The Preliminary Study Of Expression Of IL-22 In The Early Infection By Echinococcus Granulosus In Mice

ObjectiveTo observe the effect of treatment with Echinococcus granulosus protoscolices and hydatid fluid on IL-22 expression in mice splenocytes in vitro. By building of murine models of Echinococcus granulosus infection, we explored whether E. granulosus effected the expression of IL-22 by spleen cells, and then analyzed the change of CD4~+ T IL-22-producing cell during the Echinococcus granulosus infection, which have evolved the mechanisms of evading the host immune response.Methods1. In vitroSplenocytes were prepared from BALB/c mice and were co-cultured with different concentration of protoscolices or hydatid fluid for 48 h, the level of IL-22 in co-cultured supernatants were measured using ELISA. The m RNA extracted from cells and relative level of expression of IL-22 was detected using q RT-PCR. The percentages of CD4~+IL-22~+ T cells were detected at different stages(0, 12, 24, 36, 48 h after infection) by Flow Cytometry Method. Spleen cells were co-cultured with 1640 to serve as a control.2. In vivoBALB/c mice were infected with Echinococcus granulosus protoscoleces. Spleen cells were collected at Day 3, Day 6, Day 9 and Day 12 post-infection. At the different time points, the relative expression of IL-22 related m RNAs was detected using q RT-PCR, the plasma level of IL-22 related cytokines was analyzed by ELISA and the percentage of IL-22 in spleen CD4~+T cells subsets was tested by FCM.Results1. In vitroELISA and q RT-PCR indicated that Splenocytes co-cultured with 2.05mg/ml hydatid fluid proteins and1000/ml protoscolices, the IL-22 increased significantly compared with the control group. FCM analyzed that hydatid fluid and protoscolices of CD4~+IL-22~+ T cells increased obviously after 12 h.2. In vivo2. In vivo(1) Compared with the control group, ELISA indicated that the production of IFN-γ had a statistically significant increased from peripheral blood after infection 6 and 9days(P<0.05). QRT-PCR analyzed that IFN-γ and T-bet, the transcription factor of Th1 cells, were increased after infection at Day 3, Day 6, Day 9and Day 12. FCM analyzed that the numbers of CD4~+IFN-γ~+ T cells(Th1cells) had a statistically significant increased from Splenocytes at Day 6 and Day 9 post- infection(P<0.05).(2) Compared with the control group, ELISA indicated that the production of IL-17 A had a statistically significant increased from peripheral blood at Day 3, Day 6, Day 9 and Day 12 post- infection(P<0.05).QRT-PCR analyzed that IL-17 and RORγτ, the transcription factor of Th17 cells, were increased after infection at Day 3, Day 6, Day 9 and Day 12 post- infection. FCM analyzed that the numbers of CD4~+IFN-γ-IL-17~+IL-22~+ T cells(Th17cells) had a statistically significant increased from Splenocytes after infection at Day 3, Day 6, Day 9 and Day 12 post- infection.(P<0.05).(3) Compared with the control group, ELISA indicated that the production of IL-22 had a statistically significant increased from peripheral blood at Day 3, Day 6, Day 9 and Day 12 post- infection(P<0.05).q RT-PCR analyzed that IL-22 and Ahr, the transcription factor of Th22 cells, were increased at Day 3, Day6, Day 9 and Day 12 post- infection. FCM analyzed that the numbers of CD4~+IFN-γ-IL-17-IL-22~+ T cells(Th22cells) had a statistically significant increased from Splenocytes at Day 6 and Day 9 postinfection(P<0.05).(4) Compared with the control group, ELISA indicated that the production of TGF-β had a statistically significant increased from peripheral blood at Day 3, Day 6, Day 9 and Day 12 post- infection(P <0.05).QRT-PCR analyzed that IL-22R1 from liver and intestine tissues and TGF-β from spleen were increased after infection at Day 3, Day 6, Day 9 and Day 12 post- infection.Conclusion1. The secretion of IL-22 increased significantly in mice splenocytes treated with hydatid fluid and protoscoleces in vitro, suggesting that IL-22 may paly an important role in host immune response after Echinococcus granulosus infection.2. In the early stage infection by Echinococcus granulosus, IL-22 expression is unregulated in CD4~+ T cells. Th1, Th17 and Th22 cells of IL-22-producing cells may participate in host defense immunoregulation.