INFLAMMATORY BIOMARKERS IN INDUCED SPUTUM FROM YOUNG CHILDREN WITH CYSTIC FIBROSIS
Background: In adults with cystic fibrosis (CF), induced sputum (IS) is a minimally invasive alternative to bronchoalveolar lavage (BAL) to monitor airway inflammation. Here, we investigated whether IS could yield biomarkers of early disease in young children with CF. Methods: We collected IS, BAL (right middle lobe and lingula) and blood, and performed chest computed tomography (CT) scans from 2-year-olds with CF (N=11) within a single visit. Molecular biomarkers included 20 immune mediators and soluble neutrophil elastase (NE). Cellular biomarkers consisted in frequency and phenotype (including surface NE) of T cells, monocytes / macrophages and neutrophils. Results: Six mediators were correlated between IS and BAL. Eight mediators showed similar levels in IS and BAL, including GRO-alpha, IL-1alpha, IL-6, IL-8, IP-10, M-CSF and VEGF. Four mediators were higher in IS than in both BAL fractions, including ENA-78, IL-1beta, I-TAC and TRAIL. IL-10 and IFN-gamma were present in IS samples but largely undetectable in BAL. At the cellular level, T-cell frequency was lower in IS than in BAL. Monocytes / macrophages were dominant in IS and BAL with similar frequencies but differing expression of CD16 (lower in IS), CD115 and surface-associated NE (higher in IS). Meanwhile, soluble NE had lower activity in IS than in BAL. Neutrophil frequency and phenotype did not differ between IS and BAL. Importantly however, neutrophil frequency in IS correlated measures of lung damage by chest CT. Conclusions: IS collected from young children with CF yields molecular and cellular biomarkers of early airway inflammation and structural lung damage.
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SAMPLE COLLECTION: Blood, IS and BAL samples were obtained from CF patients. Blood was centrifuged at 400 x g for 10 minutes at room termperature to separate cells from plasma and plasma was removed for further processing. The blood pellet was washed with PBS+EDTA and resuspended to the original volume using PBS+EDTA. EDTA was added to BAL for a final concentration of 2.5 mM and a standard volume of 3 mL PBS+EDTA was added to each IS sample. IS and BAL samples were mechanically homogenized by passing through an 18-gauge needle and centrifuged at 800 x g for 10 minutes at 4°C. The supernatant was centrifuged at 3000 x g to yield debris-free supernatant for analysis of cytokines and enzymes. 50 uL of whole blood or 200,000 cells purified from IS and BAL in 50 uL of PBS+EDTA were stained for analysis by flow cytometry. CYTOKINES AND ELASTASE: BCA Total protein levels were quantified in IS and BAL using the Pierce copper sulfate/bicinchoninic acid (BCA) assay (ThermoFisher Scientific), with bovine serum albumin used for calibration. Protein concentrations are provided in the accompanying excel file (BCA.xlsx) Cytokines A custom assortment of 20 soluble immune mediators were selected to be measured using the U-PLEX kit from Meso Scale Diagnostics according to the manufacturer’s protocol. For values that fell below the lower limit of detection, a value of half the lower limit for that cytokine in that particular assay was assigned. These values are colored in red in the accompanying excel file (cytokines_non normalized.xlsx). To enable comparison between IS and BAL samples, mediator concentrations were normalized to total protein concentration as measured by BCA assay (BCA.xlsx). The indication of values below the limit of detection using red color was maintained after normalization in the accompanying excel file (cytokines_normalized.xlsx). NE Extracellular NE activities in IS, RML BAL and LIN BAL were measured with a Förster resonance energy transfer (FRET)-based assay using the NEmo-1 probe (Sirius Fine Chemicals SiChem GmbH). NE concentration was normalized to total protein concentration as measured by BCA assay (BCA.xlsx) FLOW CYTOMETRY: Samples were acquired on a BD LSRII or BD FACSYMPHONY and the cytometer was calibrated at each acquisition. Data were compensated and analyzed using FlowJo V9.9.5. The exported data for media fluorescent intensity and cellular frequencies can be found in the corresponding excel files for each sample type. Missing values in these data tables are for several reasons: -sample was not collected (no IS collected from subject #3) -staining panels omitted due to lack of cells obtained from a sample (approximately 200,000 cells needed for each panel) -insufficient number of events remaining after gating (we set a minimum requirement of 50 events to include for statistical analysis) PRAGMA: Four parameters of lung disease were scored using the PRAGMA-CF method (PRAGMA.xlsx)