Regulatory T cell mediated control of colitis by Dkk1

Dkk1 调节性 T 细胞介导的结肠炎控制

• 批准号: 8583635
• 负责人: Alfred LM Bothwell
• 金额: $ 23.47万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-13 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8583635
• 关键词: APC gene; Address; Adenomatous Polyposis Coli; Affect; Autoimmune Diseases; Autoimmunity; Biochemical; CD4 Positive T Lymphocytes; Cell Differentiation process; Cell Proliferation; Cells; Colitis; Colon; Complex; Crohn' s disease; Data; Disease model; Event; Failure; Homeostasis; Immune; Immune system; In Vitro; Inflammation; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Interferons; Interleukin-10; Intestines; Lead; Ligands; Lymphoid Cell; Mammalian Cell; Measures; Mediating; Mesentery; Modeling; Mus; Mutant Strains Mice; Mutation; Nuclear; Pathway interactions; Population; Property; Recombinants; Regulatory T-Lymphocyte; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Source; Spleen; T cell differentiation; T-Cell Activation; T-Cell Proliferation; T-Lymphocyte; Testing; Th1 Cells; adaptive immunity; cell type; clinical application; in vivo; inhibitor/antagonist; intestinal homeostasis; lymph nodes; prevent; programs; public health relevance; receptor; receptor expression

DESCRIPTION (provided by applicant): Accumulation of pathogenic helper CD4 T cells and failure of regulatory T cell (Treg)-mediated suppression are now considered as central to understanding the role of adaptive immunity in autoimmune diseases, including inflammatory bowel disease (IBD). Multiple mechanisms by which Tregs control intestinal inflammation have been suggested but additional mechanisms may exist particularly to maintain homeostasis in the intestinal microenvironment. Our analysis of Wnt ligand expression in distinct lymphoid cell populations revealed that Tregs express the Wnt antagonist Dickkopf1 (Dkk1) significantly higher than other major Wnt ligands, suggesting a potentially important role of Tregs secreting this potent Wnt antagonist. To pursue mechanistic studies we generated a recombinant form of Dkk1 produced in mammalian cells. The findings were notable in that the rDkk1 demonstrated the marked inhibitory effect on naive CD4 T proliferation and stimulation of Treg proliferation. Our preliminary analysis with Tregs from doubleridge mice (Dkk-1 d/d) that express very low level of Dkk-1 showed that indeed these Tregs failed to protect host from colitis by failure to suppress CD4 T cell proliferation. This study utilizing the standard T-cell mediated Inflammatory Bowel Disease (IBD) model allowed us to uncover important properties of Treg that warrant an extensive mechanistic characterization of their function. More importantly, further analysis of CD4 T cell differentiation results showed that rDkk1 inhibited Th1 cell differentiation and markedly elevates IL-10. All of these findings have lead to the hypothesis that Treg-derived Dkk1 is a potent regulator to prevent intestinal inflammation that is critical in IBD. To test thi hypothesis in Aim 1, the mechanism by which Dkk1 inhibits cell proliferation of naive CD4 T cells will be determined in vitro. Naive CD4 T cells lacking the canonical receptor will be utilize to determine whether canonical signaling is required. These cells will also be tested for function in vivo. Finally, we investigate the mechanism to inhibit Th1 polarization and the elevation of IL-10 by Dkk-1. In Aim 2, we will generate Dkk1 deficient Treg utilizing Dkk1 floxed mice. These Dkk1 deficient Treg will be evaluated functionally in the in vivo IBD model. In vitro, the stimulatory effect of Dkk-1 on Tregs will be studied regarding signal transduction pathways that may lead to Treg proliferation. Treg will also be generated that lack the Wnt canonical receptor Lrp5 for assessing the effects of Dkk1 on signaling pathways activating proliferation. We expect these studies to reveal fundamentally important but currently unrecognized properties of Tregs that are essential to understanding autoimmune disease in the intestine but are also generally relevant to autoimmunity. This will likely lead to increased efforts to target Wnt signaling for clinical applications, particularly in Crohn's disease and other autoimmune diseases.

描述（由申请人提供）：致病性辅助 CD4 T 细胞的积累和调节性 T 细胞 (Treg) 介导的抑制的失败现在被认为是了解适应性免疫在自身免疫性疾病（包括炎症性肠病 (IBD)）中的作用的核心。人们已经提出了 Tregs 控制肠道炎症的多种机制，但可能还存在其他机制，特别是维持肠道微环境的稳态。我们对不同淋巴细胞群中 Wnt 配体表达的分析表明，Treg 表达 Wnt 拮抗剂 Dickkopf1 (Dkk1) 的量显着高于其他主要 Wnt 配体，表明分泌这种有效 Wnt 拮抗剂的 Tregs 具有潜在的重要作用。为了进行机制研究，我们在哺乳动物细胞中生成了 Dkk1 的重组形式。值得注意的是，rDkk1 对幼稚 CD4 T 增殖和刺激 Treg 增殖具有显着的抑制作用。我们对表达极低水平 Dkk-1 的双脊小鼠 (Dkk-1 d/d) 的 Tregs 进行的初步分析表明，这些 Tregs 确实无法抑制 CD4 T 细胞增殖，从而无法保护宿主免受结肠炎的影响。这项利用标准 T 细胞介导的炎症性肠病 (IBD) 模型的研究使我们能够揭示 Treg 的重要特性，从而保证对其功能进行广泛的机制表征。更重要的是，进一步分析CD4 T细胞分化结果表明，rDkk1抑制Th1细胞分化并显着升高IL-10。所有这些发现都得出这样的假设：Treg 衍生的 Dkk1 是预防肠道炎症的有效调节剂，而肠道炎症对于 IBD 至关重要。 为了检验目标 1 中的假设，将在体外确定 Dkk1 抑制初始 CD4 T 细胞增殖的机制。缺乏典型受体的初始 CD4 T 细胞将被用来确定是否需要典型信号传导。这些细胞还将进行体内功能测试。最后，我们研究了Dkk-1抑制Th1极化和升高IL-10的机制。在目标 2 中，我们将利用 Dkk1 floxed 小鼠生成 Dkk1 缺陷 Treg。这些 Dkk1 缺陷的 Treg 将在体内 IBD 模型中进行功能评估。在体外，将研究Dkk-1对Tregs的刺激作用，涉及可能导致Treg增殖的信号转导途径。还将生成缺乏 Wnt 经典受体 Lrp5 的 Treg，用于评估 Dkk1 对激活增殖的信号通路的影响。我们期望这些研究能够揭示 Treg 的基本重要但目前未被认识的特性，这些特性对于了解肠道自身免疫性疾病至关重要，但通常也与自身免疫相关。这可能会导致人们加大力度针对 Wnt 信号转导进行临床应用，特别是在克罗恩病和其他自身免疫性疾病方面。