Vitamin D Receptor Regulation of Microbiota in Intestinal Epithelia

 描述(申请人提供)：我们研究的长期目标是阐明参与维持微生物体内平衡的宿主因素的分子基础，并开发通过恢复宿主-微生物关系来预防和治疗炎症性肠病(IBD)的新方法。在人类IBD和实验性结肠炎中发现维生素D及其受体VDR水平降低。在结肠炎动物模型中，VDR的下调促进了粘膜炎症的严重程度、程度和持续时间。我们已经证明，有条件地移除肠道上皮中的VDR会使小鼠更容易受到化学损伤和沙门氏菌结肠炎的影响。我们最近的肠道论文表明，肠上皮细胞VdR条件性基因敲除(VdRΔIEC)会导致生物失调。然而，肠道上皮细胞VDR如何参与维持微生物的动态平衡和先天免疫，目前仍不清楚。这项应用的目的是研究潘氏细胞中VDR缺乏和炎症状态下的生物失调的特殊后果。我们研究潘氏细胞是因为：1)潘氏细胞感知微生物并分泌抗菌肽；2)潘氏细胞的作用依赖于功能自噬途径，这在先天免疫和微生物生态中是必不可少的；以及3)IBD危险基因ATG16L1的缺失导致肠道潘氏细胞异常。有趣的是，我们首次报道ATG16L1是VDR的靶基因。肠上皮细胞VDR的缺失伴随着ATG16L1基因和蛋白水平的降低。因此，我们假设肠上皮VDR是IBD风险的决定因素，它通过作用于肠道内稳态中的潘氏细胞功能和微生物组合。我们现在已经开发了新的实验模型来研究VDR的作用，这些作用对宿主功能和肠道微生物区系有重要影响。目的1验证肠道上皮VDR决定潘氏细胞功能和自噬的工作假说，从而改变炎症中的肠道微生物区系。我们将A)阐明VDR是如何导致VDRΔIEC和VDRΔPaneth细胞小鼠的异常潘氏细胞(分化受损和AMP减少)的；B)利用有机物和VDR转基因小鼠，通过凋亡/自噬平衡来确定VDR对自噬调节因子的影响和机制；以及C)使用有机化合物和TG-I-VDR小鼠，研究VDR的过度表达是否能恢复潘氏细胞和自噬。目的2验证微生物群的调节和VDR功能恢复平衡的工作假说，微生物群会恢复Paneth细胞的功能和自噬，从而减轻结肠炎的严重程度。我们将：A)研究肠道上皮VDR在通过粪便移植或在最初无菌的小鼠中重新集落时在改变细菌种群中的作用；以及B)确定强迫肠道VDR表达在通过Paneth细胞和自噬调节在IL10/结肠炎小鼠中恢复微生物动态平衡的作用和机制。我们的研究是创新的，因为他们提供了一个统一的假设，可以潜在地解释在许多IBD和肠道疾病患者中发现的有缺陷的自噬、异常的Paneth细胞和生物失调。这些见解将导致预防和治疗IBD和肠道疾病的新策略。

 DESCRIPTION (provided by applicant): The long-term goal of our study is to elucidate the molecular basis for host factors involved in maintaining microbial homeostasis and to develop novel approaches that prevent and treat inflammatory bowel diseases (IBD) by restoring host-microbe relationships. Reduced levels of vitamin D and its receptor VDR have been found in human IBD and experimental colitis. In colitis animal models, downregulation of VDR promotes the severity, extent, and duration of mucosal inflammation. We have shown that conditional removal of VDR in the intestinal epithelium made mice more susceptible to chemical injury and Salmonella-colitis. Our recent Gut paper has demonstrated that intestinal epithelial VDR conditional knockout (VDRΔIEC) leads to dysbiosis. However, how intestinal epithelial VDR is involved in maintaining microbial homeostasis and innate immunity remains largely unknown. The objective of this application is to study special consequences of VDR deficiency in Paneth cells and dysbiosis in the inflamed states. We study Paneth cells because: 1) the Paneth cells sense microbes and secrete anti-microbial peptides (AMPs); 2) the roles of Paneth cells are dependent on a functional autophagy pathway, which is essential in innate immunity and microbial ecology; and 3) a lack of ATG16L1, an IBD risk genes, leads to abnormal Paneth cells in the intestine. Intriguingly, we are the first to report that ATG16L1 is a target gene of VDR. Th absence of intestinal epithelial VDR is accompanied by a reduction in the mRNA and protein levels of ATG16L1. Therefore, we hypothesize that intestinal epithelial VDR is a determinant of IBD risk through its actions on Paneth cell functions and microbial assemblage in intestinal homeostasis. We have now developed novel experimental models to investigate VDR actions that critically affect host functions and microbiota of the intestine. Aim 1 will test the working hypothesis that intestinal epithelial VDR determines the function of Paneth cells and autophagy, thus changing gut microbiota in inflammation. We will A) elucidate how intestinal epithelial VDR leads to abnormal Paneth cells (impaired differentiation and decreased AMPs), in VDRΔIEC and VDRΔpaneth cell mice; B) determine effects and mechanisms of VDR on autophagy regulators through apoptosis/autophagy balance, using organoids and VDR transgenic mice; and C) investigate whether over-expression of intestinal VDR restores Paneth cells and autophagy, using organoids and Tg-I-VDR mice. Aim 2 will test the working hypothesis that regulating the microbiome and restoring balance in VDR functions and the microbiome will restore function of Paneth cells and autophagy, thus reducing severity of colitis. We will: A) investigate the role of intestinal epithelial VDR in altering bacterial populations when administered via fecal transplantation or recolonization in originally germ-free mice; and B) define the effect and mechanism of forced intestinal VDR expression in restoring microbial homeostasis though Paneth cell and autophagy regulation in IL10-/- colitis mice. Our studies are innovative because they provide a unifying hypothesis that can potentially account for defective autophagy, abnormal Paneth cells, and dysbiosis found in many patients with IBD and intestinal disorders. These insights will lead to novel strategies to prevent and treat IBD and intestinal disorders.