Differential type I interferon response and primary airway neutrophil extracellular trap release in children with acute respiratory distress syndrome

Abstract

Acute respiratory distress syndrome (ARDS) is a heterogeneous condition characterized by the recruitment of large numbers of neutrophils into the lungs. Neutrophils isolated from the blood of adults with ARDS have elevated expression of interferon (IFN) stimulated genes (ISGs) associated with decreased capacity of neutrophils to kill Staphylococcus aureus and worse clinical outcomes. Neutrophil extracellular traps (NETs) are elevated in adults with ARDS. Whether pediatric ARDS (PARDS) is similarly associated with altered neutrophil expression of ISGs and neutrophil extracellular trap release is not known. Tracheal aspirate fluid and cells were collected within 72 h from seventy-seven intubated children. Primary airway neutrophils were analyzed for differential ISG expression by PCR, STAT1 phosphorylation and markers of degranulation and activation by flow cytometry. Airway fluid was analyzed for the release of NETs by myeloperoxidase-DNA complexes using an ELISA. Higher STAT1 phosphorylation, markers of neutrophil degranulation, activation and NET release were found in children with versus without PARDS. Higher NETs were detected in the airways of children with ventilator-free days less than 20 days. Increased airway cell IFN signaling, neutrophil activation, and NET production is associated with PARDS. Higher levels of airway NETs are associated with fewer ventilator-free days.

Machine Learning–Based Discovery of a Gene Expression Signature in Pediatric Acute Respiratory Distress Syndrome

“…The transcriptomic reporter assay is grounded in the idea that metabolites, pattern-associated molecular patterns, damage-associated molecular patterns, chemokines, cytokines, and inflammatory mediators within the airway fluid will elicit an immune response in a genetically homogeneous pool of donor neutrophils. Neutrophils were chosen because they are the predominant innate immune cell recruited and recovered from tracheal aspirate lavage of the airways of children with PARDS ( 19 , 20 , 42 , 43 ). Neutrophils have the greatest differential gene expression response compared with monocytes and monocyte-derived dendritic cells in response to the plasma of septic adults ( 10 ).…”

“…Tracheal aspirate samples were collected within 72 hours of endotracheal intubation with an inline Ballard suction catheter (Halyard, Alpharetta, GA) connected to a sterile Lukens trap (Covidien, Walpole, MA) using up to 5 mL of sterile saline and processed according to published protocols ( 19 , 20 ). For infants and toddlers, 1–2 mL of saline is instilled.…”

“…Cell viability was determined with trypan blue exclusion on a Countess hemocytometer. Cell purity was assessed by Shandon Kwik-Diff (Thermo Scientific, Waltham, MA) staining of cytospin cell preparations ( 20 ). Up to 1 × 10 6 cells were stored in RNALater at –80°C until RNA was extracted for gene expression analysis.…”

“…RNA was isolated from airway cells using the Nucleospin RNA II kit with on-column genomic DNA digestion according to the manufacturer’s protocol (Takara, Mountain View, CA). RNA sizing quantification and quality control were performed in the Emory Integrated Genomics Core on an Agilent 2100 bioanalyzer using Pico and Nano Agilent kits (Agilent Technologies, Santa Clara, CA) and a Tecan optical density plate reader (Tecan, Männedorf, Switzerland) to measure the concentration of the RNA ( 20 ). A low input RNA amplification kit was used.…”

Endothelial Damage in Acute Respiratory Distress Syndrome

“…Apart from the beneficial roles of PMNs at the inflamed sites, production of reactive oxygen species (ROS) and secretion of neutrophil extracellular traps (NETs), also confer to PMNs auto-damaging functions [ 143 ]. The overproduction of NETs has been shown to be involved in the inflammatory injury of lung tissues [ 144 , 145 , 146 , 147 ].…”