TGFβ-activated kinase 1-dependent microglial and macrophage responses aggravate long-term outcomes after ischemic stroke

Microglia/macrophages (Mi/MΦ) can profoundly influence stroke outcomes by acquiring functionally dominant phenotypes (pro-inflammatory or anti-inflammatory; neurotoxic or neuroprotective). Identification of the molecular mechanisms that dictate the functional status of Mi/MΦ after brain ischemia/reperfusion may reveal novel therapeutic targets for stroke. We hypothesized that activation of TGFβ-activated kinase 1 (TAK1), a key MAP3K upstream of multiple inflammation-regulating pathways, drives Mi/MΦ towards a pro-inflammatory phenotype and potentiates ischemia/reperfusion brain injury. Young adult mice were subjected to 1 h of middle cerebral artery occlusion (MCAO) followed by reperfusion. TAK1 was targeted by tamoxifen-induced Mi/MΦ-specific knockout (mKO). Mi/MΦ functional status and brain inflammatory profiles were assessed 3 days after MCAO by RNA-seq of flow cytometry-sorted brain Mi/MΦ. The results showed that TAK1 promotes ischemia/reperfusion-induced inflammation, brain injury, and maladaptive behavior by enhancing pro-inflammatory and neurotoxic Mi/MΦ responses. TAK1 mKO mice were generated by crossing the Cx3cr1 CreER mice and Map3k7 flox mice for 2 generations. Both strains were on a C57BL/6 background. The full genotype for the TAK1 mKO mice was Cx3cr1(CreER/wt);Map3k7(flox/flox). Hemizygous Cx3cr1 CreER mice served as age- and sex-matched wild-type (WT) controls (genotype Cx3cr1(CreER/wt);Map3k7(wt/wt)). TAK1 mKO mice and WT control mice received intraperitoneal injections of tamoxifen (75 mg/kg/day for 4 days) to induce TAK1 knockout. Forteen days after the initial tamoxifen injection, TAK1 mKO mice and WT control mice (8-10 weeks old, male) were subjected to transient focal cerebral ischemia induced by occlusion of the left middle cerebral artery for 1 hour, followed by reperfusion. The CD11b+CD45low (microglia) and CD11b+CD45high (macrophage) cell populations were collected by fluorescence-activated cell sorting from the left (ipsilesional) brain hemisphere 3 days after ischemic stroke. There were 4 groups: WT microglia, WT macrophage, TAK1 mKO microglia, and TAK1 mKO macrophage. In each group there were 2-3 biological replicates. For microglia, each sample was from 1 mouse brain. For macrophages, each sample was pooled from 3 brains to obtain enough cells. The sorted cells were subjected to mRNA sequencing.