Ontogeny of intestinal dendritic cells and its regulation by retinoic acid [electronic resource]

The intestines are constantly challenged by antigenic stimuli from microbiota, digested food and invading pathogens. Intestinal dendritic cells (DC) play a key role in directing appropriate immune responses to each antigen encountered. DC have the unique ability to sample and present processed antigens to T cells while providing environmental cues in the form of cytokines and active metabolites such as retinoic acid (RA) to initiate and regulate immune responses or to induce tolerance. Understanding intestinal DC development and function is important in developing vaccines and treatment for intestinal diseases. The goal of my thesis project was to elucidate the origin and specialization of intestinal DC subsets and to define the roles of RA in development and specification of these DC and their precursors. The two predominant cDC subsets in the small intestine (SI) are identified phenotypically as CD11c+MHCII+ cells that are either CD103+CD11b- (cDC1) or CD103+CD11b+ (cDC2). The two subsets differ developmentally, requiring different transcription factors, and functionally. They derive from BM-derived precursors that remain incompletely characterized. In Chapter 2, I identified a previously unidentified gut-tropic DC precursor, termed pre-[mu] DC (pre-mucosal DC). pre-[mu] DC express [alpha] 4 [beta] 7, a gut-homing receptor that targets them efficiently to the intestines, although they can also contribute to DC populations in other sites. pre-[mu] DC are 10 fold more efficient than the previously known DC precursor pre-cDC at giving rise to intestinal cDC on a per cell basis; and, unlike pre-cDC, they can also generate plasmacytoid DC (pDC). Furthermore, pre-[mu] DC have an intrinsic preference to develop into cDC1; whereas pre-cDC preferentially become cDC2. Thus pre-[mu] DC represent a specialized BM DC population with unique gut tropism and the potential to populate each of the major DC subsets in the intestines. RA plays a critical role in adaptive mucosal immunity through effects on epithelial, mesenchymal and lymphoid cells. In chapter 3, I showed that it is also important for intestinal DC development and DC subset specialization as well. RA was indispensible for pre-[mu] DC development in the bone marrow. It also directly regulated cDC1 and cDC2 development from pre-[mu] DC in situ in the gut wall. In the absence of RA signaling, cDC2 were reduced in frequency and number in the SI LP, and the cDC1 and cDC2 generated are phenotypically abnormal. I devised an in vitro culture system to model the developmental potential of pre-[mu] DC and demonstrated that RA is required, in combination with GM-CSF and Flt3L, for the generation of cDC with phenotypic and transcriptional signatures of intestinal CD103+ subsets. Interestingly, the presence of RA enhances the frequency and number of cDC2 generated from purified pre-[mu] DC, consistent with a role in fate decisions. However, my whole genome transcriptomic analyses showed that RA has broad effects on gene expression, driving transcription in in vitro-derived cDC1 and cDC2 towards patterns of physiologic gene expression. These studies indicated that the role of RA in intestine cDC development is cell-intrinsic, a finding that I confirmed in vivo using CD11c-cre.RAR403fl/fl mice, which express a dominant negative RA receptor RAR403 in DC and thus lack RAR signaling selectively in DC. Thus RA plays a critical roles in regulating intestinal cDC development both at the precursor stage by controlling pre-[mu] DC development in the bone marrow, and by driving transcriptional programs for cDC1 and cDC2 specialization in the gut wall. pre-[mu] DC are the first DC precursor identified that has a defined tissue tropism. Their identification raises the possibility that other specialized DC precursors may home to skin or lung or other sites. Comparison of pre-[mu] DC with pre-cDC reveals differences in their potential to differentiate into each of the two major functionally specialized cDC subsets in the SI. This in combination with my discovery that RA regulates pre-[mu] DC development in the bone marrow and cDC1 and cDC2 development in the gut wall may provide useful tool to manipulate intestinal cDC to aid in oral vaccination and treatment for intestinal diseases. The description of an in vitro culture system to generate intestine-like cDC will be a useful tool to study intestinal cDC development and functions, and offers the potential to generate specialized cDC subsets for immune therapy.

Advisor: Butcher Eugene primary advisor. Thesis advisor
Engleman Edgar G. advisor. Thesis advisor
Lewis David advisor. Thesis advisor
Monack Denise M. advisor. Thesis advisor