The Effect And Mechanism Of HMGB1 On Peripheral Treg/Th17 Balance In Patients With Atherosclerosis

Objective To clarify the influence of high mobility group box B1(HMGB1) on the apoptosis and differentiation of Treg and Th17 cells.To explore the mechanism of Treg/Th17 imbalance and the role of HMGB1 on the balance in peripheral blood of atherosclerosis patients.Methods(1) We collected the venous blood of atherosclerosis patients according to coronary angiography results and exclusion criteria and separated the peripheral blood mononuclear cell(PBMC).(2) We detected the frequencies of apoptotic Treg cells and apoptotic Th17 cells in atherosclerosis(AS) and normal coronary arteries(NCA) subjects by flow cytometry.(3) HMGB1 m RNA levels in PBMC were detected by qRT-PCR.(4)We examined the frequencies of Treg and Th17 cells in PBMC after stimulation of recombinant HMGB1(rHMGB1) at different levels of concentration and incubation time.(5) Treg and Th17 cells frequencies and Foxp3、RORC mRNA levels in PBMC from AS and NCA subjects were analyzed by flow cytometry and qRT-PCR after rHMGB1 stimulation.(6) The apoptosis ratios of Treg and Th17 cells in PBMC were examined by flow cytometry after rHMGB1 stimulation.(7) We separated na?ve CD4~~+T cells and analyzed the effects of rHMGB1 on differentiations of Treg and Th17 cells from na?ve CD4~~+T cells.Results(1) The frequencies of apoptotic Treg cells in AS subjects were significantly higher than in NCA subjects(p<0.01), whereas Th17 cell apoptosis ratios had no significant difference between two groups(p>0.05).(2) HMGB1 mRNA levels in PBMC from AS subjects were significantly higher than in NCA subjects(p<0.01) and were positively correlated with the ratio of apoptotic Treg cell(r=0.660,p<0.001), while had no significant relativity with Th17 cell apoptosis ratio(r=0.062,p>0.05).(3) Stimulation of rHMGB1 obviously increased the level of Th17 cells, while markedly decreased Treg cell frequency. The effect of rHMGB1 was more obvious at 100ng/ml concentration(p<0.01)and 24 h incubation time(p<0.01).(4) The Treg cell frequency(p<0.01) and Foxp3 mRNA level(p<0.01) were decreased and the Th17 cell frequency(p<0.01) and RORC mRNA level(p<0.01) were increased in PBMC from both AS and NCA subjects after r HMGB1 stimulation. The effect of rHMGB1 on AS subjects was more significant than on NCA group.(5) Stimulation of rHMGB1 obviously elevated Treg cell apoptosis ratio(p<0.01),while had no significant effect on ratio of Th17 cell apoptosis(p>0.05).(6) The frequencies of Treg cell and Th17 cell were(1.5 ± 0.45)% and(0.9 ± 0.38)% before treating na?ve CD4~~+T cells with induction conditions. The CD4~~+CD25~~+CD127-Treg cell ratio was elevated to(29.8±1.75)% after induction of TGF-β1 and IL-2 on na?ve CD4~~+T cell for 7 days. The CD4~~+IL-17~~+Th17 cell ratio was elevated to(8.0 ± 0.84)% after induction of IL-1β、IL-6 and IL-23 on na?ve CD4~~+T cell for 7 days. The Treg cell ratio was decreased to(20.6±1.41)% in rHMGB1 treatment group. IL-10 level in treatment group was lower than in control group(p<0.05). The Th17 cell ratio was elevated to(12.1 ±0.81)% in rHMGB1 treatment group. IL-17 A level in treatment group was higher than in control group(p<0.01).Conclusion(1) In vitro analysis suggests that HMGB1 can influence Treg/Th17 balance in peripheral blood of atherosclerosis patients through promoting the balance to the pro-inflammatory Th17 cell side.(2) HMGB1 can induce the apoptosis of Treg cell,probably leading to the increasing apoptosis level of Treg cell in PBMC from AS subjects.(3) HMGB1 can restrain the differentiation of Treg cell and promote Th17 cell differentiation from na?ve CD4~~+T cells.(4) HMGB1 possibly influence Treg/Th17 balance in atherosclerosis patients through inducing Treg cell apoptosis, promoting Th17 cell differentiation and inhibiting Treg cell differentiation.