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.

Figure 1

Neutrophil activation and degranulation markers in the tracheal aspirate samples from children with acute respiratory failure with and without PARDS. The gating strategy for single, living, CD66b⁺ neutrophils is shown (A–C). The total number of neutrophils in the sample (D) was determined by multiplying the total number of cells in the tracheal aspirate sample by the percentage of living CD66b⁺ cells (neutrophils) in the sample (E). The mean fluorescence intensity (MFI) of (F) CD63 (n = 17, No PARDS, n = 10, Yes PARDS), (G) sphingosine 1-phosphate receptor 3 (S1PR3) (n = 17, No PARDS, n = 10, Yes PARDS), (H) CD16 (n = 17, No PARDS, n = 10, Yes PARDS), and (I) Arginase 1 (Arg1, n = 16, No PARDS, n = 10, Yes PARDS) on the surface of airway neutrophils of children with PARDS (gray boxplots) compared with children without PARDS (white boxplots) are shown. Each circle (No PARDS) and each square (Yes PARDS) represent an individual patient. A cytospin and Diff-Quik stained processed airway sample is shown (J). Scale bars indicate 50 microns. Two-tailed Mann–Whitney U test. **p < 0.01, *p < 0.05.

Figure 2

Phosphorylated STAT-1 (P-STAT1 (Y701)) and STAT1 expression in primary airway cells in children with and without PARDS. Cells were gated on forward and side-scatter. Histograms of primary flow data along with cell counts are noted. Histogram data is scaled using the modal function in FlowJo analysis software (A). Boxplots of the mean fluorescence intensity (MFI) of P-STAT1 (B) and STAT-1 (C) along with the percent cells positive for P-STAT1 (D) or STAT1 (E) are shown. The fluorescence minus 1 (FM1) histogram was used to draw the percent positive gate for P-STAT1 and STAT-1, respectively. FM1 (gray histogram), No PARDS (circles/blue boxplot and blue histograms) and PARDS (squares/red boxplot) and red histograms). For all analyses, n = 19 (No PARDS), n = 15 (Yes PARDS). Two-tailed Mann–Whitney U test. *p < 0.05.

Figure 3

Anti-viral and interferon (IFN) stimulated gene (ISG) expression in control children and in children with and without PARDS. (A) IFIT1 (IFN Induced proteins with Tetraicopaptide repeats), n = 10 with 3 outliers removed (No PARDS), n = 7 (Yes PARDS), (B) ISG15, n = 12 with 1 outlier removed (No PARDS), n = 7 (Yes PARDS), (C) MX1, n = 13 with no outliers (No PARDS), n = 7 (Yes PARDS). Two-tailed Mann–Whitney U test. The p values are noted above the boxplots.

Figure 4

Airway neutrophil extracellular trap (NET) release by PARDS status and ventilator-free days. NET release as measured by MPO-DNA enzyme-linked immunosorbent assay (A) from cell-free tracheal aspirate samples of children without PARDS (blue boxplot, n = 33, where each circle is an individual patient) versus with PARDS (red boxplot, n = 42, where each square is an individual patient). (B) MPO-DNA complexes in tracheal aspirates were stratified by those children who did not have more than 20 ventilator-free days (VFD) (white boxplot, n = 22, where each circle is an individual patient) and those children who did have more than 20 VFD (gray boxplot, n = 53, where each square is an individual patient). Boxplots depict median (line), 25th–75th interquartile range (box edges), and min to max values (whiskers). Values are normalized to a standardized value of 1 and analyzed using a two-tailed Mann–Whitney U test, *p < 0.05, **p < 0.01.

Figure 5

Airway neutrophils are activated in intubated children with compared to children without PARDS. Surface expression of the primary granule exocytosis marker, CD63, and the lipid signaling G protein-coupled receptor, sphingosine-1-phosphate receptor 3 (S1PR3), are higher in children with versus without PARDS. The type I interferon (IFN) signaling pathway transcription factor, STAT1, is upregulated and phosphorylated, and transcript levels of ISG15 is increased in children with versus without PARDS. Neutrophil extracellular trap (NET) release is regulated by a NADPH oxidase (NOX) respiratory burst (reactive oxygen species (ROS) triggered mechanism and an intracellular calcium-dependent trigger. It is not known which trigger dominates in the airways of children with PARDS. Children with PARDS have elevated levels of NETs in their airways as detected by myeloperoxidase (MPO)-DNA complexes in our study. Elevated NET levels are associated with a higher number of ventilator-free days (VFD) over 20 days in a 28-day period (i.e. if the child survived, then they were more likely to spend ≥ 7 days endotracheally intubated and mechanically ventilated). Created with BioRender.com using the web version, which may be accessed at https://biorender.com/, with a paid individual subscription granting permission to publish in journals.