A Comparative Genomics and Transgenic Approach to Regulation of IL-10 Expression.

描述(由申请人提供)：未能正确控制炎症反应是人类疾病的常见特征。IL-10在限制炎症方面发挥着核心作用，而IL-10水平与人类的炎症性疾病密切相关。IL-10基因启动子上的单核苷酸多态(SNPs)可影响IL-10的产生水平，这些SNPs也与疾病易感性有关。这表明，个体间在调节IL-10产生方面的差异可能是决定疾病风险的关键因素。然而，由于缺乏适当的研究工具，控制人类IL-10(hIL-10)产生的机制仍然不清楚。为此，我们在第一个资助期建立了一个原则验证系统，在没有外来遗传和环境影响的情况下，对hIL-10的表达调控进行转基因建模。我们使用了IL10基因两侧的一大段人类基因组DNA，以确保赋予适当的hIL-10表达所需的调控信息将是独立的(HIL10BAC)。此外，由于hIL-10在小鼠体内具有功能，我们可以使用该模型来建立hIL-10调节与体内疾病结局之间的联系。我们已经仔细地验证了hIL-10在几个特征良好的IL-10依赖疾病模型中的表达以及在原代人类细胞中的表达。我们发现，hIL10BAC可使IL10-/-小鼠免于脂多糖毒性和结肠炎，这分别与巨噬细胞和CD4+FoxP3+Tregs产生hIL-10有关。有趣的是，hIL10BAC不能恢复IL10-/-小鼠对持续感染杜氏乳杆菌的易感性。这是因为，在hIL10BAC小鼠中，只诱导了一小部分hIL-10+Th1细胞(介导这种表型)。我们的发现表明，与小鼠IL-10相比，hIL-10处于不同的细胞类型特异性调控约束下。此外，由于hIL10BAC包含与低hIL-10产生相关的IL10启动子等位基因，这些数据表明IL10 SNP传递细胞特异性的hIL-10表达模式，从而导致疾病结局的改变。我们现在建议扩展这些发现，将重点放在控制细胞类型和等位基因特异性hIL-10表达模式的机制上，作为确定hIL-10‘S在人类疾病预后中作用的分子基础的一种手段。在目标1中，我们将定义调控边界和染色质结构如何影响与疾病发病相关的不同类型细胞中hIL-10的表达，并确定控制细胞特异性hIL-10表达的分子机制。为此，我们产生了新的hIL10BAC系，在IL10基因座上携带不同的基因组缺失。在目标2中，我们将使用携带与高IL-10产生相关的IL 10启动子等位基因的新的hIL10BAC转基因来确定SNPs在hIL-10产生和疾病结局中的作用。将在原代人类细胞中验证hIL-10 SNPs的影响，并确定等位基因特异性hIL-10表达的机制。总之，这些研究将阐明hIL-10产生的细胞和等位基因特异性调节如何与人类炎症性疾病有关。

DESCRIPTION (provided by applicant): The failure to properly control inflammatory responses is a common feature in human disease. IL-10 plays a central role in limiting inflammation and IL-10 levels are strongly linked to inflammatory disorders in humans. The levels of IL-10 production are reported to be influenced by single nucleotide polymorphisms (SNPs) in the IL10 promoter and these SNPs are also associated with disease susceptibility. This indicates that inter- individual differences in the regulation of IL-10 production are likely key factor which determines disease risk. However, the mechanisms that control human IL-10 (hIL-10) production remain unclear due to a lack of appropriate research tools. For that reason, we established a proof-of-principle system in the first funding period to transgenically model the regulation of hIL-10 expression in the absence of extraneous genetic and environmental influence. We used a large segment of human genomic DNA flanking the IL10 gene to assure that the regulatory information required to confer appropriate hIL-10 expression would be self-contained (hIL10BAC). In addition, because hIL-10 is functional in mice, we can use this model to establish the connection between hIL-10 regulation and disease outcomes in vivo. We have carefully validated hIL-10 expression in several well-characterized IL-10-dependent disease models as well as in primary human cells. We found that the hIL10BAC rescues Il10-/- mice from LPS toxicity and colitis which was associated with hIL-10 production from macrophages and CD4+FoxP3+ Tregs respectively. Interestingly, the hIL10BAC did not restore susceptibility to persistent L. Donovani infection in Il10-/- mice. This was because; only a small population of hIL-10+Th1 cells (which mediates this phenotype) were induced in hIL10BAC mice. Our findings suggest that hIL-10 is under different cell type-specific regulatory constraints compared to mouse IL-10. Furthermore, because the hIL10BAC contains an IL10 promoter allele associated with low hIL-10 production, these data suggest that IL10 SNPs impart cell-specific hIL-10 expression patterns which result in altered disease outcomes. We now propose to extend these findings to focus on the mechanims which govern cell type- and allele-specific hIL-10 expression patterns as a means to determine the molecular basis for hIL-10's role in human disease outcomes. In Aim 1, we will define how regulatory boundaries and chromatin structure impact hIL-10 expression in different cell types which are implicated in disease pathogenesis and identify the molecular mechanims which control cell-specific hIL-10 expression. For this we have generated new hIL10BAC lines carrying different genomic deletions within the IL10 locus. In Aim 2, we will determine the role of SNPs in hIL-10 production and disease outcomes using new hIL10BAC transgenics carrying an IL10 promoter allele associated with high IL-10 production. The influence of hIL10 SNPs will be validated in primary human cells and the mechanisms of allele-specific hIL-10 expression identified. Together, these studies will clarify how cell- and allele-specific regulation of hIL-10 production contributes to human inflammatory diseases.