Mucociliary clearance, characterized by mucus secretion and its conveyance by ciliary action, is a fundamental physiological process that plays an important role in host defense. Although it is known ...that ciliary activity changes with chemical and mechanical stimuli, the autoregulatory mechanisms that govern ciliary activity and mucus transport in response to normal and pathophysiological variations in mucus are not clear. We have developed a high-speed, 1-μm-resolution, cross-sectional imaging modality, termed micro-optical coherence tomography (μOCT), which provides the first integrated view of the functional microanatomy of the epithelial surface. We monitored invasion of the periciliary liquid (PCL) layer by mucus in fully differentiated human bronchial epithelial cultures and full thickness swine trachea using μOCT. We further monitored mucociliary transport (MCT) and intracellular calcium concentration simultaneously during invasion of the PCL layer by mucus using colocalized μOCT and confocal fluorescence microscopy in cell cultures. Ciliary beating and mucus transport are up-regulated via a calcium-dependent pathway when mucus causes a reduction in the PCL layer and cilia height. When the load exceeds a physiological limit of approximately 2 μm, this gravity-independent autoregulatory mechanism can no longer compensate, resulting in diminished ciliary motion and abrogation of stimulated MCT. A fundamental integrated mechanism with specific operating limits governs MCT in the lung and fails when periciliary layer compression and mucus viscosity exceeds normal physiologic limits.
Lipopolysaccharide (LPS) serves as the interface between gram-negative bacteria (GNB) and the innate immune response in respiratory epithelial cells (REC). Herein, we describe a novel biological role ...of LPS that permits GNB to persist in the respiratory tract through inducing CFTR and mucociliary dysfunction. LPS reduced cystic fibrosis transmembrane conductance regulater (CFTR)-mediated short-circuit current in mammalian REC in Ussing chambers and nearly abrogated CFTR single channel activity (defined as forskolin-activated Cl- currents) in patch clamp studies, effects of which were blocked with toll-like receptor (TLR)-4 inhibitor. Unitary conductance and single-channel amplitude of CFTR were unaffected, but open probability and number of active channels were markedly decreased. LPS increased cytoplasmic and mitochondrial reactive oxygen species resulting in CFTR carbonylation. All effects of exposure were eliminated when reduced glutathione was added in the medium along with LPS. Functional microanatomy parameters, including mucociliary transport, in human sinonasal epithelial cells in vitro were also decreased, but restored with co-incubation with glutathione or TLR-4 inhibitor. In vivo measurements, following application of LPS in the nasal cavities showed significant decreases in transepithelial Cl- secretion as measured by nasal potential difference (NPD) – an effect that was nullified with glutathione and TLR-4 inhibitor. These data provide definitive evidence that LPS-generated reactive intermediates downregulate CFTR function in vitro and in vivo which results in cystic fibrosis-type disease. Findings have implications for therapeutic approaches intent on stimulating Cl- secretion and/or reducing oxidative stress to decrease the sequelae of GNB airway colonization and infection.
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•Screening efficient antisense oligonucleotide correcting the CFTR 3849 splicing defect.•The lead antisense promotes generation of normal full-length mature CFTR protein.•The lead antisense restores ...CFTR function to wild type levels.•Efficient antisense free uptake delivery to primary respiratory patient-derived cells.•The 3849 antisense-based splicing modulation is now progressing to drug development.
Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10 kb C-to-T splicing mutation in the CFTR gene.
We have screened, in FRT cells expressing the 3849+10 kb C-to-T splicing mutation, ~30 2′-O-Methyl-modified phosphorothioate ASOs, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The effect of highly potent ASO candidates on the splicing pattern, protein maturation and CFTR function was further analyzed in well differentiated primary human nasal and bronchial epithelial cells, derived from patients carrying at least one 3849+10 kb C-to-T allele.
A highly potent lead ASO, efficiently delivered by free uptake, was able to significantly increase the level of correctly spliced mRNA and completely restore the CFTR function to wild type levels in cells from a homozygote patient. This ASO led to CFTR function with an average of 43% of wild type levels in cells from various heterozygote patients. Optimized efficiency of the lead ASO was further obtained with 2′-Methoxy Ethyl modification (2′MOE).
The highly efficient splicing modulation and functional correction, achieved by free uptake of the selected lead ASO in various patients, demonstrate the ASO therapeutic potential benefit for CF patients carrying splicing mutations and is aimed to serve as the basis for our current clinical development.
Cystic Fibrosis (CF) is caused by mutations to the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. CFTR is composed of two membrane spanning domains, two cytosolic ...nucleotide-binding domains (NBD1 and NBD2) and a largely unstructured R-domain. Multiple CF-causing mutations reside in the NBDs and some are known to compromise the stability of these domains. The ability to predict the effect of mutations on the stability of the cytosolic domains of CFTR and to shed light on the mechanisms by which they exert their effect is therefore important in CF research. With this in mind, we have predicted the effect on domain stability of 59 mutations in NBD1 and NBD2 using 15 different algorithms and evaluated their performances via comparison to experimental data using several metrics including the correct classification rate (CCR), and the squared Pearson correlation (R 2) and Spearman’s correlation (ρ) calculated between the experimental ΔT m values and the computationally predicted ΔΔG values. Overall, the best results were obtained with FoldX and Rosetta. For NBD1 (35 mutations), FoldX provided R 2 and ρ values of 0.64 and −0.71, respectively, with an 86% correct classification rate (CCR). For NBD2 (24 mutations), FoldX R 2, ρ, and CCR were 0.51, −0.73, and 75%, respectively. Application of the Rosetta high-resolution protocol (Rosetta_hrp) to NBD1 yielded R 2, ρ, and CCR of 0.64, −0.75, and 69%, respectively, and for NBD2 yielded R 2, ρ, and CCR of 0.29, −0.27, and 50%, respectively. The corresponding numbers for the Rosetta’s low-resolution protocol (Rosetta_lrp) were R 2 = 0.47, ρ = −0.69, and CCR = 69% for NBD1 and R 2 = 0.27, ρ = −0.24, and CCR = 63% for NBD2. For NBD1, both algorithms suggest that destabilizing mutations suffer from destabilizing vdW clashes, whereas stabilizing mutations benefit from favorable H-bond interactions. Two triple consensus approaches based on FoldX, Rosetta_lpr, and Rosetta_hpr were attempted using either “majority-voting” or “all-voting”. The all-voting consensus outperformed the individual predictors, albeit on a smaller data set. In summary, our results suggest that the effect of mutations on the stability of CFTR’s NBDs could be largely predicted. Since NBDs are common to all ABC transporters, these results may find use in predicting the effect and mechanism of the action of multiple disease-causing mutations in other proteins.
Objectives/Hypothesis:
Sinonasal respiratory epithelial mucociliary clearance is dependent on the transepithelial transport of ions such as Cl−. The objectives of the present study were to ...investigate the role of oxygen restriction in 1) Cl− transport across primary sinonasal epithelial monolayers, 2) expression of the apical Cl− channels cystic fibrosis transmembrane conductance regulator (CFTR) and transmembrane protein 16A (TMEM16A), and 3) the pathogenesis of chronic rhinosinusitis.
Study Design:
In vitro investigation.
Methods:
Murine nasal septal epithelial (MNSE), wild type, and human sinonasal epithelial (HSNE) cultures were incubated under hypoxic conditions (1% O2, 5% CO2). Cultures were mounted in Ussing chambers for ion transport measurements. CFTR and TMEM16A expression were measured using quantitative reverse‐transcription polymerase chain reaction (RT‐PCR).
Results:
The change in short‐circuit current (ΔISC in microamperes per square centimeter) attributable to CFTR (forskolin‐stimulated) was significantly decreased due to a 12‐hour hypoxia exposure in both MNSE (13.55 ± 0.46 vs. 19.23 ± 0.18) and HSNE (19.55 ± 0.56 vs. 25.49 ± 1.48 control; P < .05). TMEM16A (uridine triphosphate–stimulated transport) was inhibited by 48 hours of hypoxic exposure in MNSE (15.92 ± 2.87 vs. 51.44 ± 3.71 control; P < .05) and by 12 hours of hypoxic exposure in HSNE (16.75 ± 0.68 vs. 24.15 ± 1.35 control). Quantitative RT‐PCR (reported as relative mRNA levels ± standard deviation) demonstrated significant reductions in both CFTR and TMEM16A mRNA expression in MNSE and HSNE owing to airway epithelial hypoxia.
Conclusions:
Sinonasal epithelial CFTR and TMEM16A‐mediated Cl− transport and mRNA expression were robustly decreased in an oxygen‐restricted environment. These findings indicate that persistent hypoxia may lead to acquired defects in sinonasal Cl− transport in a fashion likely to confer mucociliary dysfunction in chronic rhinosinusitis.
Recently approved therapies that modulate CFTR function have shown significant clinical benefit, but recent investigations regarding their molecular mechanism when used in combination have not been ...consistent with clinical results. We employed micro-optical coherence tomography as a novel means to assess the mechanism of action of CFTR modulators, focusing on the effects on mucociliary clearance. Primary human airway monolayers from patients with a G551D mutation responded to ivacaftor treatment with increased ion transport, airway surface liquid depth, ciliary beat frequency, and mucociliary transport rate, in addition to decreased effective viscosity of the mucus layer, a unique mechanism established by our findings. These endpoints are consistent with the benefit observed in G551D patients treated with ivacaftor, and identify a novel mechanism involving mucus viscosity. In monolayers derived from F508del patients, the situation is more complicated, compounded by disparate effects on CFTR expression and function. However, by combining ion transport measurements with functional imaging, we establish a crucial link between in vitro data and clinical benefit, a finding not explained by ion transport studies alone. We establish that F508del cells exhibit increased mucociliary transport and decreased mucus effective viscosity, but only when ivacaftor is added to the regimen. We further show that improvement in the functional microanatomy in vitro corresponds with lung function benefit observed in the clinical trials, whereas ion transport in vitro corresponds to changes in sweat chloride. Functional imaging reveals insights into clinical efficacy and CFTR biology that significantly impact our understanding of novel therapies.
•CFTR missense alleles exhibit diverse mechanisms of dysfunction.•Compounds targeting specific classes of CFTR defects have been variably successful.•Methods and models utilized for studying CFTR ...variants are continually improving.•Characterizing each CFTR variant will facilitate approaches to precision medicine.•Genetic tools under development may lead to future treatments for all forms of CF.
With over 1900 variants reported in the cystic fibrosis transmembrane conductance regulator (CFTR), enhanced understanding of cystic fibrosis (CF) genotype–phenotype correlation represents an important and expanding area of research. The potentiator Ivacaftor has proven an effective treatment for a subset of individuals carrying missense variants, particularly those that impact CFTR gating. Therapeutic efforts have recently focused on correcting the basic defect resulting from the common F508del variant, as well as many less frequent missense alleles. Modest enhancement of F508del-CFTR function has been achieved by combining Ivacaftor with Lumacaftor, a compound that aids maturational processing of misfolded CFTR. Continued development of in silico and in vitro models will facilitate CFTR variant characterization and drug testing, thereby elucidating heterogeneity in the molecular pathogenesis, phenotype, and modulator responsiveness of CF.
This report describes treatment of locoregional head and neck squamous cell carcinoma (HNSCC) by an innovative, experimental strategy involving generation of a robust anti‐cancer agent ...(2‐fluoroadenine F‐Ade) following transduction by Escherichia coli purine nucleoside phosphorylase (PNP) in a small number of tumor cells. F‐Ade works by a unique mechanism of action (ablation of RNA and protein synthesis) and confers tumor regressions of otherwise refractory HNSCC in human subjects. Clinical studies have now advanced to a pivotal (registration‐directed) trial involving locoregional HNSCC, with plans to begin subject enrollment late in 2018. The present review is the first to summarize use of PNP in the context of HNSCC, and provides background regarding this emerging anti‐cancer approach.
High-mobility group box 1 (HMGB1) is a potent inflammatory mediator elevated in sepsis and rheumatoid arthritis, although its role in cystic fibrosis (CF) lung disease is unknown.
To determine ...whether HMGB1 contributes to CF lung inflammation, including neutrophil chemotaxis and lung matrix degradation.
We used sputum and serum from subjects with CF and a Scnn1b-transgenic (Scnn1b-Tg) mouse model that overexpresses beta-epithelial Na(+) channel in airways and mimics the CF phenotype, including lung inflammation. Human secretions and murine bronchoalveolar lavage fluid (BALF) was assayed for HMGB1 by Western blot and ELISA. Neutrophil chemotaxis was measured in vitro after incubation with human neutrophils. The collagen fragment proline-glycine-proline (PGP) was measured by tandem mass spectroscopy.
HMGB1 was detected in CF sputum at higher levels than secretions from normal individuals. Scnn1b-Tg mice had elevated levels of HMGB1 by Western blot and ELISA. We demonstrated that dose-dependent chemotaxis of human neutrophils stimulated by purified HMGB1 was partially dependent on CXC chemokine receptors and that this could be duplicated in CF sputum and BALF from Scnn1b-Tg mice. Neutralization by anti-HMGB1 antibody, in both the sputum and BALF-reduced chemotaxis, which suggested that HMGB1 contributed to the chemotactic properties of these samples. Intratracheal administration of purified HMGB1 induced neutrophil influx into the airways of mice and promoted the release of PGP. PGP was also elevated in Scnn1b-Tg mice and CF serum.
HMGB1 expression contributes to pulmonary inflammation and lung matrix degradation in CF airway disease and deserves further investigation as a biomarker and potential therapeutic target.
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A growing number of diseases are linked to the misfolding of integral membrane proteins, and many of these proteins are targeted for ubiquitin-proteasome-dependent degradation. One ...such substrate is a mutant form of the Cystic Fibrosis Transmembrane Conductance Regulator (F508del-CFTR). Protein folding “correctors” that repair the F508del-CFTR folding defect have entered the clinic, but they are unlikely to protect the entire protein from degradation. To increase the pool of F508del-CFTR protein that is available for correction by existing treatments, we determined a structure-activity relationship to improve the efficacy and reduce the toxicity of an inhibitor of the E1 ubiquitin activating enzyme that facilitates F508del-CFTR maturation. A resulting lead compound lacked measurable toxicity and improved the ability of an FDA-approved corrector to augment F508del-CFTR folding, transport the protein to the plasma membrane, and maintain its activity. These data support a proof-of-concept that modest inhibition of substrate ubiquitination improves the activity of small molecule correctors to treat CF and potentially other protein conformational disorders.
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