Expression of human caspase-4 in the gingival epithelium affected with periodontitis: Its involvement in Porphyromonas gingivalis-challenged gingival epithelial cells.

OBJECTIVE: Implication of human caspase-4 in periodontitis and in sensing periodontal pathogens by gingival epithelial cells (GECs) is unclear. This study aimed to determine caspase-4 and interleukin (IL)-18 expressions in gingival tissues affected with periodontitis and to investigate caspase-4 involvement in mediating innate immune responses in GECs. DESIGN: Ex vivo, caspase-4 and IL-18 expressions in gingival biopsies, obtained from healthy participants with periodontitis or clinically healthy gingiva (N = 20 each), were determined by immunohistochemistry. In vitro, caspase-4 activation in cultured GECs stimulated with Porphyromonas gingivalis or Fusobacterium nucleatum was analyzed by immunoblotting. mRNA expressions of human ß-defensin-2 (hBD-2), IL-8, and IL-18 in stimulated GECs in the presence or absence of a caspase-4 inhibitor were assayed by RT-qPCR. RESULTS: Ex vivo, compared with healthy gingival epithelium, the epithelium affected with periodontitis displayed a significant decrease in caspase-4 expression (P = 0.015), whereas IL-18 expression was significantly increased (P = 0.012). Moreover, the expression of caspase-4, but not IL-18, was found to be a predictor of periodontitis (P = 0.007). In vitro, caspase-4 was activated in cultured GECs challenged with P. gingivalis, but not F. nucleatum. mRNA upregulations of hBD-2, IL-8, and IL-18 upon P. gingivalis stimulation were significantly reduced when caspase-4 was inhibited (P < 0.05), whereas the inhibitor failed to suppress those inductions by F. nucleatum. CONCLUSIONS: Caspase-4 expression is diminished in the epithelium affected with periodontitis while that of IL-18 is enhanced. Caspase-4 activation in P. gingivalis-infected GECs upregulates the three innate immune effector molecules, suggesting a possible sensing mechanism of caspase-4 in GECs in periodontal disease pathogenesis.

Protective role of irbesartan against cyclophosphamide-induced testicular damage in rats via up-regulating PPAR-γ signaling and ameliorating NF-κB/NLRP3/IL-18 inflammatory axis.

BACKGROUND: Cancer and its therapies can impact fertility in various ways, and therefore a growing number of cancer survivors face fertility as a significant concern. The cytotoxic alkylating agent cyclophosphamide (CP) is commonly used as an antineoplastic agent; unfortunately, its use is significantly associated with male infertility and damage to the reproductive system. AIM: The present study aimed to assess the possible beneficial effects of Irbesartan (IRB) in a rat model of CP-induced testicular toxicity. MAIN METHODS: The effects of treatment were assessed by measuring peroxisome proliferator-activated receptor gamma (PPAR-γ) expression via qRT-PCR, the immunohistochemical (IHC) assessment of apoptotic markers, NOD-like receptor protein 3 (NLRP3), and nuclear factor-κB (NF-κB), determination of the count and viability of epididymal sperm, oxidative stress markers via biochemical analysis, serum testosterone, caspase-1, and interleukin-18 (IL-18) levels via ELISA, histopathological assessment, and fibrosis by Masson's trichrome (MT) stain. KEY FINDINGS: There was a significant increase in malondialdehyde (MDA), caspase-1, and IL-18 contents, NF-κB, NLRP3, Bcl-2-associated X protein (Bax), caspase-3, and MT staining in testicular tissue after CP administration compared to the normal control group. Whereas reduced glutathione (GSH), superoxide dismutase (SOD), PPAR-γ expression, B-cell lymphoma-2 (Bcl-2) staining, serum testosterone, and the count and viability of epididymal sperm were decreased compared to the normal control group. The IRB treatment has reversed CP-induced testicular toxicity. SIGNIFICANCE: It is possible to conclude that IRB revealed a significant testicular protective effect against CP via antioxidant, anti-apoptotic, and anti-inflammatory effects.

N-Acetylcysteine (NAC) Inhibits Synthesis of IL-18 in Macrophage by Suppressing NLRP3 Expression to Reduce the Production of IFN-γ from NK Cells.

BACKGROUND: N-Acetylcysteine (NAC) had exerted antioxidation and anti-inflammation effects on chronic obstructive pulmonary disease (COPD) patients. However, its effect in regulating interleukin- (IL-) 18 was not fully understood. This study was designed to evaluate the specific mechanism of NAC regulating IL-18. MATERIALS AND METHODS: A total of 112 COPD patients and 103 health individuals were recruited in the study. Cytokine level in patients' serum was measured by enzyme-linked immunosorbent assay (ELISA). A COPD mouse model was established by administration of lipopolysaccharide (LPS) and cigarette smoke. The expression of cytokines was measured by ELISA and flow cytometry. Inflammasome-related protein was measured by Western blot. RESULT: NAC could effectively improve the immune status of COPD patients as well as the COPD mouse model by downregulating proinflammation and inflammation cytokines including IL-1ß, interferon- (IFN-) γ, tumor necrosis factor- (TNF-) α, and IL-18. It also had the capability to suppress synthesis of IL-18 in macrophage to inhibit the secretion of IFN-γ from natural killer (NK) cells through influencing the inflammasome-related protein in macrophages. CONCLUSION: NAC could effectively inhibit the production of IL-18 by suppressing NLRP3 expression in macrophages to reduce the production of IFN-γ in NK cells.

Pyroptosis in the Retinal Neurovascular Unit: New Insights Into Diabetic Retinopathy.

Diabetic retinopathy (DR) is prevalent among people with long-term diabetes mellitus (DM) and remains the leading cause of visual impairment in working-aged people. DR is related to chronic low-level inflammatory reactions. Pyroptosis is an emerging type of inflammatory cell death mediated by gasdermin D (GSDMD), NOD-like receptors and inflammatory caspases that promote interleukin-1ß (IL-1ß) and IL-18 release. In addition, the retinal neurovascular unit (NVU) is the functional basis of the retina. Recent studies have shown that pyroptosis may participate in the destruction of retinal NVU cells in simulated hyperglycemic DR environments. In this review, we will clarify the importance of pyroptosis in the retinal NVU during the development of DR.

Caspase-1-Inhibitor AC-YVAD-CMK Inhibits Pyroptosis and Ameliorates Acute Kidney Injury in a Model of Sepsis.

OBJECTIVE: To observe the protective effect of AC-YVAD-CMK on sepsis-induced acute kidney injury in mice and to explore its possible mechanisms primarily. METHODS: Eighteen male C57BL/6 mice were randomly divided into sham-operated group (Control), cecal ligation and puncture group (CLP), and CLP model treated with AC-YVAD-CMK group (AC-YVAD-CMK) (n = 6 in each group). Mice were sacrificed at 24 h after operation, and blood and kidney tissue samples were collected for analyses. Histologic changes were determined microscopically following HE staining. The expression of Ly-6B and CD68 was investigated using immunohistochemistry. Serum concentrations of creatinine (sCR) and blood urea nitrogen (BUN) were measured. Serum levels of interleukin-1ß (IL-1ß), interleukin-18 (IL-18), TNF-α, and interleukin-6 (IL-6) were determined by ELISA. The expressions of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues were investigated using Western blot. Immunofluorescence staining was used to detect the expression of GSDMD protein in renal tissues. RESULTS: AC-YVAD-CMK treatment significantly alleviates sepsis-induced acute kidney injury, with decreased histological injury in renal tissues, suppresses the accumulation of neutrophils and macrophages in renal tissues, and decreased sCR and BUN level (P < 0.05). Attenuation of sepsis-induced acute kidney injury was due to the prohibited production of inflammatory cytokines and decrease expression of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues. In addition, AC-YVAD-CMK treatment significantly reduced the expression of GSDMD in renal tissues compared to those observed in controls (P < 0.05). CONCLUSIONS: We demonstrated a marked renoprotective effect of caspase-1-inhibitor AC-YVAD-CMK in a rat model of sepsis by inhibition of pyroptosis.

Genetic and commensal induction of IL-18 drive intestinal epithelial MHCII via IFNγ.

Major histocompatibility complex class II (MHCII) is dynamically expressed on intestinal epithelial cells (IECs) throughout the intestine, but its regulation remains poorly understood. We observed that spontaneous upregulation of IEC MHCII in locally bred Rag1-/- mice correlated with serum Interleukin (IL)-18, was transferrable via co-housing to commercially bred immunodeficient mice and could be inhibited by both IL-12 and IL-18 blockade. Overproduction of intestinal IL-18 due to an activating Nlrc4 mutation upregulated IEC MHCII via classical inflammasome machinery independently of immunodeficiency or dysbiosis. Immunodeficient dysbiosis increased Il-18 transcription, which synergized with NLRC4 inflammasome activity to drive elevations in serum IL-18. This IL-18-MHCII axis was confirmed in several other models of intestinal and systemic inflammation. Elevated IL-18 reliably preceded MHCII upregulation, suggesting an indirect effect on IECs, and mice with IL-18 overproduction showed activation or expansion of type 1 lymphocytes. Interferon gamma (IFNg) was uniquely able to upregulate IEC MHCII in enteroid cultures and was required for MHCII upregulation in several in vivo systems. Thus, we have linked intestinal dysbiosis, systemic inflammation, and inflammasome activity to IEC MHCII upregulation via an intestinal IL-18-IFNg axis. Understanding this process may be crucial for determining the contribution of IEC MHCII to intestinal homeostasis, host defense, and tolerance.

DHX15 is required to control RNA virus-induced intestinal inflammation.

RNA helicases play critical roles in various biological processes, including serving as viral RNA sensors in innate immunity. Here, we find that RNA helicase DEAH-box helicase 15 (DHX15) is essential for type I interferon (IFN-I, IFN-ß), type III IFN (IFN-λ3), and inflammasome-derived cytokine IL-18 production by intestinal epithelial cells (IECs) in response to poly I:C and RNA viruses with preference of enteric RNA viruses, but not DNA virus. Importantly, we generate IEC-specific Dhx15-knockout mice and demonstrate that DHX15 is required for controlling intestinal inflammation induced by enteric RNA virus rotavirus in suckling mice and reovirus in adult mice in vivo, which owes to impaired IFN-ß, IFN-λ3, and IL-18 production in IECs from Dhx15-deficient mice. Mechanistically, DHX15 interacts with NLRP6 to trigger NLRP6 inflammasome assembly and activation for inducing IL-18 secretion in IECs. Collectively, our report reveals critical roles for DHX15 in sensing enteric RNA viruses in IECs and controlling intestinal inflammation.

N-acetylserotonin alleviated the expression of interleukin-1ß in retinal ischemia-reperfusion rats via the TLR4/NF-κB/NLRP3 pathway.

This study aimed to explore the effects of N-acetylserotonin (NAS) on the expression of interleukin-1ß (IL-1ß) in the retina of retinal ischemia-reperfusion injury (RIRI) rats via the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB)/nod-like receptor pyrin domain containing 3 (NLRP3) signaling pathway. In this study, adult male Sprague Dawley rats were randomly divided into the sham, RIRI, RIRI + NAS and RIRI + TAK-242 + NAS groups. The rats in the RIRI + NAS and RIRI + TAK-242 + NAS groups were intraperitoneally injected with NAS 30 min before and after modeling. TAK-242, a selective TLR4 inhibitor, was administered by intraperitoneal injection in RIRI + TAK-242 + NAS group. The RIRI rat model was established by elevating the intraocular pressure to 110 mmHg for 60 min. The retinal structure and edema were assessed by H&E staining. The expression levels of TLR4, phosphorylated NF-κB (p-NF-κB), NLRP3, cleaved Caspase-1, and IL-1ß in the retina of each group were detected using immunohistochemistry and Western blot. The correlations of the differences of TLR4+ and cleaved Caspase-1+ with IL-1ß+ cells (between the NAS and the RIRI groups) were analyzed, using linear regression in the RIRI + NAS group. Results showed that thinner retina, more RGCs, and less TLR4+, p-NF-κB+, NLRP3+, cleaved Caspase-1+, and IL-1ß+ cells in the retina were observed in the RIRI + NAS and RIRI + TAK-242 + NAS groups compared with the RIRI group 12 h after RIRI (all P < 0.01). Western blot analysis results showed that the expression of IL-1ß in the RIRI + NAS group began to increase 6 h after RIRI, and it reached a high level 12 h after RIRI, and then decreased. And it was lower at each time point in the RIRI + NAS group than in the RIRI group, and there existed significant difference (all P < 0.01). Besides, the expression levels of TLR4, p-NF-κB, NLRP3, and cleaved Caspase-1 proteins in the RIRI + NAS and RIRI + TAK-242 + NAS groups decreased 12 h after RIRI compared with those in the RIRI group (all P < 0.01). The difference in IL-1ß+ cells was significantly correlated with those of TLR4+ and cleaved Caspase-1+ cells in the RIRI + NAS group (r2 = 0.9054 or 0.7431, P < 0.01). In conclusion, NAS could attenuate the expression of IL-1ß by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway, reduce the retina edema, and promote the survival of RGCs, thereby alleviating the retinal injury and exert its neuroprotective effect.

Manganese salts function as potent adjuvants.

Aluminum-containing adjuvants have been used for nearly 100 years to enhance immune responses in billions of doses of vaccines. To date, only a few adjuvants have been approved for use in humans, among which aluminum-containing adjuvants are the only ones widely used. However, the medical need for potent and safe adjuvants is currently continuously increasing, especially those triggering cellular immune responses for cytotoxic T lymphocyte activation, which are urgently needed for the development of efficient virus and cancer vaccines. Manganese is an essential micronutrient required for diverse biological activities, but its functions in immunity remain undefined. We previously reported that Mn2+ is important in the host defense against cytosolic dsDNA by facilitating cGAS-STING activation and that Mn2+ alone directly activates cGAS independent of dsDNA, leading to an unconventional catalytic synthesis of 2'3'-cGAMP. Herein, we found that Mn2+ strongly promoted immune responses by facilitating antigen uptake, presentation, and germinal center formation via both cGAS-STING and NLRP3 activation. Accordingly, a colloidal manganese salt (Mn jelly, MnJ) was formulated to act not only as an immune potentiator but also as a delivery system to stimulate humoral and cellular immune responses, inducing antibody production and CD4+/CD8+ T-cell proliferation and activation by either intramuscular or intranasal immunization. When administered intranasally, MnJ also worked as a mucosal adjuvant, inducing high levels of secretory IgA. MnJ showed good adjuvant effects for all tested antigens, including T cell-dependent and T cell-independent antigens, such as bacterial capsular polysaccharides, thus indicating that it is a promising adjuvant candidate.

Cytokine secretion and pyroptosis of cholesteatoma keratinocytes mediated by AIM2 inflammasomes in response to cytoplasmic DNA.

Cholesteatoma constitutes an acquired benign epidermal non­permanent bone lesion that is locally destructive and patients often relapse. Inflammasomes, which mediate the maturation and production of IL­18 and IL­1ß, resulting in pyroptosis, have been documented to serve a core function in multiple inflammatory conditions. Absent in melanoma 2 (AIM2) is an inflammasome that identifies cytoplasmic DNA and has previously been reported as a pivotal modulator of inflammatory responses. Therefore, the present study aimed to determine the expression levels of AIM2 in human cholesteatoma tissues, and elucidate its function in modulating cytokine production. The expression levels of IL­18, apoptosis­associated speck­like protein containing a CARD (ASC), IL­1ß, AIM2 and caspase­1 were markedly elevated in cholesteatoma tissues. Protein expression levels of AIM2, caspase­1 and ASC were localized in the cellular cytoplasm, primarily in the granular and prickle­cell layers in the cholesteatoma epithelium. Induction using IFN­Î³, as well as cytoplasmic DNA markedly activated the AIM2 inflammasome and elevated the release of IL­18 and IL­1ß in human cholesteatoma keratinocytes. IFN­Î³ was found to enhance poly(dA:dT)­induced pyroptosis of cells and cytokine production. The results of the present study revealed that AIM2 expressed in human cholesteatoma serves a vital function in the inflammatory response by initiating the inflammasome signaling cascade in cholesteatoma.

Riboflavin, vitamin B2, attenuates NLRP3, NLRC4, AIM2, and non-canonical inflammasomes by the inhibition of caspase-1 activity.

Riboflavin is commonly taken as a nutritional supplement, and it converts to coenzymes during the process of energy production from carbohydrates, fats, and proteins. Although riboflavin is considered to be an anti-inflammatory vitamin because of its antioxidant properties, the effects of riboflavin on inflammasome have been not reported. Inflammasome, a cytosolic surveillance protein complex, leads to the activation of caspase-1, cytokine maturation, and pyroptosis. In the present study, riboflavin attenuated the indicators of NLRP3 inflammasome activation in macrophages, such as the maturation and secretion of interleukin (IL)-1ß, IL-18, and caspase-1; the formation of Asc pyroptosome; and the cleavage of gasdermin D. In addition, the oral and peritoneal administration of riboflavin inhibited the peritoneal production of IL-1ß and IL-18 in a mouse model. Mechanistically, riboflavin prevented mitochondrial perturbations, such as mitochondrial ROS production and mitochondrial DNA release, which trigger the NLRP3 inflammasome assembly. Riboflavin was further confirmed to disrupt the activity of caspase-1, and it also inhibited the AIM2, NLRC4, and non-canonical inflammasomes. Therefore, riboflavin has both an antioxidant effect and an anti-inflammasome property that regulates the inflammatory response.

Effects of irbesartan on myocardial injury in diabetic rats: The role of NLRP3/ASC/Caspase-1 pathway.

To observe the mechanism of myocardial injury in diabetic rats after irbesartan intervention and analyze the role of nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3) inflammatory pathway. The experiment was divided into four groups: normal control group (CON), high glucose and high caloric diet group (HC), diabetes group (DM) and diabetes+irbesartan group (DM+Ir). Compared with CON group, in HC group, triglyceride, total cholesterol and fasting blood glucose levels were increased; however, there was no significant difference of the cardiac function, the degree of myocardial fibrosis, NLRP3, ASC, Caspase-1 mRNA and protein expressions and the releasing of inflammatory factors interleukin (IL)-1ß and IL-18. Compared with HC group, in DM group, triglyceride, total cholesterol, fasting blood glucose, IL-1ß and IL-18 levels, NLRP3, ASC, Caspase-1 mRNA and protein expressions and the degree of myocardial fibrosis were increased, but the cardiac function was decreased. Compared with DM group, there were no changes in total cholesterol and fasting blood glucose, the degree of myocardial fibrosis cardiac function was attenuated, NLRP3, ASC, Caspase-1 expressions, IL-1ß and IL-18 levels were reduced in DM+Ir group. The results suggested that irbesartan may exert myocardial protection by inhibiting the expression of the NLRP3/ASC/Caspase-1 pathway in diabetic rats.

Enteric Nervous System-Derived IL-18 Orchestrates Mucosal Barrier Immunity.

Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.

Inhibition of the IL-18 Receptor Signaling Pathway Ameliorates Disease in a Murine Model of Rheumatoid Arthritis.

Interleukin (IL)-18 expression in synovial tissue correlates with the severity of joint inflammation and the levels of pro-inflammatory cytokines. However, the role of the IL-18/IL-18 receptor-alpha (Rα) signaling pathway in autoimmune arthritis is unknown. Wild-type (WT) and IL-18Rα knockout (KO) mice were immunized with bovine type II collagen before the onset of arthritis induced by lipopolysaccharide injection. Disease activity was evaluated by semiquantitative scoring and histologic assessment. Serum inflammatory cytokine and anticollagen antibody levels were quantified by an enzyme-linked immunosorbent assay. Joint cytokine and matrix metalloproteinases-3 levels were determined by a quantitative polymerase chain reaction. Splenic suppressors of cytokine signaling (SOCS) were determined by Western blot analysis as indices of systemic immunoresponse. IL-18Rα KO mice showed lower arthritis and histological scores in bone erosion and synovitis due to reductions in the infiltration of CD4+ T cells and F4/80+ cells and decreased serum IL-6, -18, TNF, and IFN-γ levels. The mRNA expression and protein levels of SOCS3 were significantly increased in the IL-18Rα KO mice. By an up-regulation of SOCS, pro-inflammatory cytokines were decreased through the IL-18/IL-18Rα signaling pathway. These results suggest that inhibitors of the IL-18/IL-18Rα signaling pathway could become new therapeutic agents for rheumatoid arthritis.

Cilostazol ameliorates high free fatty acid (FFA)-induced activation of NLRP3 inflammasome in human vascular endothelial cells.

Cardiovascular disease is recognized as a leading cause of death worldwide, but the risk of death is 2-3 times higher for individuals with diabetes. NLRP3 inflammasome activation is a leading pathway of vascular damage, and new treatment methods are needed to reduce NLRP3 inflammasome expression, along with a detailed understanding of how those treatments work. In a series of assays on human vascular endothelial cells that were exposed to high concentrations of free fatty acids (FFA) to induce a diabetes-like environment, we found a significant impact of cilostazol, a vasodilator widely used to treat blood flow problems and well-tolerated medication. To our knowledge, this study is the first to demonstrate the effects of cilostazol in primary human aortic endothelial cells. We found that cilostazol significantly reduced NLRP3 inflammasome activation, as well as the activity of other related and harmful factors, including oxidative stress, expression of NADPH oxidase 4 (NOX-4), thioredoxin-interacting protein (TxNIP), high mobility group box 1 (HMGB-1), interleukin 1ß (IL-1ß) and IL-18. Cilostazol also protected the functionality of sirtuin 1 (SIRT1), which serves to restrict NLRP3 inflammasome activity, when exposure to FFAs would have otherwise impaired its function. Thus, it appears that cilostazol's mechanism of action in reducing NLRP3 inflammasome activation is an indirect one; it protects SIRT1, which then allows SIRT1 to perform its regulatory job. Cilostazol has potential as an already-available, well-tolerated preventive medication that may alleviate some of the adverse vascular effects of living with diabetes. The findings of the present study lay the groundwork for further research on the potential of cilostazol as a safe and effective treatment against diabetic endothelial dysfunction and vacular disease.

Effects of NLRP6 in Cerebral Ischemia/Reperfusion (I/R) Injury in Rats.

The NOD-like receptor protein 6 (NLRP6), an intracytoplasmic pattern recognition receptor in the nucleotide-binding domain, leucine-rich repeat-containing (NLR) innate immune receptor family, influences the inflammation reaction. The role of NLRP6 in cerebral ischemia-reperfusion (I/R) injury in rats is unclear. We explore the function of NLRP6 in cerebral I/R injury. The investigators used a middle cerebral artery occlusion/reperfusion model (MCAO) to imitate ischemic injury. We found the peak expression of NLRP6 is in 48-h post-cerebral I/R injury. The expression of NLRP6 siRNA, as well as the expression of protein and mRNA, was detected by Western blot and qRT-PCR. The degree of IL-1ß and IL-18 was assessed by ELISA. After downregulating NLRP6, the expression of IL-1ß, IL-18, cleaved Caspase-1, and myeloperoxidase (MPO) were reduced. In HE and Nissl staining, pathological injury of brain tissue after downregulating NLRP6 was improved. NLRP6 siRNA decreased the NLRP6-ASC binding states by CO-IP. NRP6 has a pro-inflammatory effect in cerebral I/R injury, which may provide a new target for the treatment of cerebral I/R injury.

Blockade of the NLRP3/Caspase-1 Axis Ameliorates Airway Neutrophilic Inflammation in a Toluene Diisocyanate-Induced Murine Asthma Model.

Multiple studies have addressed the vital role of Nod-like receptor protein 3(NLRP3)/caspase-1/IL-1ß signaling in asthma. Yet, the role of NLRP3/caspase-1 in toluene diisocyanate (TDI)-induced asthma is still obscure. The aim of this study is to investigate the role of the NLRP3/caspase-1 axis in TDI-induced asthma. Using an established murine model of TDI-induced asthma as described previously, we gave the asthmatic mice a highly selective NLRP3 inhibitor, MCC950, as well as the specific caspase-1 inhibitors VX-765 and Ac-YVAD-CHO for therapeutic purposes. Airway resistance was measured and bronchoalveolar lavage fluid was analyzed. Lungs were examined by histology, immunohistochemistry, Western blotting, and flow cytometry. TDI exposure elevated the expression of NLRP3 and caspase-1 that was coupled with increased airway hyperresponsiveness (AHR), neutrophil-dominated cell infiltration, pronounced goblet cell metaplasia, extensive collagen deposition, and increased TH2/TH17 responses. Both VX-765 and Ac-YVAD-CHO effectively inhibited the activation of caspase-1 in TDI-asthmatic mice that was accompanied by dramatic attenuation of AHR, airway inflammation, and airway remodeling, in addition to a decreased TH2 response and lower levels of IL-18 and IL-1ß. MCC950 blocked the activation of NLRP3 and downregulated protein expression of caspase-1, IL-1ß, and IL-18 in TDI-exposed mice. Furthermore, MCC950 remarkably alleviated AHR, airway inflammation, airway remodeling, and significantly suppressed TH2/TH17 responses. These findings suggested that blockade of the NLRP3/caspase-1 axis effectively prevents the progression of TDI-induced asthma and could be used as therapeutic targets for asthmatics.

Vildagliptin inhibits high free fatty acid (FFA)-induced NLRP3 inflammasome activation in endothelial cells.

Elevated free fatty acids (FFAs) are a risk factor for type 2 diabetes. Endothelial dysfunction induced by high levels of FFAs is one of the mechanisms related to the progression of diabetes. In clinical diabetes care, DPP-4 inhibitors have been shown to be effective in reducing glucose levels. In this study, we investigated the molecular mechanism of the clinically available DPP-4 inhibitor vildagliptin in the protection of FFA-induced endothelial dysfunction. Treatment of endothelial cells with vildagliptin inhibits FFA-induced cellular LDH release and generation of ROS. Vildagliptin also reverses FFA-induced reduced levels of GSH and elevated expression of the FFA-associated NAPHD oxidase protein NOX-4. Moreover, vildagliptin ameliorates the reduction in mitochondrial potential triggered by FFAs. Mechanistically, we show that vildagliptin suppresses FFA-induced expression of proteins of the NLRP3 inflammasome complex, including NLRP3, ASC, p20 and HMGB-1, and mitigates FFA-induced inactivation of the AMPK pathway. Consequently, vildagliptin inhibits production of two cytokines that are favored by NLRP3 inflammasome machinery: IL-1ß and IL-18. Finally, we demonstrate that vildagliptin ameliorates FFA-induced reduced eNOS, indicating its protective role against endothelial dysfunction. Collectively, we conclude that the protective role of vildagliptin in endothelial cells is mediated via suppression of the AMPK-NLRP3 inflammasome-HMGB-1 axis pathway. These findings imply that the anti-diabetic drug vildagliptin possesses dual therapeutic applications in lowering glucose and improving vascular function.

Ginkgolide B ameliorates NLRP3 inflammasome activation after hypoxic-ischemic brain injury in the neonatal male rat.

INTRODUCTION: Perinatal hypoxic-ischemic (HI) insult is an important cause of brain injury in neonates. The development of novel treatment strategies for neonates with HI brain injury is urgently needed. Ginkgolide B (GB) is a main component of Ginkgo biloba extracts with a long history of use in traditional Chinese medicine. However, it is unknown whether GB could play a protective role in hypoxic stress in immature animals. METHODS: Using neonatal hypoxic-ischemic (HI) brain injury model of rat pups, neurological score, infarct size, and brain edema were evaluated after HI injury. The activation of microglia and the production of IL-1ß and IL-18 were detected by immunohistochemistry and ELISA, respectively. A priming signal (NF-κB P65) and an activation signal (Caspase-1) of NLRP3 inflammasome activation were detected by western blot analyses. RESULTS: GB administrated 30 min prior to ischemia induction can improve neurological disorder, reduce infarct volume and alleviate cerebral edema. Compared with the HI groups, GB inhibited the activation of microglia and decreased the production of IL-1ß and IL-18 in neocortex. Furthermore, GB reduced NLRP3 expression mainly in microglia, and significantly inhibited the expression of Caspase-1 and the nuclear translocation of NF-κB P65, preventing NLRP3 inflammasome activation. CONCLUSIONS: GB ameliorates hypoxic-ischemic brain injury in the neonatal male rat via inhibiting NLRP3 inflammasome activation.

Procyanidin B2 protects rats from paraquat-induced acute lung injury by inhibiting NLRP3 inflammasome activation.

Paraquat is a commonly used heterocyclic herbicide and has high toxicity by causing acute lung injury. There is no effective treatment for paraquat poisoning. We evaluated the effects of procyanidin B2, a natural dietary phytochemical, on paraquat-induced lung injury in rats. Paraquat was used to induce acute lung injury of rats, which were administered with procyanidin B2. The lung injury was evaluated by measuring the lung/body weight ratio, the histology and PMNs count. The oxidative stress was assessed by detecting ROS-mediated indices in the BALF. The expression of IL-1ß and IL-18 were detected by RT-PCR and ELISA. The levels of NLRP3 inflammasome components including NLRP3, ASC and caspase-1 were detected by western blot. The lung injury in the paraquat-induced models in NLRP3 gene silenced animals was compared with the same lung injury model treated with procyanidin B2. Administration of procyanidin B2 significantly reduced paraquat-induced lung injury with lower BALF PMNs count, MPO activity, MDA level and elevated SOD activity. Procyanidin B2 suppressed expression of IL-1ß and IL-18 at both RNA and protein levels, similar to the NLRP3 gene silenced rats. Compared to paraquat-induced group, procyanidin B2 showed remarkably decreased NLRP3, ASC and caspase-1 signals in the lung tissues in a dose-dependent manner. Procyanidin B2 significantly suppressed the activation of NLRP3 inflammasome in the lung tissue induced by paraquat in the rat model. This finding revealed a novel mechanism by which procyanidin B2 exerts anti-inflammatory effects and their clinical benefits in health.