Neutrophil Gas6 expression is involved in neutrophil recognition by microglia for phagocytosis

Abstract

Background: Ischemic stroke induces a strong inflammatory response in the brain involving leukocyte infiltration. Microglia, the resident innate immune cells of the central nervous system, phagocytose infiltrating neutrophils1,2. However, the signals involved in neutrophil recognition are not well-known. The objective of this study was to investigate the involvement of the ¿eat-me¿ signal Gas6 in phagocytosis of neutrophils by microglia.

Methods: We used wild type (wt) mice, Gas6-/- mice, and DsRed reporter mice. We isolated microglia from the adult mouse brain and maintained the cells in culture for 7 days. We then exposed Gas6+/+ or Gas6-/- microglia to Gas6+/+ or Gas6-/- bone marrow-derived neutrophils, and studied the cells with a multiposition time-lapse microscopy for 10-14h. Cultures of Gas6+/+ and Gas6-/- cells were run in parallel in n=3 independent experiments with 2 replicates per genotype in each experiment. We quantified events by cell tracking with ImageJ.

Results: Compared to Gas+/+ neutrophils, phagocytosis of Gas6-/- neutrophils by either Gas+/+ or Gas6-/- microglia was reduced (40%) regardless of microglia genotype (two-way ANOVA by neutrophil genotype and microglia genotype, neutrophil genotype p< 0.0001). We also observed signs of neutrophil NETosis, i.e. apparent restructuration of the nucleus, clear expulsion of the intracellular content, increase in cell size, and loss of fluorescence, likely due to enzymatic degradation. We estimated that approximately 10% of neutrophils suffered NETosis. At this point cells looked empty, devoid of cellular organelles and lacked DNA. These structures remained in the culture but we observed that some of them (15%) were eventually engulfed by microglia.

Conclusion: Microglia recognize and phagocyte neutrophils, including neutrophils that have undergo NETosis. Neutrophil Gas6 expression mediates neutrophil recognition by microglia for phagocytosis.