To determine what microglia-secreted signals induce A1s, we next performed a screen to individually test various candidate molecules. We used immunopanning18 to prepare highly pure populations of resting (non-reactive) astrocytes (Extended Data Fig. 2a,b). We cultured purified astrocytes in serum-free conditions and tested effects of various molecules on gene expression using our microfluidic assay. As a control, we first investigated if astrocytes in culture can respond to LPS and found they do not (Extended Data Fig. 2). This was expected as rodent astrocytes lack receptors and downstream signaling components required for LPS-activation (TLR4 and MYD88)12–14. We found however, that several cytokines could induce some, but not all, A1 reactive genes. Our best inducers of a partial A1 phenotype were interleukin 1 alpha (Il-1α), tumor necrosis factor alpha (TNFα), and complement component 1, q subcomponent (C1q,). When purified astrocytes were cultured with all three cytokines, astrocytes exhibited an A1 phenotype nearly identical to the A1 phenotype induced by LPS in vivo (Fig. 1a). All three of these cytokines are highly expressed specifically by microglia13,15, again suggesting a critical role for microglia in inducing A1 reactive astrocytes.
