Effects Of TL1A On Fibroblast-like Synoviocytes In Rheumatoid Arthritis

Objective: Rheumatoid Arthritis(RA)is a chronic autoimmune disease characterized by hyperplasia and progressive joint inflammation of synovial tissue,frequently leading to cartilage and bone destruction.Although T cells,B cells and macrophages are included in RA,fibroblast-like synoviocytes(FLS)may play an important role in the pathogenesis of RA.Cytokines secreted by FLS in the lining aggravate the local inflammation of the joint,which result in cartilage destruction and bone resorption.Studies suggest that TNF bind to their specific receptors can initiate the inflammatory cascade reaction in RA,which plays a critical role in the disease.Tumor necrosis factor(TNF)-like cytokine 1A(TL1A,TNFSF15)belongs to tumor necrosis family,was correlated with the activity of RA.TNF-α and IL-1β stimulation increased TL1 A production in FLS,and TL1 A bind to its receptor DR3 could promote the expression of various proinflammatory cytokines which amplify the inflammatory effect.Nowadays,most of the studies on TL1 A in RA in vitro were targeting on T cells.However,there is no reports that concerning the TL1 A and FLS in RA.Our study will focus on the cytokines production and mechanism of RA FLS after being stimulated with TL1 A.Methods:(1)Serum of RA patients and healthy controls were collected.TL1 A expression in the serum were detected by ELISA.(2)FLS was isolated by enzymatic digestion of synovial tissues obtained from RA patients undergoing total joint replacement surgery.Supernatants and m RNA were collected after being stimulated with TL1 A.Levels of cytokines from stimulated RA FLS were measured by RT-PCR and ELISA.(3)RA FLS were incubated with TL1 A after 1h pretreatment with signaling inhibitors,m RNA expression of IL-6 were tested by RT-PCR and the concentration of IL-6 in the supernatants of cultured FLS was measured by ELISA.(4)FLS were stained with PE labelled anti-DR3,Alexa Fluor-488 labelled anti-vimentin and single cell suspensions were detected and analyzed by flow cytometer.RA FLS were preincubated with TNFR1,TNFR2 antagonists for 1h before stimulated with TL1 A.IL-6 m RNA production and expression in supernatants were detected by RT-CR and ELISA.(5)RA FLS were incubated in the present or absent of TL1 A for 48 h,and the supernatants were discarded.TL1A-stimulated FLS co-cultured with PBMC for another 72 h.The percentage of Th17 was detected using a flow cytometer.(6)Supernatants of cultured FLS at the present or absence of TL1 A were collected to test the concentration of cytokines by Ray Bio Human Cytokine Antibody Array G-Series 2000.Results:(1)Serum TL1 A levels in RA patients [(248.67 ± 92.09)pg/ml] was significantly higher than that of the healthy control [(71.99 ± 35.98)pg/ml](P<0.05).(2)FLS from four RA patients were stimulated with 50 ng/ml TL1 A for 6 hours.The results by RT-PCR showed that IL-6 and PGE2 m RNA expression was obviously higher on TL1A-stimulated RA FLS.In addition,the protein levels of IL-6 and PGE2 from FLS supernatant were measured by ELISA after FLS were cultured for 0,12,24,48 hours in the present or absent of 50 ng/ml TL1 A.In accordance with RT-PCR results,TL1A-stimulated FLS secreted remarkably higher levels of IL-6 and PGE2 compared to that of unstimulated FLS(P<0.05).(3)Several inhibitors of signaling pathway including JNK,p38,Erk1/2 and NF-κB were using in the experiment.The results showed that in the presence of IKK-16 and SP600125 IL-6 m RNA and protein expression by TL1A-stimulated FLS was obviously decreased(P<0.05).(4)DR3 expression could be not tested on the membrane of FLS.(5)TNFR(TNFR1 and TNFR2)antagonists were added in TL1A-stimulated FLS.The relative m RNA and protein expressions of IL-6 were significantly decreased when anti-TNFR2 antibody was added the TL1A-stimulated FLS.While the addition of TNFR1 antagonist did not change IL-6 expression on RA FLS.(6)Percentage of Th17 in PBMC cocultured with TL1A-stimulated FLS was significantly higher than PBMC cocultured with FLS only with increased IL-6 levels in supernatants of coculture system.While the addition of anti-IL-6 receptor antibody for the neutralization of IL-6R downregulated Th17 percentage in the coculture system of TL1A-stimulated FLS and PBMC.(7)Chemokines and growth factors production in the supernatants of TL1A-stimulated RA FLS were elevated significantly.Conclusion:(1)TL1A was capable of acting on fibroblasts to elevate the production of IL-6 via NF-κB and JNK signaling pathway.(2)TL1A could influence on RA FLS through binding to TNFR2 rather than DR3 on FLS.(3)TL1A was capable of acting on RA FLS to increase the expression of IL-6,which promoted the production of Th17.