Human IL-13 Gene Regulation and Impact of Polymorphisms

摘要 哮喘和过敏的发病机制以Th2细胞因子IL4的异常表达为标志 对Th2分化和IgE合成至关重要)和IL13(过敏症的中枢效应因子 炎症)。我们试图剖析人类Th2细胞因子基因的调节，并通过以下方式定义其机制 哪些自然多态会影响它们的表达和/或功能，从而影响过敏易感性。 在前一个资金周期中，我们研究了基于染色质的调节IL13的机制 人CD4T细胞的表达。我们确定了新的元件(HS4，HS5，HS11/12)，标记为 顺式调节功能的三个指标的趋同：DNase I超敏、DNA低甲基化和 序列守恒。同时，我们发现IL13单核苷酸多态(SNPs)与 随着对过敏/哮喘易感性的增加，并定位于HS4和HS5，IL13在 体外培养。为了推动这一领域的发展，我们随后开发了一个强大的体内模型来探索人类Th2细胞因子 调控及其通过遗传变异的调节。这个模型依赖于携带160kb BAC转基因的小鼠。 (BAC5)包括人类RAD50、IL13和IL4。人白介素13和白介素4在BAC5TG中被忠实地调控 小鼠T细胞，表明BAC5包含控制人类Th2细胞因子的所有顺式作用元件。使用 由另一笔赠款(R21A1076715，PI：Vercelli)支持，我们正在研究自然变异对 使用携带野生型或过敏相关人IL13等位基因的BAC5TG小鼠表达人IL13。 出乎意料的是，携带HS4，HS5和HS11/12 SNPs的小鼠不仅表现出显著的，尽管轻微的， 人IL-13的增加，但人IL-4的显著增加(3倍)。值得注意的是，参加 携带相同SNPs的婴儿免疫研究显示出类似的Th2细胞因子模式。这些数据 提示HS4、HS5和/或HS11/12含有主要参与人类IL4调控的新的顺式元件(S)。 这一竞争性更新应用程序基于我们的染色质分析和新的TG模型，并提出了 在体内鉴定这些新的IL13/IL4顺式调控区。我们将产生BAC5 TG小鼠，携带 Cre介导的HS4、HS5或HS11/12缺失，我们将评估这些缺失对人类的影响 Th2细胞因子的表达，Th2基因座染色质结构和染色体内的远程相互作用。我们 将把重点放在IL4和IL13上，因为IL4可能会受到重大影响，并将产生重大影响 对过敏性疾病发病机制的启示。我们将在不同的分化阶段检测CD4Th细胞 阶段和肥大细胞，尽管分布范围较小。具体目标是定义HS4的作用(目标1)和 HS5(Aim 2)，它位于IL13启动子的末端，是在新人中检测到的唯一IL13 HS位点 CD4T细胞；HS11/12，它位于IL13/IL4基因间隔区，是最强烈的 IL13HS站点(目标3)。我们的研究将首次探索人类Th2细胞因子在体内的调节，并将 为过敏性疾病的有效治疗确定新的合理靶点。

ABSTRACT The pathogenesis of asthma and allergy is marked by dysregulated expression of the Th2 cytokines IL4 (which is critical for Th2 differentiation and IgE synthesis) and IL13 (which is the central effector of allergic inflammation). We seek to dissect the regulation of human Th2 cytokine genes and define the mechanisms by which natural polymorphisms affect their expression and/or function, thereby influencing allergy susceptibility. During the previous funding cycle, we investigated the chromatin-based mechanisms that regulate IL13 expression in human CD4 T cells. We identified novel elements (HS4, HS5, HS11/12) marked by the convergence of three indicators of cis-regulatory function: DNase I hypersensitivity, DNA hypomethylation and sequence conservation. In parallel, we showed that IL13 single nucleotide polymorphisms (SNPs) associated with increased susceptibility to allergy/asthma, and located in HS4 and HS5, increase IL13 transcription in vitro. To advance the field, we then developed a powerful in vivo model to explore both human Th2 cytokine regulation and its modulation by genetic variants. This model relies on mice carrying a 160 kb BAC transgene (BAC5) encompassing human RAD50, IL13 and IL4. Human IL13 and IL4 are faithfully regulated in BAC5 TG murine T cells, suggesting BAC5 contains all the cis-acting elements that control human Th2 cytokines. With support from a separate grant (R21A1076715, PI: Vercelli), we are studying the impact of natural variants on human IL13 expression using BAC5 TG mice that carry wild-type or allergy-associated human IL13 alleles. Unexpectedly, mice with SNPs in HS4, HS5 and HS11/12 exhibited not only a significant, albeit modest, increase in human IL13, but also striking (3-fold) increases in human IL4. Notably, children enrolled in the Infant Immune Study who carry the same SNPs showed comparable Th2 cytokine patterns. These data suggest that HS4, HS5 and/or HS11/12 contain novel cis-element(s) primarily involved in human IL4 control. This competing renewal application builds on our chromatin analyses and our new TG model and proposes to characterize these novel IL13/IL4 cis-regulatory regions in vivo. We will generate BAC5 TG mice carrying Cre-mediated deletions of HS4, HS5 or HS11/12, and we will assess the impact of these deletions on human Th2 cytokine expression, Th2 locus chromatin architecture and long-range intra-chromosomal interactions. We will focus on IL4 as well as IL13 because significant effects on IL4 are likely, and would have major implications for the pathogenesis of allergic disease. We will examine CD4 Th cells at distinct differentiation stages and, albeit less extensively, mast cells. Specific objectives are to define the role of HS4 (Aim 1) and HS5 (Aim 2), which reside in the distal IL13 promoter and are the only IL13 HS sites detected in na¿ve human CD4 T cells; and to characterize HS11/12, which lies in the IL13/IL4 intergenic region and is the most intense IL13 HS site (Aim 3). Our studies will be the first to explore human Th2 cytokine regulation in vivo and will identify novel rational targets for effective treatments of allergic disease.