Regulatory T cell mediated control of colitis by Dkk1

描述（由申请人提供）：致病性辅助性CD4 T细胞的积累和调节性T细胞（Treg）介导抑制的失败现在被认为是理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。Tregs控制肠道炎症的多种机制已被提出，但可能存在其他机制，特别是维持肠道微环境的内稳态。我们对不同淋巴细胞群中Wnt配体表达的分析显示，Tregs表达Wnt拮抗剂Dickkopf1 （Dkk1）显著高于其他主要Wnt配体，这表明Tregs分泌这种有效的Wnt拮抗剂可能具有重要作用。为了进行机制研究，我们在哺乳动物细胞中产生了一种重组形式的Dkk1。值得注意的是，rDkk1对初始CD4 T增殖和刺激Treg增殖具有明显的抑制作用。我们对双桥小鼠（Dkk-1 d/d）中表达非常低水平Dkk-1的Tregs进行初步分析，结果表明这些Tregs确实无法通过抑制CD4 T细胞增殖来保护宿主免受结肠炎的侵袭。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细胞分化结果显示，rDkk1抑制Th1细胞分化，显著升高IL-10。所有这些发现都导致假设treg衍生的Dkk1是一种有效的调节剂，可以预防肠道炎症，这是IBD的关键。为了在Aim 1中验证这一假设，我们将在体外确定Dkk1抑制初始CD4 T细胞增殖的机制。缺乏典型受体的幼稚CD4 T细胞将被用来确定是否需要典型信号。这些细胞还将在体内进行功能测试。最后，我们研究了Dkk-1抑制Th1极化和IL-10升高的机制。在Aim 2中，我们将利用Dkk1粘接小鼠产生Dkk1缺陷Treg。这些Dkk1缺陷Treg将在体内IBD模型中进行功能评估。在体外，我们将研究Dkk-1对Treg的刺激作用，包括可能导致Treg增殖的信号转导途径。也会产生缺乏Wnt典型受体Lrp5的Treg，用于评估Dkk1对激活增殖的信号通路的影响。我们希望这些研究能够揭示Tregs的重要特性，这些特性对于理解肠道自身免疫性疾病至关重要，但也与自身免疫有关。这可能会增加临床应用Wnt信号的努力，特别是在克罗恩病和其他自身免疫性疾病中。

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.