Effects of Th17 cytokines on transcriptome of primary microglial cultures in vitro

Peripheral infections can result in neuropsychiatric changes in many contexts, including after recurrent Group A Streptococcus (GAS) infections in children. In a mouse model of intranasal GAS inoculation, we have previously demonstrated in vivo that mice lacking Th17 cells, or the key Th17 cytokines interleukin 17A (IL-17A) or granulocyte-macrophage colony-stimulating factor (GM-CSF), have altered microglial responses. As an attempt to determine whether these cytokines have direct effects on microglia, we cultured primary microglia and incubated them with either interferon gamma (IFNg), IL-17A or GM-CSF for 24 hours, then collected RNA for bulk sequencing. Microglia treated with IFNg or GM-CSF displayed striking transcriptional shifts, including upregulation of many inflammatory genes. IL-17A treatment did not have a noticeable effect on the microglial transcriptome, likely due to the in vitro absence of IL-17A receptors, which are expressed by microglia in vivo. Overall design: Mixed glial cultures were obtained from forebrains of P0-P2 pups. After 7-14 days, microglia were obtained using a shake off protocol and cultured in serum-free media. Cultures were incubated with 100 ng/mL of either IFNg, IL-17A or GM-CSF for 24 hours. Cells were then collected into TRIzol reagent for bulk sequencing.