The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity.

Appropriate immune responses require a fine balance between immune activation and attenuation. NLRC3, a non-inflammasome-forming member of the NLR innate immune receptor family, attenuates inflammation in myeloid cells and proliferation in epithelial cells. T lymphocytes express the highest amounts of Nlrc3 transcript where its physiologic relevance is unknown. We show that NLRC3 attenuated interferon-γ and TNF expression by CD4+ T cells and reduced T helper 1 (Th1) and Th17 cell proliferation. Nlrc3-/- mice exhibited increased and prolonged CD4+ T cell responses to lymphocytic choriomeningitis virus infection and worsened experimental autoimmune encephalomyelitis (EAE). These functions of NLRC3 were executed in a T-cell-intrinsic fashion: NLRC3 reduced K63-linked ubiquitination of TNF-receptor-associated factor 6 (TRAF6) to limit NF-κB activation, lowered phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and diminished glycolysis and oxidative phosphorylation. This study reveals an unappreciated role for NLRC3 in attenuating CD4+ T cell signaling and metabolism.

Dynamic bidirectional regulation of NLRC3 and gammaherpesviruses during viral latency in B lymphocytes.

While most NOD-like receptors (NLRs) are predominately expressed by innate immune cells, NLRC3, an inhibitory NLR of immune signaling, exhibits the highest expression in lymphocytes. The role of NLRC3 or any NLRs in B lymphocytes is completely unknown. Gammaherpesviruses, including human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV-68), establish latent infection in B lymphocytes, which requires elevated NF-κB. This study shows that during latent EBV infection of human B cells, viral-encoded latent membrane protein 1 (LMP1) decreases NLRC3 transcript. LMP1-induced-NF-κB activation suppresses the promoter activity of NLRC3 via p65 binding to the promoter. Conversely, NLRC3 inhibits NF-κB activation by promoting the degradation of LMP1 in a proteasome-dependent manner. In vivo, MHV-68 infection reduces Nlrc3 transcripts in splenocytes, and Nlrc3-deficient mice show greater viral latency than controls. These results reveal a bidirectional regulatory circuit in B lymphocytes, where viral latent protein LMP1 reduces NLRC3 expression, while NLRC3 disrupts gammaherpesvirus latency, which is an important step for tumorigenesis.

Fish gut-derived probiotic Pediococcus pentosaceus alleviates gossypol-induced intestinal inflammation by inhibiting NLRC3/NF-κB/IL-1ß signal pathway in Nile tilapia.

Cottonseed meal (CSM) and cottonseed protein concentrate (CPC) serve as protein alternatives to fish meal and soybean meal in the feed industry. However, the presence of gossypol residue in CSM and CPC can potentially trigger severe intestinal inflammation, thereby restricting the widespread utilization of these two protein sources. Probiotics are widely used to prevent or alleviate intestinal inflammation, but their efficacy in protecting fish against gossypol-induced enteritis remains uncertain. Here, the protective effect of Pediococcus pentosaceus, a strain isolated from the gut of Nile tilapia (Oreochromis niloticus), was evaluated. Three diets, control diet (CON), gossypol diet (GOS) and GOS supplemented with P. pentosaceus YC diet (GP), were used to feed Nile tilapia for 10 weeks. After the feeding trial, P. pentosaceus YC reduced the activity of myeloperoxidase (MPO) in the proximal intestine (PI) and distal intestine (DI). Following a 7-day exposure to Aeromonas hydrophila, the addition of P. pentosaceus YC was found to increase the survival rate of the fish. P. pentosaceus YC significantly inhibited the oxidative stress caused by gossypol, which was evidenced by lower reactive oxygen species (ROS) and malondialdehyde (MDA), as well as higher activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in PI and DI. Addition of P. pentosaceus YC significantly inhibited enteritis, with the lower expression of pro-inflammatory cytokines (il-1ß, il-6, il-8) and higher expression of anti-inflammatory cytokines tgf-ß. RNA-seq analysis indicated that P. pentosaceus YC supplementation significantly inhibited nlrc3 and promoted nf-κb expression in PI and DI, and the siRNA interference experiment in vivo demonstrated that intestinal inflammation was mediated by NLRC3/NF-κB/IL-1ß signaling pathway. Fecal bacteria transplantation experiment demonstrated that gut microbiota mediated the protective effect of P. pentosaceus YC. These findings offer valuable insights into the application of P. pentosaceus YC for alleviating gossypol-induced intestinal inflammation in fish.

Regulation of NF-κB signaling by NLRC (NLRC3-like) gene in the common carp (Cyprinus carpio).

Among teleost NLRs, NLR-C subfamily is a large group of proteins that were teleost-specific and evolution analysis showed that NLR-Cs are most likely to evolve from NLRC3 gene (thus also called as NLRC3Ls). Presently, although there have been rich studies investigating teleost NLRC3 and NLRC3L, the data on the regulatory mechanism was limited. In this study, immune regulation of inflammatory signaling pathway mediated by common carp NLRC3L gene (CcNLRC) has been investigated. Confocal microscopy analysis showed that CcNLRC was located in cytoplasm, and in HEK293T cells, dual-luciferase reporter assay showed the regulation of NF-κB signaling by CcNLRC, in which CcNLRC could alter/decrease RIPK2-induced activation of NF-κB. These results indicated that CcNLRC may function as a negative NLR in the regulation of inflammatory response in common carp. Our data will allow to gain more insights into the molecular mechanism of teleost specific NLR (NLRC3L).

NLRC3 deficiency promotes hypoxia-induced pulmonary hypertension development via IKK/NF-κB p65/HIF-1α pathway.

Hypoxia-induced pulmonary hypertension is a subgroup of type 3 pulmonary hypertension (PH) with the recommended treatment limited to oxygen therapy and lacks potential therapeutic targets. To investigate the role of NLRC3 in hypoxia-induced PH and its potential mechanism, we first collected lung tissues of high-altitude pulmonary hypertension (HAPH) patients. Immunohistochemistry and immunofluorescence showed that NLRC3 was downregulated and was mainly co-localized with the smooth muscle cells of the pulmonary vessels in HAPH patients. Besides, we found that NLRC3 was also expressed in endothelial cells in HAPH patients for the first time. Then, wild type (WT) and NLRC3 knockout (NLRC3-/-) mice were used to construct hypoxia models and primary pulmonary arterial smooth muscle cells (PASMCs) of rats and endothelial cells were cultured for verification. Right heart catheterization and echocardiography suggested that NLRC3 knockout promoted right ventricular systolic pressure (RVSP) up-regulation, right ventricular hypertrophy and fibrosis in hypoxia-induced mice. This study first demonstrated that NLRC3 deficiency promoted hypoxia-stimulated PASMCs proliferation, Human umbilical vein endothelial cells (HUVECs) apoptosis, migration and inflammation through IKK/NF-κB p65/HIF-1α pathway in vitro and in vivo, further promoted vascular remodeling and PH progression, which provided a new target for the treatment of hypoxia-induced PH.

NLRC3 expression in macrophage impairs glycolysis and host immune defense by modulating the NF-κB-NFAT5 complex during septic immunosuppression.

Impairment of innate immune cell function and metabolism underlies immunosuppression in sepsis; however, a promising therapy to orchestrate this impairment is currently lacking. In this study, high levels of NOD-like receptor family CARD domain containing-3 (NLRC3) correlated with the glycolytic defects of monocytes/macrophages from septic patients and mice that developed immunosuppression. Myeloid-specific NLRC3 deletion improved macrophage glycolysis and sepsis-induced immunosuppression. Mechanistically, NLRC3 inhibits nuclear factor (NF)-κB p65 binding to nuclear factor of activated T cells 5 (NFAT5), which further controls the expression of glycolytic genes and proinflammatory cytokines of immunosuppressive macrophages. This is achieved by decreasing NF-κB activation-co-induced by TNF-receptor-associated factor 6 (TRAF6) or mammalian target of rapamycin (mTOR)-and decreasing transcriptional co-activator p300 activity by inducing NLRC3 sequestration of mTOR and p300. Genetic inhibition of NLRC3 disrupted the NLRC3-mTOR-p300 complex and enhanced NF-κB binding to the NFAT5 promoter in concert with p300. Furthermore, intrapulmonary delivery of recombinant adeno-associated virus harboring a macrophage-specific NLRC3 deletion vector significantly improved the defense of septic mice that developed immunosuppression upon secondary intratracheal bacterial challenge. Collectively, these findings indicate that NLRC3 mediates critical aspects of innate immunity that contribute to an immunocompromised state during sepsis and identify potential therapeutic targets.

NLRC3 expression in dendritic cells attenuates CD4+ T cell response and autoimmunity.

NOD-like receptor (NLR) family CARD domain containing 3 (NLRC3), an intracellular member of NLR family, is a negative regulator of inflammatory signaling pathways in innate and adaptive immune cells. Previous reports have shown that NLRC3 is expressed in dendritic cells (DCs). However, the role of NLRC3 in DC activation and immunogenicity is unclear. In the present study, we find that NLRC3 attenuates the antigen-presenting function of DCs and their ability to activate and polarize CD4+ T cells into Th1 and Th17 subsets. Loss of NLRC3 promotes pathogenic Th1 and Th17 responses and enhanced experimental autoimmune encephalomyelitis (EAE) development. NLRC3 negatively regulates the antigen-presenting function of DCs via p38 signaling pathway. Vaccination with NLRC3-overexpressed DCs reduces EAE progression. Our findings support that NLRC3 serves as a potential target for treating adaptive immune responses driving multiple sclerosis and other autoimmune disorders.

Identification of a novel fusion gene NLRC3-NLRP12 in miiuy croaker (Miichthys miiuy).

Fusion gene is a new gene formed by the fusion of all or part of the sequences of two genes, it is caused by chromosome translocation, middle deletion or chromosome inversion. Numerous studies in the past have continuously shown that gene fusions are tightly associated with the occurrence and development of various diseases, especially cancer. Many fusion genes have been identified in humans. However, few fusion genes have been identified in fish. In this study, a novel NLRC3-NLRP12 fusion gene was identified in the Miichthys miiuy (miiuy croaker) by quantitative real-time PCR (qRT-PCR), PCR, and Sanger sequencing. This fusion gene is fused by two genes related to NLRs (nucleotide binding domain and oligomerization domain like receptors). We found that the expression of the NLRC3-NLRP12 fusion gene was significantly upregulated after infection with Vibrio anguillarum (V. anguillarum) or stimulation with lipopolysaccharide (LPS). In addition, the NLRC3-NLRP12 fusion gene was strongly induced by V. anguillarum infection, peaking within the kidney and liver at 12 h post infection. Further functional experiments showed that overexpression of NLRC3-NLRP12 significantly inhibited nuclear factor kappa-B (NF-κB) activation. This study suggests that the newly discovered NLRC3-NLRP12 fusion genes may play an important role in innate immunity in miiuy croaker.

NLRC3 Participates in Inhibiting the Pulmonary Inflammatory Response of Sepsis-Induced Acute Lung Injury.

Acute lung injury (ALI) progresses rapidly, is difficult to treat, and has a high fatality rate. The excessive inflammatory response is an important pathological mechanism of ALI. NLRC3 (NLR family CARD domain-containing 3), a non-inflammasome member of the NLR family, has been found that it could negatively regulates various biological pathways associated with inflammatory response, such as NF-κB (nuclear factor kappa B), PI3K (Phosphatidylinositol 3'-kinase)-Akt (protein kinase B)-mTOR (mammalian target of the rapamycin), and STING (stimulator of interferon genes) pathways, which are responsible for the progression of pulmonary inflammation and participate in regulating the pathological progression of ALI. However, the effects of NLRC3 in sepsis-induced pathological injury of lung tissue remain unclear. In this study, we aimed to investigate the potential effects of NLRC3 in the sepsis-induced ALI. To investigate whether NLRC3 participates in inhibiting the pulmonary inflammatory response of sepsis-induced ALI. Sepsis-induced ALI mice models were established by intrabronchial injection of lipopolysaccharide (LPS) or cecum ligation and puncture (CLP). The lentivirus with overexpression of NLRC3 (LV-NLRC3) and downregulation of NLRC3 (LV-NLRC3-RNAi) were transfected to LPS-induced ALI mice. The expression of NLRC3 was upregulated or downregulated in the lung tissue of sepsis-induced ALI mice. Transfection with NLRC3-overexpression lentivirus significantly decreased inflammatory response in the lung of LPS-induced ALI mice in contrast to the control group. By transfection with NLRC3-silencing lentivirus, the inflammatory response in LPS-induced ALI mice was aggravated. Our study provides evidence of the protective effect of NLRC3 in sepsis-induced ALI by inhibiting excessive inflammatory response of the lung tissue.AbbreviationsAcute lung injury: ALI; intensive care units: ICU; lipopolysaccharide: LPS; acute respiratory distress syndrome: ARDS; bronchoalveolar lavage fluid: BALF; nucleotide-binding oligomerization domain-like receptors: NLRs; NLR family CARD domain containing 3: NLRC3; nuclear factor kappa B: NF-κB; tumor necrosis factor receptor-associated factor 6: TRAF6; Phosphatidylinositol 3'-kinase: PI3K; protein kinase B: Akt; mammalian target of the rapamycin: mTOR; stimulator of interferon genes: STING; TANK-binding kinase 1: TBK1; type I interferon: IFN-I; toll-like receptors: TLRs; tumor necrosis factor: TNF; interleukin: IL; NOD-like receptor protein 3: NLRP3; enhanced green fluorescent protein: EGFP; lentivirus: LV; phosphate-buffered saline: PBS; intrabronchial: i.t.; cecum ligation and puncture: CLP; wet/dry: W/D; Real time polymerase chain reaction: RT-PCR; enzyme-linked immunosorbent assay: ELISA; hematoxylin and eosin: H&E; radio immunoprecipitation assay: RIPA; sodium dodecyl sulfate polyacrylamide gel electrophoresis: SDS-PAGE; polyvinylidene fluoride: PVDF; glyceraldehyde 3-phosphate dehydrogenase: GAPDH; bovine serum albumin: BSA; Tris buffered saline containing Tween 20: TBST; standard deviation: SD; one-way analysis of variance: ANOVA; janus kinase 2: JAK2; activators of transcription 3: STAT3; pathogen associated molecular patterns: PAMPs; danger associated molecular patterns: DAMPs.

NLRC3 attenuates antiviral immunity and activates inflammasome responses in primary grouper brain cells following nervous necrosis virus infection.

NLRC3 is identified as a unique regulatory NLR involved in the modulation of cellular processes and inflammatory responses. In this study, a novel Nod like receptor C3 (NLRC3) was functionally characterized from seven band grouper in the context of nervous necrosis virus infection. The grouper NLRC3 is highly conserved and homologous with other vertebrate proteins with a NACHT domain and a C-terminal leucine-rich repeat (LRR) domain and an N-terminal CARD domain. Quantitative gene expression analysis revealed the highest mRNA levels of NLRC3 were in the brain and gill followed by the spleen and kidney following NNV infection. Overexpression of NLRC3 augmented the NNV replication kinetics in primary grouper brain cells. NLRC3 attenuated the interferon responses in the cells following NNV infection by impacting the TRAF6/NF-κB activity and exhibited reduced IFN sensitivity, ISRE promoter activity, and IFN pathway gene expression. In contrast, NLRC3 expression positively regulated the inflammasome response and pro-inflammatory gene expression during NNV infection. NLRC3 negatively regulates the PI3K-mTOR axis and activated the cellular autophagic response. Delineating the complexity of NLRC3 regulation of immune response in the primary grouper brain cells following NNV infection suggests that the protein acts as a virally manipulated host factor that negatively regulated the antiviral immune response to augment the NNV replication.

Whole exome sequencing in unclassified autoinflammatory diseases: more monogenic diseases in the pipeline?

OBJECTIVE: Autoinflammatory diseases (AIDs) are characterized by recurrent sterile systemic inflammation attacks. More than half of the patients remain genetically undiagnosed with next-generation sequencing panels for common AIDs. In this study, we aimed to define phenotype-genotype correlations in a cohort of unclassified AID patients via whole exome sequencing (WES). METHODS: Patients with features of AIDs were included in this study followed in the Department of Pediatric Rheumatology at Hacettepe University. They were first screened for MEFV with Sanger sequencing and then WES performed for the patients with clinically insignificant results. Pre-analysis of WES data was done by considering the 13 most common AID-related genes. Further bioinformatic analysis was performed if the patient remained genetically undiagnosed. RESULTS: The median age at disease onset was 1.2 years (range 0.2-16) and at the time of study recruitment was 14 years (range 3.5-17). In our cohort, WES provided a definite or probable disease-causing variant in 4 of 11 patients (36%). Heterozygous mutations for two of these genes were previously associated with neurological defects (ADAM17, TBK1), also homozygous ADAM17 mutations were observed in one family with neonatal inflammatory skin and bowel disease. Besides, two genes (LIG4, RAG1) were associated with immunodeficiency although the patients had presented with inflammatory features. Finally, for one patient, we associated a strong candidate gene (NLRC3) with autoinflammatory features. CONCLUSION: WES strategy is cost-effective and provides substantial results for a selected group of undefined AID patients. Our results will contribute to the spectrum of unclassified AIDs.

miR-190b promotes tumor growth and metastasis via suppressing NLRC3 in bladder carcinoma.

Bladder cancer is one of the most common urogenital malignancies. However, its pathogenesis, especially molecular mechanisms remain elusive. Thus, understanding the molecular mechanisms underlying bladder cancer is important for the discovery of novel therapeutic paradigms for these diseases. In current study, we found that micro-RNA (miR)-190b is highly expressed in bladder cancer tissues and cells. Overexpression of miR-190b enhanced the proliferation, growth, migration and invasion capabilities, and angiogenesis of bladder cancer cells, whereas downregulation of miR-190b reversed these effects. Target prediction and dual luciferase reporter assays identified NLR family CARD domain containing 3 (NLRC3) as a potential target of miR-190b. Pathway analysis indicated that miR-190b promotes bladder cancer progression via the Wnt/ß-catenin and mTOR signaling pathways. Taken together, our findings imply that miR-190b acts as a critical regulator for bladder cancer development by repressing NLRC3 and partly through the Wnt/ß-catenin and mTOR pathways. Our study suggests that miR-190b may be served as a potential therapeutic target for bladder cancer treatment.

Leptin reduces ventilator-induced lung injury in rats by regulating NLRP3, NLRC4 and NLRC3.

Leptin has been linked to acute lung injury (ALI) through its regulation of immune responses. We aimed to scrutinize the effects of leptin on nucleotide oligomerization domain-like receptors containing pyrin domain 3 (NLRP3), nucleotide oligomerization domain-like receptors with caspase activation and recruitment domain 4 (NLRC4), and nucleotide oligomerization domain-like receptors with caspase activation and recruitment domain 3 (NLRC3), as an essential part of the immune system, in ventilator-induced lung injury (VILI) of rats. In the present study, pathogen-free adult male SD rats were given saline or leptin, followed by ventilation. Lung tissue samples, bronchoalveolar lavage fluids (BALF), and blood were collected four hours after installation. Notable acute lung inflammation induced by mechanical ventilation is well-characterized by a massive increase in lung injury score and wet/dry weight (W/D) ratio. We also observed VILI was associated with interleukin (IL-1ß and IL-18). Rats that received ventilation showed a decrease in the levels of NLRP3 and NLRC4, and an increased level of NLRC3. Pre-treatment with leptin could abolish all of these effects induced by VILI. It has been suggested that the regulation of NLRP3, NLRC4, and NLRC3 may underlie the protection observed during VILI by exogenous leptin.

NLRC3 inhibits PDGF-induced PASMCs proliferation via PI3K-mTOR pathway.

Few studies about nucleotide-oligomerization domain-like receptor subfamily C3 (NLRC3) in PASMCs have been conducted. This research aimed to investigate the role of NLRC3 on platelet-derived growth factor (PDGF)-induced proliferation of pulmonary artery smooth muscle cells (PASMCs) and its underlying mechanism. We found that the proliferation of PASMCs stimulated with PDGF decreased when phosphoinositide 3-kinase (PI3K) or mammalian target of rapamycin (mTOR) inhibitors pretreatment. Overexpression of NLRC3 inhibited the proliferation of PASMCs and the phosphorylation of PI3K and mTOR while knocking down NLRC3 reversed this effect. Targeted to PI3K or mTOR can also reverse the effect of NLRC3. Activation of PI3K increased the phosphorylation of mTOR while inhibition of PI3K reduced it. Our data suggest that PDGF can induce abnormal proliferation of PASMCs, and NLRC3 suppresses activation of the PI3K-mTOR signaling thus inhibits PASMCs proliferation. These findings unveiled the effect of NLRC3 as an inhibitor of the PI3K-mTOR pathway mediating protection against PASMCs proliferation.

Dihydromethysticin, a natural molecule from Kava, suppresses the growth of colorectal cancer via the NLRC3/PI3K pathway.

Dihydromethysticin (DHM), a natural compound derived from Kava, has been reported to be effective against mental disorders and some malignant tumors. However, little is known about the inhibitory effect of DHM on colorectal cancer (CRC). First, we examined the impact of DHM on human colon cancer cell lines, which demonstrated that DHM inhibits proliferation, migration, and invasion and promotes apoptosis and cell cycle arrest in colon cancer cells in vitro. Using small hairpin RNA, we inhibited nucleotide-oligomerization domain-like receptor subfamily C3 (NLRC3)/phosphoinositide 3-kinase (PI3K) pathway to elucidate the partial signaling of DHM-mediated tumor suppression. Additionally, using an ectopic human CRC model, we verified whether DHM inhibits tumor growth and angiogenesis via the NLRC3/PI3K pathway in vivo. Overall, DHM showed an inhibitory effect on CRC by altering cell proliferation, migration, invasion, apoptosis, cell cycle, and angiogenesis, possibly via the NLRC3/PI3K pathway. Thus, DHM may be a promising candidate for CRC therapy.

NLRC3 silencing accelerates the invasion of hepatocellular carcinoma cell via IL-6/JAK2/STAT3 pathway activation.

Nucleotide-binding domain, leucine-rich repeat family with a caspase activation and recruitment domain 3 (NLRC3) participates in both immunity and cancer. The aim of this study was to determine the role of NLRC3 in human hepatocellular carcinoma (HCC) and the underlying mechanisms. We collected human liver tissues from nonalcoholic steatohepatitis (NASH), HCC, and adjacent normal tissues to characterize the pattern of NLRC3 expression by real-time quantitative polymerase chain reaction and immunohistochemistry. Then, we used the HCC cell line, HuH-7, transfected with small interfering RNA to silence the NLRC3 expression. 5-Ethynyl-2'-deoxyuridine assay, scratch assay, and transwell invasion assay were used for assessing proliferation, migration, and invasion, respectively. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted to assess cell apoptosis. The expression of NLRC3 was reduced in human HCC tissues, compared with normal liver and nonalcoholic steatohepatitis tissues. After knocking down of NLRC3, the proliferation, migration, and invasion were increased in HuH-7 cells. And flow cytometry and TUNEL assay showed that HuH-7 cell apoptosis was suppressed after NLRC3 knockdown. As for the underlying mechanisms, knockdown of NLRC3 in HuH-7 cells was associated with the activation of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway under interleukin-6 (IL-6) stimulation. NLRC3 expression was downregulated in human HCC tissues. NLRC3 silencing in HuH-7 cells can promote the proliferation, migration, and invasion of hepatocellular carcinoma cells. JAK2/STAT3 pathway activation induced by IL-6 may be the underlying mechanism for HCC when NLRC3 expression is silenced. And the invasion of HuH-7 cells was partially suppressed by the STAT3 specific inhibitor (cryptotanshinone). Therefore, NLRC3 may play a significant role in HCC and might be a therapeutic target for the treatment of HCC.

NLRC3 alleviates hypoxia/reoxygenation induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6.

BACKGROUND: NOD-like receptor family CARD domain containing 3 (NLRC3) plays an important role in both innate and adaptive immunity. This study was to explore the function and related mechanisms of NLRC3 in a hypoxia/reoxygenation (H/R)-induced inflammatory response in RAW264.7 cells. METHODS: Liver ischemia-reperfusion (I/R) model in mice and H/R model in RAW264.7 cells were constructed. Western blotting was used to determine the protein expression level of NLRC3 in liver tissue and NLRC3, TRAF6, p-p65, p65, IκB-α, and the K63-linked ubiquitination level of TRAF6 in cells. The immunofluorescence assay was performed to evaluate the nuclear level of the NF-κB (p65). ELISA was conducted to measure the content of IL-1ß in serum and cell supernatant. The interaction between NLRC3 and TRAF6 in cells was analyzed by the Co-IP assay. RESULTS: The NLRC3 protein level in liver tissue was decreased with the prolongation of reperfusion time (P < 0.05). The expression of NLRC3 and IκB-α protein in RAW264.7 was decreased gradually, while the expression of p-p65 and TRAF6 proteins and K63-linked ubiquitination of TRAF6 were increased gradually with the prolongation of reoxgenation time (P < 0.05). The Co-IP assay revealed that NLRC3 and TRAF6 can bind to each other directly. However, NLRC3 had no effect on the expression of TRAF6 protein. The ubiquitination test results showed that the K63-linked ubiquitination level of TRAF6 in H/R + Lv-NLRC3 group was significantly lower than that in the H/R + negative control (NC) group (P < 0.05). Moreover, the activation of NF-κB in H/R + Lv-NLRC3 group was inhibited compared with that in the H/R + NC group, and the level of the inflammatory factor IL-1ß in the cell culture supernatant was also decreased accordingly (P < 0.05). CONCLUSIONS: NLRC3 might alleviate H/R-induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6.

NLRC3 inhibits MCT-induced pulmonary hypertension in rats via attenuating PI3K activation.

Phosphoinositide 3-kinase (PI3K) activation plays a critical role in the pulmonary vascular remodeling of pulmonary hypertension (PH). The nucleotide-oligomerization domain (NOD)-like receptor subfamily C3 (NLRC3) inhibits proliferation and inflammation via PI3K signaling in cancer. We previously showed NLRC3 was significantly reduced in PH patients, but the mechanism of function remains unclear. This study aimed to determine the potential role of NLRC3 in PH. We found that NLRC3 was downregulated in the pulmonary arteries of PH animal models and platelet-derived growth factor-BB (PDGF-BB) stimulated pulmonary arterial smooth muscle cells (PASMCs). NLRC3 pretreatment reduced right ventricular systolic pressure, attenuated pulmonary vascular remodeling and RVHI, and ameliorated proliferation, migration, and inflammation. Monocrotaline (MCT)- and PDGF-BB-mediated PI3K activation were suppressed by NLRC3 pretreatment. 740Y-P decreased the effect of NLRC3. Collectively, NLRC3 protected against MCT-induced rat PH and PDGF-BB-induced PASMC proliferation, migration, and inflammation through a mechanism involving PI3K inhibition. NLRC3 may have a therapeutic effect on PH and provide a promising therapeutic strategy for PH.

Butyrate ameliorated-NLRC3 protects the intestinal barrier in a GPR43-dependent manner.

BACKGROUND: Intestinal barrier dysfunctions are related to dysbacteriosis and chronic gut inflammation in type 2 diabetes. Although there is emerging evidence that the chronic gut inflammatory response is stimulated by nucleotide-binding oligomerization domain-like receptors (NLRs), the relationship and precise mechanism between NLRC3 and the colonic epithelial barrier remains largely elusive. METHODS: We investigated the function and mechanism of NLRC3 in the colonic tissues of diabetic mice and colonic epithelial cell lines. The regulatory mechanism between NLRC3, butyrate and tight junctions was elucidated via a transepithelial electrical resistance measurement, transmission electron microscopy, RNA interference and western blotting. RESULTS: In this study, we found that NLRC3 expression was decreased in the colonic tissues of diabetic mice. NLRC3 over-expression ameliorated colonic epithelial barrier integrity and up-regulated tight junction proteins in colonic epithelial cells. Knockdown of TRAF6 diminished NLRC3-induced ZO-1/occludin expression. In addition, we demonstrated that butyrate could stimulate NLRC3 expression in both diabetic mice and colonic epithelial cells. GPR43 on colonic epithelial cells is involved in the activation of NLRC3 induced by butyrate. CONCLUSION: Our findings demonstrated that NLCR3 could ameliorate colonic epithelial barrier integrity in diabetes mellitus in a TRAF6-dependent manner, and NLCR3 was stimulated by butyrate via binding GPR43 on colonic epithelial cells.

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.