Mucociliary clearance (MCC) results in an effective interaction between the mucus layer and the normal coordinated ciliary beating [1]. Ciliary dyskinesia, or abnormal ciliary beating, can be primary, or secondary to chronic infection or inflammation [2].
Tweetable abstract
Chronic mucus hypersecretion (CMH) is linked to increased asthma severity. Ciliary dyskinesia is present in severe asthma but CMH was not associated with a worse ciliary dysfunction, suggesting another mechanism to explain chronic cough and phlegm. https://bit.ly/3JNUgGr
To the Editor:
Mucociliary clearance (MCC) results in an effective interaction between the mucus layer and the normal coordinated ciliary beating [1]. Ciliary dyskinesia, or abnormal ciliary beating, can be primary, or secondary to chronic infection or inflammation [2].
MCC impairment is well documented in asthma [3], a chronic inflammatory disease of the airways. Mucus alterations in asthma have been described, including a greater viscosity and modification of mucins composition [3, 4]. Respiratory cilia have been poorly studied in asthma but Thomas et al. [5] demonstrated ciliary ultrastructural defects in severe asthma, and a ciliary dyskinesia in moderate and severe asthma.
Chronic cough and phlegm are reported as chronic mucus hypersecretion (CMH) [6]. In asthma, CMH is associated with increased severity [4] and recent data support a causal role for mucus plugs in the mechanism of airflow obstruction in severe asthma [7]. However, the mechanism of CMH in asthma is not well understood and it is unknown whether CMH is associated with an increased ciliary dyskinesia. In this study, we compared ciliary function in severe asthma with or without CMH.
This was a single-centre, cross-sectional study of a series of 20 adults (>18 years old) with severe asthma (according to European Respiratory Society/American Thoracic Society criteria [8]) recruited from the University Asthma Clinic of Liege between 1 January 2021 and 5 May 2021, and of 17 adult healthy controls.
We selected 10 severe asthmatics with CMH and 10 without, matched for age (mean±sd 55.8±10.3 versus 54.1±19.4 years, respectively; p=0.80). CMH was defined as cough and phlegm from the chest on most days for ≥3 consecutive months, for ≥2 consecutive years [6]. Exclusion criteria included active smoking, respiratory tract infection or oral corticosteroid use during the previous 4 weeks, and inhaled or nasal medication during the previous 24 h. Ciliated epithelial samples were obtained by nasal brushing and beating cilia were recorded using digital high-speed videomicroscopy (DHSV) at 37°C, as previously reported [9]. Ciliary function was assessed by ciliary beat frequency (CBF) and the percentage of abnormal beat pattern (CBP). The video sequences of beating cilia were reviewed at a reduced speed and the path taken by a cilium or a group of cilia during a complete beat cycle was compared with the normal beat pattern observed using DHSV. The observed movement was categorised into five distinct CBPs (normal, immotile, stiff, circular and asynchronous) [2]. For each cilium or group of cilia analysed, CBF was calculated and a distinct CBP was attributed. The percentage of abnormal CBPs was calculated ((number of abnormal CBP/total number of CBP readings for the sample) × 100)) [9]:
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Ciliary dyskinesia was defined as an abnormal CBF and/or CBP.
Demographic data, quality of life and asthma control were obtained in all severe asthmatic patients by questionnaires and medical chart review. Patients underwent spirometry, exhaled nitric oxide fraction (FENO) and nasal NO (nNO) measurement (breath-hold technique) (NIOX; Aerocrine, Sweden). Sputum was induced and processed as previously described [10].
Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, Inc.). Data were presented as mean±sd for continuous variables, median (range) for skewed distributions and n (%) for categorical variables. Comparisons used the t-test for parametric data, the Mann–Whitney test for nonparametric data, and the Fisher test for categorical variables. The study was approved by the ethics committee of CHU Liege (references 2020-174 and 2009-161).
Our results confirm that ciliary dyskinesia is increased in adults with severe asthma (n=20, age 55.0±15.2 years) compared with healthy adults (n=17, age 23.5±7.2 years), with a lower CBF (11.9±2.1 versus 14.2±2.3 Hz, p=0.002) and a higher percentage of abnormal CBPs (32.8% (15.4–55.3%) versus 17.9% (11.3–23.7%), p=0.005).
However, when comparing ciliary function between severe asthmatics with or without CMH, there was no significant difference in CBF (11.8±2.1 versus 11.9±2.2 Hz, p=0.89) or in the percentage of abnormal CBPs (30.3% (14.6–47.9%) versus 36.2% (20.7–59.1%), p=0.53).
The demographics and functional characteristics of severe asthmatic patients with or without CMH are summarised in table 1. There was no statistical difference between the groups concerning gender, body mass index, age at onset of asthma, atopic status or comorbidities. The prevalence of bronchiectasis was similar whatever the secretion status and similar to what is reported in severe asthma [11]. In our cohort of severe asthmatics, CMH was associated with a lower control and quality of life, but the difference was not statistically different. Type-2 biomarkers (FENO, sputum and blood eosinophils) were not significantly different between both groups. Mean nNO (185±93 nL·min−1) was not significantly different between the CMH and non-CMH groups.
TABLE 1.
Demographic, functional, clinical and inflammatory characteristics of severely asthmatic adults with or without chronic mucus hypersecretion
| Adults with severe asthma | |||
| With chronic mucus hypersecretion | Without chronic mucus hypersecretion | p-value | |
| Patients | n=10 | n=10 | |
| Males | n=5 | n=4 | >0.99 |
| Age, years | 55.8±10.3 | 54.1±19.4 | 0.81 |
| Age of onset, years | 12.5 (5.5–44.3) | 20.0 (4.0–47.5) | 0.90 |
| BMI, kg·m−2 | 28.3±4.2 | 28.3±3.4 | 0.97 |
| Atopy | 60% | 60% | >0.99 |
| Biologic therapy | n=8 | n=9 | >0.99 |
| Anti-IL-5 | n=7 | n=6 | 0.58 |
| Anti-IgE | n=0 | n=3 | 0.21 |
| Anti-IL-4/13 | n=1 | n=0 | 0.47 |
| FEV1, % pred | 77.3±24.0 | 69.7±20.4 | 0.46 |
| FVC, % pred | 79.1±19.7 | 80.9±16.5 | 0.83 |
| FEV1/FVC, % | 75.6±9.6 | 67.7±12.0 | 0.12 |
| FENO50, ppb | 29.5 (15.5–47.0) | 38.5 (30.3–60.8) | 0.23 |
| nNO, nL·min−1 | 169.9±105.2 | 199.4±80.8 | 0.49 |
| Sputum inflammation | n=9 | n=4 | 0.06 |
| Eosinophils, % | 5.0 (0.7–7.8) | 4.3 (0.5–10.3) | 0.85 |
| Neutrophils, % | 72.8 (58.5–81.6) | 69.6 (50.0–86.7) | 0.94 |
| Blood eosinophils per mm3 | n=8 | n=10 | 0.47 |
| 120 (63–658) | 50 (8–175) | 0.14 | |
| ACT | 16.3±6.4 | 20.2±3.7 | 0.11 |
| ACQ | 1.50 (1.00–4.03) | 1.36 (1.25–2.14) | 0.64 |
| AQLQ | 4.58±1.57 | 5.47±1.04 | 0.15 |
| Chronic rhinitis | 70% | 60% | >0.99 |
| Chronic rhinosinusitis | 60% | 50% | >0.99 |
| Nasal polyps | 30% | 50% | 0.65 |
| Bronchiectasis | 10% | 10% | >0.99 |
Data are presented as mean±sd for continuous variables or median (range) for variables with a skewed distribution, unless otherwise stated. Comparisons between groups used the t-test for parametric data, the Wilcoxon–Mann–Whitney test for nonparametric data and the Fisher test for categorical variables. A p-value of <0.050 was taken as the threshold for statistical significance. BMI: body mass index; IL: interleukin; FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity; FENO50: exhaled nitric oxide fraction measured at a flow rate of 50 mL·s−1; nNO: nasal nitric oxide; ACT: Asthma Control Test; ACQ: Asthma Control Questionnaire; AQLQ: Asthma Quality of Life Questionnaire.
In conclusion, our study confirms a higher ciliary dyskinesia in severe asthma, compared with healthy subjects. Moreover, CMH was not associated with a worse ciliary dysfunction in severe asthma.
Ciliary beating was evaluated using DHSV, a technique used to diagnose primary ciliary dyskinesia (PCD), a rare genetic disease characterised by abnormal ciliary function. DHSV has a high sensitivity and specificity to diagnose PCD when performed by experienced scientists following standardised protocols [12]. However, recent PCD diagnosis guidelines state that CBP and MCC measurements are required, as CBF alone is not sufficient [13]. Furthermore, CBP might help to confirm the pathogenicity of variants in PCD-causing genes but DHSV is not standardised across centres in order to be used as a confirmatory diagnostic test alone [13]. Interestingly, while nNO is decreased in PCD, nNO was within the normal range [14] in our severe asthmatic population.
Previous studies have described MCC impairment in asthma [3, 5] and our results confirm that ciliary beating is abnormal in adults with severe asthma. CBF was significantly lower in our severe asthmatic population than in our healthy controls but within previous published normal values [1]. One limitation of our study is the age difference between the healthy and severe asthma groups. This might explain the lower CBF in the severe asthma group but not the higher percentage of abnormal CBPs, as previous data described a CBF decrease with age from infancy to adulthood [15, 16], but also a decreased dyskinesia after 19 years of age [16].
Our understanding of CMH in different respiratory diseases remains limited [4]. 25–50% of asthmatic patients report chronic sputum production [4, 17] that may sometimes be attributed to post-nasal drips in patients combining asthma and chronic rhinosinusitis. However, in our cohort, the prevalence of rhinosinusal comorbidities was similar in the CMH and non-CMH groups, and higher than previously reported in literature [11].
CMH in asthma has been linked with a higher disease burden: a greater airflow obstruction, a lower quality of life, a poor asthma control and an increased exacerbation rate [4, 17, 18]. However, the physiopathology of CMH in asthma has poorly been studied. To our knowledge, this is the first study aiming to evaluate the relationship between CMH and ciliary function in severe asthma. Our results do not support that CMH in asthma is linked to increased ciliary dyskinesia, suggesting that another mechanism explains chronic cough and phlegm. There are few data regarding the association between CMH and the composition of mucus in asthma. Previous studies suggest a link between CMH and Th2 inflammation in asthma, with a higher blood and bronchoalveolar lavage eosinophilia [18], but in our small cohort, we did not find any significant difference in type-2 biomarkers. One limitation of our study is that most patients received biologic therapies at inclusion, mainly interleukin (IL)-5, which might modify the severity of the disease [19] as well as the relationship between type-2 biomarkers and CMH. Furthermore, diverse cytokines influence ciliary function. In particular, it has been shown that IL-5 leads to a CBF increase in vitro [20]. It would be interesting to evaluate ciliary function in severe asthmatics not treated with biologic therapies, to evaluate whether this modifies ciliary beating in vivo. To understand the mechanisms of abnormal ciliary beating in asthma, it would also be interesting to study ciliary beating in non-severe asthma and after cell culture of respiratory ciliated epithelium.
In summary, our results confirm an abnormal ciliary beating in severe asthma and suggest that CMH in asthma is not explained by an increased ciliary dyskinesia. The next step is to understand the pathophysiology of ciliary dyskinesia in asthma, which might be innate, or linked to chronic infection or inflammation.
Footnotes
Provenance: Submitted article, peer reviewed.
Author contributions: C. Kempeneers had full access to all the data in the study, and takes responsibility for the integrity of the data and the accuracy of the data analysis. C. Kempeneers contributed to study design, acquisition and analysis of the data, and wrote and submitted the manuscript. R. Bonhiver and N. Bricmont contributed to the nasal brushing sample processing and analysis. F. Guissard and C. Moermans contributed to sputum induction and processing. R. Bonhiver performed the statistical analysis. C. Kempeneers, R. Bonhiver, N. Bricmont, M. Pirotte, C. Moermans, F. Guissard, F. Schleich and S. Engleskirchen contributed to patients’ data collection. C. Kempeneers, L. Benchimol, D. Calmes and F. Schleich contributed to nasal brushings. F. Schleich and R. Louis contributed to study design and interpretation of the data. All authors participated in manuscript review, gave final approval of the manuscript, and ensured that questions related to the accuracy or integrity of any part of the work were investigated and resolved appropriately.
Ethics approval: This study was approved by the Ethics committee of CHU Liege (references 2020-174 and 2009-161). Participants gave informed consent to participate in the study before taking part.
Conflict of interest: C. Kempeneers has received consulting fees from Sanofi (international advisory board). R. Louis and F. Schleich have received educational and research grants from GSK, AstraZeneca and Chiesi; consulting fees from GSK and AstraZeneca (national and international advisory boards); and lecture fees from GSK, AstraZeneca and Chiesi. F. Schleich has received lecture fees from TEVA. R. Bonhiver, N. Bricmont, M. Pirotte, S. Engelskirchen, L. Benchimol, D. Calmes, F. Guissard, C. Moermans and M-C. Seghaye have no conflicts to declare.
Support statement: This work was supported by the Excellence of Science programme, a federal grant from the Belgian Government (EOS 0013618F). The sponsors had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript.
References
- 1.Kempeneers C, Seaton C, Chilvers MA. Variation of ciliary beat pattern in three different beating planes in healthy subjects. Chest 2017; 151: 993–1001. doi: 10.1016/j.chest.2016.09.015 [DOI] [PubMed] [Google Scholar]
- 2.Kempeneers C, Seaton C, Garcia Espinosa B, et al. Ciliary functional analysis: beating a path towards standardization. Pediatr Pulmonol 2019; 54: 1627–1638. doi: 10.1002/ppul.24439 [DOI] [PubMed] [Google Scholar]
- 3.Del Donno M, Bittesnich D, Chetta A, et al. The effect of inflammation on mucociliary clearance asthma: an overview. Chest 2000; 118: 1142–1149. doi: 10.1378/chest.118.4.1142 [DOI] [PubMed] [Google Scholar]
- 4.Martínez-Rivera C, Crespo A, Pinedo-Sierra C, et al. Mucus hypersecretion in asthma is associated with rhinosinusitis, polyps and exacerbations. Respir Med 2018; 135: 22–28. doi: 10.1016/j.rmed.2017.12.013 [DOI] [PubMed] [Google Scholar]
- 5.Thomas B, Rutman A, Hirst RA, et al. Ciliary dysfunction and ultrastructural abnormalities are features of severe asthma. J Allergy Clin Immunol 2010; 126: 722–729. doi: 10.1016/j.jaci.2010.05.046 [DOI] [PubMed] [Google Scholar]
- 6.Stott-Miller M, Müllerová H, Miller B, et al. Defining chronic mucus hypersecretion using the CAT in the SPIROMICS cohort. Int J Chron Obstruct Pulmon Dis 2020; 15: 2467–2476. doi: 10.2147/COPD.S267002 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Tang M, Elicker BM, Henry T, et al. Mucus plugs persist in asthma, and changes in mucus plugs associate with changes in airflow over time. Am J Respir Crit Care Med 2022; 205: 1036–1045. doi: 10.1164/rccm.202110-2265OC [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Chung KF, Wenzel SE, Brozek JL, et al. International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma. Eur Respir J 2014; 43: 343–373. doi: 10.1183/09031936.00202013 [DOI] [PubMed] [Google Scholar]
- 9.Bricmont N, Benchimol L, Poirrier A-L, et al. Nasal brushing sampling and processing using digital high speed ciliary videomicroscopy – adaptation for the covid-19 pandemic. J Vis Exp 2020; 165: 61949. doi: 10.3791/61949 [DOI] [PubMed] [Google Scholar]
- 10.Guiot J, Demarche S, Henket M, et al. Methodology for sputum induction and laboratory processing. J Vis Exp 2017; 130: 56612. doi: 10.3791/56612 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Schleich F, Brusselle G, Louis R, et al. Heterogeneity of phenotypes in severe asthmatics. The Belgian Severe Asthma Registry (BSAR). Respir Med 2014; 108: 1723–1732. doi: 10.1016/j.rmed.2014.10.007 [DOI] [PubMed] [Google Scholar]
- 12.Rubbo B, Shoemark A, Jackson CL, et al. Accuracy of high-speed video analysis to diagnose primary ciliary dyskinesia. Chest 2019; 155: 1008–1017. doi: 10.1016/j.chest.2019.01.036 [DOI] [PubMed] [Google Scholar]
- 13.Shoemark A, Dell S, Shapiro A, et al. ERS and ATS diagnostic guidelines for primary ciliary dyskinesia: similarities and differences in approach to diagnosis. Eur Respir J 2019; 54: 1901066. doi: 10.1183/13993003.01066-2019 [DOI] [PubMed] [Google Scholar]
- 14.Collins SA, Gove K, Walker W, et al. Nasal nitric oxide screening for primary ciliary dyskinesia: systematic review and meta-analysis. Eur Respir J 2014; 44: 1589–1599. doi: 10.1183/09031936.00088614 [DOI] [PubMed] [Google Scholar]
- 15.Ho JC, Chan KN, Hu WH, et al. The effect of aging on nasal mucociliary clearance, beat frequency, and ultrastructure of respiratory cilia. Am J Respir Crit Care Med 2001; 163: 983–988. doi: 10.1164/ajrccm.163.4.9909121 [DOI] [PubMed] [Google Scholar]
- 16.Chilvers MA, Rutman A, O'Callaghan C. Functional analysis of cilia and ciliated epithelial ultrastructure in healthy children and young adults. Thorax 2003; 58: 333–338. doi: 10.1136/thorax.58.4.333 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Hughes R, Rapsomaniki E, Janson C, et al. Frequent productive cough: symptom burden and future exacerbation risk among patients with asthma and/or COPD in the NOVELTY study. Respir Med 2022; 200: 106921. doi: 10.1016/j.rmed.2022.106921 [DOI] [PubMed] [Google Scholar]
- 18.Siroux V, Basagan X, Boudier A, et al. Identifying adult asthma phenotypes using a clustering approach. Eur Respir J 2011; 38: 310–317. doi: 10.1183/09031936.00120810 [DOI] [PubMed] [Google Scholar]
- 19.Schleich F, Graff S, Nekoee H, et al. Real-word experience with mepolizumab: does it deliver what it has promised? Clin Exp Allergy 2020; 50: 687–695. doi: 10.1111/cea.13601 [DOI] [PubMed] [Google Scholar]
- 20.Grosse-Onnebrink J, Werner C, Loges NT, et al. Effect of TH2 cytokines and interferon gamma on beat frequency of human respiratory cilia. Pediatr Res 2016; 79: 731–735. doi: 10.1038/pr.2016.8 [DOI] [PubMed] [Google Scholar]