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“μDC”存在于肠道固有层和Peyer‘s斑块(PP)以及骨髓(BM)。初步研究提供了导致以下一般假设的见解：a)μDC在表型、微环境定位和发育方面与传统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)肠道归巢的μDC是包括CD103+CDC在内的肠道DC群体的主要祖细胞，也可产生CCR9+浆细胞样树突状细胞(PDC)。D)μ树突状细胞在粘膜免疫中可能具有特殊的作用，其独特的反应和效应活性证明了这一点。因此，我们假设μDC既是肠道DC的特化祖细胞，又是肠道免疫反应的参与者。目的：1.明确μDC的前体和后代：骨髓发育和肠道DC的动态平衡。μDC和CCR9+PDC在Flt3L刺激的骨髓细胞培养中产生。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μDC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μ树突状细胞生成的影响。目的2.比较μDC与CCR9+PDC的归巢特性，并探讨其转运机制。短期归巢检测将确定μDC相对于其他部位通过血液迁移到肠道的能力，通过流式细胞术对从受体组织中回收的细胞进行定位监测。免疫组织化学将被用来询问μDC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μ树突状细胞(DC)祖细胞和免疫活性的影响。除了其祖细胞活性外，μDC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μ树突状细胞的成熟和祖细胞能力的改变，以应对TLRL配体；产生免疫刺激与调节细胞因子；以及培养或印记T细胞对呈现的抗原做出反应。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)。

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 "μDC" 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) μDC 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 μDC are key progenitors of intestinal DC populations including CD103+ cDC; and they can also give rise to CCR9+ plasmacytoid DC (pDC). d) μDC may have specialized roles in mucosal immunity as evidenced by unique TLR responses and effector activities. Thus we hypothesize that μDC are both specialized progenitors of intestinal DC, and participants in intestinal immune responses. Aims include: Aim 1. To define the precursors and progeny of μDC: bone marrow development and intestinal DC homeostasis. μDC, 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 μDC 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 μDC generation will be assessed. Aim 2. To determine the homing properties of μDC vs. CCR9+ pDC, and to define trafficking mechanisms involved. Short term homing assays will determine the ability of μDC 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 μDC 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 μDC. In addition to their progenitor activities, μDC stimulate T cells and display unique patterns of cytokine expression. μDC 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).