Novel Mucosa-homing Dendritic Cell: Development, Trafficking and Function

描述(由申请人提供)：我们的总体目标是确定一种新的肠道归巢树突状细胞(DC)的发展、运输和功能特性。这些粘膜“DC”存在于肠道固有层和Peyer‘s结(PP)以及骨髓(BM)中。初步研究提供了导致以下一般假设的见解：a)DC在表型、微环境定位和发育方面与传统的DC亚群不同。B)他们使用新奇的运输级联，从起源于骨髓的肠道回到家。C)肠道归巢DC是包括CD103 CDC在内的肠道DC群体的主要祖细胞，也可产生CCR9浆细胞样DC(PDC)。D)？DC在粘膜免疫中可能具有特殊的作用，这是由独特的TLR反应和效应器活动所证明的。因此，我们假设DC既是肠道DC的特化祖细胞，也是肠道免疫反应的参与者。目的：1.确定DC的前体和后代：骨髓发育和肠道DC的动态平衡。？DC和CCR9 PDC在Flt3L刺激的BM培养中产生。为了确定其来源，将从骨髓中分离出共同的DC前体细胞(CDP)和其他DC前体细胞，并在体外培养后通过免疫表型检测其向DC亚群的发育情况。分选的DC也将在体外和体内进行研究，以评估其祖细胞和更新潜力。将评估包括维甲酸在内的调节因子对DC生成的影响。目的2.确定DC与CCR9 PDC的归巢特性，并确定涉及的转运机制。短期归巢试验将确定DC通过血液迁移到肠道的能力，并通过流式细胞术对从受体组织中回收的细胞进行定位。免疫组织化学将被用来询问？DC是否在肠壁中定位于特定的靶微环境。为了进行比较，将并行研究CCR9 PDC。来自基因靶向小鼠的中和抗体和/或DC将被用来定义涉及的新的转运机制/级联。目的3.明确激活(TLR配体)和肠道调节因子(如维甲酸)对DC祖细胞和免疫活性的影响。DC除了具有祖细胞活性外，还能刺激T细胞并表现出独特的细胞因子表达模式。？将检测树突状细胞的成熟和祖细胞能力的改变，以应对TLR配体；产生免疫刺激与调节细胞因子；以及培养或印记T细胞对提呈抗原的反应。还将评估肠道相关因子包括维甲酸和WNTS对其祖细胞和免疫活性的调节。阐明肠道相关DC亚群的来源、功能和调控，将有助于我们了解粘膜免疫动态平衡和对感染的反应；并有可能导致新的方法来加强粘膜疫苗接种(例如针对HIV或轮状病毒)和控制病理性炎症(例如炎症性肠道疾病或乳糜泻)。 与公共卫生相关：树突状细胞(DC)是一种特殊的白细胞，它控制身体在良好(对感染的免疫力)和不良(例如，糖尿病或炎症性肠道疾病等自身免疫性疾病)免疫反应之间的平衡。我们发现了一种新的粘膜(肠)相关DC，它作为其他肠道DC的前体，可能有助于控制肠道的免疫反应。我们的目标是了解这种粘膜DC是如何发育和到达肠道的，是什么控制它分化成其他类型的DC，它如何对感染相关的刺激做出反应，以及是什么因素控制它对理想免疫反应和破坏性免疫反应的贡献。在未来，我们的发现可能会导致新的方法来加强艾滋病或腹泻疾病的疫苗接种，或者治疗炎症性肠道疾病。

DESCRIPTION (provided by applicant): Our overall goal is to define the development, trafficking, and functional properties of a novel gut-homing dendritic cell (DC). These mucosal "?DC" reside in the intestinal lamina propria and Peyer's patches (PP), and in the bone marrow (BM). Preliminary studies have provided insights leading to the following general hypotheses: a) ?DC are distinct from conventional DC subsets in phenotype, microenvironmental localization, and development. b) They use novel trafficking cascades to home to the gut from their origin in the bone marrow. c) Gut-homing ?DC are key progenitors of intestinal DC populations including CD103+ cDC; and they can also give rise to CCR9+ plasmacytoid DC (pDC). d) ?DC may have specialized roles in mucosal immunity as evidenced by unique TLR responses and effector activities. Thus we hypothesize that ?DC are both specialized progenitors of intestinal DC, and participants in intestinal immune responses. Aims include: Aim 1. To define the precursors and progeny of ?DC: bone marrow development and intestinal DC homeostasis. ?DC, and CCR9+ pDC are generated in Flt3L-stimulated BM cultures. To identify their origin, common DC progenitors (CDP) and other DC progenitors will be sorted from BM, and their development into DC subsets will be monitored by immunophenotyping after in vitro culture. Sorted ?DC will also be studied to evaluate their progenitor and renewal potential in vitro and in vivo. The effects of regulatory factors including retinoic acid on ?DC generation will be assessed. Aim 2. To determine the homing properties of ?DC vs. CCR9+ pDC, and to define trafficking mechanisms involved. Short term homing assays will determine the ability of ?DC to migrate via the blood into the intestines vs. other sites, monitoring localization by flow cytometry of recovered cells from recipient tissues. Immunohistochemistry will be used to ask whether ?DC home to specific target microenvironments in the gut wall. CCR9+ pDC will be studied in parallel for comparison. Neutralizing antibodies, and/or DC from gene-targeted mice, will be used to define novel trafficking mechanisms/cascades involved. Aim 3. To define the effects of activating (TLR ligands) and gut regulatory factors (e.g. retinoic acid) on the progenitor and immunologic activities of ?DC. In addition to their progenitor activities, ?DC stimulate T cells and display unique patterns of cytokine expression. ?DC will be assayed for maturation and altered progenitor ability in response to TLR ligands; production of immunostimulatory vs. modulatory cytokines; and education or imprinting of T cells responding to presented antigen. Regulation of their progenitor vs. immunologic activities by gut-associated factors including retinoic acid and wnts will also be assessed. Elucidating the origin, function and regulation of intestine-associated DC subsets, as proposed here, will help us understand mucosal immune homeostasis and responses to infection; and has the potential to lead to novel approaches to enhance mucosal vaccination (e.g. for HIV or rotavirus) and to control pathologic inflammation (e.g. in inflammatory bowel diseases or celiac disease). PUBLIC HEALTH RELEVANCE: Dendritic cells (DC) are specialized white blood cells that control the body's balance between good (immunity to infection) and bad (for example autoimmune diseases like diabetes or inflammatory bowel disease) immune responses. We have discovered a new kind of mucosa (intestine)- associated DC that acts as a progenitor for other intestinal DC, and that may help control immune responses in the intestines. Our goal is to understand how this mucosal DC develops and gets to the intestines, what controls its differentiation into other DC types, how it responds to infection-related stimuli, and what factors control its contribution to desirable vs. damaging immune responses. In the future our findings may lead to new approaches to enhancing vaccination for AIDS or diarrheal disease, or to treating inflammatory bowel diseases.