METHODS
Human subjects and samples. Data were collected from 11
2-year-old children at stable clinic visits. Subjects with CF were
enrolled in the IMPEDE-CF study at Emory University and Children’s
Healthcare of Atlanta (Atlanta, GA, USA). All aspects of subject
enrollment and sample collection were approved by the Emory University
Institutional Review Board (IRB00097352). Consent for sample collection
from subjects was obtained from parents on the day of the clinic visit.
Subject demographics are summarized in Table 1 . Collection of
IS, blood, chest CT scan and BAL from the RML and LIN, were completed in
that order within the same 4-hour visit. Samples were stored on ice
following collection and delivered to the laboratory for immediate
processing. Details of sample collection procedures are provided in
online supplement and Figure S1 , with sample yields indicated
in Table S1 .
Chest CT imaging and PRAGMA scoring. Chest CT scans were
obtained under general anesthesia at Children’s Healthcare of Atlanta
[22] with full inspiratory and expiratory breath hold maneuvers, and
scored using the validated PRAGMA-CF method [23]. Scoring of mucus
plugging (%MP), bronchiectasis (%Bx) and abnormal airways (%AA) after
excluding areas of atelectasis were done with inspiratory scans, and
scoring of trapped air (%TA) used expiratory scans. Total Disease
(%Dis) score was calculated by combining all abnormal areas as a
percentage of total scored CT sections after excluding areas of
atelectasis.
Total protein quantification. Total protein levels were
quantified in IS and BAL using the Pierce copper sulfate/bicinchoninic
acid (BCA) assay (ThermoFisher), with bovine serum albumin used for
calibration.
Soluble immune mediator quantification. A custom
assortment of 20 soluble immune mediators was measured in IS and BAL
using a highly sensitive chemiluminescent assay (U-PLEX; Meso Scale
Diagnostics), according to the manufacturer’s protocol. To enable
comparison between IS and BAL samples, mediator concentrations were
normalized to total protein concentration (measured using BCA assay), as
illustrated in Figures 1 and 2 .
Flow cytometry. Multicolor flow cytometry was performed
on cells from blood, BAL, and IS using previously described methods
[11]. A gating strategy was developed to identify all major
leukocyte populations in blood and airway fluids, as detailed inFigure S4 . Additional experimental details are presented in
Supplemental Methods. Consistent fluorescence output was achieved
through stringent cytometer calibration techniques, as detailed before
[24].
Extracellular NE activity. Extracellular NE activities
in IS, RML BAL and LIN BAL were measured with a Förster resonance energy
transfer (FRET) assay using the NEmo-1 probe (Sirius Fine Chemicals
SiChem GmbH), as previously described [25-27]. NE concentration was
normalized to total protein concentration (measured using BCA assay) in
the fluid.
Statistical analysis. Data were analyzed in Prism
(GraphPad, version 8) using nonparametric statistics due to the limited
number of samples, including the Wilcoxon matched-pairs signed rank test
and Spearman test for correlation. Specifically for cytokines, a value
of half the lower limit of detection or twice the upper limit of
detection was assigned for data points which fell outside the limits of
detection. Those values are clearly labeled as closed symbols in related
figures. To avoid over-representation of imputed values, statistical
comparisons of mediator concentration were not performed where more than
half the data points of a sample group were imputed values. To conduct
correlations, imputed values were removed, and correlations were
conducted using R only for mediators with at least 6 non-imputed data
points.