PU.1 expression levels regulate multiple myeloid/microglial cell functions47, including proliferation, survival and differentiation, that could also modulate AD risk. Indeed, expression of Il34 and Csf1, soluble factors that bind to Csf1r and required for microglial development and maintenance in vivo49, were elevated after knock-down of Spi1, while expression of Csf1r was reduced (Fig. 4a, 4c). Interestingly, inhibition of Csf1r in a 3xTg-AD mouse model led to a reduction in the number of microglia associated with β-amyloid plaques and improved cognition50. These findings suggest the importance of analyzing cell proliferation, survival, differentiation, and migration phenotypes with differential Spi1 expression, because Spi1 levels modulate expression of Ccl2 and Cxcl2 (Fig. 4a), which are MCP1 and MIP2α proteins that help recruitcirculating monocytes and neutrophils to the brain to promote neuroinflammation. In addition, knocking down Spi1 reduced expression of a microgliosis marker Aif1 (Iba1) along with Il1b, Nos2, Ptgs2, Arg1 and Nlrp3 (Fig. 4a, 4c), suggesting that decreased Spi1 expression may blunt the pro-inflammatory response of microglial cells to improve disease outcomes. Interestingly, expression of Cx3cr1 and Axl were elevated upon knock-down of Spi1
