Circulating Neutrophil Extracellular Traps in the Pathogenesis of Acute Chest Syndrome of Sickle Cell Disease
Authors: Vats Ravi , Tutuncuoglu Egemen , Tejero Jesus , Hillery Cheryl A , Gladwin Mark T , Sundd Prithu 
Published: November 2019

Introduction: Acute chest syndrome (ACS) is a type of acute lung injury and among the primary reasons for mortality and morbidity among Sickle Cell Disease (SCD) patients. Although epidemiologic evidence suggests that vaso-occlusion in the lung may serve as an antecedent to ACS, the cellular, molecular and biophysical mechanism of ACS is incompletely understood. Our recent findings revealed that the lung vaso-occlusion is enabled by the entrapment of embolic neutrophil-platelet aggregates in the pulmonary arterioles of transgenic humanized SCD mice. Recent evidence also suggests a role for neutrophil extracellular traps (NETs) in ACS. NETs are web-like structures of decondensed nuclear DNA decorated with citrullinated-histones (H3-cit) and neutrophil granule proteins. Interestingly, circulating nucleosomes and NETs fragments are elevated in SCD patient blood and the levels correlate with onset of ACS, however, the molecular mechanism that promotes generation of circulating NETs and the role of circulating NETs in promoting ACS remains poorly understood.
Materials and Methods: Townes knock-in humanized SS (hα/hα:βS/βS) and AS (hα/hα:βA/βS) mice were used as SCD and control mice, respectively. SS and AS mice were intravenously (IV) administered 10 µmole/kg Oxy-Hb followed by Sytox orange, FITC-dextran or fluorescent anti-mouse mAbs against Ly6G, CD49b, H3cit, and neutrophil elastase for in vivo visualization of extracellular DNA, blood vessels, neutrophils, platelets and NETs, respectively. Pulmonary microcirculation was monitored using multi-photon-excitation enabled quantitative fluorescence intravital lung microscopy (qFILM).
Results and Discussion: IV Oxy-Hb triggered the occlusion of pulmonary arterioles by neutrophil-platelet aggregates leading to loss of pulmonary blood flow in SCD but not control mice. Surprisingly, pulmonary vaso-occlusion in SCD mice was accompanied by the arrival of circulating cell free DNA (CFD) and NETs fragments into the pulmonary circulation. The cell free DNA (CFD) and NETs fragments entered the lung through the arterial circulation suggesting that they originated outside of lung. These cell free DNA (CFD) and NETs fragments contributed to lung vaso-occlusion and injury by promoting neutrophil-platelet aggregation in the lung arterioles.
Conclusion: These findings reveal for the first time that circulating cell free DNA (CFD) and NETs fragments originating outside of lung contribute to pathogenesis of ACS. Currently, experiments are underway to identify the innate immune pathways that promote circulating NETs dependent lung injury in SCD.

Disclosures
Gladwin: Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Bayer Pharmaceuticals: Other: Co-investigator.