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

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

doi:10.3389/fimmu.2021.636436

Kate M Sutton, Helen Sang + Show 5 more

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https://doi.org/10.3389/fimmu.2021.636436

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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 theCSF1R-reporter transgenic chickens (CSF1R-tg), in which cells of the MPS express a transgene under the control elements of the chickenCSF1R, 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)-associatedCSF1R-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 theCSF1R-tg. In flow cytometric analysis, diverse expression of theCSF1R-tg and MHCII was observed leading to the categorization ofCSF1R-tg cells intoCSF1R-tgdimMHCIIinter−hi,CSF1R-tghiMHCIIhi, andCSF1R-tghiMHCIIintersubpopulations. Low levels of CD80, CD40, MHCI, CD44, and Ch74.2 were expressed by theCSF1R-tghiMHCIIintercells. Functionally,in vivofluorescent bead uptake was significantly higher in theCSF1R-tghiMHCIIhiMRC1L-B+cells compared to theCSF1R-tgdimandCSF1R-tghiMHCIIinterMRC1L-B+subpopulations while LPS enhanced phagocytosis by theCSF1R-tghiMHCIIintersubpopulation. The analysis of bead localization in the spleen suggests the presence of ellipsoid-associated macrophage subsets. In addition, we demonstrated the functionality ofex vivoderivedCSF1R-tg+MRC1L-BnegcDC. Finally, RNA-seq analysis of theCSF1R-tg subpopulations demonstrated that separating theCSF1R-tghisubpopulation into CD11chiand CD11cdimcells enriched for cDC and macrophage lineages, respectively, while theCSF1R-tghiMHCIIintersubpopulation was enriched for red pulp macrophages. However, our analysis could not define the cell lineage of the heterogeneousCSF1R-tgdimsubpopulation. This detailed overview of the MPS in the chicken spleen will contribute to future research on their role in antigen uptake and presentation.

Highlights

The vertebrate spleen is a secondary lymphoid organ and despite differences in the immune system among vertebrates, the spleens basic histological architecture and its role in filtering the blood for antigens and damaged, aged cells has been conserved during evolution from fish, amphibians, birds, and mammals
At the origin of the periarteriolar lymphoid sheath (PALS), CSF1R-tg+ cells that surround the central artery express the chicken monocyte/macrophage markers, Ch74.2, which binds to an unknown epitope, MHCII and mannose receptor C1-like B [MRC1L-B formally known as KUL01, Figures 1C,D; [30]]
Within the inner regions of the peri-ellipsoid white-pulp (PWP) are ellipsoid-associated cells (EAC) that express the CSF1R-tg [33] and can be identified using the monoclonal Ch68.2 which binds to an unknown epitope [Figure 1I; [25]]

Summary

Introduction

The vertebrate spleen is a secondary lymphoid organ and despite differences in the immune system among vertebrates, the spleens basic histological architecture and its role in filtering the blood for antigens and damaged, aged cells has been conserved during evolution from fish, amphibians, birds, and mammals. The white pulp and red pulp are extensively populated with macrophages and dendritic cells (DC) with distinct origins and functions that contribute to the control of blood-borne pathogens and homeostatic processes [1]. The venous system of the red pulp gives it the capacity to filter the blood and remove old erythrocytes by red pulp macrophages. The murine splenic red pulp contains reservoirs of monocytes that are transcriptionally similar to their blood counterparts, Ly-6C+ and Ly-6C++ cells (ADGRE1−/lo CD11b+ CD11c−) that egress to sites of tissue damage [4, 5]

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