Human IL-13 Gene Regulation and Impact of Polymorphisms

摘要 哮喘和变态反应的发病机制以Th2细胞因子IL 4（其 是Th2分化和IgE合成的关键）和IL 13（其是过敏性细胞因子的中枢效应物）。 炎症）。我们试图剖析人类Th2细胞因子基因的调节，并通过以下方法确定其机制： 所述天然多态性影响它们的表达和/或功能，从而影响变态反应敏感性。 在上一个资助周期中，我们研究了调节IL 13的基于染色质的机制。 在人CD4 T细胞中表达。我们鉴定了新的元件（HS4，HS5，HS11/12），其标记为 顺式调节功能的三个指标：DNA酶I超敏反应、DNA低甲基化和 序列保守性与此同时，我们发现IL 13单核苷酸多态性（SNP）与IL 13的表达相关。 与过敏/哮喘易感性增加，并位于HS4和HS5，增加IL 13的转录， 体外为了推进这一领域，我们随后开发了一种强大的体内模型来探索人Th2细胞因子 调节及其通过遗传变异的调节。该模型依赖于携带160 kb BAC转基因的小鼠 （BAC 5），包括人RAD 50、IL 13和IL 4。人IL 13和IL 4在BAC 5 TG中被忠实地调节 表明BAC 5含有控制人Th2细胞因子的所有顺式作用元件。与 在一项单独拨款（R21 A1076715，PI：韦尔切利）的支持下，我们正在研究自然变异对 使用携带野生型或过敏相关人IL 13等位基因的BAC 5 TG小鼠进行人IL 13表达。 出乎意料的是，在HS4、HS5和HS11/12中具有SNP的小鼠不仅表现出显著的，尽管是适度的， 人IL 13的显著增加，而且人IL 4的显著（3倍）增加。值得注意的是， 携带相同SNP的婴儿免疫研究显示出相当的Th2细胞因子模式。这些数据 表明HS4、HS5和/或HS11/12含有主要参与人IL 4控制新的顺式元件。 这个竞争性的更新应用程序建立在我们的染色质分析和我们新的TG模型的基础上， 以在体内表征这些新的IL 13/IL 4顺式调节区。我们将产生携带BAC5 TG的小鼠， Cre介导的HS4、HS5或HS11/12缺失，我们将评估这些缺失对人类细胞的影响。 Th2细胞因子表达，Th2基因座染色质结构和长距离染色体内相互作用。我们 将重点放在IL4以及IL13，因为对IL4的显著影响是可能的，并且将具有重大意义。 过敏性疾病的发病机制的影响。我们将检测不同分化程度的CD4 Th细胞， 阶段和肥大细胞，尽管不太广泛。具体目标是界定HS4的作用（目标1）， HS5（Aim 2），其位于IL 13启动子的远端，并且是在幼稚人类中检测到的唯一IL 13 HS位点。 CD4 T细胞;并表征HS11/12，其位于IL 13/IL 4基因间区域，并且是最强的 IL13 HS位点（目标3）。我们的研究将是第一个探索体内人类Th 2细胞因子调节的研究，并将 确定有效治疗过敏性疾病的新型合理靶点。

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.