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

Grant Number: 5R01AI093981-04
PI Name: Butcher
Project Title: Novel Mucosa-homing Dendritic Cell: Development, Trafficking and Function

Project Abstract: Our overall goal is to define the development, trafficking, and functional properties of a novel gut-homing dendritic cell. These mucosal “µDC” reside in the intestinal lamina propria (LP) and Peyer’s patches (PP), and in the bone marrow. 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 mucosal DC populations including CD103+ cDC; and they can also give rise to CCR9+ plasmacytoid DC (pDC); d) µDC 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 homeostasis. µDC, and CCR9+ pDC are generated in Flt3L-stimulated BM cultures. To test the hypothesis that µDC arise from common DC progenitors (CDP), CDP and other DC progenitor subsets 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 intestinal compartments and lymphoid 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; education or imprinting of T cells responding to presented antigen; and regulation of their progenitor vs. immunologic activities by TLR signaling and gut-associated regulatory factors including retinoic acid and wnts. Elucidating the origin, function and regulation of intestine-associated DC subsets, as proposed here, will help us understand mucosal homeostasis; and may lead to novel approaches to enhance mucosal vaccination and to control pathologic inflammation (e.g in inflammatory bowel diseases or celiac disease).

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