Pulmonary exacerbations in early CF lung disease
Background: In chronic cystic fibrosis (CF) lung disease, neutrophilic inflammation and T-cell inhibition occur concomitantly, partly due to neutrophil-mediated release of the T-cell inhibitory enzyme Arg1. However, the onset of this tonic inhibition of T cells, and the impact of pulmonary exacerbations (PEs) on this process remains unknown. Methods: Children with CF aged 0-5 years were enrolled in a longitudinal cohort study at Emory University. Blood (n = 35) and BAL fluid (n = 18) were collected at stable outpatient clinic visits or inpatient PE hospitalizations and analyzed by flow cytometry (for immune cell presence and phenotype) and 20-plex chemiluminescence assay (for immune mediators). Patients were categorized by PE history into no prior PE, past history of PE prior to stable visit, or current PE. Results: PEs were associated with increased concentration of both pro- and anti-inflammatory mediators in BAL and increased neutrophil frequency and G-CSF in circulation. PE BAL samples showed a trend for an increased frequency of hyperexocytic GRIM neutrophils previously identified in chronic CF. When comparing the expression of the T-cell receptor associated molecule CD3 and the inhibitory programmed death-1 (PD-1) receptor on T cells from bronchoalveolar lavage (BAL) vs. blood samples, we found that CD3 was significantly decreased and PD-1 expression was increased across the board in all patients. When categorized by PE status, there was no difference in CD3 or PD-1 expression on the circulating blood T cells, but CD3 expression was decreased, and PD-1 was increased on BAL T cells from current PE samples. Conclusions: These data suggest that airway T cells are engaged during early-life PEs prior to the onset of chronic neutrophilic inflammation. However, increased neutrophil frequency in circulation and trend towards increasing frequency of GRIM neutrophils in the airway offers new insights into the mechanisms of how childhood PEs may accelerate airway inflammation and shift the balance of airway immune cells towards neutrophil dominance.
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Soluble immune mediators were quantified using the Meso Scale Diagnostics UPLEX kits Soluble neutrophil elastase activity was quantified using a FRET reporter assay Leukocyte frequencies and surface marker expression were determined using multi-color flow cytometry Section 1: PCA -data tables are provided to generate a principal component analysis of immune mediators in plasma and BAL -note that data points which were below the limit of detection are highlighted in red, indicating an imputed value of 1/2 the LLOD for that target in that specific assay Section 2: immune mediators -data tables are provided to compare concentrations of immune mediators in BAL and plasma -subjects were separated into 3 groups by history of pulmonary exacerbations (PE history): None, Prior, and Current -note that data points which were below the limit of detection are highlighted in red, indicating an imputed value of 1/2 the LLOD for that target in that specific assay Section 3: Leukocyte frequencies -frequencies of major leukocyte subsets were compared between blood and BAL for subjects of the 3 PE categories -These included neutrophils, monocytes (blood)/macrophages (BAL), and T cells -The frequency of neutrophils displaying the GRIM phenotype (CD63high, CD16low as compared to blood populations) was also compared Section 4: Leukocyte surface markers -expression of surface markers as well as surface-bound neutrophil elastase (NE) was compared for blood vs BAL, as well as by PE history