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“？DC”存在于肠固有层和派伊尔集合淋巴结（PP）以及骨髓（BM）中。初步研究提供了见解，导致以下一般假设：a）？DC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？DC是肠道DC群体的关键祖细胞，包括CD 103 + cDC;并且它们还可以产生CCR 9+浆细胞样DC（pDC）。d）？DC可能在粘膜免疫中具有特殊的作用，如独特的TLR反应和效应子活性所证明的。所以我们假设？DC是肠道DC的特化祖细胞，参与肠道免疫应答。目标包括：目标1。定义的前体和后代？DC：骨髓发育和肠道DC稳态。? DC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。解决了？还将研究DC以评估其体外和体内的祖细胞和更新潜力。包括维甲酸在内的调节因素对？将评估DC生成。目标二。以确定归巢的特性？DC vs. CCR 9 + pDC，并定义所涉及的贩运机制。短期归巢试验将决定能力？DC通过血液迁移到肠中与其他部位相比，通过流式细胞术监测从受体组织回收的细胞的定位。免疫组化将用于问是否？DC在肠壁中的特定目标微环境的家。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。确定激活（TLR配体）和肠道调节因子（如视黄酸）对祖细胞和免疫活性的影响？DC.除了他们的祖先活动，？DC刺激T细胞并显示独特的细胞因子表达模式。?将测定DC的成熟和响应TLR配体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;以及响应呈递抗原的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）。 公共卫生相关性：树突状细胞（DC）是一种特殊的白色血细胞，它控制着身体在好的（对感染的免疫力）和坏的（例如糖尿病或炎症性肠病等自身免疫性疾病）免疫反应之间的平衡。我们已经发现了一种新的粘膜（肠）相关DC，它可以作为其他肠道DC的祖细胞，并且可能有助于控制肠道中的免疫反应。我们的目标是了解这种粘膜DC如何发育并到达肠道，是什么控制其分化为其他DC类型，它如何对感染相关刺激做出反应，以及哪些因素控制其对理想免疫反应的贡献。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病的疫苗接种，或治疗炎症性肠病。

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.