Mechanisms of Spns2 in regulating neuroinflammatory activation of microglia after cerebral ischemia in rats.

Microglia, the resident innate immune cells of the brain, maintain central nervous system homeostasis but can also mediate inflammatory injury after ischemic stroke. Following ischemia, microglia can adopt pro-inflammatory M1- or reparative M2-like state; thus, regulating the balance between these phenotypes may be therapeutically relevant. Sphingosine-1-phosphate signaling regulates immune responses, angiogenesis, and endothelial barrier integrity. Enhanced sphingosine-1-phosphate signaling in activated microglia after ischemic injury may amplify inflammatory cascades. However, strategies targeting sphingosine-1-phosphate signaling are constrained by the heterogeneous expression of sphingosine-1-phosphate receptors. Spinster homolog 2 (Spns2), a sphingosine-1-phosphate exporter, may provide an alternative target, although its role in ischemic stroke remains unclear. To investigate the role of Spns2 in post-ischemic neuroinflammation, middle cerebral artery occlusion/reperfusion was induced in Spns2-deficient (Spns2-/-) and wild-type rats. BV2 microglial cells exposed to oxygen-glucose deprivation/reoxygenation were used as an in vitro model. Spns2 mRNA expression in the peri-infarct penumbra increased after middle cerebral artery occlusion/reperfusion and peaked at 24 hours. Compared with wild-type rats, Spns2-/- rats showed lower neurological deficit scores, improved motor performance, and smaller infarct volumes. In the peri-infarct penumbra, Spns2 deficiency was associated with decreased expression of pro-inflammatory markers, increased expression of anti-inflammatory markers, and attenuated microglia-associated NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome signaling. In BV2 cells, Spns2 knockdown produced similar changes in inflammatory-marker expression and reduced oxygen-glucose deprivation/reoxygenation-induced NLRP3 inflammasome-associated signaling and oxidative stress. Spns2 knockdown reduced extracellular sphingosine-1-phosphate levels in BV2-cell culture supernatants, whereas exogenous sphingosine-1-phosphate supplementation increased the expression of inflammasome-associated inflammatory mediators in Spns2-knockdown BV2 cells. RNA sequencing identified Itga5 upregulation in Spns2-/- rats and showed that differentially expressed genes between Spns2-/- and wild-type rats were enriched in the phosphoinositide 3-kinase/protein kinase B (Akt) signaling pathway. Increased Akt phosphorylation in Spns2-/- rats was further confirmed by western blotting. These findings indicate that Spns2 deficiency limits sphingosine-1-phosphate export and attenuates microglia-associated neuroinflammation after ischemic stroke. Itga5 upregulation and increased Akt phosphorylation may be associated with this neuroprotective effect. Collectively, our results provide a basis for further investigation of the Spns2-sphingosine-1-phosphate axis in ischemic stroke.