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Event: 1912

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Motile Cilia Number/Length, Decreased

Short name

The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help

Level of Biological Organization
Cellular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place. For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable. For individual/population events, the organ context is not applicable. More help

Cell term
multi-ciliated epithelial cell

Organ term

Organ term
lung epithelium

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help

Process	Object	Action
	motile cilium	decreased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help

AOP Name	Role of event in AOP	Point of Contact	Author Status	OECD Status
ox stress-mediated FOXJ1/cilia/CBF/MCC impairment	KeyEvent	Agnes Aggy (send email)	Open for comment. Do not cite

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Name
Cigarette smoke

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help

Term	Scientific Term	Evidence	Link
Danio rerio	Danio rerio		NCBI
Homo sapiens	Homo sapiens	High	NCBI
Xenopus laevis	Xenopus laevis		NCBI
Mus musculus	Mus musculus		NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help

Life stage	Evidence
During development and at adulthood	High

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help

Term	Evidence
Mixed	High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

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

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information. More help

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

Cigarette smoke

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).

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

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.

Verra, F., Escudier, E., Lebargy, F., Bernaudin, J.F., De Cremoux, H., and Bignon, J. (1995). Ciliary abnormalities in bronchial epithelium of smokers, ex-smokers, and nonsmokers. Am. J. Respir. Crit. Care Med. 151, 630-634.

Wanner, A., Salathe, M. and O'riordan, T.G. (1996). Mucociliary clearance in the airways. Am. J. Respir. Crit. Care Med. 154, 1868-1902.

Wessely, O. and Obara, T. (2008). Fish and frogs: models for vertebrate cilia signaling. Front. Biosci. 13, 1866-1880.

Yaghi, A. and Dolovich, M.B. (2016). Airway Epithelial Cell Cilia and Obstructive Lung Disease. Cells. 5, 40.