Free DNA in cystic fibrosis airway fluids correlates with airflow obstruction

Abstract

Chronic obstructive lung disease determines morbidity and mortality of patients with cystic fibrosis (CF). CF airways are characterized by a nonresolving neutrophilic inflammation. After pathogen contact or prolonged activation, neutrophils release DNA fibres decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). NETs have been described to act in a beneficial way for innate host defense by bactericidal, fungicidal, and virucidal actions. On the other hand, excessive NET formation has been linked to the pathogenesis of autoinflammatory and autoimmune disease conditions. We quantified free DNA structures characteristic of NETs in airway fluids of CF patients and a mouse model with CF-like lung disease. Free DNA levels correlated with airflow obstruction, fungal colonization, and CXC chemokine levels in CF patients and CF-like mice. When viewed in combination, our results demonstrate that neutrophilic inflammation in CF airways is associated with abundant free DNA characteristic for NETosis, and suggest that free DNA may be implicated in lung function decline in patients with CF.

Figure 1

Free NET-like DNA structures in CF lung disease. (a) Immunological characterization of free DNA structures in CF airway fluids by CLSM. Upper two panel rows: NET-like DNA structures in induced CF sputum. Blue: DAPI stains DNA-NET backbone. Red: elastase. Lower two panel rows: Blue: DAPI, red: citrullinated histones. Scale bar: 20 μm. (b) Ultrastructure of free NET-like DNA structures. Upper and middle panels: SEM images of CF airway fluids. Arrow marks bacteria entrapped in DNA-NET-like structures. Scale bar: 2 μm; Middle right panel: TEM staining of citrullinated histones in CF airway fluids (sputa). Lower panel: Ultrathin sections of CF airway DNA-NET-like structures. The NETs and the bacterial extracellular polysaccharides are visualized by the ruthenium-red-osmium-tetroxide technique. Bacteria embedded in a dense wickerwork of NETs. Arrowheads mark NETs; asterisk: bacterial extracellular polysaccharide. (c) Upper panel: free DNA structures in CF lung tissue. Red: MPO (as characteristic NET component). Blue: DAPI (DNA). Inlays mark characteristic NET-areas. Lower panel: CF airway NETs in CF BAL fluids. Red: elastase (as characteristic NET component). Blue: DAPI (DNA). (d) Costainings of DAPI, citrullinated histones, and F-actin. Scale bar: 20 μm. (e) Dead/live staining of CF airway fluids (induced sputum). Free DNA and dead bacteria appear red; vital bacteria appear green.

Figure 2

Free DNA, disease severity, and infection in CF lung disease. (a) Stratification of CF patients (n = 80). Upper left: correlation of free DNA levels with FEV₁ in sputum supernatants from CF patients. Upper right: free DNA levels in sputum supernatants from healthy controls and CF patients, stratified by FEV₁. Lower panels: free DNA levels in sputum supernatants from healthy controls and CF patients, stratified by Pseudomonas aeruginosa or Aspergillus fumigatus infection/colonization status (Leeds criteria, 0: never, 1: negative, 2: intermittent, and 3: chronic). (b) Representative CLSM images of airway fluids from one healthy individual and three different CF patients, stratified for lung function, are depicted. Scale bar: 10 μm; FEV₁: forced expiratory volume in 1 second (% of prediction). DAPI staining of nuclei and extracellular DNA strands in CF sputa. (c) Chemokine levels in sputum supernatants from healthy controls (white, n = 6) or CF patients (grey, n = 50). (d) Correlation of free DNA with CXCL2 levels in airway fluids (cell-free sputum supernatants) from CF patients (n = 50). ^∗ P < 0.05.

Figure 3

Free DNA in murine CF lung disease in vivo. (a) CXCR2 chemokines in BALF from βENaC-transgenic (βENaC-Tg, n = 19) and wild-type (WT, n = 11) mice. Levels of CXCL1 (KC) and CXCL2 (MIP-2) were quantified by ELISA. (b) Free DNA in BALs from βENaC-Tg mice. Free DNA was quantified in BALF from βENaC-Tg (filled circles, n = 19) and wild-type (empty circles, n = 11) mice. (c) Correlation of free DNA with CXCL2 levels in BAL. (d) Correlation of free DNA with lung function (FEV100). ^∗ P < 0.05.