The use of genetically humanized IL-10 mice to determine the molecular basis of a

使用基因人源化 IL-10 小鼠来确定 IL-10 的分子基础

• 批准号: 8776126
• 负责人: Jay H. Bream
• 金额: $ 40.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8776126
• 关键词: Accounting; Acute; Address; Alleles; Anatomy; Anti-Inflammatory Agents; Anti-inflammatory; Autoimmune Diseases; Bacterial Artificial Chromosomes; Binding; Cells; Chromatin; Colitis; Communicable Diseases; Data; Disease; Disease Outcome; Disease model; Disease susceptibility; Elements; Epigenetic Process; Failure; Functional RNA; Funding; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Genomic DNA; Genomics; Haplotypes; Heterogeneity; Human; IL10 gene; Immune System Diseases; Immune response; In Vitro; Inbred Mouse; Individual Differences; Infection; Inflammation; Inflammatory; Inflammatory Response; Influenza; Interleukin-10; Knowledge; Lead; Leishmania; Link; Location; Mediating; Modeling; Modification; Molecular; Mus; Outcome; Pattern; Phenotype; Play; Population; Predisposition; Production; Public Health; Publications; Regulation; Relative (related person); Reporting; Research; Resistance; Role; Sepsis; Signal Transduction; Single Nucleotide Polymorphism; Source; System; T-Lymphocyte; Testing; Th1 Cells; Tissues; Toxic effect; Transgenic Mice; Variant; base; cell type; cytokine; design; disorder risk; human disease; in vivo; insight; macrophage; mouse model; novel therapeutics; programs; promoter; public health relevance; theories; tool; transcription factor

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.

描述（由申请人提供）：未能正确控制炎症反应是人类疾病的一个常见特征。 IL-10 在限制炎症方面发挥着核心作用，IL-10 水平与人类炎症性疾病密切相关。据报道，IL-10 的产生水平受到 IL10 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 基因侧翼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是独立的 (hIL10BAC)。此外，由于 hIL-10 在小鼠体内具有功能，因此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。我们在几种已充分表征的 IL-10 依赖性疾病模型以及原代人类细胞中仔细验证了 hIL-10 表达。我们发现 hIL10BAC 可将 Il10-/- 小鼠从 LPS 毒性和结肠炎中拯救出来，这分别与巨噬细胞和 CD4+FoxP3+ Tregs 产生 hIL-10 有关。有趣的是，hIL10BAC 并没有恢复 Il10-/- 小鼠对持续性杜氏乳杆菌感染的易感性。这是因为在 hIL10BAC 小鼠中仅诱导了一小部分 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。为了测试这一点，我们培育了带有常见变异启动子等位基因 (GCC) 的新小鼠，该基因先前与高 IL-10 相关。我们证实这些等位基因分别编码低/高 IL-10，并表明 hIL10 等位基因改变杜氏乳杆菌感染的结果。我们现在建议将这些发现扩展到具有公共卫生重要性的其他炎症性疾病（败血症和流感），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 的分子基础的手段。 hIL-10 在人类疾病结果中的作用。在目标 1 中，我们将定义 hIL10 小鼠不同细胞亚群中的等位基因特异性 hIL-10 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。这些研究将共同​​阐明 hIL-10 产生的细胞和等位基因特异性调节如何导致人类炎症性疾病。