Correlation Between Plasma NLRP3, IL-1ß, and IL-18 and Diabetic Nephropathy in Patients With Type 2 Diabetes.

Context: Diabetic nephropathy (DN) is a common microvascular complication in diabetic patients. The pathogenesis of DN is complex. Inflammatory response may play a key role as a common downstream pathway. Objective: The study intended to explore the relationship between the levels of plasma nucleotide-binding oligomeric domain-like receptor protein 3 (NLRP3 inflammasome), interleukin-1ß (IL-1ß), and IL-18 and the progression of type 2 diabetic nephropathy to clarify their relationship with type 2 diabetes mellitus (T2DM) and to provide evidence for clinical treatment. Design: The research team performed a controlled observational study. Setting: The study took place at Baoding No. 1 Central Hospital in Baoding, Hebei, China. Participants: Participants were 153 patients with T2DM who received treatment at the hospital between October 2020 and October 2021. The research team allocated 30 participants without evidence of DN to the control group. Based on the DN stage, the team assigned the 123 remaining participants to one of five observation groups: (1) 32 participants with stage 1 DN to the DN1 group, (2) 31 participants with stage 2 DN to the DN2 group, (3) 30 participants with stage 3 DN to the DN3 group, (4) 30 participants with stage 4 DN to the DN4 group, and (5) 29 participants with stage 5 DN to the DN5 group. Outcome Measures: The research team measured participants' levels of "nucleotide binding oligomeric domain-like receptor protein 3" (NLRP3), interleukin-1 beta (IL-1ß), and IL-18 and used the Spearman rank correlation analysis to determine the correlation between those levels and the DN stages. Results: The levels of NLRP3 , IL-1ß and IL-18 in all the five observation groups were significantly higher than those in the control group (all P < .01). The levels were also significantly higher: (1) in the DN2, DN3, DN4, and DN5 groups than those in the DN1 group (all P < .01); (2) in the DN3, DN4, and DN5 groups than those in the DN2 group (all P < .01); (3) in the DN4 and DN5 groups than those in the DN3 group (all P < .01); and (4) in the DN5 groups than those in the DN4 group (all P < .01). The Spearman rank correlation analysis showed that the NLRP3, IL-1ß, and IL-18 levels were significantly positively correlated with the DN stage (P = .01). Conclusions: NLRP3, IL-1ß and IL-18 played an important role in the progression of T2DM, and their levels increased with the aggravation of DN. Therefore, the plasma levels of NLRP3, IL-1ß and IL-18 can be useful as indicators of the occurrence and development of DN and can provide clinical guidance for the early diagnosis of DN and for the determination and adjustment of treatment plans.

Pulmonary vascular inflammation with fatal coronavirus disease 2019 (COVID-19): possible role for the NLRP3 inflammasome.

BACKGROUND: Pulmonary hyperinflammation is a key event with SARS-CoV-2 infection. Acute respiratory distress syndrome (ARDS) that often accompanies COVID-19 appears to have worse outcomes than ARDS from other causes. To date, numerous lung histological studies in cases of COVID-19 have shown extensive inflammation and injury, but the extent to which these are a COVID-19 specific, or are an ARDS and/or mechanical ventilation (MV) related phenomenon is not clear. Furthermore, while lung hyperinflammation with ARDS (COVID-19 or from other causes) has been well studied, there is scarce documentation of vascular inflammation in COVID-19 lungs. METHODS: Lung sections from 8 COVID-19 affected and 11 non-COVID-19 subjects, of which 8 were acute respiratory disease syndrome (ARDS) affected (non-COVID-19 ARDS) and 3 were from subjects with non-respiratory diseases (non-COVID-19 non-ARDS) were H&E stained to ascertain histopathological features. Inflammation along the vessel wall was also monitored by expression of NLRP3 and caspase 1. RESULTS: In lungs from COVID-19 affected subjects, vascular changes in the form of microthrombi in small vessels, arterial thrombosis, and organization were extensive as compared to lungs from non-COVID-19 (i.e., non-COVID-19 ARDS and non-COVID-19 non-ARDS) affected subjects. The expression of NLRP3 pathway components was higher in lungs from COVID-19 ARDS subjects as compared to non-COVID-19 non-ARDS cases. No differences were observed between COVID-19 ARDS and non-COVID-19 ARDS lungs. CONCLUSION: Vascular changes as well as NLRP3 inflammasome pathway activation were not different between COVID-19 and non-COVID-19 ARDS suggesting that these responses are not a COVID-19 specific phenomenon and are possibly more related to respiratory distress and associated strategies (such as MV) for treatment.

IL-12 Contributes to the Development of Asthma by Targeting HIF-1α/NLRP3 Pathway through Runx3.

INTRODUCTION: The aim of the study was to determine the role and mechanism of runt-related transcription factor 3 (Runx3) in the development of asthma. METHODS: An asthma mouse model was constructed and validated by hematoxylin-eosin analysis of lung tissue and noninvasive enhanced pause (Penh) evaluation of airway hyperresponsiveness. Then, the levels of Runx3 and interleukin (IL)-12 in peripheral blood and lung tissue were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. By use Runx3+/- mice, the effect of Runx3 downregulation on ovalbumin (OVA)-induced asthma was investigated. After stimulated by different doses of IL-12, the expressions of Runx3, hypoxia inducible factor-1α (HIF-1α), and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in BEAS-2B cells were tested through Western blot and immunofluorescence. Subsequently, BEAS-2B cells treated with 20 ng/mL IL-12 were divided into control, Runx3 overexpression negative control, Runx3 overexpression, HIF-1α inhibitor, and Runx3 overexpression + HIF-1α agonist groups. The Western blot, immunofluorescence, and ELISA indicators were tested repeatedly. RESULTS: The increased number of inflammatory cells and Penh value confirmed the success of the asthma mouse model. IL-12 expression was significantly increased, and Runx3 was reduced in asthma mice compared with wild-type mice. Meanwhile, the level of immunoglobulin E (IgE) in serum, cytokines in bronchoalveolar lavage fluid, and IL-12, HIF-1α, NLRP3 in the lung were significantly elevated in Runx3+/- mice. With the increase of IL-12 concentration, Runx3 protein expression decreased, while HIF-1α and NLRP3 expression increased. Further mechanistic studies suggest that Runx3 ameliorates IL-12-induced BEAS-2B injury by inhibiting HIF-1α/NLRP3 pathway. CONCLUSION: These results suggested that IL-12 contributes to the development of asthma by targeting HIF-1α/NLRP3 pathway through Runx3, thus providing a novel strategy for asthma therapy.

Identification of NLRP3 as a covalent target of 1,6-O,O-diacetylbritannilactone against neuroinflammation by quantitative thiol reactivity profiling (QTRP).

Neuroinflammation plays a key etiological role in the progressive neuronal damage of neurodegenerative diseases. Our phenotypic-based screening discovered 1,6-O,O-diacetylbritannilactone (OABL, 1) from Inula britannica exhibited the potential anti-neuroinflammatory activity as well as a favorable blood-brain barrier penetration. 1 and its active derivative Br-OABL (2) with insert of Br at the C-14 position both modulated TLR4/NF-kB/MAPK pathways. However, proteome-wide identification of 1 binding proteins remains unclear. Here, we employed an adapted isoTOP-ABPP, quantitative thiol reactivity profiling (QTRP) approach, to identify and quantify thiol reactivity binding proteins in murine microglia BV-2 cells. We screened out 15 proteins co-targeted by 1 and 2, which are involved in cellular response to oxidative stress and negative regulation NF-κB transcription factor in biological processes. In site-specific profiling, NLRP3 was identified as a covalent target of 1 and 2 for the first time, and the Cys483 of NLRP3 NACHT domain was identified as one active-site of NLRP3 cysteine residues that can be covalently modified by the α-methylene-γ-lactone moiety. Furthermore, NLRP3 was validated to be directly binded by 1 and 2 by cellular thermo shift assay (CETSA) and activity-based protein profiling (ABPP), and NLRP3 functions were also verified by small interfering RNA approach. Notably, OABL treatment (i.p., 20 mg/kg/day) for 21 days reduced inflammation in 5XFAD mice brain. Together, we applied the QTRP to uncover the binding proteins of OABL in BV-2 cells, among which NLRP3 was revealed as a new covalent target of 1 and 2 against neuroinflammation.

Inflammasome formation in the lungs of patients with fatal COVID-19.

OBJECTIVE: The orf8b protein of the coronavirus SARS-CoV, analogous to SARS-CoV-2, triggers the NLRP3 inflammasome in macrophages in vitro. Deregulated inflammasome-mediated release of interleukin-1 family cytokines is important in hyper-inflammatory syndromes, like happens in SARS-CoV-2-mediated cytokine release syndrome. We propose that an intense inflammasome formation characterizes the lungs of patients with fatal COVID-19 disease due to pneumonia and acute respiratory distress syndrome (ARDS). METHODS: Samples from four patients with confirmed COVID-19 pneumonia who had been hospitalized at the Hospital of the University of Trieste (Italy) and died of ARDS and four lung samples from a historical repository from subjects who had died of cardiopulmonary arrest and had not been placed on mechanical ventilation and without evidence of pulmonary infection at postmortem examination were collected. Pathology samples had been fixed in formalin 10% at time of collection and subsequently embedded in paraffin. We conducted staining for ASC (Apoptosis-associated Speck-like protein containing a Caspase recruitment domain), NLRP3 (NACHT, LRR, and PYD domains-containing protein 3), and cleaved caspase-1. RESULTS: Intense expression of the inflammasome was detected, mainly in leukocytes, within the lungs of all patients with fatal COVID-19 in the areas of lung injury. The number of ASC inflammasome specks per high power fields was significantly higher in the lungs of patients with fatal COVID-19 as compared with the lungs of control subjects (52 ± 22 vs 6 ± 3, P = 0.0064). CONCLUSIONS: These findings identify the presence of NLRP3 inflammasome aggregates in the lungs of fatal COVID-19 pneumonia thus providing the potential molecular link between viral infection and cytokine release syndrome.

Discovery of carbon-11 labeled sulfonamide derivative: A PET tracer for imaging brain NLRP3 inflammasome.

We report herein the discovery of a positron emission tomography (PET) tracer for the (NOD)-like receptor protein 3 (NLRP3). Our recent medicinal chemistry campaign on developing sulfonamide-based NLRP3 inhibitors led to an analog, 1, with a methoxy substituent amenable to labeling with carbon-11. PET/CT imaging studies indicated that [11C]1 exhibited rapid blood-brain barrier (BBB) penetration and moderate brain uptake, as well as blockable uptake in the brain. [11C]1, thus suggesting the potential to serve as a useful tool for imaging NLRP3 inflammasome in living brains.

NLRP3 Inflammasome Expression in Gingival Crevicular Fluid of Patients with Periodontitis and Chronic Hepatitis C.

The study is aimed at assessing the impact that periodontal disease and chronic hepatitis C could have on gingival crevicular fluid levels of the NLRP3 inflammasome, caspase-1 (CASP-1), and interleukin-18 (IL-18) and at evaluating whether the increased local inflammatory reaction with clinical periodontal consequences is correlated to their upregulation. Patients were divided into four groups, according to their periodontal status and previously diagnosed hepatitis C, as follows: (i) CHC group, chronic hepatitis C patients; (ii) P group, periodontal disease patients, systemically healthy; (iii) CHC + P group, patients suffering from both conditions; and (iv) H group, systemically and periodontally healthy controls. Gingival crevicular samples were collected for quantitative analysis of the NLRP3 inflammasome, CASP-1, and IL-18. CHC + P patients expressed the worse periodontal status and the highest NLRP3, CASP-1, and IL-18 levels, the difference being statistically significant (p < 0.05). The P group patients also expressed significantly more elevated NLRP3, CASP-1, and IL-18 levels, as compared to nonperiodontal patients (CHC and H groups). Chronic hepatitis C and periodontal disease could have a significant influence on the upregulation of NLRP3 inflammasome and its components, possibly contributing to an increased local inflammatory reaction and clinical periodontal consequences.

NLRP3 inflammasome negatively regulates podocyte autophagy in diabetic nephropathy.

Inflammasome mechanisms are recognized as a key pathophysiology of diabetic nephropathy (DN). The nucleotide-oligomerization domain-like receptor 3 (NLRP3) inflammasome has attracted the most attention. Autophagy as a conserved intracellular catabolic pathway plays essential roles in the maintenance of podocytes. Although autophagy was involved in preventing excessive inflammatory responses in kidney diseases, a clear understanding of the regulation of NLRP3 inflammasome on autophagy in glomerular damage in DN is still lacking. In this study, we focused on the effect of the activation of NLRP3 inflammasome on the suppression of podocyte autophagy and aimed to investigate the role of autophagy in podocyte injury in DN. Podocyte autophagy has been confirmed to be inhibited in high-fat diet/streptozotocin (HFD/STZ)-induced DN mice, and NLRP3 has been found to be upregulated in both mice and human DN biopsies and in vitro. Activation of NLRP3 inflammasome exacerbated podocyte autophagy and reduced podocyte nephrin expression, while silencing of NLRP3 efficiently restored podocyte autophagy and ameliorated podocyte injury induced by high glucose. The results showed that NLRP3 was a negative regulator of autophagy and suggested that restoration of podocyte autophagy by inactivation of NLRP3 under high glucose could reduce podocyte injury. Proper modification of autophagy and inflammasome has the potential to benefit the kidney in DN.

Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy.

Tubulointerstitial inflammation plays a critical role in the progression of diabetic nephropathy (DN), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes contribute to renal interstitial inflammation in DN. Decreased expression of optineurin (OPTN) is also associated with the progression of DN. We investigated the role of OPTN in activation of the NLRP3 inflammasome in DN. We initially examined the renal biopsy tissues of 172 patients with type 2 DN and 32 nondiabetic patients with renal hamartoma. Expression of renal OPTN was significantly lower in patients with DN and negatively correlated with urinary levels of IL-1ß and IL-18. Confocal microscopy analysis of the biopsies indicated no NLRP3 or IL-1ß staining in OPTN-positive renal tubular epithelial cells (RTECs), indicating a negative correlation of OPTN expression with the activation of NLRP3 inflammasome. In vitro studies of murine RTECs indicated the levels of OPTN mRNA and protein decreased significantly after stimulation by a high glucose (HG) treatment. Relative to RTECs given HG, RTECs overexpressing OPTN showed significantly lower levels of NLRP3 expression, cleavage of caspase-1 and IL-1ß, and release of IL-1 ß and IL-18. Overexpression of OPTN in the presence of HG significantly increased the costaining of microtubule-associated protein 1A/1B-light chain 3-II and translocase of outer mitochondrial membrane 20 in RTECs, suggesting that OPTN enhances mitophagy. In addition, mitochondrial division inhibitor 1 blocked the inhibitory effect of OPTN overexpression on the activation of NLRP3 inflammasome in the presence of HG, indicating that OPTN overexpression inhibited NLRP3 inflammasome activation by enhancement of mitophagy. OPTN gene silencing significantly enhanced production of mitochondrial reactive oxygen species (mtROS) in the presence of HG. Compared with HG+OPTN small interfering RNA (siRNA)-treated RTECs, HG+OPTN siRNA+MitoTempo-treated RTECs attenuated NLRP3 inflammasome activation, suggesting that OPTN gene silencing activates the NLRP3 inflammasome by increasing mtROS in HG-treated RTECs. Taken together, our results demonstrate that OPTN inhibits the activation of NLRP3 inflammasome by enhancing mitophagy.-Chen, K., Feng, L., Hu, W., Chen, J., Wang, X., Wang, L., He, Y. Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy.

Hydrogen attenuates sepsis-associated encephalopathy by NRF2 mediated NLRP3 pathway inactivation.

OBJECTIVE: Sepsis-associated encephalopathy (SAE) is a major cause of mortality worldwide. Oxidative stress, inflammatory response and apoptosis participate in the pathogenesis of SAE. Nuclear factor erythroid 2-related factor 2 (Nrf2) and nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) pathway is involved in oxidative stress and inflammatory response. We reported that hydrogen gas protected against sepsis in wild-type (WT) but not Nrf2 knockout (KO) mice. Therefore, it is vital to identify the underlying cause of hydrogen gas treatment of sepsis-associated encephalopathy. METHODS: SAE was induced in WT and Nrf2 KO mice by cecal ligation and puncture (CLP). As a NLRP3 inflammasome inhibitor, MCC950 (50 mg/kg) was administered by intraperitoneal (i.p.) injection before operation. Hydrogen gas (H2)-rich saline solution (5 mL/kg) was administered by i.p. injection at 1 h and 6 h after sham and CLP operations. Brain tissue was collected to assess the NLRP3 and Nrf2 pathways by western blotting, reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. RESULTS: SAE increased NLRP3 and Nrf2 expression in microglia. MCC950 inhibited SAE-induced NLRP3 expression, interleukin (IL)-1ß and IL-18 cytokine release, neuronal apoptosis and mitochondrial dysfunction. SAE increased NLRP3 and caspase-1 expression in WT mice compared to Nrf2 KO mice. Hydrogen increased Nrf2 expression and inhibited the SAE-induced expression of NLRP3, caspase-1, cytokines IL-1ß and IL-18, neuronal apoptosis, and mitochondrial dysfunction in WT mice but not Nrf2 KO mice. CONCLUSION: SAE increased NLRP3 and Nrf2 expression in microglia. Hydrogen alleviated inflammation, neuronal apoptosis and mitochondrial dysfunction via inhibiting Nrf2-mediated NLRP3 pathway.

Estrogen receptors participate in carcinogenesis signaling pathways by directly regulating NOD-like receptors.

Increasing evidence indicates that the NOD-like receptors (NLRs) family may act as critical back-up defenses and provide synergistic responses when confronted with persistent danger. However, the precise regulatory mechanism of NLRs and the contribution of NLRs to cancer are still unknown. In our previous study, we found that estrogen receptors (ERs) have a close connection with NLRs in the inflammatory response. Here, ERs are first identified as NLRs transcription regulation factors, both regulate NLRs expression and promote inflammasome co-localization. Furthermore, we identified that NLRP3 was differentially expressed in colon normal and cancer cells, selective ERα antagonist could significantly decrease pro-inflammatory cytokines expression, suppress proliferation and promote apoptosis by inhibited NLRP3 expression and inflammasome activity. In short, the research demonstrates that ERs participate in the NLR-associated signaling pathway in cancer by directly regulating NLRs. Our results provide novel insight into ERs as therapeutic targets in NLR-related inflammation and cancer.

The selective Nlrp3 inflammasome inhibitor Mcc950 attenuates lung ischemia-reperfusion injury.

Lung ischemia-reperfusion (IR) occurs in many circumstances and leads to impaired lung function. The NACHT, LRR and PYD domains-containing protein 3 (Nlrp3) inflammasome is reportedly activated during lung IR. Mcc950 is a recently developed Nlrp3 inhibitor. The aim of our study was to test the efficacy of Mcc950 on lung IR injury and to investigate the role of reactive oxygen species (ROS) in Nlrp3 inflammasome activation using a murine lung IR model. The results of the current study confirmed that Nlrp3 was upregulated and activated during lung IR, and inhibiting oxidative stress by the ROS scavenger edaravone attenuated Nlrp3 inflammasome activation. Mcc950 pretreatment significantly alleviated IR-induced lung injury by reducing production of the proinflammatory cytokines Il-1ß and Il-18 and inhibiting neutrophil infiltration and cell apoptosis. Protein coimmunoprecipitation revealed that Mcc950 partially blocked the interaction between Nlrp3 and Nek7 (NimA-related protein kinase 7). Therefore, we conclude that ROS-dependent activation of the Nlrp3 inflammasome contributed to lung IR injury. Mcc950 significantly reduced lung IR injury by blocking Nlrp3 inflammasome activation, and the mechanism was partially attributed to inhibition of the interaction between Nlrp3 and Nek7. Thus, Mcc950 is a promising treatment for the prevention of lung IR injury.

NLRP3 in somatic non-immune cells of rodent and primate testes.

NLRP3 is part of the NLRP3 inflammasome and a global sensor of cellular damage. It was recently discovered in rodent Sertoli cells. We investigated NLRP3 in mouse, human and non-human primate (marmoset and rhesus macaque) testes, employing immunohistochemistry. Sertoli cells of all species expressed NLRP3, and the expression preceded puberty. In addition, peritubular cells of the adult human testes expressed NLRP3. NLRP3 and associated genes (PYCARD, CASP1, IL1B) were also found in isolated human testicular peritubular cells and the mouse Sertoli cell line TM4. Male infertility due to impairments of spermatogenesis may be related to sterile inflammatory events. We observed that the expression of NLRP3 was altered in the testes of patients suffering from mixed atrophy syndrome, in which tubules with impairments of spermatogenesis showed prominent NLRP3 staining. In order to explore a possible role of NLRP3 in male infertility, associated with sterile testicular inflammation, we studied a mouse model of male infertility. These human aromatase-expressing transgenic mice (AROM+) develop testicular inflammation and impaired spermatogenesis during aging, and the present data show that this is associated with strikingly elevated Nlrp3 expression in the testes compared to WT controls. Interference by aromatase inhibitor treatment significantly reduced increased Nlrp3 levels. Thus, throughout species NLRP3 is expressed by somatic cells of the testis, which are involved in testicular immune surveillance. We conclude that NLRP3 may be a novel player in testicular immune regulation.

Inflammasome Activation Characterizes Lesional Skin of Folliculitis Decalvans.

Folliculitis decalvans (FD) is a chronic inflammatory disease leading to scarring alopecia with poorly defined pathogenesis. The aim of this study was to investigate the expression of markers associated with the activation of innate immune signals, such as inflammasome (NALP1 and NALP3), interleukin (IL)-1ß and IL-8 and type I interferon (MxA). A retrospective monocentric study was conducted and included 17 patients with FD with available biopsies. Disease activity (stable vs. active) was defined clinically and histologically. Immunostaining was performed using antibodies directed against NALP1, NALP3, IL-1ß, IL-8, and MxA on FD skin biopsies. Results were compared with normal controls and lichen planopilaris. Eleven patients had active disease and 6 had stable disease. NALP1, NALP3, and IL-1ß expression were significantly increased in hair follicles in FD compared with controls and lichen planopilaris. This study highlights the predominant immune signal associated with inflammasome activation in FD, suggesting the use of IL-1ß blockade in FD.

H3 Relaxin Protects Against Myocardial Injury in Experimental Diabetic Cardiomyopathy by Inhibiting Myocardial Apoptosis, Fibrosis and Inflammation.

BACKGROUND/AIMS: Apoptosis, fibrosis and NLRP3 inflammasome activation are involved in the development of diabetic cardiomyopathy (DCM). Human recombinant relaxin-3 (H3 relaxin) is a novel bioactive peptide that inhibits cardiac injury; however, whether H3 relaxin prevents cardiac injury in rats with DCM and the underlying mechanisms are unknown. METHODS: To investigate the effect of H3 relaxin on DCM, we performed a study using H3 relaxin treatment in male Sprague-Dawley (SD) rats with streptozotocin (STZ)-induced diabetes (DM). We measured apoptosis, fibrosis and NLRP3 inflammasome markers in the rat hearts four and eight weeks after the rats were injected with STZ (65 mg/kg) by western blot analysis. Subsequently, 2 or 6 weeks after the STZ treatment, the rats were treated with H3 relaxin [2 µg/kg/d (A group) or 0.2 µg/kg/d (B group)] for 2 weeks. Cardiac function was evaluated by echocardiography to determine the extent of myocardial injury in the DM rats. The protein levels of apoptosis, fibrosis and NLRP3 inflammasome markers were used to assess myocardial injury. In addition, we determined the plasma levels of IL-1ß and IL-18 using a Milliplex MAP Rat Cytokine/Chemokine Magnetic Bead Panel kit. RESULTS: The protein expression of cleaved caspase-8, caspase-9 and caspase-3 as well as fibrosis markers increased at 4 and 8 weeks in the STZ-induced diabetic hearts compared with the levels in the control group. Furthermore, the NLRP3 inflammasome was substantially activated in STZ-induced diabetic hearts, leading to increased IL-1ß and IL-18 levels. Compared with the DM group, the A group exhibited substantially better cardiac function. The protein levels of apoptosis markers were attenuated by H3 relaxin, indicating that H3 relaxin inhibited myocardial apoptosis in the hearts of diabetic rats. The protein expression of fibrosis markers was inhibited by H3 relaxin. Additionally, the protein expression and activation of the NLRP3 inflammasome were also effectively attenuated by H3 relaxin. CONCLUSIONS: This study is the first to demonstrate that H3 relaxin plays an anti-apoptotic, anti-fibrotic and anti-inflammatory role in DCM.

Increased neutrophil expression of pattern recognition receptors during COPD exacerbations.

Previously, we observed increased serum levels of damage-associated molecular patterns (DAMPs) during COPD exacerbations. Here, gene expression of DAMP receptors was measured in peripheral blood neutrophils of COPD patients during stable disease and severe acute exacerbation. The expression of toll-like receptor (TLR)2, TLR4 and NLR family, pyrin domain-containing 3 (NLRP3) was significantly increased, while serum levels of the soluble form of the decoy receptor for advanced glycation end-product (sRAGE) were decreased during exacerbation. Together, these data indicate that increased DAMP signalling contributes to activation of neutrophils during COPD exacerbations.

[Protective effect of taxifolin on H2O2-induced H9C2 cell pyroptosis].

OBJECTIVE: To explore the effect of taxifolin on H2O2-induced pyroptosis in H9C2 cells and the possible mechanisms.
 Methods: The H9C2 cells was divided into 3 groups: a control group, a hydrogen peroxide (H2O2)group and a taxifolin group. The morphology of H9C2 cells was observed by inverted phase contrast microscope. The mitochondrial membrane potential was measured by JC-1 staining and flow cytometry. The alteration of the level of reactive oxygen species (ROS) was detected by specific mitochondrial probe. The protein levels of cysteinyl aspartate specific proteinase-1 (caspase-1)was determined by Western blot. The mRNA levels of interleukin-18 (IL-18), interleukin-1a (IL-1a), interleukin-1b (IL-1b), absent in melanoma 2 (AIM2), apoptosis-associated apeck-like protein (ASC), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)and nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain-containing protein 4 (NLRC4) were determined by reverse transcription-polymerase chain reaction (RT-PCR).
 Results: Compared with the control group, the morphology of H9C2 cells obviously changed in the H2O2-treated group, which was guadually improved in the presence of taxifolin. Compared with the control group, the mitochondrial membrane potential was markedly decreased in the H2O2-treated cells, accompanied by the increase ofROS (both P<0.05). Compared with the H2O2 group, the mitochondrial membrane potential changes in the taxifolin group was increased while the ROS was decreased, with significant difference (both P<0.05). Compared with the control group, the protein level of caspase-1 and the mRNA levels of IL-18, IL-1a, IL-1b, AIM2, ASC, NLRP3 and NLRC4 in the H2O2-treated group were significantly increased (all P<0.05), which were attenuated in the presence of taxifolin (all P<0.05).
 Conclusion: Taxifolin can protect H9C2 cells from oxidative injury, and it is able to suppress the H2O2-induced H9C2 cell pyroptosis through inhibition of AIM2, NLRP3 and NLRC4 in flammasome.

Roles of the NLRP3 inflammasome in the pathogenesis of diabetic nephropathy.

Diabetic nephropathy (DN) is a serious complication of diabetes mellitus, and persistent inflammation in circulatory and renal tissues is an important pathophysiological basis for DN. The essence of the microinflammatory state is the innate immune response, which is central to the occurrence and development of DN. Members of the inflammasome family, including both "receptors" and "regulators", are key to the inflammatory immune response. Nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) and other inflammasome components are able to detect endogenous danger signals, resulting in activation of caspase-1 as well as interleukin (IL)-1ß, IL-18 and other cytokines; these events stimulate the inflammatory cascade reaction, which is crucial for DN. Hyperglycaemia, hyperlipidaemia and hyperuricaemia can activate the NLRP3 inflammasome, which then mediates the occurrence and development of DN through the K+ channel model, the lysosomal damage model and the active oxygen cluster model. In this review, we survey the involvement of the NLRP3 inflammasome in various signalling pathways and highlight different aspects of their influence on DN. We also explore the important effects of the NLRP3 inflammasome on kidney function and structural changes that occur during DN development and progression. It is becoming more evident that NLRP3 inflammasome targeting has therapeutic potential for the treatment of DN.

Inflammasome and toll-like receptor signaling in human monocytes after successful cardiopulmonary resuscitation.

BACKGROUND: Whole body ischemia-reperfusion injury (IRI) after cardiopulmonary resuscitation (CPR) induces a generalized inflammatory response which contributes to the development of post-cardiac arrest syndrome (PCAS). Recently, pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) and inflammasomes, have been shown to mediate the inflammatory response in IRI. In this study we investigated monocyte PRR signaling and function in PCAS. METHODS: Blood samples were drawn in the first 12 hours, and at 24 and 48 hours following return of spontaneous circulation in 51 survivors after cardiac arrest. Monocyte mRNA levels of TLR2, TLR4, interleukin-1 receptor-associated kinase (IRAK)3, IRAK4, NLR family pyrin domain containing (NLRP)1, NLRP3, AIM2, PYCARD, CASP1, and IL1B were determined by real-time quantitative PCR. Ex vivo cytokine production in response to stimulation with TLR ligands Pam3CSK4 and lipopolysaccharide (LPS) was assessed in both whole blood and monocyte culture assays. Ex vivo cytokine production of peripheral blood mononuclear cells (PBMCs) from a healthy volunteer in response to stimulation with patients' sera with or without LPS was assessed. The results were compared to 19 hemodynamically stable patients with coronary artery disease. RESULTS: Monocyte TLR2, TLR4, IRAK3, IRAK4, NLRP3, PYCARD and IL1B were initially upregulated in patients following cardiac arrest. The NLRP1 and AIM2 inflammasomes were downregulated in resuscitated patients. There was a significant positive correlation between TLR2, TLR4, IRAK3 and IRAK4 expression and the degree of ischemia as assessed by serum lactate levels and the time until return of spontaneous circulation. Nonsurvivors at 30 days had significantly lower mRNA levels of TLR2, IRAK3, IRAK4, NLRP3 and CASP1 in the late phase following cardiac arrest. We observed reduced proinflammatory cytokine release in response to both TLR2 and TLR4 activation in whole blood and monocyte culture assays in patients after CPR. Sera from resuscitated patients attenuated the inflammatory response in cultured PBMCs after co-stimulation with LPS. CONCLUSIONS: Successful resuscitation from cardiac arrest results in changes in monocyte pattern recognition receptor signaling pathways, which may contribute to the post-cardiac arrest syndrome. TRIAL REGISTRATION: The trial was registered in the German Clinical Trials Register ( DRKS00009684 ) on 27/11/2015.

The NLRP3/Caspase-1/Interleukin-1ß Axis Is Active in Human Lumbar Cartilaginous Endplate Degeneration.

BACKGROUND: Modic changes are the MRI signal changes of degenerative lumbar vertebral endplate and which lead to or accelerate intervertebral disc degeneration. NLRP3, caspase-1, and interleukin-1ß (IL-1ß) play a pivotal role in the pathogenesis of many inflammatory diseases, such as osteoarthritis. However, the roles of IL-1ß and its activators caspase-1 and NLRP3 are unclear in the degenerative endplate. QUESTIONS/PURPOSES: We asked: (1) What are the degenerative changes of the histologic features and chondrogenic markers' gene expressions between the cartilaginous endplates of patients with Modic changes and trauma (control)? (2) How does the NLRP3/caspase-1/IL-1ß axis in the cartilaginous endplates of patients with Modic changes compare with control (trauma) specimens? METHODS: Surgical specimens of cartilaginous endplates were divided into Modic changes (n = 56) and the trauma control (n = 16) groups. Hematoxylin and eosin and safranin O staining of cartilaginous endplate tissues were done to evaluate the extracellular matrix. Reverse transcription-polymerase chain reaction was performed on these tissues to investigate mRNA expression of type II collagen (Col II), SOX-9, matrix metalloproteinase-3, and a disintegrin like and metalloproteinase thrombospondin type I motifs-5. NLRP3, caspase-1, and IL-1ß were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: Hematoxylin and eosin and safranin O staining showed the extracellular matrix degraded in the cartilaginous endplates of patients with Modic changes but not in the control cartilaginous endplates. Chondrogenic Col II (p = 0.024) and SOX9 (p = 0.053) were downregulated in the Modic changes group compared with the control group. In contrast to the control group, the transcriptional levels of NLRP3 (p < 0.001), caspase-1 (p = 0.054), and IL-1ß (p = 0.001) were all upregulated in the Modic changes group. CONCLUSIONS: The expression of NLRP3, caspase-1, and IL-1ß was upregulated in the patients with low back pain and Modic changes on MRI compared with patients with vertebral burst fracture without degenerative changes on MRI. The data suggest the NLRP3/caspase-1/IL-1ß axis may be implicated in lumbar cartilaginous endplate degeneration. CLINICAL RELEVANCE: The NLRP3/caspase-1/IL-1ß axis is active in cartilaginous endplates of patients with Modic changes and inflammatory cascades can exacerbate the cartilaginous endplate degeneration which may act as a trigger for intervertebral disc degeneration and low back pain. If these findings can be confirmed by others, we hope that new and effective therapy could be developed directed against this target.