Neutrophil extracellular traps regulate ischemic stroke brain injury
Frederik Denorme, … , Christian C. Yost, Robert A. Campbell
Frederik Denorme, … , Christian C. Yost, Robert A. Campbell
Published March 31, 2022
Citation Information: J Clin Invest. 2022;132(10):e154225. https://doi.org/10.1172/JCI154225.
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Research Article Hematology

Neutrophil extracellular traps regulate ischemic stroke brain injury

Abstract

Ischemic stroke prompts a strong inflammatory response, which is associated with exacerbated outcomes. In this study, we investigated mechanistic regulators of neutrophil extracellular trap (NET) formation in stroke and whether they contribute to stroke outcomes. NET-forming neutrophils were found throughout brain tissue of ischemic stroke patients, and elevated plasma NET biomarkers correlated with worse stroke outcomes. Additionally, we observed increased plasma and platelet surface–expressed high-mobility group box 1 (HMGB1) in stroke patients. Mechanistically, platelets were identified as the critical source of HMGB1 that caused NETs in the acute phase of stroke. Depletion of platelets or platelet-specific knockout of HMGB1 significantly reduced plasma HMGB1 and NET levels after stroke, and greatly improved stroke outcomes. We subsequently investigated the therapeutic potential of neonatal NET-inhibitory factor (nNIF) in stroke. Mice treated with nNIF had smaller brain infarcts, improved long-term neurological and motor function, and enhanced survival after stroke. nNIF specifically blocked NET formation without affecting neutrophil recruitment after stroke. Importantly, nNIF also improved stroke outcomes in diabetic and aged mice and was still effective when given 1 hour after stroke onset. These results support a pathological role for NETs in ischemic stroke and warrant further investigation of nNIF for stroke therapy.

Authors

Frederik Denorme, Irina Portier, John L. Rustad, Mark J. Cody, Claudia V. de Araujo, Chieko Hoki, Matthew D. Alexander, Ramesh Grandhi, Mitchell R. Dyer, Matthew D. Neal, Jennifer J. Majersik, Christian C. Yost, Robert A. Campbell

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Figure 6

Platelet-specific HMGB1 knockout blocks platelet-induced NET formation and improves stroke outcomes.

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Platelet-specific HMGB1 knockout blocks platelet-induced NET formation a...
(A and B) Platelets and neutrophils were isolated from 4 healthy donors. Platelets were activated for 15 minutes with convulxin and then incubated for 2.5 hours with neutrophils in the presence of BoxA or vehicle, after which NETs were quantified using a MPO-DNA ELISA. n = 4 per group. (C and D) Platelets were isolated from 3 HMGB1fl/fl (WT) or HMGB1fl/fl PF4-cre (KO) mice, activated with convulxin, and incubated for 2.5 hours with WT neutrophils, after which NETs were quantified using an MPO-DNA ELISA. n = 3 per group. (E–J) HMGB1fl/fl (WT; n = 9) or HMGB1fl/fl PF4-cre (KO; n = 11) mice were subjected to 1 hour of tMCAO followed by 23 hours of reperfusion. Plasma was isolated and brains were analyzed for ischemic stroke brain damage by TTC staining 24 hours after stroke onset. Upon TTC staining, live brain tissue will stain red, while dead brain tissue will remain white (outlined with black dotted line). (E) Plasma HMGB1 levels were measured by ELISA. (F) Plasma NET levels were measured by MPO-DNA complex ELISA. (G and H) Infarct size was determined by TTC staining and planimetric analysis. (I) Neurological score was measured 24 hours after stroke using Bederson’s test. (J) Motor function was assessed 24 hours after stroke using the grip test. Groups were compared by unpaired t test (B, D–F, and H) or Mann-Whitney test (I and J). *P < 0.05, **P < 0.01, ***P < 0.001.