Neutrophil Integrin α9 Impairs Efferocytosis and Worsens Long-Term Recovery After Subarachnoid Hemorrhage.

Abstract

Background: Neutrophil infiltration exacerbates brain injury after subarachnoid hemorrhage (SAH). Integrin α9, expressed on neutrophils, facilitates their adhesion and transendothelial migration, leading to aggravated inflammatory responses and neuronal apoptosis. Insufficient clearance of apoptotic neurons by microglia and infiltrating blood-derived macrophages (defective efferocytosis) contributes to persistent inflammation and poor SAH recovery. This study investigated the role of neutrophil integrin α9 in neuronal apoptosis, microglia/macrophage efferocytosis, and SAH outcomes.

Methods: Neutrophil-specific α9^-/- (α9^fl/flMrp8Cre^-/+) and littermate control (α9^fl/flMrp8Cre^-/-) mice were subjected to the endovascular perforation model to induce SAH. Sensorimotor and cognitive function were assessed for up to 4 weeks post-SAH using neurological severity score, corner and cylinder tests, Y-maze, and novel object recognition. In vitro and in vivo functional assays were conducted to assess the effect of integrin α9-dependent neutrophil transendothelial migration on efferocytosis of apoptotic neurons. Neutrophil infiltration, cerebral inflammation, neuronal apoptosis, and MMP (matrix metalloproteinase)-9 were quantified 24 hours post-SAH.

Results: Mice subjected to SAH exhibited increased integrin α9 levels on infiltrated neutrophils compared with sham surgery controls. Neutrophil-specific α9^-/- mice demonstrated improved long-term sensorimotor and cognitive recovery, reduced neutrophil infiltration, and decreased MMP-9 expression and neuronal apoptosis. Importantly, neutrophil-specific α9^-/- mice exhibited reduced brain neutrophil elastase levels and enhanced efferocytosis. Mechanistic studies have revealed that the reduced transendothelial migration of α9^-/- neutrophils directly contributed to the enhanced microglia/macrophage efferocytosis of apoptotic neurons. Pharmacological targeting of integrin α9 with macitentan significantly improved SAH outcomes by reducing neutrophil infiltration and enhancing efferocytosis. Comparable SAH outcomes in both macitentan-treated controls and neutrophil-specific α9^-/- mice suggested that the therapeutic effects of macitentan were mediated by inhibition of neutrophil integrin α9.

Conclusions: Our study revealed a novel role for neutrophil integrin α9 in sensorimotor function and cognitive recovery after SAH, suggesting it as a potential therapeutic target for SAH.