| Summary: | 博士 === 國立臺灣大學 === 生化科技學系 === 105 === The purpose of the study is to investigate the effects of folate deficiency on the maturation and function of antigen presenting cell (APC) and subsequent T cell differentiation. Macrophages and bone marrow-derived dendritic cell (BMDC) were cultured in folate sufficient (F) or folate deficient (FD) condition to examine the impact of folate deficiency on APC function and T-cell priming ability. Moreover, mice fed with AIN-76 contains 0, 2, 20 mg folic acid/kg diet (F0, F1, F10) to confirm the effects of folate deficiency on APC function and T cell differentiation in vivo.
Folate deficiency increased pro-inflammatory cytokine TNFα and IL-6 productions and expressions of CD80 and CD11b, but decreased CD86 and CD40, in LPS-stimulated RAW264.7 macrophage. Primary peritoneal exudates cell (PEC) cultured in FD medium produced higher TNFα, IL-6 and IL-12p40 by LPS stimulation, indicating folate deficiency enhanced inflammatory response of LPS-stimulated macrophage. In addition, PEC from mice fed with folate-deficient diet produced higher TNFα after LPS-stimulation but reduced IL-10 and IL-17 productions in PEC-CD4 co-culture system. Furthermore, the mouse model of LPS-induced endotoxic shock was used to determine the effects of folate deficiency on inflammatory response in vivo. The data was showed that folate deficiency reduced serum TNFα, MCP-1, IFNγ, IL-12p70, and IL-12p40 levels and then increased the survival rate of mice in the F0 group. In summary, folate deficiency increased inflammatory response of macrophage by LPS-stimulation, but diminished T cell differentiation toward Th17 and IL-10-producing cells.
FD-BMDC displayed more immature phenotype including reduced levels of MHC class II and co-stimulatory molecules CD40/CD80/CD86 and characteristics of higher apoptosis percentage and endocytic activity compared to F-BMDC. FD-BMDC produced less IL-12p70 and pro-inflammatory cytokines than F-BMDC under LPS stimulation. The immature property of FD-BMDC resulted in reduced BMDC-induced CD4+ T cell activity with lower IFNγ, IL-2, IL-10 and IL-13 productions by FD-CD4. F-BMDC was observed to reverse Th1 cytokines IFNγ and IL-2 productions of FD-CD4, suggesting the importance of folate on Th1 differentiation. In vivo study showed spDC from F0 mice expressed lower levels of MHC class II and CD80 resulted in decreased DC-induced IFNγ, IL-2 and IL-13 productions of CD4+ T cell. In addition, to investigate the effects of different period of folate deficiency or supplement on T cell responses, mice were fed with F0, F1, or F10 diet for 5, 9, and 13 weeks, respectively. The splenocytes of F0 mice had higher IL-2 while impeded other cytokines (IFNγ, IL-4, IL-5, IL-13, IL-10 and IL-17), and these inhibitory effects were found apparently at week 9. The results have shown that F0 also increased the percentage of naïve CD4+ T cell and reduced the percentage of CD4+ Foxp3+ Treg in spleen. Taken together, folate deficiency impaired DC maturation and inhibited CD4+ T cell differentiation in vivo.
To asses the immuno-modulatory effects of folate deficiency on T cell activation, the data has demonstrated that folate deficiency increased Nfat5 expression and decreased DNA methylation status on Nfat binding site in il2 promoter of EL4 mouse T lymphocyte cell line, indicating that folate deficiency might regulate gene expression by altering DNA methylation status. Thus, we observed that gene expressions of DNA methyltransferase (Dnmt) 1, Dnmt3a, and Dnmt3b were increased in splenocytes and splenic CD4+ T cells from F0 mice. Methylated DNA immuno- precipitation (MeDIP) microarray was used to screen the candidate genes. The expressions of these candidate genes from CD4+ T cells of mice fed with F0, F1, or F10 diet for 9 weeks were used. The splenocytes of F0 mice had higher naïve T-related gene Cd247 but lower activated T-related gene Icos and Lag3 expression while F10 mice reduced Socs family levels. DNA methylation status of CpG island and promoter of Icos were analyzed by EpiTYPER assay but there was no significant difference of DNA methylation status on CpG islands and promoter region of Icos, indicated that F0 reduced Icos might via other pathway.
In conclusion, folate deficiency decreases APC maturation with decreased MHC class II and co-stimulatory molecules resulting in inhibiting CD4+ T cell differentiation from naïve CD4+ T cell toward Th1, Th2, Th17 and Treg. The study demonstrates that folate is essential to maintain the responses and balances of CD4+ T cell differentiation and plays a critical role in modulating immune response.
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