TNF-α和CRT双信号对RA患者滑膜组织NLRP3炎症小体的活化作用

目的研究肿瘤坏死因子-α(TNF-α)和钙网织蛋白(CRT)双信号对类风湿性关节炎(RA)患者成纤维样滑膜细胞(FLS)中核苷酸结合寡聚化结构域受体3(NLRP3)炎症小体活化的影响。方法采用间接免疫荧光染色法检测12例RA和10例骨性关节炎(OA)患者滑膜组织NLRP3、衔接蛋白凋亡相关斑点样蛋白(ASC)的表达,并与滑膜衬里层和衬里下层FLS标志物进行共定位研究。采用胶原酶消化法分离RA患者滑膜组织中FLS并进行体外培养。分别用不同浓度的TNF-α或脂多糖(LPS)(刺激剂)处理细胞,采用免疫印迹法和实时荧光定量聚合酶链反应(qRT-PCR)检测细胞NLRP3、白细胞介素1β前体(pro-IL-1β)和白细胞介素18前体(pro-IL-18)的蛋白和mRNA表达。加入尼日利亚菌素(Nigericin)或CRT后采用免疫印迹法检测FLS中半胱氨酸天冬氨酸特异性蛋白酶1(Caspase-1)活化片段p20的表达;收集细胞培养上清液,采用酶联免疫吸附试验(ELISA)检测分泌型白细胞介素(IL)-1β和IL-18水平。结果 RA患者滑膜组织中NLRP3炎症小体的主要成分NLRP3和ASC的平均荧光强度高于OA患者(P<0.01)。NLRP3、ASC和IL-1β裂解片段(cleaved IL-1β)与RA患者滑膜衬里层FLS表面标志物PDPN和衬里下层FLS表面标志物CD248均有共定位。体外细胞实验结果显示TNF-α可以促进FLS中NLRP3和pro-IL-1β的蛋白表达;与对照组(无刺激剂)相比,二者的mRNA表达显著增加(P<0.05、P<0.001),而LPS对RA患者FLS中NLRP3炎症小体的预活化无影响。TNF-α/Nigericin或TNF-α/CRT双信号能够促进FLS中Caspase-1的活化,导致Caspase-1活化片段p20呈浓度依赖性升高;与对照组相比,TNF-α/CRT组分泌型IL-1β显著升高(P<0.05)。结论 TNF-α/CRT双信号可促进RA患者FLS中NLRP3炎症小体的活化。

TNF and CRT dual signaling for promoting NLRP3 inflammasome activation in synoviocytes for RApatients

Objective To investigate the effect of tumor necrosis factor-alpha(TNF-α)and calreticulin(CRT)dual signaling on nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome activation in fibroblast-like synoviocytes(FLS)of rheumatoid arthritis(RA).Methods Indirect immunofluorescence staining was used to determine the expressions of NLRP3 and apoptosis-associated specklike protein(ASC)in synovial tissue of 12 RA and 10 osteoarthritis(OA)patients,and co-localizations with podoplanin and CD248 were performed.RA FLS in synovial tissue of RA patients was isolated by collagenase digestion and cultured in vitro.The cells were treated with different concentrations of TNF-αor lipopolysaccharide(LPS)(stimulant),and the protein and mRNA expressions of NLRP3,pro-interleukin 1 beta(pro-IL-1β)and pro-interleukin 18(pro-IL-18)were determined by western blot and real-time fluorescence quantitative polymerase chain reaction(qRT-PCR),respectively.The supernatant of cells was discarded,and then the cells were treated with Nigericin or CRT.The expression of cysteine containing aspartate specific protease-1(Caspase-1)activated fragment p20 in FLS was determined by western blot,and the cell culture supernatant was collected for secretive interleukin(IL)-1βand IL-18 determinations by enzyme-linked immunosorbent assay(ELISA).Results The expression of NLRP3 and ASC in RA synovial tissue was higher than that in OA(P<0.01).The co-localizations of NLRP3,ASC and cleaved IL-1βwith podoplanin and CD248 were observed.In in vitro experiments,TNF-α could promote the protein expressions of NLRP3 and pro-IL-1β,and the mRNA expressions were higher than thoseof control group(treated without stimulant)(P<0.05,P<0.001),whereas LPS had no effect on the primingof NLRP3 inflammasome in RA FLS. TNF-α/Nigericin or TNF-α/CRT dual signaling can promote the activationof Caspase-1 in FLS,represented by the increasing of Caspase-1 activated fragment p20 in a dose-dependentmanner,further leading to the increasing of secretive IL-1β compared with that in control group(P<0.05).Conclusions TNF-α/CRT dual signaling promotes the activation of NLRP3 inflammasome in FLS of RA.