The Preliminary Study Of The Functional Activity And Mechanism Of Regulatory T Cell In Sepsis

ObjectionRegulatory T cell (Treg) is a class of mature T cell subset with the regulatory function, which plays an important role in maintaining immune tolerance and immune balance. The anergic and suppressive function are the most important functional properties for Tregs, Which mediate suppression of CD4+CD25-T cells by cell-cell contact and secretion of suppressor cytokines. The clinical mortality rate of patients with severe sepsis is as high as50%, more than half of which is caused by Gram-negative bacteria. Toll-like receptor4(TLR4) plays a key role in the recognition of Gram-negative bacteria and their products, and also connets innate immunity with acquired immunity. In view of the importance of TLR4in LPS signal transduction, and TLR4is one of the surface membrane molecules in Treg, whether TLR4affect the process of immune dysfunction through the intervention of Treg in sepsis incidence? This study was performed cecal ligation and puncture (CLP) on mice to induce sepsis models, regulatory T cells were isolated by immunomagnetic beads isolate system(MACS), then to explore the possible related mechanism involved of TLR4on Treg in sepsis by observed the functional activity and apoptosis of Treg, as well as the variation of TLR4’s protein and gene expression.Methods(1) Model Preparation:Specific pathogen-free C57BL/6mice were underwent cecal ligation and puncture(CLP) to cause septic mice models and then randomly divided into2equal groups:CLP group (20mice) and sham-operation group (20mice), evaluating survival rate at postoperative24h,48h and72h, respectively. In addition, postoperative24h, collected peripheral blood samples to detecte the level of cytokines, respectively.(2) CD4+CD25-T cells as well as CD4+CD25+Tregs were isolated from the spleens of mice by immunomagnetic beads, the purity of which were verified as well.(3) CD4+CD25+Tregs and CD4+CD25-T cells were isolated from mice, CD4+CD25-T cells co-cultured medium with CD4+CD25+Tregs in radio of1:1, divided into3equal groups:Teff and sham-operation group, Teff and postoperative24h group, Teff and postoperative48h group. Cells were treated with anti-CD3and anti-CD28for72h, in addition, CD4+CD25+Tregs without treat as normal group. Proliferative activity of CD4+CD25-T cells was analyzed by MTT test.(4) CD4+CD25-T cells co-cultured with CD4+CD25+Tregs for68h. The supernatants were collected for determination of cytokines levels with ELISA kits, respectively.(5) CD4+CD25+Tregs were isolated by immunomagnetic beads, which were stained with Annexin-V-FITC and Propidium iodide(PI). The apoptotic rate of Treg was analyzed by flow cytometry(FCM).(6) The gene expression between TLR4and forkhead/winged helix transcription factor3(Foxp3) of sham-operation group, postoperative24h group and postoperative48h group, were analyzed by SYBR GREEN method.(7) CD4+CD25+Tregs were isolated from sham-operation group, postoperative24h group and postoperative48h group, which were stained with Anti-Mouse CD152, Anti-mouse Foxp3FITC and Anti-mouse TLR4(CD284) respectively, then detected CD4+CD25+Tregs specific markers of forkhead/winged helix transcription factor3(Foxp3), T lymphocyte toxicity associated antigen4(CTLA-4) as well as the expression of Toll-like receptor4(TLR4).Results(1) Postoperative CLP mice performed a series of manifestations of septic symptoms, and death occurred after12hours. The mortality of CLP at72h was50%, and the immune dysfunction had occured after24h in CLP, which affirmed a stable sepsis mouse model.(2) The purity of CD4+CD25+Tregs and CD4+CD25-T cells were92.13%～98.62%and88.70%～95.33%respectively after isolated twice by magnetic beads, and the activity of Tregs exceeded96%.(3) The proliferative response of CD4+CD25-T cells activation was significant when stimulated with anti-CD3and anti-CD28, and the suppressive activity of proliferation of CD4+CD25-T cells was markedly suppressed when co-cultured with CD4+CD25+Tregs. The proliferation inhibitory rate were37.55%,53.91%and62.40%in Sham、CLP24h and CLP48h group respectively.(4) The secretion of proinflammatory cytokine IL-2, IFN-yco-cultured with CD4+CD25+Tregs was markedly decreed in comparsion with normal control group, while the secretion of anti-inflammatory cytokine IL-4, IL-10was increased(P<0.05). Compared with the Sham group, CLP24h group and CLP48h groups changes were more significantly (P<0.01), Tregs mediated proinflammatory response to the drift of the anti-inflammatory response in sepsis.(5) The apoptosis rate after CLP indicated a significant decline, and in Sham group,CLP24h and CLP48h Group were30.92±1.98%,25.06±1.59%and19.50±0.76respectively. Furthermore,CLP48h group and Sham group were statistically significant (P<0.05).(6) After sepsis the expression of Foxp3mRNA of Treg in24-48h significantly up-regulated (P<0.05or P<0.01), which was particularly evidently increased in the CLP48h group (P<0.01), meanwhile the expression of Foxp3examined by flow cytometry was significantly up-regulated after CLP24h and48h (P<0.05). After CLP24h and48h the expression of CTLA-4was significantly up-regulated (P<0.05);(7) TLR4mRNA expression was significantly up-regulated after the sepsis of24-48h (P<0.05or P<0.01), in CLP48h group wss particularly significant (P<0.01). The expression of TLR4in postoperative24h and48h were significantly up-regulated compared with the Sham group (P<0.05or P<0.01), while after CLP48h the increase was particularly evident (P<0.01).Conclusion(1) The septic models created by the application of cecal ligation and puncture were stable, reliable, and suitable for subsequent experimental studies.(2) The CD4+CD25+Tregs isolated by magnetic beads were pure and dynamic, which were suitable for the subsequent experiments.(3) The functional activity of CD4+CD25+Tregs in sepsis was significantly enhanced, which exacerbated the immune negative regulatory role in sepsis: ①Treg had a stronger inhibitory activity to suppress the proliferation of effector T cells (Teff).②Treg mediated the drift of Thl to Th2by influencing the secretion of cytokines, inhibiting the secretion of proinflammatory cytokines IL-2and TNF-alpha, at the same time promoteing anti-inflammatory cell factor IL-4and IL-10secretion.③The apoptosis rate of Tregs in sepsis was declined, the reduced number of apoptotic Treg prompted to play the functional activity of Treg increase in the number.④The phenotype expression of Foxp3and CTLA-4in Treg was significantly enhanced.(4)There was a similar trend between the gene and protein expression of Toll-like receptor4and expression of Foxp3,further the functional activity of Treg is Consistent with the change. TLR4may affect the performance of functional activity of Treg by affecting the role of Foxp3in sepsis, and the mechanism needed further study.