Cystic Fibrosis Airway Mucus Research Articles

Modeling Cystic Fibrosis Chronic Infection Using Engineered Mucus-like Hydrogels.

The airway mucus of patients with cystic fibrosis has altered properties, which create a microenvironment primed for chronic infections that are difficult to treat. These complex polymicrobial airway infections and corresponding mammalian-microbe interactions are challenging to model in vitro. Here, we report the development of mucus-like hydrogels with varied compositions and viscoelastic properties reflecting differences between healthy and cystic fibrosis airway mucus. Models of cystic fibrosis and healthy airway microenvironments were created by combining the hydrogels with relevant pathogens, human bronchial epithelial cells, and an antibiotic. Notably, pathogen antibiotic resistance was not solely dependent on the altered properties of the mucus-like hydrogels but was also influenced by culture conditions including microbe species, monomicrobial or polymicrobial culture, and the presence of epithelial cells. Additionally, the cystic fibrosis airway model showed the ability to mimic features characteristic of chronic cystic fibrosis airway infections including sustained polymicrobial growth and increased antibiotic tolerance.

Heterogeneity Governs 3D‐Cultures of Clinically Relevant Microbial Communities

AbstractThe intrinsic heterogeneity of bacterial niches should be retained in in vitro cultures to represent the complex microbial ecology. As a case study, mucin‐containing hydrogels ‐CF‐Mu3Gel ‐ are generated by diffusion‐induced gelation, bioinspired on cystic fibrosis (CF) mucus, and a microbial niche challenging current therapeutic strategies. At breathing frequency, CF‐Mu3Gel exhibits a G′ and G″ equal to 24 and 3.2 Pa, respectively. Notably, CF‐Mu3Gel exhibits structural gradients with a gradual reduction of oxygen tension across its thickness (280–194 µmol L−1). Over the culture period, a steep decline in oxygen concentration occurs just a few millimeters below the air–mucus interface in CF‐Mu3Gel, similar to those of CF airway mucus. Importantly, the distinctive features of CF‐Mu3Gel significantly influence bacterial organization and antimicrobial tolerance in mono‐ and co‐cultures of Staphylococcus aureus and Pseudomonas aeruginosa that standard cultures are unable to emulate. The antimicrobial susceptibility determined in CF‐Mu3Gel corroborates the mismatch on the efficacy of antimicrobial treatment between planktonically cultured bacteria and those in patients. With this example‐based research, new light is shed on the understanding of how the substrate influences microbial behavior, paving the way for improved fundamental microbiology studies and more effective drug testing and development.

Pulmonary bacteriophage and cystic fibrosis airway mucus: friends or foes?

For those born with cystic fibrosis (CF), hyper-concentrated mucus with a dysfunctional structure significantly impacts CF airways, providing a perfect environment for bacterial colonization and subsequent chronic infection. Early treatment with antibiotics limits the prevalence of bacterial pathogens but permanently alters the CF airway microenvironment, resulting in antibiotic resistance and other long-term consequences. With little investment into new traditional antibiotics, safe and effective alternative therapeutic options are urgently needed. One gathering significant traction is bacteriophage (phage) therapy. However, little is known about which phages are effective for respiratory infections, the dynamics involved between phage(s) and the host airway, and associated by-products, including mucus. Work utilizing gut cell models suggest that phages adhere to mucus components, reducing microbial colonization and providing non-host-derived immune protection. Thus, phages retained in the CF mucus layer result from the positive selection that enables them to remain in the mucus layer. Phages bind weakly to mucus components, slowing down the diffusion motion and increasing their chance of encountering bacterial species for subsequent infection. Adherence of phage to mucus could also facilitate phage enrichment and persistence within the microenvironment, resulting in a potent phage phenotype or vice versa. However, how the CF microenvironment responds to phage and impacts phage functionality remains unknown. This review discusses CF associated lung diseases, the impact of CF mucus, and chronic bacterial infection. It then discusses the therapeutic potential of phages, their dynamic relationship with mucus and whether this may enhance or hinder airway bacterial infections in CF.

Effects of Mucin and DNA Concentrations in Airway Mucus on Pseudomonas aeruginosa Biofilm Recalcitrance.

ABSTRACTThe pathological properties of airway mucus in cystic fibrosis (CF) are dictated by mucus concentration and composition, with mucins and DNA being responsible for mucus viscoelastic properties. As CF pulmonary disease progresses, the concentrations of mucins and DNA increase and are associated with increased mucus viscoelasticity and decreased transport. Similarly, the biophysical properties of bacterial biofilms are heavily influenced by the composition of their extracellular polymeric substances (EPS). While the roles of polymer concentration and composition in mucus and biofilm mechanical properties have been evaluated independently, the relationship between mucus concentration and composition and the biophysical properties of biofilms grown therein remains unknown. Pseudomonas aeruginosa biofilms were grown in airway mucus as a function of overall concentration and DNA concentration to mimic healthy, and CF pathophysiology and biophysical properties were evaluated with macro- and microrheology. Biofilms were also characterized after exposure to DNase or DTT to examine the effects of DNA and mucin degradation, respectively. Identifying critical targets in biofilms for disrupting mechanical stability in highly concentrated mucus may lead to the development of efficacious biofilm therapies and ultimately improve CF patient outcomes. Overall mucus concentration was the predominant contributor to biofilm viscoelasticity and both DNA degradation and mucin reduction resulted in compromised biofilm mechanical strength.IMPORTANCE Pathological mucus in cystic fibrosis (CF) is highly concentrated and insufficiently cleared from the airway, causing chronic inflammation and infection. Pseudomonas aeruginosa establishes chronic infection in the form of biofilms within mucus, and this study determined that biofilms formed in more concentrated mucus were more robust and less susceptible to mechanical and chemical challenges compared to biofilms grown in lower concentrated mucus. Neither DNA degradation nor disulfide bond reduction was sufficient to fully degrade biofilms. Mucus rehydration should remain a priority for treating CF pulmonary disease with concomitant multimechanistic biofilm degradation agents and antibiotics to clear chronic infection.

Properties of airway mucus in cystic fibrosis: their modification by changes in the activity of CFTR and after application of bicarbonate

Properties of airway mucus in cystic fibrosis: their modification by changes in the activity of CFTR and after application of bicarbonate

Hypertonic Saline in Treatment of Pulmonary Disease in Cystic Fibrosis

The pathogenesis of lung disease in cystic fibrosis is characterised by decreased airway surface liquid volume and subsequent failure of normal mucociliary clearance. Mucus within the cystic fibrosis airways is enriched in negatively charged matrices composed of DNA released from colonizing bacteria or inflammatory cells, as well as F-actin and elevated concentrations of anionic glycosaminoglycans. Therapies acting against airway mucus in cystic fibrosis include aerosolized hypertonic saline. It has been shown that hypertonic saline possesses mucolytic properties and aids mucociliary clearance by restoring the liquid layer lining the airways. However, recent clinical and bench-top studies are beginning to broaden our view on the beneficial effects of hypertonic saline, which now extend to include anti-infective as well as anti-inflammatory properties. This review aims to discuss the described therapeutic benefits of hypertonic saline and specifically to identify novel models of hypertonic saline action independent of airway hydration.

Anaerobiosis-Induced Loss of Cytotoxicity Is Due to Inactivation of Quorum Sensing in Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen of clinical importance, causes chronic airway infections in patients with cystic fibrosis (CF). Current literature suggests that pockets with reduced oxygen tension exist in the CF airway mucus. However, virulence features of this opportunistic pathogen under such conditions are largely unknown. Cell-free supernatant of the standard laboratory P. aeruginosa strain PAO1 obtained from anaerobic culture, but not aerobic culture, failed to kill A549 human airway epithelial cells. Further investigation revealed that this reduced cytotoxicity upon anaerobiosis was due to the suppressed secretion of elastase, a virulence factor controlled by P. aeruginosa quorum sensing (QS). Both a lacZ-reporter fusion assay and quantitative real-time PCR (RT-PCR) analysis demonstrated that transcription of the elastase-encoding lasB gene was substantially decreased during anaerobic growth compared with aerobic growth. Moreover, transcription of other genes controlled by the LasI/R QS system, such as rhlR, vqsR, mvfR, and rsaL, was also repressed under the same anaerobic growth conditions. Importantly, synthesis of 3-oxo-C(12)-HSL (PAI-1), an autoinducer molecule that mediates induction of the LasI/R QS system, was >22-fold decreased during anaerobic growth while C(4)-HSL (PAI-2), which mediates RhlI/R QS, was nondetectable under the same growth conditions. Transcription of the lasB gene was restored by exogenous supplementation with autoinducers, with PAI-2 more effective than PAI-1 or Pseudomonas quinolone signal (PQS) at restoring transcription of the lasB gene. Together, these results suggest that anaerobiosis deprives P. aeruginosa of the ability to regulate its virulence via QS and this misregulation attenuates the pathogenic potential of this important pathogen.

Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions.

Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2 also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways.

Airway mucus in cystic fibrosis.

Defective expression and function of the cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis (CF) airway epithelial cells are associated with airway mucus hypersecretion, inflammation and infection that begin early in life and lead, at an advanced stage of the disease, to severe airway obstruction with hyperviscous and adhesive airway mucus. Whether the abnormalities of airway mucus are already present at birth before infection is debatable. In CF, the impaired Cl(-) and HCO(3)(-) secretion associated with increased epithelial Na(+) absorption results in dehydration of airway mucus, decreased antimicrobial functions and impaired mucociliary clearance. Alterations in antibacterial peptide function, as well as the increased mucin expression and secretion (MUC 5AC and MUC 5B), are important biochemical factors responsible for the propensity for infection in CF airways. Alterations in mucin and lipid composition induce an increased viscosity and adhesiveness to the airways that can affect the mucociliary and cough transport. The increased content of pro-inflammation cytokines such as interleukin-8 (IL-8) suggest that, before infection, airway inflammation occurs very early in CF. The development of non-invasive techniques and humanised animal models (xenografts) represents a major opportunity to identify early abnormalities in CF airway mucus.