Seeing cilia: imaging modalities for ciliary motion and clinical connections

Abstract

The respiratory tract is lined with multiciliated epithelial cells that function to move mucus and trapped particles via the mucociliary transport apparatus. Genetic and acquired ciliopathies result in diminished mucociliary clearance, contributing to disease pathogenesis. Recent innovations in imaging technology have advanced our understanding of ciliary motion in health and disease states. Application of imaging modalities including transmission electron microscopy, high-speed video microscopy, and micron-optical coherence tomography could improve diagnostics and be applied for precision medicine. In this review, we provide an overview of ciliary motion, imaging modalities, and ciliopathic diseases of the respiratory system including primary ciliary dyskinesia, cystic fibrosis, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.

INTRODUCTION

The respiratory tract is continuously exposed to pathogens and particulates from inspired air and thus must continually clear debris to maintain a clean environment. Conducting airways are lined with ciliated epithelial cells and mucus-producing goblet cells (38), which together form the mucociliary clearance (MCC) apparatus and function to transport mucus and trapped matter out of the lungs through coordinated beating of motile cilia. An intact mucociliary transport (MCT) apparatus is an essential protective mechanism against a constant inoculation of potentially noxious stimuli. Delayed MCC is an underlying driver of pathogenesis in diseases such as primary ciliary dyskinesia (PCD), cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD) and may prove to be an important disease mechanism in idiopathic pulmonary fibrosis (IPF) (165). Novel imaging techniques can help characterize both innate and acquired defects in ciliary motion. Innovative application of these techniques has significant potential to aid in the diagnosis of disorders of impaired MCC and could inform treatment options in diseases of ciliary motion.

Recent advancements in quantitative imaging technologies have made in situ functional assessment of ciliary motion and MCT possible. The goal for this review is to provide an up-to-date review of 1) the biology of cilia motion, 2) classical and cutting-edge modalities to study cilia motion, and 3) the present efforts for use of cilia motion imaging to improve diagnostic modalities for motile respiratory cilia.

Cilia Structure and Function

Cilia are cellular organelles composed of protrusions of microtubule scaffolding covered by cell membrane (60). The central core structure of cilia is called an axoneme. In motile cilia of the respiratory tract, the axoneme is composed of a ring of nine interconnected microtubule doublets surrounding two microtubule singlets (9 + 2 arrangement) (106). Centrioles, nine triplets of microtubules, anchor the axoneme to the cell and are termed basal bodies when associated with an axoneme (Fig. 1A). The inner singlet microtubules are attached to each other by paired bridges, and radial spoke heads (RSH) provide attachment to the peripheral doublets. Adjacent outer doublets interact with each other via outer dynein arms (ODAs), inner dynein arms (IDAs), and nexin (46). Dynein ATPase motor proteins generate vectoral, propulsive force by causing microtubule doublets to slide against adjacent microtubule doublets, bending the cilium (43, 73, 88, 130, 135, 136, 162, 174) Table 1.

Fig. 1.

A: schematic representation of motile cilium, axoneme, and basal body. The axoneme is the central core of the cilium. In motile cilia, it comprises interconnected 9 + 2 microtubule doublets covered by cell membrane. The basal body, 9 triplets of microtubules, anchors the axoneme to cell (connection denoted by dotted line). ODA, outer dynein arm; IDA, inner dynein arm; RSH, radial head spoke. B: illustration of ciliary stroke cycle. The power stroke propels mucus and trapped particulate forward, and the cilium resets to its starting position during the recovery stroke.

Table 1.

Summary of present imaging modalities for visualizing cilia and ciliary motion

	Electron Microscopy	High-Speed Video Microscopy	µ-Optical Coherence Tomography
Resolution	50 pm	<1 μm	~1 μm
Capable of imaging live samples	No	Yes	Yes
Fixation necessary	Yes	No	No
Capable of in vivo imaging	No	No	Yes
Advantages	• Able to visualize ciliary ultrastructure and characterize subcellular defects	• Automated quantification of CBF possible	• Simultaneous recording of MCT, CBF, and other metrics of ciliary motion possible
	• Highest resolution imaging tool currently available	• Able to visualize individual cilium	• Does not require addition of exogenous dyes, beads, or contrast agents
		• Analysis of CBP and waveforms possible	• Capable of fully resolving and recording cilia motion in real time
	• Not capable of imaging live samples	• Presently limited to highly specialized centers experienced in performing HSVM analysis	• Presently limited to proprietary technology; not widely available
Disadvantages	• Extensive sample preparation necessary	• No standardized protocol established at present	• Presently not capable of three-dimensional real-time imaging of CBP
	• Not all PCD mutations cause defects in ciliary ultrastructure detectable by EM	• High degree of variability and potential for false-positive and false-negative findings	• In vivo capability limited to nasal epithelium
	• Structure-to-function relationship not yet established for most genetic defects	• Manual analysis still required for CBP	• Need for prospective clinical validation
		• Sensitivity to abnormal ciliary motion can be limited; not yet endorsed as diagnostic modality
		• Cannot capture measures of mucus transport or ciliary intercoordination

MCT, mucociliary transport; CBP, cilia beat pattern; HSVM, high-speed video microscopy; PCD, primary ciliary dyskinesia; EM, electron microscopy.

The master regulator of motile ciliogenesis is FoxJ1, a forkhead-domain transcription factor (10, 18, 64, 172). FoxJ1 programs motile cilia generation through its regulation of genes that facilitate the generation, assembly, transport, and docking of the central doublet, IDA, and ODA and control of genes that encode intraflagellar transport (IFT) proteins (18, 30, 62, 105, 159, 185). IFT is essential for the formation, growth, and maintenance of cilia because cilia lack the ability to synthesize and recycle axonemal proteins, but structural remodeling at both poles of the cilia is necessary for normal function (128, 148). Cilia grow via the assembly of new axonemal subunits to the distal “plus end” of the cilia by anterograde kinesin-II motor protein (42, 148). The retrograde motor is driven by cytoplasmic dynein ATPase, which is actually carried as IFT cargo anterograde to the tip of the cilium (128, 148). Dynein carries IFT particles retrograde to the proximal “negative end” of the cilia (128, 148). Cilia and flagella are constantly turning over, and the maintenance of organelle length is dynamically regulated by IFT (2, 97, 148).

In humans, ciliated cells of the respiratory tract may have between 200 and 300 motile cilia (157, 165). MCT relies on synchronous waves of cilia beating to generate directional fluid flow, which allows for transport of the mucus lining the respiratory tract to the oral cavity, where it can either be swallowed or expectorated (12). Directional MCC is possible because of the coordinated alignment of cell polarity across the respiratory tract. This process is regulated by the planar cell polarity (PCP) pathway, which orchestrates centriole position in dividing cells and cilia position in nondividing cells (29). In airway epithelium, PCP signaling depends on FoxJ1-regulated multiciliated cell differentiation (175).

Beyond the global function of motile cilia in mucus propulsion, individual motile cilia may also have sensory functions, similar to primary cilia (7, 108, 142), suggesting an expanded role of motile cilia in health-promoting homeostasis and response to environmental stimuli. In the same manner, some disorders of primary cilia (e.g., Joubert syndrome, Bardet-Biedl syndrome) may also have motile cilia phenotypes, including bronchiectasis and rhinosinusitis, suggesting connections between primary and motile cilia generation and function (63, 143).

Although the topic of primary cilia is beyond the scope of this review, we note that these commonalities may offer an opportunity for translation between studies on primary cilia and motile cilia. In particular, the application of state-of-the-art imaging modalities may be informative in understanding the underlying pathophysiology in disorders of both primary and motile cilia and discerning the shared mechanisms of disease.

Cilia beat frequency (CBF) in healthy people is between 12 and 15 Hz, with associated MCT rates of 4 to 20 mm/min (38). Cilia beating is characterized by power and recovery strokes, analogous to the power and recovery strokes on a rowboat or arm movements of a swimmer doing breaststroke. When the cilium is fully extended, it applies directional force to the mucus gel as a forward, power stroke. During the recovery stroke, the cilium moves back to its starting position by bending backward 90° to decrease resistance (Fig. 1B). CBF is driven in part by autocrine extracellular ATP signaling (13, 15, 35, 79, 131). ATP binds to a membrane receptor, increasing free cytosolic Ca²⁺ concentrations, thus opening. Opening of the calcium-activated K⁺ channels results in membrane depolarization, which drives cilia motion (74, 110, 160, 177).

IMAGING MODALITIES

Advances in imaging modalities have catalyzed our understanding of cilia and ciliary motion. Ciliary movement was first described as “little legs, which moved very nimbly” by Anton Van Leewenhoek in 1675 after he observed a living protozoan, using an early light microscope of his design (134). However, the utility of light microscopy for characterization of cilia has been restricted because the diameter of cilia extends to the limit of resolution of this modality (133). Early transmission electron microscopy (TEM) in the 1950s–1960s brought forth classification of ciliary ultrastructure (39) and provided the first evidence of the sliding microtubule mechanism of the axoneme (135, 136), and a clever fixation study demonstrated the metachronal wave (137). Studies now utilize a combination of imaging approaches such as TEM and wide-field and superresolution immunofluorescence (80). The introduction of high-speed video microscopy (HSVM) in the 1980s bolstered the utility of light microscopy by allowing measurement of CBF (34, 58, 132). Within the last 5 yr, the development of micron-optical coherence tomography (μOCT) has enabled comprehensive study of the intact MCC apparatus through simultaneous and direct measurement of functional microanatomy and ciliary function (90). The following sections will expand on these techniques and their derivatives, highlighting the advantages and disadvantages in the application of ciliary imaging.

Electron Microscopy

Jakus and Hall (65) employed TEM to observe cilia of paramecium in 1946, but it was Fawcett’s 1954 comparative study (39) of ciliated epithelium that initiated EM as a tool to understand organelle motion through analysis of ultrastructure in the mammalian respiratory tract. TEM passes an electron beam through a thin, fixed sample and has been a valuable tool for studying ciliated respiratory epithelium because of its ability to achieve subnanometer resolution (32, 100, 146). The wavelength of an electron beam is 100,000-fold shorter, which explains the superior resolution of TEM (178) and accounts for the superior resolution over light microscopy, which is limited to around 20 nm because of the photon wavelength in visible light spectrum.

A significant limitation of EM is that three-dimensional architecture is represented by two-dimensional micrographs. Electron tomography is a technological advancement derived from traditional TEM in which a series of TEM micrographs obtained from different angles can be reconstructed to create a single three-dimensional model (146, 147). Subtomographic averaging can be employed for imaging of symmetric structures and is valuable because it results in greater resolution (147) although it is not widely used in diagnostics. The 9 + 2 axonemal organization of motile cilia results in ninefold rotational symmetry, so subtomographic averaging can produce higher resolved three-dimensional models compared with TEM alone (146). Burgoyne and colleagues (11) generated the first three-dimensional model of human respiratory cilia via electron tomography, providing novel insight into ciliary ultrastructure.

More recently, Cryo-EM is another method that has been used to determine three-dimensional structures of biological macromolecules (61, 113). Cryo-EM does not require three-dimensional crystals of the structure of interest; rather, samples are cryo-embedded in near native conditions, which allows for direct observation of multiple conformations at practically atomic resolution (102). Knowledge gleaned from structural information has led to better understanding of ciliary motion and resultant motility defects from dynein and spoke mutations (59, 89, 111, 112, 170). Cryo-EM subtomograms has allowed for the visualization of structural polymorphisms of dynein molecules in cilia (89, 147). Integrated SNAP-tag technology and cryo-electron tomography have been applied to localize subunits of the nexin-dynein regulatory complex, which has provided insight into these regulators of axonemal dynein, which are necessary for ciliary and flagellar movement (156).

The application of EM as a diagnostic tool has historically been the gold standard for PCD diagnosis; however, the advent of other advanced imaging tools had reduced the reliance on EM to confirm a diagnosis of PCD (72). Although EM is useful for identifying defects in the ciliary ultrastructure, which is a common hallmark of PCD diagnosis, a substantial portion (>30%) of patients with PCD have normal ultrastructure (72). Thus EM may be of greater utility as a tool in a battery of diagnostic tests, including genetic testing, clinical measures such as nasal nitric oxide (nNO), and other imaging modalities such as HSVM. However, the use of EM to understand the link between observed disease phenotypes and ciliary ultrastructure remains useful and may be particularly useful for designing treatments based on known ciliary ultrastructure defects. Structure-based drug design is now incorporating cryo-EM, which will expand the range of drug targets (8) and may lead to therapies for diseases of primary cilia dysfunction, as well as secondary MCC problems with precise structural targets. This approach may enable the development of therapeutics that correct basic cellular defects, thus aiding rational drug design based on structure-function relationships in motile cilia.

High-Speed Video Microscopy

HSVM is a technique for visualizing ciliary motion in nasal epithelial samples obtained by brushing of the inferior nasal turbinate (17). Samples can be viewed immediately under a microscope with an integrated camera capable of recording video at high speeds (120–500 frames/s), which can later be reviewed in slow motion for three-dimensional analysis of ciliary function, including both beat frequency and pattern (44, 68, 77, 93). Both CBF and cilia beat pattern (CBP) are useful in the diagnosis of abnormal ciliary motion although the latter is more sensitive and specific for PCD (158). Although this technique is useful when used as part of a panel of diagnostic tests for PCD (82), specialized training and experience with the analysis of videos is required to correctly perform functional ciliary analysis, and there is substantial risk for both false-positive and false-negative results (82). Therefore, the North American Research Consortium (Genetic Disorders of Mucociliary Clearance) and the PCD Foundation recommend HSVM be performed only by select centers that are highly experienced with this diagnostic technique (82).

An advantage of HSVM is that it enables visualization of CBP, which allows for characterization of different types of aberrant ciliary motion beyond simple changes in CBF alone. This is especially important considering that there is overlap of CBF between PCD and normal subjects (72, 114, 164), therefore limiting the diagnostic utility of CBF for PCD alone. CBP analysis aims to assess metrics such as direction, coordination, and stiffness of cilia, which can each impact ciliary efficiency. For example, recognizable regular forward and recovery strokes are typically categorized as normal, whereas aberrant CBPs can be described as static cilia, almost static cilia with minimal movements, uncoordinated beating, stiff beating, or abnormal circular beating (123). These descriptions of ciliary motion are more informative than CBF alone because some abnormal patterns, such as abnormal circular beating or stiff beating, may produce defective movement of mucus without an abnormally low CBF. At present, however, the relationship between various aberrant CBPs and mucus transport is not well described. Given that abnormal ciliary motion may have varying effects on MCC rate and CBF, analysis of CBP may inform the nature of the defect. Furthermore, imaging that allows abnormal CBPs to be linked with quantitative metrics may allow prediction of clinical severity by defining the degree to which ciliary motion abnormalities confer defective MCT and ultimately how treatments affect these parameters. Presently, CBF analysis alone is insufficient to adequately distinguish PCD from non-PCD ciliopathies (93). When combined with CBP analysis, however, the sensitivity and specificity for detecting PCD are enhanced (158).

Recent efforts have been aimed at developing an automated method for analysis of CBF (31, 96, 119, 123, 149, 150). Despite some promising results with automated CBF calculation, significant issues remain with automated analysis of other important metrics, such as CBP (68). Automation of CBF calculation has been largely successful (119); however, manual analysis is still required for a valid measure of CBP (68, 123). Advancements in manual analysis have recently been made in a study on healthy subjects (68), with prioritization of carefully selecting ciliary edges to evaluate CBP. However, even within this healthy sample, only a small number of subjects (2 of 13) had normal CBP in the three profiles that were evaluated (side view, above view, and toward view) (68). Furthermore, regions were identified that possessed asynchronous CBPs, or even immotile cilia, suggesting that, even in carefully selected samples from healthy subjects, there is a lack of uniformity that limits the diagnostic utility of CBP analysis by HSVM. Importantly, the challenges observed with this manual analysis have implications for automated analysis attempts, given that automated analyses typically rely on limited regions of interest that may not be representative of each sample as a whole. Furthermore, HSVM protocols are not standardized, and variations in sampling techniques, equipment, sample processing, temperature during analysis, and evaluation criteria present a limitation that must be addressed in order for HSVM to become more useful as a diagnostic tool.

In light of present needs, future studies should focus on refining a standardized protocol for CBP analysis by HSVM. In particular, a clear set of evaluation criteria to describe potential CBP defects would be helpful in categorizing aberrant CBPs. It is possible that repeat biopsies could be necessary to enhance the value of HSVM analysis (83) although presently neither the timing of repeat studies nor the number of measurements are defined. Once established and validated in healthy control subjects, a standardized HSVM protocol can be applied to patients with a confirmed PCD diagnosis to test specificity and sensitivity of this analysis for PCD diagnosis and compare interobserver variation. A systematic approach to develop such standardized protocols is warranted before wide implementation of HSVM as a diagnostic tool for PCD and other disorders of ciliary motion (40).

Once established, a standardized protocol for HSVM, and in particular the evaluation of aberrant CBP, would allow for specific phenotypic categorization of abnormal ciliary motion based on the type of aberrant CBP. Thus therapeutic development may be aimed at correcting specific types of aberrant CBPs, much like some CF therapeutics have effectively corrected known defects of individual mutation classes, representing unique mechanisms of channel dysfunction. HSVM will be an extremely useful tool in monitoring treatment outcomes, particularly given that this technique is relatively noninvasive, and the use of a standard protocol would enable more widespread use outside of highly specialized centers and/or enable high-throughput screening algorithms for rational drug design.

Optical Coherence Tomography

OCT is an interferometry-based, noncontact technique used to image the cross-sectional structures beneath an opaque surface (41, 54). It utilizes broadband laser light, which is reflected by the components of a structure of interest. Interferometry is used to collect reflected light from subsurface structures while disregarding photons that are diffusely scattered, thus reducing the background signal while collecting reflection from the surface(s) of interest. An extension of the technology is Doppler OCT, in which ciliary movement during the stroke cycle causes a phase shift in the interferometric OCT signal allowing for visualization of CBP and quantification of CBF without the need to resolve individual cilia (66, 86). Although conventional OCT systems offer axial and transverse resolutions of 10–15 μm and 30–40 μm, respectively, an ultra-high-resolution next-generation system called μOCT has been developed, which is capable of resolving individual cells at a resolution of 2 μm × 2 μm × 1 μm (x, y, z; transverse, axial, and sagittal planes, respectively) (91). μOCT has been successfully applied in evaluation of the functional airway microanatomy, including individual cilia beating and CBP (6, 25, 90, 92).

The application of μOCT for imaging the airway epithelium presents several potential advantages over other imaging modalities. First, the airway surface liquid (ASL) and periciliary liquid (PCL) layer depths can be visualized noninvasively from the cross-sectional view generated by μOCT without disturbing the native airway surface. Second, because the natural reflectance of light back-scattered from the sample determines the contrast, the cell layers and subcellular structures can be visualized without the use of exogenous dyes or contrast agents. Finally, although most OCT systems lack sufficient resolution to fully resolve the cell layers in airway epithelia, the 1-μ resolution of the μOCT system allows for direct and fully resolved measurements of ASL, PCL, CBF, MCT, and ciliary stroke pattern. The use of μOCT to quantitatively and accurately interrogate the airway microanatomy has been validated in comparison to gold standard techniques in tissue culture (90), and this technique has since been applied to primary cell cultures grown on air-liquid interfaces (5, 6, 166), intact ex vivo human and animal respiratory tissues (6, 25, 126, 151), and in vivo porcine respiratory tract (20).

Recently, quantitative metrics of ciliary motion measured by μOCT provided differential classification of ciliary motion defects in mutations known to cause PCD (151). These metrics included motile cilia area, the effective stroke rate, an index of total ciliary workload, as well as stroke distance and the angular range of ciliary stroke, and degree of metachrony (168). These parameters are capable of differentiating distinct functional abnormalities in ciliary motion, which were linked to defective MCT in three unique mutations known to cause PCD. Presently, development of a miniaturized μOCT system capable of imaging the respiratory tract in vivo is underway (20, 25) with preliminary results denoting that a flexible bronchial probe exhibits performance comparable to that of a benchtop μOCT system (25). Future development in μOCT technology will focus on in vivo imaging capability throughout the entirety of the respiratory tract as well as three-dimensional imaging of individual cilia.

Although μOCT has several distinct advantages, there are some noted limitations given its early development stage. At present, there is no means to measure the entire structure of cilia across a three-dimensional plane. Three-dimensional imaging at the resolution provided by μOCT would significantly enhance the ability to characterize abnormalities in CBP. Presently, the application of μOCT for in vivo imaging of human patients is limited to the nasal epithelium. Even then, motion artifact is a significant concern, particularly for the analysis of MCT (87). Future efforts should aim to enhance the stability of in vivo μOCT probes, which should also aid the ability to perform μOCT during bronchoscopy and help generalize the technique beyond individual research centers.

Presently, in vivo μOCT is a proprietary technology that is still in development as both a research and clinical tool. Advancements in three-dimensional imaging capabilities and image stabilization are necessary to expand the utility of in vivo μOCT and drive its commercialization and adoption beyond its current uses. These advancements should make in vivo assessment of ciliary function possible in both clinical and research settings, greatly advancing the capability to monitor treatment outcomes, restoration of cilia beating, and coordination of cilia beating, ASL hydration, and mucus properties in the target tissues. These outcome measures will likely be highly informative for evaluating the efficacy of new drugs and the success of other treatment strategies for disorders of ciliary motion. Therefore, more widespread adoption of in vivo μOCT imaging to assess ciliary function should greatly advance treatment development and monitoring.

GENETIC AND ACQUIRED DISORDERS OF CILIA

The study of ciliopathic diseases has been greatly aided by the imaging tools described above. Presently, it is known that several different diseases can result from genetic or acquired ciliopathies. Particularly emblematic are PCD, in which the pathology is a direct result of abnormal ciliary motion, and other diseases in which ciliary motion is defective secondary to other pathologies, such as airway dehydration, aberrant mucus expression, or environmental insults. Advances in imaging capabilities to assess cilia structure, function, and motion have enhanced our understanding of the underlying pathophysiology of both inherited and acquired ciliopathies. In particular, primary disorders of ciliary motion, such as PCD, have benefitted from advanced imaging modalities, which are useful tools to either confirm or exclude a PCD diagnosis when used as part of a comprehensive diagnostic evaluation.

Primary Ciliary Dyskinesia

PCD is a genetic disorder characterized by abnormal ciliary structure or function leading to aberrant MCC (99, 145). One classic form of PCD known as Kartagener syndrome was initially described in 1933 as a triad of chronic sinusitis, bronchiectasis, and situs inversus and was linked to defects in ciliary motion and structure in 1976 by Afzelius (1, 67, 70). Mutations in a number of underlying genes have been linked to PCD, including the ODA, IDA, RSH, and central pairs (3). This broad base of potential underlying mutations leads to the heterogeneity in the clinical presentation, ciliary ultrastructure, and ciliary motility among patients (26, 70). Because of this heterogeneity, prevalence is difficult to determine with estimates ranging from 1:2,200 to 1:40,000 (76, 94, 99, 109).

Most patients (70–80%) present with neonatal respiratory distress that may be attributed incorrectly to other respiratory conditions of newborns (26, 84). Lower respiratory symptoms persist with chronic cough, usually productive, seen in 84–100% of patients (84, 107). Common findings in adults with PCD include alveolar consolidation and bronchiectasis (27). The upper airways are frequently involved as well, with persistent nasal congestion and obstruction seen at all ages. Situs inversus totalis is present in ~50% of adult patients with PCD and can be attributed to the lack of functional nodal cilia in the embryonic period causing random orientation (37). Recurrent otitis media affects nearly all children with PCD because of decreased ciliary function in the Eustachian tube and middle ear cleft although symptoms usually improve during teenage years (3, 99, 118). Infertility can affect men and women with PCD because of impaired spermatozoal motility in men and decreased ciliary function in the fallopian tubes in women (70).

PCD is typically inherited in an autosomal-recessive pattern, but in rare instances autosomal-dominant and X-linked forms have been reported (53, 101, 104). Advances in functional candidate gene testing, homozygosity mapping, and comparative computational analysis have led to the identification of at least 35 genes in which mutations cause PCD that previously could not be identified through conventional family-based genome linkage studies (52, 53, 85, 99). Most genes associated with PCD have been linked with ODA. Mutations in DNAI1 and DNAH5 account for over 30% of cases (3). Despite these advances in PCD genetics, known mutations presently only account for ~60% of documented cases (52, 53). Although many of these genetic defects have been correlated with abnormalities in TEM, HSVM, and immunofluorescence (IF), only preliminary data have correlated genotype with phenotype (27, 71, 99, 115). For example, Davis and colleagues (27) found that patients with IDA/central apparatus/microtubular disorganization ultrastructural defects, most with biallelic mutations in CCDC39 or CCDC40, had more lobes with bronchiectasis and consolidation and lower mean forced expiratory volume in 1 s (FEV₁) compared with patients with ODA or ODA + IDA ultrastructural defects.

The variability in both genetics and clinical presentation can make diagnosing PCD particularly difficult. Onset of symptoms typically occurs during the newborn period, but the median age of diagnosis is at 5.3 yr in Europe (99). Diagnosis requires a combination of studies, including nNO screening and confirmatory testing, such as HSVM analysis and TEM (32). Sensitivity and specificity for nNO measurements range between 90 and 99% and 86–96%, respectively, and it is the first-line screening test because the vast majority of patients with PCD have low or absent nNO (9, 144, 180). CBF may be measured using HSVM analysis, which can detect specific variants or groups of variants based on CBP (123). Once considered the gold standard for diagnosing PCD, TEM is limited to 83% sensitivity, detecting only defects that cause ultrastructural changes (75, 116). For example, TEM in a patient with mutations in the RSH gene RSPH9 may reveal only the normal 9 + 2 arrangement because only 38–54% of cross sections have abnormal 9 + 0 or 8 + 1 arrangements (188). Although 30% of cases have yet to be linked with identified genetic mutations, genetic testing can be considered in cases where nNO and HSVM results are normal but clinical suspicion remains high. Therefore, given that cilia are often randomly affected, advances in imaging that provide a more global assessment of ciliary function will aid in the diagnosis and characterization of PCD phenotypes. Application of three-dimensional HSVM analysis, which remains in development, should allow for global assessment of samples along the epithelial plane. Further discoveries in PCD genetics will likely improve the timing and accuracy of diagnosis. As HSVM evolves, or as new techniques such as μOCT show promise, quantifying ciliary activity and MCT may improve the identification of the underlying cause of the ciliary abnormality (151). In particular, new techniques such as μOCT, which allow for in vivo imaging of a multitude of regions of interest spanning a broad segment of the respiratory tract (i.e., nasal epithelium, trachea, and airway generations accessible by bronchoscopy), should provide the capability for a more global assessment of ciliary function. Three-dimensional applications of μOCT may also enable planar investigation of ciliary motion.

Cystic Fibrosis

CF is a rare autosomal-recessive genetic disorder caused by absent or defective CF transmembrane conductance regulator (CFTR) protein (129). CFTR is an apical anion channel (4), and diminished activity results in depleted ASL secondary to failed chloride secretion and resultant increase in sodium resorption through the epithelial sodium channel activity (47, 161). Thick, viscous mucus fails to detach (50) and accumulates on the airway surface, which results in osmotically driven dehydration of the PCL (14, 48, 92). ASL dehydration and PCL collapse negatively impact the MCT apparatus, which contributes to mucus stasis, biofilm formation and infection, inflammatory damage, and functional decline of patients with CF (14, 36, 48, 98, 161, 181).

Application of imaging techniques has elucidated cilia-dependent pathophysiological methods in CF. Schmid and colleagues (138) discovered that CFTR-dependent apical bicarbonate exchange regulates CBF, which has implications for aberrant function of the mucociliary escalator during CF exacerbations, a time when airway luminal bicarbonate significantly increases. μOCT validated the relationship between PCL and MCT, identified functional anatomic defects in CFTR(−/−) piglet trachea and primary CF human bronchial epithelial (HBE) cultures, and demonstrated that airway epithelium dynamically responds to mucus load until mucus viscosity exceeds normal physiological limits (6, 92).

Although ciliary imaging is not a primary diagnostic modality for CF, in situ visualization of ciliary motion has been applied to assess MCC in the context of pharmacological intervention (5, 22, 169). HSVM has been a key preclinical parameter for characterizing pharmacological activation of MCC via CBF in HBE and human sinonasal epithelial cultures following treatment with CFTR modulators, CFTR activators, phosphodiesterase inhibitors, and other drugs (22, 169, 171, 186, 187). Birket and colleagues (5) combined traditional ion transport techniques with μOCT functional imaging to elucidate the mechanism of combination therapy. Insight from such assessment of functional microanatomy of cilia will continue to contribute to drug development and may lead to precision medicine in which treatment decisions are influenced by drug efficacy in human cells evaluated from individual patients (103).

Chronic Obstructive Pulmonary Disease

COPD is the third leading cause of death worldwide, with increased incidence among active and former smokers (152, 163, 176). Persistent respiratory symptoms and airflow limitation are attributable to acquired airway and alveolar abnormalities. The MCT apparatus, a component of the innate immune system, which defends against inhaled toxins, can be compromised in several ways in COPD, one of which includes increased epithelial mucin stores in habitual smokers that can contribute to airflow obstruction (57, 69, 176). Moreover, recent efforts have been made to describe subphenotypes in COPD, including those with defects in MCC and shortened cilia (182, 183). Histological analysis of lung tissue in patients with COPD has also shown a strong correlation between COPD stage and small airway inflammatory mucus accumulation (51).

Separately, cigarette smoke and other environmental exposures contribute to acquired CFTR dysfunction in the respiratory tract and beyond (33, 120, 124, 125, 127), which leads to the development of a clinical phenotype similar to that of mild CF, despite the absence of disease-causing mutations in the CFTR gene. Importantly, cigarette smoke-induced CFTR dysfunction has been shown to partially but incompletely recover following the cessation of smoking (24) and appears to respond to CFTR potentiator treatment in vitro (126, 152, 154, 155, 171). MCC defects in COPD and chronic bronchitis are a potential therapeutic target that could benefit from tracking mucociliary function to measure treatment response.

Although factors such as acquired CFTR dysfunction, increased airway inflammation, and mucus accumulation contribute to MCC defects in COPD, alterations in cilia structure or function have also been implicated in disease-causing pathogenesis. Because patients with COPD have been shown to have similar characteristics between nasal and lower airway epithelium, the majority of present studies utilize the less invasive nasal epithelium to observe ciliary motion (55). Moderate to severe COPD shows depressed CBF in HSVM, with increased presence of nonciliated epithelial cells (183). Although β-agonist therapy can attenuate the decreased CBF found in COPD (117), other nontreatable factors, such as reduced cilia length, contribute to the decreased clearance in smokers with COPD (49). The multifactorial basis for decreased MCC in smokers with COPD contributes to the complexity of translating effective therapies to the clinic.

The mechanisms leading to decreased cilia length and airway inflammation in obstructive lung disease have been the subject of recent studies. Chen and colleagues (16, 21) showed increased autophagocytic markers in emphysema models, and autophagy-impaired mice have shown resistance to cigarette smoke-induced cilia shortening. Subsequent studies have suggested that DUOX1-dependent superoxide production may be regulated by autophagy proteins in airway epithelium (139). Histone deacetylase 6-mediated autophagy (78, 81) and IFT gene expression (49) have also been linked to abnormal cilia structure. Advances in cilia and MCT imaging may allow for both staging and the development of new therapies targeting inflammatory mucus accumulation and small airway obstruction.

Idiopathic Pulmonary Fibrosis

IPF is a chronic, progressive disorder characterized by fibrosing interstitial pneumonia of unknown etiology (121). The disease occurs primarily in older adults, is limited to the lungs, and is associated with either histological or radiographic pattern of usual interstitial pneumonia (UIP) (121, 173). Clinically, patients present most often in the sixth or seventh decade of life with chronic exertional dyspnea, nonproductive cough, bibasilar crackles, and finger clubbing (45, 121). Studies investigating the incidence of IPF have resulted in a wide range of estimates, ranging from 4.6:100,000 to 16.3:100,000 (23, 45, 122).

Although not classically considered a disorder of MCC, recent investigation has established that the single greatest risk factor for developing IPF is a common gain-of-function promotor variant for MUC5B, a mucin protein (141). Analysis of honeycomb cysts in IPF/UIP demonstrated expression of MUC5B by distal airway cells with pseudostratified ciliated epithelium more closely resembling proximal airway epithelium (95, 140, 167). Pulmonary expression of the ciliary proteins DNAH6, DNAH7, DNAI1, and RPGRIP1L is increased in patients with IPF (19, 179, 184), further suggesting MCC dysregulation as a propagator of disease. Finally, recent work has linked overexpression of MUC5B in the airways of murine overexpression models to reduced MCT using μOCT (153).

Combination therapy of the mucolytic N-acetylcysteine in addition to azathioprine or prednisone was shown to slow deterioration in pulmonary function in patients with IPF, which could indicate the importance of the MCC system in the progression of IPF, although the effect of these agents is protean (28). These results should be interpreted cautiously, given that combination therapy studied in the PANTHER-IPF trial (prednisone, azathioprine, and acetylcysteine or acetylcysteine alone) was inconclusive (28, 56). However, nebulized mucolytic therapy may be more effective at addressing the mucus pathway. Future research should assess ciliary functional microanatomy in the context of lung fibrosis to help elucidate the role of mucociliary dysregulation for IPF pathogenesis.

CONCLUSIONS

Imaging techniques to explore cilia structure and function and their relation to airway mucus transport have significantly advanced in recent years. Application of these technologies could allow considerations of cilia motion to be implemented into improved diagnostics of both genetic and acquired disorders of the MCT apparatus. If successfully applied, these tools could help usher in an era of new therapies, directed to improving ciliary function.

GRANTS

This work was supported by Cystic Fibrosis Foundation Grant CLANCY09YO (to G. M. Solomon) and National Institutes of Health Grants 1-KL2-TR-001419-01 (to G. M. Solomon), R35-HL-135816 and DK-072482 (to S. M. Rowe), 5-T32-HL-105346 08: PI Victor J. Thannickal (to R.-J. Shei), and 1-T32-HL-134640-01: PI Chad Steele (to J. E. Peabody).

DISCLOSURES

S. Rowe has an unlicensed patent use of micron-Optical Coherence Tomography as a diagnostic device.

AUTHOR CONTRIBUTIONS

J.E.P., R.-J.S., S.M.R., and G.M.S. conceived and designed research; J.E.P. and R.-J.S. prepared figures; J.E.P., R.-J.S., and B.M.B. drafted manuscript; J.E.P., R.-J.S., B.M.B., S.E.P., B.T., S.M.R., and G.M.S. edited and revised manuscript; J.E.P., R.-J.S., B.M.B., S.E.P., B.T., S.M.R., and G.M.S. approved final version of manuscript.