Characterization of conventional dendritic cells and macrophages in the spleen using the CSF1R-reporter transgenic chickens

Abstract

The spleen is a major site for the immunological responses to blood-borne antigens that is coordinated by cells of the mononuclear phagocyte system (MPS). The chicken spleen is populated with a number of different macrophages while the presence of conventional dendritic cells (cDC) has been described. However, a detailed characterization of the phenotype and function of different macrophage subsets and cDC in the chicken spleen is limited. Using the CSF1R-reporter transgenic chickens (CSF1R-tg), in which cells of the MPS express a transgene under the control elements of the chicken CSF1R, we carried out an in-depth characterization of these cells in the spleen. Immunohistological analysis demonstrated differential expression of MRC1L-B by periarteriolar lymphoid sheaths (PALS)-associated CSF1R-tg⁺ cells. In the chicken's equivalent of the mammalian marginal zone, the peri-ellipsoid white-pulp (PWP), we identified high expression of putative CD11c by ellipsoid-associated cells compared to ellipsoid-associated macrophages. In addition, we identified a novel ellipsoid macrophage subset that expressed MHCII, CD11c, MRC1L-B, and CSF1R but not the CSF1R-tg. In flow cytometric analysis, diverse expression of the CSF1R-tg and MHCII was observed leading to the categorization of CSF1R-tg cells into CSF1R-tg^dim MHCII^inter−hi, CSF1R-tg^hi MHCII^hi, and CSF1R-tg^hi MHCII^inter subpopulations. Low levels of CD80, CD40, MHCI, CD44, and Ch74.2 were expressed by the CSF1R-tg^hi MHCII^inter cells. Functionally, in vivo fluorescent bead uptake was significantly higher in the CSF1R-tg^hi MHCII^hi MRC1L-B⁺ cells compared to the CSF1R-tg^dim and CSF1R-tg^hi MHCII^inter MRC1L-B⁺ subpopulations while LPS enhanced phagocytosis by the CSF1R-tg^hi MHCII^inter subpopulation. The analysis of bead localization in the spleen suggests the presence of ellipsoid-associated macrophage subsets. In addition, we demonstrated the functionality of ex vivo derived CSF1R-tg⁺ MRC1L-B^neg cDC. Finally, RNA-seq analysis of the CSF1R-tg subpopulations demonstrated that separating the CSF1R-tg^hi subpopulation into CD11c^hi and CD11c^dim cells enriched for cDC and macrophage lineages, respectively, while the CSF1R-tg^hi MHCII^inter subpopulation was enriched for red pulp macrophages. However, our analysis could not define the cell lineage of the heterogeneous CSF1R-tg^dim subpopulation. This detailed overview of the MPS in the chicken spleen will contribute to future research on their role in antigen uptake and presentation.