Microglia subtypes show substrate- and time-dependent phagocytosis preferences and phenotype plasticity

Abstract

Microglia are phagocytosis-competent CNS cells comprising a spectrum of subtypes with beneficial and/or detrimental functions in acute and chronic neurodegenerative disorders. The heterogeneity of microglia suggests differences in phagocytic activity and phenotype plasticity between microglia subtypes. To study these issues, primary murine glial cultures were cultivated in the presence of serum, different growth factors and cytokines to obtain M0-like, M1-like, and M2-like microglia as confirmed by morphology, M1/M2 gene marker expression, and nitric oxide assay. Single-cell analysis after 3 hours of phagocytosis of E.coli particles or IgG-opsonized beads showed equal internalization by M0-like microglia, whereas M1-like microglia preferably internalized E.coli particles and M2-like microglia preferably internalized IgG beads, suggesting subtype-specific preferences for different phagocytosis substrates. Time-lapse live-cells imaging over 16 hours revealed further differences between microglia subtypes in phagocytosis preference and internalization dynamics. M0- and, more efficiently, M1-like microglia continuously internalized E.coli particles for 16 hours, whereas M2-like microglia discontinued internalization after approximately 8 hours. IgG beads were continuously internalized by M0- and M1-like microglia but strikingly less by M2-like microglia. M2-like microglia initially showed continuous internalization similar to M0-like microglia but again discontinuation of internalization after 8 hours suggesting that the time of substrate exposure differently affect microglia subtypes. After prolonged exposure to E.coli particles or IgG beads for 5 days all microglia subtypes showed increased internalization of E.coli particles compared to IgG beads, increased nitric oxide release and up-regulation of M1 gene markers, irrespectively of the phagocytosis substrate, suggesting phenotype plasticity. In summary, microglia subtypes show substrate- and time-dependent phagocytosis preferences and phenotype plasticity. The results suggest that prolonged phagocytosis substrate exposure enhances M1-like profiles and M2-M1 repolarization of microglia. Similar processes may also take place in conditions of acute and chronic brain insults when microglia encounter different types of phagocytic substrates.