Chicken CSF2 and IL-4-, and CSF2-Dependent Bone Marrow Cultures Represent Macrophages

Chicken bone marrow-derived macrophages (BMMQ) and dendritic cells (BMDC) are utilised as models to study the mononuclear phagocytic system (MPS). A widely used method to generate macrophages and DC in vitro is to culture bone marrow cells in the presence of colony-stimulating factor-1 (CSF1 ) to differentiate BMMQ and granulocyte-macrophage-CSF (GM-CSF, CSF2) and interleukin-4 (IL-4) to differentiate BMDC, while CSF2 alone can lead to the development of granulocyte-macrophage-CSF-derived dendritic cells (GMDC). However, in chickens, the cell lineages and their functions represented in these cultures are poorly understood. Here, we discriminated the phenotypical, functional and transcriptional differences between chicken BMMQ and BMDC along with examining the difference in DC cultures grown without IL-4 on days 2, 4, 6 and 8 of culture. BMMQ cultures develop into a morphologically homogenous cell population in contrast to the BMDC and GMDC cultures, which produce morphologically heterogeneous cell populations. At a phenotypical level, all cultures contained similar cell percentages and expression levels of MHCII, CD11c and CSF1R-transgene while MRC1L-B expression decreased over time in BMMQ. All cultures were efficient at uptake of 0.5 µm beads, but poorly phagocytosed 1 µm beads. Little difference was observed in the kinetics of phagosomal acidification across the cultures on each day of analysis. Temporal transcriptomic analysis indicated that all cultures expressed high levels of CSF3R, MERTK, SEPP1, SPI1 and TLR4, genes associated with macrophages in mammals. In contrast, low levels of FLT3, XCR1 and CAMD1, genes associated with DC, were expressed at day 2 in BMDC and GMDC after which expression levels decreased. Collectively, chicken CSF2 and IL-4- and CSF2-dependent BM cultures represent cells of the macrophage lineage rather than inducing conventional DC.