Abstract:

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）. Methodse Indirect immunofluorescence staining was used to determine the expressions of NLRP3 and apoptosis-associated speck-like 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-α or 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）-1β and 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-1β with 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 those of control group（treated without stimulant）（P<0.05,P<0.001）,whereas LPS had no effect on the priming of NLRP3 inflammasome in RA FLS. TNF-α/Nigericin or TNF-α/CRT dual signaling can promote the activation of Caspase-1 in FLS,represented by the increasing of Caspase-1 activated fragment p20 in a dose-dependent manner,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.

Key words: Tumor necrosis factor-alpha, Calreticulin, Nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome, Rheumatoid arthritis