NLR Family CARD Domain-containing Protein 4 Research Articles

Inflammasome protein scaffolds the DNA damage complex during tumor development.

Inflammasome sensors activate cellular signaling machineries to drive inflammation and cell death processes. Inflammasomes also control the development of certain diseases independently of canonical functions. Here, we show that the inflammasome protein NLR family CARD domain-containing protein 4 (NLRC4) attenuated the development of tumors in the Apcmin/+ mouse model. This response was independent of inflammasome signaling by NLRP3, NLRP6, NLR family apoptosis inhibitory proteins, absent in melanoma 2, apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1 and caspase-11. NLRC4 interacted with the DNA-damage-sensing ataxia telangiectasia and Rad3-related (ATR)-ATR-interacting protein (ATRIP)-Ewing tumor-associated antigen 1 (ETAA1) complex to promote the recruitment of the checkpoint adapter protein claspin, licensing the activation of the kinase checkpoint kinase-1 (CHK1). Genotoxicity-induced activation of the NLRC4-ATR-ATRIP-ETAA1 complex drove the tumor-suppressing DNA damage response and CHK1 activation, and further attenuated the accumulation of DNA damage. These findings demonstrate a noninflammatory function of an inflammasome protein in promoting the DNA damage response and mediating protection against cancer.

Identifying prognostic genes related PANoptosis in lung adenocarcinoma and developing prediction model based on bioinformatics analysis

Cell death-related genes indicate prognosis in cancer patients. PANoptosis is a newly observed form of cell death that researchers have linked to cancer cell death and antitumor immunity. Even so, its significance in lung adenocarcinomas (LUADs) has yet to be elucidated. We extracted and analyzed data on mRNA gene expression and clinical information from public databases in a systematic manner. These data were utilized to construct a reliable risk prediction model for six regulators of PANoptosis. The Gene Expression Omnibus (GEO) database validated six genes with risk characteristics. The prognosis of LUAD patients could be accurately estimated by the six-gene-based model: NLR family CARD domain-containing protein 4 (NLRC4), FAS-associated death domain protein (FADD), Tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD), Receptor-interacting serine/threonine-protein kinase 1 (RIPK1), Proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), and Mixed lineage kinase domain-like protein (MLKL). Group of higher risk and Cluster 2 indicated a poor prognosis as well as the reduced expression of immune infiltrate molecules and human leukocyte antigen. Distinct expression of PANoptosis-related genes (PRGs) in lung cancer cells was verified using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we evaluated the relationship between PRGs and somatic mutations, tumor immune dysfunction exclusion, tumor stemness indices, and immune infiltration. Using the risk signature, we conducted analyses including nomogram construction, stratification, prediction of small-molecule drug response, somatic mutations, and chemotherapeutic response.

Role of some inflammasomes in rheumatoid arthritis patients in Egypt

AimThis study aims to demonstrate the role of some inflammasomes genes: NLRC4 (the NLR family, CARD domain-containing protein 4), NLRP1 (NLR family, pyrin domain-containing 1), ASC (Apoptosis-associated speck-like protein containing a CARD), and CASPASE-1 in the pathogenesis of Rheumatoid arthritis (RA) in Egyptian population.Main methodsThe expression level of NLRC4, NLRP1, ASC, and CASPASE-1 within PBMCs isolated from all RA subjects by quantitative real-time PCR. GAPDH gene was used as a reference gene. Measurement of serum level of IL-1β and IL-18 was performed using ELISA.Key findingsResults showed dysregulated inflammasomes expression that may participate in the pathogenesis of the inflammatory process of the disease.SignificanceUnderstanding the role of inflammasomes in RA pathogenesis helps in finding promising therapy for the treatment and management of this disease.

Ozonated triglyceride protects against septic lethality via preventing the activation of NLRP3 inflammasome.

Sepsis is a critical dysregulated host response with high mortality and current treatment is difficult to achieve optimal efficacy. Ozone therapy has been revealed to protect infection and inflammation-related diseases due to its role in antibiotic and immunoregulatory effect. Ozonated triglyceride is a key component of ozonated oil that is one of ozone therapy dosage form. However, the potential role of ozonated triglyceride in sepsis remains unclear. This study aims to explore the effect of ozonated triglyceride on septic mouse model and the molecular mechanism. Intraperitoneal injection of lipopolysaccharide (LPS), cecal ligation and puncture (CLP) were applied to construct septic mouse model. The mouse serum was obtained for detection of cytokines, and lung tissues were collected for hematoxylin and eosin (HE) staining to evaluate the extent of lung injury in septic mouse with ozonated triglyceride treatment at different time and doses. The survival of septic mice was observed for 96 h and Kaplan-Meier analysis was used to analyze the survival rates. In addition, primary peritoneal macrophages and human acute monocytic-leukemia cell line (THP-1) were treated with inflammasome activators with or without ozonated triglyceride. The level of cytokines was detected by enzyme-linked immunosorbent assay (ELISA). The cleavage of caspase-1 and gasdermin-D (GSDMD) was detected by Western blotting. Ozonated triglyceride at different time and doses reduced the release of inflammasome-related cytokines [interleukin (IL)-1β and IL-18] (all P<0.05) but not pro-inflammatory cytokines such as IL-6 and tumor necrosis factor-α (TNF-α) in septic mice (all P>0.05). Ozonated triglyceride significantly improved the survival rate of septic mice and reduced sepsis-induced lung injury (all P<0.05). Ozonated triglyceride significantly suppressed the canonical and non-canonical activation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome (all P<0.05) but not affected absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) inflammasomes in vitro (all P>0.05). Ozonated triglyceride reduced the cleavage of caspase-1 and the downstream GSDMD. Ozonated triglyceride presents a protect effect on sepsis lethality via reducing cytokines release and sepsis-related organ injury. The mechanism is that ozonated triglyceride specifically suppresses the activation of NLRP3 inflammasome. Ozonated triglyceride is a promising candidate for sepsis treatment.

Clostridioides difficile Flagellin Activates the Intracellular NLRC4 Inflammasome.

Clostridioides difficile (C. difficile), is a major cause of nosocomial diarrhea and colitis. C. difficile flagellin FliC contributes toxins to gut inflammation by interacting with the immune Toll-like receptor 5 (TLR5) to activate nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) signaling pathways. Flagella of intracellular pathogens can activate the NLR family CARD domain-containing protein 4 (NLRC4) inflammasome pathway. In this study, we assessed whether flagellin of the extracellular bacterium C. difficile internalizes into epithelial cells and activates the NLRC4 inflammasome. Confocal microscopy showed internalization of recombinant green fluorescent protein (GFP)-FliC into intestinal Caco-2/TC7 cell line. Full-length GFP-FliC activates NLRC4 in Caco-2/TC7 cells in contrast to truncated GFP-FliC lacking the C-terminal region recognized by the inflammasome. FliC induced cleavage of pro-caspase-1 into two subunits, p20 and p10 as well as gasdermin D (GSDMD), suggesting the caspase-1 and NLRC4 inflammasome activation. In addition, colocalization of GFP-FliC and pro-caspase-1 was observed, indicating the FliC-dependent NLRC4 inflammasome activation. Overexpression of the inflammasome-related interleukin (interleukin (IL)-1β, IL-18, and IL-33) encoding genes as well as increasing of the IL-18 synthesis was detected after cell stimulation. Inhibition of I-kappa-B kinase alpha (IKK-α) decreased the FliC-dependent inflammasome interleukin gene expression suggesting a role of the NF-κB pathway in regulating inflammasome. Altogether, these results suggest that FliC internalizes into the Caco-2/TC7 cells and activates the intracellular NLRC4 inflammasome thus contributing to the inflammatory process of C. difficile infection.

Salmonella Enteritidis GalE Protein Inhibits LPS-Induced NLRP3 Inflammasome Activation.

Microbial infection can trigger the assembly of inflammasomes and promote secretion of cytokines, such as IL-1β and IL-18. It is well-known that Salmonella modulates the activation of NLRC4 (NLR family CARD domain-containing protein 4) and NLRP3 (NLR family pyrin domain-containing 3) inflammasomes, however the mechanisms whereby Salmonella avoids or delays inflammasome activation remain largely unknown. Therefore, we used Salmonella Enteritidis C50336ΔfliC transposon library to screen for genes involved in modulating inflammasomes activation. The screen revealed the galactose metabolism-related gene galE to be essential for inflammasome activation. Here, we found that inflammasome activation was significantly increased in J774A.1 cells or wild-type bone marrow-derived macrophages (BMDMs) during infection by ΔfliCΔgalE compared to cells infected with ΔfliC. Importantly, we found that secretion of IL-1β was Caspase-1-dependent, consistent with canonical NLRP3 inflammasome activation. Furthermore, the virulence of ΔfliCΔgalE was significantly decreased compared to ΔfliC in a mouse model. Finally, RNA-seq analysis showed that multiple signaling pathways related to the inflammasome were subject to regulation by GalE. Taken together, our results suggest that GalE plays an important role in the regulatory network of Salmonella evasion of inflammasome activation.

NLRC4 Deficiency Leads to Enhanced Phosphorylation of MLKL and Necroptosis.

Hosts rely on the innate immune system to clear pathogens in response to infection. Pathogen-associated molecular patterns bind to innate immune receptors and engage activation of downstream signaling to initiate a host immune response to fight infection. A key component of this innate response is programmed cell death. Recent work has highlighted significant cross-talk and functional redundancy between cell death pathways, leading to the discovery of PANoptosis, an inflammatory programmed cell death pathway dependent on PANoptosomes, which are innate immune danger-sensing complexes that activate inflammatory cell death and contain caspases with or without inflammasome components and receptor interacting protein homotypic interaction motif-containing proteins. Although PANoptosis has been characterized in response to a growing number of pathogens, inflammatory diseases, and cancer, its role and the functional consequences of PANoptotic component modulation during NLR family CARD domain-containing protein 4 (NLRC4) activation by Pseudomonas aeruginosa infection remain unknown. In this study, we show that P. aeruginosa can induce PANoptosis in mouse bone marrow-derived macrophages (BMDMs). Only the combined deletion of caspase-1, -11, -8, and RIPK3 protected mouse BMDMs from cell death. Moreover, we showed that PANoptotic components act in a compensatory manner; in the absence of NAIP5 and NLRC4 during P. aeruginosa challenge, activation of caspase-1, -3, -7, and -8 was reduced, whereas alternative cell death molecules such as RIPK1 and MLKL were activated in mouse BMDMs. Taken together, these data highlight the extensive cross-talk between cell death signaling molecules and showcase the plasticity of the system.

Hyperglycaemia-associated macrophage pyroptosis accelerates periodontal inflamm-aging.

Pyroptosis and inflamm-aging have been newly identified to be involved in diabetic periodontitis. This study aimed to elucidate whether macrophage pyroptosis plays a role in periodontal inflamm-aging by impacting the senescence of fibroblasts, as well as the potential mechanism via NLR family CARD domain-containing protein 4 (NLRC4) phosphorylation. Diabetes was induced in mice using streptozotocin. Periodontal pyroptosis and senescence were detected using immunohistochemical analysis. Prior to evaluating senescence in human gingival fibroblasts cultured with conditioned medium derived from macrophages, RAW 264.7 macrophages were confirmed to undergo pyroptosis by scanning electron microscopy and gasdermin D (GSDMD) detection. The NLRC4-related pathway was examined under hyperglycaemic conditions. Our data showed that macrophage pyroptosis induced the expression of senescent markers in vivo and in vitro. Importantly, clearance of pyroptotic macrophages rescued senescence in fibroblasts. Furthermore, GSDMD activation and pyroptosis in hyperglycaemia were found to be mediated by NLRC4 phosphorylation. Hyperglycaemia could initially induce macrophage pyroptosis and lead to cellular senescence, thereby critically contributing to periodontal pathogenesis in diabetes. In particular, NLRC4 phosphorylation could be a potential therapeutic target for the inhibition of this process.

AIM2-driven inflammasome activation in heart failure.

Interleukin-1β (IL-1β) is an important pathogenic factor in cardiovascular diseases including chronic heart failure (HF). The CANTOS trial highlighted that inflammasomes as primary sources of IL-1 β are promising new therapeutic targets in cardiovascular diseases. Therefore, we aimed to assess inflammasome activation in failing hearts to identify activation patterns of inflammasome subtypes as sources of IL-1β. Out of the four major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human HF regardless of the aetiology (ischaemic or dilated cardiomyopathy), while the NLRP1/NALP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change in HF samples. AIM2 expression was primarily detected in monocytes/macrophages of failing hearts. Translational animal models of HF (pressure or volume overload, and permanent coronary artery ligation in rat, as well as ischaemia/reperfusion-induced HF in pigs) demonstrated activation pattern of AIM2 similar to that of observed in end-stages of human HF. In vitro AIM2 inflammasome activation in human Tohoku Hospital Pediatrics-1 (THP-1) monocytic cells and human AC16 cells was significantly reduced by pharmacological blockade of pannexin-1 channels by the clinically used uricosuric drug probenecid. Probenecid was also able to reduce pressure overload-induced mortality and restore indices of disease severity in a rat chronic HF model in vivo. This is the first report showing that AIM2 and NLRC4 inflammasome activation contribute to chronic inflammation in HF and that probenecid alleviates chronic HF by reducing inflammasome activation. The present translational study suggests the possibility of repositioning probenecid for HF indications.

Up-regulation of AIM2 and TLR4 and down-regulation of NLRC4 are associated with septicemia

PurposeInnate immunity receptors play key roles in recognition of bacterial associated molecular patterns. Inflammasomes and toll like receptors (TLRs) are the important innate immunity receptors. In this project transcription levels of TLR4, a TLR member, absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4), as inflammasomes, in the patients suffering from septicemia. MethodsAIM2, NLRC4 and TLR4 mRNA levels were evaluated in the 40 patients suffering from septicemia and 40 healthy controls using Real-Time PCR technique. ResultsData analysis revealed that, although NLRC4 expression decreased, TLR4 and AIM2 levels significantly increased in the patients suffering from septicemia. Gender and infection with various bacteria did not affect expression of AIM2, NLRC4 and TLR4. ConclusionsIt appears that septicemia can be limited by immune responses in AIM2 and TLR4 dependent manner. The potential roles played by bacteria to down-regulation of NLRC4 need to be evaluated by further investigations.

Betulinic Acid Affects the Energy-Related Proteomic Profiling in Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a 5-year survival rate of <8%. Therefore, finding new treatment strategies against PDAC cells is an imperative issue. Betulinic acid (BA), a plant-derived natural compound, has shown great potential to combat cancer owing to its versatile physiological functions. In this study, we observed the impacts of BA on the cell viability and migratory ability of PDAC cell lines, and screened differentially expressed proteins (DEPs) by an LC-MS/MS-based proteomics analysis. Our results showed that BA significantly inhibited the viability and migratory ability of PDAC cells under a relatively low dosage without affecting normal pancreatic cells. Moreover, a functional analysis revealed that BA-induced downregulation of protein clusters that participate in mitochondrial complex 1 activity and oxidative phosphorylation, which was related to decreased expressions of RNA polymerase mitochondrial (POLRMT) and translational activator of cytochrome c oxidase (TACO1), suggesting that the influence on mitochondrial function explains the effect of BA on PDAC cell growth and migration. In addition, BA also dramatically increased Apolipoprotein A1 (APOA1) expression and decreased NLR family CARD domain-containing protein 4 (NLRC4) expression, which may be involved in the dampening of PDAC migration. Notably, altered expression patterns of APOA1 and NLRC4 indicated a favorable clinical prognosis of PDAC. Based on these findings, we identified potential proteins and pathways regulated by BA from a proteomics perspective, which provides a therapeutic window for PDAC.

Sphingomyelin synthase 1 mediates hepatocyte pyroptosis to trigger non-alcoholic steatohepatitis

ObjectiveLipotoxic hepatocyte injury is a primary event in non-alcoholic steatohepatitis (NASH), but the mechanisms of lipotoxicity are not fully defined. Sphingolipids and free cholesterol (FC) mediate hepatocyte injury, but their...

AIM2-driven inflammasome activation in chronic heart failure

Abstract Background Inflammation and cytokine release have been implicated in the pathogenesis of chronic heart failure (CHF). Of particular interest, Canakinumab, a monoclonal antibody against interleukin-1b (IL-1β), had provided benefit against cardiovascular events, suggesting that blockade of IL-1β secretion and signaling might be a promising new therapeutic target. Although, recent studies have provided evidence that inflammasome activation is the main contributor to IL-1β maturation, the role of inflammasome activation in CHF remains unknown. Objective Therefore, we aimed to assess inflammasome activation in myocardial samples from end-stage failing hearts. Methods Inflammasome activation was assessed by immunoblotting in left ventricular myocardial specimens harvested from patients with end-stage CHF. Furthermore, immunoblot measurements were also performed on translational animal models of CHF (e.g. rat models of permanent coronary artery ligation and transverse aortic constriction). Left ventricular monocyte and macrophage infiltration was detected by immunohistochemistry. To investigate the molecular background of inflammasome activation, a series of cell culture experiments were performed on AC16 human cardiomyocytes and THP-1 human monocytic cell lines. Results Out of the 4 major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human CHF while the NLRP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change. A similar expression pattern in AIM2 and NLRC4 was also noted in CHF animal models. Furthermore, robust infiltration of Iba1+ monocytes/macrophages was observed in human failing hearts as well as in different animal models of CHF. In vitro AIM2 inflammasome activation, as induced by transfection with double-stranded DNA [poly(deoxyadenylic-deoxythymidylic)] was reduced significantly by the pharmacological blockade of pannexin-1 channels. Conclusions AIM2 and NLRC4 inflammasome activation might contribute to chronic inflammation in CHF. Our findings suggest that pannexin-1 channels might be a promising novel target to reduce inflammasome activation. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): NVKP_16-1-2016-0017

Extracellular ASC exacerbated the recurrent ischemic stroke in an NLRP3-dependent manner

Using a photothrombotic mouse model of single stroke, we show that a single stroke onset increases the nuclear factor-κB (NF-κB), NLR family CARD domain containing protein 4 (NLRC4), and absent in melanoma 2 (AIM2) inflammasomes, as well as the mRNA levels of NLRP3. Next, using a photothrombotic mouse model of recurrent stroke, we found that recurrent strokes increased the activation of NLRP3, exacerbated the brain damage and the pro-inflammatory response in wild type (WT) mice, but not in NLRP3 knockout (NLRP3 KO) mice. Additionally, we found that apoptosis-associated speck-like protein containing a CARD (ASC) protein level surrounding the infarct area was comparatively increased, but that ASC specks outside of microglia in both the ipsilateral and contralateral of stroke site were decreased in NLRP3 KO mice relative to wild-type (WT) controls, and the number of ASC specks surrounding the second infarct area was positively correlated to the damage scores. Mechanistically, we found that recombinant ASC (RecASC) activated NLRP3 and induced pro-inflammatory responses, exacerbating the outcome of ischemic stroke, in WT mice, but not in NLRP3 KO mice. We therefore conclude that the NLRP3 inflammasome is activated by two attacks of stroke, which act together with ASC to exacerbate recurrent strokes.

NLRC4 inflammasome activation regulated by TNF-α promotes inflammatory responses in nonalcoholic fatty liver disease

The prevalence of nonalcoholic fatty liver disease (NAFLD) is high and increasing throughout the world. The intense sterile inflammation stemming from steatosis plays a significant role in the development of NAFLD, but the mechanism is unclear. We found that the NLRC4 inflammasome was activated in an in vitro cell model of NAFLD, and that the secretion of inflammatory factors IL-18, IL-1β, and tumor necrosis factor α (TNF-α) increased during this process and pyroptosis is triggered. In this study, we used NLRC4-targeting siRNA and an NLRC4-encoding plasmid to demonstrate that the increases in IL-18 and IL-1β are mainly attributable to the activation of the NLR family CARD domain-containing protein 4 (NLRC4) inflammasome, which stimulates cellular pyroptosis. NLRC4 inflammasome activation is regulated by TNF-α and accompanies the translocation of NLRC4 from the cytoplasm into the mitochondria, but the specific mechanism is unclear. In summary, the increase in TNF-α during NAFLD promotes the activation of the NLRC4 inflammasome, which increases the production of IL-18 and IL-1β and triggers pyroptosis. These exacerbate inflammation and promote disease development.

The Salmonella pathogenicity island-2 subverts human NLRP3 and NLRC4 inflammasome responses.

Inflammasomes are signaling hubs that activate inflammatory caspases to drive cytokine maturation and cell lysis. Inflammasome activation by Salmonella Typhimurium infection or Salmonella-derived molecules is extensively studied in murine myeloid cells. Salmonella-induced inflammasome signaling in human innate immune cells, is however, poorly characterized. Here, we show that Salmonella mutation to inactivate the Salmonella pathogenicity island-2 type III secretion system (SPI2 T3SS) potentiates S. Typhimurium-induced inflammasome responses from primary human macrophages, resulting in strong IL-1β production and macrophage death. Inactivation of the SPI1 T3SS diminished human macrophage responses to WT and ΔSPI2 Salmonella. Salmonella ΔSPI2 elicited a mixed inflammasome response from human myeloid cells, in which NLR family CARD-domain containing protein 4 (NLRC4) and NLR family PYRIN-domain containing protein 3 (NLRP3) perform somewhat redundant functions in generating IL-1β and inducing pyroptosis. Our data suggest that Salmonella employs the SPI2 T3SS to subvert SPI1-induced NLRP3 and NLRC4 inflammasome responses in human primary macrophages, in a species-specific immune evasion mechanism.

Extracellular ASC Exacerbated the Recurrent Ischemic Stroke in a NLRP3-Dependent Manner

Using a photothrombotic mouse model of single stroke, we show here that a single stroke onset increases the nuclear factor -κB (NF-κB), NLR family CARD domain containing protein 4 (NLRC4), and absent in melanoma 2 (AIM2) inflammasomes, as well as the mRNA levels of NLRP3. Next, using a photothrombotic mouse model of recurrent stroke, we found that recurrent strokes increased the level of NLRP3 activation and exacerbated the brain damage score and the pro-inflammatory response in wild type (WT) mice, but not in NLRP3 KO mice. Additionally, we found that apoptosis-associated speck-like protein containing a CARD (ASC) protein levels surrounding the infarct area were comparatively increased, but that ASC specks outside of microglia in both the ipsilateral and contralateral of stroke site were decreased in NLRP3 knock out (KO) mice relative to wild-type (WT) controls. Mechanistically, we found that recombinant ASC (recASC) activated NLRP3 and induced pro-inflammatory responses, exacerbating the outcome of ischemic stroke, in WT mice, but not in NLRP3 KO mice. We therefore conclude that the NLRP3 inflammasome is activated by two attacks of stroke, which act together with ASC to exacerbate recurrent strokes. Funding: This work was supported by grants from the National Natural Science Foundation of China (grant numbers: 81671102, 81572224, 81572230, 81371255 and 81772438), The National Key Research and Development Program of China, Stem Cell and Translational Research (2017YFA0105104), the National Key Clinical Department, National Key Discipline, Guangdong Provincial Key Laboratory For Diagnosis and Treatment of Major Neurological Disease (2014B030301035), the Science and Technology Planning Project of Guangdong Province, China (grant numbers: 2016A020213003, 2016B040404053, 2014B020212001, 2014A030304018, 2014B040404053, 2016B030230002 and 2017A040406007), the National Science and Technology Support Program (grant numbers: 2015BAI07B01), and the Science and Technology Planning Project of Guangzhou, China (grant numbers: 2016201604030036). Guangdong Provincial Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases（2017B030314103）; The Southern China International Cooperation Base for Early Intervention and Functional Rehabilitation of Neurological Diseases (2015B050501003); Guangzhou Clinical Research and Translational Center for Major Neurological Diseases (201604020010; Guangdong Provincial Engineering Center for Major Neurological Disease Treatment. The funding bodies had no involvement in the experimental design or the interpretation of the results. Declaration of Interest: No conflicts of interest, financial or otherwise, are declared by the authors. Ethical Approval: This study was approved by the Animal Research Committee of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China; committee’s reference number: [2013]97).

A Role for the Inflammasome in Spontaneous Preterm Labor With Acute Histologic Chorioamnionitis.

Inflammasomes are cytosolic multiprotein complexes that orchestrate inflammation in response to pathogens and endogenous danger signals. Herein, we determined whether the chorioamniotic membranes from women in spontaneous preterm labor with acute histologic chorioamnionitis (1) express major inflammasome components; (2) express caspase (CASP)-1 and CASP-4 as well as their active forms; (3) exhibit apoptosis-associated speck-like protein containing a CARD (ASC)/CASP-1 complex formation; and (4) release the mature forms of interleukin (IL)-1β and IL-18. We utilized quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunoblotting, and immunohistochemistry to determine the messenger RNA (mRNA) and protein expression of major inflammasome components, nucleotide-binding oligomerization domain (NOD) proteins, and the pro- and mature/active forms of CASP-1, CASP-4, IL-1β, and IL-18. The ASC/CASP-1 complex formation was determined using an in situ proximity ligation assay. When comparing the chorioamniotic membranes from women in spontaneous preterm labor with acute histologic chorioamnionitis to those without this placental lesion, we found that (1) the mRNA of NLR family pyrin domain-containing protein ( NLRP) 1, NLRP3, NLR family CARD domain-containing protein 4 ( NLRC4), and NOD2 were higher; (2) the NLRP3 protein was increased; (3) the mRNA and active form (p10) of CASP-1 were greater; (4) the mRNA and active form of CASP-4 were increased; (5) the mRNA and mature form of IL-1β were higher; (6) the mature form of IL-18 was elevated; and (7) ASC/CASP-1 complex formation was increased. In conclusion, spontaneous preterm labor with acute histologic chorioamnionitis is characterized by an upregulation of NLRP3 and the active form of CASP-4, as well as increased ASC/CASP-1 complex formation, which may participate in the activation of CASP-1 and the maturation of IL-1β and IL-18 in the chorioamniotic membranes. These findings provide the first evidence that supports a role for the inflammasome in the pathological inflammation implicated in spontaneous preterm labor with acute histologic chorioamnionitis.

Structural and biochemical basis for induced self-propagation of NLRC4.

Responding to stimuli, nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs) oligomerize into multiprotein complexes, termed inflammasomes, mediating innate immunity. Recognition of bacterial pathogens by NLR apoptosis inhibitory proteins (NAIPs) induces NLR family CARD domain-containing protein 4 (NLRC4) activation and formation of NAIP-NLRC4 inflammasomes. The wheel-like structure of a PrgJ-NAIP2-NLRC4 complex determined by cryogenic electron microscopy at 6.6 angstrom reveals that NLRC4 activation involves substantial structural reorganization that creates one oligomerization surface (catalytic surface). Once activated, NLRC4 uses this surface to catalyze the activation of an inactive NLRC4, self-propagating its active conformation to form the wheel-like architecture. NAIP proteins possess a catalytic surface matching the other oligomerization surface (receptor surface) of NLRC4 but not those of their own, ensuring that one NAIP is sufficient to initiate NLRC4 oligomerization.

β-arrestin1 is critical for the full activation of NLRP3 and NLRC4 inflammasomes.

Inflammasomes are multiprotein complexes that trigger the activation of caspase-1 and the maturation of IL-1β, which are critical for inflammation and control of pathogen infection. Although the function of inflammasomes in immune response and disease development is well studied, the molecular mechanism by which inflammasomes are activated and assembled remains largely unknown. In this study, we found that β-arrestin1, a key regulator of the G protein-coupled receptor signaling pathway, was required for nucleotide-binding domain and leucine-rich repeat containing (NLR) family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing protein 4 (NLRC4) inflammasome-mediated IL-1β production and caspase-1 activation, but it had no effect on absent in melanoma 2 (AIM2) inflammasome activation. Moreover, apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome, which is ASC aggregation mediating caspase-1 activation, was also impaired in β-arrestin1-deficient macrophages upon NLRP3 or NLRC4, but not AIM2 inflammasome activation. Mechanistic study revealed that β-arrestin1 specifically interacted with NLRP3 and NLRC4 and promoted their self-oligomerization. In vivo, in a monosodium urate crystal (MSU)-induced NLRP3-dependent peritonitis model, MSU-induced IL-1β production and neutrophil flux were significantly reduced in β-arrestin1 knockout mice. Additionally, β-arrestin1 deficiency rescued the weight loss of mice upon log-phase Salmonella typhimurium infection, with less IL-1β production. Taken together, our results indicate that β-arrestin1 plays a critical role in the assembly and activation of two major canonical inflammasomes, and it may provide a new therapeutic target for inflammatory diseases.