We next investigated if genetic deficiency of Il-1α, TNFα, or C1q would be sufficient to prevent A1 astrocyte reactivity in vivo. First we checked if single knock mice (Il-1α−/−, TNFα−/−, or C1q−/−) were still able to produce neuroinflammatory reactive microglia following systemic LPS injection. Using qPCR we saw microglia from these animals still had many reactive transcripts15 highly upregulated 24 h following LPS injection (Extended Data Fig. 4). We next used astrocytes purified from these same mice and used our microfluidic qPCR screen to determine whether they were reactive. Each knock-out mouse had significantly decreased A1 astrocyte reactivity (Fig. 1e). Additionally, we looked at double (Il-1α−/−TNFα−/−) and triple knock-out mice (Il-1α−/−TNFα−/−C1q−/−) and saw decreases in A1 reactivity, with triple knock-out animals having no response following systemic LPS injection (Fig. 1e). Microglia from these same knock-out mice still upregulate inflammatory mediators in response to LPS injection, but simply fail to release A1 initiators (Extended Data Fig. 4). Taken together our data show that microglia-derived Il-1α, TNFα, and C1q work together to mediate A1 reactive astrocytes.
