Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical Coherence Tomography

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified

A Pharmacologic Approach to Acquired Cystic Fibrosis Transmembrane Conductance Regulator Dysfunction in Smoking Related Lung Disease

<div><h3>Background</h3><p>Mucus stasis in chronic obstructive pulmonary disease (COPD) is a significant contributor to morbidity and mortality. Potentiators of cystic fibrosis transmembrane conductance regulator (CFTR) activity pharmacologically enhance CFTR function; ivacaftor is one such agent approved to treat CF patients with the G551D-CFTR gating mutation. CFTR potentiators may also be useful for other diseases of mucus stasis, including COPD.</p> <h3>Methods and Findings</h3><p>In primary human bronchial epithelial cells, exposure to cigarette smoke extract diminished CFTR-mediated anion transport (65.8±0.2% of control, P<0.005) and mucociliary transport (0.17±0.05 µm/sec vs. 2.4±0.47 µm/sec control, P<0.05) by reducing airway surface liquid depth (7.3±0.6 µm vs. 13.0±0.6 µm control, P<0.005) and augmenting mucus expression (by 64%, P<0.05) without altering transepithelial resistance. Smokers with or without COPD had reduced CFTR activity measured by nasal potential difference compared to age-matched non-smokers (−6.3±1.4 and −8.0±2.0 mV, respectively vs. −15.2±2.7 mV control, each P<0.005, n = 12–14/group); this CFTR decrement was associated with symptoms of chronic bronchitis as measured by the Breathlessness Cough and Sputum Score (r = 0.30, P<0.05) despite controlling for smoking (r = 0.31, P<0.05). Ivacaftor activated CFTR-dependent chloride transport in non-CF epithelia and ameliorated the functional CFTR defect induced by smoke to 185±36% of non-CF control (P<0.05), thereby increasing airway surface liquid (from 7.3±0.6 µm to 10.1±0.4 µm, P<0.005) and mucociliary transport (from 0.27±0.11 µm/s to 2.7±0.28 µm/s, P<0.005).</p> <h3>Conclusions</h3><p>Cigarette smoking reduces CFTR activity and is causally related to reduced mucus transport in smokers due to inhibition of CFTR dependent fluid transport. These effects are reversible by the CFTR potentiator ivacaftor, representing a potential therapeutic strategy to augment mucociliary clearance in patients with smoking related lung disease.</p> </div

Mucociliary transport is inhibited by CSE.

<p>(<b>A</b>) Representative photomicrograph of fully differentiated HBE cells exposed to CSE (2%, apical) or vehicle control for 24 h then fixed, sectioned, and stained with PAS. PAS staining of intracellular and extracellular mucus shows as dark red. Scale bar depicts 25 µm. (<b>B</b>) Summary data of proportion of PAS positive cells from slides shown in (A). *P = 0.05, <i>n = </i>7–10/condition. (<b>C</b>) HBE cells were exposed to CSE (2%) or vehicle control, and transport rates of 1 µm fluorescent particles monitored. Baseline measures were made 24 h preceding time 0 h, followed by application of CSE or vehicle, then monitored at the times indicated. All results are normalized to transport rate at time 0 h for each individual well. *P<0.05, **P<0.001, <i>n = </i>10 particles/well.</p

Demographics of Study Subjects.

<p>Demographics of Study Subjects.</p

μOCT measured parameters from swine trachea <i>ex vivo</i>.

<p>Number of samples <i>n</i> refers to separate measurements from a single tissue sample.</p

Cigarette smoke reduces CFTR-dependent activity and expression.

<p>(<b>A</b>) Representative tracings from primary human bronchial epithelial cells were grown at air-liquid interface until fully differentiated, followed by exposure to cigarette smoke extract (CSE 2%) on the apical surfaces for 24 h. Cells were then mounted in modified Ussing chambers and sequentially exposed to forskolin (20 µM) and CFTR_Inh-172 (10 µM) in the setting of amiloride (100 µM) and Cl^–free gluconate on the apical compartment. (<b>B–C</b>) Summary data of experiments shown outlined in (A). Change in short-circuit current (Isc) following addition of forskolin is shown as a percentage of wild-type non-CF current and absolute Isc (B). Change following 2% CSE is also shown for CFTR_Inh-172 (10 µM, C). *P<0.05, **P<0.005, <i>n = </i>4 per concentration. (<b>D</b>) Western blot of cell lysates of primary HBE cells shown in (A) harvested immediately after treatment in the Ussing chamber. CFTR bands B and C are shown by the black and white arrows, respectively. All lanes were normalized for protein concentration and no difference was observed in protein levels at baseline: vehicle control treated wells (1.79±0.37 mg/ml) vs. CSE treated wells (1.92±0.40 mg/ml; P = 0.81); α-tubulin is also shown as a loading control. This blot is representative of 3 similar experiments. (<b>E</b>) Densitometry of CFTR band C shown in (D). **P<0.01. (<b>F</b>) Surface CFTR expression was quantified by a cell surface biotinylation assay following CSE (2%) or vehicle control treatment for 24 hrs. Blot is representative of 3 similar experiments. (<b>G</b>) Densitometry of surface CFTR band shown in (F). *P<0.05. (<b>H</b>) Primary HBE cells were exposed to CSE (2%) 24 h, then RNA isolated, and quantitative RT-PCR performed in comparison to 18S RNA. *P<0.05, <i>n = </i>9/condition. (<b>I</b>) cAMP concentration in HBE cells exposed to CSE (2%) or vehicle control for 24 h prior to assay. Equivalent forskolin concentration was calculated based on a standard curve in the same cells (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039809#pone.0039809.s002" target="_blank">Fig. S2</a>). *P<0.05.</p

Comparison of μOCT and gold standard measurements in HBE cells.

<p>All error bars represent SEM. A. ASL depth measured with μOCT (7.40±1.82 µm, n = 5) and confocal microscopy (7.76±0.87 µm, n = 6). B. CBF measured with μOCT (9.32±0.27 Hz, n = 4) and Hoffman contrast microscopy (10.17±0.56 Hz, n = 4). C. MCT velocity measured with μOCT (24.22±14.88 µm/sec, n = 6) and particle-tracking fluorescence microscopy (1.91±0.62 µm/sec, n = 11). Number of measurements n refers to separate wells analyzed.</p

μOCT instrumentation schematic and axial resolution.

<p>A. System diagram. RM: reference mirror. OL. objective lens. EC: environmental chamber. AO: analog output board. G: grating. IMAQ: image acquisition board. L: camera lens. LSC: line scan camera. SMF: single mode fiber. PC: personal computer. RAID: redundant array of independent disks. CL: Camera Link cable. B. Depth profile of mirror surface, indicating axial full-width half maximum of 1.3 µm.</p

Functional anatomy of excised swine trachea.

<p>A. μOCT image. Yellow bar indicates airway surface liquid (ASL) depth and Red bar indicates PCL depth. Epithelium (ep) and lamina propria (lp) are also visible. B. H&E stained histology image illustrating cilia (c), epithelium (ep) and lamina propria (lp). Scale bar of both images: 10 µm.</p