Vitamin D Regulation of CD14 in Mammary Epithelial Cells

维生素 D 对乳腺上皮细胞 CD14 的调节

• 批准号: 8454739
• 负责人: Katrina Marie Simmons
• 金额: $ 2.88万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-24 至 2014-09-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8454739
• 关键词: Address; Agonist; Anti-Inflammatory Agents; Anti-inflammatory; Binding; Biological Response Modifiers; Brefeldin A; CD14 gene; Cell Nucleus; Cell membrane; Cells; Chemicals; Conditioned Culture Media; Confocal Microscopy; Data; Dose; Environment; Enzyme-Linked Immunosorbent Assay; Epithelial; Epithelial Cells; Flow Cytometry; GPI Membrane Anchors; Gene Expression; Genes; Genetic Transcription; Glycosylphosphatidylinositols; Human; Immune; Immune response; Immune system; Immunity; Incubated; Infection; Inflammation; Inflammatory; Inflammatory Response; Ligands; Lipopolysaccharides; Mammary gland; Measures; Mediating; Membrane; Microscopy; Molecular Weight; Nuclear; Nuclear Envelope; Outcome; Pathway interactions; Physiological; Production; Protein Binding; Protein Isoforms; Proteins; Receptor Signaling; Regulation; Relative (related person); Research; Role; Signal Pathway; Surface; Testing; Time; Tissues; Vitamin D; Vitamin D Analog; Vitamin D Deficiency; Vitamin D3 Receptor; Vitamins; Western Blotting; Work; cytokine; immune function; in vivo; inhibitor/antagonist; insight; knock-down; macrophage; prevent; response; small hairpin RNA; trafficking

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.

描述（由申请人提供）：本项目将研究维生素D对乳腺上皮细胞中先天免疫蛋白CD 14的调节。虽然维生素D在免疫细胞中的作用得到了很好的认可，但维生素D对上皮细胞免疫反应的影响尚未得到很好的研究。人乳腺上皮细胞（hTERT-HME）的初步数据表明，维生素D诱导CD 14的表达和分泌，CD 14是一种结合细菌脂多糖（LPS）的蛋白质。这项研究将阐明维生素D对乳腺上皮细胞中CD 14和先天免疫反应的影响。中心假设是维生素D通过VDR促进抗炎环境，刺激乳腺上皮细胞释放可溶性CD 14。在目标1中，我们将确定VDR是否是响应生理剂量的维生素D诱导CD 14合成所必需的，如果是，则确定VDR信号在细胞核和质膜中的相对贡献。为此目的，我们将用shRNA构建体敲低hTERT-HME细胞中的VDR，并利用选择性激活核或膜VDR活性的维生素D类似物。结果将包括qPCR法测定的CD 14基因表达和ELISA法测定的sCD 14分泌。在目标2中，我们将评估维生素D是否改变膜CD 14（mCD 14）的表达，并表征hTERT-HME细胞中响应维生素D分泌的可溶性CD 14（sCD 14）的亚型。这些细胞将用浓度为1，25 D（VDR配体）或25 D（模拟人体生理循环浓度的循环维生素D代谢物）处理。结果将包括mCD 14表达的显微镜检查和用于鉴定特定sCD 14亚型的蛋白质印迹法。由于mCD 14通过糖基磷脂酰肌醇（GPI）锚与膜相连，我们将研究基因组抑制GPI合成是否会阻断维生素D的作用。我们还将使用化学抑制剂阻断分泌途径，以确定其对维生素D诱导的sCD 14的影响。在目标3中，我们将研究乳腺细胞释放的sCD 14对维生素D的反应功能。我们将测试来自维生素D处理的hTERT-HME细胞的条件培养基对THP-1巨噬细胞免疫应答的影响。将THP-1细胞在条件培养基中孵育并用LPS攻击。通过qPCR和ELISA评估THP-1细胞的促炎细胞因子和抗炎细胞因子的表达和分泌。我们还将通过流式细胞术确定维生素D诱导的sCD 14是否改变巨噬细胞的LPS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sCD 14，并促进抗炎环境。我们将定义25 D的抗炎反应的剂量反应，这将提供洞察是否维生素D缺乏或维生素D过量可能会改变这些反应在体内。总的来说，这项工作将提高我们对维生素D在上皮组织中的免疫功能的理解。 公共卫生相关性：在过去的十年中，维生素D补充剂的使用急剧增加，但维生素D对暴露于感染和炎症因子的上皮细胞中的先天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CD 14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CD 14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中的炎症和感染。