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

使用基因人源化 IL-10 小鼠确定等位基因特异性基因表达和疾病易感性的分子基础

• 批准号: 9278093
• 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/9278093
• 关键词: 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; FOXP3 gene; Failure; Funding; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Genomic DNA; Genomics; Haplotypes; Heterogeneity; Human; IL10 gene; Immune response; In Vitro; Inbred Mouse; Individual Differences; Infection; Inflammation; Inflammatory; Inflammatory Response; Influenza; Influenza A virus; Interleukin-10; Knowledge; Lead; Leishmania; Leishmania donovani; Link; Location; Mediating; Modeling; Modification; Molecular; Mus; Outcome; Pattern; Phenotype; Play; Population; Predisposition; Production; Public Health; Publications; Regulation; Regulatory T-Lymphocyte; Reporting; Research; Resistance; Role; Sepsis; Signal Transduction; Single Nucleotide Polymorphism; Source; System; T-Lymphocyte; Testing; Th1 Cells; Tissues; Toxic effect; Transgenic Mice; Transgenic Model; Untranslated RNA; Variant; cell type; cytokine; design; disorder risk; human disease; in vivo; insight; macrophage; mouse model; novel therapeutic intervention; 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常见特征。IL-10在限制炎症方面发挥着核心作用，而IL-10水平与人类的炎症性疾病密切相关。IL-10基因启动子上的单核苷酸多态(SNPs)可影响IL-10的产生水平，这些SNPs也与疾病易感性有关。这表明，个体间在调节IL-10产生方面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)产生的机制仍然不清楚。为此，我们在第一个资助期建立了一个原则验证系统，在没有外来遗传和环境影响的情况下，对hIL-10的表达调控进行转基因建模。我们使用了IL10基因两侧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将是独立的(HIL10BAC)。此外，由于hIL-10在小鼠体内具有功能，我们可以使用该模型来建立hIL-10调节与体内疾病结局之间的联系。我们已经仔细地验证了hIL-10在几个特征良好的IL-10依赖疾病模型中的表达以及在原代人类细胞中的表达。我们发现，hIL10BAC可使IL10-/-小鼠免于脂多糖毒性和结肠炎，这分别与巨噬细胞和CD4+FoxP3+Tregs产生hIL-10有关。有趣的是，hIL10BAC不能恢复IL10-/-小鼠对持续感染杜氏乳杆菌的易感性。这是因为在hIL10BAC小鼠中只诱导了一小部分hIL-10+Th1细胞(介导这一表型)。我们推测低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，我们产生了携带共同变异启动子等位基因(GCC)的新小鼠，该等位基因以前与高IL-10有关。我们证实了这些等位基因分别编码低和高IL-10，并表明hIL10等位基因改变了杜诺瓦尼乳杆菌感染的结局。我们现在建议将这些发现扩展到其他对公共卫生具有重要意义的炎症性疾病(败血症和流感)，重点研究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‘S在人类疾病预后中作用的分子基础的一种手段。在目标1中，我们将定义hIL10小鼠不同细胞亚群中等位基因特异性hIL-10的表达，确认在人类细胞中的发现，并确定hIL10 SNPs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产生的细胞和等位基因特异性调节如何与人类炎症性疾病有关。