Introduction

Chronic Obstructive Pulmonary Disease (COPD) is currently among the top three leading causes of death globally, posing a significant public health challenge. Fungal sensitization, particularly to Aspergillus fumigatus, is increasingly reported in individuals with chronic respiratory diseases, including COPD, asthma, and bronchiectasis, where it is associated with persistent symptoms, increased disease severity, and higher mortality rates.1–6 However, the role of Aspergillus sensitization in the pathogenesis of COPD is largely unknown. Previous studies have shown that exposure to A. fumigatus extracts increases mucin production in airway epithelia7,8, and chronic exposure to A. fumigatus spores upregulates the expression of MUC5AC, and induces goblet cell hyperplasia in the airways of asthma rats.9 However, the relationship between mucus plugging and Aspergillus hypersensitivity in COPD remains unclear.

Chronic mucus hypersecretion is a key characteristic of COPD,10,11 manifesting as cough and sputum production which are associated with greater airflow obstruction, lower oxygen saturation, and worsened quality of life and all-cause mortality. Therefore, studies on risk factors for and mechanisms underlying mucus hypersecretion, a potential treatable trait in COPD, are highly desirable for targeted therapy. A visual chest computed tomography (CT) scoring system was recently developed, demonstrating that high mucus scores on CT were significantly associated with worse lung function in both asthma and COPD.12,13 Therefore, we used this scoring system to assess mucus plugging and examined its associated features, especially Aspergillus fumigatus sensitization in COPD patients. Revealing the association between Aspergillus sensitization and mucus plugging has important clinical implications, as potential interventions, such as environmental isolation or antifungal therapy, may be applied for this subgroup of patients.14

Methods Patient and Data Collection

Patients visiting the Department of Respiratory and Critical Care Medicine, Peking University Third Hospital and received measurement of blood Aspergillus fumigatus-specific IgE (Af sIgE) from Oct 1, 2018 to Sep 30, 2023, were retrospectively included for this study. The subject selection and diagnostic process are shown in Figure 1. The study and the exemption from informed consent were approved by the Clinical Research Ethics Committee of Peking University Third Hospital. Patient data confidentiality was maintained in compliance with the Declaration of Helsinki.

Figure 1 Patient selection process.

Patients were diagnosed with COPD according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria: (1) age over 40 years, (2) consistent symptoms including chronic cough, expectoration, and/or dyspnea, (3) a history of cigarette smoking (≥10 pack-years), and/or exposure to biomass fuel for at least 10 years, and/or occupational exposure to noxious particles and fumes for at least 10 years, and (4) evidence of irreversible obstructive impairment on spirometry, defined by post-bronchodilator FEV1/FVC <70%.

Af sIgE testing was performed within six months before or after the CT examination. Positive Af sIgE was defined as a level of more than 0.35 KU/L, indicating Aspergillus hypersensitivity (Phadia, Thermo Fisher Scientific, Uppsala, Sweden). Besides, according to whether the Af sIgE was greater than 0.7 KU/L, we divided participants with a positive Af sIgE into a low-sensitization group and a high-sensitization group.

The clinical data included demographics, smoking history, the number of cigarettes smoked, respiratory symptoms (cough, sputum, hemoptysis, dyspnea), comorbidities, and medication use. The laboratory data included white blood cell count (WBC) and differentials, hemoglobin (HB), serum total IgE, and specific IgE to Aspergillus fumigatus. The normal range of total IgE was 0–60 KU/L (Phadia, Thermo Fisher Scientific, Uppsala, Sweden). Lung function was measured post-bronchodilator (after reversibility testing) as forced expiratory volume in the first second in percent predicted values (FEV1% pred) and FEV1/forced vital capacity (FEV1/FVC (%)). Patients avoided active or passive smoking and strenuous exercise for 12 hours before the test, and avoided caffeinated drinks and nitrogen-rich food for 2 hours before the test.

Evaluation of Chest HRCT Mucus Plug Score Evaluation

CT images were scored for mucus plugging using the bronchopulmonary segment-based scoring system by Dunican et al.13 Mucus plugging was defined as a complete luminal occlusion of the bronchus (Figure 2), irrespective of generation or size. The lung zone within 2 cm of the costal or diaphragmatic pleura was excluded because the airways in that zone are too small to ascertain a complete occlusion by luminal plugs. A total of 18 segmental airways, including ten bronchopulmonary segments in the right lung (3, 2, and 5 segments in the upper, middle, and lower lobes, respectively) and 8 in the left lung (4 segments each in the upper and lower lobes), were evaluated. A luminal plug score was generated for each CT as an aggregate of the number of bronchopulmonary segments with luminal plugging, ranging from 1 to 18. For those without luminal plugging, the score is 0. A standard window width of 1200 hU and a level of 600 hU were used for visual bronchial wall evaluation. Two pulmonary physicians evaluated the CT scans without knowledge of the patient’s clinical data. They independently completed the assessments, and differences in readings were resolved through consensus.

Figure 2 An example of airway mucus plugs (yellow arrowheads) of one participant in this study in the Coronal plane (A), the Axial plane (B) and Sagittal plane (C).

Statistical Analysis

All statistical analyses were performed using SPSS 17.0 (Statistics Package for the Social Sciences, SPSS Inc., Chicago, IL, USA). The propensity score matching was performed using SPSS. With Aspergillus fumigatus sensitization as the dependent variable and age and gender as independent variables, the propensity scores for the two patient groups were calculated through logistic regression. The nearest-neighbor matching method was applied to match the covariates at a 1:2 ratio, with a caliper value of 0.05. Data were expressed as mean ± standard deviation (SD). Data not normally distributed were expressed as median (interquartile range, IQR). Categorical variables between different groups were analyzed using the chi-square test. Spearman correlations were used for correlation analysis. Comparisons of continuous data between two groups were performed using the independent-samples T-test (for normally distributed parameters) and the Mann–Whitney U-test (for abnormally distributed parameters). Univariate and multivariable logistic regression analyses were used to assess the relationship of mucus plugging and multiple variables. P-values less than 0.05 were considered statistically significant.

Results Demographics and Clinical Characteristics of the Patients

During the study period, 378 patients diagnosed with COPD underwent testing for blood Af sIgE, of whom 29 (7.7%) had an Af sIgE level >0.35KU/L, therefore classified as the Aspergillus hypersensitivity (AH) group. We then used propensity score matching at a ratio of 1:2 to recruit controls from the 341 patients without AH. Consequently, 29 patients of the AH+ group and 58 patients from the AH- group were included for the final analysis. Patient demographics and associated clinical characteristics are shown in Table 1.

Table 1 Characteristics of COPD Subjects with Aspergillus Hypersensitivity (AH+) and Without Aspergillus Hypersensitivity (AH-)

As expected, elevated serum total IgE levels were found in the AH+ group (1405 vs 76.75, p<0.01). Subjects in the AH +group exhibited lower FEV1% predicted (50.43 vs 64.44, p=0.008) and FEV1/FVC (%) (51.00 vs 58.6, p=0.023). Additionally, peripheral WBC and neutrophil counts and proportion of blood eosinophils exceeding 300/µL were higher in the AH+ group compared to the AH- group, although there was no difference in the number and percentage of eosinophils between the AH+ and AH- groups. The number of patients with a history of physician-diagnosed asthma or allergic diseases, such as allergic rhinitis and atopic dermatitis, was not significantly different between the two groups.

Mucus Plugging Scores

The prevalence of luminal plugging in the AH+ group was 58.6%, compared to 31.0% in the AH- group, with the difference being statistically significant (p=0.013).

Relationships Between Mucus Plug and Aspergillus Hypersensitivity

Using univariable and multivariable logistic regression models, we found that the blood neutrophil counts and Af sIgE >0.7KU/L were independently associated with mucus plugging, whereas ever smoker, FEV1%predicted, and FEV1/FVC ratio were not (Table 2).

Table 2 Univariate Logistic Regression Analysis of the Associated Factors for Mucus Plugging

Discussion

The relationship between Aspergillus fumigatus hypersensitivity and mucus plugging in COPD is poorly understood. Although Aspergillus sensitization has been associated with a higher burden of clinical symptoms and decreased lung function in COPD patients,4,14 to our knowledge, few studies have examined the role of fungal allergy in the pathogenesis of COPD. In this study, we found that COPD patients with Aspergillus sensitization were more likely to have mucus plugging on HRCT, in addition to a lower FEV1, as compared to non-sensitized patients. Interestingly, a significant association was found between higher levels of blood Af sIgE and mucus plugging, which was also associated with the blood neutrophil count.

Aspergillus sensitization was demonstrated in 7.7% of patients with COPD in our study, whereas previous studies reported a prevalence ranging from 7.9% to 21%.1,2,4–6 Several methods are available to assess Aspergillus sensitization, such as the Aspergillus skin test, serum-specific IgE to crude Aspergillus antigen extracts (used in our study), and recombinant A. fumigatus antigens (rAsp.f),4 which may account for the discrepancy.

It is notable that previous studies have shown varying results regarding the relationship between Aspergillus hypersensitivity and clinical characteristics of chronic respiratory diseases. Most studies demonstrated that fungal sensitization was associated with progressive and persistent symptoms, increased disease severity, and reduced lung function in chronic respiratory diseases1,4,14–17, though some reported no association between Aspergillus sensitization and lung function decline in COPD.1,5,6 It is proposed that COPD patients with lower lung function are more susceptible to Aspergillus sensitization,2 and sensitization accelerates the decline of lung function.18

In this study, we found that Aspergillus-sensitized COPD patients showed a higher blood neutrophil counts and were more likely to have a blood eosinophil count of more that 300/µL. The relationship between fungal sensitization and elevated eosinophil counts is expected, as eosinophils can be recruited to the lungs in response to chitin exposure from Aspergillus fumigatus, enhancing Th2-mediated immune pathology.19 Additionally, prior studies have shown that A. fumigatus induces neutrophil extracellular traps and eosinophil extracellular traps formation by eosinophils and neutrophils.20,21

Recent studies have demonstrated that mucus plugs identified on HRCT scans are frequent in patients with COPD (25%–67%) and are closely associated with clinical measures of the disease, including lung function, quality of life, emphysema on CT, airway wall thickening, and all-cause mortality.12,22 In this study, we reported mucus plugging in 40.2% of participants with COPD. Importantly, the prevalence of subjects with luminal plugging was higher in the Aspergillus-sensitized COPD group than in the non-sensitized COPD group. Multivariate analysis also demonstrated that Af sIgE more than 0.7KU/L was independently associated with mucus plugging. Although our study cannot explain the mechanisms underlying the association, possible explanations may be that A. fumigatus extracts promote the production of Muc5ac and Muc5b,7,8,23 which are predominant mucins in normal airway mucus, leading to impaired mucociliary clearance24,25 and a higher likelihood of mucus plugging. Additionally, evidence suggests that mucus-microbiome shifts occur as COPD progresses,26 and that COPD patients with very frequent exacerbations and higher mortality are characterized by Aspergillus with a concomitant increase in serum-specific IgE levels.27 It is possible that mucus and allergy in COPD also change over time, which requires further longitudinal studies to confirm. Additionally, compared to Dunican’s finding that asthma patients with mucus plugging were more likely to have sputum eosinophilia and systemic eosinophilia,13 and the sputum neutrophil percentage was higher in smoking patients with a high mucus plug score than in those with a low score,28 we found that neutrophil count was related to mucus plugging in COPD patients. One possible mechanism is that human neutrophil elastase mediates mucus hypersecretion via the tumor necrosis factor-α converting enzyme-epidermal growth factor receptor signaling pathway in vivo.29

Finally, our study was not without limitations. The sample size of this study was relatively small, and because only cross-sectional assessment was performed, we were unable to ascertain longitudinal changes in both sensitization state and clinical outcomes. Future studies are warranted to elucidate the mechanisms and pathological changes associated with Aspergillus sensitization and mucus plugging in COPD.

Conclusion

In summary, COPD patients with Aspergillus hypersensitivity exhibited poorer lung function and were more frequently observed to have mucus plugging on lung CT scans. A blood level of Af sIgE more than 0.7KU/L was related to mucus plugging, suggesting a potential role of Aspergillus sensitization in mucus hypersecretion in a subset of COPD patients.

Abbreviations

COPD, chronic obstructive pulmonary disease; IgE, immunoglobulin E; T-IgE, total IgE; A. Fumigatus, Aspergillus Fumigatus; Af sIgE, Aspergillus Fumigatus specific IgE; AH, Aspergillus hypersensitivity; ABPA, allergic bronchopulmonary aspergillosis; HRCT, high resolution computed tomography; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; GOLD, the global Initiative for chronic obstructive lung disease.

Data Sharing Statement

All data generated or analyzed during this study are available from the corresponding author upon reasonable request.

Ethics Approval and Consent to Participate

The study was approved by the Clinical Research Ethics Committees of Peking University Third Hospital (M2023784), and as it was a retrospective study, exemption from informed consent was applied and approved by the Clinical Research Ethics Committees of Peking University Third Hospital.

Funding

This study was financially supported by the National Natural Science Foundation of China (82370047).

Disclosure

The authors declare that they have no conflicts of interest in this work.

References

1. Jin J, Liu X, Sun Y. The prevalence of increased serum ige and aspergillus sensitization in patients with COPD and their association with symptoms and lung function. Respir. Res. 2014;15:1––12.

2. Everaerts S, Lagrou K, Dubbeldam A, et al. Sensitization to aspergillus fumigatus as a risk factor for bronchiectasis in COPD. COPD. 2017;12:2629–2638. doi:10.2147/COPD.S141695

3. Halpin DMG, Celli BR, Criner GJ, et al. The GOLD summit on chronic obstructive pulmonary disease in low- and middle-income countries. Int j Tuberc Lung Dis. 2019;23(11):1131–1141. doi:10.5588/ijtld.19.0397

4. Tiew PY, Narayana JK, Quek MSL, et al. Sensitisation to recombinant aspergillus fumigatus allergens and clinical outcomes in COPD. Eur Respir J. 2023;61(1):2200507. doi:10.1183/13993003.00507-2022

5. Agarwal R, Hazarika B, Gupta D, Aggarwal AN, Chakrabarti A, Jindal SK. Aspergillus hypersensitivity in patients with chronic obstructive pulmonary disease: COPD as a risk factor for ABPA? Med Mycol. 2010;48(7):988–994. doi:10.3109/13693781003743148

6. Hammond EE, McDonald CS, Vestbo J, Denning DW. The global impact of aspergillus infection on COPD. BMC Pulm Med. 2020;20(1):241. doi:10.1186/s12890-020-01259-8

7. Oguma T, Asano K, Tomomatsu K, et al. Induction of mucin and MUC5AC expression by the protease activity of aspergillus fumigatus in airway epithelial cells. J Immunol. 2011;187(2):999–1005. doi:10.4049/jimmunol.1002257

8. Wu X, Lee B, Zhu L, Ding Z, Chen Y. Exposure to mold proteases stimulates mucin production in airway epithelial cells through Ras/Raf1/ERK signal pathway. PLoS One. 2020;15(4):e0231990. doi:10.1371/journal.pone.0231990

9. Gao FS, Gao YY, Liu MJ, Liu YQ. Chronic aspergillus fumigatus exposure upregulates the expression of mucin 5AC in the airways of asthmatic rats. Exp Lung Res. 2012;38(5):256–265. doi:10.3109/01902148.2012.676705

10. Charriot J, Volpato M, Petit A, Vachier I, Bourdin A. Methods of sputum and mucus assessment for muco-obstructive lung diseases in 2022: time to “unplug” from our daily routine! Cells. 2022;11(5):812. doi:10.3390/cells11050812

11. Boucher RC. Muco-obstructive lung diseases. N Engl J Med. 2019;380(20):1941–1953. doi:10.1056/NEJMra1813799

12. Okajima Y, Come CE, Nardelli P, et al. Luminal plugging on chest CT scan. Chest. 2020;158(1):121–130. doi:10.1016/j.chest.2019.12.046

13. Dunican EM, Elicker BM, Gierada DS, et al. Mucus plugs in patients with asthma linked to eosinophilia and airflow obstruction. J Clin Investig. 2018;128(3):997–1009. doi:10.1172/JCI95693

14. Tiew PY, Fws K, Pang SL, et al. Environmental fungal sensitisation associates with poorer clinical outcomes in COPD. Eur Respir J. 2020;56(2):2000418. doi:10.1183/13993003.00418-2020

15. Fairs A, Agbetile J, Hargadon B, et al. IgE sensitization to aspergillus fumigatus is associated with reduced lung function in asthma. Am J Respir Crit Care Med. 2010;182(11):1362–1368. doi:10.1164/rccm.201001-0087OC

16. Fungal Culture and Sensitisation in Asthma, Cystic fibrosis and chronic obstructive pulmonary disorder: what does it tell us? | mycopathologia. Available from: https://link.springer.com/article/10.1007/s11046-014-9804-y. Accessed May8, 2024.

17. Bafadhel M, Mckenna S, Agbetile J, et al. Aspergillus fumigatus during stable state and exacerbations of COPD. Eur Respir J. 2014;43(1):64–71. doi:10.1183/09031936.00162912

18. Fillaux J, Brémont F, Murris M, et al. Assessment of aspergillus sensitization or persistent carriage as a factor in lung function impairment in cystic fibrosis patients. Scand J Infect Dis. 2012;44(11):842–847. doi:10.3109/00365548.2012.695454

19. O’Dea EM, Amarsaikhan N, Li H, et al. Eosinophils are recruited in response to chitin exposure and enhance Th2-mediated immune pathology in aspergillus fumigatus infection. Infect Immun. 2014;82(8):3199–3205. doi:10.1128/IAI.01990-14

20. Shin SH, Ye MK, Lee DW, Choi MH, Geum SY. Aspergillus enhances eosinophil and neutrophil extracellular DNA trap formation in chronic rhinosinusitis. IJMS. 2023;24(24):17264. doi:10.3390/ijms242417264

21. Muniz VS, Silva JC, Braga YAV, et al. Eosinophils release extracellular DNA traps in response to Aspergillus fumigatus. J Allergy Clin Immunol. 2018;141(2):571–585.e7. doi:10.1016/j.jaci.2017.07.048

22. Diaz AA, Orejas JL, Grumley S, et al. Airway-occluding mucus plugs and mortality in patients with chronic obstructive pulmonary disease. JAMA. 2023;329(21):1832. doi:10.1001/jama.2023.2065

23. Shin SH, Ye MK, Kim JK. Effects of fungi and eosinophils on mucin gene expression in rhinovirus-infected nasal epithelial cells. Allergy Asthma Immunol Res. 2014;6(2):149. doi:10.4168/aair.2014.6.2.149

24. Hill DB, Long RF, Kissner WJ, et al. Pathological mucus and impaired mucus clearance in cystic fibrosis patients result from increased concentration, not altered pH. Eur Respir J. 2018;52(6):1801297. doi:10.1183/13993003.01297-2018

25. Radicioni G, Ceppe A, Ford AA, et al. Airway mucin MUC5AC and MUC5B concentrations and the initiation and progression of chronic obstructive pulmonary disease: an analysis of the SPIROMICS cohort. Lancet Respir Med. 2021;9(11):1241–1254. doi:10.1016/S2213-2600(21)00079-5

26. Meldrum OW, Donaldson GC, Narayana JK, et al. Accelerated lung function decline and mucus-microbe evolution in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2024;210:298––310. doi:10.1164/rccm.202306-1060OC

27. Tiew PY, Dicker AJ, Keir HR, et al. A high-risk airway mycobiome is associated with frequent exacerbation and mortality in COPD. Eur Respir J. 2021;57(3):2002050. doi:10.1183/13993003.02050-2020

28. Dunican EM, Elicker BM, Henry T, et al. Mucus plugs and emphysema in the pathophysiology of airflow obstruction and hypoxemia in smokers. Am J Respir Crit Care Med. 2021;203(8):957–968. doi:10.1164/rccm.202006-2248OC

29. Zhao J, Yang T, Qiao W, Ye Y, Zhang J, Luo Q. Human neutrophil elastase mediates MUC5AC hypersecretion via the tumour necrosis factor-α converting enzyme-epidermal growth factor receptor signalling pathway in vivo. ORL. 2021;83(5):310–318. doi:10.1159/000509982