The use of genetically humanized IL-10 mice to determine the molecular basis of allele-specific gene expression and disease susceptibility

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，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.在IllO-/-小鼠中的杜氏感染。这是因为在hIL-10 BAC小鼠中仅诱导了一小群hIL-10+ Th 1细胞（其介导该表型）。我们假设低T细胞IL-10由其启动子等位基因（ATA）编码。为了测试这一点，我们产生了新的小鼠携带一个共同的变体启动子等位基因（GCC），以前与高IL-10。我们证实这些等位基因分别编码低/高IL-10，并表明hIL-10等位基因改变结果为L。Donovani感染我们现在建议将这些发现扩展到其他具有公共卫生重要性的炎症性疾病（脓毒症和流感），并重点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，并作为确定hIL-10的分子基础的一种手段。在人类疾病结果中的作用。在目标1中，我们将定义hIL-10小鼠不同细胞亚群中等位基因特异性hIL-10的表达，确认人类细胞中的发现，并确定hIL-10 SNP如何影响疾病。在目标2中，我们将确定控制细胞特异性hIL-10表达的分子机制。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. We hypothesized that low T cell IL-10 was encoded by its promoter allele (ATA). To test this we generated new mice bearing a common variant promoter allele (GCC), previously associated with high IL-10. We confirmed these alleles encode for low/high IL-10 respectively and show that hIL10 alleles change the outcome to L. donovani infection. We now propose to extend these findings to other inflammatory disease of public health importance (sepsis and influenza) and to focus on the mechanisms which govern cell type- and allele-specific hIL-10 expression patterns and as a means to determine the molecular basis for hIL-10's role in human disease outcomes. In Aim 1, we will define allele-specific hIL-10 expression in different cell subsets of hIL10 mice, confirm findings in human cells, and determine how hIL10 SNPs effect disease. In Aim 2 we will identify the molecular mechanisms which control cell-specific hIL-10 expression. Together, these studies will clarify how cell- and allele-specific regulation of hIL-10 production contributes to human inflammatory diseases.