A novel neutrophil-like in vitro model for the study of human neutrophil polarization

Abstract

Background and Aims : Neutrophils are key players in the activation and regulation of innate and adaptive immunity. As mediators of the inflammatory response in cardiovascular diseases, the polarity of neutrophils towards pro- and anti-inflammatory phenotypes has recently been advanced. Considering that their lifespan is very limited and long-term in vitro studies are challenging, our aim was to develop and characterize a representative model of human neutrophil-like cells.Methods: Human HL-60 promyeloblasts were cultured for 5 days in complete media supplemented with 1.3% DMSO, then assessed for proliferation, nuclear segmentation by cytochemistry, expression of cell-surface markers by flow cytometry and differentiation markers by qPCR. Polarization of the neutrophil-like cells was induced for 48h towards the pro-inflammatory phenotype (N1) in the presence of LPS and IFNγ, whereas IL-4 was used for the anti-inflammatory phenotype (N2), then assessed for viability and gene expression.Results: Promyeloblasts differentiation was confirmed by down-regulation of TERT expression, strong FPR1 and Integrinβ3 induction (∼300-fold and ∼25-fold over control, respectively) and cell surface expression of CD11b and CD18. Equally, neutrophil-like cells exhibited nuclear segmentation, decrease in proliferation and maintained similar viability in culture. Polarization towards N1 phenotype was confirmed by up-regulation of pro-inflammatory genes MCP-1, IL-1β, TNFα and IRF7, whereas N2 markers CD206 and TGM2 were highly induced following IL-4 stimulation. Cellular viability remained comparable under both treatments.Conclusions: Promyeloblast differentiation by DMSO generates neutrophil-like cells that can be polarized towards N1/N2 phenotypes. These resemble subsets of native neutrophils and can be employed for long-term in vitro studies. Background and Aims : Neutrophils are key players in the activation and regulation of innate and adaptive immunity. As mediators of the inflammatory response in cardiovascular diseases, the polarity of neutrophils towards pro- and anti-inflammatory phenotypes has recently been advanced. Considering that their lifespan is very limited and long-term in vitro studies are challenging, our aim was to develop and characterize a representative model of human neutrophil-like cells. Methods: Human HL-60 promyeloblasts were cultured for 5 days in complete media supplemented with 1.3% DMSO, then assessed for proliferation, nuclear segmentation by cytochemistry, expression of cell-surface markers by flow cytometry and differentiation markers by qPCR. Polarization of the neutrophil-like cells was induced for 48h towards the pro-inflammatory phenotype (N1) in the presence of LPS and IFNγ, whereas IL-4 was used for the anti-inflammatory phenotype (N2), then assessed for viability and gene expression. Results: Promyeloblasts differentiation was confirmed by down-regulation of TERT expression, strong FPR1 and Integrinβ3 induction (∼300-fold and ∼25-fold over control, respectively) and cell surface expression of CD11b and CD18. Equally, neutrophil-like cells exhibited nuclear segmentation, decrease in proliferation and maintained similar viability in culture. Polarization towards N1 phenotype was confirmed by up-regulation of pro-inflammatory genes MCP-1, IL-1β, TNFα and IRF7, whereas N2 markers CD206 and TGM2 were highly induced following IL-4 stimulation. Cellular viability remained comparable under both treatments. Conclusions: Promyeloblast differentiation by DMSO generates neutrophil-like cells that can be polarized towards N1/N2 phenotypes. These resemble subsets of native neutrophils and can be employed for long-term in vitro studies.