Airway Surface Liquid Research Articles

TRPV4 Channel Modulators as Potential Drug Candidates for Cystic Fibrosis.

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in defective chloride ion channels. This leads to thick, dehydrated mucus that severely disrupts mucociliary clearance in the respiratory system and triggers infection that eventually is the cause of death of CF patients. Current therapeutic strategies primarily focus on restoring CFTR function, blocking epithelial sodium channels to prevent mucus dehydration, or directly targeting mucus to reduce its viscosity. Among the ion channels expressed in ciliated bronchial epithelial cells, the transient receptor potential vanilloid 4 (TRPV4) channel emerges as a significant channel in CF pathogenesis. Activation of TRPV4 channels affects the regulation of airway surface liquid by modulating sodium absorption and intracellular calcium levels, which indirectly influences CFTR activity. TRPV4 is also involved in the regulatory volume decrease (RVD) process and enhances inflammatory responses in CF patients. Here, we combine current findings on TRPV4 channel modulation as a promising therapeutic approach for CF. Although limited studies have directly explored TRPV4 in CF, emerging evidence indicates that TRPV4 activation can significantly impact key pathological processes in the disease. Further investigation into TRPV4 modulators could lead to innovative treatments that alleviate severe respiratory complications and improve outcomes for CF patients.

BOS-318 treatment enhances elexacaftor-tezacaftor-ivacaftor (ETI)-mediated improvements in airways hydration and mucociliary transport

BackgroundCFTR triple modulator therapy, elexacaftor-tezacaftor-ivacaftor (ETI) has transformed care for people with cystic fibrosis (pwCF) who have eligible mutations. It is, however, not curative. Response to treatment also varies and lung disease although slowed, remains progressive. We have previously demonstrated that inhibition of the epithelial sodium channel (ENaC) by selective furin inhibition offers an alternative, mutation-agnostic approach to enhance airways hydration and restore mucociliary transport (MCT) in CF. Inhibition of furin therefore, offers a potential therapeutic strategy for those ineligible, intolerant or non-responsive to ETI and may provide a further opportunity for clinical benefit for those currently treated with ETI. The aim of this study was to determine the impact of furin inhibition on ETI responses to assess its utility as an adjunct therapy.MethodsDifferentiated primary human CF bronchial epithelial cells (CF HBECs) were treated with the highly selective furin inhibitor, BOS-318 and ETI. Ion channel function was measured using a 24-channel Transepithelial Current Clamp (TECC-24) system and airways surface hydration was investigated by measuring airway surface liquid (ASL) height and MCT rate.ResultsThe presence of BOS-318 had no effect on the ability of ETI to stimulate CFTR-mediated Cl−secretion but contributed a reduced Na+transportviarobust inhibition of ENaC. This altered ion transport profile effected an improved ASL height and MCT rate which were significantly greater than improvements observed with ETI alone, demonstrating the benefits of the dual approach.ConclusionsSelective furin inhibition has potential to further improve clinical outcomes for all pwCF and offers opportunity as an adjunct to improve responses to currently available CFTR modulator therapies.

The pH-sensitive translocation of V-ATPase in the small airway of cystic fibrosis pigs.

In contrast to pig large airways, the pH of airway surface liquid (ASL) in pig small airways is regulated by CFTR mediated HCO-3 secretion and the vacuolar-type H+ ATPase (V-ATPase) proton secretion. We hypothesized that in Cystic Fibrosis (CF), the ASL pH of small airways is acidic, and the V-ATPase is internalized. We quantified proton secretion during the addition of an alkaline test solutions by measuring changes in a pH dependent fluorescent dye generated by porcine small airway epithelia in the absence and presence of bafilomycin A1. The pH-dependent translocation of V-ATPase in ex vivo and in vivo preparations was measured using immunolocalization of V-ATPase. We found that bafilomycin sensitive proton secretion stopped when the ASL pH was less than 7.10. In non-CF pigs and mice, we found that V-ATPase was localized in the apical membrane, and internalized when the lungs were instilled with a pH 6.8 solution.Studies where we immediately fixed lungs from pigs revealed apical V-ATPase detection in non-CF piglets and less apical detection in CF piglets. Our data suggest that V-ATPase in small airways is internalized when the ASL pH is acidic. The decrease in apical localization of V-ATPase in CF pigs is consistent with an acidic ASL pH.

IL-13 induces loss of CFTR in ionocytes and reduces airway epithelial fluid absorption.

The airway surface liquid (ASL) plays a crucial role in lung defense mechanisms, and its composition and volume are regulated by the airway epithelium. The cystic fibrosis transmembrane conductance regulator (CFTR) is abundantly expressed in a rare airway epithelial cell type called an ionocyte. Recently, we demonstrated that ionocytes can increase liquid absorption through apical CFTR and basolateral barttin/chloride channels, while airway secretory cells mediate liquid secretion through apical CFTR channels and basolateral NKCC1 transporters. Th2-driven (IL-4/IL-13) airway diseases, such as asthma, cause goblet cell metaplasia, accompanied by increased mucus production and airway secretions. In this study, we investigate the effect of IL-13 on chloride and liquid transport performed by ionocytes. IL-13 treatment of human airway epithelia was associated with reduced epithelial liquid absorption rates and increased ASL volume. Additionally, IL-13 treatment reduced the abundance of CFTR-positive ionocytes and increased the abundance of CFTR-positive secretory cells. Increasing ionocyte abundance attenuated liquid secretion caused by IL-13. Finally, CFTR-positive ionocytes were less common in asthma and chronic obstructive pulmonary disease and were associated with airflow obstruction. Our findings suggest that loss of CFTR in ionocytes contributes to the liquid secretion observed in IL-13-mediated airway diseases.

Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations.

Hyper IgE syndrome (STAT3-HIES), also known as Job's syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES syndrome is characterized by chronic pulmonary infection and inflammation, suggesting impairment of pulmonary innate host defense. To identify airway epithelial host defense defects consequent to STAT3 mutations that, in addition to reported mutant STAT3 immunologic abnormalities, produce pulmonary infection. STAT3-HIES sputum was evaluated for biochemical/biophysical properties. STAT3-HIES excised lungs were harvested for histology; bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific mutation (R382W), expressed by lentiviruses, and a STAT3 knockout, generated by CRISPR/Cas9, were maintained in normal human bronchial epithelia under basal or inflammatory (IL1β) conditions. Effects of STAT3 deficiency on transcriptomics, and epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed. Mucus concentrations and viscoelasticity were increased in STAT3-HIES sputum. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1β expression. STAT3 deficiency impaired CFTR-dependent fluid and mucin secretion, inhibited expression of antimicrobial peptides, cytokines, and chemokines, and acidified airway surface liquid at baseline and post-IL1β exposure in vitro. Notably, mutant STAT3 suppressed IL1R1 expression. STAT3 mutations also inhibited ciliogenesis in vivo and impaired mucociliary transport in vitro, a process mediated via HES6 suppression. Administration of a γ-secretase inhibitor increased HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells. STAT3 dysfunction leads to multi-component defects in airway epithelial innate defense, which, in conjunction with STAT3-HIES immune deficiency, contributes to chronic pulmonary infection.

Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator correctors VX-445 and VX-121.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The large-conductance calcium-activated potassium channel (BKCa, KCa1.1) is also critical for maintaining lung airway surface liquid (ASL) volume. Here, we show that the class 2 (C2) CFTR corrector VX-445 (elexacaftor) induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121, a corrector under clinical evaluation. Whole-cell patch-clamp recordings verified that CFTR correctors potentiated BKCa activity from both primary HBEs and HEK cells stably expressing the α subunit (HEK-BK cells). Furthermore, excised patch-clamp recordings from HEK-BK cells verified direct action on the channel and demonstrated a significant increase in open probability. In mouse mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced firing frequency in primary rat hippocampal and cortical neurons. We raise the possibilities that C2 CFTR correctors gain additional clinical benefit by activation of BKCa in the lung yet may lead to adverse events through BKCa activation elsewhere.

Epithelial responses to CFTR modulators are improved by inflammatory cytokines and impaired by antiinflammatory drugs.

Cystic fibrosis (CF) is a genetic disorder that disrupts CF transmembrane conductance regulator (CFTR) anion channels and impairs airway host defenses. Airway inflammation is ubiquitous in CF, and suppressing it has generally been considered to improve outcomes. However, the role of inflammation in people taking CFTR modulators, small-molecule drugs that restore CFTR function, is not well understood. We previously showed that inflammation enhances the efficacy of CFTR modulators. To further elucidate this relationship, we treated human ΔF508-CF epithelia with TNF-α and IL-17, two inflammatory cytokines that are elevated in CF airways. TNF-α+IL-17 enhanced CFTR modulator-evoked anion secretion through mechanisms that raise intracellular Cl- (Na+/K+/2Cl- cotransport) and HCO3- (carbonic anhydrases and Na+/HCO3- cotransport). This enhancement required p38 MAPK signaling. Importantly, CFTR modulators did not affect CF airway surface liquid viscosity under control conditions but prevented the rise in viscosity in epithelia treated with TNF-α+IL-17. Finally, antiinflammatory drugs limited CFTR modulator responses in TNF-α+IL-17-treated epithelia. These results provide critical insights into mechanisms by which inflammation increases responses to CFTR modulators. They also suggest an equipoise between potential benefits and limitations of suppressing inflammation in people taking modulators, call into question current treatment approaches, and highlight a need for additional studies.

Metachrony drives effective mucociliary transport via a calcium-dependent mechanism.

The mucociliary transport apparatus is critical for maintaining lung health via the coordinated movement of cilia to clear mucus and particulates. A metachronal wave propagates across the epithelium when cilia on adjacent multiciliated cells beat slightly out of phase along the proximal-distal axis of the airways in alignment with anatomically directed mucociliary clearance. We hypothesized that metachrony optimizes mucociliary transport (MCT) and that disruptions of calcium signaling would abolish metachrony and decrease MCT. We imaged bronchi from human explants and ferret tracheae using micro-optical coherence tomography (µOCT) to evaluate airway surface liquid depth (ASL), periciliary liquid depth (PCL), cilia beat frequency (CBF), MCT, and metachrony in situ. We developed statistical models that included covariates of MCT. Ferret tracheae were treated with BAPTA-AM (chelator of intracellular Ca2+), lanthanum chloride (nonpermeable Ca2+ channel competitive antagonist), and repaglinide (inhibitor of calaxin) to test calcium dependence of metachrony. We demonstrated that metachrony contributes to mucociliary transport of human and ferret airways. MCT was augmented in regions of metachrony compared with nonmetachronous regions by 48.1%, P = 0.0009 or 47.5%, P < 0.0020 in humans and ferrets, respectively. PCL and metachrony were independent contributors to MCT rate in humans; ASL, CBF, and metachrony contribute to ferret MCT rates. Metachrony can be disrupted by interference with calcium signaling including intracellular, mechanosensitive channels, and calaxin. Our results support that the presence of metachrony augments MCT in a calcium-dependent mechanism.NEW & NOTEWORTHY We developed a novel imaging-based analysis to detect coordination of ciliary motion and optimal coordination, a process called metachrony. We found that metachrony is key to the optimization of ciliary-mediated mucus transport in both ferret and human tracheal tissue. This process appears to be regulated through calcium-dependent mechanisms. This study demonstrates the capacity to measure a key feature of ciliary coordination that may be important in genetic and acquired disorders of ciliary function.

Apical pH regulates calcium-activated chloride secretion across human airway epithelia

Anion secretion across airway epithelia contributes to host defense by increasing airway surface liquid (ASL) volume and pH. Anion channels in the apical membrane of airway epithelia include Ca2+-activated Cl− channels (CaCCs) and cystic fibrosis transmembrane conductance regulator (CFTR) channels. Mutations in the CFTR gene that encode nonfunctional CFTR channels cause cystic fibrosis (CF); thus, activating CaCCs may restore anion secretion in people with CF. ATP secreted into the ASL activates purinergic receptors, leading to an increase in intracellular Ca2+ (Cai2+) and the opening of CaCCs. Data from docking simulations suggest that the protonation state of ATP influences its affnity for purinergic receptors, therefore we tested the hypothesis that ASL pH influences the effcacy of ATP to activate CaCCs in airway epithelia. In saline solutions buffered with 5 mM ATP, titration experiments revealed a pKa of ~6.9 at 37°C. Thus, ATP exists in its protonated ATP3− and deprotonated ATP4− in ASL that can range from pH 6 to pH 7.5. To determine the effect of pH on ATP-stimulated Cl− secretion through CaCCs, we measured Cl− secretion across CF epithelia generated by several ATP concentrations with apical pH-6.00, -6.70, and -7.40 solutions. As pH became alkaline, the EC50 for the relationship between [ATP] and Cl− secretion decreased. Using the Henderson-Hasselbalch equation and the pKa for ATP, we determined the relationship between ATP4− and Cl− secretion. This analysis revealed that regardless of apical pH values, the relationship between [ATP4−] and Cl− secretion aligned to a single experimental fit. These data suggest that pH regulates the CaCC response by deprotonating ATP. To examine the pH dependence of purinergic receptor activation, we measured ATP-induced Cai2+ responses in acutely dissociated human airway epithelial cells loaded with FURA-2 and perfused with pH-7.40 or -6.50 solutions. Separate experiments measuring intracellular pH in pH-7.40 or 6.50 solutions informed Kd values for FURA-2 experiments. With an [ATP] that induces similar levels of Cl− secretion from pH 6.00 to pH 7.40 (100 μM ATP), we observed similar Cai2+ responses for pH-7.40 and -6.50 solutions. However, when we repeated the experiment with an [ATP] that induces pH-sensitive Cl− secretion (10 μM ATP), we observed larger Ca2+ responses with pH-7.40 solutions than with pH-6.50 solutions. These data indicate that pH influences purinergic receptor activation near the pKa of ATP, a finding consistent with ATP4− activating purinergic receptors. These data have implications in CF and airway inflammation. In CF, decreased CFTR-mediated HCO−3 secretion acidifies the ASL, thus a large portion of ATP will exist in the protonated ATP3− species requiring a higher total [ATP] to open CaCCs. Inflammatory cytokines that cause airway inflammation increase the ASL pH, therefore a larger portion of ATP will exist in the deprotonated ATP4− species requiring less total [ATP] to open CaCCs. Cystic Fibrosis Foundation Research Development Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of electrogenic and electroneutral monocarboxylate transport in airway clearance

Introduction: Monocarboxylates can be transported by the electrogenic Na+-coupled system (SMCTs/SLC5 family) and the electroneutral H+-coupled system (MCTs/SLC16 family). Previously, MCTs have been described in human bronchial epithelium but there is no evidence of SMCTs activity in airways. We describe here for first time that SMCT1/SCL5A8 is functionally expressed in mouse tracheal epithelium. We hypothesized that monocarboxylate transport activity might downregulate airway clearance in mouse tracheas as increased Na+-absorption reduce airway surface liquid (ASL), and increased H+-secretion acidify ASL. Methods: Short-circuit current ( ISC) recordings were performed in bicarbonate buffer with and without Na+, videomicroscopy of particle track speed (PTS) and mucus transport speed were performed in mouse tracheas. Animals (C57BL6/J) were housed at CECs-Animal facility under controlled temperature, humidity and free access to water and food. All protocols were approved (IACUC, #CECs-2022-04), in accordance with relevant regulations. Results: 10 mM apical L-lactate, D-lactate, pyruvate, propionate or acetate induced a negative current ( ISC~ -35 μA·cm−2; n=4-5 each group) and reduced (p&lt;0.05 rank-sum test) when Na+ was replaced. L-lactate or pyruvate, transported by SMCTs and MCTs increased PTS (2.1±0.1 control vs 3.28±0.4 μm·s−1* for L-lactate and 3.31±0.1 μm·s−1* for pyruvate; n=3 each group; * indicates p&lt;0.05 rank-sum test) and inhibited by 30 μM of the MCT inhibitor AR-C155858 (1.9±0.1 μm·s−1). D-lactate, transported by SMCTs only, did not affected PTS (2.03±0.1 μm·s−1; p&gt;0.05; n=3 each group). We observed a reduction of amiloride-sensitive Na+-absorptive currents in tissues incubated with L-lactate compared to non-incubated ( ISC -17±4 μA·cm−2 vs -5±4 μA·cm−2; p&lt;0.05 rank-sum test; n=4 each group). Finally, analysis of mucus transport determined that L- but not D-lactate increased the speed of mucus, that was prevented when tissues were incubated with AR-C155858 (control 1.06 ± 0.05 μm·s−1 vs L-lactate 1.24 ± 0.08 μm·s−1*, vs L-lactate + AR-C155858 1.02 ± 0.06 μm·s−1; * indicates p&lt;0.05 ANOVA on ranks; n=4 each group). Real time PCR determined that the only SMCT expressed in airway epithelium is SLC5A8. Conclusion: SLC5A8 Na+-dependent D-lactate transport didn't affect mucus clearance. This is explained by the fact that ENaC Na+ absorption competes with SMCTs for basolateral Na+ transport, suggesting that both ENaC and SLC5A8 localize in the same cells. On the other hand, monocarboxylates that increase PTS and mucus speed transport might be explained by MCT-dependent H+ removal (alkalization) from ASL. Since alkalization of the airway surface can restore airway clearance, the use of MCTs transportable substrates might help alleviate mucostasis in muco-obstructive diseases. Funded by FONDECYT 1221257 (C.A.F.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Adenylate cyclase 6 targets in Mucociliary Clearance

Background. Mucociliary clearance (MCC) is a frontline airway defense mechanism that relies on the appropriate interactions between the epithelium, ciliary beat frequency, and the quantity and quality of mucus. MCC is severely impaired in cystic fibrosis (CF) and drives lung function decline and ultimate respiratory failure and death. A great need remains to identify the key molecular players that regulate MCC in CF outside the scope of FDA approved CFTR therapy and to specifically target MCC dysfunction in CF using small molecules. Results. We discover a novel key role of cAMP-synthesizing enzyme Adenylate cyclase 6 (AC6) in MCC via (a) regulation of CFTR chloride channels in the secretory cells that generate airway surface liquid (ASL) component of MCC and (b) AC6 mediated regulation of ciliary beat frequency in the ciliated cells that is required for the mechanical clearance. Specific activators of AC6 (C20: Top candidate) were developed and improved MCC process ex vivo in CF explant tissues and cells. Together these mechanisms and small molecule approach that improve airway fluid balance and cilia function could be beneficial in CF and other respiratory diseases. Experimental Approach. We performed CFTR functional assessment in normal and CF patient derived airway epithelial cells by adapting patch-sequencing approach and using the specific activators of AC6. The effect of AC6 activators on MCC will be evaluated ex vivo in patient derived lung explant tissues , in vivo (micro-CT) in CF rat models and in vitro (ciliary beat frequency and bead flow measurements) in air-liquid interface airway epithelial cultures. Overall, these studies will be mechanistic and translational involving human disease specimens, animal models, drug development and computational analysis. Significance of the results. The proposed research can improve our understanding of epithelial biology and advance activators of AC6 as therapeutic agents to treat mucus clearance defects. These findings can be leveraged to modulate CFTR function and cilia-generate flow, presenting a potential therapeutic avenue for conditions that involve CFTR and cilia dysregulation associated with several respiratory diseases. National Institutes of Health (NIH) DK080834 and P30-DK117467 and HL147351 (to APN). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

SERS Microsensors for the Study of pH Regulation in Cystic Fibrosis Patient-Derived Airway Cultures.

Acidification of the airway surface liquid in the respiratory system could play a role in the pathology of Cystic Fibrosis, but its low volume and proximity to the airway epithelium make it a challenging biological environment in which to noninvasively collect pH measurements. To address this challenge, we explored surface enhanced Raman scattering microsensors (SERS-MS), with a 4-mercaptobenzoic acid (MBA) pH reporter molecule, as pH sensors for the airway surface liquid of patient-derived in vitro models of the human airway. Using air-liquid interface (ALI) cultures to model the respiratory epithelium, we show that SERS-MS facilitates the optical measurement of trans-epithelial pH gradients between the airway surface liquid and the basolateral culture medium. SERS-MS also enabled the successful quantification of pH changes in the airway surface liquid following stimulation of the Cystic Fibrosis transmembrane conductance regulator (CFTR, the apical ion channel that is dysfunctional in Cystic Fibrosis airways). Finally, the influence of CFTR mutations on baseline airway surface liquid pH was explored by using SERS-MS to measure the pH in ALIs grown from Cystic Fibrosis and non-Cystic Fibrosis donors.

Recessively Inherited Deficiency of Secreted WFDC2 (HE4) Causes Nasal Polyposis and Bronchiectasis.

Rationale: Bronchiectasis is a pathological dilatation of the bronchi in the respiratory airways associated with environmental or genetic causes (e.g., cystic fibrosis, primary ciliary dyskinesia, and primary immunodeficiency disorders), but most cases remain idiopathic. Objectives: To identify novel genetic defects in unsolved cases of bronchiectasis presenting with severe rhinosinusitis, nasal polyposis, and pulmonary Pseudomonas aeruginosa infection. Methods: DNA was analyzed by next-generation or targeted Sanger sequencing. RNA was analyzed by quantitative PCR and single-cell RNA sequencing. Patient-derived cells, cell cultures, and secretions (mucus, saliva, seminal fluid) were analyzed by Western blotting and immunofluorescence microscopy, and mucociliary activity was measured. Blood serum was analyzed by electrochemiluminescence immunoassay. Protein structure and proteomic analyses were used to assess the impact of a disease-causing founder variant. Measurements and Main Results: We identified biallelic pathogenic variants in WAP four-disulfide core domain 2 (WFDC2) in 11 individuals from 10 unrelated families originating from the United States, Europe, Asia, and Africa. Expression of WFDC2 was detected predominantly in secretory cells of control airway epithelium and also in submucosal glands. We demonstrate that WFDC2 is below the limit of detection in blood serum and hardly detectable in samples of saliva, seminal fluid, and airway surface liquid from WFDC2-deficient individuals. Computer simulations and deglycosylation assays indicate that the disease-causing founder variant p.Cys49Arg structurally hampers glycosylation and, thus, secretion of mature WFDC2. Conclusions: WFDC2 dysfunction defines a novel molecular etiology of bronchiectasis characterized by the deficiency of a secreted component of the airways. A commercially available blood test combined with genetic testing allows its diagnosis.

How Does Airway Surface Liquid Composition Vary in Different Pulmonary Diseases, and How Can We Use This Knowledge to Model Microbial Infections?

Growth environment greatly alters many facets of pathogen physiology, including pathogenesis and antimicrobial tolerance. The importance of host-mimicking environments for attaining an accurate picture of pathogen behaviour is widely recognised. Whilst this recognition has translated into the extensive development of artificial cystic fibrosis (CF) sputum medium, attempts to mimic the growth environment in other respiratory disease states have been completely neglected. The composition of the airway surface liquid (ASL) in different pulmonary diseases is far less well characterised than CF sputum, making it very difficult for researchers to model these infection environments. In this review, we discuss the components of human ASL, how different lung pathologies affect ASL composition, and how different pathogens interact with these components. This will provide researchers interested in mimicking different respiratory environments with the information necessary to design a host-mimicking medium, allowing for better understanding of how to treat pathogens causing infection in these environments.

SPLUNC1 as a biomarker of pulmonary exacerbations in children with cystic fibrosis

BackgroundShort palate, lung, and nasal epithelium clone 1 (SPLUNC1) is an innate defence protein that acts as an anti-microbial agent and regulates airway surface liquid volume through inhibition of the epithelial sodium channel (ENaC). SPLUNC1 levels were found to be reduced in airway secretions of adults with cystic fibrosis (CF). The potential of SPLUNC1 as a biomarker in children with CF is unknown. MethodsWe quantified SPLUNC1, interleukin-8 (IL-8) and neutrophil elastase (NE) in sputum of CF children treated with either intravenous antibiotics or oral antibiotics for a pulmonary exacerbation (PEx)s, and in participants of a prospective cohort of CF children with preserved lung function on spirometry, followed over a period of two years. ResultsSputum SPLUNC1 levels were significantly lower before compared to after intravenous and oral antibiotic therapy for PEx. In the longitudinal cohort, SPLUNC1 levels were found to be decreased at PEx visits compared to both previous and subsequent stable visits. Higher SPLUNC1 levels at stable visits were associated with longer PEx-free time (hazard ratio 0.85, p = 0.04). SPLUNC1 at PEx visits did not correlate with IL-8 or NE levels in sputum or forced expiratory volume in one second (FEV1) but did correlate with the lung clearance index (LCI) (r=-0.53, p < 0.001). ConclusionSPLUNC1 demonstrates promising clinometric properties as a biomarker of PEx in children with CF.

Apical dehydration impairs the cystic fibrosis airway epithelium barrier via a β1-integrin/YAP1 pathway.

Defective hydration of airway surface mucosa is associated with lung infection in cystic fibrosis (CF), partly caused by disruption of the epithelial barrier integrity. Although rehydration of the CF airway surface liquid (ASL) alleviates epithelium vulnerability to infection by junctional protein expression, the mechanisms linking ASL to barrier integrity are unknown. We show here the strong degradation of YAP1 and TAZ proteins in well-polarized CF human airway epithelial cells (HAECs), a process that was prevented by ASL rehydration. Conditional silencing of YAP1 in rehydrated CF HAECs indicated that YAP1 expression was necessary for the maintenance of junctional complexes. A higher plasma membrane tension in CF HAECs reduced endocytosis, concurrent with the maintenance of active β1-integrin ectopically located at the apical membrane. Pharmacological inhibition of β1-integrin accumulation restored YAP1 expression in CF HAECs. These results indicate that dehydration of the CF ASL affects epithelial plasma membrane tension, resulting in ectopic activation of a β1-integrin/YAP1 signaling pathway associated with degradation of junctional proteins.

Specialized proresolving mediator resolvin E1 corrects the altered cystic fibrosis nasal epithelium cilia beating dynamics

In cystic fibrosis (CF), impaired mucociliary clearance leads to chronic infection and inflammation. However, cilia beating features in a CF altered environment, consisting of dehydrated airway surface liquid layer and abnormal mucus, have not been fully characterized. Furthermore, acute inflammation is normally followed by an active resolution phase requiring specialized proresolving lipid mediators (SPMs) and allowing return to homeostasis. However, altered SPMs biosynthesis has been reported in CF. Here, we explored cilia beating dynamics in CF airways primary cultures and its response to the SPMs, resolvin E1 (RvE1) and lipoxin B4 (LXB4). Human nasal epithelial cells (hNECs) from CF and non-CF donors were grown at air-liquid interface. The ciliary beat frequency, synchronization, orientation, and density were analyzed from high-speed video microscopy using a multiscale Differential Dynamic Microscopy algorithm and an in-house developed method. Mucins and ASL layer height were studied by qRT-PCR and confocal microscopy. Principal component analysis showed that CF and non-CF hNEC had distinct cilia beating phenotypes, which was mostly explained by differences in cilia beat organization rather than frequency. Exposure to RvE1 (10 nM) and to LXB4 (10 nM) restored a non-CF-like cilia beating phenotype. Furthermore, RvE1 increased the airway surface liquid (ASL) layer height and reduced the mucin MUC5AC thickness. The calcium-activated chloride channel, TMEM16A, was involved in the RvE1 effect on cilia beating, hydration, and mucus. Altogether, our results provide evidence for defective cilia beating in CF airway epithelium and a role of RvE1 and LXB4 to restore the main epithelial functions involved in the mucociliary clearance.

Akt activator SC79 stimulates antibacterial nitric oxide generation in human nasal epithelial cells in vitro.

The role of Akt in nasal immunity is unstudied. Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) expressed in epithelial ciliated cells. Nitric oxide (NO) production by ciliated cells can have antibacterial and antiviral effects. Increasing nasal NO may be a useful antipathogen strategy in chronic rhinosinusitis (CRS). We previously showed that small-molecule Akt activator SC79 induces nasal cell NO production and suppresses IL-8 via the transcription factor Nrf-2. We hypothesized that SC79 NO production may additionally have antibacterial effects. NO production was measured using fluorescent dye DAF-FM. We tested effects of SC79 during co-culture of Pseudomonas aeruginosa with primary nasal epithelial cells, using CFU counting and live-dead staining to quantify bacterial killing. Pharmacology determined the mechanism of SC79-induced NO production and tested dependence on Akt. SC79 induced dose-dependent, Akt-dependent NO production in nasal epithelial cells. The NO production required eNOS and Akt. The NO released into the airway surface liquid killed P. aeruginosa. No toxicity (LDH release) or inflammatory effects (IL8 transcription) were observed over 24h. Together, these data suggest multiple immune pathways are stimulated by SC79, with antipathogen effects. This in vitro pilot study suggests that a small-molecule Akt activator may have clinical utility in CRS or respiratory other infection settings, warranting future in vivo studies.

Dynamic measurement of airway surface liquid volume with an ex vivo trachea-chip.

The volume and composition of airway surface liquid (ASL) is regulated by liquid secretion and absorption across airway epithelia, controlling the pH, solute concentration, and biophysical properties of ASL in health and disease. Here, we developed a method integrating explanted tracheal tissue with a micro-machined device (referred to as "ex vivo trachea-chip") to study the dynamic properties of ASL volume regulation. The ex vivo trachea-chip allows real-time measurement of ASL transport (Jv) with intact airway anatomic structures, environmental control, high-resolution, and enhanced experimental throughput. Applying this technology to freshly excised tissue we observed ASL absorption under basal conditions. The apical application of amiloride, an inhibitor of airway epithelial sodium channels (ENaC), reduced airway liquid absorption. Furthermore, the basolateral addition of NPPB, a Cl- channel inhibitor, reduced the basal rate of ASL absorption, implicating a role for basolateral Cl- channels in ASL volume regulation. When tissues were treated with apical amiloride and basolateral methacholine, a cholinergic agonist that stimulates secretion from airway submucosal glands, the net airway surface liquid production shifted from absorption to secretion. This ex vivo trachea-chip provides a new tool to investigate ASL transport dynamics in pulmonary disease states and may aid the development of new therapies targeting ASL regulation.

PM2.5 Exposure Inhibits Transepithelial Anion Short-circuit Current by Downregulating P2Y2 Receptor/CFTR Pathway.

Fine particulate matter (PM2.5) can damage airway epithelial barriers. The anion transport system plays a crucial role in airway epithelial barriers. However, the detrimental effect and mechanism of PM2.5 on the anion transport system are still unclear. In this study, airway epithelial cells and ovalbumin (OVA)-induced asthmatic mice were used. In transwell model, the adenosine triphosphate (ATP)-induced transepithelial anion short-circuit current (Isc) and airway surface liquid (ASL) significantly decreased after PM2.5 exposure. In addition, PM2.5 exposure decreased the expression levels of P2Y2R, CFTR and cytoplasmic free-calcium, but ATP can increase the expressions of these proteins. PM2.5 exposure increased the levels of Th2-related cytokines of bronchoalveolar lavage fluid, lung inflammation, collagen deposition and hyperplasisa of goblet cells. Interestingly, the administration of ATP showed an inhibitory effect on lung inflammation induced by PM2.5. Together, our study reveals that PM2.5 impairs the ATP-induced transepithelial anion Isc through downregulating P2Y2R/CFTR pathway, and this process may participate in aggravating airway hyperresponsiveness and airway inflammation. These findings may provide important insights on PM2.5-mediated airway epithelial injury.