Seasonal Antimicrobial Activity of the Airway: Post-Hoc Analysis of a Randomized Placebo-Controlled Double-Blind Trial.

BACKGROUND: It is widely unknown why respiratory infections follow a seasonal pattern. Variations in ultraviolet B (UVB) light during seasons affects cutaneous synthesis of vitamin D3. Serum vitamin D concentration influences the expression of airway surface liquid (ASL) antimicrobial peptides such as LL-37. OBJECTIVE: We sought to determine the effect of seasons on serum vitamin D levels and ASL antimicrobial activity. METHODS: Forty participants, 18-60 years old, were randomized 1:1 to receive 90 days of 1000 IU vitamin D3 or placebo. We collected ASL via bronchoscopy and measured serum 25(OH) vitamin D from participants before and after intervention across seasons. We measured ASL antimicrobial activity by challenging samples with bioluminescent Staphylococcus aureus and measured relative light units (RLUs) after four minutes. We also investigated the role of LL-37 using a monoclonal neutralizing antibody. RESULTS: We found that participants, prior to any intervention, during summer-fall (n = 20) compared to winter-spring (n = 20) had (1) decreased live bacteria after challenge (5542 ± 175.2 vs. 6585 ± 279 RLU, p = 0.003) and (2) higher serum vitamin D (88.25 ± 24.25 vs. 67.5 ± 45.25 nmol/L, p = 0.026). Supplementation with vitamin D3 increased vitamin D levels and restored ASL antimicrobial activity only during the winter-spring. The increased ASL antimicrobial activity seen during the summer-fall was abrogated by adding the LL-37 neutralizing antibody. CONCLUSION: ASL kills bacteria more effectively during the summer-fall compared to the winter-spring. Supplementation of vitamin D during winter-spring restores ASL antimicrobial activity by increasing the expression of antimicrobial peptides including LL-37.

Airway surface liquid from smokers promotes bacterial growth and biofilm formation via iron-lactoferrin imbalance.

BACKGROUND: Smoking is a leading cause of respiratory infections worldwide. Tobacco particulate matter disrupts iron homeostasis in the lungs and increases the iron content in the airways of smokers. The airway epithelia secrete lactoferrin to quench iron required for bacteria to proliferate and cause lung infections. We hypothesized that smokers would have increased bacterial growth and biofilm formation via iron lactoferrin imbalance. METHODS: We collected bronchoalveolar lavage (BAL) samples from non-smokers and smokers. We challenged these samples using a standard inoculum of Staphylococcus aureus and Pseudomonas aeruginosa and quantified bacterial growth and biofilm formation. We measured both iron and lactoferrin in the samples. We investigated the effect of supplementing non-smoker BAL with cigarette smoke extract (CSE) or ferric chloride and the effect of supplementing smoker BAL with lactoferrin on bacterial growth and biofilm formation. RESULTS: BAL from smokers had increased bacterial growth and biofilm formation compared to non-smokers after both S. aureus and P. aeruginosa challenge. In addition, we found that samples from smokers had a higher iron to lactoferrin ratio. Supplementing the BAL of non-smokers with cigarette smoke extract and ferric chloride increased bacterial growth. Conversely, supplementing the BAL of smokers with lactoferrin had a concentration-dependent decrease in bacterial growth and biofilm formation. CONCLUSION: Cigarette smoking produces factors which increase bacterial growth and biofilm formation in the BAL. We propose that smoking disrupts the iron-to-lactoferrin in the airways. This finding offers a new avenue for potential therapeutic interventions to prevent respiratory infections in smokers.

Effect of vitamin D3 on the antimicrobial activity of human airway surface liquid: preliminary results of a randomised placebo-controlled double-blind trial.

INTRODUCTION: Vitamin D3 supplementation has been reported to prevent lung infections and increase the gene expression of antimicrobial peptides such as cathelicidin. We investigated the effect of vitamin D3 supplementation on the antimicrobial activity of airway surface liquid (ASL) in human subjects. Since smoking can increase the risk of respiratory infections, we also investigated the effect of smoking in the cathelicidin response to vitamin D3 in human airway epithelia in vitro. METHODS: This study is a subanalysis of single-centre community-based randomised placebo-controlled double-blind trial. Participants were randomised to receive 1000 international units per day of oral vitamin D3 or identical placebo for 90 days. Blood and ASL samples were collected preintervention and postintervention. 105 participants were originally enrolled, 86 completed the trial, and due to low protein concentration in the samples, 40 participants were finally analysed. Our primary outcome was ASL antimicrobial activity. We also considered secondary outcomes including changes in serum concentration of 25-hydroxyvitamin D3 (25(OH)D3), 1,25-hydroxyvitamin D3, calcium and parathyroid hormone (PTH). In addition, we studied the effect of cigarette smoke extract (CSE) exposure to primary human airway epithelial cell cultures on the gene expression of cathelicidin in response to vitamin D3 and expression of CYP27B1 (1-alpha hydroxylase), responsible for vitamin D3 activation. RESULTS: Vitamin D3 supplementation significantly increased both ASL antimicrobial activity and serum concentration of 25(OH)D3. In a subgroup analysis, we found that smokers did not increase their baseline antimicrobial activity in response to vitamin D3. Exposure to CSE on human airway epithelia decreased baseline CYP27B1 gene expression and cathelicidin response to 25(OH)D3. CONCLUSION: Vitamin D3 supplementation for 90 days increases ASL antimicrobial activity. Data from this preliminary study suggest that smoking may alter the ability of airway epithelia to activate vitamin D3 and increase the gene expression of cathelicidin antimicrobial peptide. TRIAL REGISTRATION NUMBER: NCT01967628; Post-results.

Cystic Fibrosis Transmembrane Conductance Regulator Potentiation as a Therapeutic Strategy for Pulmonary Edema: A Proof-of-Concept Study in Pigs.

OBJECTIVES: To determine the feasibility of using a cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor (VX-770/Kalydeco, Vertex Pharmaceuticals, Boston, MA), as a therapeutic strategy for treating pulmonary edema. DESIGN: Prospective laboratory animal investigation. SETTING: Animal research laboratory. SUBJECTS: Newborn and 3 days to 1 week old pigs. INTERVENTIONS: Hydrostatic pulmonary edema was induced in pigs by acute volume overload. Ivacaftor was nebulized into the lung immediately after volume overload. Grams of water per grams of dry lung tissue were determined in the lungs harvested 1 hour after volume overload. MEASUREMENTS AND MAIN RESULTS: Ivacaftor significantly improved alveolar liquid clearance in isolated pig lung lobes ex vivo and reduced edema in a volume overload in vivo pig model of hydrostatic pulmonary edema. To model hydrostatic pressure-induced edema in vitro, we developed a method of applied pressure to the basolateral surface of alveolar epithelia. Elevated hydrostatic pressure resulted in decreased cystic fibrosis transmembrane conductance regulator activity and liquid absorption, an effect which was partially reversed by cystic fibrosis transmembrane conductance regulator potentiation with ivacaftor. CONCLUSIONS: Cystic fibrosis transmembrane conductance regulator potentiation by ivacaftor is a novel therapeutic approach for pulmonary edema.

Effects of Coal Fly Ash Particulate Matter on the Antimicrobial Activity of Airway Surface Liquid.

BACKGROUND: Sustained exposure to ambient particulate matter (PM) is a global cause of mortality. Coal fly ash (CFA) is a byproduct of coal combustion and is a source of anthropogenic PM with worldwide health relevance. The airway epithelia are lined with fluid called airway surface liquid (ASL), which contains antimicrobial proteins and peptides (AMPs). Cationic AMPs bind negatively charged bacteria to exert their antimicrobial activity. PM arriving in the airways could potentially interact with AMPs in the ASL to affect their antimicrobial activity. OBJECTIVES: We hypothesized that PM can interact with ASL AMPs to impair their antimicrobial activity. METHODS: We exposed pig and human airway explants, pig and human ASL, and the human cationic AMPs ß-defensin-3, LL-37, and lysozyme to CFA or control. Thereafter, we assessed the antimicrobial activity of exposed airway samples using both bioluminescence and standard colony-forming unit assays. We investigated PM-AMP electrostatic interaction by attenuated total reflection Fourier-transform infrared spectroscopy and measuring the zeta potential. We also studied the adsorption of AMPs on PM. RESULTS: We found increased bacterial survival in CFA-exposed airway explants, ASL, and AMPs. In addition, we report that PM with a negative surface charge can adsorb cationic AMPs and form negative particle-protein complexes. CONCLUSION: We propose that when CFA arrives at the airway, it rapidly adsorbs AMPs and creates negative complexes, thereby decreasing the functional amount of AMPs capable of killing pathogens. These results provide a novel translational insight into an early mechanism for how ambient PM increases the susceptibility of the airways to bacterial infection. https://doi.org/10.1289/EHP876.

Electrolyte transport properties in distal small airways from cystic fibrosis pigs with implications for host defense.

While pathological and clinical data suggest that small airways are involved in early cystic fibrosis (CF) lung disease development, little is known about how the lack of cystic fibrosis transmembrane conductance regulator (CFTR) function contributes to disease pathogenesis in these small airways. Large and small airway epithelia are exposed to different airflow velocities, temperatures, humidity, and CO2 concentrations. The cellular composition of these two regions is different, and small airways lack submucosal glands. To better understand the ion transport properties and impacts of lack of CFTR function on host defense function in small airways, we adapted a novel protocol to isolate small airway epithelial cells from CF and non-CF pigs and established an organotypic culture model. Compared with non-CF large airways, non-CF small airway epithelia cultures had higher Cl(-) and bicarbonate (HCO3 (-)) short-circuit currents and higher airway surface liquid (ASL) pH under 5% CO2 conditions. CF small airway epithelia were characterized by minimal Cl(-) and HCO3 (-) transport and decreased ASL pH, and had impaired bacterial killing compared with non-CF small airways. In addition, CF small airway epithelia had a higher ASL viscosity than non-CF small airways. Thus, the activity of CFTR is higher in the small airways, where it plays a role in alkalinization of ASL, enhancement of antimicrobial activity, and lowering of mucus viscosity. These data provide insight to explain why the small airways are a susceptible site for the bacterial colonization.