Journal
INFLAMMATION RESEARCH
Volume 68, Issue 7, Pages 597-611Publisher
SPRINGER BASEL AG
DOI: 10.1007/s00011-019-01244-w
Keywords
NLRP3 inflammasome; Calreticulin; Human antigen R; Tumor necrosis factor-; Rheumatoid arthritis; Fibroblast-like synoviocytes
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Funding
- Natural Science Foundation of Tianjin City [14JCYBJC25600]
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ObjectiveThe present study was undertaken to validate whether TNF- and calreticulin (CRT) serve as dual signaling to activate nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) inflammasome in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and HUVECs. The effect of human antigen R (HuR) in NLRP3 inflammasome activation was also explored in RA FLS.MethodsImmunofluorescence was used to determine the expression of NLRP3 and adaptor protein apoptosis associated speck-like protein containing a CARD (ASC) in RA synovial tissue and HuR location in RA FLS. Western blot and quantitative real-time PCR were employed to measure the priming effect of NLRP3 inflammasome in cells and HuR expression in synovial tissue. The concentrations of IL-1 and IL-18 were detected by enzyme linked immunosorbent assay. Immunohistochemistry was used to visualize the expression of HuR in synovial tissue. HuR knockdown in RA FLS was achieved by siRNA-mediated gene silencing.ResultsHigher expression of NLRP3 and ASC in RA synovial tissue than those in osteoarthritis was detected. The staining of NLRP3, ASC and cleaved IL-1 were observed in FLS and vascular endothelial cells in RA synovium. Expression of NLRP3 and pro-IL-1 in RA FLS and HUVECs treated with TNF- was increased. The pro-IL-18 expression was also enhanced in HUVECs, but not in RA FLS. TNF-/CRT dual stimulation of cells gave rise to caspase-1 p20 expression and the secretion of IL-1. The secreted IL-18 was also elevated in HUVECs but not in RA FLS. HuR expression was significantly elevated in RA synovial tissue. TNF- initiated the nucleocytoplasmic shuttling of HuR in both FLS and HUVECs. The knockdown of HuR in FLS incubated with TNF- led to reduced caspase-1 p20 protein expression and further resulted in decreased secretion of IL-1 in the presence of CRT.ConclusionsTNF-/CRT dual signaling induced NLRP3 inflammasome activation, which could be suppressed by HuR knockdown presumably due to the block of HuR translocating from nucleus to cytoplasma.
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