| Objective Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by chronic and invasive joint synovitis.Various pro-inflammatory cytokines are increased and participate in the pathogenesis of RA.TNF-αinitiates and maintains the inflammation of the inflamed joint and is a predominant cytokine in RA pathogenesis.In addition,our earlier study reported that the expression of calreticulin(CRT)was increased in RA patients and that serum CRT levels were positively correlated with disease activity in RA patients.Nucleotide binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3)inflammasome is a cytoplasmic protein complex,which can recognize a variety of pathogen-associated molecular patterns(PAMPs)and damage-associated molecular patterns(DAMPs),and further triggers the secretion of interleukin.The activation of NLRP3 inflammasome was found in the monocytes of peripheral blood of patients with RA,which was represented by significantly elevated Caspase-1 IL-1β and IL-18.The molecular mechanism of NLRP3 inflammasome activation has not yet been fully elucidated.This study was undertaken to explore the effect of TNF-α/CRT dual signaling on NLRP3 inflammasome activation in fibroblast-like synoviocytes(FLS)and vascular endothelial cells in RA and the potential molecular mechanism,which may provide a novel target for clinical treatment of RA.Methods 1.The synovial tissue specimens of patients undergoing arthroscopy and knee arthroplasty at the Tianjin Hospital Joint Surgery Center were collected for this study,including 12 patients with RA and 10 patients with OA.The expression and localization of NLRP3 and ASC in RA and OA synovium were observed by immunohistochemistry.2.FLS and primary umbilical vein endothelial cells(HUVECs)in synovial tissue of RA were isolated and cultured in vitro.Western blot was used to detect the expression of NLRP3,pro-IL-1β and pro-IL-18 d in cell lysate and the production of Caspase-1 p20 in cell culture supernatant,as well as the expression of Hu R in RA and OA synovial tissue.3.Real-time quantitative PCR(q RT-PCR)was used to evaluate the m RNA levels of NLRP3,pro-IL-1β and pro-IL-18 in RA FLS and HUVECs.4.Enzyme-linked immunosorbent assay(ELISA)was used to detect the release of IL-1β and IL-18 in cell culture supernatant.5.Immunohistochemical staining(IHC)was used to measure the expression of Hu R molecules in RA and OA synovium.6.The expression and localization of Hu R in RA FLS and HUVECs were analyzed by immunofluorescence assay(ICC).7.Hu R knockdown in RA FLS was achieved by si RNA-mediated gene silencing.Results 1.The expression of NLRP3 and ASC in RA synovial tissue(0.095±0.005;0.076±0.003)was higher than those in OA(0.075±0.002,t=3.450,p=0.003;0.060±0.004,t=3.544,p=0.002).Expression of NLRP3,ASC and cleaved-IL-1βwas observed in both FLS and vascular endothelial cells in RA synovium.2.TNF-α up-regulated the protein expression of NLRP3 and pro-IL-1β in RA FLS and HUVEC,and the expression of pro-IL-18 in HUVEC was also increased,but there was no change in RA FLS.m RNA expression of NLRP3 and pro-IL-1β was significantly increased in TNF-α-stimulated RA FLS(3.123±0.263,p<0.05;3.697±0.142,p<0.001)compared with those in control group(0.687±0.148;0.727±0.119).There was no significant difference in the expression of pro-IL-18(0.430±0.079,0.390±0.137,p>0.05).In TNF-α stimulated HUVEC,the m RNA expression of all three increased significantly(1.920±0.271,p<0.05;0.993±0.153,p<0.001;1.107±0.222,p<0.01)compared with those in control groups(0.707±0.148,0.0833±0.023,0.303±0.160).3.TNF-α/CRT dual signal promoted the production of Caspase-1 p20 in RA FLS and HUVEC.Meanwhile,TNF-α/CRT stimulation caused a significant increase of secretive IL-1β in RA FLS [(222.9±25.66)pg/ml;(502.3±28.40)pg/ml,p<0.05]and HUVEC [(221.8±14.47)pg/ml,335.9±25.87,p<0.05] compared with the those in control group.Secretive IL-18 was also significantly elevated in HUVEC culture supernatant [(37.91±11.17)pg/ml,(246.3±37.21)pg/ml,p<0.05],but not in RA FLS [(36.13±10.71)pg/ml,(55.87±9.468)pg/ml,p>0.05].4.Immunohistochemistry(0.190±0.009,0.023±0.002,t=18.14,p<0.0001),Western Blot and q RT-PCR(4.397±0.255,1.463±0.119,t=10.43,p=0.0005)results showed that protein and m RNA expression of Hu R in RA synovial tissue was significantly higher than that in OA.5.Western Blot and cellular immunofluorescence showed that TNF-α could not induce the change of Hu R protein expression in RA FLS and HUVEC,but it led to a nucleocytoplasmic shuttling of Hu R.6.Down-regulation of Hu R led to reduced expression of NLRP3 protein and decreased m RNA stability in RA FLS pre-incubated with TNF-α,which further caused a decrease of Caspase-1 p20 and IL-1β secretion in the presence of TNF-α/CRT stimulation.Conclusions 1.TNF-α/CRT dual signaling is capable of inducing activation of NLRP3 inflammasome in RA FLS and HUVECs.2.Hu R nucleoplasmic shuttling probably mediated the upregulation of protein expression and m RNA stability of NLRP3 in RA FLS,thereby promoting TNF-α/CRT dual signaling-mediated activation of NLRP3 inflammasome. |
