| AbstractObjective:The present study was performed to investigate the effect of high mobility group box-1 protein(HMGB1)on immunological function of regulatory T cells(Tregs)in mice and its potential receptor mechanism in vitro,and to identify whether Toll-like receptor 4(TLR4)is critically involved in regulating immunological activity of CD4~+CD25~+Tregs(regulatory T cells)in vivo.Methods:(1)CD4~+CD25~+Tregs were isolated from the spleens of male C3H/HeN(TLR4 wild type)mice by magnetic beads,then the purity of these cells were detected.CD4~+CD25~+Tregs were seeded on 96-well cell culture plates supplemented with 1640 culture solution containing 10%calf serum.(2)By treatment with or without anti-mouse TLR4 antibody,the time- and dose-dependent responses between HMGB1 stimulation and cytotoxic T lymphocyte-associated antigen 4(CTLA-4)as well as forkhead/winged helix transcription factor 3(Foxp3)expressions on CD4~+CD25~+Tregs were studied. CD4~+CD25~+Tregs were divided into two parts as follows:experiments with dose-and time-dependent response.In the time-dependent study,CD4~+CD25~+Tregs were divided into four subgroups named normal control group,24 hours(h)group, 48 h group,and 72 h group;In dose-dependent study,CD4~+CD25~+Tregs were divided into four subgroups named normal control group,10ng/ml group, 100ng/ml group,and 1000ng/ml group,respectively(n=4).After stimulated with different dosage of HMGB1 for various length of time,the expressions of CTLA-4 and Foxp3 of Tregs were determined by means of flow cytometry. Meanwhile cells supernatant was collected to examine the concentration of IL-10 by ELISA.(3)10μg or 20μg HMGB1 was administered i.p in C3H/HeN(TLR4 wild type)and C3H/HeJ(TLR4 mutant type)mice(n=6).Mice were sacrificed at 48h after injection,and splenic CD4~+CD25~+Tregs were harvested aseptically following another 12 h culture,then CTLA-4 and Foxp3 expressed on CD4~+CD25~+Tregs were determined and cells supernatants were collected.Results:(1)The purity of CD4~+CD25~+Tregs were over 91%after being isolated twice by magnetic beads,and the activity of CD4~+CD25~+Treg exceeded 97%.(2)After stimulation with HMGB1,the expression of TLR4 in CD4~+CD25~+Treg was markedly decreased(p＜0.05 or p＜0.01),starting at doses as low as 10ng/ml and with a maximal response at 100 ng/ml or 1000ng/ml(P＜0.01). The effects of HMGB1 on CD4~+CD25~+Treg were also time-dependent,with a significant decrease in the expression of TLR4 at 24 h,48 h and 72 h.(3) Compared to normal control group,the expression of CTLA-4 and Foxp3 on CD4~+CD25~+Tregs stimulated by 100ng/ml HMGB1 were significantly down-regulated at 24 h,48 h,and 72 h,respectively(P＜0.01),especially at 48 h and 72 h.Meanwhile,markedly decreased expressions of CTLA-4 and Foxp3 were found by treated with HMGB1 at 48 h in various doses(10,100 and 1000 ng/ml) (P＜0.01).Pretreatment with TLR4 antibody,however,CTLA-4 and Foxp3 expressions on CD4~+CD25~+Tregs were significantly enhanced by HMGB1 (100ng/ml)stimulation at different time points(24 h,48 h and 72 h),respectively (P＜0.05 or P＜0.01).Treatment with different doses of HMGB1 for 48 h,CTLA-4 and Foxp3 expressions on CD4~+CD25~+Tregs were much higher in 100 ng/ml HMGB1 group than that in normal controls(P＜0.01).(4)With in vivo study,the CTLA-4 and Foxp3 expressions in two gene types mice were both in a similar levels(P＞0.05);While HMGB1 was administered i.p in C3H/HeN mice and C3H/HeJ mice,it was revealed a significant distinction in the expression of CTLA-4 and Foxp3 on two gene types mice.Compared to normal controls,the expressions of CTLA-4 and Foxp3 on CD4~+CD25~+Tregs were markedly decreased in C3H/HeN mice with a maximal response at a dose of 20μtg(P＜0.01). However,the expressions of CTLA-4 and Foxp3 on CD4~+CD25~+Tregs were significantly increased in C3H/HeJ mice with a maximal response at a dose of 10μg(P＜0.01).Conclusions:(1)The CD4~+CD25~+Tregs isolated by magnetic beads were pure and suitable for the subsequent experiments.(2)Stimulation with HMGB1 could decrease the expression of TLR4 on cellular membrane of CD4~+CD25~+Tregs, indicating TLR4 might regulate the immunological activity of CD4~+CD25~+Tregs. (3)In comparison to changes in immunological activity of CD4~+CD25~+Tregs with or without TLR4 antibody treatment in vitro,we concluded that TLR4 might play a negatively regulatory role in Treg immunological activity.(4)In comparison to changes in immunological activity of CD4~+CD25~+Tregs in two gene types mice when stimulated with HMGB1 in vivo,it was suggested that TLR4 can markedly down-regulate CTLA-4 and Foxp3 expressions on CD4~+CD25~+Tregs,thereby negatively influencing the immunological activity of Tregs. |
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