Pilot study of inflammatory biomarkers in matched induced sputum and bronchoalveolar lavage from 2-year-olds with cystic fibrosis

Abstract

Background: In this pilot study, we investigated whether induced sputum (IS) could serve as a viable alternative to bronchoalveolar lavage (BAL) and yield robust inflammatory biomarkers in toddlers with cystic fibrosis (CF) featuring minimal structural lung disease. 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), all within a single visit. Inflammatory biomarkers included 20 soluble immune mediators and neutrophil elastase (NE), as well as frequency and phenotype of T cells, monocytes / macrophages and neutrophils. Results: At the molecular level, nine mediators showed similar levels in IS and BAL (CXCL1, CXCL8, IL-1, IL-1RA, IL-6, CCL2, CXCL10, M-CSF, VEGF-A), four were higher in IS than in BAL (CXCL5, IL-1, CXCL11, TNFSF10) and two were present in IS but undetectable in BAL (IL-10, IFN-). Meanwhile, soluble NE had lower activity in IS than 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). Neutrophil frequency and phenotype did not differ between IS and BAL. Conclusions: IS collected from 2-year-olds with CF yields biomarkers of early airway inflammation with good agreement with BAL notably with regards to molecular and cellular outcomes related to neutrophils and monocytes/macrophages.

Pulmonary exacerbations in early cystic fibrosis lung disease are marked by strong modulation of CD3 and PD-1 on luminal T cells

“…Previously, we showed that neutrophilic inflammation in CF airways, particularly their release of Arg1, contributes to inhibition of T-cell activity and their eventual exclusion from the airway lumen ( 13 ). Early in disease, however, T cells can still comprise 5-10% of total leukocytes obtained from BAL ( 18 ). We used flow cytometry to conduct a basic phenotypic analysis of T cells in blood vs, BAL and between the three groups of study visits reflecting PE history.…”

“…As such, our reported findings are an important basis of future studies to make use of such rare and valuable samples. More frequent collection of airway samples will provide more data on the earliest inflammatory mechanisms and resolution of PEs and may be enabled by expanded use of induced sputum collection, which can be done in young children with CF and has many similarities as well some differences with BAL ( 18 ). Another limitation of this study is the lack of control groups, which prevents us to state decisively whether observed changes in CF PEs are CF-driven or PE-driven.…”

“…Blood and BAL sample yields are summarized in Table S1 . BAL samples were processed as described previously ( 18 ). In brief, the sample was homogenized by passing through an 18-gauge needle for 12 cycles.…”

“…Analysis of leukocytes in blood and BAL fluid by flow cytometry was performed as described previously, using a gating strategy to identify neutrophils, monocytes/macrophages, and T cells ( 18 ). The gating strategy with representative subject-matched blood and BAL is shown in Figure S2 .…”

“…All compensation, gating, and calculation of median fluorescence intensity (MFI) and cell frequencies was performed using FlowJo V9.9.5 (BD). A threshold of at least 50 events after all gating steps was established for populations to be considered reliable for analysis of fluorescence parameters, as we demonstrated before in published analyses of blood, sputum and BAL ( 12 , 18 , 22 , 23 ). Staining panels are detailed in Table S4 .…”