AUTHOR=Sutton Kate M. , Morris Katrina M. , Borowska Dominika , Sang Helen , Kaiser Pete , Balic Adam , Vervelde Lonneke 

TITLE=Characterization of Conventional Dendritic Cells and Macrophages in the Spleen Using the CSF1R-Reporter Transgenic Chickens

JOURNAL=Frontiers in Immunology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2021.636436

DOI=10.3389/fimmu.2021.636436

ISSN=1664-3224

ABSTRACT=<p>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 <italic>CSF1R</italic>-reporter transgenic chickens (<italic>CSF1R</italic>-tg), in which cells of the MPS express a transgene under the control elements of the chicken <italic>CSF1R</italic>, 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 <italic>CSF1R</italic>-tg<sup>+</sup> 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 <italic>CSF1R</italic>-tg. In flow cytometric analysis, diverse expression of the <italic>CSF1R</italic>-tg and MHCII was observed leading to the categorization of <italic>CSF1R</italic>-tg cells into <italic>CSF1R</italic>-tg<sup>dim</sup> MHCII<sup>inter−hi</sup>, <italic>CSF1R</italic>-tg<sup>hi</sup> MHCII<sup>hi</sup>, and <italic>CSF1R</italic>-tg<sup>hi</sup> MHCII<sup>inter</sup> subpopulations. Low levels of CD80, CD40, MHCI, CD44, and Ch74.2 were expressed by the <italic>CSF1R</italic>-tg<sup>hi</sup> MHCII<sup>inter</sup> cells. Functionally, <italic>in vivo</italic> fluorescent bead uptake was significantly higher in the <italic>CSF1R</italic>-tg<sup>hi</sup> MHCII<sup>hi</sup> MRC1L-B<sup>+</sup> cells compared to the <italic>CSF1R</italic>-tg<sup>dim</sup> and <italic>CSF1R</italic>-tg<sup>hi</sup> MHCII<sup>inter</sup> MRC1L-B<sup>+</sup> subpopulations while LPS enhanced phagocytosis by the <italic>CSF1R</italic>-tg<sup>hi</sup> MHCII<sup>inter</sup> subpopulation. The analysis of bead localization in the spleen suggests the presence of ellipsoid-associated macrophage subsets. In addition, we demonstrated the functionality of <italic>ex vivo</italic> derived <italic>CSF1R</italic>-tg<sup>+</sup> MRC1L-B<sup>neg</sup> cDC. Finally, RNA-seq analysis of the <italic>CSF1R</italic>-tg subpopulations demonstrated that separating the <italic>CSF1R</italic>-tg<sup>hi</sup> subpopulation into CD11c<sup>hi</sup> and CD11c<sup>dim</sup> cells enriched for cDC and macrophage lineages, respectively, while the <italic>CSF1R</italic>-tg<sup>hi</sup> MHCII<sup>inter</sup> subpopulation was enriched for red pulp macrophages. However, our analysis could not define the cell lineage of the heterogeneous <italic>CSF1R</italic>-tg<sup>dim</sup> subpopulation. This detailed overview of the MPS in the chicken spleen will contribute to future research on their role in antigen uptake and presentation.</p>