Ischemia/Reperfusion Induces Interferon-Stimulated Gene Expression in Microglia

Ischemic preconditioning induces cortical microglial proliferation and a transcriptomic program of robust cell cycle activation

“…Other specific proliferation associated genes of note that were markedly upregulated in cortical microglia by IPC included the microglial/macrophage homeostasis factor/mitogen colony stimulating factor 1 ( csf1 ) and the gene encoding the microglial/macrophage in vivo activation marker translocator protein ( tspo ). As we reported previously (McDonough et al, ) IPC also induces significant microglial expression of multiple type 1 ISGs of which several are specifically associated with cellular proliferation, including chemokine cxcl10 and interferon regulatory factor 7 ( irf7 ) and which are found in Table . Bioinformatic gene ontology analysis revealed that the top molecular and cellular function categories for regulation were for genes associated with cell cycle, cellular assembly and organization, DNA replication/recombination/repair, and cellular growth/proliferation (Table ).…”

“…To investigate the effects of IPC on the microglial transcriptome we performed 15 min tMCAO and then acutely isolated and analyzed gene expression in cortical microglia 72 hr following reperfusion at the peak of IPC‐mediated neuroprotection in mice (Zhang et al, ). As previously described (McDonough et al, ; McDonough & Weinstein, ; Su et al, ) we used ex vivo flow cytometry sorting to isolate microglia from the cerebral cortex ipsilateral or contralateral (Figure a) to the prior tMCAO (or sham) surgery for RNA extraction and subsequent microarray analysis (see Section 2). Our microglial isolation protocol using ex vivo flow cytometry allows us to distinguish microglia from infiltrating macrophage subsets, neutrophils, and other resident CNS cell types based on a panel of cell surface markers (Figure a; McDonough & Weinstein, ).…”

“…Ipsilateral and contralateral cortices were dissected out separately and then mechanically and enzymatically dissociated as previously described (McDonough et al, ; Su et al, ). Depletion of myelin and positive selection of myeloid cells with a CD11b microbead column (Miltenyi Biotec) were performed as described (McDonough et al, ; Su et al, ). Myeloid enriched cell suspension was stained with a panel of fluorochrome conjugated antibodies (BD Pharmingen, San Diego, CA; eBioscience, Inc., San Diego, CA; Fisher Scientific, Kent, WA) against microglia, macrophage, and neutrophil markers as well as 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride (DAPI; Sigma‐Aldrich, St. Louis, MO) and flow cytometrically sorted on a BD ARIA II cell sorter directly into TRIzol LS reagent (Invitrogen).…”

“…Myeloid enriched cell suspension was stained with a panel of fluorochrome conjugated antibodies (BD Pharmingen, San Diego, CA; eBioscience, Inc., San Diego, CA; Fisher Scientific, Kent, WA) against microglia, macrophage, and neutrophil markers as well as 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride (DAPI; Sigma‐Aldrich, St. Louis, MO) and flow cytometrically sorted on a BD ARIA II cell sorter directly into TRIzol LS reagent (Invitrogen). Microglia were defined as the population of live (DAPI negative ) cells that were Ly6C negative , Ly6G negative , F4/80 positive , CD45 low/intermediate as we have previously reported (McDonough et al, ; Su et al, ). Cellular identity of sorted cortical microglia was confirmed by our transcriptional analysis on extracted microglial RNA showing high levels of expression (>95% of all genes in microarray) for multiple known microglia specific genes (Butovsky et al, ) including P2ry12 , Fcrls , Tmem119 , Hexb , and Tgfbr1 in both control (sham‐operated) and experimental (tMCAO) WT mice.…”

“…Multiple preconditioning stimuli have converged on the requirement for type I interferon stimulated genes (ISGs) for establishing preconditioning (Stevens et al, ). We have demonstrated that type I IFN signaling specifically in myeloid cells is required for IPC‐mediated axonal protection in white matter (Hamner et al, ), and in a separate study focusing on transcriptomal profiling of microglial gene expression following in vivo exposure to an IPC stimulus we demonstrated that the cortical microglia transcriptome showed marked type I IFN receptor (IFNAR1)‐dependent expression of ISGs (McDonough et al, ). These studies provide cell type‐specific context to earlier studies implicating type 1 IFN signaling in unsorted whole‐brain tissue.…”