The Regulation Of IL-34 On Th17 Cells And Its Role In The Pathogenesis Of Rheumatoid Arthritis

Objective: Rheumatoid arthritis(RA)is a chronic highly disabling autoimmune disease with synovial joint as the main target tissue.Abnormal inflammation and immune response are the main pathogenesis of RA patients.Cytokines play a key role in driving synovial cell activation leading to joint failure.Recent studies have shown that Interleukin(IL)-34 is expressed in synovial fibroblasts and subsynovial and endometrial layers in patients with arthritis and is significantly associated with the severity of synovitis.Significantly elevated IL-34 levels may be a valid marker of RA activity.However,the specific mechanism of IL-34 in RA is still unclear.T helper cells(T helper cell,Th)17 can secrete cytokines IL-17.IL-17 is a kind of promote the inflammatory cytokines.It can promote joint local inflammation,cause a large number of cytokines released,such as Tumor necrosis factor alpha(Tumor necrosis factor alpha,TNF-a),IL-1,IL-6 and these cytokines in RA all play a role in inflammation and bone destruction.IL-17 is highly expressed in synovial tissues and synovial fluid of RA patients,and its expression level is closely related to the degree of RA joint damage.Whether IL-34 plays a role in RA by regulating Th17 cells has not been reported at home and abroad.In this study,exogenous recombinant human IL-34 acted on peripheral blood mononuclear cell(PBMC)and fibroblast-like synoviocytes(FLS)in RA patients to observe the effect of IL-34 on proliferation of different Th cell subsets in PBMC.To observe the effect of IL-34 on FLS proliferation and apoptosis.The effects of IL-34 on FLS secreted inflammatory cytokines and angiogenesis related cells were studied to investigate the role of IL-34 in the pathogenesis of RA.At the same time,we applied IL-17 inhibitor to block the effect of IL-17 in RA,and observed whether IL-34 affected the pathogenesis of RA by regulating IL-17,which provided important ideas for further research on the mechanism of action of IL-34 and the treatment of RA.Methods: 1.Peripheral blood of RA patients was collected.PBMC was extracted and stimulated with different concentrations of IL-34 to collect cells and superscript for detection.Flow cytometry was used to detect the percentage of Th1,Th2,Th17,and Regulatory T(Treg)cells.Realtime polymerase chain reaction(RT-PCR)and Western blot were used to detect the mRNA and protein expression levels of Th1,Th2,Th17,Treg cell transcription factor T-bet,GATA-3,Retinoic acid-related orphan receptor(ROR)? t and transcription factor forkhead box P3(Foxp3)in tissues.The levels of Th1 cytokine interferon-gamma(IFN?),Th2 cytokine IL-4,Th17 cytokine IL-17 A and Treg cytokine IL-10 in supernatant were determined by Eenzyme-linked immunosorbent assay(ELISA).2.FLS were stimulated by IL-34 at different concentrations in RA patients.The proliferation and apoptosis of FLS were detected by MTT and flow cytometry,and the proliferation and apoptosis of FLS were further detected by IL-34 under the action of the IL-17 inhibitor Plumbagin(PB).3.Stimulating RA-FLS at different concentrations of IL-34:RT-PCR was used to detect the mRNA expression of the inflammatory factors TNF-a,IL-6,IL-17,vascular endothelial growth factor(VEGF)and hypoxia-inducible factor-1a(HIF-1a).Western blot was used to detect the expression of TNF-a,IL-6,IL-17,and VEGF,HIF-1a proteins.ELISA detected the levels of inflammatory cytokines TNF-a,IL-6,IL-17,and angiogenic factors VEGF and HIF-1a in the supernatant.4.The addition of IL-17 inhibitors and different concentrations of IL-34 stimulated RA-FLS: RT-PCR was used to detect the mRNA expression of inflammatory factors TNF-a,IL-6,IL-17 and angiogenic factors VEGF and HIF-1a.Western blot was used to detect the expression of TNF-a,IL-6,IL-17,and VEGF,HIF-1a proteins.ELISA detected the levels of inflammatory cytokines TNF-a,IL-6,IL-17,and angiogenic factors VEGF and HIF-1a in the supernatant.Results: 1.After treatment of PBMC in RA patients with different concentrations of IL-34(0,25,50,100 ng/ml)(1)The proportion of Th17 was significantly increased.The difference was statistically significant,and there was a concentration dependence.Th1,Th2 and Treg did not change significantly.(2)The mRNA and protein levels of ROR? t and IL-17 A were significantly increased,and there was a concentration dependence.T-bet,GATA-3,Foxp3 and IL-10 mRNA and protein levels did not significantly change.(3)The level of IL-17 A in the supernatant was significantly increased,and there was a concentration dependence.The levels of IFN-? and IL-10 did not change significantly.2.After FLS stimulation with different concentrations of IL-34:(1)MTT method was used to detect the proliferation of FLS,and it was found that IL-34 could promote the proliferation of RA-FLS in a concentration-dependent manner.However,the inhibitor of IL-17(PB)can antagonize the proliferation promoting effect of IL-34 on RA-FLS.(2)The apoptosis of FLS was detected by the method of flow cytometry.It was found that IL-34 could inhibit the apoptosis of RA-FLS in a concentration-dependent manner.However,the inhibitor of IL-17(PB)can antagonize the apoptotic effect of IL-34 on RA-FLS.3.After FLS stimulation with different concentrations of IL-34:(1)The mRNA and protein expressions of the inflammatory cytokines TNF-a,IL-6 and IL-17 were significantly increased,and the secretion levels of TNF-a,IL-6 and IL-17 in the supernatant were significantly increased,and there was a concentration dependence.However,the IL-17 inhibitor PB can antagonize the above effects of TNF-a,IL-6 and HIF-1a.(2)The mRNA and protein expressions of angiogenesis related cytokines VEGF and HIF-1a were significantly increased,which were concentration-dependent.The secretion level of VEGF in the supernatant of cells increased significantly and was concentration-dependent.PB antagonized the above effects of IL-34.Conclusion: 1.IL-34 promoted the proliferation of Th17 cells and the secretion of IL-17 in PBMC.2.IL-34 promoted the proliferation of RA-FLS and inhibited its apoptosis.IL-17 inhibitor blocks the regulatory effect of IL-34 on RA-FLS proliferation and apoptosis in RA.3.IL-34 can promote the secretion of RA-FLS inflammatory factors and angiogenic factors,and IL-34 may affect the function of RA-FLS by regulating IL-17.4.IL-34 may become a potential target for the treatment of RA patients.