Effect Of Cysticercus Cellulosae Excretory Secretory Antigen On T Cell Immune Response Of Piglets

Objective:Cysticercus cellulosae was collected to prepare excretory secretory antigen(ESA)and crude antigen(CA)following establishment of pigs cysticercosis animal model.The effect of cysticercus cellulosae ESA on T cell immune response of piglets was studied with cysticercus cellulosae CA as control,the aim was to play an experimental foundation for revealing the immune pathogenesis of cysticercosis and the development of vaccines.Methods:Healthy piglets were orally fed with gravid proglottids of Taenia solium.Cysticercus cellulosae was collected to prepare ESA and CA by dissecting the infected piglets after 2～3 months.Peripheral blood mononuclear cell(PBMC)of normal piglets was stimulated with cysticercus cellulosae ESA and CA respectively to analyze T cell immune response induced by these two antigens:(1)Under phytohaemagglutinin(PHA)treatment,the PBMC was treated with cysticercus cellulosae ESA(40μg/m L)and CA(40μg/m L)respectively,and concanavalin A(Con A)was used as positive control.The change of the number of CD4~+and CD8~+T lymphocyte was detected by flow cytometry;(2)PBMC was stimulated with cysticercus cellulosae ESA and CA respectively,and the expression frequency of Foxp3~+,CD4~+Foxp3~+,CD8~+Foxp3~+,CD4~+CD8~+Foxp3~+,CD4~-CD8~-Foxp3~+Treg were analysed by flow cytometry,and the m RNA relative expression level of Foxp3 and Helios genes was measured by RT-q PCR;(3)In the presence or absence of lipopolysaccharide(LPS),PBMC was stimulated with ESA and CA severally,ELISA was used to detect the secretion level of IL-10 in the culture supernatant.Further,the cell was collected and the expression frequency of IL-10~+,CD4~+IL-10~+,CD8~+IL-10~+,CD4~+CD8~+IL-10~+,CD4~-CD8~-IL-10~+,Foxp3~+IL-10~+cell were tested by flow cytometry;(4)The effect of cysticercus cellulosae ESA and CA on the differentiation of initial CD4~+Th cell was further analysed.First,the marrow-derived DC of piglets was isolated and inducted,then stimulated with cysticercus cellulosae ESA and CA.The expression of CD80,CD86 and MHCⅡof DC surface markers was detected using flow cytometry.The expression levels of IL-6,IL-10,IL-12 and TNF-ɑin the culture supernatant were analyzed by ELISA;(5)Subsequently,the CD4~+T cell was isolated from the spleen of piglets and co-cultured with immature DC,then added cysticercus cellulosae ESA and CA respectively.The secretion levels of IFN-γ,IL-4 and IL-17 in the co-culture supernatant were measured by ELISA.Results:In this study,the immune cells of piglets were stimulated with different antigens separately based on the successful preparation of cysticercus cellulosae ESA and CA.These results were as follows:(1)Both cysticercus cellulosae ESA and CA could induce a decrease in the ratio of CD4~+/CD8~+T lymphocyte,the induction effect of the former was significantly higher than that of the latter(P<0.05).Cysticercus cellulosae ESA could induce an increase in the number of CD8~+T lymphocyte,while CA could induce a decrease in the number of CD4~+T lymphocyte and an increase in the number of CD8~+T lymphocyte;(2)Both ESA and CA could induce an increase in the number of Foxp3~+cell(P<0.05).Cysticercus cellulosae ESA could induce an increase in the number of CD4~+Foxp3~+and CD8~+Foxp3~+Treg cells,while CA only induced an increase in the number of CD4~+Foxp3~+Treg cell.In addition,both of them could stimulate the down-regulation of Foxp3 and Helios m RNA relative expression levels(P<0.05);(3)Both cysticercus cellulosae ESA and CA could stimulate the increase of IL-10 secretion level and up-regulation the expression of IL-10~+lymphocyte.CD4~+and CD4~-CD8~-T lymphocytes were the main source cells of IL-10 expression induced by cysticercus cellulosae ESA,while CA only induced the expression of CD4~+IL-10~+T lymphocyte(P<0.05).Moreover,cysticercus cellulosae ESA could significantly inhibit the expression of Foxp3~+IL-10~+lymphocyte(P<0.05);(4)Cysticercus cellulosae CA could significantly up-regulate the expression of DC surface markers CD80,CD86,MHCⅡand could increase the secretion levels of IL-6,IL-10,IL-12 and TNF-ɑcompared with ESA(P<0.05);(5)Both cysticercus cellulosae ESA and CA could induce higher secretion level of IL-4 significantly higher than that of IFN-γand IL-17.Compared with cysticercus cellulosae CA,ESA could induce a lower secretion level of various cytokines(P<0.05).Conclusions:Both cysticercus cellulosae ESA and CA could induce a change in the ratio of CD4~+/CD8~+T cell,and ESA could induce the expression of Foxp3~+Treg cell and the secretion level of IL-10 was higher than that of CA.It might be related to ESA secreted by cysticercus cellulosae during parasitic process and played a role in the host immune system.In addition,both cysticercus cellulosae CA and ESA could significantly induce Th2 type immune response.In conclusion,the regulation of T cell immune response by cysticercus cellulosae ESA and CA might be one of the mechanisms of cysticercus cellulosae exerted immune evasion.