Abstract Body: BACKGROUND: Neutrophil infiltration plays a pivotal role in exacerbating brain injury following subarachnoid hemorrhage (SAH). Integrin α9, highly expressed on activated neutrophils, facilitates their adhesion and transendothelial migration. Once infiltrated, neutrophils aggravate inflammatory response and can impair microglial efferocytosis. Defective efferocytosis may results in accumulation of apoptotic cells, persistent inflammation, and resultant poor recovery following SAH. Despite the known role of integrin α9 in promoting cerebral inflammation, the impact neutrophil integrin α9 on microglial efferocytosis and subsequent SAH outcomes remains unknown.
Hypothesis: Integrin α9 dependent neutrophil transendothelial migration impedes microglial efferocytosis, promotes cerebral inflammation, and mediates poor SAH recovery.
METHODS: Neutrophil specific integrin α9 knockout mice (α9^fl/flMrp8Cre^+/-) and littermate controls (α9^fl/flMrp8Cre^-/-) were subjected to the endovascular perforation model to induce SAH. Sensorimotor tests (modified neurological severity score, corner, and cylinder test) and cognitive function tests (Y-maze and novel object recognition test) were performed up to 4-weeks. Neutrophil infiltration, inflammation, apoptosis, and blood brain barrier (BBB) disruption were quantified 24-hr post-SAH. In vitro and in vivo functional assays were carried out to assess the neutrophil integrin α9-dependent effects on microglial efferocytosis.
RESULTS: Mice with SAH exhibited increased integrin α9 levels on infiltrated neutrophils as compared to mice with sham-surgery. α9^fl/flMrp8Cre^+/- mice showed improved sensorimotor and cognitive recovery (up to 4-weeks) (Fig 1), reduced neutrophil infiltration, BBB damage, neuronal apoptosis as compared to controls. α9^fl/flMrp8Cre^+/- mice exhibited reduced brain neutrophil elastase levels along with enhanced microglial efferocytosis of apoptotic neurons. In vitro mechanistic studies revealed that reduced transendothelial migration of α9^-/- neutrophils directly contributed to the enhanced microglial efferocytosis of apoptotic neurons (Fig 2). Pharmacological targeting of integrin α9 with macitentan significantly improved SAH outcomes, reduced neutrophil infiltration, BBB damage, neuronal apoptosis, and showed enhanced microglial efferocytosis (Fig 3).
CONCLUSIONS: Our study uncovered a previously unrecognized role of neutrophil integrin α9 in microglial efferocytosis and post-SAH sensorimotor and cognitive recovery.