PU.1 target genes are implicated in various biological processes of myeloid cells that may modulate AD risk. For example, a microglial gene network for pathogen phagocytosis has been previously implicated in the etiology of AD18. We modulated levels of PU.1 by Spi1 cDNA overexpression or shRNA knock-down in BV2 mouse microglial cells, and used zymosan bioparticles labeled with pHrodo (a pH-sensitive dye that emits a fluorescent signal when internalized in acidic vesicles during phagocytosis) to measure pathogen engulfment. Analysis of zymosan uptake by flow cytometry revealed that phagocytic activity is augmented in BV2 cells overexpressing PU.1 (Fig. 3a), while knock-down of PU.1 resulted in decreased phagocytic activity (Fig. 3a). We confirmed overexpression and knock-down of PU.1 expression levels by western blotting and qPCR (Fig. 3). Phagocytic activity was not changed in untransfected cells when analyzed by flow cytometry (Supplementary Fig. 8d, 8e, 8f, 8g). These data suggest that modulation of PU.1 expression levels significant changes microglial phagocytic activity in response to fungal targets (mimicked by zymosan).
