Chicken CSF2 and IL-4-, and CSF2-dependent bone marrow cultures differentiate into macrophages over time

Abstract

Chicken bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDC) are utilized 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 BMMΦ 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 DC (GMDC). However, in chickens, the MPS cell lineages and their functions represented by these cultures are poorly understood. Here, we decipher the phenotypical, functional and transcriptional differences between chicken BMMΦ and BMDC along with examining differences in DC cultures grown in the absence of IL-4 on days 2, 4, 6 and 8 of culture. BMMΦ cultures develop into a morphologically homogenous cell population in contrast to the BMDC and GMDC cultures, which produce morphologically heterogeneous cell cultures. At a phenotypical level, all cultures contained similar cell percentages and expression levels of MHCII, CD11c and CSF1R-transgene, whilst MRC1L-B expression decreased over time in BMMΦ. All cultures were efficiently able to uptake 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 + IL-4- and CSF2-dependent BM cultures represent cells of the macrophage lineage rather than inducing conventional DC.

Delineation of chicken immune markers in the era of omics and multicolor flow cytometry

“…Functional assays involving the assessment of phagocytosis can be integrated into flow cytometry experiments. Using commercially available fluorescent beads, which can be labeled with antigens such as LPS or inactive avian influenza virus, pH-sensitive pHrodo-labeled bioparticles, such as Salmonella or E. coli, or CFSE-labeled dead cells, can be utilized to determine the efficiency and specificity of chicken macrophage phagocytosis or effectorcytosis (10, [68][69][70]. Performing phagocytosis assays at 4°C, a temperature commonly referred to as "on ice, " should be used to assess specific binding or adhesion of particles to cell surfaces without allowing active internalization (phagocytosis) to occur.…”

“…For example, 4°C control does not prevent bone marrow-derived macrophages from phagocytosing pH-sensitive pHrodo-labeled bioparticles. Instead, an actin polymerization inhibitor, cytochalasin D, inhibited the uptake of these bioparticles ( 70 ). The differential expression of surface markers, such as CD40 and MHC class II, can be an indicator of cell activation.…”