A study led by researchers from the Chinese Academy of Sciences-Beijing that also involved other research hospitals and universities in China has alarmingly found that the SARS-CoV-2 ORF10 proteins are able to cause the degradation of ciliary proteins and cause the loss of motile cilia on the host epithelial cells.
The study findings were published in the peer reviewed Journal of Cell Biology.
Cilia are microtubule-based cellular organelles that support a wide variety of essential functions in diverse cellular processes (Reiter and Leroux, 2017; Spassky and Meunier, 2017). Cilia dysfunction is associated with human diseases including anosmia, hearing loss, bronchiectasis, and asthma, among others (Reiter and Leroux, 2017).
Cilium shrinking has been detected in SARS-CoV-2 infected HAE cells (Zhu et al., 2020a); there are also reports that mucus accumulation and smell and taste impairments – which could be caused by cilia dysfunction – are common clinical and pathological features of COVID-19 (Bagheri et al., 2020; He et al., 2020; Li et al., 2020c; Makaronidis et al., 2020; Yan et al., 2020a).
A recent study of SARS-CoV-2 infection within a reconstituted human bronchial epithelium model showed a rapid loss of the ciliary layer, with axoneme loss and misorientation of the remaining basal bodies. Further analysis of SARS-CoV-2 infection in Syrian hamsters revealed the loss of motile cilia in vivo (Robinot et al., 2021).
We also found that the SARS-CoV-2 infection in hACE2 mice displayed damaged cilia layers in bronchioles (Fig. 8, A and B). A genome-wide association study (GWAS) of 1,778 COVID-19 cases showed that variants in two genes required for ciliogenesis (DNAH7 and CLUAP1) are genetic risk loci for increased COVID-19 mortality (Hu et al., 2021).
Further, the levels of master regulators of cilia biogenesis and maintenance (e.g., FOXJ1, RFX3, NEK10, and DNAI2) are aberrantly downregulated in ciliated cells upon SARS-CoV-2 infection (He et al., 2020; Robinot et al., 2021). In the present study, we demonstrate that SARS-CoV-2 ORF10 interacts with ZYG11B, which stimulates the ubiquitination activity of CUL2ZYG11B (Fig. 1).
Enhanced CUL2ZYG11B activity causes increased ubiquitination and subsequent proteasome-mediated degradation of multiple cilium-related proteins (including IFT46), thereby impairing the cilia biogenesis and maintenance in cells (Figs. 2 and 3).
Recently, Yan et al. (2021) reported that three residues (W522, D526, and N567) of substrates are required for specific recognition of the Gly/N-degron by CRL2ZYG11B (Yan et al., 2021). We found that the mutation of key recognition elements in ZYG11B did not affect the interaction of ZYG11B and IFT46 (Fig. S4 J). CUL2ZYG11B’s interaction with the C2 domain of IFT46 promotes IFT46 ubiquitination (Fig. 5 B).
Therefore, the interaction of ZYG11B with IFT46 was independent on its recognition of the Gly/N-degron. In addition, the exposure of the respiratory tract of hACE2-humanized mice to Spike-lentivirus-ORF10 results in multiple pathogenic phenotypes, which are similar to the phenomena of SARS-CoV-2 infection in hACE2 mice (Figs. 7 and 8).
Our study therefore demonstrates that SARS-CoV-2 ORF10 hijacks CUL2ZYG11B to cause cilia dysfunction through the elimination of IFT46 and uncovers a pathological mechanism of SARS-CoV-2 that connects cilia dysfunction to reported COVID-19 symptoms (Fig. 9 C).
A recent study of HEK293T cells reported that ORF10 expression does not affect the degradation of known substrate proteins of the CUL2ZYG11B or CUL2ZER1 complexes, and that study dismissed the idea of a pathogenic impact from ORF10 study (Mena et al., 2021).
We found the ORF10 level can account for the differences between our study and Mena et al. (2021) results (Fig. S2, I–K), and the protein level of ORF10 does affect the stability of some cilium-related proteins (Fig. S2, L and M). The Global Protein Stability (GPS) system also showed that the high level of ORF10 indeed enhanced the CUL2ZYG11B activity (Fig. S2 N).
In addition, we confirmed that the 38-residue SARS-CoV-2 ORF10 can bind ZYG11B, and our study advances further to show that SARS-CoV-2 ORF10 can stimulate the E3 ligase activity of CUL2ZYG11B. The binding of ORF10 and ZYG11b presents a plausible explanation for the proteasomal degradation hijacking we observed to cause severe defects in cilia biogenesis and maintenance.
The impact of ORF10 in promoting the ubiquitination activity of the CUL2ZYG11B complex deepens our understanding of the molecular mechanisms underlying the interaction between host cells and SARS-CoV-2, and additionally presents a candidate target for developing potential therapies to treat COVID-19. Since the knockdown of Zyg11b and the overexpression of IFT46 could partially rescue the ciliogenesis defects in ORF10 expressed cells (Fig. 4 K and Fig. S3, I–K), so it is worth noting that there may be other substrates or pathways of ORF10 participating in ciliogenesis.
Inhibiting CUL2ZYG11B by some drugs and/or supplementing IFT46 (or other cilia related proteins) might be useful in fighting this coronavirus. Given that the ORF10-ZYG11B interaction is essential for the cilia-dysfunction-related symptoms of COVID-19, some specific targeting agents that disrupt the interaction between ORF10 and ZYG11B might be considered as potential treatments.
The human airway, a dichotomous hollow tube branching structure of up to 23 generations from trachea to the alveoli, is lined by a continuous layer of pseudostratified epithelium comprised of approximately 1010 cells covering a surface area of 2500 cm2 (1). The airway epithelium is composed of 4 major cell types lining a continuous basement membrane, including ciliated, secretory, undifferentiated intermediate and basal cells (2). The basal cells function as the stem/progenitor cells, responding to cell senescence and injury by generating intermediate undifferentiated cells, which then differentiate to ciliated and secretory cells in a ratio of 7–8 to 1 in both the large (0 to 5 generations) and small airways (≥6 generations).
The ciliated and secretory cells are the first line of defense against inhaled pathogens and particulates. This includes tight junctions linking the cells, providing a physical barrier; receptors that sense the environment signaling the cells to secrete defense-related molecules in response to inhaled pathogens, particulates and xenobiotics; and importantly, the mucociliary escalator, a layer of fluid and mucins lining the epithelium that functions to clean the airways by moving continuously from the lower respiratory tract cephalad, where it is insensibly swallowed (2–4).
The effectiveness of the mucociliary escalator depends on hydration, mucins produced by secretory cells and on the coordinated function of the ciliated cells that provides the force and direction of the escalator (4). When there is dysfunction of the mucociliary escalator, the defenses of the epithelium are markedly weakened, resulting in lung disease.
The focus of this review is on the role of ciliated cells and cilia per se in the normal function of the airway epithelium and how abnormalities in the structure and function of airway cilia result in disease. To do so, we will first summarize what is known about human airway ciliated cells and cilia, provide an overview of the role of the ciliated cells in the mucociliary escalator and detail how airway cilia structure and function are assessed in humans. This will provide the background to describe the current state of knowledge of the inherited and acquired disorders of airway cilia dysfunction.
Airway Cilia Structure
Cilia are evolutionarily conserved hair-like cellular organelles that project from the cell surface (20). The basic structure consists of a centriole-derived, microtubule core termed the axoneme that protrudes continuously from the plasma membrane (Figure 3) (21). Cilia are subdivided into two classes, immotile or motile, based on the physical ultrastructural characteristics of the axoneme (21–23). Airway cilia are in the motile cilia class, as are cilia found on ciliated cells of sinuses, brain ventricle ependyma, oviducts and epididymal ducts (24–26). Immotile cilia (referred to as “primary cilia”) are solitary structures on most cell types where they function to sense the environment (20, 24, 27).
Airway cilia have components typical for motile cilia (see Figure 3 for an overview and Supplemental Information for further details). Proteomic analysis of cilia isolated from in vitro generated human airway epithelial cells on air-liquid interface culture identified >200 axonemal proteins (28). Some, such as α and β tubulin, are conserved with axonemal proteins identified in other motile cilia, but some are unique, including the sperm or testis associated proteins SPA17 and SPAG6 and retinitis pigmentosa protein 1 known to associate with photoreceptor axonemal structures (28). Characterization of the transcriptome of murine tracheal epithelial cells identified similarities among components of motile and primary cilia (29), and many genes identified as part of the mouse ciliated cell transcriptome correspond to human airway cilia proteins, suggesting conservation across species.
Inherited Disorders of Cilia Dysfunction
The most important inherited disorders of airway cilia dysfunction are primary cilia dyskinesia and cystic fibrosis; other inherited cilia-related disorders are very rare.
Primary Ciliary Dyskinesia
The classic disorder of respiratory cilia dysfunction is primary ciliary dyskinesia (PCD), an autosomal recessive disorder of motile cilia (Table I) (25). The symptoms and signs of this disorder were recognized long before the mechanism was understood (66). It was first referred to as Kartagener syndrome, the triad of chronic sinusitis, bronchiectasis, and situs inversus (25, 66, 67). Subsequently, male infertility was noted to be associated with Kartagener syndrome, and dynein arm defects were observed in both the spermatozoa and respiratory epithelial cells, leading to the syndrome being named “immotile cilia syndrome” (68). The disorder was renamed PCD when it was recognized that a subgroup of patients with ciliary motility but ineffective mucociliary clearance manifest the same clinical syndrome (67, 69). The clinical manifestations of PCD include chronic otitis media, transient hearing loss/speech delays, nasal congestion, chronic sinusitis, recurrent lower respiratory tract infection, bronchiectasis, male infertility, defects in organ laterality (50% of cases) and in newborns, neonatal respiratory disorders.
Organ laterality in embryogenesis is determined by the normal rotary motion of a single specialized cilium found on each of the cells in the ventral node which defines right-left symmetry in the developing embryo (25). Without normal directional motion of this specialized cilium, organ placement is random, which is why situs inversus is found in approximately 50% of individuals with PCD.
Cilia structural defects A variety of cilia structural defects observed by electron microscopy are associated with the PCD clinical phenotype. The most common are absent or short outer dynein arms, or an outer dynein arm and inner dynein arm defect (25). Isolated inner dynein arm defects are uncommon and when causative are typically associated with abnormalities in the central apparatus of the cilia such as microtubular disorganization or abnormally placed outer doublets (25).
PCD is also associated with reduced cilia beat frequency, most prominent in patients with dynein arm defects (70). Ciliary wave form is dyskinetic in PCD, further contributing to the lack of effective mucociliary transport (70). Airway particle clearance is prolonged to 1 wk in PCD patients as compared to 12 hr in normal nonsmokers (71).
Genetic basis of PCD The genetic basis can be identified in approximately 65% of PCD patients (Table I) (25). Many mutations lead to defects in all cilia, but others produce structural abnormalities in only a fraction of cilia or exhibit no ultrastructural defects. A commercial genetic test for 60 mutant alleles of DNAI1 and DNAH5 is available (72). Most (85%) mutations implicated in PCD are loss-of-function mutations (25). The majority are rare variants found only in a single family or patient (25).
The typical causative genes for PCD encode ciliary components with mutations in specific genes having predictable effects on cilia ultrastructure. Genes coding for protein components of the outer or inner dynein arm lead to defects in those structures, and mutations in genes encoding cytoplasmic proteins that participate in ciliary assembly lead to defects in both the outer and inner dynein arms. Because the dynein arms function as ATP-dependent motors for ciliary movement, defects in these structures result in ciliary dyskinesia. Genes coding for components of the central pair microtubules and radial spokes lead to defects in those structures, which can lead to abnormalities in both ciliary beat frequency and beat coordination (73).
Because the specialized cilium present in the ventral node that controls organ laterality has no central complex, mutations leading to central complex defects do not produce laterality abnormalities. A mutation in cyclin O (CCNO) is causative of defective mucociliary clearance and bronchiectasis (74). CCNO mutant cells are defective in centriole generation and placement and affected individuals have reduced numbers of airway cilia. The cilia that were present did contain axonemal proteins and affected individuals did not exhibit laterality defects.
Clinical manifestations Diagnosis is challenging due to heterogeneity in clinical symptoms and severity, cilia ultrastructural abnormalities identified by electron microscopy, and by variability in the phenotype associated with the causative mutations (67). Typical initial screening tests include nasal epithelial assessment of ciliary motion and mucociliary transport using a saccharine taste test or radioisotope clearance (67, 75). The diagnosis of PCD is conventionally confirmed by identification of ciliary ultrastructural defects by electron microscopy. However, 30% of patients exhibit normal ultrastructure (25). Other diagnostic modalities include assessment of nasal ciliary beat frequency and motility and measurements of nasal nitric oxide, which is low in PCD (25). Fluorescent labeled antibodies may be used to evaluate for absence of specific cilia proteins (25). Caution must be taken to carry out functional and structural studies when the affected individual has had no recent infection to avoid false positive findings due to secondary ciliary dyskinesia (67).
Lung disease in PCD results from defective mucociliary clearance and worsens over time due to repeated respiratory tract infections (25). Nearly all adults develop bronchiectasis. Lung function testing generally reveals an obstructive ventilatory defect with or without air trapping; mixed obstructive and restrictive patterns are also observed (67). Management centers on airway clearance therapies, use of antibiotics for lung infections, routine immunizations, and avoidance of tobacco smoke exposure (25). Surgical resection of severe localized disease is rarely indicated and lung transplant is an option for end-stage disease (67).
Cystic fibrosis (CF) is an inherited disorder due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The disorder predominantly affects Caucasians with an incidence of 1 in 2500 newborns of Northern European descent (76). Nearly 2000 CFTR mutations have been identified (76, 77). Although affected infants are born with seemingly normal lungs, chronic lung disease develops as a result of abnormal mucociliary clearance, leading to repeated infections and the development of bronchiectasis (78).
The CFTR gene encodes a cAMP-regulated chloride channel expressed apically in epithelial cells (76). The channel modulates chloride secretion and also regulates other membrane proteins including the epithelial sodium channel (ENaC) (76). Because both CFTR and ENaC control water movement through the epithelium, CFTR dysfunction leads to increased fluid absorption, dehydration of the epithelial surface, and altered mucin concentration in abnormal airway mucus (76, 79). A “gel-on-brush” model of the periciliary layer postulates that airway mucus sits atop a dense “brush” of tethered macromolecules in the periciliary layer that function to prevent mucus from penetrating the periciliary space (9). In this model, if the airway surface is sufficiently dehydrated, as in CF, the mucus layer compresses the periciliary brush and cilia, interfering with mucus clearance. Alterations in CFTR function may also contribute to lung disease via abnormal modulation of epithelial inflammation and altered bicarbonate transport (76).
The abnormal mucociliary clearance in individuals with CF results from the abnormal biophysical properties of airway mucus, not primarily from ciliopathy (76, 80). Experimental animal data suggest that the most important factor controlling mucus clearance efficiency is airway surface hydration (76). In CF, the increased absorption of Na+ and reduced secretion of Cl− lead to reduced water content in both the mucus layer and the periciliary layer (79). This leads to a mucus layer that is highly adhesive (79, 80). The failure to clear these abnormal secretions is evident shortly after birth with bronchiolar mucus plugs detected within 48 hr of birth in CF neonates (81). In addition to the abnormal hydration status, mucus hypersecretion eventually occurs in response to recurrent infection and persistent inflammation; this further worsens the physical properties and the clearance of mucus (79). CF epithelial cells alone, without submucosal glands, generate abnormal airway surface liquid; this may be an explanation for the presence of CF lung disease even in distal airways that lack submucosal glands (82).
Electron microscopy of airway cilia from patients with CF shows alterations similar to those seen in a control group of individuals with chronic bronchitis, including compound cilia, excess cytoplasmic matrix, and an abnormal number or arrangement of microtubular doublets (83). With progressive disease, the airways develop squamous metaplasia and dysplasia, regions of missing cilia, cilia with missing inner dynein arms, abnormal numbers and location of microtubular doublets, compound cilia, single microtubules in place of normal doublets, multiple cilia occupying the central area, and detachment of the axonemal membrane from the groups of cytoplasmic filaments (84).
CF was historically considered a fatal childhood disease, but with improved therapies, including antibiotics and airway clearance treatments, the average life expectancy is now 37 years (76). Because of the abnormal mucus characteristics underlying the disease, treatments to reduce mucus adhesivity are efficacious in CF (80). In part, the “sticky” nature of CF mucus results from high concentrations of DNA derived from neutrophils recruited in response to the chronic infection (85). The use of aerosol recombinant DNAse as an effective therapy in CF is directed toward reducing the adhesive properties of the mucus layer (85). In the subset of CF caused by genotype G551D, a missense mutation found in 4 to 5% of CF patients that affects the function of CFTR channels at the cell surface (86), treatment with a CFTR potentiator, ivacaftor, leads to sustained improvements in lung function, weight, and sweat chloride concentration (a measure of CFTR activity), and decreased exacerbations and chest symptoms. Ivacaftor works by increasing the amount of time that activated CFTR channels at the cell surface remain open and increases the chloride transport activity of G551D-CFTR protein (86).
Other Inherited Disorders
Individuals with α1-antitrypsin deficiency with “pure” emphysema have normal mucociliary clearance (63, 87), although α1-antitrypsin deficiency is associated with bronchiectasis (88). In Usher syndrome, a rare autosomal recessive disorder characterized by sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa with progressive visual loss, case reports have noted bronchiectasis with impaired mucociliary clearance but no ultrastructural ciliary abnormalities (89).
Disorders of primary cilia may also have lung manifestations. Jeune syndrome (asphyxiating thoracic dystrophy), is an autosomal recessive disease with skeletal abnormalities as well as variable hepatic, pancreatic and retinal manifestations (90, 91). Respiratory failure is the most common cause of death due to restrictive disease caused by the chest wall abnormalities. While the genetics are incompletely understood, mutations in both DYNC1H1, a dynein heavy chain gene, and IFT80, involved in intraflagellar transport, have been linked to Jeune syndrome (90, 91). Roifman syndrome, a rare complex of bone dysplasia, growth retardation, retinal dystrophy and humoral immunodeficiency associated with recurrent respiratory infections, has been suggested as a possible ciliopathy of primary cilia (92).
Acquired Disorders of Airway Cilia
Abnormalities of mucociliary clearance, and consequent reduced host defenses of the lung, are a common theme in many acquired lung disorders. In many of these disorders the airway cilia demonstrate acquired structural and/or functional abnormalities with associated abnormalities in mucociliary clearance.
Smoking has long been recognized to suppress mucociliary clearance in most smokers and there is documented slowing of mucociliary clearance immediately after smoking cigarettes (93, 94). Individuals with bronchitis have reduced mucociliary clearance (95). Smoking cessation improves measurements of nasal mucociliary clearance compared to baseline values obtained prior to smoking cessation (96).
Examination of the airway ciliated cells of both male and female smokers shows patches of atypical nuclei and missing cilia (97). These abnormalities increase with increasing intensity of smoking behavior, and are more frequent in smokers using high tar/nicotine cigarettes (98). Smoking induces expression of epidermal growth factor (EGF) in ciliated cells, which may shift basal cell fate toward a squamous phenotype and suppress ciliated cell differentiation (99). Healthy smokers have shorter cilia in the large and small airways compared to nonsmokers, with further shortening observed in smokers with COPD (49, 50). Smoking is associated with suppression of a number of genes in the airway epithelium, likely contributing to slowing the process of regenerating cilia (Table I).
Electron microscopic assessment of cilia ultrastructure demonstrates that smokers with chronic bronchitis have greater numbers of ciliary abnormalities compared to nonsmokers, including compound cilia and giant cilia, and other abnormalities in the microtubules and the axonemal 9 + 2 pattern of organization and in cilia orientation (100–102). These abnormalities may persist after smoking cessation (103).
Data on the effect of smoking on cilia beat frequency are conflicting. Exposure to cigarette smoke extract (104) or direct cigarette smoke (105) leads to reduced cilia beat frequency in in vitro models of human airway epithelium. In contrast, nasal epithelium grown in culture demonstrated increased cilia beat frequency in samples from smokers or those with passive exposure compared to nonsmokers (106). In one study utilizing freshly obtained nasal samples, there was no difference in cilia beat frequency in smokers, nonsmokers, or nonsmokers who acutely smoked two cigarettes (107). No difference was found in cilia beat frequency of resected tissue from cancer surgery in smokers vs nonsmokers (108) or in samples obtained by biopsy (109). A study including smokers with lung disease found that cilia beat frequency was unaltered in healthy smokers compared to nonsmokers, though decreased in smokers with moderate to severe COPD (46). However, a contradictory study found increased cilia beat frequency in nasal biopsies from both active and passive smokers compared to nonsmokers (110).
There is a reduction in exhaled NO observed in smokers and smoking cessation is associated with an increase in exhaled NO; given that NO is important for normal ciliary beating, this may be one mechanism for the effect of cigarette smoking on ciliary motion (111). In vitro, cigarette smoke up-regulates airway epithelial expression of IL-8, which does not decrease ciliary beat frequency directly, but does abrogate the increase in ciliary beat frequency induced by beta-agonists (112).
Passive smoke exposure may also be associated with ciliary abnormalities. Children undergoing sinus surgery who were exposed to environmental tobacco smoke were found to have reduced regrowth of cilia following the procedure compared to children without passive smoke exposure (113). Examination of nasal mucosa in children passively exposed to smoke showed both patchy and generalized loss of cilia on a background of other epithelial abnormalities (114). Adenoid explants from children with exposure to secondhand smoke showed a blunted cilia beat frequency response to ciliary stimulants ex vivo (115). This is consistent with data showing an increased risk of respiratory disease in children exposed to passive cigarette smoke (113). Adults exposed to passive smoking have reduced nasal mucociliary clearance compared to nonsmokers, though to a lesser extent than active smokers (116).
Use of Alternative Tobacco Products and Illicit Drugs
Limited data exist on cilia abnormalities in users of alternative tobacco products and illicit drugs. Users of marijuana more frequently have regions of cilia loss and goblet cell hyperplasia, which may correlate with increased mucus secretion and altered mucociliary clearance, than do nonsmokers (117, 118). Cocaine use is also associated with focal loss of airway cilia (117). The effects of alternative tobacco product use, including shisha (waterpipe, hookah) or electronic cigarettes, on cilia structure or function are not known.
Several studies link environmental exposure to airborne pollutants to cilia abnormalities and dysfunction. Nasal biopsies from Mexico City residents exposed to high levels of air pollution showed patches of short cilia and regions of cilia loss (119). Healthy nonsmokers exposed to ozone at 0.4 ppm or sulfur dioxide at 0.75 ppm showed no abnormalities in cilia structure assessed in nasal samples, though experimental animals exposed to substantially higher concentrations of ozone (4 ppm) demonstrated blebbing and vesiculation of ciliary membranes as well as disruption of trachea cilia structure (120). Exposure of cultured human bronchial epithelial cells to diesel exhaust particles, but not to NO2, reduces cilia beat frequency (121). Compounds present in indoor air pollution, including formaldehyde, acrolein, and ammonia have effects on cilia beating and structure as well as mucus flow (122). Workplace exposures have also been linked to cilia dysfunction, including cadmium (reduced cilia beat frequency), nickel (reduced cilia beat frequency, cell damage and disorganized cilia), hairspray (reduced mucociliary clearance in the nose and trachea of hairdressers), and wood dust (decreased nasal mucociliary clearance and loss of ciliated epithelium) (122).
Mucociliary clearance is impaired in COPD in association with increased vulnerability to respiratory tract infections (87). This decrease in clearance is attributed to a shortening of cilia caused by cigarette smoke as well as airway epithelial dysfunction (123). Respiratory cilia are shorter in healthy smokers than in nonsmokers, and even shorter in smokers with COPD than in smokers without evidence of airway disease (49, 50). Cilia beating is impaired in nasal cilia from individuals with COPD (46), even after smoking cessation (124).
A new concept in COPD cilia dysfunction is that of ciliophagy, the consumption of cilia components by autophagic mechanisms (125). Lam and colleagues (123) demonstrated increased lung autophagic flux in the setting of cigarette smoke, and that mice deficient in autophagy mechanisms resisted smoke-induced shortening of cilia and smoke-induced impairment of mucociliary clearance. Cigarette smoke exposure also increased the turnover of cilia proteins by autophagy, a process mediated by cytosolic deacetylase HDAC6 (123). In this model, administration of tubastatin A, an inhibitor of HDAC6, or 4-phenyl butyric acid, a chemical chaperone, prior to cigarette smoke exposure protected mice from cigarette smoke-induced reduction in mucociliary clearance (123, 125). Similarly, blockage of autophagy enhances primary cilia growth and cilia-associated signaling during normal nutritional conditions (126) and autophagic degradation of a ciliopathy protein, OFD1 (oral-facial-digital syndrome 1), at centriolar satellites promotes primary cilium biogenesis (127).
Bronchiectasis is characterized by a localized, irreversible dilation of segments of the bronchial tree in association with loss of airway smooth muscle and elastic fibers (128). The most common cause is infection, but it is also associated with inhalation of toxic gases, aspiration of stomach contents and drug use (128). Interestingly, other than bronchiectasis associated with PCD, the incidence of cilia ultrastructural defects is not significantly different in idiopathic bronchiectasis compared to normal controls (129). Cilia orientation in individuals with idiopathic bronchiectasis is typically not different compared to controls (129, 130). Conflicting data exist regarding whether ciliary beat frequency in idiopathic bronchiectasis is reduced compared to normal controls (129, 131). The addition of sputum from patients with bronchiectasis to nasal epithelial fragments suspended in tissue culture medium results in reduced ciliary beat frequency, but improves after treatment with antibiotics (132). It has been proposed that defective signaling of sensory proteins in motile cilia may lead to ciliopathy and development of bronchiectasis (133).
Mucociliary clearance is impaired in asthma (134). Autopsy specimens from cases of fatal asthma show loss of cilia, shedding of the bronchial epithelium and failure of clearance of mucus with bronchial plugging (134, 135). Many functional studies in asthma subjects have documented impaired mucociliary clearance (136–139), and abnormal clearance of secretions is clinically apparent to asthma patients (136).
While alterations in characteristics of mucus play a role, changes in cilia structure and function also contribute to reduced mucociliary clearance in asthma. The shedding of ciliated epithelium observed at autopsy (134, 135) is also observed in cells obtained via biopsy and cells shed from the airway epithelium and recovered by bronchoalveolar lavage (140). Electron microscopy of epithelial biopsies in both children and adults with asthma shows damage to ciliated cells, with vacuolization of the endoplasmic reticulum and mitochondria, loss of cilia, and abnormal cilia structure (141, 142). In contrast, ultrastructural examination of nasal biopsies from patients with aspirin-exacerbated respiratory disease (aspirin sensitivity, nasal polyposis and asthma) shows no ciliary abnormalities (143). Ciliary beat frequency is reduced in moderate and severe asthma compared to controls (144) and the ciliary beat direction is disorganized (142). Subjects with moderate and severe asthma have more dyskinetic and immotile cilia than controls, while cilia length is unchanged in asthmatics as compared to controls (144). Related to these abnormalities in function, subjects with severe asthma have more ciliary disorientation, ciliary depletion, and microtubular defects compared to both normal controls and subjects with mild asthma (144). Sputum from asthma patients has an inhibitory effect on cilia beating in bronchial epithelial explants (145) A number of mediators implicated in asthma, including prostaglandins, bradykinin, prostacyclin and leukotriene D4 increase cilia beat frequency (56, 146), while varying effects on ciliary beat frequency have been reported for histamine and leukotriene C4 (56, 146, 147).
The Th2-type cytokine interleukin-13 (IL-13) is found at increased levels in the airways of asthmatics and is a key mediator of the epithelial abnormalities of asthma (148). In culture models of mucociliary differentiation, IL-13 promotes goblet cell differentiation and reduced numbers of ciliated cells (149). IL-13 is associated with modulation of expression and localization of ezrin, which anchors basal bodies to the apical cytoskeleton, and IL-13 exposure interferes with the apical localization of ezrin, leading to reduced numbers of basal bodies (16, 149, 150). The addition of IL-13 to differentiated airway epithelium in culture reduces both the number of ciliated cells and the number of cilia per cell (16). In cultured airway epithelium, IL-13 also decreases and eventually eliminates ciliary beat frequency (149). These effects appear to be mediated by an IL-13-induced decrease in expression of FoxJ1, via a decrease in FoxJ1 promoter activity, and of ezrin (16). Using a candidate gene approach, Kovacic et al (151) found that variants in KIF3A, a member of the kinesin family of microtubular motors critical to intraflagellar transport, were significantly associated with asthma. Kim and colleagues (152) found an association in a Korean population between KIF3A polymorphisms and aspirin-associated respiratory disease.
Acute and Chronic Infection
Both infectious microorganisms and the immune/inflammatory response to infection can alter airway cilia function, leading to impaired mucociliary clearance and retained secretions (153). Recurrent bronchitis is associated with loss of ciliated cells in children (154). A number of microorganisms impair cilia function by mechanisms including reducing ciliary beat frequency, disrupting ciliary coordination and inducing ciliary dyskinesia (155, 156). Some bacterial pathogens specifically target ciliated cells for adherence, including Actinobacillus pleuropneumonia, Pseudomonas aeruginosa, Moraxella catarrhalis, Mycoplasma pneumonia, Mycoplasma hyopneumoniae, and Bordatella species (155). Epithelial samples from chronically infected patients with bronchiectasis show a disruption of ciliary orientation associated with decreased mucociliary clearance although cilia ultrastructure and ciliary beat frequency remain normal (157). Lung samples from patients infected with respiratory syncytial virus show epithelial damage and loss of cilia associated with decreased expression of FoxJ1, findings replicated in a mouse model (156).
In addition to the effect of the microorganism itself, the host response to infection may also contribute to deficient mucociliary clearance. Human neutrophil elastase causes epithelial disruption and at high concentrations reduces ciliary beat frequency (153). Reactive oxygen species generated by polymorphonuclear leukocytes, especially H2O2, decrease ciliary beat frequency (158).
Interstitial Lung Disease
Limited data are available on cilia structure and function in interstitial lung disease. Mucociliary clearance is normal in subjects with asbestosis, sarcoidosis, and pulmonary fibrosis (159, 160). Transcriptional profiling of lung tissue samples from 119 subjects with idiopathic pulmonary fibrosis (IPF) categorized these subjects into two distinct groups, defined by expression of cilia-related genes, a finding validated in an independent cohort of 111 IPF patients (161). Interestingly, patients with high expression of these cilia-related genes had more microscopic honeycombing, but not fibroblastic foci, and had higher expression of MUC5B and MMP7.
Infection plays an important role in early death after lung transplant and may also contribute to the development of bronchiolitis obliterans syndrome, a leading cause of later deaths post-transplant (162). Mucociliary clearance is impaired early after lung transplant (163, 164). Studies of ciliary beat frequency in adults have had mixed findings, with some studies showing reduced ciliary beat frequency in transplanted bronchi (165) while others did not (163, 166, 167). In a study in children post-lung transplant for cystic fibrosis as well as for non-suppurative lung disease, significant ultrastructural abnormalities in the epithelium were observed 7 to 12 months post-transplant below the anastomosis as compared to above, including loss of ciliated cells, ciliated cells with loss of cilia, and ciliary disorientation (168).
Bone Marrow Transplant
Bronchiolitis obliterans is a common pulmonary complication following stem cell transplantation and an important cause of death following transplant (169). In a study of nasal cilia morphology and function in 36 Chinese patients after allogeneic stem cell transplant, reduced ciliary beat frequency was observed in the transplant patients compared to controls, with a greater reduction in ciliary beat frequency in patients with bronchiolitis obliterans (170). Assessment of 19 Chinese patients both before and after stem cell transplant showed reduced ciliary beat frequency both before and after transplant compared to age- and sex-matched controls but showed no correlation between ciliary abnormalities and pulmonary dysfunction (171).
The use of invasive mechanical ventilation for the treatment of respiratory failure may induce airway epithelial injury and cilia dysfunction. In acutely intubated patients, reduced mucociliary clearance was associated with duration of mechanical ventilation, smoking status, and isolation of pathogenic bacteria in the tracheobronchial tree (172). Airway epithelial injury including cilia loss is found in experimental animals undergoing various modes of mechanical ventilation, with some differences in pattern of injury depending on ventilator mode (173, 174). In contrast to invasive mechanical ventilation, in a group of patients using nasal continuous positive airway pressure for obstructive sleep apnea, no decrement in nasal clearance was observed (175).
Little is known about cilia function in patients with sepsis or shock. Absence of ciliary motility has been reported post-mortem in patients dying of sepsis or multiorgan system failure (176).
Effect of Drugs
A variety of drugs have an effect on airway cilia function. Activation of axonemal cAMP-dependent protein kinase A (PKA) increases cilia beat frequency by phosphorylation of dynein light chains (35). β-agonists, including salmeterol and salbutamol, raise intracellular cAMP levels and thereby increase cilia beat frequency (177). Consistent with this observation, inhalation of salbutamol increases mucociliary clearance in normals and those with chronic bronchitis (178). Similarly, methylxanthines (theophylline, aminophylline) inhibit phosphodiesterase and increase cAMP, leading to ciliary stimulation (179). The PDE4 inhibitor roflumilast increases cilia beat frequency in vitro and reverses the decrease in cilia beat frequency observed with treatment with cigarette smoke extract (180). The cholinergic agents acetylcholine and pilocarpine increase cilia beat frequency (181) and conversely, anticholinergic drugs reduce cilia beat frequency (182). Topical application of corticosteroids to airway epithelium in culture results in a small increase in cilia beat frequency (181). Prolonged inhaled steroid treatment in asthmatics has been shown to reverse epithelial damage seen in the same subjects prior to steroid treatment, including areas of epithelium that showed loss of cilia (183). Inhaled beclomethasone in patients with COPD does not affect mucociliary clearance (184). Sisson and colleagues (185) found that alcohol rapidly stimulates cilia beating through both nitric oxide- and cAMP-dependent mechanisms and have suggested that this pathway is downregulated by chronic alcohol exposure leading to chronic cilia dysfunction.
Aspirin decreases mucociliary clearance, although whether this effect is due to changes in water transport and airway secretions, or to changes in ciliary beating and coordination, is not clear (186). Volatile anesthetics depress cilia beat frequency in cultured rat tracheal cells, with halothane and isoflurane having a greater effect than sevoflurane (187). N-acetylcysteine reduces cilia beat frequency in cultured nasal epithelium but no effect was seen in nasal epithelium of CF patients taking the drug (188).
Because of the role of primary cilia in cell cycle regulation, primary cilia may be important in tumorigenesis, and genes important in cilia formation and function exhibit dysregulated expression in multiple tumor types (189). Few data exist, however, on airway cilia structure and function in the setting of lung cancer, though the histologic progression from normal histology to dysplasia to lung cancer is characterized by changes including loss of cilia (190). Less commonly, lung cancer variants with ciliated epithelium are reported (191–194). Loss of ciliary function does not appear to predispose to lung cancer and lung cancer in primary ciliary dyskinesia is rare (195). Mucociliary clearance has been reported to be slower in individuals with lung cancer than in those with chronic bronchitis and no cancer, matched for smoking history (196). Expression of ciliated cell genes has been found to be markedly decreased in a “basal cell-high” lung adenocarcinoma subset, which is characterized by increased expression of airway basal cell-related genes and associated with smoking status, higher frequency of KRAS mutations and a particularly aggressive clinical phenotype (197).
Most data on cilia function in the setting of HIV infection is focused on nasal cilia. Nasal mucociliary clearance was evaluated in a cohort of HIV+ individuals compared to HIV− controls; no subjects had active nasal symptoms or sinusitis at the time of the study. Findings included reduced mucociliary clearance in HIV+ individuals, with a trend toward worsening mucociliary clearance with progression of HIV infection to AIDS as well as a trend toward worsened mucociliary clearance in those subjects with a history of recurrent sinus infections (198). A subsequent study corroborated altered mucociliary clearance and documented abnormalities in cilia axonemal structure in HIV+ individuals, but the majority of the individuals studied had respiratory infections at the time of evaluation, which may have impacted the results, since acute infection is known to reversibly affect cilia structure and function (199). HIV+ individuals have reduced nasal NO compared to controls, which may correlate with reduced cilia function (200). Guiafenesin treatment in HIV+ individuals may reduce nasal symptoms but is not associated with improvement in mucociliary clearance (201).
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4465242/