葉酸缺乏影響抗原呈獻細胞功能與CD4+ T細胞分化之研究

Abstract

本研究欲探討葉酸缺乏如何影響抗原呈獻細胞 (atigen presenting cell, APC) 的成熟與功能，進而影響T細胞分化。分別以正常葉酸 (F) 或葉酸缺乏 (folate deficiency, FD) 培養基培養巨噬細胞與骨髓樹突細胞 (bone marrow derived dendritic cell, BMDC)，探討葉酸缺乏對APC成熟與誘導T細胞分化能力的影響，並以動物實驗驗證葉酸缺乏對小鼠體內巨噬細胞與脾臟DC (splenic DC, spDC) 功能以及T細胞反應的影響。 葉酸缺乏顯著增加RAW264.7巨噬細胞株LPS刺激後促發炎細胞激素TNF與IL-6分泌量，促進細胞表面CD80與CD11b表現，但降低CD86與CD40表現量。此外，葉酸缺乏環境下會增加初代腹腔細胞 (peritoneal exudates cell, PEC) LPS刺激後分泌TNFα、IL-6與IL-12p40，顯示葉酸缺乏促進巨噬細胞in vitro發炎反應。接著給予BALB/c小鼠餵食13週標準葉酸 (F1, 2 mg/kg diet) 或缺乏葉酸 (F0, 0 mg/kg diet) 飼料，F0小鼠PEC在LPS刺激下分泌較高量TNFα，但部分抑制CD4+ T細胞分化反應，不影響Th1與Th2，但顯著減少IL-10與IL-17分泌量。以LPS致急性發炎小鼠模式探討葉酸缺乏 (F0) 或添加葉酸 (F10, 20 mg/kg diet) 對全身性發炎反應的影響，F0小鼠血清TNFα、MCP-1、IL-12p70、IL-12p40與IFNγ濃度皆降低，同時有較長的存活率，F10小鼠有較低血清IFNγ與IL-12p40濃度但不影響存活率。由以上結果，葉酸缺乏促進巨噬細胞促發炎細胞激素分泌，但降低誘導CD4+ T細胞IL-17與IL-10分泌量。 相較正常葉酸培養的F-BMDC，葉酸缺乏的FD-BMDC表現較不成熟的表現型。FD-BMDC有較高的細胞吞噬能力，顯著降低的MHC class II與協同刺激分子CD40、CD80、CD86表現量，並分泌顯著較少量的IL-12p70、IL-12p40與促發炎細胞激素。FD-BMDC與FD-CD4共培養下顯著抑制T細胞分泌IFNγ、IL-2、IL-13與IL-10，F-BMDC則可恢復FD-CD4的IFNγ與IL-2分泌能力，但不會回復IL-13與IL-10分泌量，顯示正常DC功能對Th1分化的重要性。葉酸缺乏13週小鼠spDC有較低的MHC class II與CD80表現量，與CD4+ T細胞共培養會降低IFNγ、IL-2與IL-13分泌量，抗原呈獻能力較差。此外，餵食小鼠F0、F1、F10飼料5、9、13週，探討葉酸缺乏或補充時間對T細胞反應的影響：F0促進脾臟細胞分泌IL-2但抑制IFNγ、IL-4、IL-5、IL-13與IL-10，以9週抑制結果最顯著，同時F0小鼠脾臟CD4+ naïve T比率上升並降低Foxp3+ Treg比率；F10則降低9和13週脾臟細胞IFNγ分泌量。顯示葉酸缺乏會影響DC活性，抑制naïve CD4+ T細胞分化成Th1、Th2與Treg。 接著欲探討葉酸缺乏影響T細胞活化的機制，利用EL4小鼠T淋巴細胞株發現葉酸缺乏會促進Nfat5表現，並同時降低Il2啟動子上Nfat結合處的甲基化程度，因而促進IL-2分泌量，顯示葉酸缺乏可透過DNA甲基化影響基因表現。因此，進一步分析飲食葉酸對小鼠脾臟與CD4+ T細胞的DNA甲基轉移酵素 (DNA methyl-transferase, Dnmt) 家族表現的影響，結果F0小鼠Dnmt1、Dnmt3a與Dnmt3b表現量均增加。以methylated DNA immunoprecipitation (MeDIP) microarray分析並挑選DNA甲基化程度受影響的候選基因，發現餵食9週F0小鼠CD4+ T細胞的naïve T細胞相關基因Cd247表現量上升，activated T細胞相關基因Icos與Lag3表現量下降，F10小鼠則顯著抑制Socs家族基因表現。進一步以bisulfite轉換及EPiTYPER分析T細胞活化與Treg分化相關基因Icos是否受到甲基化調控，結果顯示Icos啟動子與CpG島的甲基化程度無顯著影響，表示葉酸缺乏透過其他途徑抑制Icos表現。 綜合以上結果，葉酸缺乏降低APC成熟與協同刺激分子表現，抑制naïve CD4+ T細胞分化成Th1、Th2、Th17與Treg，表示葉酸維持CD4+ T細胞反應與平衡扮演重要調節角色。

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.