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Th1和Th2亚型。前者被证明对细胞免疫有贡献，后者对体液免疫有贡献。根据这一理论，Th1和Th2细胞平衡受损被认为是导致免疫疾病的原因，包括过敏性疾病。Th1和Th2细胞通过与成熟的树突状细胞(DC)接触而极化，初始T细胞是驻留在免疫前线的抗原提呈细胞。只有DC才能激活初始T细胞。当DC分泌IL-12时，初始T细胞成为Th1细胞，而当它们分泌IL-4时，它们成为Th2细胞。因此，DC分泌什么样的细胞因子是非常重要的。先前的研究表明，码血产生的DC比成人外周血分泌的IL-12量更少。作者认为，这与婴儿的免疫状态有关，因为婴儿的细胞免疫能力较弱，容易发生变态反应性疾病。目的…更多：我们假设过敏组和脐带血DC分泌IL-12的减少可能是由于IL-12基因启动子区的甲基化，而过敏状态可能归因于该区域去甲基化的延迟。为了阐明这一点，我们检测了单核细胞IL-12基因启动子区的甲基化状态，单核细胞是DC的前体，分别从健康成人(HA)和过敏性成人(AA)的外周血和正常婴儿(NI)的脐带血中提取。提取基因组DNA。用硫酸氢法测定IL-12p35基因启动子区域的甲基化状态。结果：在密码子起始前的-388、-385、-375、-352、-331、-326、-322位，3组患者外周血单核细胞中IL-12p35的甲基化比例有显著差异。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：IL-12p35基因启动子区低甲基化可能与过敏患儿和正常婴儿IL-12p35基因表达抑制有关。较少

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