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

描述（由申请人提供）：未能适当控制炎症反应是人类疾病的常见特征。IL-10在限制炎症中起着核心作用，并且IL-10水平与人类的炎性疾病密切相关。据报道，IL-10产生水平受IL-10启动子中的单核苷酸多态性（SNP）的影响，并且这些SNP也与疾病易感性相关。这表明IL-10产生调节的个体间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IL-10（hIL-10）产生的机制仍不清楚。出于这个原因，我们在第一个资助期建立了一个原理验证系统，以在没有外来遗传和环境影响的情况下对hIL-10表达的调控进行转基因建模。我们使用了IL-10基因侧翼的一大段人基因组DNA，以确保赋予适当的hIL-10表达所需的调控信息是自包含的（hIL-10 BAC）。此外，由于hIL-10在小鼠中具有功能性，我们可以使用该模型来建立hIL-10调节与体内疾病结果之间的联系。我们已经仔细验证了hIL-10的表达在几个良好的特征IL-10依赖性疾病模型，以及在原代人类细胞。我们发现hIL-10 BAC拯救IL-10-/-小鼠免于LPS毒性和结肠炎，这分别与来自巨噬细胞和CD 4 + FoxP 3 + T细胞的hIL-10产生有关。有趣的是，hIL 10 BAC并不能恢复对持续性L. IllO-/-小鼠中的Donovani感染。这是因为在hIL-10 BAC小鼠中仅诱导了一小部分hIL-10+ Th 1细胞（介导该表型）。我们的研究结果表明，hIL-10是在不同的细胞类型特异性的监管约束相比，小鼠IL-10。此外，由于hIL 10 BAC含有与低hIL-10产生相关的IL 10启动子等位基因，这些数据表明IL 10 SNP赋予细胞特异性hIL-10表达模式，其导致改变的疾病结果。我们现在建议将这些发现扩展到关注控制细胞类型和等位基因特异性hIL-10表达模式的机制，以确定hIL-10在人类疾病结局中作用的分子基础。在目标1中，我们将定义调控边界和染色质结构如何影响hIL-10在不同类型的细胞中的表达，这些细胞类型与疾病的发病机制有关，并确定控制细胞特异性hIL-10表达的分子机制。为此，我们已经产生了在IL 10基因座内携带不同基因组缺失的新的hIL 10 BAC系。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hIL-10 BAC转基因来确定SNP在hIL-10产生和疾病结果中的作用。将在原代人细胞中验证hIL-10 SNP的影响，并鉴定等位基因特异性hIL-10表达的机制。总之，这些研究将阐明hIL-10产生的细胞特异性和等位基因特异性调节如何有助于人类炎症性疾病。

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.