New neutrophil models for studying host-fungal pathogen interactions

Abstract

Neutrophils are terminally differentiated phagocytic cells that represent the first line of defense against pathogens. The study of neutrophils in vitro is difficult owing to a short life span. Immortalized cell line models fail to recapitulate all the effector functions of primary cells. Furthermore, the study of murine neutrophil function in vivo is limited by the lack of a model system that can be genetically manipulated (e.g. gene knockout or overexpression) ex vivo. We describe here the development and use of conditionally immortalized granulocyte/macrophage progenitor cells (GMP) capable of unlimited expansion, genetically tractable, and that can be transplanted into mice to study neutrophil function in vivo. These GMP cell lines were derived from UBC-GFP mice by the transduction of an MSCVneo ER-HoxB8 retrovirus such that factor-dependent cell lines emerge in the presence of stem cell factor and b-estradiol (E2). Upon removal of E2 in vitro or transplantation into irradiated recipients, these GMP precursor cell lines differentiate normally to fully functional neutrophils over the course of 4–5 days. Terminal effector function was confirmed by a) flow cytometry, b) phagocytosis, c) superoxide generation, d) transepithelial migration, and e) NET formation. More importantly, these GMP-derived neutrophils were also capable of fungicidal activity. Of particular note, transplantation of these cells into neutropenic mice challenged with lethal dose of the pathogenic yeast C. albicans, showed prolongation of survival. Overall, this model closely recapitulates primary neutrophils, while providing an inexhaustible and genetically tractable supply of cells, a much-needed tool for the study of neutrophil function.