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

描述（由申请人提供）：不能正确控制炎症反应是人类疾病的共同特征。IL-10在限制炎症中起着核心作用，IL-10水平与人类炎症性疾病密切相关。据报道，IL-10的产生水平受IL-10启动子中的单核苷酸多态性（snp）的影响，这些snp也与疾病易感性相关。这表明个体间IL-10产生调节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）产生的机制仍不清楚。因此，我们在第一个资助期建立了一个原理验证系统，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IL10基因侧面的一大块人类基因组DNA，以确保授予适当的il -10表达所需的调控信息是自包含的（hIL10BAC）。此外，由于hIL-10在小鼠体内具有功能，我们可以利用该模型在体内建立hIL-10调控与疾病结局之间的联系。我们仔细验证了il -10在几种具有良好特征的il -10依赖性疾病模型以及人原代细胞中的表达。我们发现，hIL10BAC可以分别从巨噬细胞和CD4+FoxP3+ Tregs产生的hIL-10毒性和结肠炎中拯救Il10-/-小鼠。有趣的是，hIL10BAC并没有恢复Il10-/-小鼠对持续L. Donovani感染的易感性。这是因为；在hIL10BAC小鼠中，只有一小部分hIL-10+Th1细胞（介导这种表型）被诱导。我们的研究结果表明，与小鼠IL-10相比，IL-10受到不同细胞类型特异性调控约束。此外，由于hIL10BAC含有与低il -10产生相关的il -10启动子等位基因，这些数据表明，il -10 snp传递细胞特异性il -10表达模式，从而导致疾病结果的改变。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，作为确定hIL-10在人类疾病结果中作用的分子基础的手段。在Aim 1中，我们将定义调控边界和染色质结构如何影响不同细胞类型中与疾病发病机制有关的hIL-10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位点内携带不同的基因组缺失。在Aim 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hIL10BAC转基因来确定snp在IL-10产生和疾病结局中的作用。hIL10 snp的影响将在原代人细胞中得到验证，并确定等位基因特异性hIL-10表达的机制。总之，这些研究将阐明细胞和等位基因特异性调节hIL-10的产生如何促进人类炎症性疾病。

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.