Studies On The Effects Of Freund's Adjuvant And Aluminum Hydroxide Adjuvant On Tregs And CD4~+T Cell Relative Genes In Spleen Of Mice

Adjuvants are used to boost the potency and longevity of specific immune response to antigens. As a strong agent in improving the body's specific immune response, adjuvants are able to stimulate a strong and lasting immune response where the local or systemic of body, which induces different cytokines which have an effect on the local or systemic CD4+T cell subsets balance. Changes in the immune balance caused by adjuvants may have some negative impacts on the immune system, which might cause a certain degree of immunity disease or suppress the immune response to other pathogens. With the type of vaccination and inoculation times increasing in the livestock industry in our country, which results in a corresponding increase in the use of adjuvant. The situation is even more prominent especially to evaluate the effects of adjuvants on systemic immune system.Although numerous reports indicated that adjuvants had a significant effect on the immune system of Thl/Th2 immune balance, there were less studies on a newly found CD4+CD25+regulatory T cells (Tregs) and Th17 cells which following the Thl and Th2. Little information is available on cytokine induction by adjuvant only. To comprehensively understand the effects of adjuvant on CD4+T cell subsets, Freund's complete adjuvant and Al (OH)3 adjuvant were used to evaluate their effects on Tregs and gene expression profile in spleen of Balb/c mice. Thirty Balb/c mice were randomly assigned to three groups; CFA group, AHA group and control group. Three groups were administered intraperitoneally with 200μL CFA (emulsified with PBS in a ratio of 1:1), AHA(1 mg/mL) or PBS respectively. All mice were necropsied at Day 14. This studies might provide a theoretical basis for assessing the impact of adjuvants on immune system. The main research contents and results as follows:The frequencies of CD4+Foxp3+T cells, CD4+CD25- Foxp3+T cells and CD4+CD25+Foxp3+T cells in splenocytes were assessed by flow cytometry and Foxp3 mRNA levels were detected using reverse transcription-PCR (RT-PCR) and immunohistochemical analysis. The result showed that CFA decreased the proportion of CD4+T cell in splenocytes and promoted the percent of CD4+Foxp3+T cells, CD4+CD25-Foxp3+T cells and CD4+CD25+Foxp3+T cells in CD4+T cells in splenocytes on day 14 (p< 0.01). But the levels of Foxp3 mRNA were no difference among the three groups(p> 0.05). AHA has no effect on foregoing index(p> 0.05). Results from the figures of flow cytometry analysis, there were a group of cells with CD4 negative, and with large side-angle scattering and the cells number was significantly increased in Freund's adjuvant group compared with the other groups. Based on the above characteristics, these cells might be macrophages.Eighty-four gene expression profiles which related to Thl/Th2/Th3 were detected by real-time RT-PCR gene chip technology. The results showed that the expressions of genes BCL6, CCL5, CCR3, CCR4, CD28, CD4, CD80, CD86, CSF2, CTLA4, CXCR3, GATA3, ICOS, IFNG, IL-10, IL-12RB2, IL-18, IL-18BP, IL-1R1, IL-2, IL-27RA, IL-2RA, IL-4, IL-4RA, IRF1, JAK1, MAF, MAPK9, NFATC1, NFATC3, NFKB1, PTPRC, SOCS5, STAT1 and STAT4 were strongly down-regulated in Freund's adjuvant group; but IL-17, IL-23A, CEBPB, GFI1, IL-9, SPP1 and YY1 gene were significantly up-regulated. The gene expression levels of other genes was not significant. There was only IL-2 expression increased significantly in Aluminum hydroxide adjuvant group compared with the control group.The serum concentration of TGF-p and IL-17 of mice treated with Freund's adjuvant and aluminum hydroxide adjuvant were detected by ELISA. The results showed that the serum levels of TGF-βand IL-17 in were increased significantly in Freund's adjuvant group compared with the control group, and there was no significant difference between the aluminum hydroxide adjuvant group and the control group. Aluminum hydroxide adjuvant has no effect on the spleen index and the percentage of macrophages in spleen.The above results indicated that the Freund's adjuvant could significantly increase the percentage of macrophages in the spleen, Tregs spleen index and the serum concentration of IL-17 IL-23. It showed some inhibition functions on the levels of gene expression involvement with Thl, Th2, and total CD4+T cells to a certain extent. The effect of aluminum hydroxide adjuvant on the index were not significant different. We suggested that CFA could improve the level of immune system for antigen-specific immune response, but the overall function of the immune system might be inhibited to a certain extent.