Observations of, and Insights into, Cystic Fibrosis Mucus Heterogeneity in the Pre-Modulator Era: Sputum Characteristics, DNA and Glycoprotein Content, and Solubilization Time

Abstract

Airway obstruction with chronic inflammation and infection are major contributors to the lung damage and mortality of cystic fibrosis (CF). A better understanding of the congested milieu of CF airways will aid in improving therapeutic strategies. This article retrospectively reports our observations, and discusses insights gained in the handling and analysis of CF sputa. CF and non-CF mucus samples were surveyed for morphological features by electron microscopy and analyzed for the macromolecular dry weight (MDW), total protein, lipid, carbohydrate, and DNA. Mucus character was investigated with chemical solubilization time as a comparative tool. CF mucus appeared distinctly thick, viscous, and heterogeneous, with neutrophils as the dominant immune cell. CF sputum DNA content varied markedly for and between individuals (~1–10% MDW), as did solubilization times (~1–20 h). CF Sputum DNA up to 7.1% MDW correlated positively with solubilization time, whereas DNA >7.1% MDW correlated negatively. 3D analysis of CF sputa DNA, GP, and solubilization times revealed a dynamic and predictive relationship. Reflecting on the heterogeneous content and character of CF mucus, and the possible interplay in space and time in the respiratory tract of polymeric DNA and mucous glycoproteins, we highlight it’s potential to affect infection-related airway pathologies and the success of therapeutic interventions.

Engineering in vitro models of cystic fibrosis lung disease using neutrophil extracellular trap inspired biomaterials

“…The final concentration of sNET was based upon reported concentrations of extracellular DNA in CF sputum samples, which span a wide range of up to B10 mg ml À1 but average around 3 mg ml À1 . 27,28,[37][38][39] In contrast, one study reported healthy human mucus contains an average extracellular DNA concentration of 0.006 AE 0.003 mg ml À1 . 37 Previous studies have found that approximately 50% of the measured extracellular DNA in CF sputum is in complexation with proteins indicating the formation of NETs.…”

“…27,28,[37][38][39] In contrast, one study reported healthy human mucus contains an average extracellular DNA concentration of 0.006 AE 0.003 mg ml À1 . 37 Previous studies have found that approximately 50% of the measured extracellular DNA in CF sputum is in complexation with proteins indicating the formation of NETs. Therefore the final concentration of sNETs used in this study was 1.5 mg ml À1 (0.15% w/v).…”

Insights on Pseudomonas aeruginosa Carbohydrate Binding from Profiles of Cystic Fibrosis Isolates Using Multivalent Fluorescent Glycopolymers Bearing Pendant Monosaccharides

“…It should also be respected in therapeutics design that, as discussed above, the varied permutations of the condition of the host, as well as the adaptations of the colonizing microbes and the treatments being received, are likely to keep the infectious landscape ever-changing [8,20,43,133]. The P. aeruginosa variability and fluidity in the expression or lack of expression of virulence features and structures are expected to complicate the development of targeted agents [10,11,15,20,24,26,29,36,58,63,88,90,116,117,131,136].…”

“…As multi-drug resistance and conditions of antibiotic tolerance continue to increase globally among P. aeruginosa and other major pathogens responsible for drug-resistant nosocomial infections, new approaches and tools to complement the existing repertoire of anti-microbial agents are urgently needed [8,12,27,[35][36][37][38][39][40]. Successful adjunctive therapies for CF airway infection, ideally, will accommodate the heterogeneity of the biological features and activities of P. aeruginosa in the CF population and within the individual host over time, and the variability of the host pulmonary status [1,6,[16][17][18][19][20]28,30,33,34,[41][42][43][44][45]. Tactics and adjunctive agents developed to counter CF Pseudomonas respiratory infections are anticipated to also benefit other problematic host infections [12,23,26,35,37,40,46,47], and aid in attempts at antibiotic conservation.…”

Challenges and opportunities in the development of novel antimicrobial therapeutics for cystic fibrosis

“…CF sputum provides a nutrient-rich environment for microbial growth. Protein, lipid, carbohydrate, and free-DNA concentration are all increased in PwCF, alongside iron, which is sequestered by lactoferrin, transferrin and ferritin [10,11]. CF sputum has a decreased pH, which inhibits the function of secreted antimicrobials and may similarly impair target-site activity of prescribed antibiotics [11].…”