pro-interleukin-1β and pro-interleukin-18 into active forms, which are central mediators of the inflammatory response; inflammasomes can also induce pyroptosis, a type of cell death. The NLRP3 protein is involved in a variety of inflammatory pathologies, including neurological and autoimmune disorders, lung diseases, atherosclerosis, myocardial infarction, and many others. Different functional foods may have preventive and therapeutic effects in a wide range of pathologies in which inflammasome proteins are activated. In this review, we have focused on curcumin and evidenced its therapeutic potential in inflammatory diseases such as neurodegenerative diseases, respiratory diseases, and arthritis by acting on the inflammasome.

USP9X promotes lipopolysaccharide‐stimulated acute lung injury by deubiquitination of NLRP3

Alveolar epithelial cells (AECs) function as a vital defense barrier avoiding the invasion of exogenous agents and preserving the functional and structural integrity of lung tissues, while damage/breakdown of this airway epithelial barrier is frequently associated with the pathogenesis of acute lung injury (ALI). NOD-like receptor family, pyrindomain-containing 3(NLRP3) inflammasome activation-associated pyroptosis is involved in the development of ALI. Yet, how the activity of NLRP3 inflammasome is regulated in the context of ALI remains unknown. Herein we hypothesized that USP9X, an important deubiquitinase, participates in modulating the activation of NLRP3 inflammasome, thereby affecting the phenotypes in a lipopolysaccharide(LPS)-stimulated AEC model. Human pulmonary AECs were subjected to LPS/adenosine triphosphate (ATP) treatment to induce NLRP3 inflammasome activation and cell pyroptosis. Knockdown and overexpression of USP9X were applied to validate the function of USP9X. Inhibitors of proteinase and protein synthesis, as well as approach of co-immunoprecipitation coupled with Western blot, were utilized to explore the molecular mechanism. LPS/ATP challenge resulted in pronouncedly increased pyroptosis of AECs, activation of NLRP3 inflammasome and release of interleukin (IL)-1β and IL-18 cytokines, while downregulation of USP9X could reverse these alterations. USP9X was found to have marked impact on NLRP3 protein instead of mRNA level. Furthermore, increased ubiquitination of NLRP3 was observed upon downregulating USP9X. Additionally, the inhibitory effect of USP9X downregulation was reversed by NLRP3 overexpression, while the promoting impact of USP9X overexpression was dampened by NLRP3 inhibitor in terms of cell pyroptosis and cytokine secretion. USP9X modulated the activity of NLRP3 inflammasome and pyroptosis of AECs via its deubiquitination function.

Development of novel oridonin analogs as specifically targeted NLRP3 inflammasome inhibitors for the treatment of dextran sulfate sodium-induced colitis

Abnormal activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome is closely associated with a variety of inflammatory diseases. Herein, we describe the discovery and optimization of a series of NLRP3 inflammasome inhibitors based on the oridonin skeleton. These inhibitors exhibited moderate to potent inhibitory activity against interleukin 1β (IL-1β) release. Compound E6 showed the strongest inhibitory activity and better safety range against IL-1β (IC50 = 0.45 ± 0.02 μM, selectivity index = 36.49). Compared with oridonin, the activity and selectivity index of compound E6 increased 11.5 and 7.2 times, respectively. Compound E6 also exhibited broad-spectrum activity and specificity. Compound E6 mainly reduced the release of IL-1β by targeting the NLRP3 protein, thereby inhibiting the NLRP3-caspase 1-gasdermin D (GSDMD), as well as inhibiting the caspase 4-GSDMD signaling pathway. Further studies revealed an important therapeutic effect of E6 on dextran sulfate sodium-induced colitis. Compound E6 may be promising for the treatment of NLRP3-related diseases including inflammatory bowel disease.

NLRP3 inflammasome activation contributes to the cognitive decline after cardiac surgery.

Perioperative neurocognitive disorders (PND) are a common complication of cardiac surgery in elderly patients. The etiopathogenesis of PND is not clear. Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, a macromolecular protein complex, regulates inflammation by inducing the release of proinflammatory cytokines interleukin (IL)-1β and IL-18. Studies have demonstrated a close link between the NLRP3 inflammasome and central nervous system diseases. Nevertheless, the involvement of NLRP3 inflammasome in the causation of PND occurring after cardiac surgery is unclear. This study aimed to investigate the association of serum NLRP3 level with PND. We performed a retrospective study, enrolled 75 patients undergoing elective cardiac surgery and evaluated their cognitive functions one day before and 7 days after surgery. PND were determined according to the International Study of Postoperative Cognitive Dysfunction studies. Demographics and perioperative parameters were recorded. Perioperative serum NLRP3 protein, IL-1β, and IL-18 levels were monitored. The PND incidence in our cohort was 33.33%. NLRP3 protein levels were significantly increased in all patients at each postoperative time-point after general anesthesia and cardiac surgery under cardiopulmonary bypass. Patients showing cognitive dysfunction had higher serum NLRP3 protein, caspase-1, IL-1β, and IL-18 levels immediately after the operation. Variables associated with the incidence of early PND were included in the regression models. After adjusting for confounding variables, high serum NLRP3 protein level at the end of the operation and old age were identified as independent predictors of PND. High serum NLRP3 protein level at the completion of cardiac surgery was associated with a higher risk of PND seven days after surgery. The study was registered at Clinicaltrials.gov (registration number: NCT04191642).

Arsenic Activates the NLRP3 Inflammasome and Disturbs the Th1/Th2/Th17/Treg Balance in the Hippocampus in Mice.

Arsenic exerts neurotoxicity and immunomodulatory effects. Studies have shown that the nervous system is not considered to be an immune-privileged site. However, the effect of arsenic-induced neuroimmune toxicity has rarely been reported. We aimed to investigate the toxic effects of arsenic on the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and the Th1/Th2/Th17/Treg balance in the brain tissue of mice. Mice were exposed to NaAsO2 (0, 2.5, 5, and 10mg/kg) for 24h. Our results showed that 10mg/kg arsenic exposure significantly decreased brain and hippocampal indices (p < 0.05). The mRNA and protein levels of the blood‒brain barrier (BBB) tight junction protein occludin were decreased in the 5 and 10mg/kg arsenic-treated groups. Compared with those in the control group, NLRP3 protein levels in 10mg/kg arsenic-treated mice, caspase-1 protein levels in 2.5, 5, and 10mg/kg arsenic-treated mice, and IL-1β protein levels in 5 and 10mg/kg arsenic-treated mice were increased in the hippocampus (p < 0.05). In addition, arsenic induced a hippocampal inflammatory response by upregulating the mRNA levels of the proinflammatory factors IL-6 and TNF-α and downregulating the mRNA level of the anti-inflammatory factor IL-10. Moreover, arsenic decreased the mRNA levels of the Th1 and Th2 transcription factors T-bet and GATA3 and the cytokines IFN-γ and IL-4 and increased the mRNA levels of the Th17 transcription factor RORγt and the cytokine IL-22 (p < 0.05). Collectively, our study demonstrated that arsenic could induce immune-inflammatory responses by regulating the NLRP3 inflammasome and CD4+ T lymphocyte differentiation. These results provide a novel strategy to block the arsenic-induced impairment of neuroimmune responses.

Desmosterol suppresses macrophage inflammasome activation and protects against vascular inflammation and atherosclerosis

Cholesterol biosynthetic intermediates, such as lanosterol and desmosterol, are emergent immune regulators of macrophages in response to inflammatory stimuli or lipid overloading, respectively. However, the participation of these sterols in regulating macrophage functions in the physiological context of atherosclerosis, an inflammatory disease driven by the accumulation of cholesterol-laden macrophages in the artery wall, has remained elusive. Here, we report that desmosterol, the most abundant cholesterol biosynthetic intermediate in human coronary artery lesions, plays an essential role during atherogenesis, serving as a key molecule integrating cholesterol homeostasis and immune responses in macrophages. Depletion of desmosterol in myeloid cells by overexpression of 3β-hydroxysterol Δ24-reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol, promotes the progression of atherosclerosis. Single-cell transcriptomics in isolated CD45+CD11b+ cells from atherosclerotic plaques demonstrate that depletion of desmosterol increases interferon responses and attenuates the expression of antiinflammatory macrophage markers. Lipidomic and transcriptomic analysis of invivo macrophage foam cells demonstrate that desmosterol is a major endogenous liver X receptor (LXR) ligand involved in LXR/retinoid X receptor (RXR) activation and thus macrophage foam cell formation. Decreased desmosterol accumulation in mitochondria promotes macrophage mitochondrial reactive oxygen species production and NLR family pyrin domain containing 3 (NLRP3)-dependent inflammasome activation. Deficiency of NLRP3 or apoptosis-associated speck-like protein containing a CARD (ASC) rescues the increased inflammasome activity and atherogenesis observed in desmosterol-depleted macrophages. Altogether, these findings underscore the critical function of desmosterol in the atherosclerotic plaque to dampen inflammation by integrating with macrophage cholesterol metabolism and inflammatory activation and protecting from disease progression.

NLRP3 Inflammasome Assembly in Neutrophils Is Supported by PAD4 and Promotes NETosis Under Sterile Conditions.

Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of human disorders. While NETosis is known to be regulated by peptidylarginine deiminase 4 (PAD4), the role of the NLRP3 inflammasome in NETosis was not addressed. Here, we establish that under sterile conditions the cannonical NLRP3 inflammasome participates in NETosis. We show apoptosis-associated speck-like protein containing a CARD (ASC) speck assembly and caspase-1 cleavage in stimulated mouse neutrophils without LPS priming. PAD4 was needed for optimal NLRP3 inflammasome assembly by regulating NLRP3 and ASC protein levels post-transcriptionally. Genetic ablation of NLRP3 signaling resulted in impaired NET formation, because NLRP3 supported both nuclear envelope and plasma membrane rupture. Pharmacological inhibition of NLRP3 in either mouse or human neutrophils also diminished NETosis. Finally, NLRP3 deficiency resulted in a lower density of NETs in thrombi produced by a stenosis-induced mouse model of deep vein thrombosis. Altogether, our results indicate a PAD4-dependent formation of the NLRP3 inflammasome in neutrophils and implicate NLRP3 in NETosis under noninfectious conditions in vitro and in vivo.

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl- concentration.

The impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) activity induces intracellular chloride (Cl- ) accumulation. The anion Cl- , acting as a second messenger, stimulates the secretion of interleukin-1β (IL-1β), which starts an autocrine positive feedback loop. Here, we show that NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) are indirectly modulated by the intracellular Cl- concentration, showing maximal expression and activity at 75mM Cl- , in the presence of the ionophores nigericin and tributyltin. The expression of PYD and CARD domain containing (PYCARD/ASC) remained constant from 0 to 125mM Cl- . The CASP1 inhibitor VX-765 and the NLRP3 inflammasome inhibitor MCC950 completely blocked the Cl- -stimulated IL-1β mRNA expression and partially the IL-1β secretion. DCF fluorescence (cellular reactive oxygen species, cROS) and MitoSOX fluorescence (mitochondrial ROS, mtROS) also showed maximal ROS levels at 75mM Cl- , a response strongly inhibited by the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase (NOX) inhibitor GKT137831. These inhibitors also affected CASP1 and NLRP3 mRNA and protein expression. More importantly, the serum/glucocorticoid regulated kinase 1 (SGK1) inhibitor GSK650394, or its shRNAs, completely abrogated the IL-1β mRNA response to Cl- and the IL-1β secretion, interrupting the autocrine IL-1β loop. The results suggest that Cl- effects are mediated by SGK1, in which under Cl- modulation stimulates the secretion of mature IL-1β, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1β itself, through autocrine signalling.

Heme Oxygenase-1 in Macrophages Impairs the Perfusion Recovery After Hindlimb Ischemia by Suppressing Autolysosome-Dependent Degradation of NLRP3.

[Figure: see text].

NLRP3 inflammasome of renal tubular epithelial cells induces kidney injury in acute hemolytic transfusion reactions.

BackgroundBlood transfusion, a common basic supporting therapy, can lead to acute hemolytic transfusion reaction (AHTR). AHTR poses a great risk to patients through kidney function damage in a short time. Previous reports found that heme from destroyed red blood cells impaired kidney function, and NLR family pyrin domain containing 3 (NLRP3) inflammasome was augmented in case of kidney injury. However, the detailed mechanism regarding whether NLRP3 inflammasome is involved in kidney function injury in AHTR is not fully understood yet.MethodsHemolysis models were established by vein injection with human blood plasma or mouse heme from destroyed red blood cells. The injured renal tubular epithelial cells (RTECs) were evaluated by tubular damage markers staining in hemolysis models and in primary RTECs in vitro. The activation of NLRP3 inflammasome in RTECs by hemes was investigated by Western blot, ELISA, scanning electron microscopy, immunofluorescent staining, flow cytometry, and hemolysis models. NLRP3 gene knockout mice were employed to confirm these observations in vitro and in vivo. The binding between a novel inhibitor (66PR) and NLRP3 was affirmed by molecule docking and co‐immunoprecipitation. The rescue of 66PR on kidney function impairment was explored in murine hemolysis models.ResultsWe found that heme could activate NLRP3 inflammasome in RTECs to induce kidney function injury. NLRP3 gene knockout could prevent the damage of RTECs caused by hemes and recover kidney function in AHTR. Moreover, NLRP3 inflammasome chemical inhibitor, 66PR, could bind to NLRP3 protein and inhibit inflammasome activation in RTECs, which consequently relieved the injury of RTECs caused by hemes, and alleviated kidney function damage in the AHTR model.ConclusionsHemes could activate NLRP3 inflammasome in RTECs, and a novel NLRP3 inflammasome inhibitor named 66PR relieved kidney function damage in AHTR. Our findings provided a new possible strategy to treat kidney function failure in AHTR.

Macrophage inflammatory state in Type 1 diabetes: triggered by NLRP3/iNOS pathway and attenuated by docosahexaenoic acid.

Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease characterized by insulin-producing pancreatic β-cell destruction and hyperglycemia. While monocytes and NOD-like receptor family-pyrin domain containing 3 (NLRP3) are associated with T1D onset and development, the specific receptors and factors involved in NLRP3 inflammasome activation remain unknown. Herein, we evaluated the inflammatory state of resident peritoneal macrophages (PMs) from genetically modified non-obese diabetic (NOD), NLRP3-KO, wild-type (WT) mice and in peripheral blood mononuclear cells (PBMCs) from human T1D patients. We also assessed the effect of docosahexaenoic acid (DHA) on the inflammatory status. Macrophages from STZ-induced T1D mice exhibited increased inflammatory cytokine/chemokine levels, nitric oxide (NO) secretion, NLRP3 and iNOS protein levels, and augmented glycolytic activity compared to control animals. In PMs from NOD and STZ-induced T1D mice, DHA reduced NO production and attenuated the inflammatory state. Furthermore, iNOS and IL-1β protein expression levels and NO production were lower in the PMs from diabetic NLRP3-KO mice than from WT mice. We also observed increased IL-1β secretion in PBMCs from T1D patients and immortalized murine macrophages treated with advanced glycation end products and palmitic acid. The present study demonstrated that the resident PMs are in a proinflammatory state characterized by increased NLRP3/iNOS pathway-mediated NO production, up-regulated proinflammatory cytokine/chemokine receptor expression and altered glycolytic activity. Notably, ex vivo treatment with DHA reverted the diabetes-induced changes and attenuated the macrophage inflammatory state. It is plausible that DHA supplementation could be employed as adjuvant therapy for treating individuals with T1D.