Development and characterization of a bovine monocyte-derived macrophage cell line

Abstract

Abstract Monocytes are components of the innate arm of the immune response and are one of the first lines of defense again invading pathogens. However, they also serve as host cells for intracellular pathogens such as Mycobacteria, Brucella, and Salmonella. Monocytes represent only a small percentage of circulating leukocytes, thus harvesting sufficient quantities of them for in vitro experiments can be costly, time consuming, and can vary among cattle. It was our objective to develop a monocyte-derived macrophage cell line that could be utilized for studies involving macrophages. Monocytes were obtained from the whole blood of a cross-bred steer by density gradient centrifugation, and monocytes purified by adherence. After extended culture, a population emerged spontaneously that proliferated in culture and could be easily detached from the tissue culture vessels using tryspin-EDTA. These proliferating cells were tested for cell surface determinants indicative of monocyte/macrophage lineage, as well as bactericidal and phagocytic activity. Furthermore, whole genome sequencing on both the cell line and whole blood from the donor steer was performed to determine how they align with the bovine whole genome build, and to identify any genetic mutations. This bovine monocyte-derived macrophage cell line has been passaged over 25 times, resembles macrophages in culture, expresses the CD markers CD14/16, C172a, CD11b, is phagocytic and bactericidal, produces IFN in response to TLR/RLR ligands, and appears to fall into the M2 macrophage category. There is a dearth of bovine macrophage cell lines available to veterinary researchers, and this cell line should be a useful tool for the in vitro study of numerous macrophagetrophic pathogens.