HIF-1α aggravated traumatic brain injury by NLRP3 inflammasome-mediated pyroptosis and activation of microglia.

Hypoxia inducible factor 1 alpha (HIF-1α) is involved in regulating the biological functions of neuronal death after traumatic brain injury (TBI), and attaches importance in the inflammatory response, but its potential mechanism is still unknown. Our study aimed to explore the regulatory mechanism between HIF-1α and NLRP3 inflammasome after TBI. Male mice underwent controlled cortical impact (CCI) or sham-operated procedures. Brain water content and blood-brain barrier permeability were measured at the indicated time after TBI. The behavioral performance, ELISA, immunofluorescence, and western blot analysis were used to determine whether HIF-1α specifically targeted TBI-induced pyroptosis. We discovered that TBI-induced brain injury caused by external mechanical forces is characterized by edema and blood-brain barrier disorder, and the release of IL-1ß, IL-18, and LDH and upregulation of HIF-1α expression, reaching the peak on the third day post-TBI. In addition, HIF-1α accumulated NLRP3 inflammasome-mediated pyroptosis and activation of microglia. The protein expressions of NLRP3, GSDMD, GSDMD-N, pro-caspase 1, and cleaved caspase 1 were markedly increased in the injured cortex, which were restored to normal levels by the interference of HIF-1α. The inactivation of HIF-1α conferred neuroprotection and alleviated brain injury after TBI. HIF-1α was implicated in TBI-induced brain injury, aggravated NLRP3 inflammasome -mediated pyroptosis, and the activation of microglia, which provided a potential target for treating TBI.

Peripheral blood expression levels of inflammasome complex components in two different focal epilepsy syndromes.

BACKGROUND: Although the role of inflammation in epilepsy pathogenesis has been extensively investigated, the inflammasome complex, a key component of neuroinflammation, has been understudied in epilepsy patients. METHODS: To better understand the involvement of this system in epilepsy, levels of inflammasome complex components (NLRP1, NLRP3, CASP1, ASC), end-products of inflammasome complex activity [IL-1ß, IL-18, nitric oxide synthase (NOS) isoforms] and other inflammatory factors (NFκB, IL-6, TNF-α) were measured in peripheral blood of patients with focal epilepsy of unknown cause (FEoUC) (n = 47), mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (n = 35) and healthy controls using real time qPCR and/or ELISA. RESULTS: Inflammasome complex associated factors were either downregulated or unchanged in epilepsy patients. Likewise, flow cytometry studies failed to show an increase in ratios of NLRP3-expressing CD3+ and CD14+ peripheral blood mononuclear cells (PBMC) in epileptic patients. Anti-neuronal antibody positive epilepsy patients showed increased NLRP1 and neuronal NOS mRNA expression levels, whereas patients under poly-therapy showed reduced serum inflammasome levels. FEoUC patients demonstrated increased PBMC NFκB mRNA expression levels and serum IL-1ß and IL-6 levels. Both MTLE-HS and FEoUC patients displayed higher ratios of NFκB-expressing CD14+ PBMC than healthy controls. CONCLUSIONS: Although previous clinical studies have implicated increased inflammasome complex expression levels in epilepsy, our results indicate suppressed inflammasome complex activity in the peripheral blood of focal epilepsy patients. Alternatively, the IL-6-NFκB signaling pathway, appears to be activated in focal epilepsy, suggesting that factors of this pathway might be targeted for future theranostic applications.

Structural Biology of NOD-Like Receptors.

The nucleotide-binding domain (NBD) and leucine-rich repeat (LRR) containing (NLR) proteins are a large family of intracellular immune receptors conserved in both animals and plants. Mammalian NLRs function as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) or host-derived danger associated molecular patterns (DAMPs). PAMP or DAMP perception activates NLRs which consequently recruit pro-caspase-1 directly or indirectly. These sequential events result in formation of large multimeric protein complexes termed inflammasomes that mediate caspase-1 activation for pyroptosis and cytokine secretion. Recent structural and biochemical studies provide significant insights into the acting mechanisms of NLR proteins. In this chapter, we review and discuss these studies concerning autoinhibition, ligand recognition, activation of NLRs, and assembly of NLR inflammasomes.

AIM2 Inflammasome Assembly and Signaling.

AIM2 (absent in melanoma 2) is a cytoplasmic sensor of double-stranded DNA from pathogens or damaged cellular organelles. It recruits ASC (apoptosis-associated specklike protein containing a CARD) and caspase-1 to form the AIM2 inflammasome, activate caspase-1, and elicit inflammatory responses via cytokine maturation and pyroptotic cell death. Structural studies from X-ray crystallography, NMR, and cryo-EM have revealed many details in AIM2 inflammasome activation, assembly, and regulation. Many principles learned from AIM2 inflammasome also apply to other inflammasomes. In this chapter, we discuss the interactions between dsDNA and AIM2-like receptors, between AIM2 and adaptor protein ASC, and between ASC and caspase-1 with the focus on helical filament assembly formed by PYD and CARD domains.

MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and specific small-molecule inhibitor of the NLRP3 pathway, but its molecular target is not defined. Here, we show that MCC950 directly interacts with the Walker B motif within the NLRP3 NACHT domain, thereby blocking ATP hydrolysis and inhibiting NLRP3 activation and inflammasome formation.

Altered expression of inflammasomes in Hirschsprung's disease.

AIM OF THE STUDY: The pathogenesis of Hirschsprung's disease-associated enterocolitis (HAEC) is poorly understood. Inflammasomes are a large family of multiprotein complexes that act to mediate host immune responses to microbial infection and have a regulatory or conditioning influence on the composition of the microbiota. Inflammasomes and the apoptosis-associated speck-like protein (ASC) lead to caspase-1 activation. The activated caspase-1 promotes secretion of pro-inflammatory cytokines (IL-1ß and IL-18) from their precursors (pro-IL-1ß and pro-IL-18). Inflammasomes have been implicated in a host of inflammatory disorders. Among the inflammasomes, NLRP3, NLRP12 and NLRC4 are the most widely investigated. Knock-out mice models of inflammasomes NLRP3, NLRP12, NLRC4, caspase-1 and ASC are reported to have higher susceptibility to experimental colitis. The purpose of this study was to investigate the expression of NLRP3, NLRP12, NLRC4, caspase-1, ASC, pro-IL-1ß and pro-IL-18 in the bowel specimens from patients with HSCR and controls. METHODS: Pulled-through colonic specimens were collected from HSCR patients (n = 6) and healthy controls from the proximal colostomy of children with anorectal malformations (n = 6). The gene expression of NLRP3, NLRP12, NLRC4, caspase-1, ASC, pro-IL-1ß and pro-IL-18 was assessed using qPCR. The protein distribution was assessed using immunofluorescence and confocal microscopy. MAIN RESULTS: qRT-PCR analysis revealed that NLRP3, NLRP12, NLRC4, ASC and pro-IL-1ß gene expressions was significantly downregulated in the aganglionic and ganglionic colon of patients with HSCR compared to controls. Confocal microscopy revealed a markedly decreased expression of NLRP3, NLRP12, NLRC4 and ASC protein in the colonic epithelium of aganglionic and ganglionic bowel of patients with HSCR compared to controls. CONCLUSIONS: To our knowledge, this is the first study analyzing NLRP3, NLRP12, NLRC4, ASC and pro-IL-1ß gene expressions in patients with HSCR. Decreased expression of NLRP3, NLRP12, NLRC4, ASC and pro-IL-1ß in the aganglionic and ganglionic bowel may increase susceptibility of HSCR patients to develop HAEC.

Selective NLRP3 inflammasome inhibitor reduces neuroinflammation and improves long-term neurological outcomes in a murine model of traumatic brain injury.

The nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated inflammatory response has emerged as a prominent contributor to the pathophysiological processes of traumatic brain injury (TBI). Recently, a potent, selective, small-molecule NLRP3 inflammasome inhibitor, MCC950, was described. Here, we investigated the effect of MCC950 on inflammatory brain injury and long-term neurological outcomes in a mouse model of TBI. Male C57/BL6 mice were subjected to TBI using the controlled cortical impact injury (CCI) system. Western blotting, flow cytometry, and immunofluorescence assays were utilized to analyze post-traumatic NLRP3 inflammasome expression and determine its cellular source. We found that NLRP3 inflammasome expression was significantly increased in the peri-contusional cortex and that microglia were the primary source of this expression. The effects of MCC950 on mice with TBI were then determined using post-assessments including analyses of neurological deficits, brain water content, traumatic lesion volume, neuroinflammation, blood-brain barrier (BBB) integrity, and cell death. MCC950 treatment resulted in a better neurological outcome after TBI by alleviating brain edema, reducing lesion volume, and improving long-term motor and cognitive functions. The therapeutic window for MCC950 against TBI was as long as 6 h. Furthermore, the neuroprotective effect of MCC950 was associated with reduced microglial activation, leukocyte recruitment, and pro-inflammatory cytokine production. In addition, MCC950 preserved BBB integrity, alleviated TBI-induced loss of tight junction proteins, and attenuated cell death. Notably, the efficacy of MCC950 was abolished in microglia-depleted mice. These results indicate that microglia-derived NLRP3 inflammasome may be primarily involved in the inflammatory response to TBI, and specific NLRP3 inflammasome inhibition using MCC950 may be a promising therapeutic approach for patients with TBI.

NLRP3 Inflammasome Is Involved in Q-VD-OPH Induced Necroptosis Following Cerebral Ischemia-Reperfusion Injury.

Necroptosis is a manner of caspase-independent cell death,which accounts for delayed ischemic cerebral injury, and can be used as a novel tool to expand the treatment time window in ischemic cerebral injury. Q-VD-OPH, a novel pan caspase inhibitor, has been identified as an inducer of necroptosis. In this study, we determined the optimal dose of Q-VD-OPH, which induces necroptosis in rats by the middle cerebral artery occlusion, followed by reperfusion. Furthermore, we report that the NLRP3 inflammasome is involved in necroptosis, with levels of NLRP3 inflammasome proteins as well as inflammatory cytokines, such as IL-1ß, being elevated. We also demonstrated that NLRP3 was not only expressed in microglia and vascular endothelial cell, but also in neurons when necroptosis is induced with Q-VD-OPH. Inhibition of NLRP3 by glyburide strongly suppressed the expression of NLRP3 inflammasome proteins and IL-1ß, and markedly reduced brain tissue damage. Our findings provide evidence that pretreatment with Q-VD-OPH suppresses apoptosis and induces necroptosis in the cerebral ischemia-reperfusion model. We also identified that the NLRP3 inflammasome plays an important role in neuronal necroptosis, and that NLRP3 inflammasome deficiency reduces brain tissue damage after cerebral ischemia-reperfusion injury in rats.

Adipose-derived stem cells ameliorate colitis by suppression of inflammasome formation and regulation of M1-macrophage population through prostaglandin E2.

Inflammatory bowel disease (IBD) is an idiopathic disease caused by a dysregulated immune response to intestinal microbes in an individual with a genetic predisposition. Therefore, alleviation of inflammation is very important to treat IBD. Mesenchymal stem cells (MSCs) have been highlighted as new candidates for treating autoimmune disease based on their immunomodulatory properties. In this study, we investigated the anti-inflammatory mechanism and therapeutic effects of adipose tissue-derived MSCs (ASCs) using THP-1 macrophages and dextran sodium sulfate (DSS)-induced mice with chronic colitis. LPS-treated THP-1 cells expressed mRNA of CD11b, an M1 macrophage marker, at day 2. However, THP-1 co-cultured with ASCs expressed mRNA of CD206, CD68, CCL18, legumain, and IL-10, markers of M2 macrophages. In THP-1 cells co-cultured with ASCs, precursor (pro)-IL-1ß, Cox-2, and NLRP3 increased dramatically compared to LPS-treated THP-1 cells. Secretion of IL-1ß and IL-18 was significantly inhibited by ASCs, but PGE2 production was highly increased in co-culture conditions of THP-1 and ASCs. IL-18 secretion was inhibited by PGE2 treatment, and PGE2 inhibited inflammasome complex (ASC/Cas-1/NLRP3) formation in THP-1 cells. In the DSS-induced chronic colitis model, ASCs ameliorated colitis by decreasing the total number of macrophages and the M1 macrophage population. Our results suggest that ASCs can suppress the inflammatory response by controlling the macrophage population, and ASCs may be therapeutically useful for the treatment of IBD.

Overexpression of NLRP3, NLRC4 and AIM2 inflammasomes and their priming-associated molecules (TLR2, TLR4, Dectin-1, Dectin-2 and NFκB) in Malassezia folliculitis.

The activation of NLRP3, NLRC4 and AIM2 inflammasomes is pivotal for innate immunity against some pathogenic fungi, but their role in the pathogenesis of Malassezia folliculitis (MF) remains unclear. The objective of the study was to determine the expression of 4 canonical inflammasomes (NLRP1, NLRP3, NLRC4 and AIM2) and their priming-associated molecules (TLR2, TLR4, Dectin-1, Dectin-2 and NFκB) in MF lesion. Expression of NLRP1, NLRP3, NLRC4, AIM2, caspase-1, IL-1ß, TLR2, TLR4, Dectin-1, Dectin-2 and NFκB was detected by immunohistochemistry in skin lesion of 23 MF patients and normal skin of 12 healthy subjects. Furthermore, NLRP1, NLRP3, NLRC4, AIM2, caspase-1 and IL-1ß mRNA was measured by quantitative real-time PCR (qRT-PCR) in 12 MF cases and 10 controls. Immunohistochemical analysis revealed that NLRP3, NLRC4, AIM2, Casp-1, IL-1ß, TLR2, TLR4, Dectin-1, Dectin-2 and NFκB expression was up-regulated in the epidermis and dermal inflammatory cells of MF lesion compared with control skin (P < .01-.05), but NLRP1 expression was not different between both groups (P > .05). qRT-PCR showed that levels of NLRP3, Casp-1 and IL-1ß mRNA were significantly increased (P < .01-.05), whereas those of NLRP1, NLRC4 and AIM2 mRNA were slightly augmented compared to control skin (P > .05). Our observation suggests that simultaneous activation of NLRP3, NLRC4 and AIM2 inflammasomes may play an important role in the pathogenesis of MF.

TNF regulates transcription of NLRP3 inflammasome components and inflammatory molecules in cryopyrinopathies.

The NLRP3 inflammasome is a protein complex responsible for caspase-1-dependent maturation of the proinflammatory cytokines IL-1ß and IL-18. Gain-of-function missense mutations in NLRP3 cause the disease spectrum known as the cryopyrin-associated periodic syndromes (CAPS). In this study, we generated Nlrp3-knockin mice on various KO backgrounds including Il1b/Il18-, caspase-1-, caspase-11- (Casp1/11-), and Tnf-deficient strains. The Nlrp3L351P Il1b-/- Il18-/- mutant mice survived and grew normally until adulthood and, at 6 months of age, exhibited marked splenomegaly and leukophilia. Injection of these mice with low-dose LPS resulted in elevated serum TNF levels compared with Nlrp3L351P Casp1/11-/- mice and Il1b-/- Il18-/- littermates. Treatment of Nlrp3A350V mice with the TNF inhibitor etanercept resulted in all pups surviving to adulthood, with normal body and spleen/body weight ratios. Nlrp3A350V Tnf-/- mice showed a similar phenotypic rescue, with marked reductions in serum IL-1ß and IL-18, reduced myeloid inflammatory infiltrate in the skin and spleen, and substantial decreases in splenic mRNA expression of both inflammasome components (Nlrp3, Pycard, pro-Casp1) and pro-cytokines (Il1b, Il18). Likewise, we observed a reduction in the expression of both pro-Casp1 and pro-Il1b in cultured Nlrp3A350V Tnf-/- BM-derived DCs. Our data show that TNF is an important transcriptional regulator of NLRP3 inflammasome components in murine inflammasomopathies. Moreover, these results may have therapeutic implications for CAPS patients with partial responses to IL-1-targeted therapies.

NLRC3 protein inhibits inflammation by disrupting NALP3 inflammasome assembly via competition with the adaptor protein ASC for pro-caspase-1 binding.

Inflammasomes are multiprotein complexes that sense pathogen-associated and danger-associated molecular patterns and induce inflammation in cells. The NALP3 inflammasome is tightly regulated by recently discovered control mechanisms, but other modulators still remain to be characterized. NLR family CARD-containing 3 (NLRC3) protein, a caspase recruitment domain (CARD)-containing member of the nucleotide oligomerization domain-like receptor (NLR) family, was found to down-regulate the NF-κB pathway and stimulator of interferon genes (STING)-dependent cytokine secretion. However, the effect of NLRC3 on the NALP3 inflammasome or other inflammasomes is still unknown. We hypothesized that NLRC3 might inhibit NALP3 inflammasome complex assembly. Toward this end, we tested whether NLRC3 overexpression or knockdown influences NALP3 activity in human monocyte and HEK293FT cells when the complex is ectopically reconstituted. We found that NLRC3 indeed decreases NALP3-induced IL-1ß maturation and secretion, pro-caspase-1 cleavage, and speck formation by apoptosis-associated speck-like protein containing a CARD (ASC) protein in response to NALP3 activators. We also show that endogenous NLRC3 interacts with both ASC and pro-caspase-1 but not with NALP3, disrupts ASC speck formation through its CARD, and impairs the ASC and pro-caspase-1 interaction. Moreover, the NLRC3 CARD alone could dampen IL-1ß secretion and ASC speck formation induced by NALP3 mutants associated with autoinflammatory diseases. In conclusion, we show here that, besides its role in the inhibition of the NF-κB pathway, NLRC3 interferes with the assembly and activity of the NALP3 inflammasome complex by competing with ASC for pro-caspase-1 binding.

The NLRP3 Inflammasome is Involved in the Pathogenesis of Parkinson's Disease in Rats.

The etiology and pathogenesis of Parkinson's disease (PD) are complicated and have not been fully elucidated, but an important association has been identified between inflammation and PD. In this study, we investigated the role of the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain-containing (NLRP) 3 inflammasome, consisting of NLRP3, caspase-1 and cytokines of the IL-1 family, in lipopolysaccharide (LPS)-induced and 6-hydroxydopamine (6-OHDA)-induced PD rats. Microinjection of different doses of caspase-1 inhibitor (Ac-YVAD-CMK, 300 or 1200 ng/rat) was performed for seven consecutive days. Then, rotational behavior, the number of dopamine (DA) neurons in the substantia nigra pars compacta (SNc), and the mRNA and protein expression levels of NLRP3 inflammasome components were measured 14 days after the microinjection setup was established. Results showed that high mRNA and protein expression levels of NLRP3 inflammasome components were observed in the injected side of the LPS- and 6-OHDA-induced PD rats; Ac-YVAD-CMK inhibited the mRNA and protein expression of NLRP3 inflammasome components in both LPS- and 6-OHDA-induced PD rats. Moreover, the number of rotations was significantly decreased, and the number of DA neurons in the SNc improved. Our data indicate that the NLRP3 inflammasome participates in the pathogenesis of PD and that inhibiting the downstream pathway of the NLRP3/caspase-1/IL-1ß axis can alleviate the occurrence of PD symptoms, providing a new basis for the prevention and treatment of PD.

Rutin protects endothelial dysfunction by disturbing Nox4 and ROS-sensitive NLRP3 inflammasome.

High glucose induced endothelial dysfunction is blamed for initiation of vascular complication in Type 2 diabetes mellitus. Rutin has been described in possessing comprehensive pharmacological activities, but the mechanism involved in endothelial protection through regulating oxidative stress and damage is still ambiguous. In the present study, rutin was evaluated in high glucose stimulated human umbilical vein endothelial cells (HUVECs) and high glucose diet-treated SD rats were applied to explored the molecular mechanism in rutin counteracting oxidative stress and damage. Firstly, 30 and 100µM rutin effectively increased HUVECs viability in high glucose challenge. Then we found that rutin could dose-dependently reduced high glucose mediated mRNA and protein expressions of Nox4. With Nox4 and Nox2 inhibitors, we further confirm that Nox4, but not Nox2, was responsible for reactive oxygen species (ROS) production in high glucose environment. Moreover, rutin and Nox4 inhibitor significantly ameliorated ROS generation and TXNIP, NLRP3, caspase-1 and IL-1ß proteins expression in vivo. Furthermore, rutin substantially recovered nitric oxide production in HUVECs under high glucose condition. However, rutin could not inhibited inflammasome protein expressions and improved nitric oxide production in Nox4-overexpressed HUVECs under high glucose challenges. Finally, we found that rutin restored phenylephrine-mediated contractions and acetylcholine induced relaxations in aortic tissue of high glucose diet treated rats. In vitro, expressions of TXNIP, NLRP3, and caspase-1 in aortic tissue of high glucose diet treated rats were decreased under rutin administration. In summary, rutin may protect endothelial dysfunction through inhibiting Nox4 responsive oxidative stress and ROS-sensitive NLRP3 signaling pathway under high glucose stress both in vivo and vitro.

Upregulation of NLRP3 inflammasome components in Mooren's ulcer.

PURPOSE: Mooren's ulcer (MU) is a peripheral corneal ulceration of presumed autoimmune etiology. NLRP3 inflammasome has been shown to be involved in a variety of autoimmune and auto-inflammatory diseases. However, the role of NLRP3 inflammasome in MU has not been investigated. Here, we evaluate the expression of NLRP3 inflammasome and its downstream inflammatory factors in human MU. METHODS: Conjunctival biopsy specimens were obtained from seven patients with MU and six healthy donors. The removed conjunctivas were histopathologically evaluated for NLRP3 inflammasome component expression using antibodies directed against NLRP3, Caspase-1 (CASP1), and Interleukin-1ß (IL-1ß). Quantitative real-time PCR was used to measure the mRNA expression of NLRP3 and IL-1ß, and the protein expressions of NLRP3, pro-CASP1, CASP1, and IL-1ß were detected by Western blotting. RESULTS: NLRP3 and IL-1ß mRNA expression showed higher levels in the MU group than in healthy controls. Western-blot and immunofluorescence analysis also showed that basal expression of NLRP3 inflammasome components (NLRP3, CAPS1, and IL-1ß) was elevated in patients with MU compared with healthy controls. Most importantly, we found that the cleavaged form of CASP1 and IL-1ß was significantly increased in MU patients compared with healthy donors, which indicates that the upregulation of NLRP3 inflammasome was probably responsible for the enhanced IL-1ß production in MU patients. CONCLUSIONS: This study demonstrated that the expression of the NLRP3-CASP1-IL-1ß signaling pathway was markedly increased in the conjunctival lesions of patients with MU, suggesting the involvement of NLRP3 inflammasome in the onset and development of the inflammation in MU.

Elevated levels of triglycerides and vldl-cholesterol provoke activation of nlrp1 inflammasome in endothelial cells.

BACKGROUND: Soluble stimuli present in the plasma of patients with peripheral arterial disease (PAD) are capable of directly stimulating intracellular signalling in endothelium. Oxidized-LDL (oxLDL) induces NLRP3 inflammasome activation in macrophages. However, it is not clear how lipid profile affect NLRP1 inflammasome gene expression in endothelial cells. In this study, the effect of cholesterol and TG of plasma of patients with PAD on NLRP1 inflammasome gene expression in human arterial endothelial cells (HAECS) was assessed. METHODS: We included 113 patients with symptomatic PAD. HAECs were stimulated for 2h using the plasma samples of the study participants. The NLRP1 quantification of the transcription was carried out on the 7500 real-time PCR system using the Taqman® Universal PCR Master Mix and Assays on demand. Relative quantification of the NLRP1 expression was carried out using the ΔΔCt (threshold cycle) comparative method. RESULTS: Plasma from patients with elevated VLDL-cholesterol levels (>33.6mg/dL, the median value of the sample) provoked a higher expression of NLRP1 inflammasome in HAECs (RQ=1.15±0.23 vs. 1.05±0.69; p=0.045), as well as plasma from patients with elevated TGs levels (>168mg/dL, the median value of the sample) (RQ=1.15±0.23 vs. 1.05±0.69; p=0.045). A positive correlation was found between NLRP1 inflammasome expression and VLDL-cholesterol plasma levels (r=0.4; p<0.001) as between NLRP1 inflammasome expression and TG levels (r=0.4; p<0.001). CONCLUSIONS: Plasma TG and VLDL cholesterol of patients with atherosclerosis, manifested as PAD, promote the in vitro NLRP1 inflammasome expression in HAECs.

Expression of Inflammasome-Associated Proteins in Human Oropharyngeal Squamous Cell Carcinoma.

Inflammasomes, large protein complexes typically consisting of a Nod-like receptor (NLR), adapter protein apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1, are postulated to be activated in response to danger signals arising from tumors. Inflammasomes are thought to have critical but contrasting roles through facilitating antitumor immunity and inducing oncogenic factors. However, the role and function of inflammasomes in oropharyngeal carcinoma remain unclear. We analyzed nine specimens of oropharyngeal squamous cell carcinoma (SCC) and determined the expression of NLRP3, ASC, interleukin (IL)-1ß, IL-18 and caspase-1 in the specimens with and without human papilloma virus (HPV) infection using immunohistochemistry, and analyzed the correlations between the altered expression of these proteins and clinicopathological factors of oropharyngeal SCC. We found strong expression of NLRP3, ASC, IL-1ß, IL-18 and caspase-1 in human oropharyngeal SCC and weak or no expression of these proteins in normal tonsils. Furthermore, the distribution of mindbomb E3 ubiquitin protein ligase 1 and inflammasome-associated proteins in oropharyngeal SCC was not significantly different; there was no correlation between the expression of inflammasome-associated proteins and HPV infection. These findings suggest that inflammasomes in oropharyngeal SCC play a key role through facilitating antitumor immunity and the possibility of new roles for inflammasomes in the oropharynx.

Pyoderma gangrenosum and its syndromic forms: evidence for a link with autoinflammation.

Pyoderma gangrenosum is a rare inflammatory neutrophilic dermatosis manifesting as painful ulcers with violaceous, undermined borders on the lower extremities. It may occur in the context of classic syndromes like PAPA (pyogenic arthritis, pyoderma gangrenosum and acne) and SAPHO (synovitis, acne, pustulosis, hyperostosis, osteitis), as well as in a recently described entity named PASH (pyoderma gangrenosum, acne and suppurative hidradenitis). Pyoderma gangrenosum has recently been included within the spectrum of autoinflammatory diseases, which are characterized by recurrent episodes of sterile inflammation, without circulating autoantibodies and autoreactive T cells. In PAPA syndrome, different mutations involving the PSTPIP1 gene, via an increased binding affinity to pyrin, induce the assembly of inflammasomes. These are molecular platforms involved in the activation of caspase 1, a protease that cleaves inactive prointerleukin (pro-IL)-1ß to its active isoform IL-1ß. The overproduction of IL-1ß triggers the release of a number of proinflammatory cytokines and chemokines, which are responsible for the recruitment and activation of neutrophils, leading to neutrophil-mediated inflammation. In SAPHO syndrome, the activation of the PSTPIP2 inflammasome has been suggested to play a role in inducing the dysfunction of the innate immune system. Patients with PASH have recently been reported to present alterations of genes involved in well-known autoinflammatory diseases, such as PSTPIP1, MEFV, NOD2 and NLRP3. Pyoderma gangrenosum and its syndromic forms can be regarded as a single clinicopathological spectrum in the context of autoinflammation.

Inhibition of the NLRP3 inflammasome limits the inflammatory injury following myocardial ischemia-reperfusion in the mouse.

BACKGROUND: Successful reperfusion is the most effective strategy to reduce ischemic injury in acute myocardial infarction (AMI). Ischemic injury, however, also triggers a secondary ischemia-independent injury, known as reperfusion injury, contributing to the overall infarct size. We hypothesize that inhibition of the Nod-like Receptor Protein-3 (NLRP3) inflammasome limits infarct size following myocardial ischemia/reperfusion (I/R), by inhibiting the inflammatory component of the reperfusion injury. METHODS: CD-1 male mice underwent transient ligation of the left anterior descending coronary artery for 30 or 75min followed by reperfusion. Infarct size was measured at 1, 3 and 24h. A NLRP3 inflammasome inhibitor (NLRP3inh) or vehicle was administrated immediately at time of reperfusion or with a delay of 1 or 3h of reperfusion. RESULTS: A time-dependent increase in infarct size was measured at 1, 3, and 24h after reperfusion (11±2%, 30±5% and 43±4% of the area at risk respectively; P<0.001 for trend). NLRP3 myocardial expression was significantly increased at 24h and 6h vs 3h (P<0.01). Administration of the NLRP3inh at reperfusion did not reduce infarct size at 3h, while it significantly reduced infarct size at 24h (-56% vs vehicle, P<0.01). The NLRP3inh given 1h after reperfusion also significantly decreased caspase-1 activity and infarct size measured at 24h, whereas the NLRP3inh did not when given with a delay of 3h. CONCLUSIONS: Pharmacological inhibition of the NLRP3 inflammasome within 1h of reperfusion limits the secondary inflammatory injury and infarct size following myocardial ischemia-reperfusion in the mouse.

Effects of aging in the expression of NOD-like receptors and inflammasome-related genes in oral mucosa.

The molecular changes underlying the higher risk of chronic inflammatory disorders during aging remain incompletely understood. Molecular variations in the innate immune response related to recognition and interaction with microbes at mucosal surfaces could be involved in aging-related inflammation. We developed an ontology analysis of 20 nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) and seven inflammasome-related genes (IRGs) in healthy and inflamed/periodontitis oral mucosal tissues from young, adolescent, adult, and aged non-human primates (Macaca mulatta) using the GeneChip(®) Rhesus Macaque Genome array. Validation of some of the significant changes was done by quantitative reverse transcription-polymerase chain reaction. The expression of NLRB/NAIP, NLRP12, and AIM2 increased with aging in healthy mucosa whereas NLRC2/NOD2 expression decreased. Although higher expression levels of some NLRs were generally observed with periodontitis in adult mucosal tissues (e.g. NLRB/NAIP, NLRP5, and NLRX1), various receptors (e.g. NLRC2/NOD2 and NLRP2) and the inflammasome adaptor protein ASC, exhibited a significant reduction in expression in aged periodontitis tissues. Accordingly, the expression of NLR-activated innate immune genes, such as HBD3 and IFNB1, was impaired in aged but not adult periodontitis tissues. Both adult and aged tissues showed significant increase in interleukin-1ß expression. These findings suggest that the expression of a subset of NLRs appears to change with aging in healthy oral mucosa, and that aging-related oral mucosal inflammation could involve an impaired regulation of the inflammatory and antimicrobial response associated with downregulation of specific NLRs and IRGs.