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型肠道归巢树突状细胞（DC）的发育、运输和功能特性。这些粘膜“μDC”存在于肠固有层和派伊尔集合淋巴结（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）μDC在表型、微环境定位和发育方面不同于常规DC亚群。B）它们使用新的贩运级联从它们在骨髓中的起源回到肠道。c）肠道归巢μDC是肠道DC群体（包括CD 103 + cDC）的关键祖细胞;并且它们还可以产生CCR 9+浆细胞样DC（pDC）。d）μDC可能在粘膜免疫中具有特殊作用，如独特的TLR应答和效应物活性所证明的。因此，我们假设μDC既是肠道DC的特化祖细胞，又参与肠道免疫应答。目标包括：目标1。明确μDC的前体和子代：骨髓发育和肠道DC稳态。μDC和CCR 9 + pDC在Flt 3L刺激的BM培养物中产生。为了鉴定它们的来源，将从BM中分选出共同DC祖细胞（CDP）和其他DC祖细胞，并且在体外培养后通过免疫表型分析来监测它们发育成DC亚群。还将研究分选的μDC以评估其体外和体内的祖细胞和更新潜力。将评估包括视黄酸在内的调节因子对μDC生成的影响。目标二。确定μDC与CCR 9 + pDC的归巢特性，并定义所涉及的运输机制。短期归巢试验将确定μDC通过血液迁移到肠道与其他部位的能力，通过流式细胞术监测从受体组织回收的细胞的定位。免疫组织化学将用于询问μDC是否归巢于肠壁中的特定靶微环境。将平行研究CCR 9 + pDC以进行比较。中和抗体和/或来自基因靶向小鼠的DC将用于定义所涉及的新的运输机制/级联。目标3：明确TLR配体和维甲酸等肠道调节因子对μDC祖细胞和免疫活性的影响。除了它们的祖细胞活性，μDC刺激T细胞并显示独特的细胞因子表达模式。将测定μDC的成熟和对TLR配体应答的改变的祖细胞能力;免疫刺激性与调节性细胞因子的产生;以及对呈递抗原应答的T细胞的教育或印记。还将评估肠道相关因子（包括视黄酸和wnts）对其祖细胞与免疫活性的调节。 如本文所提出的，阐明精氨酸相关DC亚群的起源、功能和调节将有助于我们理解粘膜免疫稳态和对感染的反应;并且有可能导致新的方法来增强粘膜疫苗接种（例如针对HIV或轮状病毒）和控制病理性炎症（例如在炎性肠病或乳糜泻中）。

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).