Abstract 18: Targeting Neutrophil Extracellular Traps Improves Ischemic Stroke Outcomes

Abstract

Rationale: Ischemic stroke prompts an inflammatory response which is associated with worse outcomes. Classic anti-inflammatory strategies were unsuccessful in clinical trials, implying other mechanisms contribute to injurious inflammation in stroke. In response to stimuli, neutrophils can release DNA web-like structures called neutrophil extracellular traps (NETs). Recently, a role for NETs in cardiovascular disease has emerged. Here, we studied whether NETs contribute to ischemic stroke outcomes. Methods: Markers of NET formation were assessed in brain tissue and plasma from ischemic stroke patients. For murine studies, we used male and female mice that were subjected to transient middle cerebral artery occlusion. Stroke outcomes were assessed 24 hours or 7 days after stroke. Results: NETs were found in brain tissue from deceased ischemic stroke patients. Ischemic stroke patients had significantly increased plasma biomarkers of NET formation including citrullinated histone H3 (p<0.0001) and MPO-DNA complexes (p<0.001) compared to matched controls. NET biomarkers positively correlated with worse stroke outcomes at discharge (p<0.05) while they did not correlate with stroke severity at admission. To target NET formation in ischemic stroke, we investigated the therapeutic potential of a recently discovered neonatal NET inhibitory factor (nNIF). nNIF specifically blocks NET formation in human and murine neutrophils without affecting other critical neutrophil functions. Mice prophylactically treated with nNIF had significantly reduced brain and plasma NETs after stroke while cerebral neutrophil recruitment remained unaffected. The reduction in NET formation was associated with significantly reduced neuronal apoptosis and smaller brain infarcts (p<0.0001). Furthermore, nNIF treated mice had improved neurological behavior and motor function, and enhanced 7-day survival after ischemic stroke (p<0.001). Importantly, these results were confirmed in diabetic mice and >18-month-old mice and nNIF was still effective when administered therapeutically, 1 hour after stroke onset. Conclusions: Our results support a pathological role for NETs in ischemic stroke and warrant further investigation into nNIF to improve stroke outcomes.