猪囊尾蚴排泄分泌抗原LRRC15蛋白对仔猪T细胞免疫应答的影响

Effect of cysticercus cellulosae excretory secretory antigen LRRC15 protein on T-cell immune response in piglets

目的观察猪囊尾蚴排泄分泌抗原LRRC15蛋白对仔猪T细胞免疫应答的影响方法用LRRC15蛋白刺激健康仔猪外周血PBMC和脾脏CD4^(+)T细胞,分析T细胞免疫应答水平:①在PHA处理下,以RPMI 1640、ESA、ConA为对照组,用LRRC15蛋白刺激PBMC,流式检测CD4^(+)、CD8^(+)T淋巴细胞数量比例变化,以及CD4^(+)CD25^(+)Foxp3^(+)、CD8^(+)CD25^(+)Foxp3^(+)Treg细胞表达水平;②在有和无LPS存在下,用LRRC15蛋白刺激PBMC,ELISA检测培养上清中IL-10含量;③分别于加入LRRC15蛋白后24、48、72 h检测初始CD4^(+)Th细胞分化情况。分离仔猪脾脏CD4^(+)T细胞并与未成熟髓源性DC共培养,采用ELISA方法检测加入LRRC15蛋白24、48、72 h培养上清中的IL-4、IL-5、IL-10、IL-17、IFN-γ水平。结果LRRC15蛋白刺激PBMC后,CD4^(+)T淋巴细胞数量比例显著增加(P<0.05),并诱导CD4^(+)CD25^(+)Foxp3^(+)、CD8^(+)CD25^(+)Foxp3^(+)Treg表达(均P<0.05),但不能诱导IL-10的分泌。LRRC15蛋白分别作用于CD4^(+)T细胞24、48、72 h,PBMC细胞IL-4、IL-17分泌水平显著降低(均P<0.05);LRRC15蛋白诱导24与48 h相比,PBMC细胞分泌IL-5水平升高(P<0.05);诱导72 h的IL-5水平与48 h时比较差异无统计学意义(P<0.05);LRRC15蛋白诱导IFN-γ水平升高(P<0.05),诱导24、48、72 h时的分泌水平差异无统计学意义(P>0.05);LRRC15蛋白于CD4^(+)T细胞24、48、72 h均不能诱导IL-10分泌(均P>0.05)。结论LRRC15蛋白可诱导仔猪PBMC中CD4^(+)/CD8^(+)T细胞免疫失衡,诱导Treg细胞表达,通过非依赖性IL-10途径发挥免疫抑制作用,并诱导早期Th1型免疫应答和后期Th1/Th2混合型免疫应答。

Objective The purpose of this study is to examine the effect of pig cysticercus excretory-secretory antigen LRRC15 protein on the immune response of piglet T cells.Methods LRRC15 protein was used to stimulate healthy piglet peripheral blood mononuclear cells(PBMC)and spleen CD4^(+)T cells.The level of T cell immune response was analyzed as follows:①PBMC was stimulated with LRRC15 protein under PHA treatment,with RPMI 1640,ESA,and ConA as control groups.The changes in CD4^(+)and CD8^(+)lymphocyte cell ratio and the expression level of CD4^(+)CD25^(+)Foxp3^(+)and CD8^(+)CD25^(+)Foxp3^(+)Treg cells were detected by flow cytometry.②PBMC was stimulated with LRRC15 protein with and without LPS.The IL-10 content in the culture supernatant was detected by ELISA.③The initial differentiation of CD4^(+)Th cells was detected at 24,48,and 72 hours after adding LRRC15 protein.CD4^(+)T cells from piglet spleen were isolated and co-cultured with immature myeloid dendritic cells(DC),and the levels of IL-4,IL-5,IL-10,IL-17,and IFN-γin the culture supernatant at 24,48,and 72 hours after adding LRRC15 protein were detected by ELISA.Results After stimulation with the LRRC15 protein,there was a significant increase in the proportion of CD4^(+)T lymphocytes(P<0.05),and it also induced the expression of CD4^(+)CD25^(+)Foxp3^(+)and CD8^(+)CD25^(+)Foxp3^(+)Tregs(both P<0.05).However,it was unable to induce the secretion of IL-10.The LRRC15 protein acted on CD4^(+)T cells separately for 24,48,and 72 hours,and it significantly reduced the secretion levels of IL-4 and IL-17 in PBMC cells(both P<0.05).Compared to LRRC15 induction for 24 and 48 hours,the secretion level of IL-5 in PBMC cells increased significantly(P<0.05)while there was no statistical difference in the secretion level of IL-5 induced for 72 hours and 48 hours(P<0.05).The LRRC15 protein induced an increase in IFN-γlevel(P<0.05),and there was no statistical difference in the secretion level after 24,48,and 72 hours of induction(P>0.05).Additionally,the LRRC15 protein could not induce the secretion of IL-10 in CD4^(+)T cells after stimulation for 24,48,and 72 hours(all P>0.05).Conclusion The LRRC15 protein can cause an immune imbalance of CD4^(+)/CD8^(+)T cells in the PBMC of piglets,as well as induce the expression of Treg cells.It exerts immunosuppressive effects through a pathway that is not dependent on IL-10 and induces both an early Th1-type immune response and a late Th1/Th2 mixed-type immune response.