ETD001: A novel inhaled ENaC blocker with an extended duration of action in vivo.

BACKGROUND: Inhibiting ENaC in the airways of people with cystic fibrosis (pwCF) is hypothesized to enhance mucociliary clearance (MCC) and provide clinical benefit. Historically, inhaled ENaC blockers have failed to show benefit in pwCF challenging this hypothesis. It is however unknown whether the clinical doses were sufficient to provide the required long duration of action in the lungs and questions whether a novel candidate could offer advantages where others have failed? METHODS: Dose-responses with the failed ENaC blockers (VX-371, BI 1265162, AZD5634, QBW276) together with ETD001 (a novel long acting inhaled ENaC blocker) were established in a sheep model of MCC and were used to predict clinically relevant doses that would provide a long-lasting enhancement of MCC in pwCF. In each case, dose predictions were compared with the selected clinical dose. RESULTS: Each of the failed candidates enhanced MCC in the sheep model. Translating these dose-response data to human equivalent doses, predicted that substantially larger doses of each candidate, than were evaluated in clinical studies, would likely have been required to achieve a prolonged enhancement of MCC in pwCF. In contrast, ETD001 displayed a long duration of action (≥16 h) at a dose level that was well tolerated in Phase 1 clinical studies. CONCLUSIONS: These data support that the ENaC blocker hypothesis is yet to be appropriately tested in pwCF. ETD001 has a profile that enables dosing at a level sufficient to provide a long duration of action in a Phase 2 clinical study in pwCF scheduled for 2024.

Response to Elexacaftor/Tezacaftor/Ivacaftor in people with cystic fibrosis with the N1303K mutation: Case report and review of the literature.

Cystic fibrosis (CF) is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Thousands of CFTR mutations have been identified, but only a fraction are known to cause CF, with the most common being the prototypical class II CFTR mutation F508del. Elexacaftor-Tezacaftor-Ivacaftor (ETI) is a CFTR modulator that significantly increases ppFEV1 and reduces exacerbation frequencies. It is indicated for people with CF (pwCF) 2 years or older with at least one copy of F508del or one copy of the other 177 CFTR mutations that are responsive to ETI based on clinical or in vitro data. N1303K is the second most common class II mutation in the U.S. but is not yet FDA-approved for CFTR modulator therapy. However, N1303K is very similar to the F508del mutation and reveals variable in vitro responses to ETI. Theratyping provides an opportunity to consider ETI therapy for pwCF with mutations currently not approved by the FDA. We describe the case of an adult CF patient with W1282X and N1303K CFTR mutations and advanced CF lung disease (ACFLD) and declining lung function in which ETI was started after theratyping of nasal cells showed a meaningful response to ETI (current enhanced to over 10% of WT CFTR). The patient experienced clinical improvement with a 5% improvement in ppFEV1 and 10% increase in weight. However, there was no change in sweat chloride and the increase in ppFEV1 was less than what has been described for ACFLD patients with more typical ETI-amenable mutations. However, the response was in line with a few other cases described in the literature. This suggests a partial functional CFTR rescue like first-generation modulators for F508del. Thus, pwCF with N1303K CFTR variant could be considered for ETI eligibility.

The combination of propylene glycol and vegetable glycerin e-cigarette aerosols induces airway inflammation and mucus hyperconcentration.

Despite concerns over their safety, e-cigarettes (e-cigs) remain a popular tobacco product. Although nicotine and flavors found in e-cig liquids (e-liquids) can cause harm in the airways, whether the delivery vehicles propylene glycol (PG) and vegetable glycerin (VG) are innocuous when inhaled remains unclear. Here, we investigated the effects of e-cig aerosols generated from e-liquid containing only PG/VG on airway inflammation and mucociliary function in primary human bronchial epithelial cells (HBEC) and sheep. Primary HBEC were cultured at the air-liquid interface (ALI) and exposed to e-cig aerosols of 50%/50% v/v PG/VG. Ion channel conductance, ciliary beat frequency, and the expression of inflammatory markers, cell type-specific markers, and the major mucins MUC5AC and MUC5B were evaluated after seven days of exposure. Sheep were exposed to e-cig aerosols of PG/VG for five days and mucus concentration and matrix metalloproteinase-9 (MMP-9) activity were measured from airway secretions. Seven-day exposure of HBEC to e-cig aerosols of PG/VG caused a significant reduction in the activities of apical ion channels important for mucus hydration, including the cystic fibrosis transmembrane conductance regulator (CFTR) and large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. PG/VG aerosols significantly increased the mRNA expression of the inflammatory markers interleukin-6 (IL6), IL8, and MMP9, as well as MUC5AC. The increase in MUC5AC mRNA expression correlated with increased immunostaining of MUC5AC protein in PG/VG-exposed HBEC. On the other hand, PG/VG aerosols reduced MUC5B expression leading overall to higher MUC5AC/MUC5B ratios in exposed HBEC. Other cell type-specific markers, including forkhead box protein J1 (FOXJ1), keratin 5 (KRT5), and secretoglobin family 1A member 1 (SCGB1A1) mRNAs, as well as overall ciliation, were significantly reduced by PG/VG exposure. Finally, PG/VG aerosols increased MMP-9 activity and caused mucus hyperconcentration in sheep in vivo. E-cig aerosols of PG/VG induce airway inflammation, increase MUC5AC expression, and cause dysfunction of ion channels important for mucus hydration in HBEC in vitro. Furthermore, PG/VG aerosols increase MMP-9 activity and mucus concentration in sheep in vivo. Collectively, these data show that e-cig aerosols containing PG/VG are likely to be harmful in the airways.

SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in cell culture and in mice.

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in these drug targets is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein encoded as a small domain at the N terminus of nonstructural protein 3. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and IFN-stimulated gene expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.

Transport of Designed Ankyrin Repeat Proteins through reconstituted human bronchial epithelia and protection against SARS-CoV-2.

Clinical studies have proven antiviral effectiveness of treatment with a Designed Ankyrin Repeat Protein (DARPin) specific against the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). More information on transport mechanisms and efficiency to the site of action is desirable. Transepithelial migration through air-liquid interface (ALI) cultures of reconstituted human bronchial epithelia (HBE) was assessed by Enzyme-Linked Immunosorbent Assays and Confocal Laser Scanning Microscopy for different DARPin designs in comparison to a monoclonal antibody. Antiviral efficacy against authentic SARS-CoV-2, applied apically on HBE, was investigated based on viral titers and genome equivalents, after administration of therapeutic candidates on the basal side. Transepithelial translocation of all DARPin candidates and the monoclonal antibody was efficient and dose dependent. Small DARPins and the antibody migrated more efficiently than larger molecules, indicating different transport mechanisms involved. Microscopic analyses support this, demonstrating passive paracellular transport of smaller DARPins and transcellular migration of the larger molecules. All therapeutic candidates applied to the basal side of HBE conferred effective protection against SARS-CoV-2 infection. In summary, we have shown that DARPins specific against SARS-CoV-2 translocate across intact airway epithelia and confer effective protection against infection and viral replication.

E-cigarette aerosols of propylene glycol impair BK channel activity and parameters of mucociliary function.

Propylene glycol (PG) is a common delivery vehicle for nicotine and flavorings in e-cigarette (e-cig) liquids and is largely considered safe for ingestion. However, little is known about its effects as an e-cig aerosol on the airway. Here, we investigated whether pure PG e-cig aerosols in realistic daily amounts impact parameters of mucociliary function and airway inflammation in a large animal model (sheep) in vivo and primary human bronchial epithelial cells (HBECs) in vitro. Five-day exposure of sheep to e-cig aerosols of 100% PG increased mucus concentrations (% mucus solids) of tracheal secretions. PG e-cig aerosols further increased the activity of matrix metalloproteinase-9 (MMP-9) in tracheal secretions. In vitro exposure of HBECs to e-cig aerosols of 100% PG decreased ciliary beating and increased mucus concentrations. PG e-cig aerosols further reduced the activity of large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. We show here for the first time that PG can be metabolized to methylglyoxal (MGO) in airway epithelia. PG e-cig aerosols increased levels of MGO and MGO alone reduced BK activity. Patch-clamp experiments suggest that MGO can disrupt the interaction between the major pore-forming BK subunit human Slo1 (hSlo1) and the gamma regulatory subunit LRRC26. PG exposures also caused a significant increase in mRNA expression levels of MMP9 and interleukin 1 beta (IL1B). Taken together, these data show that PG e-cig aerosols cause mucus hyperconcentration in sheep in vivo and HBECs in vitro, likely by disrupting the function of BK channels important for airway hydration.

Nebulized Menthol Impairs Mucociliary Clearance via TRPM8 and MUC5AC/MUC5B in Primary Airway Epithelial Cells.

Flavorings enhance the palatability of e-cigarettes (e-cigs), with menthol remaining a popular choice among e-cig users. Menthol flavor remains one of the only flavors approved by the United States FDA for use in commercially available, pod-based e-cigs. However, the safety of inhaled menthol at the high concentrations used in e-cigs remains unclear. Here, we tested the effects of menthol on parameters of mucociliary clearance (MCC) in air-liquid interface (ALI) cultures of primary airway epithelial cells. ALI cultures treated with basolateral menthol (1 mM) showed a significant decrease in ciliary beat frequency (CBF) and airway surface liquid (ASL) volumes after 24 h. Menthol nebulized onto the surface of ALI cultures similarly reduced CBF and increased mucus concentrations, resulting in decreased rates of mucociliary transport. Nebulized menthol further increased the expression of mucin 5AC (MUC5AC) and mRNA expression of the inflammatory cytokines IL1B and TNFA. Menthol activated TRPM8, and the effects of menthol on MCC and inflammation could be blocked by a specific TRPM8 antagonist. These data provide further evidence that menthol at the concentrations used in e-cigs could cause harm to the airways.

Role of Secondary Organic Matter on Soot Particle Toxicity in Reconstituted Human Bronchial Epithelia Exposed at the Air-Liquid Interface.

Secondary organic matter (SOM) formed from gaseous precursors constitutes a major mass fraction of fine particulate matter. However, there is only limited evidence on its toxicological impact. In this study, air-liquid interface cultures of human bronchial epithelia were exposed to different series of fresh and aged soot particles generated by a miniCAST burner combined with a micro smog chamber (MSC). Soot cores with geometric mean mobility diameters of 30 and 90 nm were coated with increasing amounts of SOM, generated from the photo-oxidation of mesitylene and ozonolysis of α-pinene. At 24 h after exposure, the release of lactate dehydrogenase (LDH), indicating cell membrane damage, was measured and proteome analysis, i.e. the release of 102 cytokines and chemokines to assess the inflammatory response, was performed. The data indicate that the presence of the SOM coating and its bioavailability play an important role in cytotoxicity. In particular, LDH release increased with increasing SOM mass/total particle mass ratio, but only when SOM had condensed on the outer surface of the soot cores. Proteome analysis provided further evidence for substantial interference of coated particles with essential properties of the respiratory epithelium as a barrier as well as affecting cell remodeling and inflammatory activity.

Vegetable glycerin e-cigarette aerosols cause airway inflammation and ion channel dysfunction.

Vegetable glycerin (VG) and propylene glycol (PG) serve as delivery vehicles for nicotine and flavorings in most e-cigarette (e-cig) liquids. Here, we investigated whether VG e-cig aerosols, in the absence of nicotine and flavors, impact parameters of mucociliary function in human volunteers, a large animal model (sheep), and air-liquid interface (ALI) cultures of primary human bronchial epithelial cells (HBECs). We found that VG-containing (VG or PG/VG), but not sole PG-containing, e-cig aerosols reduced the activity of nasal cystic fibrosis transmembrane conductance regulator (CFTR) in human volunteers who vaped for seven days. Markers of inflammation, including interleukin-6 (IL6), interleukin-8 (IL8) and matrix metalloproteinase-9 (MMP9) mRNAs, as well as MMP-9 activity and mucin 5AC (MUC5AC) expression levels, were also elevated in nasal samples from volunteers who vaped VG-containing e-liquids. In sheep, exposures to VG e-cig aerosols for five days increased mucus concentrations and MMP-9 activity in tracheal secretions and plasma levels of transforming growth factor-beta 1 (TGF-ß1). In vitro exposure of HBECs to VG e-cig aerosols for five days decreased ciliary beating and increased mucus concentrations. VG e-cig aerosols also reduced CFTR function in HBECs, mechanistically by reducing membrane fluidity. Although VG e-cig aerosols did not increase MMP9 mRNA expression, expression levels of IL6, IL8, TGFB1, and MUC5AC mRNAs were significantly increased in HBECs after seven days of exposure. Thus, VG e-cig aerosols can potentially cause harm in the airway by inducing inflammation and ion channel dysfunction with consequent mucus hyperconcentration.

The CFTR Amplifier Nesolicaftor Rescues TGF-ß1 Inhibition of Modulator-Corrected F508del CFTR Function.

Highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulators have led to dramatic improvements in lung function in many people with cystic fibrosis (PwCF). However, the efficacy of CFTR modulators may be hindered by persistent airway inflammation. The cytokine transforming growth factor-beta1 (TGF-ß1) is associated with worse pulmonary disease in PwCF and can diminish modulator efficacy. Thus, strategies to augment the CFTR response to modulators in an inflammatory environment are needed. Here, we tested whether the CFTR amplifier nesolicaftor (or PTI-428) could rescue the effects of TGF-ß1 on CFTR function and ciliary beating in primary human CF bronchial epithelial (CFBE) cells. CFBE cells homozygous for F508del were treated with the combination of elexacaftor/tezacaftor/ivacaftor (ETI) and TGF-ß1 in the presence and absence of nesolicaftor. Nesolicaftor augmented the F508del CFTR response to ETI and reversed TGF-ß1-induced reductions in CFTR conductance by increasing the expression of CFTR mRNA. Nesolicaftor further rescued the reduced ciliary beating and increased expression of the cytokines IL-6 and IL-8 caused by TGF-ß1. Finally, nesolicaftor augmented the F508del CFTR response to ETI in CFBE cells overexpressing miR-145, a negative regulator of CFTR expression. Thus, CFTR amplifiers, but only when used with highly effective modulators, may provide benefit in an inflamed environment.

Persistence of airway inflammation in smokers who switch to electronic cigarettes.

As opposed to smoking cessation with nicotine-replacement therapy and/or varenicline, nicotine-containing e-cigarette use does not improve some airway inflammatory markers. https://bit.ly/3FyqIt9.

Responses of reconstituted human bronchial epithelia from normal and health-compromised donors to non-volatile particulate matter emissions from an aircraft turbofan engine.

Health effects of particulate matter (PM) from aircraft engines have not been adequately studied since controlled laboratory studies reflecting realistic conditions regarding aerosols, target tissue, particle exposure and deposited particle dose are logistically challenging. Due to the important contributions of aircraft engine emissions to air pollution, we employed a unique experimental setup to deposit exhaust particles directly from an aircraft engine onto reconstituted human bronchial epithelia (HBE) at air-liquid interface under conditions similar to in vivo airways to mimic realistic human exposure. The toxicity of non-volatile PM (nvPM) from a CFM56-7B26 aircraft engine was evaluated under realistic engine conditions by sampling and exposing HBE derived from donors of normal and compromised health status to exhaust for 1 h followed by biomarker analysis 24 h post exposure. Particle deposition varied depending on the engine thrust levels with 85% thrust producing the highest nvPM mass and number emissions with estimated surface deposition of 3.17 × 109 particles cm-2 or 337.1 ng cm-2. Transient increase in cytotoxicity was observed after exposure to nvPM in epithelia derived from a normal donor as well as a decrease in the secretion of interleukin 6 and monocyte chemotactic protein 1. Non-replicated multiple exposures of epithelia derived from a normal donor to nvPM primarily led to a pro-inflammatory response, while both cytotoxicity and oxidative stress induction remained unaffected. This raises concerns for the long-term implications of aircraft nvPM for human pulmonary health, especially in occupational settings.

Losartan ameliorates TGF-ß1-induced CFTR dysfunction and improves correction by cystic fibrosis modulator therapies.

Highly effective modulator therapies dramatically improve the prognosis for those with cystic fibrosis (CF). The triple combination of elexacaftor, tezacaftor, and ivacaftor (ETI) benefits many, but not all, of those with the most common F508del mutation in the CF transmembrane conductance regulator (CFTR). Here, we showed that poor sweat chloride concentration responses and lung function improvements upon initiation of ETI were associated with elevated levels of active TGF-ß1 in the upper airway. Furthermore, TGF-ß1 impaired the function of ETI-corrected F508del-CFTR, thereby increasing airway surface liquid (ASL) absorption rates and inducing mucus hyperconcentration in primary CF bronchial epithelial cells in vitro. TGF-ß1 not only decreased CFTR mRNA, but was also associated with increases in the mRNA expression of TNFA and COX2 and TNF-α protein. Losartan improved TGF-ß1-mediated inhibition of ETI-corrected F508del-CFTR function and reduced TNFA and COX2 mRNA and TNF-α protein expression. This likely occurred by improving correction of mutant CFTR rather than increasing its mRNA (without an effect on potentiation), thereby reversing the negative effects of TGF-ß1 and improving ASL hydration in the CF airway epithelium in vitro. Importantly, these effects were independent of type 1 angiotensin II receptor inhibition.

The SARS-CoV-2 Transcriptome and the Dynamics of the S Gene Furin Cleavage Site in Primary Human Airway Epithelia.

The spike (S) polypeptide of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary that contains a furin cleavage site (FCS), 682RRAR↓S686 Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, it acquired deletions surrounding the FCS. We studied the viral transcriptome in Vero cell-derived SARS-CoV-2-infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability of the FCS. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the FCS region: a 12 amino acid deletion (678TNSPRRAR↓SVAS689) that contains the underlined FCS and a 5 amino acid deletion (675QTQTN679) that is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions from 11 infected HAE-ALI cultures of both healthy and lung disease donors revealed that the selective pressure for the FCS maintains the FCS stably in 9 HAE-ALI cultures but with 2 exceptions, in which the FCS deletions are retained at a high rate of >40% after infection of ≥13 days. Our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is likely donor dependent.IMPORTANCE Polarized human airway epithelia at an air-liquid interface (HAE-ALI) are an in vitro model that supports efficient infection of SARS-CoV-2. The spike (S) protein of SARS-CoV-2 contains a furin cleavage site (FCS) at the boundary of the S1 and S2 domains which distinguishes it from SARS-CoV. However, FCS deletion mutants have been identified in patients and in vitro cell cultures, and how the airway epithelial cells maintain the unique FCS remains unknown. We found that HAE-ALI cultures were capable of suppressing two prevalent FCS deletion mutants (Δ678TNSPRRAR↓SVAS689 and Δ675QTQTN679) that were selected during propagation in Vero cells. While such suppression was observed in 9 out of 11 of the tested HAE-ALI cultures derived from independent donors, 2 exceptions that retained a high rate of FCS deletions were also found. Our results present evidence of the donor-dependent properties of human airway epithelia in the evolution of the FCS during infection.

An Open Label Trial to Assess Safety of Losartan for Treating Worsening Respiratory Illness in COVID-19.

Rationale: Coronavirus disease 2019 (COVID-19) can cause disruption of the renin-angiotensin system in the lungs, possibly contributing to pulmonary capillary leakage. Thus, angiotensin receptor blockers (ARBs) may improve respiratory failure. Objective: Assess safety of losartan for use in respiratory failure related to COVID-19 (NCT04335123). Methods: Single arm, open label trial of losartan in those hospitalized with respiratory failure related to COVID-19. Oral losartan (25 mg daily for 3 days, then 50 mg) was administered from enrollment until day 14 or hospital discharge. A post-hoc external control group with patients who met all inclusion criteria was matched 1:1 to the treatment group using propensity scores for comparison. Measures: Primary outcome was cumulative incidence of any adverse events. Secondary, explorative endpoints included measures of respiratory failure, length of stay and vital status. Results: Of the 34 participants enrolled in the trial, 30 completed the study with a mean age SD of 53.8 ± 17.7 years and 17 males (57%). On losartan, 24/30 (80%) experienced an adverse event as opposed to 29/30 (97%) of controls, with a lower average number of adverse events on losartan relative to control (2.2 vs. 3.3). Using Poisson regression and controlling for age, sex, race, date of enrollment, disease severity at enrollment, and history of high-risk comorbidities, the incidence rate ratio of adverse events on losartan relative to control was 0.69 (95% CI: 0.49-0.97) Conclusions: Losartan appeared safe for COVID-19-related acute respiratory compromise. To assess true efficacy, randomized trials are needed.

Losartan reduces cigarette smoke-induced airway inflammation and mucus hypersecretion.

The aim was to determine whether losartan reduces cigarette smoke (CS)-induced airway inflammation and mucus hypersecretion in an in vitro model and a small clinical trial. Primary human bronchial epithelial cells (HBECs) were differentiated at the air-liquid interface (ALI) and exposed to CS. Expression of transforming growth factor (TGF)-ß1 and the mucin MUC5AC, and expression or activity of matrix metalloproteinase (MMP)-9 were measured after CS exposure. Parameters of mucociliary clearance were evaluated by measuring airway surface liquid volumes, mucus concentrations, and conductance of cystic fibrosis transmembrane conductance regulator (CFTR) and large conductance, Ca2+-activated and voltage-dependent potassium (BK) channels. Nasal cells were collected from study participants and expression of MUC5AC, TGF-ß1, and MMP-9 mRNAs was measured before and after losartan treatment. In vitro, CS exposure of HBECs caused a significant increase in mRNA expression of MUC5AC and TGF-ß1 and MMP-9 activity and decreased CFTR and BK channel activities, thereby reducing airway surface liquid volumes and increasing mucus concentrations. Treatment of HBECs with losartan rescued CS-induced CFTR and BK dysfunction and caused a significant decrease in MUC5AC expression and mucus concentrations, partially by inhibiting TGF-ß signalling. In a prospective clinical study, cigarette smokers showed significantly reduced mRNA expression levels of MUC5AC, TGF-ß1, and MMP-9 in the upper airways after 2 months of losartan treatment. Our findings suggest that losartan may be an effective therapy to reduce inflammation and mucus hypersecretion in CS-induced chronic airway diseases.

Hyperglycaemia in cystic fibrosis adversely affects BK channel function critical for mucus clearance.

Large-conductance, Ca2+-activated, voltage-dependent K+ (BK) channel function is critical for adequate airway hydration and mucociliary function. In airway epithelia, BK function is regulated by its γ-subunit, leucine-rich repeat-containing protein 26 (LRRC26). Since patients with cystic fibrosis (CF)-related diabetes mellitus (CFRD) have worse lung function outcomes, this study determined the effects of hyperglycaemia on BK function in CF bronchial epithelial (CFBE) cells in vitro and evaluated the correlation between glycaemic excursions and mRNA expression of LRRC26 in the upper airways of CF and CFRD patients.CFBE cells were redifferentiated at the air-liquid interface (ALI) in media containing either 5.5 mM or 12.5 mM glucose. BK activity was measured in an Ussing chamber. Airway surface liquid (ASL) volume was estimated by meniscus scanning and inflammatory marker expression was measured by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). CF patients were assessed by 7 days of continuous glucose monitoring (CGM). LRRC26 mRNA expression was measured by quantitative real-time PCR from nasal cells obtained at the end of glucose monitoring.BK currents were significantly decreased in CFBE cells cultured under high glucose. These cells revealed significantly lower ASL volumes and increased inflammation, including the receptor for advanced glycation endproducts (RAGE), compared to cells cultured in normal glucose. In vivo, nasal cell expression of LRRC26 mRNA was inversely correlated with hyperglycaemic excursions, consistent with the in vitro results.Our findings demonstrate that hyperglycaemia induces inflammation and impairs BK channel function in CFBE cells in vitro These data suggest that declining lung function in CFRD patients may be related to BK channel dysfunction.

A long noncoding RNA antisense to ICAM-1 is involved in allergic asthma associated hyperreactive response of airway epithelial cells.

Epithelial cells of the conducting airways are a pivotal first line of defense against airborne pathogens and allergens that orchestrate inflammatory responses and mucociliary clearance. Nonetheless, the molecular mechanisms responsible for epithelial hyperreactivity associated with allergic asthma are not completely understood. Transcriptomic analysis of human airway epithelial cells (HAECs), differentiated in-vitro at air-liquid interface (ALI), showed 725 differentially expressed immediate-early transcripts, including putative long noncoding RNAs (lncRNAs). A novel lncRNA on the antisense strand of ICAM-1 or LASI was identified, which was induced in LPS-primed HAECs along with mucin MUC5AC and its transcriptional regulator SPDEF. LPS-primed expression of LASI, MUC5AC, and SPDEF transcripts were higher in ex-vivo cultured asthmatic HAECs that were further augmented by LPS treatment. Airway sections from asthmatics with increased mucus load showed higher LASI expression in MUC5AC+ goblet cells following multi-fluorescent in-situ hybridization and immunostaining. LPS- or IL-13-induced LASI transcripts were mostly enriched in the nuclear/perinuclear region and were associated with increased ICAM-1, IL-6, and CXCL-8 expression. Blocking LASI expression reduced the LPS or IL-13-induced epithelial inflammatory factors and MUC5AC expression, suggesting that the novel lncRNA LASI could play a key role in LPS-primed trained airway epithelial responses that are dysregulated in allergic asthma.

Oxidative stress-induced inflammation in susceptible airways by anthropogenic aerosol.

Ambient air pollution is one of the leading five health risks worldwide. One of the most harmful air pollutants is particulate matter (PM), which has different physical characteristics (particle size and number, surface area and morphology) and a highly complex and variable chemical composition. Our goal was first to comparatively assess the effects of exposure to PM regarding cytotoxicity, release of pro-inflammatory mediators and gene expression in human bronchial epithelia (HBE) reflecting normal and compromised health status. Second, we aimed at evaluating the impact of various PM components from anthropogenic and biogenic sources on the cellular responses. Air-liquid interface (ALI) cultures of fully differentiated HBE derived from normal and cystic fibrosis (CF) donor lungs were exposed at the apical cell surface to water-soluble PM filter extracts for 4 h. The particle dose deposited on cells was 0.9-2.5 and 8.8-25.4 µg per cm2 of cell culture area for low and high PM doses, respectively. Both normal and CF HBE show a clear dose-response relationship with increasing cytotoxicity at higher PM concentrations. The concurrently enhanced release of pro-inflammatory mediators at higher PM exposure levels links cytotoxicity to inflammatory processes. Further, the PM exposure deregulates genes involved in oxidative stress and inflammatory pathways leading to an imbalance of the antioxidant system. Moreover, we identify compromised defense against PM in CF epithelia promoting exacerbation and aggravation of disease. We also demonstrate that the adverse health outcome induced by PM exposure in normal and particularly in susceptible bronchial epithelia is magnified by anthropogenic PM components. Thus, including health-relevant PM components in regulatory guidelines will result in substantial human health benefits and improve protection of the vulnerable population.