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.

Keywords: efferocytosis; integrins; microglia; neutrophils; subarachnoid hemorrhage.

Figure 1.. Neutrophil integrin α9 expression on infiltrated neutrophils and sensorimotor and cognitive recovery following SAH.

A, Schematic of experimental design. SAH was induced in male WT mice using the endovascular perforation model. B and C, Representative cross-sectional immunofluorescence images of the ipsilateral cortex (day 1, post-SAH) from each group. B, Ly-6G[1A8] (green), TL Dye594 (red) and DAPI (blue). Magnification 20X, Scale bar: 50 μm. Right, quantification. n=6, Mann Whitney test. C, Ly-6G[1A8] (green) with α9 (red). Right, quantification. n=6, Mann Whitney test. D Modified Neurological Severity Score (mNSS) performed on day 7, 14 and 28. n=9, two-way ANOVA followed by corrected method of Benjamini and Yekutieli. E Corner and cylinder tests performed on day 7, 14 and 28. n=9, two-way ANOVA followed by corrected method of Benjamini and Yekutieli. F, Y-maze and Novel Object Recognition (NOR) at day 28 post-SAH. n=9, unpaired t-test. Each data point presented represents an individual mouse. Data are mean ± SEM, all individual points and p values are shown.

Figure 2.. Genetic deletion of neutrophil integrin α9 resulted in improved SAH recovery.

A, Schematic of experimental design. SAH was induced in male neutrophil-specific α9^−/− and and α9^fl/fl mice using the endovascular perforation model. B Modified Neurological Severity Score (mNSS), C, corner and D, cylinder tests performed on day 7, 14 and 28. n=16, two-way ANOVA followed by corrected method of Benjamini and Yekutieli, E, Y-maze. F, Novel Object Recognition (NOR) at day 28. n=14–15, unpaired t-test. Each data point presented represents an individual mouse. Data are mean ± SEM, all individual points and p values are shown.

Figure 3.. Mice with neutrophil integrin α9 deficiency exhibited reduced neutrophil infiltration, attenuated neuroinflammation, and reduced MMP-9 following SAH.

A to C, Representative cross-sectional immunofluorescence images (left) of the ipsilateral cortex (day 1, post-SAH) from each group. A, Ly-6G[1A8] (green) and DAPI (blue). B, p-NF-κB (red) and DAPI (blue). C, MMP-9 (pink) and DAPI (blue). Right, quantification. n=6, unpaired t-test. D, Representative image and quantification of terminal deoxynucleotide transferase-deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL; red) and neuron (NeuN; green) staining the ipsilateral cortex (day 1, post-SAH) from each group. n=6, unpaired t-test. Each data point presented represents an individual mouse. Data are mean ± SEM, all individual points and p values are shown, Magnification 20X, Scale bar: 100 μm.

Figure 4.. Integrin α9 dependent neutrophil transendothelial migration impairs efferocytosis.

A, Schematic of experimental design, B, Representative cross-sectional immunofluorescence images (left) showing in-situ efferocytosis in the ipsilateral cortex (day 1, post-SAH) from each group. White arrowhead indicates NeuN⁺ TUNEL⁺ signals not colocalized with IBA1⁺ cells, Yellow Arrow indicates TUNEL⁺ NeuN⁺ signals colocalized with IBA1⁺ cells. Magnification 20X. C, Quantification of efferocytosis index. Efferocytosis index was define as: microglial associated apoptotic neurons/free apoptotic neurons. n=6, unpaired t-test. Magnification 60X, Scale bar: 100 μm. D, Representative cross-sectional immunofluorescence images (left) showing IBA1 positive MerTK expression in the ipsilateral cortex (day 1, post-SAH) from each group. Right, quantification. n=6, unpaired t-test, Magnification 20X, Scale bar: 100 μm. E, schematic of experimental design for efferocytosis assay. F, Representative image of in vitro efferocytosis indicating apoptotic Neuro2A (red) engulfed by BV2 cells (mouse microglial cells). Right, quantification, Magnification 20X, Scale bar: 100 μm. n=5, Mann Whitney test. Each data point presented represents an independent experiment or an individual mouse. Data are mean ± SEM, all individual points and p values are shown. Figure 4E was created with BioRender.com.

Figure 5.. Pharmacological inhibition of integrin α9 improves sensorimotor and cognitive function.

A, Schematic of experimental design. Macitentan was administered to mice as oral suspension in sterile PBS at the dose of 5 mg/kg (30 minutes post-SAH and then once a day for 3 days). B, Modified Neurological Severity Score (mNSS), C, corner and D, cylinder tests performed on day 7, 14 and 28. n=15–17, two-way ANOVA followed by corrected method of Benjamini and Yekutieli, E, Y-maze. F, Novel Object Recognition (NOR) at day 28. n=14–16, unpaired t-test. Each data point presented represents an individual mouse. Data are mean ± SEM, all individual points and p values are shown.

Figure 6.. Pharmacological inhibition of integrin α9 improves SAH recovery by reducing neuroinflammation and enhancing efferocytosis

A-B, Representative cross-sectional immunofluorescence images of the ipsilateral cortex (day 1, post-SAH) from each group. A, Top, Ly-6G[1A8] (green) and DAPI (blue). Bottom, p-NF-κB (red) and DAPI (blue). B, Top, MMP-9 (pink) and DAPI (blue). Bottom, TUNEL (red) and neuron (NeuN; green). n=6, unpaired t-test, Magnification 20X, Scale bar: 100 μm, n=6. C, schematic of experimental design. D, Representative image of in vitro efferocytosis. n=6, unpaired t-test. E, Representative cross-sectional immunofluorescence images (left) showing IBA1 positive MerTK expression in the ipsilateral cortex (day 1, post-SAH) from each group. Right, quantification unpaired t-test. Magnification 20X, Scale bar: 100 μm. Each data point presented represents an independent experiment or an individual mouse. Data are mean ± SEM, all individual points and p values are shown. Figure 6C was created with BioRender.com.

Figure 7.. Macitentan improved SAH outcomes by inhibiting neutrophil integrin

α9 A, Schematic of experimental design. Macitentan was administered to both, neutrophil-specific α9^−/− mice and α9^fl/fl mice as oral suspension in sterile PBS at the dose of 5 mg/kg (30 minutes post-SAH and then once a day for 3 days). B, Modified Neurological Severity Score (mNSS), C, corner and D, cylinder tests performed on day 7, 14 and 28. n=10, two-way ANOVA followed by corrected method of Benjamini and Yekutieli, E, Y-maze. F, Novel Object Recognition (NOR) at day 28. N = 9–10, unpaired t-test. Each data point presented represents an independent experiment or an individual mouse. Data are mean ± SEM, all individual points and p values are shown.