Adiponectin Mediates The Pathogenesis Of Rheumatoid Arthritis By Promoting The Secretion Of IL-6 From RA FLSs And Up-regulating The Production Of Follicular Helper T Cells

Part 1 The effect of Adiponectin on Tfh cells productionObjectiveTo determine whether AD has the effect on promoting the production of Tfh cells.MethodsPurified CD4+T cells were obtained by Magnetic activate cell sorting(MACS),then the expression of AdipoR1 on Tfh cells(CD4+CXCR5+PD-1+)was detected by flow cytometry.Purified CD4+T cells were cultured with anti-CD3 mAb(5 ?g/ml)and anti-CD28 mAb(3 ?g/ml)in the presence or absence of AD(5 ?g/ml or 25 ?g/ml).After 72 hours culture,the frequencies of Tfh cells were measured by flow cytometry.Purified CD4+T cells were cultured with serum-free,2%fatal bovine serun(FBS),5%FBS respectively in the presence or absence of AD(5 ?g/ml or 25 ?g/ml)for 72 hours,the frequencies of Tfh cells were measured by flow cytometry.siRNA-AdipoR1 were transfected into CD4+T cells,then cultured in the presence or absence of AD(5 ?g/ml or 25 ?g/ml)for 72 hours,the frequencies of Tfh cells were measured by flow cytometry.Fibroblast-like synoviocytes(FLSs)were obtained from the synovial tissue of RA patients or OA patients who underwent therapeutic synovectomy or arthroplasty,RA FLSs or OA FLSs were stimulated with different concentrations of AD(5 ?g/ml or 25 ?g/ml)for 72 hours,the supernatant was discarded and the FLSs were washed with PBS twice.Then CD4+T cells were added into the plate in the presence of anti-CD3 mAb(5 ?g/ml)and anti-CD28 mAb(3 ?g/ml)for 72h in a cell-to-cell contact stystem,the frequencies of Tfh cells were measured by flow cytometry.ResultsFirstly,FACS showed that AdipoR1 was exactly expressed on Tfh cells.However,no change of Tfh cells was observed after stimulation with AD either in the frequency or absolute number of Tfh cells.Secondly,no matter in serum-free,2%or 5%FBS DMEM medium,no change was observed among the three groups.Finally,after siRNA specifically targeting AdipoR1 expression on CD4+T cells,there was still no difference in the frequency of Tfh cells after stimulation of AD.However,we found that the frequency of Tfh cells was remarkedly upregulated in AD-stimulated RA FLSs cocultured with HC CD4+T cells system,and no change was observed on the frequency of Tfh cells in AD-stimulated OA FLSs cocultured with HC CD4+T cells system.ConclusionsIn vitro,AD had no directly effect on Tfh production,and AD promoted Tfh production indirectly via stimulating RA FLSs.Part 2 Adiponectin stimulated RA FLSs secreting IL-6 to induce Tfh production in RAObjectiveTo determine the specific mechanism how AD regulated Tfh cells production via stimulating RA FLSs.MethodsRA FLSs were firstly stimulated by AD for 72 h,and then co-cultured with HC CD4+T cells in a cell-to-cell contact or transwell system respectively.After 72 h,the frequencies of Tfh cells were measured by flow cytometry and the supernatants of each experiment group were collected and the levels of above soluble factors were detected by protein microarray or ELISA.In the above culture stystem,anti-IL-6 antibody and/or anti-IL-21 antibody was added to the co-culture system for 72 h,the frequencies of CD4+CXCR5+PD-1+(Tfh)cells were measured by flow cytometry.RA FLSs and siRNA-AdipoR1-RA FLSs were stimulated with AD,the level of IL-6 in the culture supernatants were measured by ELISA.ResultsThere was no difference in Tfh cells frequency between cell-to-cell contact and transwell system.Consistent with increased frequency of Tfh cells,IL-6 and IL-21 levels were significantly enhanced in the supernatant of AD-stimulated RA FLSs and CD4+T cells co-culture system.Neutralizing IL-6 but not IL-21 significantly downregulated the frequency of Tfh cells.The IL-6 protein expression were markedly elevated in the supernant of RA FLSs after stimulation with AD and downregulated after interfering with AdipoR1 expression on RA FLSs.ConclusionsThis result indicated that the AD-stimulated RA FLSs promoted Tfh cells production mainly by the secretion of soluble factors,and IL-6 was the mainly soluble factors by AD-stimulated RA FLSs that mediated Tfh cells generation.Part 3 Local injection of AD promoted the arthritis severity and Tfh cell expansion in CIA miceObjectiveTo observe the role of AD in aggravating arthritis severity and upregulating Tfh cells production in collagen-induced arthritis(CIA)mice.MethodsCIA mice were intraarticularly injected with AD(10 ?g AD in 10 ?l PBS)into knee joints on day 17,day 20 and day 23 post first CII-immunization.The arthritis symptoms,the clinical scores of arthritis severity in CIA mice with or without AD treatment was assayed.Tfh cells populations in knee joints were detected by flow cytometry.The mRNA expression of Tfh cells transcription factors and functional factors such as Bcl-6,Blimp-1,IL-6 and IL-21in the joint tissues was measured by PCR analysis.ResultsLocal AD-treated CIA mice exhibited earlier onset of arthritis and higher arthritis scores.The CD4+CXCR5+PD-1+T cells were significantly enhanced in the joint of local AD-treated CIA mice compared to untreated CIA mice.mRNA expression of Tfh cells transcription factors and functional factors such as Bcl-6,IL-6 and IL-21 in the joint were significantly upregulated and the expression of Blimp-1 was downregulated after AD injection.ConclusionsThese results suggested that AD was the triggering factors responsible for enhanced Tfh cells production during CIA development.