Soluble Mediators, Not Cilia, Determine Airway Surface Liquid Volume in Normal and Cystic Fibrosis Superficial Airway Epithelia

A key aspect of the lung's innate defense system is the ability of the superficial epithelium to regulate airway surface liquid (ASL) volume to maintain a 7-μm periciliary liquid layer (PCL), which is required for cilia to beat and produce mucus flow. The mechanisms whereby airway epithelia regulate ASL height to ≥7 μm are poorly understood. Using bumetanide as an inhibitor of Cl− secretion, and nystatin as an activator of Na+ absorption, we found that a coordinated “blending” of both Cl− secretion and Na+ absorption must occur to effect ASL volume homeostasis. We then investigated how ASL volume status is regulated by the underlying epithelia. Cilia were not critical to this process as (a) ASL volume was normal in cultures from patients with primary ciliary dyskinesia with immotile cilia, and (b) in normal cultures that had not yet undergone ciliogenesis. However, we found that maneuvers that mimic deposition of excess ASL onto the proximal airways, which occurs during mucociliary clearance and after glandular secretion, acutely stimulated Na+ absorption, suggesting that volume regulation was sensitive to changes in concentrations of soluble mediators in the ASL rather than alterations in ciliary beating. To investigate this hypothesis further, we added potential “soluble mediators” to the ASL. ASL volume regulation was sensitive to a channel-activating protein (CAP; trypsin) and a CAP inhibitor (aprotinin), which regulated Na+ absorption via changes in epithelial Na+ channel (ENaC) activity in both normal and cystic fibrosis cultures. ATP was also found to acutely regulate ASL volume by inducing secretion in normal and cystic fibrosis (CF) cultures, while its metabolite adenosine (ADO) evoked secretion in normal cultures but stimulated absorption in CF cultures. Interestingly, the amount of ASL/Cl− secretion elicited by ATP/ADO was influenced by the level of CAP-induced Na+ absorption, suggesting that there are important interactions between the soluble regulators which finely tune ASL volume

E-Liquid Autofluorescence can be used as a Marker of Vaping Deposition and Third-Hand Vape Exposure

In the past 5 years, e-cigarette use has been increasing rapidly, particularly in youth and young adults. Due to the novelty of e-cigarettes (e-cigs) and e-cigarette liquids (e-liquids), research on their chemo-physical properties is still in its infancy. Here, we describe a previously unknown and potentially useful property of e-liquids, namely their autofluorescence. We performed an emission scan at 9 excitation wavelengths common to fluorescent microscopy and found (i) that autofluorescence differs widely between e-liquids, (ii) that e-liquids are most fluorescent in the UV range (between 350 and 405 nm) and (iii) fluorescence intensity wanes as the emission wavelength increases. Furthermore, we used the autofluorescence of e-liquids as a marker for tracking e-cig aerosol deposition in the laboratory. Using linear regression analysis, we were able to quantify the deposition of a "vaped" e-liquid onto hard surfaces. Using this technique, we found that every 70 mL puff of an e-cigarette deposited 0.019% e-liquid (v/v) in a controlled environment. Finally, we vaped a surface in the laboratory and used our method to detect e-cig aerosol third-hand exposure. In conclusion, our data suggest that e-cigarette autofluorescence can be used as a marker of e-cigarette deposition

Tobacco exposure inhibits SPLUNC1-dependent antimicrobial activity

Background: Tobacco smoke exposure impairs the lung´s innate immune response, leading to an increased risk of chronic infections. SPLUNC1 is a secreted, multifunctional innate defense protein that has antimicrobial activity against Gram negative organisms. We hypothesize that tobacco smoke-induced SPLUNC1 dysfunction contributes to the observed defect in innate immunity in tobacco smokers and that this dysfunction can be used as a potential biomarker of harm. Methods: We collected sputum from never-smokers and otherwise healthy smokers. We performed Western blotting to determine SPLUNC1 levels and determined antimicrobial activity against nontypeable Haemophilus influenzae. An in vitro exposure model was utilized to measure the effect of tobacco exposure on human bronchial epithelial culture (HBEC) antimicrobial activity against H. influenzae. The direct effects of cigarette and little cigar smoke exposure on SPLUNC1 function was determined using 24 h growth measurements and LPS binding assays. Results: H. influenzae growth in cigarette smoker´s sputum was significantly greater compared to never-smokers sputum over 24 h. HBEC supernatants and lysates contained significantly higher numbers of H. influenzae following chronic cigarette and little cigar smoke exposure compared to air-exposed controls. Furthermore, SPLUNC1´s antimicrobial activity and LPS-binding capability against both H. influenzae and P. aeruginosa was attenuated following cigarette and little cigar exposure. Conclusions: These data suggest that cigarette and little cigar exposure impairs SPLUNC1´s antimicrobial ability and that this inhibition may serve as a novel biomarker of harm that can be used to assess the toxicity of commercial tobacco products.Fil: Moore, Patrick J.. University of North Carolina; Estados UnidosFil: Sesma, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Alexis, Neil E.. University of North Carolina; Estados UnidosFil: Tarran, Robert. University of North Carolina; Estados Unido

Mammalian short palate lung and nasal epithelial clone 1 (SPLUNC1) in pH-dependent airway hydration

The epithelia that line the conducting airways are the lung’s first point of contact with inhaled pathogens and toxicants. As such, they are known to play an important role in the lung’s innate defense system, which includes (i) the production of airway surface liquid (ASL) that helps cleanse the airways through the physical removal of pathogens and toxicants on the mucociliary escalator and (ii) the secretion of anti-microbial proteins into the ASL to kill inhaled pathogens. Interestingly, the recently crystallized short palate lung and nasal epithelial clone 1 (SPLUNC1) protein appears to be a multi-functional protein. That is, it not only acts as an anti-microbial agent, but also modulates ASL homeostasis by acting as an endogenous inhibitor of the epithelial Na+ channel (ENaC). This review will focus on the latter function of SPLUNC1, and will discuss new structural and physiological data regarding SPLUNC1’s failure to function as a regulator of ASL hydration in CF airways

Short Palate, Lung, and Nasal Epithelial Clone 1 Has Antimicrobial and Antibiofilm Activities against the Burkholderia cepacia Complex

ABSTRACT The opportunistic bacteria of the Burkholderia cepacia complex (Bcc) are extremely pathogenic to cystic fibrosis (CF) patients, and acquisition of Bcc bacteria is associated with a significant increase in mortality. Treatment of Bcc infections is difficult because the bacteria are multidrug resistant and able to survive in biofilms. Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) is an innate defense protein that is secreted by the upper airways and pharynx. While SPLUNC1 is known to have antimicrobial functions, its effects on Bcc strains are unclear. We therefore tested the hypothesis that SPLUNC1 is able to impair Bcc growth and biofilm formation. We found that SPLUNC1 exerted bacteriostatic effects against several Bcc clinical isolates, including B. cenocepacia strain J2315 (50% inhibitory concentration [IC 50 ] = 0.28 μM), and reduced biofilm formation and attachment (IC 50 = 0.11 μM). We then determined which domains of SPLUNC1 are responsible for its antimicrobial activity. Deletions of SPLUNC1's N terminus and α6 helix did not affect its function. However, deletion of the α4 helix attenuated antimicrobial activity, while the corresponding α4 peptide displayed antimicrobial activity. Chronic neutrophilia is a hallmark of CF lung disease, and neutrophil elastase (NE) cleaves SPLUNC1. However, we found that the ability of SPLUNC1 to disrupt biofilm formation was significantly potentiated by NE pretreatment. While the impact of CF on SPLUNC1-Bcc interactions is not currently known, our data suggest that understanding this interaction may have important implications for CF lung disease

Cathepsin B contributes to Na + hyperabsorption in cystic fibrosis airway epithelial cultures: Cathepsin B and ENaC

In cystic fibrosis (CF) lung disease, the absence of functional CF transmembrane conductance regulator results in Cl−/HCO3− hyposecretion and triggers Na+ hyperabsorption through the epithelial Na+ channel (ENaC), which contribute to reduced airway surface liquid (ASL) pH and volume. Prostasin, a membrane-anchored serine protease with trypsin-like substrate specificity has previously been shown to activate ENaC in CF airways. However, prostasin is typically inactive below pH 7.0, suggesting that it may be less relevant in acidic CF airways. Cathepsin B (CTSB) is present in both normal and CF epithelia and is secreted into ASL, but little is known about its function in the airways. We hypothesized that the acidic ASL seen in CF airways may stimulate CTSB to activate ENaC, contributing to Na+ hyperabsorption and depletion of CF ASL volume. In Xenopus laevis oocytes, CTSB triggered α- and γENaC cleavage and induced an increase in ENaC activity. In bronchial epithelia from both normal and CF donor lungs, CTSB localized to the apical membrane. In normal and CF human bronchial epithelial cultures, CTSB was detected at the apical plasma membrane and in the ASL. CTSB activity was significantly elevated in acidic ASL, which correlated with increased abundance of ENaC in the plasma membrane and a reduction in ASL volume. This acid/CTSB-dependent activation of ENaC was ameliorated with the cell impermeable, CTSB-selective inhibitor CA074, suggesting that CTSB inhibition may have therapeutic relevance. Taken together, our data suggest that CTSB is a pathophysiologically relevant protease that activates ENaC in CF airways

Airway hydration and COPD

Chronic obstructive pulmonary disease (COPD) is one of the prevalent causes of worldwide mortality and encompasses two major clinical phenotypes, i.e., chronic bronchitis (CB) and emphysema. The most common cause of COPD is chronic tobacco inhalation. Research focused on the chronic bronchitic phenotype of COPD has identified several pathological processes that drive disease initiation and progression. For example, the lung’s mucociliary clearance (MCC) system performs the critical task of clearing inhaled pathogens and toxic materials from the lung. MCC efficiency is dependent on: (i) the ability of apical plasma membrane ion channels such as the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na+ channel (ENaC) to maintain airway hydration; (ii) ciliary beating; and, (iii) appropriate rates of mucin secretion. Each of these components is impaired in CB and likely contributes to the mucus stasis/accumulation seen in CB patients. This review highlights the cellular components responsible for maintaining MCC and how this process is disrupted following tobacco exposure and with CB. We shall also discuss existing therapeutic strategies for the treatment of chronic bronchitis and how components of the MCC can be used as biomarkers for the evaluation of tobacco or tobacco-like-product exposure

Liquid movement across the surface epithelium of large airways

The cystic fibrosis conductance regulator CFTR gene is found on chromosome 7 (Kerem et al., 1989; Riordan et al., 1989) and encodes for a 1,480 amino acid protein which is present in the plasma membrane of epithelial cells (Anderson et al., 1992). This protein appears to have many functions, but a unifying theme is that it acts as a protein kinase C- and cyclic AMP-regulated Cl- channel (Winpenny et al., 1995; Jia et al., 1997). In the superficial epithelium of the conducting airways, CFTR is involved in Cl- secretion (Boucher, 2003) and also acts as a regulator of the epithelial Na+ channel (ENaC) and hence Na+ absorption (Boucher et al., 1986; Stutts et al., 1995). In this chapter, we will discuss the regulation of these two ion channels, and how they can influence liquid movement across the superficial airway epithelium

In Vivo Airway Surface Liquid Cl− Analysis with Solid-State Electrodes

The pathogenesis of cystic fibrosis (CF) airways disease remains controversial. Hypotheses that link mutations in CFTR and defects in ion transport to CF lung disease predict that alterations in airway surface liquid (ASL) isotonic volume, or ion composition, are critically important. ASL [Cl−] is pivotal in discriminating between these hypotheses, but there is no consensus on this value given the difficulty in measuring [Cl−] in the “thin” ASL (∼30 μm) in vivo. Consequently, a miniaturized solid-state electrode with a shallow depth of immersion was constructed to measure ASL [Cl−] in vivo. In initial experiments, the electrode measured [Cl−] in physiologic salt solutions, small volume (7.6 μl) test solutions, and in in vitro cell culture models, with ≥93% accuracy. Based on discrepancies in reported values and/or absence of data, ASL Cl− measurements were made in the following airway regions and species. First, ASL [Cl−] was measured in normal human nasal cavity and averaged 117.3 ± 11.2 mM (n = 6). Second, ASL [Cl−] measured in large airway (tracheobronchial) regions were as follows: rabbit trachea and bronchus = 114.3 ± 1.8 mM; (n = 6) and 126.9 ± 1.7 mM; (n = 3), respectively; mouse trachea = 112.8 ± 4.2 mM (n = 13); and monkey bronchus = 112.3 ± 10.9 mM (n = 3). Third, Cl− measurements were made in small (1–2 mm) diameter airways of the rabbit (108.3 ± 7.1 mM, n = 5) and monkey (128.5 ± 6.8 mM, n = 3). The measured [Cl−], in excess of 100 mM throughout all airway regions tested in multiple species, is consistent with the isotonic volume hypothesis to describe ASL physiology

Measurement of the Airway Surface Liquid Volume with Simple Light Refraction Microscopy

In the cystic fibrosis (CF) lung, the airway surface liquid (ASL) volume is depleted, impairing mucus clearance from the lung and leading to chronic airway infection and obstruction. Several therapeutics have been developed that aim to restore normal airway surface hydration to the CF airway, yet preclinical evaluation of these agents is hindered by the paucity of methods available to directly measure the ASL. Therefore, we sought to develop a straightforward approach to measure the ASL volume that would serve as the basis for a standardized method to assess mucosal hydration using readily available resources. Primary human bronchial epithelial (HBE) cells cultured at an air–liquid interface develop a liquid meniscus at the edge of the culture. We hypothesized that the size of the fluid meniscus is determined by the ASL volume, and could be measured as an index of the epithelial surface hydration status. A simple method was developed to measure the volume of fluid present in meniscus by imaging the refraction of light at the ASL interface with the culture wall using low-magnification microscopy. Using this method, we found that primary CF HBE cells had a reduced ASL volume compared with non-CF HBE cells, and that known modulators of ASL volume caused the predicted responses. Thus, we have demonstrated that this method can detect physiologically relevant changes in the ASL volume, and propose that this novel approach may be used to rapidly assess the effects of airway hydration therapies in high-throughput screening assays