Does cessation of Il-1α, TNFα, and C1q signaling enable A1 reactive astrocytes in vitro to revert back to resting astrocytes or is the A1 phenotype stable? To find out, we removed all three cytokines from A1 cultures, and added neutralizing antibodies to all three to make sure they were fully inhibited. After 7 days, we assessed levels of A1 transcripts and found the A1 phenotype remained. As a proof of principal, we also investigated if additional molecules could revert A1s to a non-reactive phenotype. We tested the anti-inflammatory cytokine TGFβ and FGF (as it has been previously shown that astrocyte activation is suppressed in the injured brain by FGF signaling19). We grew A1s in culture, then treated with TGFβ or FGF and found both significantly decreased reactive astrocyte transcript levels (Fig. 1d, Extended Data Fig. 3). Whether or not there are additional signaling processes that can revert A1s in vivo is an important question for future studies.
