Vitamin D Receptor Regulation of Microbiota in Intestinal Epithelia

 描述（由申请人提供）：我们研究的长期目标是阐明参与维持微生物稳态的宿主因子的分子基础，并开发通过恢复宿主-微生物关系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的下调促进粘膜炎症的严重性、程度和持续时间。我们已经表明，有条件地去除肠上皮中的VDR使小鼠更容易受到化学损伤和沙门氏菌结肠炎。我们最近的Gut论文已经证明肠上皮VDR条件性敲除（VDRΔIEC）导致生态失调。然而，肠上皮VDR如何参与维持微生物稳态和先天免疫仍然是未知的。本申请的目的是研究潘氏细胞中VDR缺乏的特殊后果和炎症状态下的生态失调。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微生物肽（AMP）; 2）潘氏细胞的作用依赖于功能性自噬途径，这在先天免疫和微生物生态学中是必不可少的; 3）缺乏ATG 16 L1（IBD风险基因）导致肠道中的潘氏细胞异常。有趣的是，我们是第一个报道ATG 16 L1是VDR的靶基因。肠上皮VDR的缺乏伴随着ATG 16 L1的mRNA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，通过其对潘氏细胞功能和肠道内稳态中微生物聚集的作用。我们现在已经开发了新的实验模型来研究VDR对宿主功能和肠道微生物群的影响。目的1将测试肠上皮VDR决定潘氏细胞和自噬的功能，从而改变炎症中的肠道微生物群的工作假设。我们将A）在VDRΔIEC和VDRΔ潘氏细胞小鼠中阐明肠上皮VDR如何导致异常潘氏细胞（分化受损和AMP减少）; B）使用类器官和VDR转基因小鼠，通过细胞凋亡/自噬平衡确定VDR对自噬调节剂的作用和机制;和C）使用类器官和Tg-I-VDR小鼠，研究肠VDR的过表达是否恢复潘氏细胞和自噬。目的2将测试工作假设，即调节微生物组并恢复VDR功能的平衡，微生物组将恢复潘氏细胞和自噬的功能，从而降低结肠炎的严重程度。我们将：A）研究通过粪便移植或重新定植给予原本无菌小鼠时肠上皮VDR在改变细菌种群中的作用;和B）定义强制肠道VDR表达在通过潘氏细胞和自噬调节恢复微生物稳态中的作用和机制IL 10-/-结肠炎小鼠。我们的研究是创新的，因为它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，异常的潘氏细胞和生态失调。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.