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Key Event Title
Motile Cilia Number/Length, Decreased
|Level of Biological Organization|
|multi-ciliated epithelial cell|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Oxidative stress [MIE] Leading to Decreased Lung Function [AO]||KeyEvent||Karsta Luettich (send email)||Open for comment. Do not cite|
|During development and at adulthood||High|
Key Event Description
Motile cilia are microtubule-based organelles that protrude from the cell surface and generate directional flow of fluid with coordinated beating. 50% to 80% of human respiratory epithelium is comprised of ciliated cells covered with multiple motile cilia that move mucus (together with mucus-trapped substances) upward for clearing the airways (Yaghi and Dolovich, 2016). The ciliated airway epithelial cells are typically covered by more than hundred motile cilia (Bustamante-Marin and Ostrowski, 2017). On average, 150 motile cilia were counted per ciliated human epithelial cell in the study by Mao et al. (Mao et al., 2018). In an earlier report, 200 motile cilia per ciliated cell in human trachea is mentioned (Wanner et al., 1996), and, in a more recent study, a range of 100 to 600 ciliary precursors were counted in fully differentiated mouse tracheal epithelial cells correlated with increasing surface area (Nanjundappa et al., 2019). Cilia are 6–7 µm long and 0.2–0.3 µm in diameter (Brooks and Wallingford, 2014; Yaghi and Dolovich, 2016). Ciliated cell density and the motile cilia length and number per cell correlate with ciliary beating frequency which is routinely used as a predictor of the mucociliary clearance efficiency (King, 2006). Morphological changes of airway cilia are expected to impact multiple motile cilia functional integrity. This key event represents the decrease in the numbers or absence of motile cilia or reduction in length of motile cilia.
How It Is Measured or Detected
Acetylated tubulin is a common ciliary marker (Kim et al., 2013; Piperno and Fuller, 1985), and apical acetylated tubulin staining with subsequent microscope image scoring is a frequently used method of cilia detection and enumeration (Johnson et al., 2018; Mao et al., 2018; Stubbs et al., 2008). Staining of beta-tubulin IV, a protein enriched in motile cilia, is another common method of cilia detection (Brekman et al., 2014; Milara et al., 2012). Ciliated cells can also be identified by the presence of axonemal structures on the cell surface using scanning electron microscopy (Gomperts et al., 2007). Mature cilia numbers could be deduced from ciliary precursors in immunofluorescence assays: ciliary precursors can be calculated from three-dimensional superresolution structured illumination microscopy (3D-SIM) images using e.g. a spot detection tool (Nikon Elements AR 4 Software) (Nanjundappa et al., 2019).
For cilia length measurement, the ciliated cells/tissue needs to be stained (Diff-Quik: Dade Behring stain, hematoxylin and eosin staining, labelling with antibodies for ciliary markers such as alpha-tubulin), visualized by microscopy and cilia length quantified (using e.g. ImageJ software or MetaMorph Microscopy Automation & Image Analysis Software) (Brekman et al., 2014; Leopold et al., 2009b; Li et al., 2014). Generally, multiple measurements of one sample and multiple sample preparations of cells/tissues are imaged for reliable quantitation.
Domain of Applicability
The ultrastructural features of human and other mammalian respiratory epithelial cilia and those from lower animals (e.g. flatworms and mollusks) are remarkably similar (Meunier and Azimzadeh, 2016; Wanner et al., 1996). The master regulators of multiciliated cell differentiation, such as NOTCH, GEMC1, MCIDAS, FOXJ1, RFX2/3 are conserved throughout vertebrates (e.g. mammals, Xenopus, zebrafish) and multiple motile cilia across these organisms are functionally similar in generating fluid flow through coordinated beating (Choksi et al., 2014; Meunier and Azimzadeh, 2016; Wessely and Obara, 2008).
The motile cilia numbers reach adult levels in the mouse airway epithelium at day 21 after birth (Rawlins et al., 2007; Toskala et al., 2005). At birth, there is no discernable cilia-generated airway fluid flow in mice (Francis et al., 2009). Between postnatal days 3 and 7 the flow is established in trachea correlating with the increase in the density of ciliated cells in the tracheal epithelia (Francis et al., 2009). After airway fluid flow establishment, the KE is applicable to all life stages.
Evidence for Perturbation by Stressor
Cilia length was reduced in endobronchial biopsies and airway brushings of smokers (average 30 pack-years) compared to nonsmokers (Leopold et al., 2009).
Exposure of human bronchial epithelial cells cultured at the air-liquid interface to 1, 3, and 6% cigarette smoke extract (from the basolateral side) between days 5 and 28 of differentiation significantly shortened the average cilia length of day 28 ALI cultures to 5.7, 5.5, and 4.9 µm, respectively, compared an average cilia length of 6.7 µm in untreated cultures. Continuous treatment of differentiated cultures with 3 and 6% cigarette smoke extract between days 28 and 42 showed that ciliated cells in the untreated day 42 cultures had longer cilia than day 28 cultures (ca. +1.5 µm), whereas in the presence of 3 and 6% of CSE, this elongation of cilia was suppressed (+0.5 µm and -0.5 µm, respectively) (Brekman et al., 2014).
Apical exposure of mouse tracheal epithelial cells differentiated at the air-liquid interface to cigarette smoke from 3R4F research cigarettes at a total particular matter concentration of 50 and 100 mg/m3 for 10 min resulted in cilia shortening (approx. -20% and -50%, respectively) and complete loss of cilia (approx. -25% and -60% of ciliated cells, respectively) at 24 h post-exposure (Lam et al., 2013).
Mean cilia length in the large airway epithelium was 7% shorter in healthy smokers (32.5+10 pack-years) compared to nonsmokers (7.09 vs 7.63 µm), 12% shorter in COPD smokers (39+21 pack-years) compared to healthy smokers (6.16 vs 7.09 µm), and 19% shorter in COPD smokers as compared to nonsmokers. In the small airway epithelium, mean cilia length was 9% shorter in healthy smokers relative to nonsmokers (6.49 vs 7.15 µm), 6% shorter in COPD smokers relative to healthy smokers (6.05 vs 6.49 µm), and 15% shorter in COPD smokers compared to nonsmokers (Hessel et al., 2014).
Exposure of mouse nasal septal epithelial cells to cigarette smoke condensate at concentrations >30 µg/mL for the first 15 days growing at the air-liquid interface inhibited ciliogenesis (ciliated area: 89.9+8.0% in untreated vs 48.8+10.0% [30 µg/mL] and 37.5+12.0% [100 µg/mL]) and resulted in cilia shortening (not quantified) (Tamashiro et al., 2009).
Whole-body exposure of female C57BL/6 mice to mainstream and sidestream cigarette smoke from 1R1 reference cigarettes at 150 mg/m3 total particular matter for 2 h per day, 5 days per week, for up to 1 year resulted in some areas of sparse or detached ciliated cells by month 6 and an almost complete loss of ciliated cells by 12 months (Simet et al., 2010).
In a small cohort study in adults with adults with chronic sputum production, current and former smokers had a higher frequency of axonemal ultrastructural abnormalities (16.53 ± 2.66% and 17.66 ± 6.99%, respectively) than non-smokers and controls (5.18 ± 0.9% and 0.7% ± 0.2%, respectively) (Verra et al., 1994).
Brekman, A., Walters, M.S., Tilley, A.E. and Crystal, R.G. (2014). FOXJ1 prevents cilia growth inhibition by cigarette smoke in human airway epithelium in vitro. Am. J. Respir. Cell Mol. Biol. 51, 688-700.
Brooks, E.R. and Wallingford, J.B. (2014). Multiciliated cells. Curr. Biol. 24, R973-982.
Bustamante-Marin, X.M. and Ostrowski, L.E. (2017). Cilia and Mucociliary Clearance. Cold Spring Harb. Persp. Biol. 9, a028241.
Choksi, S.P., Lauter, G., Swoboda, P. and Roy, S. (2014). Switching on cilia: transcriptional networks regulating ciliogenesis. Development 141, 1427-1441.
Francis, R.J., Chatterjee, B., Loges, N.T., Zentgraf, H., Omran, H. and Lo, C.W. (2009). Initiation and maturation of cilia-generated flow in newborn and postnatal mouse airway. Am. J. Physiol. Lung Cell. Mol. Physiol. 296, L1067-1075.
Gomperts, B.N., Kim, L.J., Flaherty, S.A. and Hackett, B.P. (2007). IL-13 Regulates Cilia Loss and foxj1 Expression in Human Airway Epithelium. Am. J. Respir. Cell Mol. Biol. 37, 339-346.
Hessel, J., Heldrich, J., Fuller, J., Staudt, M.R., Radisch, S., Hollmann, C., et al. (2014). Intraflagellar Transport Gene Expression Associated with Short Cilia in Smoking and COPD. PLoS ONE 9, e85453.
Johnson, J.A., Watson, J.K., Nikolic, M.Z. and Rawlins, E.L. (2018). Fank1 and Jazf1 promote multiciliated cell differentiation in the mouse airway epithelium. Biol. Open 7, bio033944.
Kim, G.W., Li, L., Ghorbani, M., You, L. and Yang, X.J. (2013). Mice lacking alpha-tubulin acetyltransferase 1 are viable but display alpha-tubulin acetylation deficiency and dentate gyrus distortion. J. Biol. Chem. 288, 20334-20350.
King, M. (2006). Physiology of mucus clearance. Paediatr. Respir Rev. 7, S212-214.
Lam, H.C., Cloonan, S.M., Bhashyam, A.R., Haspel, J.A., Singh, A., Sathirapongsasuti, J.F., et al. (2013). Histone deacetylase 6–mediated selective autophagy regulates COPD-associated cilia dysfunction. J. Clin. Invest. 123(12), 5212-5230.
Leopold, P.L., O'mahony, M.J., Lian, X.J., Tilley, A.E., Harvey, B.-G. and Crystal, R.G. (2009). Smoking is associated with shortened airway cilia. PloS ONE 4, e8157.
Li, Y.Y., Li, C.W., Chao, S.S., Yu, F.G., Yu, X.M., Liu, J., et al. (2014). Impairment of cilia architecture and ciliogenesis in hyperplastic nasal epithelium from nasal polyps. J. Allergy Clin. Immunol. 134, 1282-1292.
Mao, S., Shah, A.S., Moninger, T.O., Ostedgaard, L.S., Lu, L., Tang, X.X., et al. (2018). Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling. Proc. Natl. Acad. Sci. U. S. A. 115, 1370-1375.
Meunier, A. and Azimzadeh, J. (2016). Multiciliated Cells in Animals. Cold Spring Harb. Perspect. Biol. 8, a028233.
Milara, J., Armengot, M., Bañuls, P., Tenor, H., Beume, R., Artigues, E., et al. (2012). Roflumilast N-oxide, a PDE4 inhibitor, improves cilia motility and ciliated human bronchial epithelial cells compromised by cigarette smoke in vitro. Brit. J. Pharmacol. 166, 2243-2262.
Nanjundappa, R., Kong, D., Shim, K., Stearns, T., Brody, S.L., Loncarek, J., et al. (2019). Regulation of cilia abundance in multiciliated cells. Elife 8, e44039.
Piperno, G. and Fuller, M.T. (1985). Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J. Cell Biol. 101, 2085-2094.
Rawlins, E.L., Ostrowski, L.E., Randell, S.H. and Hogan, B.L. (2007). Lung development and repair: contribution of the ciliated lineage. Proc. Natl. Acad. Sci. U. S. A. 104, 410-417.
Simet, S.M., Sisson, J.H., Pavlik, J.A., Devasure, J.M., Boyer, C., Liu, X., et al. (2010). Long-term cigarette smoke exposure in a mouse model of ciliated epithelial cell function. Am. J. Respir. Cell Mol. Biol. 43, 635-640.
Stubbs, J.L., Oishi, I., Izpisua Belmonte, J.C. and Kintner, C. (2008). The forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish embryos. Nat. Genet. 40, 1454-1460.
Tamashiro, E., Xiong, G., Anselmo-Lima, W.T., Kreindler, J.L., Palmer, J.N., and Cohen, N.A. (2009). Cigarette smoke exposure impairs respiratory epithelial ciliogenesis. Am. J. Rhinol. Allergy 23, 117-122.
Toskala, E., Smiley-Jewell, S.M., Wong, V.J., King, D. and Plopper, C.G. (2005). Temporal and spatial distribution of ciliogenesis in the tracheobronchial airways of mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 289, L454-459.
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Yaghi, A. and Dolovich, M.B. (2016). Airway Epithelial Cell Cilia and Obstructive Lung Disease. Cells. 5, 40.