A Study On The Relationship Between Th17、CD4~+CD25~+Treg And ICOS/ICOSL In Lymph Fluid Of Asthmatic Rat

Object:To determine the effect of the ICOS/ICOSL signaling pathway to the number of the Th17, CD4+CD25+Foxp3+Treg and the secretion of IL-17,IL-10and TGF-β1in lymph fluid and blood of the asthma rat.Method:Build the asthma model. The blood and lymph fluid were collected from the rats at0h、24h、48h after the last challenge, the MNC from lymph and blood were isolated and seeded in24well plate, after96h-cocultured with anti-ICOSAb to block, the percentage of the Th17and CD4+CD25+Foxp3+Treg were detected by FCM(flow cytometer), while the levels of the IL-17、IL-10and TGF-β1in supernatant were detected by enzyme-linked immune-sorbent assay.Result:1. The asthma model was copied successfully and the lymph drainage patterns were established.2. In vitro culture system, the percentage of Th17in the lymph were significantly higher than that in blood in each group at different time point; The percentage of Th17in the MNC from lymph and blood in cases of asthma group were significantly higher compare to the control group at different time point (P<0.05); The percentage of Th17in the MNC from lymph in anti-ICOSAb group were lower than the asthma group, as compare to the control group, there were no significant differences; The percentage of Th17in the MNC from blood in anti-ICOSAb were lower than the asthma group, but higher than control group.3. In vitro culture system, the levels of IL-17in the supernatant of the MNC from the lymph fluid and blood in the anti-ICOSAb was lower than the asthma group at Oh, and there were no differences compare to the control group; At24h,48h after the last challenge, The content of IL-17in the supernatant of the MNC from the blood in the anti-ICOSAb group were significant lower than the asthma, and there were no differences compare to the control group; While there were no difference between each group that from lymph fluid.4. In vitro culture system, the percentage of CD4+CD25+Foxp3+Treg in the MNC from lymph were significantly higher than that from blood in each group(P<0.05); The percentage of CD4+CD25+Foxp3+Treg in the MNC from lymph and blood in cases of asthma group were significantly lower compare to the control group (P<0.05); The percentage of CD4+CD25+Foxp3+Treg in the MNC from lymph and blood in the anti-ICOSAb group obviously decreases compare with the asthma group (P<0.05).5. At Oh after the last challenge, the level of IL-10in the supernatant of MNC from lymph and blood in the anti-ICOSAb group were significantly lower than that of the control and asthma group(P<0.05), there were no significant differences between the asthma group and the control group; At24h-.48h after the last challenge and there were no significant differences in the supernatant of the mixed lymphocyte from the blood and lymph fluid among each group; There were no significant differences in the different group, at different time point between lymph fluid and blood.6. In vitro culture system, there were no significant differences of TGF-01expression in the supernatant of MNC from lymph and blood in each group at different time points.Conclusion:1-. The number of Th17in the MNC from lymph and blood in asthma rats was higher than the control, blocking the ICOS/ICOSL signaling pathway with anti-ICOSAb could reduce the number of Th17from lymph and blood in bronchial asthmatic rat, meanwhile inhibit the Th17from the lymph fluid and blood in bronchial asthmatic rat secreting IL-17at Oh after the last challenge, in addition restrain the MNC from blood in asthmatic rats secreting IL-17at24h,48h after the last challenge. 2、The percentage of CD4+CD25+Foxp3+Treg in the MNC from lymph and blood in cases of asthma group were significantly lower compare to the control group, blocking the ICOS/ICOSL signaling pathway by anti-ICOSAb could exacerbate the deficiency of CD4+CD25+Foxp3+Treg from lymph and blood in bronchial asthmatic rat, meanwhile inhibit the CD4+CD25+Foxp3+Treg secreting IL-10at Oh after the last challenge, but have no significant effect on the secretion of TGF-β1.3、Regardless of asthma group or normal control group, the number of Th17and CD4+CD25+Foxp3+Treg in lymph were significantly higher than that in blood, indicate the immune function of lymph circulation may be a more sensitive indicator reflecting the change of immune status in the body..