David B. Hill

A Periciliary Brush Promotes the Lung Health by Separating the Mucus Layer from Airway Epithelia

Sticky Mucus? Mucus—experienced, for example, in the form of a runny nose or productive cough—is one of the tools the body uses to expel or prevent the uptake of foreign matter. In a number of diseases, a failure of the normal mucus-control system leads to obstructions of the airways and respiratory problems. Button et al. (p. 937; see the Perspective by Dickey) examine the existing gel-on-liquid model, where the mucus is thought to sit on a watery periciliary layer around the beating lung cilia that has been used to explain the flow of mucus. A gel-on-brush model is proposed where the mucus sits on a brushlike periciliary layer. The key elements of this layer are membrane-tethered macromolecules that cause normal flow and clearance of mucus. When dehydrated, the interface is disrupted, preventing normal mucus motion. The lung is protected by a brushlike biopolymer that contributes to mucus flow and can trigger muco-obstructive diseases. Mucus clearance is the primary defense mechanism that protects airways from inhaled infectious and toxic agents. In the current gel-on-liquid mucus clearance model, a mucus gel is propelled on top of a “watery” periciliary layer surrounding the cilia. However, this model fails to explain the formation of a distinct mucus layer in health or why mucus clearance fails in disease. We propose a gel-on-brush model in which the periciliary layer is occupied by membrane-spanning mucins and mucopolysaccharides densely tethered to the airway surface. This brush prevents mucus penetration into the periciliary space and causes mucus to form a distinct layer. The relative osmotic moduli of the mucus and periciliary brush layers explain both the stability of mucus clearance in health and its failure in airway disease.

A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms

A vexing problem in cystic fibrosis (CF) pathogenesis has been to explain the high prevalence of Pseudomonas aeruginosa biofilms in CF airways. We speculated that airway surface liquid (ASL) hyperabsorption generates a concentrated airway mucus that interacts with P. aeruginosa to promote biofilms. To model CF vs. normal airway infections, normal (2.5% solids) and CF-like concentrated (8% solids) mucus were prepared, placed in flat chambers, and infected with an ≈5 × 103 strain PAO1 P. aeruginosa. Although bacteria grew to 1010 cfu/ml in both mucus concentrations, macrocolony formation was detected only in the CF-like (8% solids) mucus. Biophysical and functional measurements revealed that concentrated mucus exhibited properties that restrict bacterial motility and small molecule diffusion, resulting in high local bacterial densities with high autoinducer concentrations. These properties also rendered secondary forms of antimicrobial defense, e.g., lactoferrin, ineffective in preventing biofilm formation in a CF-like mucus environment. These data link airway surface liquid hyperabsorption to the high incidence of P. aeruginosa biofilms in CF via changes in the hydration-dependent physical–chemical properties of mucus and suggest that the thickened mucus gel model will be useful to develop therapies of P. aeruginosa biofilms in CF airways.

A Biophysical Basis for Mucus Solids Concentration as a Candidate Biomarker for Airways Disease

In human airways diseases, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), host defense is compromised and airways inflammation and infection often result. Mucus clearance and trapping of inhaled pathogens constitute key elements of host defense. Clearance rates are governed by mucus viscous and elastic moduli at physiological driving frequencies, whereas transport of trapped pathogens in mucus layers is governed by diffusivity. There is a clear need for simple and effective clinical biomarkers of airways disease that correlate with these properties. We tested the hypothesis that mucus solids concentration, indexed as weight percent solids (wt%), is such a biomarker. Passive microbead rheology was employed to determine both diffusive and viscoelastic properties of mucus harvested from human bronchial epithelial (HBE) cultures. Guided by sputum from healthy (1.5–2.5 wt%) and diseased (COPD, CF; 5 wt%) subjects, mucus samples were generated in vitro to mimic in vivo physiology, including intermediate range wt% to represent disease progression. Analyses of microbead datasets showed mucus diffusive properties and viscoelastic moduli scale robustly with wt%. Importantly, prominent changes in both biophysical properties arose at ∼4 wt%, consistent with a gel transition (from a more viscous-dominated solution to a more elastic-dominated gel). These findings have significant implications for: (1) penetration of cilia into the mucus layer and effectiveness of mucus transport; and (2) diffusion vs. immobilization of micro-scale particles relevant to mucus barrier properties. These data provide compelling evidence for mucus solids concentration as a baseline clinical biomarker of mucus barrier and clearance functions.

Monitoring airway mucus flow and ciliary activity with optical coherence tomography.

Muco-ciliary transport in the human airway is a crucial defense mechanism for removing inhaled pathogens. Optical coherence tomography (OCT) is well-suited to monitor functional dynamics of cilia and mucus on the airway epithelium. Here we demonstrate several OCT-based methods upon an actively transporting in vitro bronchial epithelial model and ex vivo mouse trachea. We show quantitative flow imaging of optically turbid mucus, semi-quantitative analysis of the ciliary beat frequency, and functional imaging of the periciliary layer. These may translate to clinical methods for endoscopic monitoring of muco-ciliary transport in diseases such as cystic fibrosis and chronic obstructive pulmonary disease (COPD).

Effects of guaifenesin, N-acetylcysteine, and ambroxol on MUC5AC and mucociliary transport in primary differentiated human tracheal-bronchial cells

BackgroundTherapeutic intervention in the pathophysiology of airway mucus hypersecretion is clinically important. Several types of drugs are available with different possible modes of action. We examined the effects of guaifenesin (GGE), N-acetylcysteine (NAC) and ambroxol (Amb) on differentiated human airway epithelial cells stimulated with IL-13 to produce additional MUC5AC.MethodsAfter IL-13 pre-treatment (3 days), the cultures were treated with GGE, NAC or Amb (10–300 μM) in the continued presence of IL-13. Cellular and secreted MUC5AC, mucociliary transport rates (MTR), mucus rheology at several time points, and the antioxidant capacity of the drugs were assessed.ResultsIL-13 increased MUC5AC content (~25%) and secretion (~2-fold) and decreased MTR, but only slightly affected the G’ (elastic) or G” (viscous) moduli of the secretions. GGE significantly inhibited MUC5AC secretion and content in the IL-13-treated cells in a concentration-dependent manner (IC50s at 24 hr ~100 and 150 μM, respectively). NAC or Amb were less effective. All drugs increased MTR and decreased G’ and G” relative to IL-13 alone. Cell viability was not affected and only NAC exhibited antioxidant capacity.ConclusionsThus, GGE effectively reduces cellular content and secretion of MUC5AC, increases MTR, and alters mucus rheology, and may therefore be useful in treating airway mucus hypersecretion and mucostasis in airway diseases.

Establishment of Respiratory Air–Liquid Interface Cultures and Their Use in Studying Mucin Production, Secretion, and Function

Primary cultures of human airway bronchial airways represent a valuable tool in understanding the roles of the epithelium, cilia, and the mucus layer in coordinating the clearance of mucus from the airways. The ability to obtain cells from both normal and diseased populations (such as cystic fibrosis and Chronic obstructive pulmonary disease (COPD)) allows researchers to investigate the disease phenotype on these processes. Furthermore, such cultures have provided investigators with a vast source of native airway mucus, devoid of external biological processes that occur in vivo, for biochemical and rheological studies. The primary goal of this chapter is to describe the culturing and use of human airway cultures grown under an in vivo-like air-liquid interface for use in a variety of mucus and mucociliary studies.

Forces Required of Kinesin during Processive Transport through Cytoplasm

The purpose of this paper is to deduce whether the maximum force, steplike movement, and rate of ATP consumption of kinesin, as measured in buffer, are sufficient for the task of fast transport of vesicles in cells. Our results show that moving a 200-nm vesicle in viscoelastic COS7 cytoplasm, with the same steps as observed for kinesin-driven beads in buffer, required a maximum force of 16 pN and work per step of 1 +/- 0.7 ATP, if the drag force was assumed to decrease to zero between steps. In buffer, kinesin can develop a force of 6-7 pN while consuming 1 ATP/step, comparable to the required values. As an alternative to assuming that the force vanishes between steps, the measured COS7 viscoelasticity was extrapolated to zero frequency by a numerical fit. The force required to move the bead then exceeded 75 pN at all times and peaked briefly to 92 pN, well beyond the measured capabilities of a single kinesin in buffer. The work per step increased to 7 +/- 5 ATP, greatly exceeding the energy available to a single motor.

Probing biological nanotopology via diffusion of weakly constrained plasmonic nanorods with optical coherence tomography

Significance Many diseases are characterized by nanostructural changes in connective fibers and soluble proteins, which can indicate or drive disease progression. Noninvasive methods sensitive to nanotopological changes in 3D tissue models can elucidate biophysical changes associated with disease progression. Nanoparticles probe their environment via their diffusion, which is impacted by the size and connectivity of pores into which they freely diffuse. Here, we show that optical coherence tomography provides depth-resolved imaging of gold nanorods (GNRs) to infer local biological nanotopology. We demonstrate the broad potential of this method by sensing changes in diffusion of GNRs in 3D models of mammary ECM and pulmonary mucus. Biological materials exhibit complex nanotopology, i.e., a composite liquid and solid phase structure that is heterogeneous on the nanoscale. The diffusion of nanoparticles in nanotopological environments can elucidate biophysical changes associated with pathogenesis and disease progression. However, there is a lack of methods that characterize nanoprobe diffusion and translate easily to in vivo studies. Here, we demonstrate a method based on optical coherence tomography (OCT) to depth-resolve diffusion of plasmon-resonant gold nanorods (GNRs) that are weakly constrained by the biological tissue. By using GNRs that are on the size scale of the polymeric mesh, their Brownian motion is minimally hindered by intermittent collisions with local macromolecules. OCT depth-resolves the particle-averaged translational diffusion coefficient (DT) of GNRs within each coherence volume, which is separable from the nonequilibrium motile activities of cells based on the unique polarized light-scattering properties of GNRs. We show how this enables minimally invasive imaging and monitoring of nanotopological changes in a variety of biological models, including extracellular matrix (ECM) remodeling as relevant to carcinogenesis, and dehydration of pulmonary mucus as relevant to cystic fibrosis. In 3D ECM models, DT of GNRs decreases with both increasing collagen concentration and cell density. Similarly, DT of GNRs is sensitive to human bronchial-epithelial mucus concentration over a physiologically relevant range. This novel method comprises a broad-based platform for studying heterogeneous nanotopology, as distinct from bulk viscoelasticity, in biological milieu.

Polarization-modulated differential-interference contrast microscopy with a variable retarder.

A liquid-crystal variable retarder inserted into a differential-interference contrast video microscope switches image highlights into shadows and vice versa in alternate frames. Synchronous computation and display of the difference between alternate frames yield a stream of images with doubled contrast and reduced fixed-position noise because of the automatic background subtraction. The measured signal-to-noise ratio (SNR) peaks when the modulation +/- Gamma of the retarder equals the phase shift delta of the sample. A Jones calculus model of the central ray in the polarization-modulated differential-interference contrast microscope yields SNR = (sin Gamma sin delta)/((1 - cos Gamma cos delta)N), where N is the rms time-dependent photon noise. This expression fits the experiments closely for 1.8 degrees < or = Gamma < or = 115 degrees.

Model Comparison and Assessment for Single Particle Tracking in Biological Fluids

ABSTRACT State-of-the-art techniques in passive particle-tracking microscopy provide high-resolution path trajectories of diverse foreign particles in biological fluids. For particles on the order of 1 μm diameter, these paths are generally inconsistent with simple Brownian motion. Yet, despite an abundance of data confirming these findings and their wide-ranging scientific implications, stochastic modeling of the complex particle motion has received comparatively little attention. Even among posited models, there is virtually no literature on likelihood-based inference, model comparisons, and other quantitative assessments. In this article, we develop a rigorous and computationally efficient Bayesian methodology to address this gap. We analyze two of the most prevalent candidate models for 30-sec paths of 1 μm diameter tracer particles in human lung mucus: fractional Brownian motion (fBM) and a Generalized Langevin Equation (GLE) consistent with viscoelastic theory. Our model comparisons distinctly favor GLE over fBM, with the former describing the data remarkably well up to the timescales for which we have reliable information. Supplementary materials for this article are available online.