Vitamin D Regulation of CD14 in Mammary Epithelial Cells

描述(申请人提供)：目前的项目将研究乳腺上皮细胞中维生素D对天然免疫蛋白CD14的调节。虽然维生素D在免疫细胞中的作用已得到很好的认识，但维生素D对上皮细胞免疫反应的影响还没有得到很好的研究。人乳腺上皮(hTERT-HME)细胞的初步数据表明，维生素D诱导CD14的表达和分泌，CD14是一种结合细菌脂多糖(LPS)的蛋白质。这项拟议的研究将阐明维生素D对CD14和乳腺上皮细胞天然免疫反应的影响。中心假说是，维生素D通过VDR刺激乳腺上皮细胞释放可溶性CD14，从而促进抗炎环境。在目标1中，我们将确定VDR是否是诱导CD14合成所必需的，以响应生理剂量的维生素D，如果是的话，VDR信号在细胞核和质膜中的相对贡献。为此，我们将用shRNA结构敲除hTERT-HME细胞中的VDR，并利用维生素D类似物选择性地激活核或膜VDR的活性。结果将包括定量聚合酶链式反应检测CD14基因的表达和酶联免疫吸附试验检测CD14的分泌。在目标2中，我们将评估维生素D是否改变膜CD14(MCD14)的表达，并表征hTERT-HME细胞中因维生素D而分泌的可溶性CD14(SCD14)的异构体。这些细胞将被浓度为1，25D(VDR配体)或25D(循环中的维生素D代谢物，将模拟人体的生理循环浓度)处理。结果将包括mCD14表达的显微镜和Western blotting以确定特定的sCD14亚型。由于mCD14是通过糖基磷脂酰肌醇(GPI)锚拴在膜上的，因此我们将研究基因抑制GPI合成是否能阻断维生素D的作用。我们还将使用化学抑制剂来阻断分泌途径，以确定其对维生素D诱导的sCD14的影响。在目标3中，我们将研究乳腺细胞释放的sCD14对维生素D的反应。我们将测试来自经维生素D处理的hTERT-HME细胞的条件培养液对THP-1巨噬细胞免疫反应的影响。THP-1细胞将在条件培养液中孵育，并用内毒素攻击。THP-1细胞的促炎和抗炎细胞因子的表达和分泌将通过定量聚合酶链式反应和酶联免疫吸附试验进行评估。我们还将通过流式细胞术确定维生素D诱导的sCD14是否改变巨噬细胞与内毒素的结合或释放。我们预计，拟议的研究将证明维生素D触发乳腺上皮细胞分泌sCD14，并促进抗炎环境。我们将定义25D在抗炎反应方面的剂量反应，这将为体内维生素D缺乏或过量可能改变这些反应提供洞察力。总体而言，这项工作将加强我们对维生素D在上皮组织中的免疫功能的理解。 与公共健康相关：维生素D补充剂的使用在过去十年中大幅增加，但维生素D对暴露于感染性和炎症性介质的上皮细胞中的天然免疫系统的影响尚不清楚。该项目将确定维生素D如何通过调节免疫蛋白CD14来改变乳腺上皮细胞的免疫环境。了解维生素D如何通过乳腺上皮细胞中的CD14调节免疫环境，将有助于深入了解维生素D状态如何影响屏障上皮组织的炎症和感染。

DESCRIPTION (provided by applicant): The current project will investigate the regulation of an innate immunological protein, CD14, by vitamin D in mammary epithelial cells. Although actions of vitamin D in immune cells are well recognized, effects of vitamin D on the immune responses of epithelial cells have not been well studied. Preliminary data with human mammary epithelial (hTERT-HME) cells demonstrated that vitamin D induces expression and secretion of CD14, a protein that bind bacterial lipopolysaccharide (LPS). The proposed study will clarify the effect of vitamin D on CD14 and the innate immune response in mammary epithelial cells. The central hypothesis is that vitamin D promotes an anti-inflammatory environment through VDR to stimulate of soluble CD14 release from mammary epithelial cells. In Aim 1 we will determine if VDR is required for the induction of CD14 synthesis in response to physiological doses of vitamin D and, if so, the relative contributions of VDR signaling in the nucleus and at the plasma membrane. For this aim we will knock down VDR in the hTERT- HME cells with shRNA constructs and utilize vitamin D analogs that selectively activate nuclear or membrane VDRs activity. Outcomes will include CD14 gene expression by qPCR and sCD14 secretion by ELISA. In Aim 2 we will assess whether vitamin D alters the expression of membrane CD14 (mCD14) and characterize the isoforms of soluble CD14 (sCD14) secreted in response to vitamin D in hTERT-HME cells. These cells will be treated with concentrations of 1,25D (the VDR ligand) or 25D (the circulating vitamin D metabolite that will mimic physiological circulating concentrations in humans). Outcomes will include microscopy for mCD14 expression and western blotting to identify specific sCD14 isoforms. Because mCD14 is tethered to the membrane by a glycosylphosphatidylinositol (GPI) anchor, we will examine whether genomically inhibiting GPI synthesis blocks the effects of vitamin D. We will also use a chemical inhibitor to block the secretory pathway to determine its impact on vitamin D-induced sCD14. In Aim 3 we will investigate the functionality of sCD14 released from mammary cells in response to vitamin D. We will test the effects of conditioned media derived from vitamin D-treated hTERT-HME cells on immune responses of THP-1 macrophages. The THP-1 cells will be incubated in the condition media and challenged with LPS. The expression and secretion of pro- and anti- inflammatory cytokines by THP-1 cells will be assessed by qPCR and ELISA. We will also determine if the vitamin D-induced sCD14 alters binding or release of LPS from the macrophage by flow cytometry. We anticipate that the proposed studies will demonstrate that vitamin D triggers mammary epithelial cells to secrete sCD14 and promotes an anti-inflammatory environment. We will define the dose response of 25D with respect to anti-inflammatory responses, which will provide insight into whether vitamin D deficiency or vitamin D excess might alter these responses in vivo. Overall this work will enhance our understanding of the immunological functions of vitamin D in epithelial tissues. PUBLIC HEALTH RELEVANCE: Use of vitamin D supplements has increased dramatically over the past ten years yet the effects of vitamin D on the innate immune system in epithelial cells, which are exposed to infectious and inflammatory agents, are poorly defined. This project will determine how vitamin D alters the immune environment in mammary epithelial cells through regulation of the immunological protein CD14. Understanding how vitamin D modulates the immune environment through CD14 in mammary epithelial cells will provide insight into how vitamin D status impacts inflammation and infections in barrier epithelial tissues.