Abstract
Background:
Mucociliary clearance is a main defense mechanism of the airway and is impaired in ciliary dyskinesia. The objective of this study is to evaluate the prevalence of chronic rhinosinusitis (CRS) and its characteristics in bronchiectasis patients suspected of harboring ciliary dyskinesia.
Methods:
Bronchiectasis patients referred to Rhinology clinic for nasal brush biopsy (NBB) were included in this study. NBB was performed using a curettage technique whereby ciliated epithelial cells were obtained from the surface of the inferior nasal turbinate. Results of transmission electron microscopy findings, primary ciliary dyskinesia (PCD) gene (35 genes) analyses (Invitae, CA) and sinus CT scans were reviewed.
Results:
Twenty-three patients (age:54+/−2.9) were referred for NBB between 2015 and 2018. Thirteen patients (56.5%) met criteria for diagnosis of CRS. Nineteen patients had ciliary ultrastructural defects. The most common finding was compound cilia (n=11, 47.8%). Five patients (21.7%) had central microtubule defects (CMD) with higher FEV1 at the time of referral than those without CMD (CMD+:91+/−3.7%, CMD-:73.5+/−5.7%, p=0.023). Of 15 subjects with a PCD gene panel, 67% (9/15) carried at least one gene associated with PCD. Only 1 patient reached diagnosis of PCD. Approximately 50% of non-PCD carriers had smoking history (p<0.05). Lund-Mackey scores did not significantly differ between PCD and non-PCD carriers (p=0.72).
Conclusions:
Nearly half of bronchiectasis patients referred for NBB had concurrent CRS. The presence of ciliary abnormalities was not amplified in bronchiectasis patients with CRS compared to those without CRS. Extrinsic factors may be related to ciliary structural abnormalities in non-PCD gene carriers.
Keywords: primary ciliary dyskinesia, acquired ciliary dyskinesia, sinusitis, ultrastructure, electron microscopy, bronchiectasis, chronic rhinosinusitis, mucociliary clearance
INTRODUCTION
Bronchiectasis is characterized by permanent dilation of the bronchi and bronchioles as a result of elastic and smooth muscle tissue breakdown. It generally occurs as a result of infection, although non-infectious factors may contribute to the development of this condition.1 Interestingly, the incidence of chronic rhinosinusitis (CRS) is increased in idiopathic bronchiectasis patients.1,2 Between 50–70% of patients with bronchiectasis will have both subjective and objective findings of CRS.2,3 The close association between upper and lower airway disease has led to exploration of potential common pathophysiologic mechanisms.
The upper and lower respiratory tract is lined with ciliated epithelial cells (Figure 1A).4 Healthy ciliated cells beat in a coordinated fashion to drive mucociliary clearance.5 Disruptions in the ultrastructural architecture of the cilia, either primary or acquired, can cause abnormal mucociliary transit.6 Defective mucociliary clearance leads to recurrent bacterial infections in the lower airways, ultimately leading to the development of dysregulated inflammation, tissue injury, and bronchiectasis.7 Mucus stasis within the sinonasal cavities confers a similar environment of chronic inflammation and infection.8
Figure 1.
Transmission electron microscopic findings of nasal cilia from Nasal Brush Biopsy
A. Normal 9+2 cilia ultrastructure
B. Compound (fused) cilia
C. Peripheral microtubule abnormality (9+2+1)
D. Central microtubule abnormality (9+0)
The ultrastructural findings in secondarily acquired ciliary dyskinesia are quite different than those that are identified in primary ciliary dyskinesia. The most common acquired defects include compound, or fused cilia, and cilia with peripheral microtubule anomalies.9 Less common findings are the absence of 1 or both of the central pair of microtubules, supernumerary microtubules, and swollen membranes.4,10 The objective of the current study is to examine the prevalence of CRS and its clinical characteristics in bronchiectasis patients referred specifically for nasal brush biopsy (NBB) by pulmonology and to evaluate the ultrastructural abnormalities and primary ciliary dyskinesia (PCD) gene carrier status in this population.
METHODS
Subjects
The Institutional Review Board at the University of Alabama at Birmingham provided approval for this study. Adult patients with idiopathic non-cystic fibrosis bronchiectasis diagnosed by high-resolution computed tomography (CT) scans of the chest were referred to the sinus clinic at the University of Alabama at Birmingham after reporting subjective sinonasal complaints. All patients underwent evaluation for sinusitis, including sinus CT scan and inferior turbinate brush biopsy to further workup their bronchiectasis. Data collected included forced expiratory volume in 1 second (FEV1) at the time of referral and results from a 35 gene panel surveying for primary ciliary dyskinesia (Invitae INC, CA). CRS was diagnosed based on the criteria outlined in the International Consensus Statement on Allergy and Rhinology: Rhinosinusitis.11
Brush biopsy
Ciliated cells from the inferior turbinate were obtained by a curettage technique with the patient awake in clinic, described by Olin et al.12 The bronchial biopsy brushes (2mm cytology brush, Olympus Inc.) were immediately immersed in a cold fixative consisting of 2% glutaraldehyde, 2% paraformaldehyde, and 0.5% tannic acid. The specimens were then transferred to the Children’s Hospital of Alabama Pathology department for imaging with transmission electron microscopy.
Statistical Analysis
Statistical analyses were conducted using Excel 2016 and GraphPad Prism 6.0 software (La Jolla, Ca) with significance set at P < 0.05. Continuous variables were compared by performing t-test, while categorical variables were analyzed by χ2 test or fisher’s exact test. Data is expressed as mean +/− standard deviation.
RESULTS
Twenty-three subjects were referred for evaluation of sinusitis and brush biopsy between Jan. 2015 and Dec. 2018 (Table 1). The average age of the subjects was 54 +/− 2.9 years (16 females, 7 males). Ultrastructural ciliary abnormalities were identified in 82.6% (19/23) of subjects. Compound cilia and cilia with peripheral microtubule abnormalities were equally high in prevalence (47.8% vs 47.8%). The two most common findings were compound cilia (47.8%) and a 9+2+1 configuration (34.8%), while other defects in the peripheral microtubule made up the remainder (13.0%) (Figure 1B and 1C). Five patients had central microtubule defects (CMD) (Figure 1D), and were observed to have higher FEV1 at the time of referral than those without CMD (CMD present:91+/−3.7% vs CMD absent:73.5+/−5.7%, p=0.023). Nine patients (39.1%) had more than one pathologic finding, most commonly compound cilia in addition to peripheral microtubule abnormalities.
Table 1.
Demographics of Patients
| Demographics | All (n=23) |
|---|---|
| Female sex (%) | 69.57 |
| Age (yrs) | 53.04 |
| Ultrastructural abnormality (%) | 82.61 |
| Compound cilia (%) | 47.83 |
| Central MT defect (%) | 21.74 |
| Peripheral MT defect (%) | 47.83 |
| Previous Sinus surgery (%) | 47.83 |
| CRS (%) | 56.52 |
| Lund-Mackay | 3.43 |
| FEV1 (% predicted) | 76% |
| PCD gene carrier (%) | 60 (from n=15) |
MT: microtubule, CRS: chronic rhinosinusitis, FEV1: forced expiratory volume in 1 second, PCD: primary ciliary dyskinesia
A PCD gene panel was obtained in 15 patients; others elected not to proceed with genetic testing by the pulmonologists. Surprisingly 60% (9/15) carried at least one gene associated with PCD (Table 2). More than 85% of patients with positive PCD gene panel had ultrastructural abnormalities from NBB. The most common (n=2) gene defect was the Dynein Axonemal Heavy Chain 8 (DNAH8) heterozygous mutation. One patient was officially diagnosed with PCD, with heterozygous mutation of DNAH11. PCD was diagnosed based on her clinical picture and decreased nasal nitric oxide. 9+2+2 and 9+2+3 peripheral tubular defects and 8+2 central tubular defect were only observed in patients with PCD gene mutations. 50% (3/6) of patients without PCD gene mutations had a history of cigarette smoking (Fisher’s exact test, p = 0.044). Even though statistical significance was lacking, patients with PCD gene defects had higher total number of ultrastructural defects per patient than those without PCD gene defect (Gene+: 1.78 +/− 0.43, Gene-: 1.33 +/− 0.21, p=0.38). Lund-Mackay scores did not significantly differ between PCD carriers and non-PCD carriers (Gene+:2.9+/−1.2, Gene-:2.3+/−0.8, p=0.72) (Table 2). Notably, patients who carried more than one gene mutation were found to have significantly higher Lund-Mackay scores than non-carriers and those who carried a single gene mutation (Multiple: 6.0 +/− 4.3, Single: 1.3 +/− 1.2, None: 1.2 +/− 1.2, p=0.05 (Figure 2).
Table 2.
Characteristics based on the Presence of PCD gene
| PCD Gene + (n=9) |
PCD Gene − (n=6) |
p-value | |
|---|---|---|---|
| Lund-Mackay | 2.89 | 2.33 | 0.71 |
| Ultrastructural abnormality (%) | 88.8 | 100 | >0.99 |
| Compound cilia (%) | 66.7 | 50.0 | 0.62 |
| Central MT defect (%) | 22.2 | 20 | >0.99 |
| Peripheral MT defect (%) | 55.6 | 50 | >0.99 |
| Total Number of Ultrastructural abnormalities per patient | 1.78 | 1.33 | 0.38 |
| Cigarette Smoking History (%) | 0 | 50 | 0.044* |
Figure 2.
Lund-Mackay scores were found to be higher in carriers of multiple mutations when compared to carriers of a single mutation or no mutation at all (** p=0.05).
CRS was diagnosed in 56.5% (13/23) of subjects (Table 3). Eleven subjects (47.8%) had at least one prior sinus surgery. As anticipated, Lund-Mackay scores were significantly different between CRS and non-CRS patients (5.7 vs 0.5, p=0.01). There was no difference in the presence of ultrastructural ciliary abnormalities between the CRS and non-CRS group (84.6% vs. 80.0%, p=1.00). The CRS cohort tended to have a higher prevalence of central microtubule defects, but this did not meet significance (30.8% vs. 10.0%, p=0.34). Percentage of predicted FEV1 was also not significantly different between CRS and non-CRS individuals (82% vs. 70%, p=0.23). Interestingly both groups carried PCD gene mutations (p=0.61). No significant difference was noted in bacterial colonization from sputum samples between the CRS and the non-CRS cohorts (69.2% vs 80.0%, p=0.66). There was no difference in colonization rates when comparing individual microbes (Table 4).
Table 3.
Characteristics with and without Presence of CRS
| CRS (n=13) |
Non-CRS (n=10) |
p-value | |
|---|---|---|---|
| Female sex (%) | 84.6 | 50.0 | 0.1688 |
| Age (yrs) | 54.0 | 52.0 | 0.8073 |
| Lund-Mackay | 5.7 | 0.5 | 0.0103* |
| Ultrastructural abnormality (%) | 84.6 | 80.0 | 1 |
| Compound cilia (%) | 46.2 | 50.0 | 1 |
| Central MT defect (%) | 30.8 | 10.0 | 0.3394 |
| Peripheral MT defect (%) | 61.5 | 40.0 | 0.6802 |
| Previous Sinus surgery (%) | 53.8 | 40.0 | 0.6802 |
| FEV1 (% predicted) | 82% | 70% | 0.23 |
| PCD gene carrier (%) | 75.00 (n=8) | 57.14 (n=7) | 0.6084 |
CRS: chronic rhinosinusitis, FEV1: forced expiratory volume in 1 second, PCD: primary ciliary dyskinesia,
< p.05
Table 4.
Respiratory Microbiology
| Bacterial Colonization (Sputum) | All (n=23) | CRS (n=13) |
Non-CRS (n=10) |
p-value |
|---|---|---|---|---|
| Chronic colonization (%) | 73.9 | 69.2 | 80.0 | 0.66 |
| Gram positive (%) | 47.8 | 53.8 | 40.0 | 0.6802 |
| Staphylococcus aureus (%) | 34.8 | 30.8 | 40.0 | 0.685 |
| Streptococcus pneumoniae (%) | 13.0 | 23.1 | 0.0 | 0.2292 |
| Gram negative (%) | 69.6 | 53.8 | 90.0 | 0.0886 |
| Haemophilus parainfluenzae (%) | 47.8 | 38.5 | 60.0 | 0.4136 |
| Pseudomonas aeruginosa (%) | 34.8 | 30.8 | 40.0 | 0.685 |
| P. aeruginosa – mucoid (%) | 21.7 | 23.1 | 20.0 | 1 |
| Escherichia coli (%) | 13.0 | 15.4 | 10.0 | 1 |
| Moraxella catarhalis (%) | 8.7 | 7.7 | 10.0 | 1 |
| Stenotrophomonas maltophilia (%) | 8.7 | 0.0 | 20.0 | 0.1779 |
| Enterobacter cloacea (%) | 8.7 | 7.7 | 10.0 | 1 |
DISCUSSION
The prevalence of CRS in bronchiectasis patients is reported to be between 50–70%, which is similar to what was found in this cohort.2 Given the frequent association, it has been suggested that these two diseases originate from a similar pathophysiologic mechanism.13 Acquired ciliary dysfunction as a result of extrinsic factors that alter ciliary architecture and interfere with normal ciliary function has been investigated as a potential etiology.14 Based on our analysis, approximately 50% of non-PCD gene carriers had history of cigarette smoking, which could have caused the acquired ciliary defects. Indeed, prior studies have recognized that bronchiectasis patients with significant smoking history have more severe clinical disease, higher incidence of exacerbations, and increased risk of death from respiratory causes.15 It is also interesting to notice that a significant number of carriers of PCD gene mutations have ultrastructural defects, and even gene carriers without CRS demonstrate ultrastructural ciliary abnormalities. This suggests that secondary inflammation may not be the cause of ciliary dysfunction in those individuals with PCD gene carriers.
The ultrastructural findings in secondarily acquired ciliary dyskinesia are quite different than those found in primary ciliary dyskinesia. The most common acquired defects include compound, or fused cilia, and cilia with peripheral microtubule anomalies.9 This is similar to what was observed in our patient cohort. However, there is a lack of understanding regarding the impact of specific acquired ciliary abnormalities on MCC. Interestingly, the presence of acquired ciliary abnormalities was not amplified in bronchiectasis patients with CRS compared to those without CRS. These findings suggest two theories: 1) ciliary ultrastructural changes alone may not be sufficient to precipitate the pathogenesis of CRS in bronchiectasis; 2) based on a high prevalence of genetic abnormalities, these ultrastructural changes are merely a manifestation of the disease process.
PCD is often considered in the workup of idiopathic bronchiectasis, and recent advances with genetic testing have identified 35 genes associated with primary ciliary dyskinesia.16 In the current investigation, a number of subjects heterozygous for genetic mutations associated with PCD were identified. Similar to the higher prevalence of CRS noted in individuals heterozygous for cystic fibrosis mutations,17–20 patients who are carriers of PCD genetic mutations could be predisposed to develop chronic upper and lower airway disease and inflammation. In the current study, patients who carried multiple genetic mutations were found to have higher Lund-Mackay scores than those with a single or no mutation. Future higher powered studies investigating PCD gene carrier status as it relates to CRS may better delineate the role of genetics in disease pathogenesis.
Chronic bacterial colonization of the lower airways is also common in bronchiectasis patients. Because several classes of bacteria produce byproducts that impair ciliary beat frequency (Table 5),14 we explored whether these pathogens were more common in patients with both CRS and bronchiectasis when compared to those with isolated lower airway disease. There was no increase in prevalence of ciliotoxic pathogens in the CRS plus bronchiectasis cohort suggesting a multifactorial pathogenesis of ciliary dysfunction in these patients.
Table 5.
Byproducts from Ciliotoxic Pathogens
| Ciliotoxic Pathogens | Byproducts |
|---|---|
| Pseudomonas aeruginosa | Pyocyanin |
| Haemophilus influenzae | lipooligosaccharide |
| Haemophilus influenzae | protein D |
| Streptococcus pneumoniae | pneumolysin |
| Streptococcus pneumoniae | hydrogen peroxide |
Limitations of this study include the retrospective nature and small sample size. Larger patient cohorts will increase the power of this study and could further delineate phenotypic differences between CRS plus bronchiectasis patients versus isolated bronchiectasis patients. Additionally, further studies incorporating control groups (CRS without bronchiectasis and normal controls) may identify differences in cilia ultrastructure and gene mutations among these groups.
CONCLUSION
Nearly half of bronchiectasis patients referred for NBB had concurrent CRS. The presence of ciliary abnormalities was not amplified in bronchiectasis patients with CRS compared to those without CRS. A significant portion of patients who carry PCD gene mutations had ultrastructural defects even without CRS, suggesting inflammation may not be etiology of ciliary defects in these patients. Extrinsic factors (e.g. cigarette smoking), however, may be related to acquired ciliary structural abnormalities in non-PCD gene carriers.
ACKNOWLEDGEMENTS
This work was supported by National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute (1 R01 HL133006-04) and National Institute of Diabetes and Digestive and Kidney Diseases (5P30DK072482-04, CF Research Center Pilot Award) to B.A.W. and John W. Kirklin Research and Education Foundation Fellowship Award, American Rhinologic Society New Investigator Award, and Cystic Fibrosis Foundation Research Development Pilot grant (ROWE15R0) to D.Y.C,.
Funding Support: This work was supported by National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute (1 R01 HL133006-04) to B.A.W., National Institute of Diabetes and Digestive and Kidney Diseases (5P30DK072482-02) to S.M.R., and Cystic Fibrosis Foundation Research Development Pilot grant (ROWE15R0) to D.Y.C.
Footnotes
Disclosures: All authors have read and approved the manuscript. Dr. Bradford A. Woodworth is a consultant for Cook Medical and Smith and Nephew. Manuscript was presented at the Rhinoworld 2019, Chicago, Il, June 9, 2019.
Level of Evidence: NA.
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