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.
Neutrophil extracellular traps (NETs) are important components of innate immunity. Neonatal neutrophils (PMNs) fail to form NETs due to circulating NET-Inhibitory Peptides (NIPs) -cleavage fragments of alpha-1-antitrypsin (A1AT). However, how fetal and neonatal blood NIPs are generated remains unknown. The placenta expresses High-Temperature Requirement serine protease A1 (HTRA1) during fetal development, which can cleave A1AT. We hypothesized that placentally expressed HTRA1 regulates the formation of NIPs and that NET competency changed in PMNs isolated from neonatal HTRA1-knock out mice (HTRA1-/-). We found that umbilical cord blood plasma has elevated HTRA1 levels compared to adult plasma, and that recombinant and placenta-eluted HTRA1 cleaves A1AT to generate an A1AT cleavage fragment (A1ATM383S-CF) of similar molecular weight to previously identified NIPs that block NET formation by adult neutrophils. We demonstrated that neonatal mouse pup plasma contains A1AT fragments which inhibit NET formation by PMNs isolated from adult mice, indicating that NIP generation during gestation is conserved across species. LPS-stimulated PMNs isolated from HTRA1+/+ littermate control pups exhibit delayed NET formation following birth. However, plasma from HTRA1-/- pups had no detectable NIPs and PMNs from HTRA1-/- pups became NET competent earlier after birth compared to HTRA1+/+ littermate controls. Finally, in the cecal slurry model of neonatal sepsis, A1ATM383S-CF improved survival in C57BL/6 pups by preventing pathogenic NET formation. Our data indicate that placentally expressed HTRA1 is a serine protease that cleaves A1AT in utero to generate NIPs that regulate NET formation by human and mouse PMNs.
Purpose of review In this review, we will describe how the combined ability of platelets and neutrophils to interact with each other drives ischemic stroke brain injury. Recent findings Neutrophils are one of the first cells to respond during ischemic stroke. Although animals stroke models have indicated targeting neutrophils improves outcomes, clinical trials have failed to yield successful strategies. Platelets play a critical role in recruiting neutrophils to sites of injury by acting as a bridge to the injured endothelium. After initial platelet adhesion, neutrophils can rapidly bind platelets through P-selectin and glycoprotein Ibα. In addition, recent data implicated platelet phosphatidylserine as a novel key regulator of platelet-neutrophil interactions in the setting of ischemic stroke. Inhibition of procoagulant platelets decreases circulating platelet-neutrophil aggregates and thereby reduces infarct size. Platelet binding alters neutrophil function, which contributes to the injury associated with ischemic stroke. This includes inducing the release of neutrophil extracellular traps, which are neurotoxic and pro-thrombotic, leading to impaired stroke outcomes. Summary Platelet-neutrophil interactions significantly contribute to the pathophysiology of ischemic stroke brain injury. Better understanding the mechanisms behind their formation and the downstream consequences of their interactions will lead to improved therapies for stroke patients.
Background Treatment of neonatal peritonitis and sepsis is challenging. Following infection, neutrophils elaborate neutrophil extracellular traps (NETs)—extracellular lattices of decondensed chromatin decorated with antimicrobial proteins. NETs, however, can augment pathogenic inflammation causing collateral damage. We hypothesized that NET inhibition would improve survival in experimental neonatal infectious peritonitis. Methods We induced peritonitis in 7 to 10-day-old mice by intraperitoneal injection with cecal slurry. We targeted NETs by treating mice with neonatal NET-Inhibitory Factor (nNIF), an endogenous NET-inhibitor; Cl-amidine, a PAD4 inhibitor; DNase I, a NET degrading enzyme, or meropenem (an antibiotic). We determined peritoneal NET and cytokine levels and circulating platelet–neutrophil aggregates. Survival from peritonitis was followed for 6 days. Results nNIF, Cl-amidine, and DNase I decreased peritoneal NET formation and inflammatory cytokine levels at 24 h compared to controls. nNIF, Cl-amidine, and DNase I decreased circulating platelet–neutrophil aggregates, and NET-targeting treatments significantly increased survival from infectious peritonitis compared to controls. Finally, nNIF administration significantly improved survival in mice treated with sub-optimal doses of meropenem even when treatment was delayed until 2 h after peritonitis induction. Conclusions NET inhibition improves survival in experimental neonatal infectious peritonitis, suggesting that NETs participate pathogenically in neonatal peritonitis and sepsis. Impact Neutrophil extracellular trap formation participates pathogenically in experimental neonatal infectious peritonitis. NET-targeting strategies improve outcomes in a translational model of neonatal infectious peritonitis. NET inhibition represents a potential target for drug development in neonatal sepsis and infectious peritonitis.
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