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）：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-/-小鼠对持续多诺瓦氏杆菌感染的易感性。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）。我们假设低T细胞IL-10是由其启动子等位基因（ATA）编码的。为了验证这一点，我们产生了带有共同变异启动子等位基因（GCC）的新小鼠，该等位基因先前与高IL-10相关。我们证实了这些等位基因分别编码低/高IL-10，并表明hIL10等位基因改变了L. donovani感染的结果。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），并将重点放在控制细胞类型和等位基因特异性il -10表达模式的机制上，并作为确定il -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hIL10小鼠不同细胞亚群中等位基因特异性的hIL10表达，确认人类细胞中的发现，并确定hIL10 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. 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.