A Novel System for the Study of Neutrophil-Fungal Interactions

Background: Neutrophils are a terminally-differentiated phagocytic cell critical in the protection against bacterial and fungal pathogens. The study of neutrophils and their function in vitro has been hampered by the fact that primary neutrophils are cumbersome to isolate and have a short life-span, and that immortalized cell line models of neutrophils fail to recapitulate all the terminal effector functions of primary cells. Furthermore, the study of murine neutrophil function in vivo has been limited by the lack of a model system that can be genetically manipulated (e.g. gene knockout or overexpression) ex vivo prior to transplantation to examine the in vivo consequence of such manipulation.

Methods: Here we describe the development and use of conditionally immortalized GMP cell lines that are capable of unlimited expansion, are genetically tractable, and that can be transplanted into recipient mice to study the function of mature and terminally differentiated neutrophils in vivo. These GMP cell lines were derived from the bone marrow of 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 (SCF) and beta-estradiol (E2). Upon removal of beta-estradiol 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.

Results: Terminal effector function was confirmed by (a) flow cytometry, (b) phagocytosis, (c) superoxide generation, (d) chemotaxis, and (e) NET formation. More importantly, these GMP-derived neutrophils were also capable of phagocytosis and killing of fungal pathogens including Candida glabrata and Candida albicans. Of particular note, transplantation of these cells into neutropenic mice challenged with lethal dose of Candida species showed prolongation of survival, confirming in vivo functionality.

Conclusion: Overall, this model closely recapitulates primary neutrophils, but provides an inexhaustible and genetically tractable supply of cells that should provide researchers with a much-needed tool for investigating the molecular mechanisms governing neutrophil-fungal pathogenesis.