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。前者细胞已被证明有助于细胞免疫，后者有助于体液免疫。根据该理论，破坏Th1和Th2细胞的平衡被认为会导致免疫疾病，包括过敏性疾病。Th1和Th2细胞通过与成熟树突状细胞（dc）接触而从幼稚T细胞极化，成熟树突状细胞是驻扎在免疫前线的抗原呈递细胞。只有dc可以激活幼稚T细胞。当dc分泌IL-12时，幼稚T细胞变成Th1细胞，而当dc分泌IL-4时，幼稚T细胞变成Th2细胞。因此，DCs分泌何种细胞因子是非常重要的。先前已经证明，与来自成人外周血的dc相比，来自代码血的dc分泌的IL-12较少。作者认为，这与婴儿的免疫状态有关，婴儿的细胞免疫力较弱，容易发生过敏性疾病。目的：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基因启动子区域的甲基化，而过敏状态可能是由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基因启动子区域的甲基化状态，单核细胞是dc的前体，分别来自健康成人（HA）和过敏成人（AA）的外周血以及正常婴儿（NI）组的脐带血。方法：用MACS系统纯化三组单核细胞，分别用cd14抗体纯化。提取基因组dna。采用二硫酸盐测序法研究IL-12p35基因启动子区甲基化状态。采用Real time RT-PCR检测HA组和AA组培养和LPS刺激的外周血单核细胞IL-12p35 mRNA水平。结果：在起始密码子前-388、-385、-375、-352、-331、-326、-322位的7个CpGs中，三组间甲基化率存在显著差异。与我们的预期相反，AA组和NI组的甲基化比率明显低于HA组。HA组与AA组IL-12p35 mRNA表达水平无显著差异。结论：提示IL-12p35启动子区域的低甲基化可能与过敏患者和正常婴儿IL-12p35基因表达的抑制有关。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