Targeting Neutrophil α9 Improves Functional Outcomes After Stroke in Mice With Obesity-Induced Hyperglycemia

Abstract

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: α9^Neu-KO (α9^fl/flMRP8Cre⁺) and littermate control α9^WT (α9^fl/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 α9^WT obese mice). Obese α9^Neu-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.

Figure 1:. Stroke upregulated α9 expression.

(A) Schematic of experimental design. (B) Representative immunoblots and densitometric analysis of α9 expression in neutrophils following stroke onset in wild-type (WT) mice fed a chow diet (CD) and high-fat diet (HFD). n=6,6. (C) Representative flow cytometry and fold change in mean fluorescence intensity (MFI) of α9 expression. n=8,7,8,7. (D) Fold change in MFI of Neutrophil CD11b (Left) and elastase level (right) following stroke onset. n=6,6,6,6. (E) Representative immunoblot of α9 from the bone-marrow-derived neutrophils of the α9^WT and α9^Neu-KO mice. #1 and #2 represent samples from two individual mice and β-actin as a loading control. n=4,4. Data are mean ± SD. Statistical analysis: two-way ANOVA followed by Holm-Sidak multiple comparisons test (B-D). NS: non-significant.

Figure 2.. Neutrophil-specific deletion of α9 improves stroke outcome in male obese mice.

(A) Schematic of experimental design. (B) Representative T2-MRI images (left) from one mouse on day 2 and mean infarct (middle) of each genotype. White (demarcated by yellow dots) is the infarct area. n=14,14. Right: Modified Neurological Severity Score (mNSS) in the same cohort of mice up to weeks 4 (a higher score indicates a better outcome). n=13,11 (week 1) & 11,10 (week 2-4). (C-E) Sensorimotor recovery in the same cohort of mice as analyzed by motor strength in the hanging-wire test (C), fall latency in the accelerated rota-rod test (D), and right turn ratio in the corner test (E). n=13,11 (week 1) & 11,10 (week 2-4). (F) Survival (%) up to day 28. Data are mean ± SD (infarct) and median ± range (functional outcome). Statistical analysis: unpaired t-test (infarct), two-way ANOVA followed by Holm-Sidak multiple comparisons test (functional outcome). The comparison of survival curves was evaluated by the log-rank (Mantel-Cox) test. NS: non-significant.

Figure 3.. Neutrophil α9^−/− obese mice exhibited improved local cerebral blood flow and reduced post-ischemia/reperfusion thrombosis.

(A) Left: Representative images were taken using laser speckle imaging of the cortical region's regional cerebral blood flow (CBF). Right: Quantification at different time points. n=8,8. (B) Representative Western blots of brain homogenates and densitometric analysis of platelets (CD4-positive) and fibrin(ogen) from the infarcted and peri-infarcted areas. β-Actin was used as a loading control. n=5,5. (C) Representative microphotographs of thrombus growth in FeCl₃-injured carotid arteries as visualized by upright intravital microscopy. Platelets were labeled with calcein green. White lines delineate the arteries. Right: Mean time to complete occlusion. n=10,10. (D) The tail bleeding time was determined by the time taken for the initial cessation of bleeding after transection, n=8,8. Data are mean ± SD. Statistical analysis: two-way ANOVA followed by Holm-Sidak multiple comparisons test (A), Mann-Whitney test (B-D). NS: non-significant.

Figure 4.. Neutrophil-specific deletion of α9 limits post-ischemic inflammation and NETosis.

(A) Quantification of elastase, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) levels in peripheral neutrophils 6 hours post-reperfusion by ELISA. n=6,6. (B) Plasma level of MPO-DNA complexes (left) and DNase activity (middle) 6 hours post-reperfusion. n=6,6. (C) BM derived neutrophils were stimulated with pFn or cFn (20 μg/ml) for 5 mins. Fluorometric quantitation of intracellular calcium flux using Fura-2 AM loaded neutrophils. n= 6,6, 6, 6. (D) BM derived neutrophils were stimulated with pFn or cFn (20 μg/ml) for 15 min (ERK1/2) and 3 h (PAD4). Left: Representative immunoblots and densitometric analysis of p-ERK1/2 and PAD4 in neutrophils lysates. β-Actin was used as a loading control. Right: Quantification. n=6,6,6,6. (E) BM derived neutrophils were stimulated with pFn or cFn (20 μg/ml). NETosis (left) was detected by SYTOX green fluorescence. Quantification of MPO-DNA complexes (right) by ELISA. n=6,6,7,6. (F) U0126 (10 μM) (left) and GSK484 (10 μM) (right) were added 30 min before cFn incubation. NETosis was detected by SYTOX green fluorescence. n=6,6,6,6,6,6. Data are mean ± SD. Statistical analysis: unpaired t-test (A), one-way ANOVA followed by Dunnett’s multiple comparisons tests (B), two-way ANOVA followed by Holm-Sidak multiple comparisons test (C-F). NS: non-significant.

Figure 5.. Fn-EDA contributes to neutrophil α9-mediated stroke exacerbation in obese mice.

(A) Schematic of experimental design. (B) Representative T2-MRI images (left) from one mouse of each genotype on day 2 and mean infarct (middle) of each genotype. White (demarcated by yellow dots) is the infarct area. n=11,13,7,12. Right: Modified Neurological Severity Score (mNSS) in the same cohort of mice at day 7. n=10,12,7,12. (C-E) Sensorimotor recovery in the same cohort of mice as analyzed by motor strength in the hanging-wire test (C), fall latency in the accelerated rota-rod test (D), and right turn ratio in the corner test (E). n=10,12,7,12. (F) Survival (%) up to day 28. Data are mean ± SD (infarct) and median ± range (functional outcome). Statistical analysis: two-way ANOVA followed by Holm-Sidak multiple comparisons test. The comparison of survival curves was evaluated by the log-rank (Mantel-Cox) test. NS: non-significant.

Figure 6.. Anti-integrin α9 antibody-treated obese mice exhibited improved long-term stroke outcomes.

(A) Schematic of experimental design. (B) Representative T2-MRI from one mouse of each group on day 2 (left) and corrected mean infarct of each genotype (middle). White (demarcated by yellow dots) is the infarct area. n=14,14. Modified Neurological Severity Score (mNSS) in the same cohort of mice up to weeks 4 (right) n=12,14. (C-E) Sensorimotor recovery in the same cohort mice as analyzed by motor strength in the hanging-wire test (C), fall latency in the accelerated rota-rod test (D), and right turn ratio in the corner test (E). n=12,14. (F) Survival rates up to day 28. Data are represented as mean ± SD (infarct) and median ± range (functional outcome). Statistical analysis: unpaired t-test (infarct area), two-way ANOVA followed by Holm-Sidak multiple comparisons test (functional outcome). The comparison of survival curves was evaluated by the log-rank (Mantel-Cox) test. NS: non-significant.