Small Airway Mucus Plugging in Severe Asthma: Insights and Implications

Asthma is a common, chronic obstructive airway disease characterized by inflammation, airway hyper-reactivity, and mucus plugging.1 Recent studies have demonstrated that mucus plugging is a key feature in severe asthma and the most common driver of fatal asthma events.2,3,4 The understanding of the role of mucus plugging in asthma has been enhanced by thoracic CT imaging studies of large airways.

These cross-sectional imaging studies have demonstrated that: 1) over half of severe asthmatics in the Severe Asthma Research Program (SARP) cohort have persistent CT-defined large airway mucus plugging despite inhaled corticosteroid therapy; 2) the amount of mucus plugging correlates with airflow limitation; and 3) the mucus plugs are long-lived within a given pulmonary segment.3,4

However, in asthma, along with most muco-obstructive diseases, mucus obstruction in small airways are sites of early and severe disease.5 These small airways are not effectively visualized by CT. Clinical studies, including the ATLANTIS study, have reported an association between small airways dysfunction and both asthma control and exacerbations.6 Importantly, the molecular pathophysiology and the characteristics of mucus plugging in small airways disease in asthma are not well-characterized.7

Our study aims to better understand molecular characteristics of the airway epithelium in the peripheral lung. We expand on previous fatal asthma morphology studies by adding VATS biopsies of severe, steroid-resistant asthmatics and using innovative technologies, including spatial transcriptomics and multiplex immunostaining, to define the small airway molecular asthmatic phenotype.8,9,10, 11

We hypothesized that asthmatic small airways would exhibit mucus plugging with high expression of MUC5AC, the secreted mucin known to be upregulated in the large airway in asthma.12,13 To test this hypothesis, we characterized small airways in tissues from fatal asthma cases and biopsies from steroid-resistant severe asthmatics. Using AB-PAS staining, we showed that there is a high mucus plug burden in bronchioles in both cohorts.

We measured mucin expression and mucus plugging by MUC5AC and MUC5B using RNA in situ hybridization (RNA-ISH) and immunohistochemistry (IHC), which showed increased MUC5AC expression into small airways of asthmatics. We found that small airways disease is strikingly heterogeneous in terms of the expression of MUC5AC, both between different airways in a single lung segment and even within a single airway.

To better characterize the epithelial heterogeneity of MUC5AC expression in asthmatic bronchioles, we performed spatial transcriptomics on fatal and severe asthma bronchiolar epithelia. We utilized this technology to measure gene expression differences between MUC5AC expressing epithelia (MUC5AC-High) and epithelia not expressing MUC5AC (MUC5AC-Low).

We compared each of these epithelial regions with control epithelia from organ donors not accepted for transplant and lobectomy specimens. We identified that, in addition to an increase in goblet cell genes and a loss of normal small airway secretory cell genes, MUC5AC-High epithelia expressed increased early ciliated cells and basal cells.

We further investigated the basal cells in the MUC5AC-High epithelia, by performing spatial transcriptomics segmented on the basal cell marker, cytokeratin 5. (KRT5). In MUC5AC-High regions, basal cells expressed high levels of genes associated with a type 2 inflammatory signature, namely IL-33, ALOX15, and POSTN.14 These findings suggest that the basal cells in MUC5AC-high epithelium are primed to differentiate into MUC5AC-expressing goblet cells.

Finally, we used multiplex immunostaining to identify whether epithelial heterogeneity of MUC5AC expression was associated with accumulation of immune cells. These studies revealed that specific populations of immune cells did not co-localize with MUC5AC niches, suggesting the importance of epithelial-intrinsic dysfunction in the mucus plugging characteristic of bronchiolar disease in asthma.

Our ongoing work supported by the ALA/AAAAI Allergic Respiratory Diseases Award focuses on better understanding the specific cell type alterations in asthma using single cell sequencing and chromatin accessibility in freshly isolated cells and cell culture from asthmatic individuals. These studies aim to identify important changes in gene expression and epigenetics of asthmatic goblet cells and basal progenitor cells. Understanding the mechanisms driving goblet cell metaplasia and subsequent MUC5AC-dominant mucus plugging in small airways will reveal potential targets for modulating small airway dysfunction in asthma.

References:

1. Papi A, Brightling C, Pedersen SE, Reddel HK. Asthma. Lancet. 2018;391(10122):783-800. doi:10.1016/S0140-6736(17)33311-1
2. Dunican EM, Watchorn DC, Fahy JV. Autopsy and Imaging Studies of Mucus in Asthma. Lessons Learned about Disease Mechanisms and the Role of Mucus in Airflow Obstruction. Ann Am Thorac Soc. 2018;15(Suppl 3):S184-S191. doi:10.1513/AnnalsATS.201807-485AW
3. 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(9):1036-1045. doi:10.1164/rccm.202110-2265OC
4. Dunican EM, Elicker BM, Gierada DS, et al. Mucus plugs in patients with asthma linked to eosinophilia and airflow obstruction. J Clin Invest. 2018;128(3):997-1009. doi:10.1172/JCI95693
5. Usmani OS, Han MK, Kaminsky DA, et al. Seven pillars of small airways disease in asthma and COPD: supporting opportunities for novel therapies. Chest. 2021;160(1):114-134. doi:10.1016/j.chest.2021.03.047
6. Kraft M, Richardson M, Hallmark B, et al. The role of small airway dysfunction in asthma control and exacerbations: a longitudinal, observational analysis using data from the ATLANTIS study. Lancet Respir Med. 2022;10(7):661-668. doi:10.1016/S2213-2600(21)00536-1
7. van den Bosch WB, James AL, Tiddens HAWM. Structure and function of small airways in asthma patients revisited. Eur Respir Rev. 2021;30(159). doi:10.1183/16000617.0186-2020
8. Kuyper LM, Paré PD, Hogg JC, et al. Characterization of airway plugging in fatal asthma. Am J Med. 2003;115(1):6-11. doi:10.1016/s0002-9343(03)00241-9
9. Schworer S, Okuda K, Dang H, et al. Mucus plugging and regional heterogeneity are features of asthmatic small airways. Journal of Allergy and Clinical Immunology. 2023;151(2):AB223. doi:10.1016/j.jaci.2022.12.694
10. Schworer S, Okuda K, Dang H, et al. Transcriptional pathways in small airway basal cells define niches of goblet cell metaplasia in asthma. In: C19. CHARTING THE LUNG: INNOVATION IN MULTIOMICS. American Thoracic Society; 2024:A5015-A5015. doi:10.1164/ajrccm-conference.2024.209.1_MeetingAbstracts.A5015
11. Schworer S, Okuda K, Dang H, et al. Steroid-resistant Severe Asthma and Fatal Asthma Small Airways Are Defined by Mucus Plugging and Heterogeneous MUC5AC Expression. In: C19. EMERGING MECHANISMS IN COPD AND ASTHMA. American Thoracic Society; 2023:A4535-A4535. doi:10.1164/ajrccm-conference.2023.207.1_MeetingAbstracts.A4535
12. Woodruff PG, Modrek B, Choy DF, et al. T-helper type 2-driven inflammation defines major subphenotypes of asthma. Am J Respir Crit Care Med. 2009;180(5):388-395. doi:10.1164/rccm.200903-0392OC
13. Bonser LR, Zlock L, Finkbeiner W, Erle DJ. Epithelial tethering of MUC5AC-rich mucus impairs mucociliary transport in asthma. J Clin Invest. 2016;126(6):2367-2371. doi:10.1172/JCI84910
14. Christenson SA, Steiling K, van den Berge M, et al. Asthma-COPD overlap. Clinical relevance of genomic signatures of type 2 inflammation in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2015;191(7):758-766. doi:10.1164/rccm.201408-1458OC