In support of this hypothesis, we also demonstrate that changes in PU.1 expression levels alter phagocytic activity in BV2 mouse microglial cells (Fig. 3, Supplementary Fig. 8). Knock-down of PU.1 expression reduced engulfment of zymosan, whereas overexpression of PU.1 increased engulfment of zymosan, a Toll-like receptor 2 (TLR2) agonist that mimics fungal pathogens. This is in line with previous data showing decreased uptake of Aβ42 (also a TLR2 agonist) in primary microglial cells isolated from adult human brain tissue and transfected with siRNA targeting SPI147. Interestingly, several AD-associated genes (e.g., CD33, TYROBP, TREM2, TREML2, CR1, ABCA7, APOE, CLU/APOJ) have been shown to be involved in the phagocytic clearance of pathogens or host-derived cellular material (e.g., β-amyloid, apoptotic cells, myelin debris, lipoproteins, etc.), suggesting a strong link between perturbation of microglial phagocytosis and AD pathogenesis. In addition to Cd33, Tyrobp, Apoe and Clu/ApoJ, several genes with roles in phagocytosis are dysregulated by altering Spi1 expression, i.e. Cd36, Fcgr1, P2ry12, Itgam, Cx3cr1, Axl, Ctsb (Fig. 4a, 4b, 4c), suggesting a collective and coordinated effect of Spi1 on the phagocytic activity of BV2 cells.
