An Approach to Investigate the Pathogenesis of Allergy in Children Using Epigenetic Status Analysis

背景：辅助性T细胞可分为Th 1和Th 2两种亚型。前一种细胞已被证明有助于细胞免疫，后者有助于体液免疫。基于这一理论，Th 1和Th 2细胞平衡受损被认为会导致免疫性疾病，包括过敏性疾病。Th 1和Th 2细胞通过与成熟的树突状细胞（DC）接触而从幼稚T细胞极化，树突状细胞是位于免疫前线的抗原递呈细胞。只有DC可以激活初始T细胞。当DC分泌IL-12时，初始T细胞成为Th 1细胞，而当它们分泌IL-4时，它们成为Th 2细胞。因此，DCs分泌何种细胞因子是非常重要的，以前已经证明，与来自成人外周血的DCs相比，来自代码血的DCs分泌较少量的IL-12。作者认为这与婴幼儿的免疫状态有关，婴幼儿细胞免疫力较弱，易发生过敏性疾病。 关于我们 E：我们推测，过敏组和脐血DC分泌IL-12的减少可能是由于IL-12基因启动子区甲基化所致，而过敏状态可能归因于该区域去甲基化延迟。为了阐明这一点，我们检测了正常成人（HA）、过敏性成人（AA）和正常婴儿（NI）外周血单核细胞（DC前体细胞）IL-12基因启动子区甲基化状态。提取基因组DNA。采用硫酸氢盐测序法检测IL-12 p35基因启动子区甲基化状态。结果：HA组和AA组起始密码子前-388、-385、-375、-352、-331、-326、-322位点的7个CpG甲基化率有显著性差异（P <0.05）;与我们的预期相反，AA和NI组的甲基化比率显著低于HA组。结论：过敏患儿和正常婴儿IL-12 p35基因启动子区甲基化水平降低可能与IL-12 p35基因表达抑制有关。少

Back ground: Helper T cells can be classified into Th1 and Th2 subtype. The former cells have been shown to contribute to cellular immunity, the latter to humoral immunity. Based on the theory, impairment of balancing Th1 and Th2 cells is thought to cause immune diseases, including allergic diseases. Th1 and Th2 cells are polarized from naive T cells by getting in contact with mature dendritic cells (DCs), which are antigen presenting cells resident in front line of immunity. Only DCs can activate naive T cells. When DCs secrete IL-12, naive T cells become Th1 cells, while when they secrete IL-4, they become Th2 cells. Therefore, it is very important what kind of cytokines DCs secrete.It has been previously demonstrated that DCs generated from code blood secrete less amounts of IL-12 compared to those from adult peripheral blood. The authors suggested that this is correlated with the immune status of infants, who have weak cellular immunity and develop allergic diseases easily.Objectiv … More e : We hypothesized that less secreted IL-12 by DCs from both allergy group and cord blood would be due to methylation of the promoter region of IL-12 gene, and that allergic status might be attributed to delay of demethylation of the region. To elucidate it, we examined the methylation status in the promoter region of IL-12 gene of monocytes, which are precursors of DCs, prepared from peripheral blood from healthy adults (HA) and allergic adults (AA) and from cord blood from normal infants (NI) group.Methods : Monocytes were purified by using MACS system with an antibody for CD 14 from each of three groups. Genomic DNAs were extracted. Methylation status in the promoter region of IL-12p35 gene was investigated with bisulphate-sequencing analysis method. Real time RT-PCR reactions were performed to determine IL-12p35 mRNA level in cultured and LPS stimulated monocytes of peripheral blood from HA and AA group.Results : Of 7 CpGs at -388, -385, -375,-352,-331, -326, -322 position prior to start codon, there were significant differences in methylated ratio among three groups. Contrary to our expectations, methylated ratios were significantly lower in AA and NI groups than in HA group. There was no difference in IL-12p35 mRNA level between HA and AA groups.Conclusion : These results suggest that hypo-methylation in the promoter region might be associated with suppression of gene expression of IL-12p35 in allergic patients and normal infants. Less