Relationship: 2440: CFTR Function, Decreased leads to ASL Height, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Phylogenetic analysis of CFTR DNA sequences across multiple species suggests a close evolutionary relationship between human and primate CFTR, followed by rabbit, guinea pig, equine, ovine, and bovine CFTR, whereas rodent CFTR DNA largely diverges from the human DNA (Chen et al., 2001). Of note, CFTR ion permeability differs from species to species (Higgins, 1992). For example, murine CFTR displays reduced channel activity compared with its human counterpart, while ovine CFTR exhibits higher ATP sensitivity, greater single-channel conductance and larger open probability than human CFTR. Moreover, sensitivity to pharmacological agents able to potentiate or block CFTR gating varies greatly from species to species (Bose et al., 2015). Therefore, results from animal studies are not directly transferable to human.  

To date, ASL has been investigated in several species including mice, rats, guinea pigs, ferrets, cats, dogs, cows, monkeys, and humans. Although most studies provide data on its composition rather than its height, it is reasonable to assume that regulation of ASL height is equally critical to MCC across these species. 

CFTR dysfunction as a consequence of inherited CFTR gene defects is studied in pediatric as well as adult cystic fibrosis patients. Acquired CFTR dysfunction following inhalation exposures (e.g. to cigarette smoke) may also apply to both pediatric and adult populations, depending on the setting and type of exposure, and this also applies to decreased ASL height. 

To our knowledge, the role of gender has not been systematically evaluated in acquired CFTR dysfunction and its impact on ASL height. It is thought that the observed suppression of CFTR expression and impairment of CFTR function in cigarette smokers is a contributing factor to the pathogenesis of chronic obstructive pulmonary disease (COPD)(Dransfield et al., 2013; Raju et al., 2016). The main risk factor for COPD is cigarette smoking, and COPD is more common in men than in women, which may be directly related to the higher prevalence of smoking in men, although this gender gap is closing (Hitchman and Fong, 2011; Ntritsos et al., 2018; Syamlal et al., 2014). Nevertheless, the available clinical evidence in support of this AOP suggests that there is no remarkable gender difference.
 

Key Event Relationship Description

Serous and glandular secretions of the airway epithelium contribute to the ASL, and epithelial ion channel (e.g. CFTR, ENaC, CaCC, BK) function is critical to normal ASL homeostasis. Should the PCL decrease in depth, liquid will be absorbed from the mucus layer until the necessary depth is restored. Conversely, the mucus layer will absorb surplus PCL to reduce any increase in its depth. The regulation of these reabsorption processes is complex and not fully elucidated (Boucher, 2004). Experimental evidence suggests that the balance between Na⁺ absorption and Cl⁻ secretion mediated by ENaC and CFTR plays a major role, with the ion channels affecting each other’s activity (increased CFTR activity leads to decreased ENaC activity and vice versa) (Boucher, 2003; Boucher, 2004; Schmid et al., 2011). Mechanistic studies with selective CFTR and ENaC inhibitors suggest that the sensors for regulating ASL height lie within the ASL itself (Boucher, 2003; Hobbs et al., 2013). Additionally, ATP, adenosine and other purinergic receptor agonists, adenylate cyclase and cyclic adenosine monophosphate (cAMP)-dependent protein kinases acting on CFTR and/or ENaC ensure that the ASL height is adjusted to the appropriate height, resulting in maintenance of PCL depth at approximately the length of cilia (Antunes and Cohen, 2007). If the CFTR-ENaC interaction is perturbed, the airways become “dehydrated” (i.e., the ASL height decreases), resulting in slowing or inhibition of cilia movement and impaired MCC (Munkholm and Mortensen, 2014).  

Evidence Supporting this KER

As a major Cl^– channel in the respiratory epithelium, CFTR levels and function are vital for maintenance of ASL homeostasis. In vitro studies on the effects of cigarette smoke exposure on human lung primary cells and cell lines showed a reduction in ASL height, associated with decreased CFTR levels (Hassan et al., 2014; Rasmussen et al., 2014; Xu et al., 2015; Ghosh et al., 2017) and decreased Cl^– current (Lambert et al., 2014; Raju et al., 2016). Moreover, pharmaceutical stimulation and inhibition of CFTR function and expression directly increased and decreased ASL height, respectively (Song et al., 2009; Van Goor et al., 2009; Van Goor et al., 2011; Tuggle et al., 2014). 

Impaired function of the CFTR and ENaC ion channels results in enhanced Na⁺ absorption and reduced Cl⁻ secretion, and as a consequence, reduced ASL height. This phenomenon is well-known from studies in models of cystic fibrosis and acquired CFTR deficiency, even though the exact mechanism of the interaction between these two channels remains to be elucidated (Boucher, 2003; Hassan et al., 2014; Raju et al., 2016a; Rasmussen et al., 2014; Tarran et al., 2001a; Woodworth, 2015; Zhang et al., 2013). Additionally, evidence from studies with pharmacological agents that enhance CFTR expression and/or function or perturb the interaction between CFTR and ENaC provide further support for strong biological plausibility of this KER (Lambert et al., 2014; Van Goor et al., 2009; Van Goor et al., 2011).

Addition of 2% cigarette smoke extract (CSE) to differentiated primary human bronchial epithelial cells (HBECs) stimulated with forskolin acutely decreased Cl⁻ transport, which was associated with reduced ASL height and cilia beat frequency (Raju et al., 2016a).

Exposure of differentiated primary HBECs to cigarette smoke for up to 120 h significantly reduced plasma membrane CFTR expression and ASL height (Hassan et al., 2014) in one study, and reduced intracellular cAMP levels, Cl⁻ conductance, ASL volume and cilia beat frequency in another study (Schmid et al., 2015). 

Exposure of primary HBECs grown in monolayers to cigarette smoke caused a dose- and time-dependent decrease in forskolin-stimulated Cl⁻ transport, which could be partially restored by treatment with roflumilast via a cAMP-dependent mechanism. Smoke exposure also decreased ASL height, and this effect could also be ameliorated by roflumilast (Lambert et al., 2014).

Treatment of immortalized 16HBE14o- airway epithelial cells with 10% cigarette smoke extract for 48 h resulted in significant reduction in CFTR total and cell surface protein expression as well as in ASL height (Xu et al., 2015). 

Repeated exposure of differentiated primary HBECs to smoke from little cigars significantly decreased CFTR protein expression and ASL height (Ghosh et al., 2017).

CFTR protein expression was also reduced following exposure of baby hamster kidney cells expressing human CFTR (BHK^CFTR) or Calu-3 lung cancer cells to cigarette smoke, resulting in cigarette smoke-induced increases in intracellular Ca²⁺, CFTR internalization and subsequent reduction in ASL height (Rasmussen et al., 2014).

In excised pig and human trachea preparations, addition of amiloride and CFTR activators increased ASL height. In pig trachea, CFTR inhibition prevented the increase in ASL height produced by forskolin/3-isobutyl-1-methylxanthine (IBMX). This study did not investigate dose or time responses (Song et al., 2009). 

Experiments with pharmacological compounds shown to enhance CFTR cell surface expression or channel open probability in primary human bronchial epithelial cells demonstrated enhanced Cl− currents and increased ASL height (Van Goor et al., 2009; Van Goor et al., 2011).

ASL height in the excised tracheas of rats without functional CFTR expression was approx. half that of wild-type animals (Tuggle et al., 2014).

Exposure of differentiated primary HBECs to supernatant from mucopurulent material (SMM) of cystic fibrosis airways decreased amiloride-sensitive Cl− current in CFTR wild-type but not CFTR mutant HBECs. Forskolin-induced Cl⁻ conductance was absent in treated CFTR mutant HBECs and enhanced in wild-type HBECs, where this effect was both dose- and time-dependent. Enhanced UTP-induced currents were seen in HBECs, independent of CFTR genotype. SMM treatment furthermore induced an increase in ASL height in CFTR wild-type but not mutant HBECs (Abdullah et al., 2018).

Knockdown of SPLUNC1, an allosteric regulator of ENaC, in human bronchial epithelial cells significantly reduced ENaC currents and rapidly and significantly reduced ASL height (Garcia-Caballero et al., 2009). SPLUNC1 is degraded in airway epithelia of cystic fibrosis patients, resulting in failure of ENaC internalization and, consequently, increased ENaC activity. Addition of recombinant SPLUNC1 or sputum supernatants from healthy subjects significantly elevated ASL height in HBECs (Hobbs et al., 2013; Webster et al., 2018).
Knock-down of β-ENaC mRNA in HBECs resulted in significantly reduced amiloride-sensitive short-circuit (Na+) currents and significantly increased ASL height (Gianotti et al., 2013). Knockdown of ENaC mRNA in BMI-1 transduced cystic fibrosis BECs resulted in nearly 50% reduction in amiloride short-circuit currents, a ca. 1.5 fold increase in ASL height and increased cilia beat frequency (Tagalakis et al., 2018).
Inhibition of channel activating proteases, including prostasin, matriptase, and furin, in ΔF508 cystic fibrosis airway epithelia by QUB-TL1 increased intracellular ENaC, decreased  ENaC-mediated Na⁺ transport and increased ASL height (Reihill et al., 2016).
In excised tracheas and bronchi from mice overexpressing the β-ENaC subunit in lower airway epithelia, amiloride-sensitive Na⁺ transport was increased ca. 2- to 3-fold, and ASL was significantly depleted (Mall et al., 2004).
 

The process of reabsorption of excess liquid to regulate ASL height is also known as “isosmotic volume hypothesis” or “isotonic volume transport/mucus clearance hypothesis” and implies that CFTR assumes a critical role in regulating ASL height by inhibiting ENaC activity (Ganesan et al., 2013; Matsui et al., 1998). However, an alternative, opposing hypothesis exists, the “hypotonic hypothesis” which states “that normal airway epithelia are covered by an ASL with a [NaCl] sufficiently low (≤ 50 mM NaCl) to activate defensins and create an antimicrobial “shield” on airway surfaces”, and there is evidence to both support and refute it (Cowley et al., 1997; Goldman et al., 1997; Jayaraman et al., 2001; Knowles et al., 1997; Landry and Eidelman, 2001; Matsui et al., 1998; Tarran et al., 2001a; Tarran et al., 2001b; Verkman et al., 2003). Other studies suggest the involvement of additional ion channels such as alternative chloride channels (Grasemann et al., 2007) and cyclic nucleotide-gated cation channels, particularly in the alveolar epithelium (Schwiebert et al., 1997; Wilkinson et al., 2011) in the regulation of ASL height.
In addition, one study showing that instillation of Pseudomonas aeruginosa-laden agarose beads into excised swine tracheas significantly increased ASL height, and that this increase could be blocked by pre-incubation with the CFTR inhibitor CFTRinh172 (100 μM, 30 minutes) (Luan et al., 2014) presents an inconsistency with the available evidence presented here.

Quantitative Understanding of the Linkage

While there are convincing quantitative data in support of this KER, it becomes clear from the review of the evidence that the downstream KE can only be modulated to a certain extent (e.g., maximal decrease in ASL height did not exceed 50% in the majority of studies), independent of the extent of change in the upstream KE. In addition, the available temporal data indicates that acute exposures predominantly cause a transient, rather than a lasting change in the downstream KE. Since chronic treatment data are not available, we judge our quantitative understanding as being moderate.

Treatment of fully differentiated primary human bronchial epithelial cells (HBECs) with 2% cigarette smoke extract (CSE; bubbling 10 puffs of smoke from one 3R4F reference into 1 mL DMSO, at 2 s/10 mL puff, 10 puffs over 3 min; defined as 100%) for 20 minutes reduced CFTR channel activity by 50% and ASL by approx. 2-fold (Raju et al., 2016). 

Apical treatment of primary HBECs grown in monolayers with 2% CSE (not further described) for 24 h decreased forskolin-induced Cl⁻ currents by ca. 20% and ASL height by approx. 25%, and this could be counteracted by co-treatment with 10 µM ivacaftor, a CFTR potentiator known to significantly augment cAMP-mediated ion transport activity (Sloane et al., 2012). 

Exposure of primary HBECs to cigarette smoke (5 min, ca. 12 puffs at 1 puff every 30 s; generated according to ISO standards) resulted in efficient removal of CFTR from the plasma membrane and a ca. 2-fold reduction in ASL height (Xu et al., 2015).

Exposure of primary HBECs, differentiated at the air-liquid interface, to cigarette smoke from 1 cigarette (ten 35-mL puffs, 2R4F reference cigarette) nearly abolished responses of the transepithelial electric potential difference Vt to ADO (i.e., blocking the ADO-A2b-cAMP-CFTR- active ion transport) and significantly decreased ASL volume/height by approx. 2-fold after 30 min (Clunes et al., 2012).

Exposure of fully differentiated primary HBECs to 30 puffs of whole smoke from 2 cigarettes (generated according to ISO standards) every day for 5 days (120 h) resulted in a ca. 40% reduction in CFTR expression and approx. 50% reduction in ASL height (Hassan et al., 2014). 

Exposure of primary human airway epithelial cells grown in monolayers to whole smoke (3R4F reference cigarette; inExpose exposure system; 3L/min) resulted in significant reduction of CFTR Cl⁻ currents (ca. 40% for a 30-min exposure) and significantly decreased ASL depth from 11.4± 4.1 to 5.6± 2.0 µm (Lambert et al., 2014).

Exposure of fully differentiated primary HBECs to smoke from 1 cigarette or little cigar every day for 5 days (1 × 35 ml puff per 30 second, up to a butt length of 36 mm) significantly reduced CFTR protein expression by ca. 2- to 4-fold and ASL height by 10 to 20% (Ghosh et al., 2017). 

Cell surface CFTR protein expression was reduced by ca. 70% following exposure of baby hamster kidney cells expressing human CFTR (BHK^CFTR) to cigarette smoke for 10 min (3R4F reference cigarette, 1 puff per min according to ISO standards). This was accompanied by a significant reduction in ASL height by approx. 50% (Rasmussen et al., 2014).

Treatment of primary HBECs from a cystic fibrosis patient with the ΔF508 mutation, grown as monolayer at the air-liquid interface, with the CFTR corrector VX-809 for 48 h increased CFTR maturation by ca. 8-fold and enhanced Cl⁻ transport by approx. 4-fold, from 1.9±0.4 to 7.8±1.3 μA/cm². VX-809 treatment for 5 days increased ASL height from 4.5±0.2 to 6.7±0.5 μm. Addition of 3 μM VX-770 further increased the ASL height to 9.2±0.2 μm (Van Goor et al., 2011). Treatment of primary HBECs from a G551D/ΔF508 cystic fibrosis patient, grown as monolayer at the air-liquid interface, with the CFTR potentiator VX-770 (10 µM) for 72 h dose-dependently increased forskolin-mediated Cl⁻ currents by ca. 10-fold to 27±2 μA/cm² and ASL volume to 125% that of controls (Van Goor et al., 2009).

ASL depth in the excised tracheas of rats without functional CFTR expression was approx. half that of wild-type animals (Tuggle et al., 2014).

Knockdown of mRNAs for the α- and β-ENaC subunits resulted in a ca. 70% decrease in amiloride-sensitive currents and a significant increase in ASL height from 6.8±0.5 and 7.4±0.5 µm to 9.8±0.6 and 9.6±0.8 µm in non-CF and CF epithelia, respectively (Gianotti et al., 2013).

Knockdown of α-ENaC mRNA in BMI1-transduced cystic fibrosis bronchial epithelial cells resulted in ca. 50% reduction in protein expression, reduction in amiloride-sensitive short-circuit current from 11.5 (siRNA control) to 6.4 μA/cm² and increase in ASL height from 7.9 (siRNA control) to 12.1 μm (Tagalakis et al., 2018).

Overexpression of the β-ENaC subunit in mouse airways increased basal and amiloride-sensitive short-circuit currents approx. 2-fold (excised tracheas; compared to wild-type) and significantly reduced ASL height in bronchi and tracheas (by approx. 2 µm) (Mall et al., 2004).
 

Treatment of fully differentiated primary HBECs with 2% CSE (bubbling 10 puffs of smoke from one 3R4F reference into 1 mL DMSO, at 2 s/10 mL puff, 10 puffs over 3 minutes; defined as 100%) for 24 h decreased total CFTR expression and cell surface CFTR expression by approx. 20 and 25%, respectively, but treatment for 20 min did not. A 50% reduction in CFTR channel activity occurred immediately after addition of CSE and lasted for at least 20 minutes. A 2-fold reduction in ASL height was seen after 20 minutes, and ASL height was only partially restored at 1 h after CSE treatment  (Raju et al., 2016a).

Following exposure of primary HBECs, differentiated at the air-liquid interface, to cigarette smoke from 1 cigarette (ten 35-mL puffs, 2R4F reference cigarette), ASL volume/height was significantly decreased by approx. 2-fold after 30 min. This decrease lasted for >2.5 h, and ASL height was restored at 4 h post-exposure (Clunes et al., 2012).
Exposure of BHK^CFTR cells to cigarette smoke for 10 min (3R4F reference cigarette, 1 puff per min according to ISO standards) resulted in a reduction in ASL height by approx. 50% within 30 min; the decrease lasted for up to 1 h post-exposure (Rasmussen et al., 2014).

Exposure of fully differentiated primary human HBECs to 30 puffs of whole smoke from 2 cigarettes (generated according to ISO standards) was sufficient to decrease ASL height by approx. 50% within 1 h of exposure, and following daily exposure for another 4 days, ASL height remained at around this level (Hassan et al., 2014).

Exposure of fully differentiated primary HBECs to whole smoke from four 3R4F reference cigarettes (generated according to ISO standard 3308; Vitrocell VC10 exposure system) resulted in a small, non-significant increase in ASL volume 4 h post-exposure. ASL volume decreased to baseline levels 7 h post-exposure and continued to drop below baseline levels until 24 h post-exposure (Schmid et al., 2015).

ASL height of primary HBECs dropped within 30 min of exposure to cigarette smoke (5 min, ca. 12 puffs at 1 puff every 30 seconds). ASL height stayed at that reduced level up to until 70 min post-exposure (Xu et al., 2015).

The maximum effect of VX-809 treatment on Cl⁻ currents of primary human bronchial epithelial cells, grown as monolayer at the air-liquid interface, occurred following 24 h, and Cl– transport returned to uncorrected levels within 48 h of compound washout (concurrent ASL data not available) (Van Goor et al., 2011).
 

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Relationship: 2441: ASL Height, Decreased leads to CBF, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

The airway surface liquid (ASL) is a liquid layer on the apical side of the respiratory epithelium, reportedly between 5 to 100 μm in depth (Widdicombe and Widdicombe, 1995), and consists of an inner aqueous periciliary liquid layer (PCL) that spans the length of cilia and the outer gel-like mucus layer. Under physiological conditions, ASL composition and height are regulated via vectorial transport of electrolytes, driven by transepithelial transport and apical secretion of Cl⁻ by (predominantly) CFTR, resulting in passive H₂O secretion and, consequently, increased ASL height. Absorption of Na⁺ at the apical side by ENaC and ENaC’s interaction with the basolateral Na⁺/K⁺-ATPase exchanging Na⁺ for K⁺ leads to net absorption of Na⁺, which in turn drives fluid absorption and therefore decreases ASL height (Althaus, 2013; Hollenhorst et al., 2011). Decreased ASL height or ASL dehydration, if not rebalanced, results in cilia collapse and thereby effectively hinders coordinated ciliary beating (Knowles and Boucher, 2002; Matsui et al., 1998; Tarran et al., 2001)

Evidence Supporting this KER

Concurrent ASL height and CBF decreases were noted in human 3D airway epithelial cultures following exposure to cigarette smoke (Åstrand et al., 2014; Xu et al., 2015) and following the addition of large dextran molecules, low-melting point agarose or endogenous mucus (Button et al., 2012). Treatment of human airway epithelial with an ENaC inhibitor prevented the cigarette smoke effect on ASL height and CBF (Åstrand et al., 2014). In addition, treatment of cystic fibrosis airway cultures with a CFTR-modifying drug increased both ASL height and CBF (Van Goor et al., 2009). 

Boucher states that “Morphological studies of normal cultures under these steady-state conditions reveal that the 7 µm height is optimal for the extension and beating of cilia and, therefore, is physiologically suited to efficient mucociliary clearance.” (Boucher R., 2003). The link between decreased ASL height and reduced cilia beating has been established in multiple in vitro and in vivo studies (Van Goor et al., 2009; Xu et al., 2015; Zhang et al., 2014), and even though the evidence does not describe causality between these two events, this KER is biologically plausible (Button et al., 2012; Mall, 2008).

When increasing the osmotic modulus in human bronchial epithelial cells (differentiated at the air-liquid interface) by treatment with large dextran molecules, low-melting point agarose or endogenous mucus, ASL height decreased and cilia collapsed. Although the cilia were still beating, they did not do so at their full height (Button et al., 2012).

Although the empirical evidence suggests a link between decreased ASL height and reduced cilia beating, causality between the two KEs has not been proven nor has this KER been systematically examined or quantified yet.

Quantitative Understanding of the Linkage

The evidence provided here stems from studies reporting on the effects of stressors such as cigarette smoke on both ASL height and CBF. Although the empirical evidence suggests a link between decreased ASL height and reduced ciliary beating, causality between the two KEs has not been proven nor has this KER been systematically examined or quantified yet. Our quantitative understanding of this KER is therefore poor (weak).

Osmotic compression of the ASL between 300 and 800 Pa (using large dextran molecules, endogenous mucin or low-melting point agarose) had minimal effects on cilia height or cilia beating. At osmotic pressures exceeding 800 Pa, the ASL became compressed from 7 to less than 2 µm, and cilia height decreased to approx. the same extent (consequently, cilia were not beating at full height) (Button et al., 2012).

Exposure of primary human airway epithelial cells to cigarette smoke (5 min, ca. 12 puffs at 1 puff every 30 seconds) resulted in a ca. 2-fold reduction in ASL height. CBF decreased from 4.19 ± 0.24 Hz (in air control) to 1.28 ± 0.06 Hz. Replenishment of the ASL by addition of 50 µL PBS restored CBF in air- and smoke-exposed cultures (6.04 ± 0.3 Hz vs 6.82 ± 0.37 Hz) (Xu et al., 2015).

Treatment of murine nasal septal epithelia with Sinupret, a phytomedicine, significantly increased ASL depth from 5.25±0.38 to 9.14±0.42 µm and increased the mean CBF from 1.52±0.10 to 2.05±0.15 when applied apically, from 0.99±0.04 to 1.37±0.09 when applied basally, and from 1.53±0.09 to 2.17±0.12 when applied to both compartments  (Zhang et al., 2014).

An experimental compound targeting ENaC termed “compound A” dose-dependently increased ASL height in ASL-depleted cultures (absorptive mode analysis). Following exposure to cigarette smoke (1 2R4F cigarette, ISO smoking regimen), ASL decreased by approx. 4 µm within 30 min compared to air controls, and this could be prevented by a 2.5-h pre-treatment with 1 µM compound A. In the same cultures, CBF was significantly decreased by more than 1 Hz following cigarette smoke exposure, whereas pre-treatment with compound A completely prevented this (Åstrand et al., 2014).

ASL height of primary human airway epithelial cells dropped within 30 min of exposure to cigarette smoke (5 min, ca. 12 puffs at 1 puff every 30 s). ASL height stayed at that reduced level up until 70 min post-exposure. Significant decreases in CBF in cigarette smoke-exposed cultures were seen 3 h post-exposure (Xu et al., 2015).

Treatment of primary human bronchial epithelial cells from a cystic fibrosis patient with the G551D/ΔF508 genotype, grown as monolayer at the air-liquid interface, with the CFTR potentiator VX-770 (10 µM) for 72 h increased ASL height by approx. 25%, and treatment for 5 days more than doubled CBF (Van Goor et al., 2009).

Exposure of human bronchial epithelial cells to cigarette smoke decreased ASL height by approx. 4 µm within 30 min, whereas pre-treatment with 1 µM compound A prevented this decrease. When compound A was added 30 min after exposure to cigarette smoke, ASL height returned to normal levels significantly more quickly. In the same exposed cultures CBF was decreased by more than 1 Hz within 1 h (Åstrand et al., 2014).

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References

• Althaus, M. (2013). ENaC inhibitors and airway re-hydration in cystic fibrosis: state of the art. Curr. Mol. Pharmacol. 6(1), 3-12.
• Åstrand, A.B., Hemmerling, M., Root, J., Wingren, C., Pesic, J., Johansson, E., et al. (2014). Linking increased airway hydration, ciliary beating, and mucociliary clearance through ENaC inhibition. Am. J. Physiol. Lung Cell. Mol. Physiol. 308(1), L22-L32.
• Boucher, R. (2003). Regulation of airway surface liquid volume by human airway epithelia. Pflügers Arch. 445(4), 495-498. 
• Button, B., Cai, L.-H., Ehre, C., Kesimer, M., Hill, D.B., Sheehan, J.K., et al. (2012). A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia. Science 337(6097), 937-941. 
• Hollenhorst, M.I., Richter, K., and Fronius, M. (2011). Ion transport by pulmonary epithelia. Biomed. Res. Int. 2011(2011), 174306.
• Knowles, M.R., and Boucher, R.C. (2002). Mucus clearance as a primary innate defense mechanism for mammalian airways. J. Clin. Investig. 109(5), 571-577.
• Mall, M.A. (2008). Role of cilia, mucus, and airway surface liquid in mucociliary dysfunction: lessons from mouse models. J. Aerosol Med. Pulm. Drug Deliv. 21(1), 13-24.
• Matsui, H., Grubb, B.R., Tarran, R., Randell, S.H., Gatzy, J.T., Davis, C.W., et al. (1998). Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95(7), 1005-1015.
• Tarran, R., Grubb, B., Parsons, D., Picher, M., Hirsh, A., Davis, C., et al. (2001). The CF salt controversy: in vivo observations and therapeutic approaches. Mol. Cell 8(1), 149-158.
• Van Goor, F., Hadida, S., Grootenhuis, P.D.J., Burton, B., Cao, D., Neuberger, T., et al. (2009). Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc. Natl. Acad. Sci. U. S. A. 106(44), 18825-18830. 
• Widdicombe, J., and Widdicombe, J. (1995). Regulation of human airway surface liquid. Respir. Physiol. 99(1), 3-12.
• Xu, X., Balsiger, R., Tyrrell, J., Boyaka, P.N., Tarran, R., and Cormet-Boyaka, E. (2015). Cigarette smoke exposure reveals a novel role for the MEK/ERK1/2 MAPK pathway in regulation of CFTR. Biochim. Biophys. Acta 1850(6), 1224-1232.
• Zhang, S., Skinner, D., Hicks, S.B., Bevensee, M.O., Sorscher, E.J., Lazrak, A., et al. (2014). Sinupret Activates CFTR and TMEM16A-Dependent Transepithelial Chloride Transport and Improves Indicators of Mucociliary Clearance. PloS one 9(8), e104090. doi: 10.1371/journal.pone.0104090.

Relationship: 2442: CBF, Decreased leads to MCC, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

Synchronized ciliary action transports mucus from the distal lung to the mouth, where it is swallowed or expectorated (Munkholm and Mortensen, 2014). In addition to ASL and mucus properties, the speed of ciliary movement, and hence the effectiveness of mucociliary clearance (MCC), is dependent on ciliary amplitude and beat frequency (Rubin, 2002). CBF itself is influenced by several factors, including changes in the physical and chemical properties of the ASL (especially the periciliary fluid), structural modulation in the cilia, concentration of cyclic nucleotides cAMP and cGMP, and intracellular calcium (Ca²⁺). Aside from genetic defects leading to ciliopathies, there is ample evidence for prolonged exposure to noxious agents, such as cigarette smoke, nitrogen oxide and sulfur dioxide, playing a major role in decreasing CBF and hampering efficient MCC.

Evidence Supporting this KER

A decrease in CBF resulting from sulfur dioxide exposure reduced mucociliary clearance in dogs (Yeates et al., 1997) and mucociliary activity in guinea pig tracheas (Knorst et al., 1994). In rats, formaldehyde inhalation exposure resulted in lower numbers of ciliated cells, while ciliary activity and mucus flow rates were decreased in a dose and time-dependent manner (Morgan et al., 1986). In humans, CBF positively correlates with nasal mucociliary clearance time (Ho et al., 2001), and bronchiectasis patients have lower nasal CBF and slower mucociliary transport (MCT) (Rutland and Cole, 1981). Administration of nebulized CBF inhibitors and enhancers quantifiably decreased or increased mucociliary clearance, respectively (Boek et al., 1999; Boek et al., 2002). Increased CBF and MCT was also noted in human sinonasal epithelial cell cultures treated with Myrtol®, an essential oil distillate (Lai et al., 2014) and in sheep tracheas and human airway epithelial cultures subjected to temperature changes (Kilgour et al., 2004; Sears et al., 2015). Exposures of frog palate epithelia to formaldehyde and PM10 reduced MCC and mucociliary transport, but only formaldehyde-treated epithelia showed decreases in CBF (Morgan et al., 1984; Macchione et al., 1999; Fló-Neyret et al., 2001).
Ex vivo treatment of sheep trachea with acetylcholine and epinephrine increased CBF, but only acetylcholine increased surface liquid velocity, while both parameters were decreased upon incubation with platelet-activating factor (Seybold et al., 1990). 

Ciliary function and mucus transport are invariably linked to effective mucus transport along the mucociliary escalator. Studies in animal models of ciliopathies and in individuals with genetic disorders causing cilia defects demonstrate that absent or asynchronous cilia beating results in defective mucus clearance from the lungs, consequently leading to respiratory infections that may be chronic recurrent in nature and ultimately lead to declining lung function (Knowles et al., 2013; Munkholm and Mortensen, 2014; Tilley et al., 2015). Similarly, indirect effects of airway inflammation, caused for example by respiratory infections or allergies, are known to be responsible for changes in cilia beating and hence mucus clearance (Almeida-Reis et al., 2010; Hisamatsu and Nakajima, 2000; Maurer et al., 1982). Finally, airway epithelial injury following exposure to inhalation toxicants can also damage cilia and inhibit cilia function and thereby impair MCC (Iravani and Van As, 1972; Wanner et al., 1996). Therefore, this KER is biologically plausible. 

A decrease in CBF resulting from sulfur dioxide exposure reduced mucociliary clearance in dogs (Yeates et al., 1997) and mucociliary activity in guinea pig tracheas (Knorst et al., 1994). In rats, formaldehyde inhalation exposure resulted in lower numbers of ciliated cells, while ciliary activity and mucus flow rates were decreased in a dose and time-dependent manner (Morgan et al., 1986). In humans, CBF positively correlates with nasal mucociliary clearance time (Ho et al., 2001), and bronchiectasis patients have lower nasal CBF and slower mucociliary transport (MCT) (Rutland and Cole, 1981). Administration of nebulized CBF inhibitors and enhancers quantifiably decreased or increased mucociliary clearance, respectively (Boek et al., 1999; Boek et al., 2002). Increased CBF and MCT was also noted in human sinonasal epithelial cell cultures treated with Myrtol®, an essential oil distillate (Lai et al., 2014) and in sheep tracheas and human airway epithelial cultures subjected to temperature changes (Kilgour et al., 2004; Sears et al., 2015). Exposures of frog palate epithelia to formaldehyde and PM10 reduced MCC and mucociliary transport, but only formaldehyde-treated epithelia showed decreases in CBF (Morgan et al., 1984; Macchione et al., 1999; Fló-Neyret et al., 2001).

The available evidence does not interrogate the direct relationship between CBF and MCC, but rather evaluates both outcomes in parallel. However, because of the intrinsic linkage of cilia function and MCC, we find the empirical evidence in support of this KER to be moderate.
Ex vivo treatment of sheep trachea with acetylcholine and epinephrine increased CBF, but only acetylcholine increased surface liquid velocity, while both parameters were decreased upon incubation with platelet-activating factor (Seybold et al., 1990). 

Although ciliary function is considered a primary determinant for effective MCC (Duchateau et al., 1985; Gizurarson, 2015), there is evidence that suggests that MCC can be impeded by other factors that do not affect CBF. For example, nasal CBF in cigarette smokers regularly exhaling through the nose was not significantly different from that of nonsmokers, although they exhibited significantly longer nasomuciliary clearance times compared to nonsmokers. Possible explanations offered for this discrepancy were a potential loss of cilia in the nasal epithelium or increased mucus viscoelasticity (Stanley et al., 1986). Similarly, formaldehyde exposure of rats resulted in decreased cilia numbers and slower mucus flow rates (Morgan KT et al., 1986). On the other hand, there are a number of pharmacological compounds that improve mucociliary clearance through reductions in mucus viscosity, but have no effect on CBF (Jiao and Zhang, 2019), or through increases in CBF, but have no effect on mucociliary clearance (Phillips et al., 1990).

Quantitative Understanding of the Linkage

There are several studies providing insights into the negative effect of inhalation exposures on CBF and MCC, that are in line with the current thinking on how these two KEs connect. Additionally, pharmacological studies demonstrated that stimulation of CBF typically results in stimulation of MCC. However, since most studies usually evaluated the KEs in parallel, and even though some results support both dose response and temporal sequence of the KEs, none of the available data affirms causal linkage between CBF and MCC. Our understanding of the evidence is therefore moderate. 

CBF decreased sequentially with increasing SO₂ doses in dogs. CBF decreased from 6.3 ± 0.2 (SE) Hz at baseline to 5.7 ± 0.2 Hz at 5.5 ppm SO₂. Five ppm SO₂ delivered to both the trachea and tracheobronchial airways for 20 min also caused a marked decrease in mean bronchial mucociliary clearance from 53.7 ± 5.7% to 32.8 ± 7.7% after 90 min (Yeates et al., 1997).

The effects of 30-min exposure to SO₂ on mucociliary activity (MCA) and ciliary beat frequency (CBF) were studied in 31 guinea pig tracheas. A 63% reduction in mean MCA and statistically insignificant changes in CBF were recorded at concentrations of 2.5 ppm SO₂. Higher SO₂ concentrations caused further impairment of MCA as well as a dose-dependent decrease in CBF: At 5 ppm SO2, CBF decreased by 45%, at 12.5 ppm by 72%. The maximum decrease in MCA (81%) was observed with 7.5 ppm SO₂; the highest SO₂ concentration did not decrease MCA further. The decrease in MCA was associated with an impairment of CBF only at SO₂ concentrations ≥5.0 ppm (Knorst et al., 1994b).

Administration of a nebulized CBF inhibitor (0.9% NaCl) to 15 healthy volunteers significantly decreased mucociliary transport (MCT) from 7.9±1.5 mm/min (SEM) to 4.5±1.6 mm/min. Salbutamol, a CBF enhancer, significantly increased MCT from 8.0±1.4 to 12.5±1.1 mm/min (Boek et al., 2002; Boek et al., 1999).

Cooling human airway epithelial cultures grown at the air-liquid interface from 37°C to 25°C over the course of approx. 20 min decreased CBF from 12 to 6 Hz and mucociliary transport (MCT) from 140 to 90 µm/s. Extending the range of temperature tested, CBF was found to increase by 0.49±0.06 Hz for every temperature increase by 1°C, and this was mirrored by an increase in MCT. MCT increased on average between 5 and 11 µm/s for every Hz increase in CBF. This study also showed that CBF decreased with increasing mucin concentration, dropping from 12.4 Hz at 2% bovine submaxillary mucin (BSM) to 10.1 Hz at 8% BSM, concurrent with a ca. 70% reduction in MCT. In addition, treatment with 10 µM basolateral forskolin reproducibly increased CBF by 19.3±2.1% and MCT by 24.4±3.1% over baseline (Sears et al., 2015). In sheep trachea CBF and mucus transport velocity (MTV) were 9.8±2.7 beats/s and 5.7±2.6 mm/min, respectively, at baseline. Temperature reductions from 37°C to 34°C caused a progressive decline in CBF (ca. –20% at 2 h and –90% at 4 h) and MTV (ca. –50% at 2 h and –90% at 4 h), which was further exacerbated by additional temperature decreases (30°C; CBF: ca. –75% at 2 h; MTV: –80% at 2 h) (Kilgour et al., 2004).

Frog palate preparations were incubated with 1.25, 2.5 and 5.0 ppm formaldehyde. At formaldehyde doses of 2.5 and 5 ppm, CBF decreased by ca. 25% compared to baseline within 30 min and by 35-50% within 60 min (Fló-Neyret et al., 2001).
Incubation of frog palates with PM10 from Sao Paolo, Brazil, for up to 120 min did not affect CBF but decreased MCT at concentrations ≥1000 pg/m3 (Macchione et al., 1999)
In freshly excised sheep tracheas, a 60-min incubation with 10 µM platelet-activating factor caused a 6% decrease in CBF and a dose-dependent decrease in surface liquid velocity, reaching a maximum of 63% (Seybold et al., 1990).

In patients with bronchiectasis, nasal CBF was 12.8±1.3 Hz and nasal clearance time was 31.8± 18.4 min. In comparison, in healthy controls, nasal CBF was 14.0±1.3 Hz and nasal clearance time was 17.6± 8.3 min (Rutland and Cole, 1981).

Following basolateral treatment of human sinonasal epithelial cell cultures grown at the air-liquid interface with  Myrtol®, a phytopharmaceutical mixture of distillates of rectified essential oils of eucalyptus, sweet orange, myrtle, and lemon as the active ingredients, increased CBF in a dose-dependent manner, with a maximum stimulation with 0.1% of 48±7% after 30 min. The same concentration caused a 46±16% increase in MCT at 40 min (Lai et al., 2014).

In New Zealand white rabbits exposed to 3 ppm NO₂ for 24 h, the average CBF decreased from 764 beats/min to 692 beats/min, and the transport velocity decreased from 5.23 mm/min to 3.03 mm/min (Kakinoki, 1998).

A 20-minute exposure of dogs to SO₂ caused a decrease in mean bronchial MCC after 90 min (Yeates et al., 1997).

Frog palate epithelia were incubated with 1.25, 2.5 and 5.0 ppm formaldehyde. At formaldehyde doses of 2.5 and 5 ppm, CBF decreased by ca. 25% compared to baseline within 30 min and by 35-50% within 60 min (Fló-Neyret et al., 2001).

Incubation of freshly excised sheep tracheas with 10 µM platelet-activating factor caused a maximal decrease in CBF of 6% after 60 min and decrease in surface liquid velocity of ca. 30% at 20 min, ca. 50% at 40 min and 63% after 60 min (Seybold et al., 1990).

Following basolateral treatment of human sinonasal epithelial cell cultures grown at the air-liquid interface with different concentrations of Myrtol®, CBF increased rapidly within the first 30 min and then declined thereafter. The maximum response for MCT was seen after 40 min (Lai et al., 2014).

Physiological factors such as age, sex, posture, sleep, and exercise were shown to affect MCC, although study findings are not always concordant (Houtmeyers et al., 1999). MCC and CBF, for example, were observed to decrease with age in several species in numerous studies (e.g. guinea pigs, mice, and human) (Bailey et al., 2014; Grubb et al., 2016; Ho et al., 2001; Joki and Saano, 1997; Paul et al., 2013; Yager et al., 1978), but evidence by (Agius et al., 1998) suggests that age does not have a major effect on CBF.

Unknown

References

• Agius, A.M., Smallman, L.A., and Pahor, A.L. (1998). Age, smoking and nasal ciliary beat frequency. Clin. Otolaryngol. Allied Sci. 23(3), 227-230. 
• Almeida-Reis, R., Toledo, A.C., Reis, F.G., Marques, R.H., Prado, C.M., Dolhnikoff, M., et al. (2010). Repeated stress reduces mucociliary clearance in animals with chronic allergic airway inflammation. Respiratory physiology & neurobiology 173(1), 79-85.
• Bailey, K.L., Bonasera, S.J., Wilderdyke, M., Hanisch, B.W., Pavlik, J.A., DeVasure, J., et al. (2014). Aging causes a slowing in ciliary beat frequency, mediated by PKCε. American journal of physiology. Lung cellular and molecular physiology 306(6), L584-L589. doi: 10.1152/ajplung.00175.2013.
• Boek, W.M., Graamans, K., Natzijl, H., van Rijk, P.P., and Huizing, E.H. (2002). Nasal Mucociliary Transport: New Evidence for a Key Role of Ciliary Beat Frequency. Laryngoscope 112(3), 570-573. 
• Boek, W.M., Keleş, N., Graamans, K., and Huizing, E.H. (1999). Physiologic and hypertonic saline solutions impair ciliary activity in vitro. Laryngoscope 109(3), 396-399.
• Duchateau, G.S., Merkus, F.W., Zuidema, J., and Graamans, K. (1985). Correlation between nasal ciliary beat frequency and mucus transport rate in volunteers. The Laryngoscope 95(7), 854-859.
• Fló-Neyret, C., Lorenzi-Filho, G., Macchione, M., Garcia, M.L.B., and Saldiva, P.H.N. (2001). Effects of formaldehyde on the frog's mucociliary epithelium as a surrogate to evaluate air pollution effects on the respiratory epithelium. Braz. J. Med. Biol. Res. 34, 639-643.
• Gizurarson, S. (2015). The effect of cilia and the mucociliary clearance on successful drug delivery. Biological and Pharmaceutical Bulletin, b14-00398.
• Grubb, B.R., Livraghi-Butrico, A., Rogers, T.D., Yin, W., Button, B., and Ostrowski, L.E. (2016). Reduced mucociliary clearance in old mice is associated with a decrease in Muc5b mucin. American journal of physiology. Lung cellular and molecular physiology 310(9), L860-L867.
• Hisamatsu, K.-i., and Nakajima, M. (2000). Pranlukast protects leukotriene C4- and D4-induced epithelial cell impairment of the nasal mucosa in vitro. Life Sciences 67(22), 2767-2773. 
• Ho, J.C., Chan, K.N., Hu, W.H., Lam, W.K., Zheng, L., Tipoe, G.L., et al. (2001). The Effect of Aging on Nasal Mucociliary Clearance, Beat Frequency, and Ultrastructure of Respiratory Cilia. Am. J. Respir. Crit. Care Med. 163(4), 983-988. 
• Houtmeyers, E., Gosselink, R., Gayan-Ramirez, G., and Decramer, M. (1999). Regulation of mucociliary clearance in health and disease. European Respiratory Journal 13(5), 1177-1188.
• Iravani, J., and Van As, A. (1972). Mucus transport in the tracheobronchial tree of normal and bronchitic rats. The Journal of pathology 106(2), 81-93.
• Jiao, J., and Zhang, L. (2019). Influence of Intranasal Drugs on Human Nasal Mucociliary Clearance and Ciliary Beat Frequency. Allergy Asthma Immunol Res 11(3), 306-319. 
• Joki, S., and Saano, V. (1997). Influence of ageing on ciliary beat frequency and on ciliary response to leukotriene D4 in guinea‐pig tracheal epithelium. Clinical and experimental pharmacology and physiology 24(2), 166-169.
• Kakinoki, Y.O., Ayaki Tanaka, Yushi Washio, Koji Yamada, Yoshiaki Nakai, Kazuhiro Morimoto, Yasushi (1998). Nitrogen dioxide compromises defence functions of the airway epithelium. Acta Otolaryngol. 118(538), 221-226.
• Kilgour, E., Rankin, N., Ryan, S., and Pack, R. (2004). Mucociliary function deteriorates in the clinical range of inspired air temperature and humidity. Intensive Care Med. 30(7), 1491-1494.
• Knorst, M.M., Kienast, K., Riechelmann, H., Müller-Quernheim, J., and Ferlinz, R. (1994). Effect of sulfur dioxide on mucociliary activity and ciliary beat frequency in guinea pig trachea. Int. Arch. Occup. Environ. Health 65(5), 325-328.
• Knowles, M.R., Daniels, L.A., Davis, S.D., Zariwala, M.A., and Leigh, M.W. (2013). Primary ciliary dyskinesia. Recent advances in diagnostics, genetics, and characterization of clinical disease. American journal of respiratory and critical care medicine 188(8), 913-922.
• Lai, Y., Dilidaer, D., Chen, B., Xu, G., Shi, J., Lee, R.J., et al. (2014). In vitro studies of a distillate of rectified essential oils on sinonasal components of mucociliary clearance. Am. J. Rhinol. Allergy 28(3), 244-248.
• Macchione, M., Oliveira, A.P., Gallafrio, C.T., Muchão, F.P., Obara, M.T., Guimarães, E.T., et al. (1999). Acute effects of inhalable particles on the frog palate mucociliary epithelium. Environ. Health Perspect. 107(10), 829-833.
• Maurer, D., Sielczak, M., Oliver Jr, W., Abraham, W., and Wanner, A. (1982). Role of ciliary motility in acute allergic mucociliary dysfunction. Journal of Applied Physiology 52(4), 1018-1023.
• Morgan, K., Patterson, D., and Gross, E. (1986). Responses of the nasal mucociliary apparatus of F-344 rats to formaldehyde gas. Toxicol. Appl. Pharmacol. 82(1), 1-13.
• Munkholm, M., and Mortensen, J. (2014). Mucociliary clearance: pathophysiological aspects. Clin. Physiol. Funct. Imaging 34(3), 171-177.
• Paul, P., Johnson, P., Ramaswamy, P., Ramadoss, S., Geetha, B., and Subhashini, A.S. (2013). The Effect of Ageing on Nasal Mucociliary Clearance in Women: A Pilot Study. ISRN Pulmonology 2013, 5. 
• Phillips, P.P., McCaffrey, T.V., and Kern, E.B. (1990). The in vivo and in vitro effect of phenylephrine (Neo Synephrine) on nasal ciliary beat frequency and mucociliary transport. Otolaryngology—Head and Neck Surgery 103(4), 558-565.
• Rubin, B.K. (2007). Mucus structure and properties in cystic fibrosis. Paediatr Respir Rev 8(1), 4-7. 
• Rutland, J., and Cole, P.J. (1981). Nasal mucociliary clearance and ciliary beat frequency in cystic fibrosis compared with sinusitis and bronchiectasis. Thorax 36(9), 654-658.
• Sears, P.R., Yin, W.-N., and Ostrowski, L.E. (2015). Continuous mucociliary transport by primary human airway epithelial cells in vitro. Am. J. Physiol. Lung Cell. Mol. Physiol. 309(2), L99-L108. 
• Seybold, Z.V., Mariassy, A.T., Stroh, D., Kim, C.S., Gazeroglu, H., and Wanner, A. (1990). Mucociliary interaction in vitro: effects of physiological and inflammatory stimuli. J. Appl. Physiol. 68(4), 1421-1426. doi: 10.1152/jappl.1990.68.4.1421.
• Stanley, P., Wilson, R., Greenstone, M., MacWilliam, L., and Cole, P. (1986). Effect of cigarette smoking on nasal mucociliary clearance and ciliary beat frequency. Thorax 41(7), 519-523.
• Tilley, A.E., Walters, M.S., Shaykhiev, R., and Crystal, R.G. (2015). Cilia dysfunction in lung disease. Annu Rev Physiol 77, 379-406. 
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• Yager, J., Chen, T.-M., and Dulfano, M.J. (1978). Measurement of frequency of ciliary beats of human respiratory epithelium. Chest 73(5), 627-633.
• Yeates, D.B., Katwala, S.P., Daugird, J., Daza, A.V., and Wong, L.B. (1997). Excitatory and inhibitory neural regulation of tracheal ciliary beat frequency (CBF) activated by ammonia vapour and SO2. Ann. Occup. Hyg. 41, 736-744.

Relationship: 2443: MCC, Decreased leads to Decreased lung function

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

It is very well known that patients suffering from motile ciliopathies, such as primary ciliary dyskinesia, have impaired or absent MCC and lower lung function (reduced FEV1 and FVC) compared to their healthy counterparts (Halbeisen et al., 2018; Marthin et al., 2010; Wallmeier et al., 2020). In cystic fibrosis patients, MCC is impaired by two main mechanisms: 1) decreased airway hydration and 2) changes in mucus chemical and viscoelastic properties. This causes mucus build-up which can lead to mucus plugging in the airways and consequently leads to decreased lung function over time (Kerem et al., 2014; Mossberg et al., 1978; Regnis et al., 1994; Robinson and Bye, 2002; Szczesniak et al., 2017; Wanner et al., 1996). Mucus plugging due to decreased MCC is also considered a major cause of airway obstruction and airflow limitation in COPD patients (Dunican et al., 2021; Okajima et al., 2020) and asthmatics (Kuyper et al., 2003; Maxwell, 1985).

Evidence Supporting this KER

Changes in MCC rate are typically paralleled by effects on lung function in several studies where both endpoints have been assessed. In patients with primary ciliary dyskinesia, absence of cilia motion prevents normal MCC and consequently, lung function is reduced (Denizoglu Kulli et al., 2020). In cystic fibrosis patients, the ASL is depleted resulting in impaired MCC (Boucher, 2004). Although the known CFTR genotypes can result in a variety of phenotypes (Derichs, 2013), clinical data indicate that some specific gene defects, such as the p.Phe508del variant, are more frequently associated with decreased lung function indices (e.g. FEV1 % predicted, FVC % predicted, FEF25-75) (Kerem et al., 1990; Johansen et al., 1991; Schaedel et al., 2002). Both cigarette smoking and occupational exposure to biomass fumes led to slower MCC and reduced FEV1 % predicted and FEV1/FVC (Ferreira et al., 2018). Nasomucociliary clearance was slower in COPD smokers compared to former smokers with COPD or to nonsmokers (Ito et al., 2015). Allergen challenge in asthma patients resulted in both reduced MCC and FEV1, which could be reversed by inhalation of hypertonic saline solution (Alexis et al., 2017). In cystic fibrosis patients, treatment with mucolytic agents (Laube et al., 1996; McCoy et al., 1996; Quan et al., 2001; Elkins et al., 2006; Amin et al., 2011; Donaldson et al., 2018) or a CFTR potentiator (Rowe et al., 2014) improved both MCC and lung function (FEV1, FVC and FEF25-75).

Lung function is known to decrease with age, and several studies showed that mucus transport rates also decrease in older compared to younger individuals (Goodman et al., 1978; Uzeloto et al., 2021). Impaired MCC is also seen in chronic smokers, even prior to a clinically significant drop in lung function and the detection of small airway disease (Clunes et al., 2012a; Goodman et al., 1978; Lourenço et al., 1971; Uzeloto et al., 2021; Vastag et al., 1986), and in patients with obstructive lung disease and hence, poor lung function (Cruz et al., 1974; Vastag et al., 1986). Adult asthmatics also displayed decreased mucus transport rates/velocities in addition to decreased lung function (Ahmed et al., 1981; Bateman et al., 1983; Foster et al., 1982; Mezey et al., 1978).
In patients with primary ciliary dyskinesia, absence of cilia motion prevents normal MCC and consequently, lung function is reduced (Denizoglu Kulli et al., 2020). In cystic fibrosis patients, the ASL is depleted resulting in impaired MCC (Boucher, 2004a). Although the known CFTR genotypes can result in a variety of phenotypes (Derichs, 2013), clinical data indicate that some specific gene defects, such as the p.Phe508del variant, are more frequently associated with decreased lung function indices (e.g. FEV1 % predicted, FVC % predicted, FEF25-75) (Johansen et al., 1991; Kerem et al., 1990; Schaedel et al., 2002). Unsurprisingly, results from studies with pharmacological agents aimed at restoring CFTR function do not only indicate enhanced MCC but also support improvements in lung function (Bennett et al., 2018; Donaldson et al., 2018; Rowe S. M. et al., 2014a). While the available data link these two KEs, causal evidence is not always available, and some inference is present. Therefore, we judge the biological plausibility of this KER as moderate.

Occupational exposure to biomass combustion products resulted in slower MCC and reduced FEV1 % predicted and FEV1/FVC (Ferreira et al., 2018).

Compared to healthy controls, current smokers without airway obstruction and current smokers with COPD exhibited longer saccharin transit times, indicative of impaired MCC, and lower FEV1 % predicted and FEV1/FVC (Uzeloto et al., 2021). Similarly, nasomucociliary clearance was slower in COPD smokers compared to former smokers with COPD or to nonsmokers (Ito et al., 2015). Additionally, mucus plug density—assessed by CT imaging—and mucoid (rather than watery) consistency were inversely related to FEF25–75% and associated with increased RV/TLV (Kesimer et al., 2018).

Asthma patients responded to allergen challenge with a reduction in both MCC and FEV1 (Bennett et al., 2011; Mezey et al., 1978), which could be rescued by inhalation with hypertonic saline solution (Alexis et al., 2017).

Multiple studies interrogating the effect of mucolytic agents such as hypertonic saline solution or recombinant DNase on mucus transport rates or mucus clearance in patients with cystic fibrosis report improvements in both, mucus transport velocities or rates and lung function indices, including FEV1, FVC and FEF25-75 (Amin et al., 2011; Donaldson et al., 2018; Elkins et al., 2006; Laube et al., 1996; McCoy et al., 1996; Quan et al., 2001).

Both MCC and lung function (FEV1, FVC and FEF25-75) improved in cystic fibrosis patients treated with ivacaftor, a CFTR potentiator that increases the channel open probability (Rowe et al., 2014b).

Some studies with mucolytics such as N-acetylcysteine, bromhexine, theophylline/ambroxol or sorebrol demonstrated improved MCC was connected with small improvements in lung function (FEV1, FVC and FEV1/FVC) in patients with chronic bronchitis (Aylward et al., 1980; Castigiioni and Gramolini, 1986; Thomson et al., 1974; Würtemberger et al., 1988).
 

Genetic defects leading to motile ciliopathies or defects in CFTR function are linked to impaired MCC. However, because of the genetic variety, not every defect, for example in the CFTR gene, also expresses an overt pulmonary phenotype. Other factors, such as low-level chronic inflammation may drive lung pathology by pathways independent of MCC. This might also explain the absence of differences in MCC between healthy smokers and smokers with COPD (Fleming et al., 2019).
Not all studies looking to elucidate the effect of mucolytics on MCC report an improvement of lung function, even though mucus transport rates or tracheobronchial clearance significantly improve. These studies include, for example, some on the effects of hypertonic saline solution, NAC, ambroxol and 2-mercapto-ethane sulphonate (Clarke et al., 1979; Ericsson et al., 1987; Millar et al., 1985; Robinson et al., 1997; Würtemberger et al., 1988). This could be, at least in part, related to the fact that a sudden drop in lung function served as an indicator of patient distress in these studies, and interventions were halted when they occurred to ensure patient safety (Robinson et al., 1996). Another reason could be related to the mechanisms underlying mucus solubilization that may be completely independent of lung function.
MCC is only one means by which mucus can be cleared from the lungs. Another one is cough clearance, and it is highly dependent on the properties of the ASL, in particular the ASL height (Knowles and Boucher, 2002).

Quantitative Understanding of the Linkage

The available data, though not causally linking decreases in MCC with decreased lung function, provide a good insight into the importance of the physiological role of MCC in maintaining normal lung function. In at least some studies, impairment of MCC correlated with the drop in FEV1 or FEF25-75. Although clinically valuable benefits can be seen in studies with pharmacological agents such as mucolytics and CFTR modifying drugs, they do not cover a wide range of dose responses nor are they supportive of the KER causality. Therefore, we judge our quantitative understanding as moderate.

Sixteen Brazilian sugarcane workers aged 25±4 years, with a BMI of 24±3 kg/m2, with exhaled CO of 2.1±1.5 ppm, were examined during the non-harvest season and during the sugarcane burning harvest season. There was a non-significant decrease in saccharin transit time (from 8±1 min to 3±1 min) and a significant decrease  FEV1/FVC ratio (from 88.62±5.68 to 84.90±6.47) and %FEV1 (from 92.19±13.24 to 90.44±12.76) during harvest compared with the non-harvest season (Ferreira et al., 2018).

12 (6M/6F) mild allergic, non-smoking asthmatics ages 20–39 with skin sensitivity to house dust mites (HDM) and normal baseline lung function (FEV1 %pred > 80, FEV1/FVC ratio >0.70) inhaled sequential doses of inhaled HDM extract (D. farinae, Greer®, Lenoir, NC) delivered as 5 inhalations from a Devilbiss 646 nebulizer (mass median aerodynamic diameter of 5 um, GSD = 2.0). Five of the 12 patients responded to the allergen challenge with >10% reductions in FEV1 % predicted and reduction in whole lung MCC as evidenced by increased retention rates (mean Central TB Ave120Ret increased from 0.69 to 0.79 for baseline vs. allergen challenge respectively). This reduction in MCC significantly correlated with the post challenge 24 hour FEV1 (Bennett et al., 2011).

Treatment of patients with chronic bronchitis with bromhexine (3 x 16 mg/day) for 14 days resulted in mean changes in FEV1, FVC and FEV1/FVC of + 0.047 L + 0.033 L and +0.6%, respectively, with MCC at 6 h being 6.8% greater after treatment compared to baseline (Thomson et al., 1974).

Treatment of patients with chronic bronchitis with ambroxol alone (2 x 30 mg/day) or with theophyllin (2 x 400 mg/day) and ambroxol (2 x 30 mg/day) for 7 days MCC/h improved from 18.3 ± 11.1% to 23.3 ± 13% and 29.6 ± 15.7%, respectively whereas lung function remained nearly unchanged with FEV1 predicted of 86.0 ± 9.78 at baseline vs 83.7 ± 9.27 (ambroxol only) and 83.1 ± 11.07 (combination) (Würtemberger et al., 1988).

Treatment of chronic bronchitics with N-acetylcysteine (4 mg/day by metered dose inhaler) for 16 weeks significantly improved sputum viscosity (-0.53 vs -0.67; differences between medians to placebo: 0-14 (-0.77 0.64)) and minimally improved FVC (3.0±0.21 vs 2.9± 0.18 L/s) and PEF (356.7 ±29.64 L/min vs 354.6±25.07) but not FEV1 (1.9±0.18 vs 2.0± 0.13 L/s) (Dueholm et al., 1992).

Treatment of asthmatics with salmeterol improved tracheobronchial clearance rates (AUC: 333±24%h vs 347±30%h in placebo) as well as FEV1 (76 ± 8), FVC (100 ± 5) and PEF % predicted (100 ± 7) compared to placebo (73 ± 8; 95 ± 5; 94 ± 7) (Hasani et al., 2003).

Treatment of mild-to-moderate bronchitics with 42 µg salmeterol slightly enhanced whole lung clearance in 2 hr (not significant; C10–2= 25±11% vs 22±10% in placebo), significantly increased mean  peripheral lung clearance (C10–2= 22±9% vs 17±10% in placebo) and significantly increased FEV1 %pred and FEF25–75 at 2 h compared to baseline (93±18%predicted, 2.45 ± 1.08 L/s vs 88±19%predicted, 2.27 ± 0.98 L/s in placebo) (Bennett et al., 2006).

Sputum induction by inhalation of hypertonic saline solution (5%) in asthmatics at 6 hr following challenge with LPS significantly improved FEV % predicted by approx. 20% and was accompanied by a ca. 6-fold increase in whole lung clearance (from 0.1 %/min to 0.6%/min) (Alexis et al., 2017).

133 cystic fibrosis patients (age (mean [SD]) was 21.1 (11.4) years and 46.4% were female. All participants had one copy of the G551D mutation, and 72.2% were compound heterozygous with F508del on the other allele.) completed a 6-month course of ivacaftor. Lung function improved from baseline FEV1% predicted of 82.6 (25.6) to 90.1 (25.0) (mean change, 6.7; 95% CI, 4.9–8.5). In a subgroup of 22 patients, particle clearance from the whole right lung was markedly increased. Average clearance through 60 minutes at 1 month post-treatment was more than twice the baseline value, reflecting substantially improved MCC (Rowe et al., 2014b).
Inhalation of hypertonic saline solution (7%, 4 mL twice daily for 48 weeks) by cystic fibrosis patients improved FVC (by 82 mL; 95 percent confidence interval, 12 to 153) and FEV1 (by 68 mL; 95 percent confidence interval, 3 to 132) values, but not FEF25–75 (Elkins et al., 2006).

In cystic fibrosis patients that inhaled hypertonic saline solution without amiloride twice a day over a period of 14 days one-hour mucus clearance rates improved from baseline (9.3±1.6%) to 14.0±2.0% and increased FEV1 by 6.2%. FVC and FEF25-75 also improved by 1.8% and 13.1%, respectively (Donaldson et al., 2006).

Dornase alfa (recombinant human DNase) is currently used as a mucolytic to treat pulmonary disease in cystic fibrosis. It reduces mucus viscosity in the lungs, promoting improved clearance of secretions (Yang and Montgomery, 2021). In children with cystic fibrosis (mean: 8.4 yrs of age with FEV1 ≥95% predicted) treated with dornase alfa for 96 weeks, FEV1 % predicted improved by 3.2 ± 1.2, FVC % predicted improved by 0.7 ± 1.0, and FEF25-75 % predicted improved by 7.9 ± 2.3 compared to placebo (Quan et al., 2001). In young patients with cystic fibrosis (6-18 yrs of age with FEV1 ≥80% predicted) treated with dornase alfa for 96 weeks, FEF25-75 % predicted improved by 6.1±10.34 compared to placebo (Amin et al., 2011). In 10 adult cystic fibrosis patients receiving 2.5 mg rhDNase twice a day for 6 days, FEV1 and FVC increased by an average of 9.4 ± 3.5% and 12.7 ± 2.6%, respectively, as compared with a decrease of 1.8 ± 1.7% and an increase of 0.4±1.1% in the placebo group, respectively, although there were no significant changes in MCC (Laube et al., 1996). In 320 cystic fibrosis patients (7 to 57 yrs of age), dornase alfa treatment at 2.5 mg/day for 12 weeks (McCoy et al., 1996).

Saccharin transit times (a marker of nasal MCC) were higher in healthy current smokers and COPD smokers than in healthy controls (10.87 [7.29–17] min and 16.47 [8.25–20.15] min, respectively, vs 8.52 [5.54–13.91] min). These groups also differed in their lung function indices: FEV1 % predicted was 101.4 ± 12.37 in healthy controls, 96.41 ± 12.3 in healthy current smokers, and 67.96 ± 24.02 in COPD smokers. FVC % predicted was 103.1 ± 13.45 in healthy controls, 97.51 ± 12.88 in healthy current smokers, and 90.33 ± 29.27 in COPD smokers. FEV1/FVC % predicted was 82.15 [78.5–85] in healthy controls, 82.20 [79.2–84.1] in healthy current smokers, and 61.1 [55.3–67.2] in COPD smokers (Uzeloto et al., 2021).
Saccharin transit time of smokers with COPD (16.5 [11–28] min, median [interquartile range 25–75%]) was slightly longer than that of current smokers (15.9 [10 –27] min), and both were longer compared with exsmokers with COPD (10.2 [6 –12] min) and nonsmokers (8 [6 –16] min). Lung function parameters for the groups were as follows: nonsmokers, FEV1/FVC 0.84 ± 0.09, FEV1 % predicted  103.2 ± 11.5, FVC % predicted 102.2 ± 13.3; current smokers, FEV1/FVC 0.76 ± 0.05, FEV1 % predicted  90.7 ± 7.4, FVC % predicted 96.3 ± 13.9; former smokers with COPD, FEV1/FVC 0.49 ± 0.08, FEV1 % predicted  46.8 ± 12.6, FVC % predicted 76.8 ± 18.5; current smokers with COPD, FEV1/FVC 0.66 ± 0.16, FEV1 % predicted  48.7 ± 16.8, FVC % predicted 71.7 ± 13.0 (Ito et al., 2015).
 

Six asymptomatic patients with bronchial asthma and a history of allergic pollenosis and episodic bronchospasm consistent with ragweed hypersensitivity wer challenged by inhalation of an aqueous, short ragweed antigen extract (Greer Laboratories, Lenoir, N.C.), diluted with a phosphate-buffered saline solution. Mean tracheal mucus velocity (TMV) decreased to 72% of baseline immediately after challenge when specific airway conductance (SGaw), and FEV1 showed a maximal decrease, with a further decrease to 47% of baseline after 1 h, when SGaw and FEV1 had returned to baseline values (Mezey et al., 1978).

Treatment of chronic bronchitics with N-acetylcysteine (3 x 200 mg/day) for 4 weeks significantly decreased sputum thickness, increased sputum pourability from 650% glycerol time (at baseline) to 320% glycerol time on day 21 and PEFR on days 28 (+5%), 35 (+6%) and 42 (+7%) and FEV1 on days 21 (+2%), 28(+3%), 35 (+4%) and 42 (+5%) compared to baseline (ca. 33% predicted and 28% predicted, respectively) (Aylward et al., 1980).

Treatment of mild-to-moderate bronchitics with 42 µg salmeterol slightly enhanced whole lung clearance in 2 hr (not significant; C10–2= 25±11% vs 22±10% in placebo), significantly increased mean  peripheral lung clearance (C10–2= 22±9% vs 17±10% in placebo) and significantly increased FEV1 %pred and FEF25–75 at 2 h compared to baseline (93±18%predicted, 2.45 ± 1.08 L/s vs 88±19%predicted, 2.27 ± 0.98 L/s in placebo) , and significantly increased FEV1 %pred and FEF25–75 at both 1 (92±19%predicted, 2.44 ± 1.14 L/s) and 2 h (93±18%predicted, 2.45 ± 1.08 L/s) compared to baseline (pre-dose; 90 ± 20%predicted, 2.16 ± 0.92 L/s) (Bennett et al., 2006).

In cystic fibrosis patients on a 6-month ivacaftor regimen, FEV1% improvement was detectable as soon as the 1-month follow-up visit (mean change, 6.7; 95% CI, 5.2–8.3) (Rowe et al., 2014b). MCC remained at elevated level at the month 3 visit (Donaldson et al., 2018).

One-hour mucus-clearance rates in cystic fibrosis patients receiving hypertonic saline with placebo were significantly faster than in the group receiving hypertonic saline with amiloride (14.0±2.0 vs. 7.0±1.5 %), and the durability of response following the inhalation of hypertonic saline with placebo was ≥8 hours (Donaldson et al., 2006).
 

Invariably, if mucus viscosity increases (independent of whether that results from increased mucus production (hypersecretion), depletion of the ASL or another cause) and MCC decreases, another mechanism comes into action to clear excess mucus: cough clearance. Cough constitutes a “backup” host defense by which acutely or chronically accumulated mucus is expelled through forceful, high-velocity airflow (Button et al., 2018; King, 2006). Our current understanding of the mechanical principles and biology of cough suggest that failure of cough clearance may also be a contributor to decreased lung function.

Unknown

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• Schaedel, C., de Monestrol, I., Hjelte, L., Johannesson, M., Kornfält, R., Lindblad, A., et al. (2002). Predictors of deterioration of lung function in cystic fibrosis. Pediatr. Pulmonol. 33(6), 483-491. 
• Szczesniak, R., Heltshe, S.L., Stanojevic, S., and Mayer-Hamblett, N. (2017). Use of FEV(1) in cystic fibrosis epidemiologic studies and clinical trials: A statistical perspective for the clinical researcher. J. Cyst. Fibros. 16(3), 318-326. 
• Thomson, M., Pavia, D., Gregg, I., and Stark, J. (1974). Bromhexine and mucociliary clearance in chronic bronchitis. British journal of diseases of the chest 68, 21-27.
• Uzeloto, J.S., Ramos, D., Silva, B.S.d.A., Lima, M.B.P.d., Silva, R.N., Camillo, C.A., et al. (2021). Mucociliary Clearance of Different Respiratory Conditions: A Clinical Study. Int Arch Otorhinolaryngol 25(1), e35-e40. 
• Vastag, E., Matthys, H., Zsamboki, G., Köhler, D., and Daikeler, G. (1986). Mucociliary clearance in smokers. European journal of respiratory diseases 68(2), 107-113.
• Wallmeier, J., Nielsen, K.G., Kuehni, C.E., Lucas, J.S., Leigh, M.W., Zariwala, M.A., et al. (2020). Motile ciliopathies. Nature Reviews Disease Primers 6(1), 1-29.
• Würtemberger, G., Michaelis, K., and Matthys, H. (1988). [Additive action of theophylline and ambroxol on bronchial clearance?]. Prax Klin Pneumol 42 Suppl 1, 300-303.

Relationship: 2444: ASL Height, Decreased leads to Mucus Viscosity, Increased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

The phenomenon of ASL volume changes determining mucus viscosity is well described in the cystic fibrosis literature. In patients with this genetic defect, impaired CFTR function results in ASL depletion and mucus hyperviscosity. Mechanistically, the imbalance of Cl^– and HCO3^– secretion and increased Na+ absorption by the airway epithelium results in dehydration of airway mucus, making it more viscous and adhesive (Knowles and Boucher, 2002; Mall et al., 2004; Puchelle et al., 2002; Tarran, 2004). Studies with transgenic mice overexpressing βENaC in the airways corroborate the link between ASL hydration and mucociliary impairment as evidenced by the increased incidence of airway mucus plugging (Mall et al., 2004; Mall, 2008). 
Increased mucus viscosity may also play a significant role in asthma and chronic bronchitis, although the mechanisms are less well explored. In a ferret model of cigarette smoke-induced COPD, Lin et al. identified ASL depletion as one of the drivers of increased mucus viscosity and decreased MCC (Lin et al., 2020). The authors also showed that mucus from COPD patients, obtained from 3D organotypic airway epithelial cultures from different smoking donors with COPD, was significantly more viscous than that of healthy, non-smoking individuals and smokers without disease (Lin et al., 2020). Considering the known effects of cigarette smoke exposure on the ASL height (Hassan et al., 2014; Lambert et al., 2014; Raju et al., 2016; Schmid et al., 2015; Xu et al., 2015), this links decreased ASL height to increased mucus viscosity in the context of chronic bronchitis. 
 

Evidence Supporting this KER

In patients with cystic fibrosis, impaired CFTR function results in ASL depletion and mucus hyperviscosity (Knowles and Boucher, 2002; Puchelle et al., 2002; Mall et al., 2004; Tarran, 2004). This has been confirmed experimentally in pig and rat models of this disease (Birket et al., 2014; Birket et al., 2016; Birket et al., 2018). Studies with transgenic mice overexpressing βENaC in the airways also corroborate the link between ASL dehydration and increased mucus viscosity, evidenced by the increased incidence of airway mucus plugging [129, 195]. In a ferret model of cigarette smoke-induced COPD, ASL depletion was shown to be one of the drivers of increased mucus viscosity and decreased MCC (Lin et al., 2020). The same study also showed that mucus from COPD patients, obtained from 3D organotypic airway epithelial cultures from different smoking donors with COPD, is significantly more viscous than that from healthy, non-smoking individuals and smokers without disease (Lin et al., 2020).

There are only few studies that report ASL height and mucus viscosity, and although studies on cystic fibrosis in animal models or human cell cultures show the dependencies between these two KEs, the causal evidence is sparse. However, because the underlying mechanism is well-described and translatable across different species and is amenable to positive modulation by e.g. CFTR drugs, we consider the biological plausibility of this KER to be moderate.

ASL height was inversely related to mucociliary transport (MCT) rate, an outcome of particle tracking measurements and hence an indicator of mucus viscosity, in tracheas of CFTR-deficient piglets. Complementary studies in airway cell monolayer cultures also identified elevated effective viscosity of the ASL in cystic fibrosis compared to non-cystic fibrosis cultures (Birket et al., 2014).

ASL height was decreased and mucus viscosity was increased in human airway epithelial cultures from cystic fibrosis donors grown at the air-liquid interface (Birket et al., 2014). Treatments with CFTR function-restoring drugs improve both ASL height and mucus viscosity (Birket et al., 2016).

Deficiency of Cftr in rats results in decreased ASL height and increased mucus viscosity compared to their wild-type littermates (Birket et al., 2018).

Exposure of rats to nebulized N-acetylcysteine, assumed to function as a mucolytic (i.e., to decrease mucus viscosity), for 20 or 90 min increased the viscoelastic properties of mucus and decreased mucus transport speed (Lorenzi et al., 1992).

Computational modeling of the MCT process indicated that mucus velocity is not only dependent on the cilia beat frequency and the numbers of cilia, but also on ASL height (Jayathilake et al., 2015; Lee et al., 2011).
 

At least one study in primary cultures of human bronchial epithelial cells grown at the air-liquid interface found mucus viscosity to be higher in cultures from cystic fibrosis donors compared to those from healthy donors, but did not observe differences in ASL height (Derichs et al., 2011).
In asthmatics, airway mucus is very viscous, and its viscosity increases even more in acute exacerbations (Innes et al., 2009). However, unlike in individuals with cystic fibrosis or chronic bronchitis, changes in ASL height because of impaired anion transport might not be the primary cause for increased mucus viscosity in patients with asthma. Instead a more acidic ASL may lead to improper unpacking of secreted mucins and tethering of mucins to the epithelial surface, where they form protein tangles that result in mucus airway plugs (Abdullah et al., 2017; Bonser and Erle, 2017; Bonser et al., 2016; Evans et al., 2015; Shimura et al., 1988; Tang et al., 2016). Alternatively, the composition of mucus may be altered by production of more acidic mucins thereby skewing the pH balance of mucus itself (Gearhart and Schlesinger, 1989; Holma, 1989; Kim et al., 2014). In addition, mucus viscosity is also affected by the presence of DNA, lipids and proteins other than mucins, such as lactoferrin, albumin and immunoglobulins, all of which may be present in higher amounts in the presence of airway inflammation (Puchelle et al., 2002; Rogers, 2004). 

Quantitative Understanding of the Linkage

There are a small number of studies reporting on ASL and mucus viscosity that provide insights into the quantitative relationship between these two KEs. Some of these studies use pharmacological agents with well described effects on ASL height and mucus viscosity. However, most studies evaluated the KEs in parallel, without interrogating dose responses and/or time responses. Therefore, we only have a rudimentary quantitative understanding of this KER, leading us to judge this as weak.

In primary human bronchial epithelial cultures grown at the air-liquid interface, the ASL depth of cystic fibrosis cells was significantly lower than that of non-cystic fibrosis cells (2.4±0.6 µm vs. 6.7±0.2 µm) and mucus viscosity was significantly higher (80.6±26.5 cP vs. 12.0±3.6 mm/min) by particle-tracking microrheology. FRAP indicated an increased half-life of fluorescence recovery (evidence of increased viscosity) of approx. 16 s in cystic fibrosis cultures vs ca. 11 s in non-cystic fibrosis cultures (Birket et al., 2014).

ASL height in the tracheas of ^-/^- CFTR piglets was significantly lower than in wild-type littermates (3.2±0.8 µm vs. 6.5±0.2 µm) and mucus viscosity was significantly higher by FRAP, which indicated an increased half-life of approx. 13 s in CFTR-deficient tracheas vs ca. 9.5 s in wild-type controls (Birket et al., 2014). Treatment of normal adult pig trachea with 100 µM bumetanide to block HCO3^– transport reduced ASL height from 7.8±0.5 µm (untreated) to 6.4±0.4 µm and mucus viscosity from approx. 600 cP (untreated) to 2000 cP at frequencies between 0.5 and 10 Hz (Birket et al., 2014).

In tracheas of Cftr-deficient rats, ASL depths were diminished in both basal and stimulated conditions, from weaning until at least 6 months of age (WT 1 month 22.9 ± 4.6 μm, 6 months 43.6 ± 12.5 μm vs. KO 1 month 6.9 ± 0.7 μm, 6 month 19.5 ± 4.8 μm). Under baseline conditions at 1 month of age, effective viscosity was no different in KO tracheae compared with WT tracheae (1.76 ± 1.0 cP WT vs. 1.90 ± 1.7 cP KO). At 3 months, effective viscosity of KO airway mucus increased slightly but was still no different than that of WT airway mucus (5.12 ± 1.0 cP WT vs. 10.72 ± 2.7 cP KO). By 6 months of age, KO tracheal mucus was 20-fold more viscous compared with WT littermates (2.91 ± 0.9 cP WT vs. 65.09 ± 3.6 cP KO) (Birket et al., 2018).

Treatment of primary bronchial epithelial cell monolayer cultures from G551D/F508del cystic fibrosis patients with ivacaftor, a CFTR potentiator, at concentrations ≥ 100 nM for 24 h increased ASL height from ca. 6 to 17.5 µm, which was similar to the ASL height of non-cystic fibrosis cultures (18.03 ± 1.6 µm). The half-life of time to recovery measured by FRAP shortened from 12.39 ± 1.3 to 7.57 ± 0.8 s, indicative of decreased mucus viscosity. Effective viscosity of cells treated with ivacaftor (600 cP) was significantly lower than control (2,600 cP) at the physiological frequency of 0.9 Hz (Birket et al., 2016).

Treatment of primary bronchial epithelial cell monolayer cultures from homozygous F508del cystic fibrosis patients with the combination of 10 µM ivacaftor and 3 µM C18 (a ivacaftor homolog) with 20 µM forskolin significantly increased ASL height (23.4 ±  2.6 vs. 9.01 ± 1.4 µm C18 + forskolin, 10.99 ± 1.7 µm ivacaftor + forskolin; 13.03 ± 2.8 µm forskolin alone, and 12.53 ± 2.3 µm vehicle). Effective mucus viscosity effective in situ was reduced from ca. 1200 cP (vehicle) to ca. 100 cP by 48-h treatment only with the combination of ivacaftor and C18 (Birket et al., 2016).

No data

Unknown

Unknown

References

• Abdullah, L.H., Evans, J.R., Wang, T.T., Ford, A.A., Makhov, A.M., Nguyen, K., et al. (2017). Defective postsecretory maturation of MUC5B mucin in cystic fibrosis airways. JCI Insight 2(6), e89752. 
• Birket, S.E., Chu, K.K., Houser, G.H., Liu, L., Fernandez, C.M., Solomon, G.M., et al. (2016). Combination therapy with cystic fibrosis transmembrane conductance regulator modulators augment the airway functional microanatomy. Am. J. Physiol. Lung Cell. Mol. Physiol. 310(10), L928-L939.
• Birket, S.E., Chu, K.K., Liu, L., Houser, G.H., Diephuis, B.J., Wilsterman, E.J., et al. (2014). A functional anatomic defect of the cystic fibrosis airway. Am. J. Respir. Crit. Care Med. 190(4), 421-432.
• Birket, S.E., Davis, J.M., Fernandez, C.M., Tuggle, K.L., Oden, A.M., Chu, K.K., et al. (2018). Development of an airway mucus defect in the cystic fibrosis rat. JCI Insight 3(1). e97199.
• Bonser, L.R., and Erle, D.J. (2017). Airway mucus and asthma: the role of MUC5AC and MUC5B. Journal of clinical medicine 6(12), 112.
• Bonser, L.R., Zlock, L., Finkbeiner, W., and Erle, D.J. (2016). Epithelial tethering of MUC5AC-rich mucus impairs mucociliary transport in asthma. J Clin Invest 126(6), 2367-2371. 
• Derichs, N., Jin, B.-J., Song, Y., Finkbeiner, W.E., and Verkman, A.S. (2011). Hyperviscous airway periciliary and mucous liquid layers in cystic fibrosis measured by confocal fluorescence photobleaching. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 25(7), 2325-2332. 
• Evans, C.M., Raclawska, D.S., Ttofali, F., Liptzin, D.R., Fletcher, A.A., Harper, D.N., et al. (2015). The polymeric mucin Muc5ac is required for allergic airway hyperreactivity. Nat Commun 6, 6281. 
• Gearhart, J.M., and Schlesinger, R.B. (1989). Sulfuric acid-induced changes in the physiology and structure of the tracheobronchial airways. Environmental health perspectives 79, 127-136. 
• Hassan, F., Xu, X., Nuovo, G., Killilea, D.W., Tyrrell, J., Da Tan, C., et al. (2014). Accumulation of metals in GOLD4 COPD lungs is associated with decreased CFTR levels. Respir. Res. 15(1), 69.
• Holma, B. (1989). Effects of inhaled acids on airway mucus and its consequences for health. Environmental health perspectives 79, 109-113. 
• Innes, A.L., Carrington, S.D., Thornton, D.J., Kirkham, S., Rousseau, K., Dougherty, R.H., et al. (2009). Ex vivo sputum analysis reveals impairment of protease-dependent mucus degradation by plasma proteins in acute asthma. American journal of respiratory and critical care medicine 180(3), 203-210.
• Jayathilake, P.G., Le, D.V., Tan, Z., Lee, H.P., and Khoo, B.C. (2015). A numerical study of muco-ciliary transport under the condition of diseased cilia. Comput. Methods Biomech. Biomed. Engin. 18(9), 944-951. 
• Kim, D., Liao, J., and Hanrahan, J.W. (2014). The buffer capacity of airway epithelial secretions. Frontiers in Physiology 5(188). doi: 10.3389/fphys.2014.00188.
• Knowles, M.R., and Boucher, R.C. (2002). Mucus clearance as a primary innate defense mechanism for mammalian airways. J. Clin. Investig. 109(5), 571-577.
• Lambert, J.A., Raju, S.V., Tang, L.P., McNicholas, C.M., Li, Y., Courville, C.A., et al. (2014). Cystic fibrosis transmembrane conductance regulator activation by roflumilast contributes to therapeutic benefit in chronic bronchitis. Am. J. Respir. Cell Mol. Biol. 50(3), 549-558.
• Lee, W., Jayathilake, P., Tan, Z., Le, D., Lee, H., and Khoo, B. (2011). Muco-ciliary transport: effect of mucus viscosity, cilia beat frequency and cilia density. Comput. Fluids 49(1), 214-221.
• Lin, V.Y., Kaza, N., Birket, S.E., Kim, H., Edwards, L.J., LaFontaine, J., et al. (2020). Excess mucus viscosity and airway dehydration impact COPD airway clearance. Eur. Respir. J. 55(1), 1900419. 
• Lorenzi, G., Böhm, G., Guimarães, E., Vaz, C., King, M., and Saldiva, P. (1992). Correlation between rheologic properties and in vitro ciliary transport of rat nasal mucus. Biorheology 29(4), 433-440.
• Mall, M.A. (2008). Role of cilia, mucus, and airway surface liquid in mucociliary dysfunction: lessons from mouse models. J. Aerosol Med. Pulm. Drug Deliv. 21(1), 13-24.
• Mall, M., Grubb, B.R., Harkema, J.R., O'Neal, W.K., and Boucher, R.C. (2004). Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. Nat. Med. 10(5), 487.
• Puchelle, E., Bajolet, O., and Abély, M. (2002). Airway mucus in cystic fibrosis. Paediatr. Respir. Rev. 3(2), 115-119. 
• Raju, S.V., Lin, V.Y., Liu, L., Mcnicholas, C.M., Karki, S., Sloane, P.A., et al. (2016). The Cftr Potentiator Ivacaftor Augments Mucociliary Clearance Abrogating Cftr Inhibition by Cigarette Smoke. Am. J. Respir. Cell Mol. Biol. 56(1), 99-108.
• Rogers, D.F. (2004). Airway mucus hypersecretion in asthma: an undervalued pathology? Current opinion in pharmacology 4(3), 241-250.
• Schmid, A., Baumlin, N., Ivonnet, P., Dennis, J.S., Campos, M., Krick, S., et al. (2015). Roflumilast partially reverses smoke-induced mucociliary dysfunction. Respir. Res. 16(1), 135. 
• Shimura, S., Sasaki, T., Sasaki, H., and Takishima, T. (1988). Chemical properties of bronchorrhea sputum in bronchial asthma. Chest 94(6), 1211-1215.
• Tang, X.X., Ostedgaard, L.S., Hoegger, M.J., Moninger, T.O., Karp, P.H., McMenimen, J.D., et al. (2016). Acidic pH increases airway surface liquid viscosity in cystic fibrosis. J Clin Invest 126(3), 879-891. 
• Tarran, R. (2004). Regulation of airway surface liquid volume and mucus transport by active ion transport. Proc. Am. Thorac. Soc. 1(1), 42-46.
• Xu, X., Balsiger, R., Tyrrell, J., Boyaka, P.N., Tarran, R., and Cormet-Boyaka, E. (2015). Cigarette smoke exposure reveals a novel role for the MEK/ERK1/2 MAPK pathway in regulation of CFTR. Biochim. Biophys. Acta 1850(6), 1224-1232. 

Relationship: 2445: Mucus Viscosity, Increased leads to CBF, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

Under physiological conditions, the viscosity of mucus has been shown to range from 1 to 100 Pa.s under low shear rate conditions and from 0.01 to 1 Pa.s under high shear rate conditions. Mucus viscoelastic properties, whether altered by airway dehydration or mucus hypersecretion, directly influence MCC. Toxicant exposures, such as to nicotine, as well as inflammation can also affect the physicochemical properties of mucus (Chen et al., 2014). Increased mucus viscosity in turn decreases CBF and slows transport of mucus on the mucociliary escalator. 

Evidence Supporting this KER

Several studies have shown that there is an optimal range of viscoelastic mucus properties that facilitates efficient MCC and that changes in mucus viscosity beyond that optimal range impact CBF and alter MCC. Studies in humans, mice, hamsters, horses and frogs have shown that increased mucus viscosity correlates with a decrease in CBF (King, 1979; Gheber et al., 1998; Matsui et al., 1998; Andrade et al., 2005; González et al., 2016; Kikuchi et al., 2017; Birket et al., 2018). 

Mucus viscoelastic properties, whether altered by airway dehydration or mucus hypersecretion, directly influence MCC. In fact, there is an inverse relationship between mucus viscosity and CBF and mucus transport/MCC, as demonstrated in several in vivo and ex vivo studies. A large proportion of these studies have employed (bio)polymers or other large organic molecules to mimic the mucus layer in the airways and the increase in its viscosity. In addition, some of these studies have shown that decreased mucus viscosity may also result in impairment of MCC. Therefore, a causal link is only tentatively supported. Because cilia function, ASL height, and mucus properties are intricately linked to each other as evidenced by cystic fibrosis studies, we consider the plausibility as moderate.

Exposure of primary cultures of hamster oviductal ciliated cells to increased viscous loading reduced the CBF (Andrade et al., 2005).

The tracheal samples from mice were used to measure ciliary beat frequencies and beat amplitudes of ciliary motion in viscous culture media over the range of η= 0.9–303.8 mPa.s. The CBF decreased with increasing viscosity, up to about 32.0 mPa.s, while it was nearly constant above 32.0 mPa.s (Kikuchi et al., 2017). In tracheal samples from mice, CBF decreased with increasing viscosity, up to about 32.0 mPa.s, while it was nearly constant above 32.0 mPa.s. CBF were calculated from the averaged cycles of beat velocity, with 14.8 ± 3.0 Hz and 9.0 ± 2.4 Hz  at η = 0.9 (0% methylcellulose solution) and 32.0 mPa.s (0.3% methylcellulose solution), respectively (Kikuchi et al., 2017).

When the viscosity of medium 199 was increased from 7.8 to 58 mP by adding polyvinylpyrrolidone, CBF of bronchial epithelial cell explants was decreased by ca. 10% from the control value. Medium viscosity of 87 millipoises decreased CBF to 25% from the control value (Luk and Dulfano, 1983).

Treatment of primary bronchial epithelial cell monolayer cultures from G551D/F508del cystic fibrosis patients with 10 µM ivacaftor, a CFTR potentiator, at concentrations ≥ 100 nM for 24 hr and 10 µM forskolin decreased mucus viscosity from 2600 cP to 600 cP at the physiological frequency of 0.9 Hz and increased CBF from ca. 3 Hz to ca. 5 Hz (Birket et al., 2016).

Epithelial cell monolayers from explants of pediatric adenoid tissues were used to assess the impact of viscosity on CBF. A decrease in CBF was observed immediately after the viscosity of the medium was increased, with a greater decrease in CBF in cultures exposed to 20% dextran (González et al., 2016).
 

Studies interrogating the link between CBF and/or mucus viscosity and MCC found the optimal range of viscoelastic mucus properties to be between 10 and 30 cP and 11 to 25 dyn/cm2 (Chen and Dulfano, 1978; King, 1979; King, 2006; King et al., 1997). These studies also documented that both increases and decreases in mucus viscosity beyond that optimal range impact CBF and decrease and increases, respectively, MCC. A large proportion of these studies utilize (bio)polymers or other large organic molecules to mimic the mucus layer in the airways and increases in its viscosity. Therefore, there may be limitations to the translatability of these findings. 

There is at least one study showing that increased mucus viscosity not only slows CBF, but also alters cilia beat metachrony, with medium viscosities in the range of 30–1500 cP increasing metachronal wave velocities by up to 50% and changes in wave direction in cultured frog esophagus (Gheber et al., 1998; Stafanger et al., 1987).
CBF also appears to be, at least in part, autoregulated by ciliated respiratory cells, which adjust cilia beating to differences in viscous load via a mechanosensory mechanism (Johnson et al., 1991).
 

Quantitative Understanding of the Linkage

The bulk of quantitative data supports the inverse relationship between mucus viscosity and MCC, either via slowing of cilia beating or decreased mucus transport speed. In particular, studies mimicking changes in mucus viscosity by using (bio)polymers or large molecules such as dextran provide insights into the dose-response effects of increasing mucus viscosity on mucociliary transport rates. They do, however, suggest that the effects are transient in nature, at least in ex vivo and in vitro systems. These studies also indicate that there is an optimal range of viscoelastic mucus properties that facilitates efficient MCC and that changes in mucus viscosity beyond that optimal range impact CBF and alter MCC. Because MCC can both decrease and increase dependent on mucus viscosity and because not all studies provide evidence of a causal relationship between these two KEs, we judge our quantitative understanding moderate.

Exposure of primary cultures of hamster oviductal ciliated cells to increased viscous loading reduced the CBF (Andrade et al., 2005).

The tracheal samples from mice were used to measure ciliary beat frequencies and beat amplitudes of ciliary motion in viscous culture media over the range of η= 0.9–303.8 mPa.s. The CBF decreased with increasing viscosity, up to about 32.0 mPa.s, while it was nearly constant above 32.0 mPa.s (Kikuchi et al., 2017).

When the viscosity of medium 199 was increased from 7.8 to 58 millipoises (by adding polyvinylpyrrolidone), CBF of bronchial epithelial cell explants was decreased by ca. 10% from the control value. Medium viscosity of 87 millipoises decreased CBF to 25% from the control value (Luk and Dulfano, 1983).

Treatment of primary bronchial epithelial cell monolayer cultures from G551D/F508del cystic fibrosis patients with 10 µM ivacaftor, a CFTR potentiator, at concentrations ≥ 100 nM for 24 hr and 10 µM forskolin decreased mucus viscosity (from 2600 cP to 600 cP) at the physiological frequency of 0.9 Hz and increased CBF (from ca. 3 Hz to ca. 5 Hz) (Birket et al., 2016).

Epithelial cell monolayers from explants of pediatric adenoid tissues were used to assess the impact of viscosity on CBF. A decrease in CBF was observed immediately after the viscosity of the medium was increased, with a greater decrease in CBF in cultures exposed to 20% dextran (González et al., 2016).
 

Within the first 10 min, the CBF of human oviductal cells dropped ~35% within the range of 2–37 cP (2–15% dextran solutions), but no further decrease was observed at higher viscosities in the range of 37–200 cP (15–30% dextran solutions) (Andrade et al., 2005).

The CBF decreased with increasing viscosity, up to about 32.0 mPa.s, while it was nearly constant above 32.0 mPa.s. The beat frequencies were calculated from the averaged cycles of beat velocity, 14.8 ± 3.0 Hz and 9.0 ± 2.4 Hz (0% methylcellulose solution) and 32.0 mPa.s (0.3% methylcellulose solution), respectively (Kikuchi et al., 2017).

In epithelial cell monolayers from explants of pediatric adenoid tissues , a decrease in CBF was observed immediately after the viscosity of the medium was increased, with a greater decrease in CBF in cultures exposed to 20% dextran. When cultures, prior to viscosity change, were treated with TNFa, CBF decreased furthermore only in culture exposed to 10% dextran. This effect of TNFa occurs in the first 10 min of viscous load, then TNFa-treated cells seem to adjust the CBF to control values (González et al., 2016).

Unknown

Unknown

References

• Andrade, Y.N., Fernandes, J., Vázquez, E., Fernández-Fernández, J.M., Arniges, M., Sánchez, T.M., et al. (2005). TRPV4 channel is involved in the coupling of fluid viscosity changes to epithelial ciliary activity. J. Cell Biol. 168(6), 869-874.
• Birket, S.E., Chu, K.K., Houser, G.H., Liu, L., Fernandez, C.M., Solomon, G.M., et al. (2016). Combination therapy with cystic fibrosis transmembrane conductance regulator modulators augment the airway functional microanatomy. Am. J. Physiol. Lung Cell. Mol. Physiol. 310(10), L928-L939.
• Birket, S.E., Davis, J.M., Fernandez, C.M., Tuggle, K.L., Oden, A.M., Chu, K.K., et al. (2018). Development of an airway mucus defect in the cystic fibrosis rat. JCI Insight 3(1), e97199. 
• Chen, E.Y., Sun, A., Chen, C.-S., Mintz, A.J., and Chin, W.-C. (2014). Nicotine alters mucin rheological properties. American Journal of Physiology-Lung Cellular and Molecular Physiology 307(2), L149-L157.
• Chen, T., and Dulfano, M. (1978). Mucus viscoelasticity and mucociliary transport rate. The Journal of laboratory and clinical medicine 91(3), 423-431.
• Gheber, L., Korngreen, A., and Priel, Z. (1998). Effect of viscosity on metachrony in mucus propelling cilia. Cell motility and the cytoskeleton 39(1), 9-20.
• González, C., Droguett, K., Rios, M., Cohen, N.A., and Villalón, M. (2016). TNFα Affects Ciliary Beat Response to Increased Viscosity in Human Pediatric Airway Epithelium. Biomed. Res. Int. 2016, 3628501.
• Johnson, N.T., Villalón, M., Royce, F.H., Hard, R., and Verdugo, P. (1991). Autoregulation of beat frequency in respiratory ciliated cells. The American review of respiratory disease 144, 1091-1094.
• Kikuchi, K., Haga, T., Numayama-Tsuruta, K., Ueno, H., and Ishikawa, T. (2017). Effect of fluid viscosity on the cilia-generated flow on a mouse tracheal lumen. Ann. Biomed. Eng. 45(4), 1048-1057.
• King, M. (1979). Interrelation between mechanical properties of mucus and mucociliary transport: effect of pharmacologic interventions. Biorheology 16(1-2), 57-68.
• King, M. (2006). Physiology of mucus clearance. Paediatr. Respir. Rev. 7 Suppl 1, S212-214. doi: 10.1016/j.prrv.2006.04.199.
• King, M., Dasgupta, B., Tomkiewicz, R.P., and Brown, N.E. (1997). Rheology of cystic fibrosis sputum after in vitro treatment with hypertonic saline alone and in combination with recombinant human deoxyribonuclease I. American journal of respiratory and critical care medicine 156(1), 173-177.
• Luk, C.K., and Dulfano, M.J. (1983). Effect of pH, viscosity and ionic-strength changes on ciliary beating frequency of human bronchial explants. Clin Sci (Lond) 64(4), 449-451.
• Matsui, H., Grubb, B.R., Tarran, R., Randell, S.H., Gatzy, J.T., Davis, C.W., et al. (1998). Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95(7), 1005-1015.
• Stafanger, G., Bisgaard, H., Pedersen, M., Mørkassel, E., and Koch, C. (1987). Effect of N-acetylcysteine on the human nasal ciliary activity in vitro. European journal of respiratory diseases 70(3), 157-162.

Relationship: 2446: Mucus Viscosity, Increased leads to MCC, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

Under physiological conditions, the viscosity of mucus has been shown to range from 1 to 100 Pa.s under low shear rate conditions and from 0.01 to 1 Pa.s under high shear rate conditions. Mucus viscoelastic properties, whether altered by airway dehydration or mucus hypersecretion, directly influence mucociliary clearance (MCC). Toxicant exposures as well as inflammation can also affect the physicochemical properties of mucus (Chen et al., 2014). Increased mucus viscosity in turn decreases CBF and slows transport of mucus on the mucociliary escalator. 

Evidence Supporting this KER

Mucus viscoelastic properties, whether altered by airway dehydration or mucus hypersecretion, directly influence MCC. Studies in cystic fibrosis models and those on mimicking changes in mucus viscosity by using (bio)polymers or large molecules such as dextran have indicated a dose-response effect of increasing mucus viscosity on mucociliary transport rates, although these changes are transient in nature in ex vivo and in vitro systems (Birket et al., 2018; Fernandez-Petty et al., 2019). Increased mucus viscosity also has a negative impact on MCC in horses with recurrent airway obstruction (Gerber et al., 2000). Conversely, inhalation of hypertonic saline solution decreases mucus viscosity and enhances MCC in cystic fibrosis patients (Robinson et al., 1997).

Mucus viscoelastic properties, whether altered by airway dehydration or mucus hypersecretion, directly influence MCC. In fact, there is an inverse relationship between mucus viscosity and CBF and mucus transport/MCC, as demonstrated in several in vivo and ex vivo studies. A large proportion of these studies have employed (bio)polymers or other large organic molecules to mimic the mucus layer in the airways and the increase in its viscosity. In addition, some of these studies have shown that decreased mucus viscosity may also result in impairment of MCC. Therefore, a causal link is only tentatively supported. Because cilia function, ASL height, and mucus properties are intricately linked to each other as evidenced by cystic fibrosis studies, we consider the biological plausibility of this KER moderate.

Exposure of primary cultures of hamster oviductal ciliated cells to increased viscous loading reduced the CBF, reaching a new stable value within the first 10 min (Fig. 1 a).The CBF dropped 35% within the range of 2–37 cP (2–15% dextran solutions), but no further decrease was observed at higher viscosities in the range of 37–200 cP (15–30% dextran solutions) (Andrade et al., 2005).

In tracheal and bronchial sections of mice, the CBF decreased with increasing viscosity up to about 32.0 mPa s, while it was nearly constant above 32.0 mPa s. The amplitude of the ciliary beat was kept at ~1.5 lm regardless of the viscosity (Kikuchi et al., 2017).

In control cultures of human nasopharyngeal pediatric airway ciliated cells, a decrease in CBF was observed immediately after the viscosity of the medium was increased, with a greater decrease in CBF in cultures exposed to 20% dextran (González et al., 2016).

CBF in bronchial biopsies from patients undergoing bronchoscopy for diagnostic purposes was inhibited by increased viscosity of the polyvinylpyrrolidone-supplemented medium (Luk and Dulfano, 1983).

In cultured ciliary cells from the frog esophagus photoelectric measurements were performed in the viscosity range of 1–2000 cP. In solutions of viscosity >30 cP. the decrease in CBF was more pronounced but the general trend of a relatively moderate decrease in the frequency at higher viscosity applies for the whole viscosity range (Gheber et al., 1998).

Sputum from chronic bronchitis patients placed on mucus-depleted frog palates moved slower if their Newtonian viscosity exceeded 1000-3000 P (Chen TM and Dulfano, 1978).

In human cystic fibrosis airway epithelial cultures grown at the air-liquid interface, ASL height was lower than that in non-cystic fibrosis cultures thereby increasing mucus viscosity, and mucus velocity was lower than that in non-cystic fibrosis cultures (Matsui et al., 1998).

In tracheas of 6-month old Cftr-deficient rats, mucus viscosity was higher than that of their wild-type littermates and mucus transport was significantly slowed down (Birket S. E. et al., 2018).

Polycarbophil and Carbopol 1342 polymers of increasing viscosity caused ever greater reductions MCT rates [cm/min] across bovine trachea (Shah and Donovan, 2007a). A similar observation was made for the transport of anionic polysaccharide gels of increasing viscosity and linear polyanionic samples across the frog palate (Lin et al., 1993; Shah and Donovan, 2007b).

In a study on children with cystic fibrosis, the mucus dry weights had a borderline significant inverse correlation with the average percent clearance through the first 60 minutes (MCC60) (Laube et al., 2020).

Mucus from patients with chronic bronchial inflammatory diseases treated with 600 mg/day of Sobrerol (decreases viscosity of sputum) traveled faster the pre-established distance on frog palate  (‘reference’ speed of MCT) than from mucus from untreated patients (Allegra et al., 1981).

Acetylcysteine reduced the viscosity of human tracheobronchial secretions in vitro (Sheffner et al., 1964). In non-smokers, treatment with 0.6 g N-acetylcysteine/day po. for 60 days increased mucociliary clearance rates that returned to baseline values after a washout period (Todisco et al., 1985).

When hypertonic saline is added to mucus/sputum, its viscosity is markedly reduced (Elkins and Bye, 2011), and this greatly enhances its transportability in bovine trachea (King et al., 1997; Wills and Cole, 1995; Wills et al., 1998). Inhalation of hypertonic saline solution increased MCC in asthmatic and healthy subjects (Daviskas et al., 1996), adult patients with cystic fibrosis (Robinson et al., 1997; Robinson et al., 1996) and improved MCT times in patients with acute/chronic sinusitis and those having undergone sinus surgery (Talbot et al., 1997).

Inhaled mannitol reduced the viscosity of the sputum in asthmatics with mucus hypersecretion (Daviskas et al., 2007). Inhaled mannitol also improved MCC in asthmatics and patients with cystic fibrosis and bronchiectasis (Daviskas et al., 1997; Daviskas et al., 2008; Daviskas et al., 2010).

Poly(acetyl, arginyl) glucosamine (PAAG) improved the viscoelasticity of sputum. The treatment of cystic fibrosis bronchial epithelial cell culture monolayers with sputum samples that were treated with PAAG had MCT rates significantly faster than those treated with PBS (Fernandez-Petty et al., 2019).

A three times daily dose of 16 mg bromhexine, which modifies the physicochemical characteristics of mucus/is a mucolytic agent (Zanasi et al., 2017), for 14 days resulted in improved MCC in subjects with chronic bronchitis (Thomson et al., 1974).

Studies interrogating the link between CBF and/or mucus viscosity and MCC found the optimal range of viscoelastic mucus properties to be between 10 and 30 cP and 11 to 25 dyn/cm2 (Chen and Dulfano, 1978; King , 1979; King, 2006; King et al., 1997). These studies also documented that both increases and decreases in mucus viscosity beyond that optimal range impact CBF and decrease and increases, respectively, MCC. A large proportion of these studies utilize (bio)polymers or other large organic molecules to mimic the mucus layer in the airways and increases in its viscosity. Therefore, there may be limitations to the translatability of these findings. 
There is at least one study showing that increased mucus viscosity not only slows CBF, but also alters cilia beat metachrony, with medium viscosities in the range of 30–1500 cP increasing metachronal wave velocities by up to 50% and changes in wave direction in cultured frog esophagus (Gheber et al., 1998; Stafanger et al., 1987).
CBF also appears to be, at least in part, autoregulated by ciliated respiratory cells, which adjust cilia beating to differences in viscous load via a mechanosensory mechanism (Johnson et al., 1991).

Quantitative Understanding of the Linkage

The bulk of quantitative data supports the inverse relationship between mucus viscosity and MCC, either via slowing of cilia beating or decreased mucus transport speed. In particular, studies mimicking changes in mucus viscosity by using (bio)polymers or large molecules such as dextran provide insights into the dose-response effects of increasing mucus viscosity on mucociliary transport rates. They do, however, suggest that the effects are transient in nature, at least in ex vivo and in vitro systems. These studies also indicate that there is an optimal range of viscoelastic mucus properties that facilitates efficient MCC and that changes in mucus viscosity beyond that optimal range impact CBF and alter MCC. Because MCC can both decrease and increase dependent on mucus viscosity and because not all studies provide evidence of a causal relationship between these two KEs, we judge our quantitative understanding moderate.

In tracheas of 6-month old Cftr-deficient rats, mucus was 20-fold more viscous than that of their wild-type littermates (2.91 ± 0.9 cP WT vs. 65.09 ± 3.6 cP KO) and mucus transport rate was significantly slowed down (ca. 0.8 mm/min WT vs ca. 0.3 mm/min KO). Following addition of sodium bicarbonate, which is known to improve airway hydration and hence decrease mucus viscosity, at concentrations of 23 mM, 69 mM, 92 mM, and 115 mM to the apical surface of Cftr-deficient rat tracheas ex vivo (6-month old animals) increased MCT rates in a dose-dependent manner, up to ca. 2.5 mm/min (Birket et al., 2018).

Sputum dry weights more than 151.0 mg/mL were linked with 6.8 (3.3‐17.7) median percent MCC60, and sputum dry weights less than 151.0 mg/mL were linked with 13.2 (6.2‐28.6) median percent MCC60. Percent MCC60 was borderline significantly higher in children with dry weights less than 151.0 mg/mL than in children with dry weights more than 151.0 mg/mL (Laube et al., 2020).

Adult CF patients receiving hypertonic saline via Omron-NE-U06 ultrasonic nebulizer exhibited increased MCC as evidenced by increased Tc particle clearance rates. The amount cleared at 90 min on the control day was 12.7% (95% confidence interval (CI) 9.8 to 17.2) compared with 19.7% (95% CI 13.6 to 29.5) for 3% hypertonic saline, 23.8% (95% CI 15.9 to 36.7) for 7% hypertonic saline and 26.0% (95% CI 19.8 to 35.9) for 12% hypertonic saline (Robinson et al., 1997).

Treatment with 100-500 μg/mL poly(acetyl, arginyl) glucosamine (PAAG) for 2 h significantly reduced the dynamic viscosity of CF sputum at low shear rates. Effective viscosities measured at a shear rate of 0.8 s^–1 indicated that the treatment effect was particularly prominent in CF sputum samples that exhibited high dynamic viscosity at baseline (359 ± 561 Pa•s for sputum treated with PBS compared with 62 ± 97 Pa•s for sputum treated with PAAG). When PAAG-treated (250 μg/mL) sputum was added to trachea sputum, it was more rapidly transported in a homogenous fashion than untreated sputum (3.91 ± 1.89 mm/min PAAG vs 1.62 ± 0.56 mm/min PBS) (Fernandez-Petty et al., 2019).

In bronchial epithelial cultures grown at the air-liquid interface, treatment with between 250 and 500 μg/mL PAAG caused a 2-log reduction in effective viscosity across all frequency ranges (597.0 ± 56.1 cP for PBS control versus 2.84 ± 0.11 cP PAAG at 1.0 Hz) and a 57% increase in MCT rate in cells treated with PAAG (250 μg/mL) compared with the PBS control (Fernandez-Petty et al., 2019).

Using Cftr^–/– rats aged 6 months in which delayed MCT occurs due to abnormally viscous mucus (Birket et al., 2018), PAAG (250 μg/mL × 20 mL over 45 minutes) or glycerol vehicle control was nebulized once daily for 14 days. Mucus transport increased approx. 3.5-fold in PAAG-treated rats compared to vehicle-treated rats, achieving approximately 44% of MCT rates in normal rats (Fernandez-Petty et al., 2019).

At 24 h following environmental challenge (i.e., stabling in stalls with straw as bedding and hay as feed) of horses with recurrent airway obstruction, the viscoelasticity of mucus increased ca. 3-fold (to log G* averaging 3.0 ± 0.3 dyn/cm² vs 2.38 ± 0.11 dyn/cm² in controls) and mucociliary clearance index (MCI) decreased to 0.69 ± 0.05 (vs 0.92 ± 0.06 in controls) (Gerber et al., 2000).

In dogs receiving a high dose of methacholine chloride (16-32 mg/mL) acutely, mucus viscosity increased by 203 ± 23% from control, while mucus transport rate on frog palates decreased to 79 ± 6% of control (King, 1979).
 

Within the first 10 min following addition of 2–15% dextran, the CBF of human oviductal cells dropped ~35% within the range of 2–37 cP. No further decrease was observed at higher viscosities (15–30% dextran solutions) in the range of 37–200 cP (Andrade et al., 2005).

Following addition of sodium bicarbonate, which is known to improve airway hydration and hence decrease mucus viscosity, at concentrations of 23 mM, 69 mM, 92 mM, and 115 mM to the apical surface of Cftr-deficient rat tracheas ex vivo (6-month old animals) increased MCT rates in a dose-dependent manner, up to ca. 2.5 mm/min. The peak effect of bicarbonate addition occurred at 20 minutes after addition and returned to baseline by 35 minutes after addition, consistent with the short half-life of bicarbonate at the surface of the airway (Birket et al., 2018).

At 24 h following environmental challenge (i.e., stabling in stalls with straw as bedding and hay as feed) of horses with recurrent airway obstruction, the viscoelasticity of mucus increased ca. 3-fold (to log G* averaging 3.0 ± 0.3 dyn/cm2 vs 2.38 ± 0.11 dyn/cm2 in controls) and mucociliary clearance index (MCI) decreased to 0.69 ± 0.05 (vs 0.92 ± 0.06 in controls). Significant changes in mucus viscoelasticity and MCI were only observed at 24 and 48 h, but not at 6 h post-challenge (Gerber et al., 2000).

In dogs receiving a high dose of methacholine chloride (16-32 mg/mL) acutely, mucus viscosity increased, while mucus transport rate on frog palates decreased at approx. 2 to 5 min post-treatment (King, 1979).

Unknown

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References

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Relationship: 2447: FOXJ1 Protein, Decreased leads to Motile Cilia Number/Length, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

Forkhead box J1 (FOXJ1) is a master regulator of motile ciliogenesis which is necessary and also sufficient to program cells to grow functional motile cilia (Vij et al., 2012; Zhou and Roy, 2015). Studies in different model organisms have shown that the loss of FOXJ1 results in a loss of motile cilia (Brody et al., 2000; Chenet al., 1998; Stubbs et al., 2008; Vij et al., 2012).

Evidence Supporting this KER

Homozygous null mutation of Foxj1 results in complete absence of cilia in mouse respiratory epithelium (Chen et al., 1998; Brody et al., 2000). In a previous study, wild-type mice had approximately 20% heavily ciliated cells in the proximal pulmonary epithelium, while explanted Foxj1^-/- mouse trachea had no ciliated cells (Gomperts et al., 2004). Loss of FOXJ1 orthologs FoxJ1–4 in flatworm Schmidtea mediterranea results in loss of ciliation of the ventral epithelium which closely resembles the human airway epithelium (Rompolas et al., 2009; Vij et al., 2012). Loss of Foxj1 activity in Xenopus and zebrafish—through antisense morpholino oligonucleotides—reduces cilia formation, while, conversely, ectopic Foxj1 overexpression results in formation of multiple motile cilia (Stubbs et al., 2008; Yu et al., 2008). There is a strong correlation between FOXJ1 and expression of the FOXJ1 ciliogenesis program genes in zebrafish, Xenopus and mouse cells (Abedalthagafi et al., 2016).
Treatment with cigarette smoke extract downregulates FOXJ1 mRNA and protein expression, which is accompanied by a reduction in cilia length and number in human bronchial epithelial cells in vitro (Milara et al., 2012; Brekman et al., 2014). This can be prevented by overexpression of FOXJ1 (Brekman et al., 2014) or treatment with roflumilast N-oxide, which reduces intracellular free radical levels and increases FOXJ1 mRNA and protein expression (Milara et al., 2012).

The requirement of FOXJ1 for cells to grow functional motile cilia was demonstrated in 1998 in a mouse model study where targeted disruption of FOXJ1 resulted in the absence of motile cilia in the respiratory epithelium, oviduct, haploid sperm, and choroid plexus (Chen et al., 1998). Subsequently, many research groups consistently showed FOXJ1 requirement for cilia growth in various model organisms (Brody et al., 2000; Gomperts et al., 2007; Stubbs et al., 2008; Vij et al., 2012; Yu et al., 2008). In addition, overexpression of FOXJ1 in ectopic locations prompted cilia growth (Stubbs et al., 2008; Yu et al., 2008), and FOXJ1 overexpression could rescue cigarette smoke extract-caused cilia growth suppression in human airway epithelium (Brekman et al., 2014). The causal association of FOXJ1 to ciliogenesis gene expression program was computationally reinforced (Abedalthagafi et al., 2016). Taken together, the empirical support for this KER based on the research in the motile ciliogenesis field implies a high (strong) confidence for the biological plausibility of the linkage.

Two research groups independently showed that homozygous null mutation of Foxj1 causes complete absence of cilia in the respiratory airways of mice (Brody et al., 2000; Chen et al., 1998).

Loss of Foxj1a activity in zebrafish (antisense morpholino oligonucleotides designed to block Foxj1a protein translation) compromises formation of motile cilia (Yu et al., 2008). In the same study, Foxj1a ectopic expression in the neural tube (through hyperactivation of the hedgehog pathway using dominant negative PKA) triggered motile cilia development (Yu et al., 2008).

In Xenopus, FoxJ1 morpholino oligo treatments dose-dependently reduced cilia formation, and FoxJ1 overexpression in ectopic locations was sufficient to drive multiciliogenesis (Stubbs et al., 2008). In addition, authors also used a zebrafish model where FOXJ1 morpholino resulted in cilia number and length reduction (Stubbs et al., 2008).

The closest ortholog of mammalian FOXJ1 in the flatworm Schmidtea mediterranea is FoxJ1-4 which is expressed in the motile ciliated cells of the ventral epithelium. Flatworm ventral epithelium closely resembles the mammalian airway epithelium (Rompolas et al., 2009; Vij et al., 2012). S. mediterranea deficient for foxJ1-4 (RNAi) lost the ciliation of the ventral epithelium (Vij et al., 2012).

Abedalthagafi et al. defined the genes that comprise the FOXJ1 ciliogenesis program using public mRNA expression profiling data from Xenopus, zebrafish and mouse cells. A strong correlation of FOXJ1 expression and the expression of the FOXJ1 ciliogenesis program was confirmed (Abedalthagafi et al., 2016).

Cigarette smoke extract (CSE) down-regulated FOXJ1 mRNA and protein levels and cilia length in differentiating human airway basal cells in air–liquid interface (ALI) cultures, while lentivirus-mediated FOXJ1 expression prevented CSE-elicited cilia growth suppression (Brekman et al., 2014).

Wild-type mouse tracheas cultured at air-liquid interface demonstrated nearly 20% of the proximal pulmonary epithelial cells to be ciliated with abundant cilia on each cell. In explanted Foxj1^-/- tracheas no ciliated epithelial cells were observed (Gomperts et al., 2004).

Exposure of differentiated human bronchial epithelial cells to CSE decreased FOXJ1 expression alongside with reduction of the average number of cells with cilia motility. Roflumilast N-oxide (which reduced intracellular reactive oxygen species levels) prevented the CSE-elicited decrease in the expression levels of Foxj1 mRNA and protein. Concurrently, roflumilast N-oxide partly prevented the loss in cells with cilia motility (Milara et al., 2012).

FOXJ1 overexpression increased the percentage of ciliated cells in mouse trachea organ culture 1.51-fold (Johnson et al., 2018).
 

Foxj1 overexpression failed to promote ciliogenesis in mouse polarized epithelial cell lines and primary cultured alveolar epithelial cells (You et al., 2004). Also, the overexpression of Foxj1 in wild-type airway epithelial cells did not enhance the total number of ciliated cells. However, delivery of Foxj1 to null cells resulted in cilia formation (You et al., 2004).

Quantitative Understanding of the Linkage

There is ample empirical evidence of FOXJ1 requirement for motile cilia formation, where complete removal of FOXJ1 results in cilia loss, and different levels of FOXJ1 downregulation result in proportional reduction in cilia number and length. Accordingly, FOXJ1 overexpression leads to higher cilia numbers. Based on these data of strong causality between upstream and downstream KEs, we judge our quantitative understanding to be high.

Complete removal of FOXJ1 by means of homologous recombination in mouse embryonic stem cells resulted in absence of cilia in mouse airways (as well as in other typically multiciliated tissues such as oviduct, haploid sperm, choroid plexus and epithelial cells of the brain but not in embryonic node) (Brody et al., 2000; Chen J. et al., 1998).

Newly fertilized zebrafish eggs were injected with antisense morpholino oligonucleotides designed to block Foxj1a protein translation. Motile cilia numbers were severely reduced in Kupffer’s vesicle (KV), the floor plate and pronephric ducts 14 and 24 hpf (Yu et al., 2008).

Downregulation of Xenopus FoxJ1 produced a dose-dependent defect in skin cilia formation. When 20 ng or 40 ng morpholino oligonucleotides were injected, cilia formed, but were reduced in number and shortened in length. After injection of 75 ng morpholino oligos, most cilia were lost. Cilia length decreased from ~11 microns to 4 microns (Stubbs et al., 2008). Morpholino oligo knockdown of zebrafish FoxJ1 caused a two-fold decrease in the number of KV cilia and a 3.5-fold decrease in the average length of KV cilia (Stubbs et al., 2008).

RNAi against Schmidtea mediterranea foxJ1-4 substantially reduced the expression levels of foxJ1-4 which lead to almost complete loss of motile cilia (Vij et al., 2012).

In the presence of CSE, in FOXJ1 overexpressing human airway epithelial cells the average cilia length was significantly higher (5.2 μm) than in lentivirus-control–infected cells (4.1 μm) (Brekman et al., 2014). CSE was obtained from one Marlboro Red commercial cigarette bubbled in 12.5 ml of differentiation medium that was then 0.2 mm pore filtered. The absorbance was measured at 320 nm on a spectrophotometer and the optical density of 1 was defined as 100%. Homozygous FOXJ1 mutant mice were obtained by mating foxj1^+/- male and female animals. The explanted trachea of the foxj1^-/- mice harbors no motile cilia in contrast to wild-type trachea (Gomperts et al., 2004).

Exposure of differentiated human bronchial epithelial cells to 10% CSE decreased FOXJ1 expression by about 40% at 24 h and 70% at 72 h exposure. The smoke of one 2R4F research cigarette was bubbled into a flask containing 25 mL of pre-warmed (37°C) differentiation medium using a respiratory pump model (Harvard Apparatus Rodent Respirator 680, Harvard Apparatus, Holliston, MA, USA) that generates three puffs min−1; 35 mL per each puff of 2 s duration with a volume of 0.5 cm above the filter. The CS solution was filtered (0.22 µm pore size) to remove particles and the tar phase. The resulting sterile solution was defined as 100% CSE and used within 30 min of preparation. Exposure to CSE concentration- and time-dependently reduced the average number of cells with cilia motility, which was significant after 3 days of incubation with CSE at 2.5% (about 30% inhibition), and reached a maximum of about 75% inhibition versus control after 7 days of incubation with CSE at 10% (Milara et al., 2012). Roflumilast N-oxide at 2 nM or 1 µM concentration-dependently prevented the decrease in the expression levels of Foxj1 mRNA and protein following 3 days of exposure of differentiated bronchial epithelial cells to CSE at 10%. Concurrently, roflumilast N-oxide partly prevented the loss in cells with cilia motility (Milara et al., 2012).

Electroporation using negative control (GFP-only) plasmid resulted in 45±1.4% (mean±s.e.m.) GFP+ ciliated cells in mouse trachea organ culture. FOXJ1 significantly increased the percentage of GFP+ ciliated cells to 68±3.6% (1.51-fold) (Johnson et al., 2018). 

14- or 24-hr treatment with antisense morpholino oligonucleotides designed to block Foxj1a protein translation results in absence of motile cilia in zebrafish (Yu et al., 2008).

Xenopus embryos were injected with FOXJ1 morpholino oligos at two-cell stage (1.5 h of embryo life) and the embryos were analyzed at stage 26 (1 day, 5 h and 30 min of embryo life) for cilia phenotype (Stubbs et al., 2008).

Schmidtea mediterranea worms received three feedings of foxJ1-4 RNAi (2 days in between feeds) and were analyzed for cilia phenotype 14 days after the last feed. RNAi against Schmidtea mediterranea foxJ1-4 substantially reduced the expression levels of foxJ1-4 which lead to almost complete loss of motile cilia (Vij et al., 2012).

Exposure of differentiated human bronchial epithelial cells to CSE concentration- and time-dependently reduced the average number of cells with cilia motility, which was significant after 3 days of incubation with CSE at 2.5% (about 30% inhibition), and reached a maximum of about 75% inhibition versus control after 7 days of incubation with CSE at 10% (Milara et al., 2012).

Regulatory factor X3 (RFX3) is a transcriptional co-activator of FOXJ1 (Didon et al., 2013) and is involved in motile cilia biogenesis (El Zein et al., 2009). Fluctuations in RFX3 levels can modulate the outcome that the upstream KE has on the downstream KE. 

Unknown

References

• Abedalthagafi, M.S., Wu, M.P., Merrill, P.H., Du, Z., Woo, T., Sheu, S.H., et al. (2016). Decreased FOXJ1 expression and its ciliogenesis programme in aggressive ependymoma and choroid plexus tumours. J. Pathol. 238(4), 584-597.
• 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(5), 688-700.
• Brody, S.L., Yan, X.H., Wuerffel, M.K., Song, S.K., and Shapiro, S.D. (2000). Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice. Am. J. Respir. Cell Mol. Biol. 23(1), 45-51.
• Chen, J., Knowles, H.J., Hebert, J.L., and Hackett, B.P. (1998). Mutation of the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in an absence of cilia and random left-right asymmetry. J. Clin. Investig. 102(6), 1077-1082. 
• Didon, L., Zwick, R.K., Chao, I.W., Walters, M.S., Wang, R., Hackett, N.R., et al. (2013). RFX3 Modulation of FOXJ1 regulation of cilia genes in the human airway epithelium. Respir. Res. 14(1), 70-70. 
• El Zein, L., Ait-Lounis, A., Morle, L., Thomas, J., Chhin, B., Spassky, N., et al. (2009). RFX3 governs growth and beating efficiency of motile cilia in mouse and controls the expression of genes involved in human ciliopathies. J Cell Sci 122(Pt 17), 3180-3189. 
• Gomperts, B.N., Gong-Cooper, X., and Hackett, B.P. (2004). Foxj1 regulates basal body anchoring to the cytoskeleton of ciliated pulmonary epithelial cells. J. Cell Sci. 117(Pt 8), 1329-1337. 
• 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(3), 339-346. 
• 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(4). bio033944. 
• 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. Br. J. Pharmacol. 166(8), 2243-2262. 
• Rompolas, P., Patel-King, R.S., and King, S.M. (2009). Schmidtea mediterranea: a model system for analysis of motile cilia. Methods Cell Biol. 93, 81-98. 
• 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(12), 1454-1460. 
• Vij, S., Rink, J.C., Ho, H.K., Babu, D., Eitel, M., Narasimhan, V., et al. (2012). Evolutionarily ancient association of the FoxJ1 transcription factor with the motile ciliogenic program. PLoS Genet. 8(11), e1003019. d
• You, Y., Huang, T., Richer, E.J., Schmidt, J.-E.H., Zabner, J., Borok, Z., et al. (2004). Role of f-box factor foxj1 in differentiation of ciliated airway epithelial cells. American Journal of Physiology-Lung Cellular and Molecular Physiology 286(4), L650-L657. 
• Yu, X., Ng, C.P., Habacher, H., and Roy, S. (2008). Foxj1 transcription factors are master regulators of the motile ciliogenic program. Nat. Genet. 40(12), 1445-1453.
• Zhou, F., and Roy, S. (2015). SnapShot: Motile Cilia. Cell 162(1), 224-224 e221. 

Relationship: 2448: Motile Cilia Number/Length, Decreased leads to CBF, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

The cilia in the respiratory epithelium beat in a coordinated fashion at a frequency of approximately 10 to 16 Hz, propelling mucus upwards (Joki et al., 1998; Smith et al., 2012). Many factors, including cilia length, number, structure, orientation as well as mucus viscosity, temperature, pH, chemicals, airway surface liquid height, exposure to bacterial and viral pathogens have been shown to affect ciliary function (Clary-Meinesz et al., 1998; Ho et al., 2001b; Jing et al., 2017; Joki et al., 1998; Kanthakumar et al., 1996; Mall, 2008; Smith et al., 2012; Snyder et al., 2017). Alteration from normal physiological conditions and from healthy cilia number/length/structure typically reduces the cilia beat frequency (CBF) (Clary-Meinesz et al., 1998; Jayathilake et al., 2012).

Evidence Supporting this KER

In Chlamydomonas, ciliary motion is directly related to the length of the cilia (Bottier et al., 2018). Similar observations have been made in zebrafish, where modulation of cilia length and number by FOR20 (centrosomal protein 20) deletion/knockdown directly impairs ciliary motility (Xie et al., 2019). There is also a positive correlation between cilia number and CBF in sinusitis patients (Joki et al., 1998), while cilia number, length and orientation correlate positively with mucociliary transport rate in patients with recurrent or longstanding respiratory infections (Toskala et al., 1995; Joki et al., 1998). Comparisons of strips of normal and disrupted ciliated epithelium have shown that CBF is decreased in the latter (Thomas et al., 2009).
Mathematical models and simulations have shown that periciliary liquid and mucus velocity are directly affected by cilia number and length (Lee et al., 2011; Jayathilake et al., 2012; Jayathilake et al., 2015).
 

Many research groups have shown positive correlations between cilia number/length and ciliary function in studies dating as early as 1995 . In some cases, the authors measured mucociliary clearance as the endpoint, thus the evidence does not describe causality between cilia number/length and CBF. However, the commonly held assumption is that reduced ciliary function, i.e., reduced CBF, leads to decreased mucociliary clearance. Based on the evidence, we judge the biological plausibility of this KER as moderate.

Cilia beating frequency analysis in sinusitis patient samples shows positive correlation between cilia number and CBF (Joki et al., 1998).

Cilia study from patients with recurrent or longstanding respiratory infections demonstrates correlation between mucociliary transport rate and cilia number, length, and orientation (Toskala et al., 1995).

According to numerical method formulated by Jayathilake et al., the average periciliary liquid layer (PCL) velocity decreases when cilia are shortened (Jayathilake et al., 2015; Jayathilake et al., 2012).

A study of Chlamydomonas cilia motion concludes that cilia shorter than 4 µm show variable or no periodicity of beating. For cilia longer than 4 µm, the ratio of frequency estimated from body motion to frequency of cilia motion is very close to 1.0, as expected. In short (2 µm, 4 µm) cilia, the ratio was significantly different from 1; apparently, even if the short cilium beats periodically, the frequency of beating is not reliably transmitted to the body motion (Bottier et al., 2018).

In a simulation study by Lee et al., it was calculated that an increase in cilia number increases the mucus velocity (Lee et al., 2011).

In zebrafish, deletion or knockdown of FOR20 causes reduced number and length of cilia. Ciliary motility is impaired in the animals with reduced FOR20 (Xie et al., 2019).

Cilia beating frequency measurements on normal ciliated or disrupted epithelial strips of tissue revealed lower CBF for tissues with cilia disruptions (Thomas et al., 2009).

Although the majority of studies discuss the effect of shorter cilia on ciliary function, there are also reports on longer than normal cilia impairing cilia function (Jayathilake et al., 2015; Li et al., 2014b). A range of factors other than cilia length and number can influence cilia beat frequency. Often a combination of two or more factors affect ciliary function making it difficult to discern the impact of an individual factor on CBF (Toskala et al., 1995; Xie et al., 2019).

Quantitative Understanding of the Linkage

Cilia beating frequency correlates with cilia size and numbers such that higher number of motile cilia with a healthy length show efficient CBF and reduction of cilia number and/or length result in proportionate reduction of CBF. In some studies, mucociliary clearance was measured as an indicator of CBF. Based on evidence presented here, we judge the quantitative understanding to be moderate.

A study in nasal mucosa samples from sinusitis patients demonstrates that reduced cilia numbers account for decreased CBF. In normally ciliated samples the CBF was 11.2±3.7 Hz, in samples with some ciliated cells CBF was 8.9±6.3 Hz, and in samples with no detectable cilia CBF was 2.1±3.8 Hz (Joki S. et al., 1998).

Patients with recurrent or longstanding respiratory infections were divided into 3 groups based on mucociliary transport rates (MTR). The group with slowest MTR had the biggest number of non-ciliated columnar cells in the nasal mucosa, and the highest number of short and disoriented cilia. Loss of ciliated cells was seen in 50% of specimens with good MTR (7 to 10.8 mm per minute), in 71% with moderate MTR (3 to 6.9 mm per minute) and in 86% with poor MTR (0 to 2.9 mm per minute). Percentage of short cilia was 1% in the first group, 6% in the second, and 10% in the third. In this study, ciliary disorientation also contributed to the low MTR (Toskala et al., 1995).

Jayathilake et al. developed a two-dimensional numerical model for computing how the length of cilia is affecting fluid flow. In the model, the cilia length was reduced by 5%, 15%, 25%, and 35% from 6 µm while other parameters were kept unchanged. The average PCL velocity in stream-wise direction decreases when cilia are shortened . When the cilia are shortened by 10%, the average stream-wise PCL velocity is reduced by about 11%, while the average span-wise PCL velocity is reduced by about 62% (Jayathilake et al., 2012). In a similar study by the same group, the length of cilia was defined as 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130% and 140% of the length of the healthy cilia (i.e., 6 µm). The mucus velocity reaches its maximum value when the ciliary length is around the length of the healthy cilia (or standard length of 6 µm) (Jayathilake et al., 2015).

Cilia shorter than 4 µm have anomalies in the ciliary beating periodicity. Cilia shorter than 2 µm were never found to beat periodically. Cilia between 2 and 4 µm in length exhibited more variable periodicity. Cilia with lengths between 4 and 12 µm beat periodically with conserved frequency (Bottier et al., 2018).

Mucus velocity increased in concordance with cilia number increase. Mean mucus velocity increased from 26.82 µm/s to 49.53 µm/s when cilia number grew from 10 to 24 (Lee et al., 2011).

FOR20 deletion or knockdown in zebrafish reduced the number and length of cilia. Cilia in FOR20 morphants had impaired ciliary motility. When cilia length was reduced from an average 5.5 µm to an average 3 µm due to FOR20 depletion, about 80% cilia displayed consistently paralyzed or arrhythmically beating pattern (Xie et al., 2019).

Nasal brush biopsy samples from pediatric patients with primary ciliary dyskinesia were analyzed. The median CBF was 13.4 Hz for normal ciliated epithelia, 11.4 Hz for the ciliated edge with minor projections and 8.7 Hz for the ciliated edge with major projections (Thomas et al., 2009).

No data

Unknown

Unknown

References

• Bottier, M., Thomas, K., Dutcher, S., and Bayly, P. (2018). (Preprint) How does cilium length affect beating? bioRxiv, 474346. 
• Clary-Meinesz, C., Mouroux, J., Cosson, J., Huitorel, P., and Blaive, B. (1998). Influence of external pH on ciliary beat frequency in human bronchi and bronchioles. Eur. Respir. J. 11(2), 330-333.
• Ho, J.C., Chan, K.N., Hu, W.H., Lam, W.K., Zheng, L., Tipoe, G.L., et al. (2001). The Effect of Aging on Nasal Mucociliary Clearance, Beat Frequency, and Ultrastructure of Respiratory Cilia. Am. J. Respir. Crit. Care Med. 163(4), 983-988.
• Jayathilake, P.G., Le, D.V., Tan, Z., Lee, H.P., and Khoo, B.C. (2015). A numerical study of muco-ciliary transport under the condition of diseased cilia. Comput. Methods Biomech. Biomed. Engin. 18(9), 944-951. 
• Jayathilake, P.G., Tan, Z., Le, D.V., Lee, H.P., and Khoo, B.C. (2012). Three-dimensional numerical simulations of human pulmonary cilia in the periciliary liquid layer by the immersed boundary method. Comput. Fluids 67, 130-137.
• Jing, J.C., Chen, J.J., Chou, L., Wong, B.J.F., and Chen, Z. (2017). Visualization and Detection of Ciliary Beating Pattern and Frequency in the Upper Airway using Phase Resolved Doppler Optical Coherence Tomography. Sci. Rep. 7(1), 8522-8522. 
• Joki, S., Toskala, E., Saano, V., and Nuutinen, J. (1998). Correlation between ciliary beat frequency and the structure of ciliated epithelia in pathologic human nasal mucosa. Laryngoscope 108(3), 426-430. 
• Kanthakumar, K., Taylor, G., Cundell, D., Dowling, R., Johnson, M., Cole, P., et al. (1996). The effect of bacterial toxins on levels of intracellular adenosine nucleotides and human ciliary beat frequency. Pulm. Pharmacol. 9(4), 223-230.
• Lee, W., Jayathilake, P., Tan, Z., Le, D., Lee, H., and Khoo, B. (2011). Muco-ciliary transport: effect of mucus viscosity, cilia beat frequency and cilia density. Comput. Fluids 49(1), 214-221.
• 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(6), 1282-1292. 
• Mall, M.A. (2008). Role of cilia, mucus, and airway surface liquid in mucociliary dysfunction: lessons from mouse models. J. Aerosol Med. Pulm. Drug Deliv. 21(1), 13-24.
• Smith, C.M., Djakow, J., Free, R.C., Djakow, P., Lonnen, R., Williams, G., et al. (2012). ciliaFA: a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software. Cilia 1(1), 14. 
• Snyder, R.J., Hussain, S., Tucker, C.J., Randell, S.H., and Garantziotis, S. (2017). Impaired Ciliogenesis in differentiating human bronchial epithelia exposed to non-Cytotoxic doses of multi-walled carbon Nanotube. Part. Fibre Toxicol. 14(1), 1-14. 
• Thomas, B., Rutman, A., and O'Callaghan, C. (2009). Disrupted ciliated epithelium shows slower ciliary beat frequency and increased dyskinesia. Eur. Respir. J. 34(2), 401-404. 
• Toskala, E., Nuutinen, J., Rautiainen, M., and Torkkeli, T. (1995). The correlation of mucociliary transport and scanning electron microscopy of nasal mucosa. Acta Otolaryngol. 115(1), 61-65. 
• Xie, S., Jin, J., Xu, Z., Huang, Y., Zhang, W., Zhao, L., et al. (2019). Centrosomal protein FOR20 is essential for cilia-dependent development in zebrafish embryos. FASEB J. 33(3), 3613-3622. 

Relationship: 2449: Oxidative Stress leads to CFTR Function, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Available evidence indicates that this KER is applicab le to human, mouse and pig, independent of life stage and gender.

Key Event Relationship Description

Exposure to inhaled oxidants (such as cigarette smoke) leads to decreased CFTR gene and protein expression as well as CFTR internalization, thereby reducing or abolishing open probabilities, short-circuit currents and subsequently ASL height/volume (Cantin et al., 2006a; Cantin et al., 2006b; Clunes et al., 2012; Rasmussen et al., 2014; Sloane et al., 2012). Decreased CFTR mRNA expression was previously linked to reduced mRNA stability following treatment with tert-butylhydroquinone (BHQ) (Cantin et al., 2006a) as well as increased intracellular Ca²⁺, which is thought to activate protein kinases, thereby decreasing transcription rates (Bargon et al., 1992a; Bargon et al., 1992b; Rasmussen et al., 2014). Other evidence suggests that the STAT1 pathway is involved in CFTR down-regulation following ozone exposure or in the presence of interferon-γ (Kulka et al., 2005; Qu et al., 2009). In addition, transcriptional activation of an antioxidant response element in the CFTR promoter by Nrf2 was shown to regulate CFTR gene expression in airway epithelial cells under oxidative stress conditions, leading to an upregulation of transcript levels in the short-term, but a decline in the long-term (Zhang et al., 2015). On the post-transcriptional level, CFTR function was shown to be affected in multiple ways due to oxidative stress: For example, cell surface CFTR expression was drastically diminished in airway epithelial cells following cigarette smoke exposure, involving a change in protein solubility and trafficking to a perinuclear aggresome-like structure rather than to lysosomes or the proteasome (Bodas et al., 2017; Clunes et al., 2012). ATP depletion as a surrogate for lung ischemia also resulted in decreased CFTR expression at the plasma membrane. This was shown to be a consequence of irreversible actin filament depolymerization, which resulted in loss of cell polarity and relocalization of CFTR to the cytoplasm (Brézillon et al., 1997)  .

Evidence Supporting this KER

Inducers of oxidative stress such as cigarette smoke reduced CFTR expression at both the RNA (Cantin et al., 2006a; Cantin et al., 2006b; Qu et al., 2009; Rennolds et al., 2010) and protein (Cantin et al., 2006b; Qu et al., 2009; Rennolds et al., 2010; Sloane et al., 2012; Hassan et al., 2014; Rasmussen et al., 2014; Xu et al., 2015) level in vitro. CFTR protein expression was lower in the airways of smokers compared to non-smokers (Dransfield et al., 2013). In some of these studies, an accompanying decrease in Cl– conductance was also observed (Qu et al., 2009; Rennolds et al., 2010; Sloane et al., 2012). There are many studies that support a direct link between oxidative stress and decreased CFTR function in vitro, ex vivo, in vivo and in human subjects. Human primary epithelial cells and cell lines of respiratory epithelial origin have consistently decreased conductance of Cl– and other ions following exposure to cigarette smoke and other oxidants (Cantin et al., 2006b; Schwarzer et al., 2008; Raju et al., 2013; Lambert et al., 2014; Schmid et al., 2015; Raju et al., 2016; Chinnapaiyan et al., 2018), which could be reversed upon antioxidant treatment (Raju et al., 2013; Lambert et al., 2014; Schmid et al., 2015). Similar observations were made under hypoxic conditions (Brézillon et al., 1997; Zhang et al., 2013; Woodworth, 2015). Antioxidants could also increase Cl– conductance and anion transport in the absence of oxidant treatment or hypoxia induction in human and murine respiratory cells in vitro and in ex vivo tissues (Azbell et al., 2010; Alexander et al., 2011; Conger et al., 2013). Healthy smokers and smokers with COPD have reduced Cl– conductance (Sloane et al., 2012; Dransfield et al., 2013) and increased sweat chloride concentrations (Raju et al., 2013; Courville et al., 2014).

A link between CFTR and chronic bronchitis, which shares some of the features of cystic fibrosis lung disease, was proposed more than 20 years ago. Gene association studies, however, found no clear link between CFTR genotype and COPD except for hereditary disseminated bronchiectasis (Artlich et al., 1995; Joos et al., 2002), leading to the ion channel being of more interest in cystic fibrosis than chronic bronchitis research for some time. More recent evidence points toward a mechanism of acquired CFTR dysfunction in the context of cigarette smoking, which remains the leading risk factor for COPD. Multiple studies clearly demonstrated that CFTR Cl⁻ conductance is significantly inhibited following cigarette smoke exposure in vitro and in vivo, resulting in reductions of ASL height/volume which ultimately impair MCC (Cantin. et al., 2006b; Clunes et al., 2012; Courville et al., 2014; Dransfield et al., 2013; Hassan et al., 2014; Raju et al., 2013; Rasmussen et al., 2014; Sailland et al., 2017; Schmid et al., 2015). Although there appear to be different schools of thought as to how cigarette smoke modulates CFTR (directly or indirectly), the available evidence on the inhibitory effects of cigarette smoke on CFTR anion transport is conclusive. Considering the similarities between cigarette smoke exposure-related oxidative stress in the airways and oxidative stress arising from e.g. the exposure to other inhalation toxicants and pathogens, the described mechanisms are likely to apply to other stressors eliciting an imbalance in the lung’s redox state. Moreover, studies with antioxidants such as NAC, resveratrol, genistein and hesperidin confirm the role of oxidative stress in modulating CFTR function (Alexander et al., 2011; Conger et al., 2013; Raju et al., 2013; Woodworth, 2015; Zhang et al., 2013). Taken together, the described cause-effect relationship is biologically plausible, and our confidence in biological plausibility is strong.

CFTR transcript and protein levels were reduced in Calu-3 lung cancer cells exposed to the gas phase of cigarette smoke (Cantin et al., 2006b; Rasmussen et al., 2014), immortalized human bronchial epithelial 16HBE14o- cells treated with 10% cigarette smoke extract (Hassan et al., 2014; Xu et al., 2015), and differentiated primary human bronchial epithelial cells exposed to cigarette smoke extract (Sloane et al., 2012) and whole cigarette smoke (Hassan et al., 2014). Consequently, CFTR membrane localization was decreased as was channel function (i.e., cAMP-dependent ¹²⁵I efflux or CFTR Cl⁻ conductance).

Ozone exposure decreased CFTR mRNA and protein expression in immortalized human bronchial epithelial 16HBE14o- cells and in tracheas of Wistar rats, leading to a reduction in forskolin-stimulated CFTR Cl⁻ conductance  (Qu et al., 2009).

Cadmium (Cd) decreased CFTR protein expression in Calu-3 lung cancer cells in a dose- and time-dependent manner, but only transiently affected CFTR gene expression. However, reduced CFTR expression at the plasma membrane was associated with a reduction in CFTR Cl⁻ conductance (Rennolds et al., 2010). 

Acrolein exhibited a complex dose-dependent response with respect to CFTR-mediated Cl⁻ transport in primary murine nasal septal epithelia: At 100 µM acrolein, Cl⁻ currents increased, whereas 300 µM acrolein reduced forskolin-induced total apical Cl⁻ secretion and 300 µM acrolein abolished all Cl⁻ transport. These effects were independent of cAMP, suggesting that channel activation was not PKA/cAMP phosphorylation-dependent (Alexander et al., 2012). Acute acrolein exposure also decreased cAMP-mediated CFTR ion transport in human bronchial epithelial cells grown in monolayers and in Calu-3 lung cancer cells, where the response was dose-dependent. Repeated, low-level exposure to acrolein (2.5 – 10 ng/mL for 7 days) had a similar effect on CFTR function and was shown to be unrelated to modulation of CFTR expression. However, pretreatment with the antioxidant N-acetylcysteine could prevent acrolein-induced CFTR inhibition (Raju et al., 2013).

There is currently only limited knowledge about the mechanism by which oxidative stress may affect CFTR expression and function. At least one study indicates that CFTR channel gating is decreased following exposure to acrolein, possibly by protein carbonylation (Raju et al., 2013). Other studies reports that cell surface CFTR expression was drastically diminished in airway epithelial cells following cigarette smoke exposure, involving a change in protein solubility and trafficking to a perinuclear aggresome-like structure rather than to lysosomes or the proteasome (Bodas et al., 2017; Clunes et al., 2012b).  

Quantitative Understanding of the Linkage

There is a ample quantitative evidence that provides an insight into this KER, with respect to both a response-response relationship and the temporal relationship between the two KEs across different test systems. Based on the evidence presented here, we judge our quantitative understanding to be high.

CFTR Gene & Protein Expression

In Calu-3 cells exposed to cigarette smoke (drawn into a syringe and injected into the 5-L exposure chamber at a rate of 35 mL/min) for 10 min every 2 h for a total of four exposures CFTR mRNA expression decreased by approx. half, from 0.87 ± 0.03 to 0.47 ± 0.03 units (CFTR/GAPDH ratio; northern blot). CFTR protein expression decreased from 2.07 ± 0.24 to 1.24 ± 0.14 units (CFTR/actin ratio; western blot) (Cantin et al., 2006b).

Exposure of fully differentiated primary human bronchial epithelial cells (HEBCs) to 30 puffs of whole smoke from 2 cigarettes (generated according to ISO standard) every day for 5 days (total exposure 120 hr) resulted in a ca. 40% reduction in CFTR expression. Exposure of 16HBE14o- airway epithelial cells to cigarette smoke extract (CSE; smoke from one non-filtered Camel cigarette was bubbled using a peristaltic pump apparatus into 10 mL of complete culture media, defined as 100%) led to a dose-dependent decrease in CFTR protein expression which, with approx. –70% from baseline, was significant for concentrations ≥ 10% (Hassan et al., 2014).

Apical treatment of fully differentiated primary HBECs with 2% CSE (not further described) for 24 h resulted in approx. 30% reduction in CFTR mRNA expression, 50% reduction in total CFTR protein and 30% reduction in CFTR protein cell surface expression (Sloane et al., 2012).

Treatment of 16HBE14o- immortalized HBECs with 10% CSE (smoke from one non-filtered Camel cigarette was bubbled using a peristaltic pump apparatus into 10 mL of complete culture media, defined as 100%) resulted in more than 50% reduction of CFTR total and cell surface protein expression. Co-treatment with 10 mM NAC prevented this decrease in CFTR expression (0.5 mM NAC was not effective) (Xu et al., 2015).

Treatment of fully differentiated primary HBECs with 2% CSE (bubbling 10 puffs of smoke from one 3R4F reference into 1 mL DMSO, at 2 s/10 mL puff, 10 puffs over 3 min; defined as 100%) for 24 h decreased total CFTR expression and cell surface CFTR expression by approx. 20 and 25%, respectively (Raju et al., 2016a).

CFTR protein expression was significantly lower in endobronchial biopsies of healthy smokers (33 [12-54] pack-years; CFTR/tubulin ratio: 0.70), smokers with COPD (52.5 [35-147] pack-years; CFTR/tubulin ratio: 0.50-0.88), and former smokers (45 [39-80] pack-years; CFTR/tubulin ratio: 1.75) with COPD than in healthy non-smokers (0 pack-years; CFTR/tubulin ratio: 4.09 – 4.49) (Dransfield et al., 2013).

Exposure of Calu-3 lung cancer cells to 50 µM cadmium sulfate for 1 or 3 days significantly reduced CFTR protein expression by approx. 20 and 50%, respectively, whereas exposure to lower doses of cadmium (2 µM) for 3 days did not affect CFTR protein levels (Rennolds et al., 2010).

At 4 hours post-exposure to 1.5 ppm ozone for 30 min, CFTR mRNA and protein expression were more than 2-fold decreased (CFTR/GAPDH ratio: 1.35±0.3 (control) vs 0.46±0.07; CFTR/actin ratio: 0.81±0.02 vs 0.33±0.02) in 16HBE14o- airway epithelial cells (Qu et al., 2009).

CFTR Channel Function

Treatment of T84 cells with 100 µM tert-butylhydroquinone (BHQ) for 6 h caused oxidative stress, evidenced by a significant increase in intracellular glutathione concentrations (44.8 ± 0.6 vs. 32.3 ± 0.3 nmol/10⁶ cells at 0 hr) and reduced peak cAMP-dependent ¹²⁵I effluxes in a dose-dependent manner; however, only at BHQ concentrations ≥ 300 µM was the reduction significant (Cantin et al., 2006a).

In murine nasal septal epithelia grown at the air-liquid interface, resting Cl⁻ secretion was maximally stimulated by 100 µM acrolein (15.9±2.2 vs. 2.4±0.8 μA/cm² (control)), whereas forskolin-sensitive ion current was inhibited by 300 μM (13.3±1.2 vs. 19.9±1.0 μA/cm² (control); max. and significant) and completely eliminated by 500 μM acrolein (Alexander et al., 2012).

Following exposure of primary HBECs to 3.2 μg/mL acrolein for 24 h, forskolin-stimulated ion currents were inhibited by approx. 50%. Noticeable inhibition occurred at concentrations above 2 μg/ml, and maximum inhibition was seen with ca. 5 μg/mL acrolein; higher concentrations did not further decrease ion currents. Repeated exposure to 10 ng/mL acrolein for 7 days also resulted in an approx. 50% decrease in forskolin-stimulated ion currents (Raju et al., 2013).

Exposure of fully differentiated primary HBECs to whole smoke from four 3R4F reference cigarettes (generated according to ISO standard 3308; Vitrocell VC10 exposure system) resulted in a significant decrease in Cl⁻ currents (2.1±0.26 vs 4.8±0.71 μA/cm² vs 4.8 ± 0.71 μA/cm² (control)) (Schmid et al., 2015).

Resveratrol dose-dependently increased CFTR-mediated anion transport in murine sinonasal epithelial cells, with no effect seen for 50 μM, maximal increase (while not affecting total stimulation) seen for 100 μM, and slight inhibitory effects seen for concentrations ≥200 μM (Alexander et al., 2011; Woodworth, 2015). Forskolin-stimulated Cl⁻ currents increased in murine (14.2±1.5 vs. 0.8±0.2 mA/cm² (control)), human (17.4±.7 vs. 1.0±0.2 mA/cm² (control)(Woodworth, 2015), 15.69±2.66 vs. 2.49±0.98 mA/cm² (control)(Alexander et al., 2011)) and porcine (6.8±0.3 vs. 1.1±0.3 mA/cm² (control)) sinonasal epithelial cells following treatment with 100 μM resveratrol. Resveratrol treatment (100 μM) also restored CFTR Cl⁻ transport in hypoxic murine sinonasal epithelial cells (11.51±0.23 vs. 0.2±0.05 mA/cm² (control)) and human sinonasal epithelial cells (10.8±0.7 vs. 0.3±0.05 mA/cm² (control)) (Woodworth, 2015; Zhang et al., 2013).
In C57B/L6 mice perfused with 100 μM resveratrol, mean nasal potential difference polarization increased to −4.0±1.87 mV (vs. −0.93±1.69 mV, control), which was slightly higher than results with forskolin (−1.65±1.78 mV), but not significant (Alexander et al., 2011).

Genistein (50 μM) enhanced basal CFTR-mediated anion transport in human sinonasal epithelial cells (23.1±1.8 vs. 0.7±0.2 μA/cm² (control)), but had no effect on forskolin-sensitive current (Conger et al., 2013).

Hesperidine dose-dependently increased CFTR-mediated anion transport in murine sinonasal epithelial cells, with the maximum responses seen for 2 mM (16.67±0.43 µA/cm²) which was not further investigated due to the precipitation of hesperidine. Instead, 1 mM hesperidine was used as it also significantly increased CFTR Cl⁻ currents (mouse cells: 13.51±0.77 vs. 4.4±0.66 µA/cm² (control); human cells: 12.28±1.08 vs. 0.69±0.32 µA/cm² (control)). In C57B/L6 mice perfused with 1 mM hesperidine, mean nasal potential difference polarization was increased (−2.3±1.0 mV vs. −0.8±0.8 mV (control)), similar to results with forskolin (−1.9±1.4 mV) (Azbell et al., 2010).

Basal lower airway potential difference (LAPD) was lower in healthy smokers (-7.71±0.88 mV; 33 [12-54] pack-years) and COPD smokers (-7.33±1.30 mV; 52.5 [32-147] pack-years) than in former smokers with COPD (no data; 45 [39-80] pack-years) or healthy non-smokers (-12.61±1.94 mV)(Dransfield et al., 2013).

Healthy smokers, current and former smokers with COPD had lower mean sweat chloride than healthy non-smokers (51.3±4.4 [31.08±14 pack-years], 41.9±3.4 [38±19 pack-years], and 39.0±5.4 [44±19 pack-years], respectively, vs. 53.6±3.3 mmol/L). The association between sweat chloride and pack-years was significant (Courville et al., 2014).

Healthy smokers and smokers with COPD exhibited significantly lower nasal potential difference than healthy non-smokers in response to isoproterenol (-6.3±1.4 [33.2; 10-78 pack-years] and -8.0±2.0 [55.1; 35-78 pack-years], respectively, vs. -15.2±2.7 mV (control)) (Sloane et al., 2012).

Treatment of wild-type CFTR expressing HEK293 cells with 1% CSE (bubbling 10 puffs of smoke from one 3R4F reference into 1 mL DMSO, at 2 s/10 mL puff, 10 puffs over 3 min; defined as 100%) decreased CFTR channel-open probability by 59% (Raju et al., 2016a).

Single channel recordings in apical membrane patches of murine sinonasal epithelial cells demonstrated that 100 µM resveratrol significantly enhanced channel-open probability  (0.329± 0.116 vs. 0.119±0.059 NPo/N (control)) (Woodworth, 2015).

In T84 cells exposed to 100 µM BHQ, intracellular GSH levels were significantly increased from 6 hours onwards (from 32.3±0.3 to 44.8±0.6 nmol/10⁶ cells), up to 24 hours (from 45.5±0.9 to 94.9±2.5 nmol/10⁶ cells), whereas CFTR mRNA expression was significantly decreased over time (from 8.2±0.8 to 1.8±0.3 CFTR/GAPDH mRNA ratio at 6 hours). As a consequence, CFTR protein expression was significantly decreased following treatment of T84 cells with 100 µM BHQ for 24 hours (from 101.3±4.6 to 84.7±4.1 CFTR/actin protein, density as % control) (Cantin et al., 2006a). 

Exposure of T84 cells to cigarette smoke condensate (CSC; prepared by drawing 35 mL/min cigarette smoke for 5 min into a syringe and injecting this smoke into a tonometer containing 10 mL culture media; defined as 100%) for 10 min resulted in a significant increase in GCLC mRNA expression (from 0.69±0.08 to 2.44± 0.25 GCLC/GAPDH mRNA ratio) and decrease in CFTR mRNA expression (from 0.57±0.04 to 0.21±0.02 CFTR/GAPDH mRNA ratio) at 6 hours post-exposure and a significant increase in GSH at 24 hours post-exposure (from 41.7±1.0 to 89.8±1.5 nmol/mg protein) (Cantin et al., 2006b).

Exposure of immortalized 16HBE14o- airway epithelial cells to 10% cigarette smoke extract (CSE; cigarette smoke from one non-filtered cigarette was bubbled using a peristaltic pump apparatus into 10 mL of complete culture media; defined as 100%) led to an approx. 50% decrease in CFTR mRNA expression and approx. 70% decrease in CFTR protein expression after 24 hours, with little change at later time points (Hassan et al., 2014).

Treatment of Calu-3 lung cancer cells with 50 µM cadmium sulfate significantly reduced CFTR protein expression after 1 day, with maximum decrease observed after 3 days (54 ± 5% of control). Exposure to lower doses of cadmium (2 µM) required 5-day treatment before CFTR protein levels were affected (Rennolds et al., 2010).

Treatment of fully differentiated primary HBECs with 2% CSE (bubbling 10 puffs of smoke from one 3R4F reference into 1 mL DMSO, at 2 s/10 mL puff, 10 puffs over 3 minutes; defined as 100%) for 24 hours decreased total CFTR expression and cell surface CFTR expression by approx. 20 and 25%, respectively, but treatment for 20 minutes did not. A 50% reduction in CFTR channel activity occurred immediately after addition of CSE and lasted for at least 20 minutes (Raju et al., 2016a).

Calu-3 lung cancer cells exposed to cigarette smoke (drawn into a syringe and injected into the 5-L exposure chamber at a rate of 35 mL/min) for 10 min every 2 h for a total of four exposures were loaded with ¹²⁵I for 1 hour, prior to stimulation with isobutyl methylxanthine, forskolin, and dibutyryl cAMP. cAMP-dependent anion efflux was significantly decreased compared to controls within 3 min of stimulation and remained significantly decreased for a further 3 min (Cantin et al., 2006b).

In untreated monolayers of CFTR-corrected CFBE41o- airway epithelial cells, cAMP-stimulated Cl^– currents remained at stimulated levels for 2 to 3 h, whereas currents were inhibited by 86.0±5.8 and 40.0±2.7% in cells treated with 100 μM pyocyanin or 100 μM H₂O₂, respectively, in the same time period. The effect of pyocyanin occurred at a faster rate than that of H₂O₂; washout of the compounds partly restored cAMP-stimulated Cl⁻ currents (Schwarzer et al., 2008).

Exposure of fully differentiated primary HBECs to whole smoke (3R4F reference cigarette; inExpose exposure system) resulted in a time-dependent decrease in CFTR short-circuit currents, with significant differences (approx. 15% reduction) from control after 10 min of exposure. Maximal reduction (ca. 50%) was seen after 30 min of exposure (Lambert et al., 2014).

Culture of fully differentiated primary human and murine sinonasal epithelial cells (HSNECs; MSNECs) in 1% O₂ for 12 or 24 h significantly reduced CFTR-mediated (forskolin-sensitive) Cl⁻ current: HSNECs, 19.55±0.56 mA/cm² (12 h); 17.67±1.13 mA/cm² (24 h) vs. 25.49±1.48 mA/cm² (control); MSNECs, 13.55±0.46 mA/cm² (12 h); 12.75±0.07 mA/cm² (24 h) vs. 19.23±0.18 mA/cm² (control). Transfer of cultures to physiologic O₂ conditions (21%) restored CFTR ion currents (HSNECs, 25.12±1.24 mA/cm²) after 24 h (Woodworth, 2015).

CFTR has also been implicated in transmembrane glutathione transport (Linsdell and Hanrahan, 1998; Roum et al., 1993). Multiple studies suggest that oxidative injury of the lungs, e.g. following inhalation exposures or infections, can be effectively counteracted, if not prevented, by CFTR-mediated elevations of ASL glutathione levels (Day et al., 2004; Gould et al., 2010; Jungas et al., 2002; Kariya et al., 2007; Velsor et al., 2001). The antioxidant properties of glutathione may temporarily delay the acquisition of CFTR dysfunction by neutralizing reactive oxygen species that would otherwise contribute to downregulation of gene and protein expression.

Not known

References

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Relationship: 2450: Oxidative Stress leads to FOXJ1 Protein, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

Oxidative stress (such as that caused by cigarette smoke exposure or irradiation) leads to decreased forkhead box J1 (FOXJ1) gene and protein expression, as well as to decreased FOXJ1 target gene expression (Brekman et al., 2014; Garcia-Arcos et al., 2016; Ishikawa and Ito, 2017; Milara et al., 2012; Valencia-Gattas et al., 2016). FOXJ1 is a key factor of multiple motile cilia assembly in the respiratory airways (Zhou and Roy, 2015). Thus oxidative stress blocks the multiple ciliogenesis program in the airway epithelium.

Evidence Supporting this KER

Cigarette smoke-induced oxidative stress downregulates FOXJ1 expression at both the gene and protein levels in human lung cells in vitro (Milara et al., 2012; Brekman et al., 2014; Valencia-Gattas et al., 2016; Ishikawa and Ito, 2017). Oxidative stress induced by human respiratory syncytial virus reduces FOXJ1 mRNA levels, which can be restored by treatment with antioxidants or the phosphodiesterase 4 inhibitor roflumilast N-oxide (Akaike et al., 1990; Geiler et al., 2010; Mata et al., 2012). In mice, thoracic irradiation results in free radical generation and subsequent reduction in FOXJ1 mRNA expression (Bernard et al., 2012). Many genes that are transcriptionally regulated by FOXJ1 are also downregulated following exposure to cigarette smoke, which implies a reduction in FOXJ1 transcriptional activity (Brekman et al., 2014).

The negative association between cigarette smoke exposure and FOXJ1 levels in airways was shown in multiple studies and can be estimated as a strong linkage. Yet, the notion that oxidative stress as a result of cigarette smoke exposure is leading to decreased FOXJ1 levels is not well demonstrated. As a complex mixture of thousands of chemicals, cigarette smoke exposure could lead to reduced FOXJ1 levels via different routes. Indirect evidence, such as antioxidant molecules that restore cigarette smoke exposure-reduced FOXJ1 levels, as well evidences from other oxidative stress generating insults that decrease FOXJ1 levels add confidence to this KER. However, studies showing a link between oxidative stress generating agents and reduced FOXJ1 levels are scarce. Collectively, the empirical evidence and uncertainties of the linkage imply a moderate ranking for the KER.

Whole cigarette smoke exposed normal human bronchial epithelial cells (HBECs) had significantly lower FoxJ1 protein levels than air treated controls (measured by immunofluorescence). In addition, FOXJ1 mRNA levels were reduced in whole smoke-exposed differentiating and differentiated cells (TaqMan quantitative RT-PCR). (Valencia-Gattas et al., 2016).

Treatment with cigarette smoke extract (CSE) significantly down-regulated FOXJ1 mRNA and protein levels in differentiating human airway basal cells in air–liquid interface (ALI) cultures (TaqMan quantitative RT-PCR, Western analysis) (Brekman et al., 2014).

Treatment with cigarette smoke extract (CSE) provokes a reduction of FOXJ1 gene and protein expression in differentiated human bronchial epithelial cells (HBECs) through IL13-mediated mechanism (RT-qPCR, Western analysis) (Milara et al., 2012). Treatment with roflumilast N-oxide (which reduced intracellular reactive oxygen species levels) prevents FOXJ1 loss in CSE treated cells (Milara et al., 2012).

Exposure of 3D co-cultures with HBECs and fibroblasts to whole cigarette smoke decreased FOXJ1 gene expression in a concentration-dependent manner (TaqMan quantitative RT-PCR) (Ishikawa Shinkichi and Ito, 2017).

Human respiratory syncytial virus (RSV) infections involve reactive oxygen intermediates (ROIs) that cause cellular damage (Akaike et al., 1990; Mata et al., 2012). Treatment with the free radical scavenger N-acetylcysteine (NAC) reduces the RSV inflammatory response (Geiler et al., 2010). RSV infection reduced FOXJ1 gene expression (RT-PCR), which was restored in a dose-dependent manner by NAC treatment (Mata et al., 2012). In another study FOXJ1 mRNA levels were consistently low after RSV infection and were restored with Roflumilast N-oxide (Mata et al., 2013).

Thoracic irradiation reduces FOXJ1 mRNA levels in mouse lungs (Bernard et al., 2012). Irradiation causes excessive levels of free radicals and associated lipid peroxidation, damage to DNA, proteins, leading to wide-spread cellular damage (Azzam et al., 2012; Koc et al., 2003; Rodrigues-Moreira et al., 2017; Shirazi et al., 2013).

The expression of cilia-related transcription factor genes, including FOXJ1, RFX2, and RFX3 was significantly down-regulated by CSE treatment. The expression of cilia motility and structural integrity genes, including DNAI1, DNAH5, DNAH9, DNAH10, DNAH11, and SPAG6 was also significantly down-regulated by CSE treatments (Brekman et al., 2014). Many of these genes (RFX2, RFX3, DNAI1, DNAH9, DNAH11, SPAG6) are transcriptionally regulated by FOXJ1 (Causal biological network database, 2019). The downregulation of FOXJ1-controlled genes infer reduced FOXJ1 transcription factor activity. Indeed, overexpression of FOXJ1 led to partial restoration of CSE treatment-induced downregulation of cilia-related genes (Brekman et al., 2014).

Schamberger et al. did not find any alterations in FOXJ1 mRNA levels or FOXJ1 target gene (DNAI1, DNALI1, SPAG6, TEKT1) transcription upon 2.5% or 5% CSE exposure of HBECs for 28 days. However, in this study, cigarette smoke exposure reduced ciliated cell numbers (Schamberger et al., 2015).

The evidences listed suggest several mechanisms on how oxidative stress could lead to decreased FOXJ1 levels, including EGFR-, MCIDAS- or IL-13-mediated mechanisms. Most of the studies, however, do not corroborate on how oxidative stress mechanistically leads to reduced FOXJ1 levels. Since there are several other factors (GMNC, NOTCH, ULK4 etc.) known to regulate FOXJ1 levels, further pathways might be involved in passing the oxidative stress signal to FOXJ1.
 

Quantitative Understanding of the Linkage

High oxidative stress causes reduction in FOXJ1 levels measured at 24 h and for up to 15 days after exposure to an oxidative stress-causing agent. The data on cigarette smoke-reduced FOXJ1 levels are convincing. Indirect evidence such as antioxidants restoring CS-reduced FOXJ1 levels suggest that oxidative stress plays a major role in the CS-induced effects (Milara et al., 2012). However, given the complexity of the CS mixtures (Baumung et al., 2016), we cannot exclude that factors other than oxidative stress are involved in FOXJ1 level reduction. Other sources of oxidative stress such as RSV infection or GY radiation reduce FOXJ1 levels to similar degree as CS-caused FOXJ1 reduction. Based on the available evidence, we classify the quantitative understanding of this KER as moderate.

Normal HBECs were exposed to whole cigarette smoke from 3R4F research grade cigarettes using the Vitrocell® VC 10® Smoking Robot (35-mL puff volume, 2 s duration and 1 min between puffs or air as a control). For differentiated cells, treatment was done every 2 d for 5 d (3 exposures) and samples were collected 48 h after treatment. Differentiating cells were exposed 3 times per week to smoke from 1 cigarette, and samples were collected after 14, 21 and 27 days. FOXJ1 protein and mRNA level changes were decreased 2.5-fold in differentiated and 2-fold in differentiating NHBE cells. There was a significantly lower percentage of FoxJ1 positive cells in the WCS exposed cells at 27 d of differentiation (4.3 +/- 4.2% vs. 13.0 +/- 7.3%, air)  (Valencia-Gattas et al., 2016).

CSE was obtained from one Marlboro Red commercial cigarette bubbled in 12.5 mL of differentiation medium that was then filtered (0.2-µm pore filter). The absorbance was measured at 320 nm on a spectrophotometer, and the optical density of 1 was defined as 100%. HBECs were differentiated at the air-liquid interface while being exposed to 0, 3, and 6% CSE between days 5 and 28. 3% and 6% CSE treatment reduced FOXJ1 mRNA levels to approx. 65% and 55% of control levels, respectively. Treatment of differentiating cultures with 3% CSE reduced FOXJ1 protein levels by 2-fold by day 28 (Brekman et al., 2014).

The smoke of one 2R4F research cigarette was bubbled into a flask containing 25 mL of pre-warmed (37°C) differentiation medium using a respiratory pump model (Harvard Apparatus Rodent Respirator 680, Harvard Apparatus, Holliston, MA, USA) that generates three puffs min⁻¹; 35 mL per each puff of 2 s duration with a volume of 0.5 cm above the filter. The solution was then filtered (0.22 µm pore size) to remove particles and the tar phase. The resulting sterile solution was defined as 100% CSE and used within 30 min of preparation. Treatment of differentiated human bronchial epithelial cells with 10% CSE decreased FOXJ1 expression by about 40% at 24 h and 70% at 72 h exposure (Milara et al., 2012).

3R4F reference cigarettes were smoked in accordance with the ISO smoking protocol (35-mL puffs of 2 s each minute). Whole CS, generated by a VC10 smoking robot, was released into a mixing device in 2.8-s exhaust and diluted with humidified clean air at 1.0 L/min dilution flow. Diluted smoke was introduced into the CULTEX RFS module and guided into the exposure chamber (5 mL/min) using a vacuum pump. FOXJ1 mRNA levels were reduced to 60% and 40% of the control after exposure to CS from 1 or 4 cigarettes, respectively (Ishikawa and Ito, 2017).

RSV infection elicits ROI-mediated effects manifested by changes in the expression of NRF2 and HMOX-1 genes (approx. 6- and 9-fold increase at day 15 post-RSV infection, respectively), H₂O₂ generation (7-fold increase in intracellular levels at day 15 post-infection) and severe reduction in total antioxidant capacity. These data together indicate the presence of oxidative stress following infection which leads to decreased FOXJ1 mRNA levels (ca. 25% of control at 15 days post-RSV infection (Mata et al., 2012) and ca. 45% of control at 10 days after RSV infection (Mata et al., 2013).

FOXJ1 mRNA levels were reduced by 50% in murine lungs 14 days after thoracic irradiation at 15 Gy (Bernard et al., 2012).

Whole cigarette smoke exposure from one 3R4F research grade cigarette using the Vitrocell® VC 10® Smoking Robot (35-mL puff volume, 2 s duration and 1 min between puffs or air as a control) once a day on alternate days for 5 days decreased FOXJ1 mRNA levels by 2.5-fold in differentiated HBECs (Valencia-Gattas et al., 2016).

Whole cigarette smoke exposure from one 3R4F research grade cigarette using the Vitrocell® VC 10® Smoking Robot (35-mL puff volume, 2 s duration and 1 min between puffs or air as a control) for 3 times per week for 4 weeks (27 days) decreased FOXJ1 mRNA levels by 2-fold in differentiating HBECs (Valencia-Gattas et al., 2016).

Treatment of differentiating HBECs between days 5 and 28 with 3% CSE reduced FOXJ1 protein levels by 2-fold by day 28. Treatment of differentiating HBECs between days 5 and 28 with 6% CSE reduced FOXJ1 protein levels by approx. 55% by day 28 (Brekman et al., 2014).

Treatment of differentiated human bronchial epithelial cells with 10% CSE decreased FOXJ1 expression by about 40% at 24 h and 70% at 72 h (Milara et al., 2012). 

Repeated exposure of 3D bronchial epithelial cultures to whole smoke of 4 cigarettes (every other day, treatment started on ALI culture day 7) resulted in 2.5-fold decrease of FOXJ1 mRNA levels by day 21 (Ishikawa and Ito, 2017).

At 15 days post-RSV infection, FOXJ1 mRNA levels were four-fold reduced compared to untreated samples (Mata et al., 2012). In another study from the same research group, FOXJ1 mRNA levels were reduced to 45% of the FOXJ1 levels in uninfected sample 10 days post-RSV infection (Mata et al., 2013).

At 7 days and 14 day after 15 GY thoracic irradiation, FOXJ1 mRNA levels were reduced to approx. 70% and 50% of controls, respectively (Bernard et al., 2012).

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References

• Akaike, T., Ando, M., Oda, T., Doi, T., Ijiri, S., Araki, S., et al. (1990). Dependence on O2- generation by xanthine oxidase of pathogenesis of influenza virus infection in mice. J. Clin. Investig. 85(3), 739-745. 
• Azzam, E.I., Jay-Gerin, J.P., and Pain, D. (2012). Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury. Cancer Lett 327(1-2), 48-60. 
• Baumung, C., Rehm, J., Franke, H., and Lachenmeier, D.W. (2016). Comparative risk assessment of tobacco smoke constituents using the margin of exposure approach: the neglected contribution of nicotine. Sci Rep 6, 35577.
• Bernard, M.E., Kim, H., Rajagopalan, M.S., Stone, B., Salimi, U., Rwigema, J.C., et al. (2012). Repopulation of the irradiation damaged lung with bone marrow-derived cells. In Vivo 26(1), 9-18.
• 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(5), 688-700.
• Garcia-Arcos, I., Geraghty, P., Baumlin, N., Campos, M., Dabo, A.J., Jundi, B., et al. (2016). Chronic electronic cigarette exposure in mice induces features of COPD in a nicotine-dependent manner. Thorax 71(12), 1119-1129. 
• Ishikawa, S., and Ito, S. (2017). Repeated whole cigarette smoke exposure alters cell differentiation and augments secretion of inflammatory mediators in air-liquid interface three-dimensional co-culture model of human bronchial tissue. Toxicol. In Vitro 38, 170-178.
• Koc, M., Taysi, S., Buyukokuroglu, M.E., and Bakan, N. (2003). Melatonin protects rat liver against irradiation-induced oxidative injury. J Radiat Res 44(3), 211-215. 
• Mata, M., Martinez, I., Melero, J.A., Tenor, H., and Cortijo, J. (2013). Roflumilast inhibits respiratory syncytial virus infection in human differentiated bronchial epithelial cells. PLoS One 8(7), e69670. 
• Mata, M., Sarrion, I., Armengot, M., Carda, C., Martinez, I., Melero, J.A., et al. (2012). Respiratory syncytial virus inhibits ciliagenesis in differentiated normal human bronchial epithelial cells: effectiveness of N-acetylcysteine. PloS one 7(10), e48037.
• 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. Br. J. Pharmacol. 166(8), 2243-2262. 
• Rodrigues-Moreira, S., Moreno, S.G., Ghinatti, G., Lewandowski, D., Hoffschir, F., Ferri, F., et al. (2017). Low-Dose Irradiation Promotes Persistent Oxidative Stress and Decreases Self-Renewal in Hematopoietic Stem Cells. Cell Rep 20(13), 3199-3211. 
• Schamberger, A.C., Staab-Weijnitz, C.A., Mise-Racek, N., and Eickelberg, O. (2015). Cigarette smoke alters primary human bronchial epithelial cell differentiation at the air-liquid interface. Scientific reports 5, 8163.
• Shirazi, A., Mihandoost, E., Ghobadi, G., Mohseni, M., and Ghazi-Khansari, M. (2013). Evaluation of radio-protective effect of melatonin on whole body irradiation induced liver tissue damage. Cell J 14(4), 292-297.
• Valencia-Gattas, M., Conner, G.E., and Fregien, N.L. (2016). Gefitinib, an EGFR Tyrosine Kinase inhibitor, Prevents Smoke-Mediated Ciliated Airway Epithelial Cell Loss and Promotes Their Recovery. PloS one 11(8), e0160216. 
• Zhou, F., and Roy, S. (2015). SnapShot: Motile Cilia. Cell 162(1), 224-224 e221. 

Relationship: 2451: Oxidative Stress leads to CBF, Decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

Because the lung interfaces with the external environment, it is frequently exposed to airborne oxidant gases and particulates, and thus prone to oxidant-mediated cellular damage (Ciencewicki et al., 2008). Oxidant stress—through the action of exogenous and endogenous free radicals, such as super oxides, hydroxyl radicals, and hydrogen peroxides—is a common factor in lung inflammation and various respiratory diseases. The presence of redox-sensitive proteins in motile cilia suggests that oxidant stresses may impact ciliary function negatively (Price and Sisson, 2019). Indeed, exposure of human or rodent ciliated airway epithelial cells to hydrogen peroxide, acetaldehyde, ozone or cigarette smoke—all of which are known to cause oxidative stress—decreases CBF in a dose- and time-dependent manner (Bayram et al., 1998; Burman and Martin, 1986; Gosepath et al., 2000; Hastie et al., 1990; Helleday et al., 1995; Kienast et al., 1994; Knorst et al., 1994a; Min et al., 1999; Simet et al., 2010).

Evidence Supporting this KER

Experimental studies in vitro have shown that exposure of ciliated respiratory cells directly or indirectly to sources of oxidative stress leads to decreased CBF (Burman and Martin, 1986; Wilson et al., 1987; Feldman et al., 1994; Yoshitsugu et al., 1995; Min et al., 1999), which can be reversed by treatment with antioxidants (Schmid et al., 2015). Cigarette smoke condensate, a known inducer of oxidative stress, also causes a decrease in CBF in vitro (Cohen et al., 2009), while, in human subjects exposed to different oxygen levels, oxygen stress causes a decrease in nasal CBF (Stanek et al., 1998).
 

One mode of antimicrobial defense in the airway epithelium is generation of free radicals by neutrophils and monocytes/macrophages. Some microbes have also been shown to produce oxidants in significant amounts, e.g. H₂O2 production by pneumococcus. Several studies have shown that oxidants, irrespective of the source (microbial or host-derived) inhibit ciliary function. Additionally, there is a large body of experimental evidence demonstrating that exposures to environmental oxidants, including volatile aldehydes, peroxides, sulfur dioxide, nitric dioxide and Diesel exhaust particles have a detrimental impact on ciliary function. Therefore, this KER is plausible.

Tretament with H₂O₂ causes a dose-dependent decline in ciliary beat frequency (CBF) in tracheal rings from male Sprague-Dawley rats (Burman and Martin, 1986).

Exposure of ciliated epithelial cells to enzymatically generated oxidants (xanthine/xanthine oxidase) was accompanied by ciliary slowing, which was maximal at the end of the experiment (4 h). After 4 h, the reduction in CBF was 37.4%. Catalase alone, or in combination with SOD, completely protected the epithelial strips from oxidant-mediated ciliary dyskinesia. Exposure of respiratory epithelium to glucose/glucose oxidase resulted in similar effects to those obtained with the xanthine/xanthine oxidase system, with 38% and 57% CBF reduction after 4 h in systems containing 25 and 100 mU of glucose oxidase, respectively. Treatments with H₂O₂ and HOCl at concentrations of ≥100 µM caused dose-dependent ciliary dyskinesia after 4 h (Feldman et al., 1994).

Marked ciliary slowing was observed after exposure of human nasal epithelial cells to free radicals within the first 5 min. There was a significant difference in CBF between experimental and control groups. Pretreatment with 300 U/mL SOD or with 5 mM 3-ABA prevented CBF changes. (Min et al., 1999).

Pyocyanin dissolved in PBS produced gradual slowing of CBF in strips of normal human nasal ciliated epithelium without recovery. Ciliary dyskinesia was observed only late in the experiments when ciliostasis and epithelial disruption were also noted. In contrast, 1-hydroxyphenazine (1-hp) produced rapid onset of ciliary slowing, dyskinesia, and immediate ciliostasis but also some recovery of ciliary beating during the experiment (Wilson et al., 1987).

Baseline CBF was analyzed in human sinonasal epithelial cells for 4 min (time 0) followed by administration of either cigarette smoke condensate (CSC) or DMSO for 3 min. Forskolin was then administered to the apical surface and stimulated CBF measured. Following addition of forskolin, stimulated CBF was significantly decreased in the CSC-exposed group compared to DMSO controls (Cohen et al., 2009).

Treatment of fully differentiated normal human bronchial epithelial cells with 100 nM roflumilast raised the CBF at 1 h after exposure. Subsequent air or cigarette smoke exposure increased CBF significantly roflumilast-treated cultures (Schmid et al., 2015).

In superficial mucosae of the inferior nasal turbinates from non-smoking healthy volunteers exposed to three different oxygen concentrations—21%, 60% and 95%—for 2 h, CBF dose-dependently increased. Extending exposure to extreme oxygen concentrations to 4 h, however, decreased CBF, which is indicative of “oxygen stress” (Stanek et al., 1998).

Within 2 min of exposure of human respiratory epithelial cell monolayers to 10 µM H₂O₂, a decrease in CBF was observed. All cilia stopped moving within 10 min without obvious surface structural change in the ciliated cells. Catalase significantly reduced the ciliotoxic effect of H₂O₂ (Yoshitsugu et al., 1995).

Several studies show that oxidants decrease CBF which can be reversed by addition of antioxidants, suggesting a direct effect. However, there is evidence suggesting that oxidant-mediated decreases in CBF cannot be prevented by addition of antioxidants. For example, a polycyanin-induced decrease in CBF in human nasal epithelium could be reversed by treatment with isobutylmethylxanthine and forskolin, both of which increase intracellular cAMP, and also by the cAMP analog dibutyryl cAMP, while antioxidants did not seem to have any effect on CBF (Kanthakumar et al., 1993). Like polycyanin, two other P. aeruginosa toxins, 1-hydroxyphenazine (1-HP) and rhamnolipid reduced CBF which was associated with a decrease in intracellular adenosine nucleotides (Kanthakumar et al., 1996). 

Inconsistent with several studies, there are studies that suggest that exposure to cigarette smoke does not inhibit CBF. A study involving 56 human subjects (27 non-smokers and 29 smokers) showed no differences in CBF between the 2 groups. However, a decrease in nasal mucociliary clearance was observed in smokers who exhaled smoke through their noses (Stanley et al., 1986). 

While several studies have shown age dependence of CBF, we found evidence that suggests otherwise  (Agius et al., 1998). 

Quantitative Understanding of the Linkage

Several studies in various species, including humans and rodents, provide evidence in support of this KER. The empirical evidence confirms both a dose- and time-dependence between the upstream KE/MIE and the downstream KE. Our quantitative understanding of this KER is therefore strong.

Treatment of human nasal ciliated epithelial cells with 0.4 mM xanthine + 100 mU/mL xanthine oxidase—producing 159 ± 4.0 µM/h H₂O₂—decreased CBF by ca. 1 Hz at 1 h and ca. 2.5 Hz (37.4%) at 4 h. Catalase alone (500 U/mL), or in combination with superoxide dismutase (SOD; 300 U/mL ) completely protected the cells from oxidant-mediated ciliary dyskinesia (Feldman et al., 1994).

Treatment of human nasal ciliated epithelial cells with 5 mM glucose + 25 mU/mL glucose oxidase— producing 114 ± 7.7 µM/h H₂O₂—decreased CBF by ca. 2 Hz at 1 h and ca. 4 Hz (38%) at 4 h. The decline in CBF was even larger with 57% (approx. 6 Hz) at 4 h when 100 mU/mL glucose oxidase was used (producing 322 ± 11.5 µM/h H₂O₂). Catalase alone (500 U/mL) completely protected the cells from oxidant-mediated ciliary dyskinesia (Feldman et al., 1994).

Treatment of human nasal ciliated epithelial cells with H₂O₂ at concentrations ≥100 µM dose-dependently decreased CBF in human nasal ciliated epithelial cells, with 100 µM causing a 22.4% reduction and the maximal decrease (51.6%) seen with 500 µM H₂O₂ at 4 h. Adding 100 mU/mL MPO to 150 µM H₂O₂ enhanced the H₂O₂-mediated decrease in CBF (control:11.7 ±0.6 Hz; H₂O₂: 8.2 ± 1.1 Hz, 30% decrease; H₂O₂ + MPO: 5.4±0.2 Hz, 53.8% decrease). (Feldman et al., 1994).

Treatment of human nasal ciliated epithelial cells with HOCl at concentrations ≥100 µM dose-dependently decreased CBF in human nasal ciliated epithelial cells, with 100 µM causing a 26.1% reduction and the maximal decrease (100%) seen with 500 µM HOCl at 4 h (Feldman et al., 1994).

Treatment of human nasal epithelial cells with 0.4 mM xanthine + 100 mU/mL xanthine oxidase decreased CBF by ca. 50% within 2 min. Addition of 300 U/mL SOD abolished this effect (Min et al., 1999).

Treatment of human nasal epithelial cells with 10 mM H₂O₂ decreased CBF to 36.5 ±4.4% of baseline within 5 min, with a maximal decrease in CBF of 100% seen after 10 min, whereas 1 mM H₂O₂ had no effect on CBF. Treatment of human nasal ciliated epithelial cells with 0.8 mM xanthine + 100 mU/mL xanthine oxidase transiently increased CBF by 12.1±1.0% from baseline. When xanthine concentration was increased to 4 and 8 mM, CBF decreased by 26.8±1.7 and 25.6±1.5%, respectively (Yoshitsugu et al., 1995).

Treatment of bovine ciliated bronchial epithelial cells with acetaldehyde, an oxidative stressor, decreased CBF in a dose-dependent manner. Significant slowing of ciliary beating by ca. 50% was observed with concentrations as low as 15-30 µM, and ciliary beating was completely abrogated at concentrations > 250 µM. Ciliary beating also decreased following treatment with 15-30 µM propionaldehyde (40-50% of control), butyraldehyde (35-65% of control), isobutyraldehyde (20-40% of control), and benzaldehyde (80-90% of control) (Sisson et al., 1991).

Exposure of rabbit tracheal explants to formaldehyde dose-dependently decreased CBF. At 66 µg formaldehyde/cm³, CBF decreased from 12.6 to 11.8 Hz; at 33 µg formaldehyde/cm³, CBF decreased from 13.0 to 10.9 Hz (Hastie et al., 1990).

Exposure of guinea pig trachea to SO₂ at concentrations of 2.5-12.5 ppm for 30 min dose-dependently decreased CBF. Exposure to 2.5 ppm SO₂ caused a small, non-significant decrease in mean CBF, and exposure to 5 ppm SO₂ caused a 45% decrease. The greatest decrease (72 %) in mean CBF was recorded after exposure to 12.5 ppm SO₂ (Knorst et al., 1994a).

Exposure of human nasal epithelial cells (cultured in Ringer’s solution) to SO₂ at concentrations of 2.5-12.5 ppm for 30 min dose-dependently decreased CBF. Exposure to 2.5 ppm yielded a 42.8% decrease, whereas exposure to 12.5 ppm yielded a 96.5% decrease in CBF (Kienast et al., 1994).

A 20-min exposure to NO₂, a known air pollutant, at concentrations of 1.5 or 3.5 ppm did not affect CBF in healthy human subjects at 45 min post-exposure (Helleday et al., 1995).

Exposure of human bronchial epithelial cells from healthy volunteers to 10, 50, and 100 µg/mL Diesel exhaust particles (DEP) significantly decreased CBF by 15.9%, 31.0%, and 55.5%, respectively, from baseline after 24 h (Bayram et al., 1998).

A 4-week exposure of human nasal epithelial cells to 100 µg/m³ ozone had no effect on CBF, whereas 5- and 10-times that concentration significantly decreased CBF (-11.1% at 500 µg/m³; -33.3% at 1000 µg/m³) (Gosepath et al., 2000).

Baseline CBF in tracheal rings from C57Bl/6 mice exposed to cigarette smoke (whole body exposure to mainstream and sidestream cigarette smoke via inhalation from 1R1 reference cigarettes, at 150 mg/m³ total particulate matter for 2 h/day, 5 days/week, for up to 1 year) for 1.5 to 3 months was slightly, but not significantly, increased (∼1 Hz). After 6 months of smoke exposure, however, baseline CBF significantly decreased (∼2–3 Hz) (Simet et al., 2010).
 

Treatment of human nasal ciliated epithelial cells with 0.4 mM xanthine + 100 mU/mL xanthine oxidase decreased CBF over time, with a noticeable decrease by ca. 1 Hz at 1 h and a maximal decrease of 37.4% reached at 4 h (Feldman et al., 1994).

Treatment of human nasal ciliated epithelial cells with 5 mM glucose + 25 mU/mL glucose oxidase decreased CBF by ca. 2 Hz at 1 h and a maximal decrease of ca. 4 Hz (38%) at 2 h, that did not change until the end of the experiment at 4 h. When 100 mU/mL glucose oxidase was used, CBF decreased by ca. 2 Hz at 1 h, 4 Hz at 2 h, 5.5 Hz at 3 h, reaching a maximum of 57% (approx. 6 Hz) at 4 h (Feldman et al., 1994).

Treatment of human nasal epithelial cells with 0.4 mM xanthine + 100 mU/mL xanthine oxidase decreased CBF maximally by ca. 50% within 2 min, after which it began to increase again, reaching approx. 80% of the baseline value after 30 min (Min et al., 1999).

Treatment of human nasal ciliated epithelial cells with 0.8 mM xanthine + 100 mU/mL xanthine oxidase transiently increased CBF by 12.1±1.0% from baseline within 15 s, after which it rapidly returned to baseline levels (within 30 min). When xanthine concentrations were increased to 4 and 8 mM, CBF decreased by 26.8±1.7 and 25.6±1.5%, respectively (Yoshitsugu et al., 1995).

Treatment of bovine ciliated bronchial epithelial cells with acetaldehyde reduced CBF rapidly, with a significant drop in CBF occurring within 30 s and a maximal decrease by 3 min (Sisson et al., 1991).

Exposure of rabbit tracheal explants to formaldehyde time-dependently decreased CBF: At 66 µg/cm³, CBF decreased from 12.6 to 11.8 Hz immediately upon addition of HCHO to complete cessation of beating by 10 min. At 33 µg/cm³, CBF decreased from 13.0 to 10.9 Hz by 30 min (Hastie et al., 1990).

At 24 h following a 4-h exposure of healthy human subjects to 3.5 ppm NO₂, there was a significant elevation in CBF from 12.4±0.9 Hz (at baseline, pre-exposure) to 13.8±0.8 Hz (Helleday et al., 1995).

Exposure of human bronchial epithelial cells to DEP significantly decreased CBF from 2 h onward after incubation with 50 to 100 µg/mL DEP and from 6 hours onward after incubation with 10 µg/mL DEP (Bayram et al., 1998).

A 4-week exposure of human nasal epithelial cells to ozone significantly reduced CBF, with effects becoming noticeable at the higher concentrations (-7.1% at 500 µg/m³; 
-10.3% at 1000 µg/m³) after 2 weeks of exposure and a maximal decrease after 4 weeks (-11.1% at 500 µg/m³; -33.3% at 1000 µg/m³) (Gosepath et al., 2000).

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References

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• Gosepath, J., Schaefer, D., Brommer, C., Klimek, L., Amedee, R.G., and Mann, W.J. (2000). Subacute effects of ozone exposure on cultivated human respiratory mucosa. American journal of rhinology 14(6), 411-418.
• Hastie, A.T., Patrick, H., and Fish, J.E. (1990). Inhibition and recovery of mammalian respiratory ciliary function after formaldehyde exposure. Toxicology and applied pharmacology 102(2), 282-291.
• Helleday, R., Huberman, D., Blomberg, A., Stjernberg, N., and Sandstrom, T. (1995). Nitrogen dioxide exposure impairs the frequency of the mucociliary activity in healthy subjects. European Respiratory Journal 8(10), 1664-1668.
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• Knorst, M.M., Kienast, K., Riechelmann, H., Müller-Quernheim, J., and Ferlinz, R. (1994). Effect of sulfur dioxide on mucociliary activity and ciliary beat frequency in guinea pig trachea. Int. Arch. Occup. Environ. Health 65(5), 325-328. 
• Min, Y.-G., Ohyama, M., Lee, K.S., Rhee, C.-S., Oh, S.H., Sung, M.-W., et al. (1999). Effects of free radicals on ciliary movement in the human nasal epithelial cells. Auris, nasus, larynx 26(2), 159-163.
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• Schmid, A., Baumlin, N., Ivonnet, P., Dennis, J.S., Campos, M., Krick, S., et al. (2015). Roflumilast partially reverses smoke-induced mucociliary dysfunction. Respir. Res. 16(1), 135. 
• 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. American journal of respiratory cell and molecular biology 43(6), 635-640.
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