Background: Obesity-induced hyperglycemia is a significant risk factor for stroke. Integrin α9β1 is expressed on neutrophils and stabilizes adhesion to the endothelium via ligands, including Fn-EDA (fibronectin containing extra domain A) and tenascin C. Although myeloid deletion of α9 reduces susceptibility to ischemic stroke, it is unclear whether this is mediated by neutrophil-derived α9. We determined the role of neutrophil-specific α9 in stroke outcomes in a mice model with obesity-induced hyperglycemia.
Methods: α9Neu-KO (α9fl/flMRP8Cre+) and littermate control α9WT (α9fl/flMRP8Cre-) mice were fed on a 60% high-fat diet for 20 weeks to induce obesity-induced hyperglycemia. Functional outcomes were evaluated up to 28 days after stroke onset in mice of both sexes using a transient (30 minutes) middle cerebral artery ischemia. Infarct volume (magnetic resonance imaging) and postreperfusion thrombo-inflammation (thrombi, fibrin, neutrophil, phospho-nuclear factor kappa B [p-NFκB], TNF [tumor necrosis factor]-α, and IL [interleukin]-1β levels, markers of neutrophil extracellular traps) were measured post 6 or 48 hours of reperfusion. In addition, functional outcomes (modified Neurological Severity Score, rota-rod, corner, and wire-hanging test) were measured for up to 4 weeks.
Results: Stroke upregulated neutrophil α9 expression more in obese mice (P<0.05 versus lean mice). Irrespective of sex, deletion of neutrophil α9 improved functional outcomes up to 4 weeks, concomitant with reduced infarct, improved cerebral blood flow, decreased postreperfusion thrombo-inflammation, and neutrophil extracellular traps formation (NETosis) (P<0.05 versus α9WT obese mice). Obese α9Neu-KO mice were less susceptible to thrombosis in FeCl3 injury-induced carotid thrombosis model. Mechanistically, we found that α9/cellular fibronectin axis contributes to NETosis via ERK (extracellular signal-regulated kinase) and PAD4 (peptidyl arginine deiminase 4), and neutrophil α9 worsens stroke outcomes via cellular fibronectin-EDA but not tenascin C. Obese wild-type mice infused with anti-integrin α9 exhibited improved functional outcomes up to 4 weeks (P<0.05 versus vehicle).
Conclusions: Genetic ablation of neutrophil-specific α9 or pharmacological inhibition improves long-term functional outcomes after stroke in mice with obesity-induced hyperglycemia, most likely by limiting thrombo-inflammation.
Keywords: hyperglycemia; ischemic stroke; mice; neutrophils; obesity; risk factor.