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

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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-tgdim MHCIIinter-hi, CSF1R-tghi MHCIIhi and CSF1R-tghi MHCIIinter subpopulations. Low levels of CD80, CD40, MHCI, CD44 and Ch74.2 were expressed by the CSF1R-tghi MHCIIinter cells. Functionally, in vivo fluorescent bead uptake was significantly higher in the CSF1R-tghi MHCIIhi MRC1L-B+ cells compared to the CSF1R-tgdim and CSF1R-tghi MHCIIinter MRC1L-B+ subpopulations while LPS enhanced phagocytosis by the CSF1R-tghi MHCIIinter 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-Bneg cDC. Finally, RNA-seq analysis of the CSF1R-tg subpopulations demonstrated that separating the CSF1R-tghi subpopulation into CD11chi and CD11cdim cells enriched for cDC and macrophage lineages, respectively while the CSF1R-tghi MHCIIinter subpopulation was enriched for red pulp macrophages. However our analysis could not define the cell lineage of the heterogeneous CSF1R-tgdim subpopulation. This detailed overview of the MPS in the chicken spleen will contribute to future research on their role in antigen uptake and presentation.
Original languageEnglish
JournalFrontiers in Immunology
Early online date9 Mar 2021
DOIs
Publication statusE-pub ahead of print - 9 Mar 2021

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