Hydroxychloroquine Prevents Collagen-induced Arthritis By Down Regulation Of T Follicular Helper Cells

BackgroundHydroxychloroquine(HCQ)was developed from Quinine by adding a hydroxyl group at the end of the side chain and has been known for its effect on malaria.Later,HCQ was shown to have benefit effects for autoimmune diseases,including systemic lupus erythematosus and rheumatoid arthritis,etc.Because of its remarkable effect and good safety,HCQ has commonly been included in long-term protocols to reduce the recurrence of the disease and organ damage.Until now,the major mechanisms of HCQ on the immune system include;(1)inhibition of antigen presentation function;(2)decreasing macrophage-mediated cytokine production,including IL-1 and IL-6;(3)inhibition of T and B cell activation by interference with calcium signaling;(4)binding and stabilizing DNA and blocking of UV light cutaneous reactions;(5)affecting the migration of inflammatory cells,etc.A few years ago,some in vitro studies demonstrated that HCQ downregulated the proportion of Th17 cells and their cytokine production in SLE and RA patient.T follicular helper(Tfh)cells is a new T cell subset discovered about ten years ago.Tfh cells are located inside lymphoid follicles,and have a unique phenotype marker CXCR5.Bcl-6 as a master regulator in Tfh cells differentiation.Tfh cells produce IL-21 that is an important cytokine for Tfh cells differentiation and for the development of germinal center.The main function of Tfh cells is to support the differentiation of GC B cells into B memory cells and plasma cells that can produce protective or pathogenic antibodies.The subset was found to expand in many autoimmune diseases and correlated with symptoms,inflammatory indexes,titres of serum antibodies or disease activity.And it has proved to be dysfunctional or defected in immunodeficiencies diseases.Whether and how HCQ affects Tfh cells remain unknown.In order to answer these questions,we planed experiments to investigate the relationship between HCQ and Tfh cells in vivo and in vitro.Objective1.To find out a proper concentration of HCQ intervention with PBMCs in vitro.2.To study whether HCQ can influence Tfh cells in vitro.3.To further investigate the possible mechanisms of HCQ on Tfh cells.4.To examine the effect of HCQ on Tfh cells from peripheral blood,lymph nodes or spleen in C57BL/6 mice.5.To determine whether Hydroxychloroquine prevents collagen-induced arthritis by down regulation of T follicular helper cells.Methods1.Purification and culture of PBMCsPBMCs were isolated from blood according to the manufacturer's instructions,cultured in RPMI-1640 supplemented with 10%fetal bovine serum and penicillin(100U/ml)/streptomycin(100U/ml)and incubated at 37? in 5%CO2 incubator.2.Determination of the proper concentration of HCQ and culture timeApoptosis and necrosis rates of PBMCs in different concentrations of HCQ were examined by flow cytometry and the cytotoxic effect on PBMCs by MTT,to determine the proper concentration of HCQ and culture time.3.Assessment of Tfh cells by flow cytometryAfter treatment with HCQ,PBMCs were harvested and labeled with CD4,CXCR5 monoclonal antibodies,incubated for 20-30 minutes and assessed the proportions of Tfh cells by flow cytometry.4.Exploration of the possible mechanisms of HCQ on Tfh cellsPBMCs were harvested and cultured with medium,rIL-21(50ng/ml),or rIL-21(50ng/ml)+HCQ(25?M/ml)to investigate whether IL-21 plays a role in the effect of HCQ on Tfh cells.5.Investigation of the effect of HCQ on Tfh cells from peripheral blood,spleen and lymph nodes in C57BL/6 miceFirstly,C57BL/6 mice were divided into two groups randomly and one group was administrated with HCQ(80mg/Kg)intragastrically while the control group with saline.Then,at day 3 and day 7,the blood,spleen and lymph node cells were collected.Finally,assessed the proportions of Tfh cells by flow cytometry.6.Examination of the effect of HCQ on Tfh cells from peripheral blood,lymph nodes in CIA modelFirstly,DBA mice were randomly divided into three groups including control group,CIA group and CIA+HCQ group.HCQ(80mg/Kg)was administrated intragastrically.Then,at day 35,the blood,lymph node cells were collected and the proportion of Tfh cells assessed by flow cytometry.To determine whether Hydroxychloroquine prevents collagen-induced arthritis by down regulation of T follicular helper cells.7.Arthritis was scored via a visual assessment scoring(VAS)system for the degree of inflammation on a scale from 0 to 4:0,no evidence of erythema and swelling;1,erythema on one or two toes or mild swelling confined to wrist or ankle joint;2,erythema and moderate swelling extending from ankle to the mid foot;3,erythema and swelling extending from ankle to metatarsal joints;4,erythema and severe swelling encompassing the ankle,foot,and digits.The clinical score for each mouse was determined by summing the scores for all individual limbs.StatisticsData were analyzed using SPSS 16.0 software.Results from in vitro experiments were analyzed by Wilcoxon matched pairs test,while results in vivo experiments with Student's t test or one way ANOVA.P<0.05 was considered significant.Results1.About 1-2×106 PBMCs were isolated from 1ml peripheral blood..Newly isolated PBMCs survived more than 99%,as assessed by flow cytometry.2.In vitro,incubation with HCQ for 24h or 48h,apoptosis rates were increased in PBMCs incubated with the concentrations of 50?M and 75?M,while a concentration of 25?M HCQ had no significant effect as compared to control group.Similarly,the results from MTT indicated that the survival rate was about 50%of the control at the concentrations of 50?M and 75?M.Therefore,we chosed 25?M HCQ for the subsequent experiments.After incubation with HCQ at the concentration of 25?M for 72h,the necrosis rate of PBMCs significantly increased compared to normal control.So 24h or 48h incubation time was chosed for the later experiments.3.PBMCs obtained from RA and AS patients who were new onset or withdrawal of more than three months were cultured in vitro with medium control or with HCQ for 24 and 48 hours.The results showed a significantly decreased proportion of Tfh cells when treated with HCQ as compared to normal controls.4.HCQ reversed the proliferation of Tfh cells induced by rIL-21.rIL-21 significantly increased the proportion of Tfh cells,while HCQ reversed the up regulation Tfh subset.5.HCQ down regulated the proportions of Tfh cells in peripheral blood,spleen in C57BL/6 mice.The results from day 3 indicated that the proportion of Tfh cells in spleen significantly reduced compared to the control group(P<0.01,n=4).The proportions of Tfh cells in peripheral blood and spleen at day 7 significantly reduced compared to the control group(peripheral blood(P<0.01),spleen(P<0.05),control group n=4,HCQ group n=6).7.In CIA model,the proportions of Tfh cells in peripheral blood and lymph nodes were significantly increased,while HCQ can reduced the proportion of Tfh cells in peripheral blood,and HCQ treatment CIA mice showed a dramatic decrease in morbidity and arthritis scores compared with the vehicle-treated CIA mice.Conclusions1.HCQ at the concentration of 25?M is the optimum concentration for intervention with PBMCs and the proper incubation time are 24h or 48h.2.HCQ down regulates the proportion of Tfh cells in vitro,through the blocking of IL-21 signaling pathway.3.HCQ down regulates the proportions of Tfh cells in peripheral blood,spleen in C57BL/6 mice.4.In CIA model,the proportions of Tfh cells in peripheral blood and lymph nodes were significantly increased,and Hydroxychloroquine prevents collagen-induced arthritis by down regulation of T follicular helper cells.