MUC5AC-rich mucus plugs in asthma are tethered to an epithelium that is buckled to form mucosal folds. We used histology and confocal imaging to characterize the mucin protein profile and histologic features of plugged and unplugged airways from patients with asthma (103 airways), patients with COPD (50 airways, disease control), and age-matched lung disease–free individuals acting as controls (lung disease–free control) airways (48 airways) (Figure 1). We found no mucus plugs in the airways of disease-free control lungs, whereas mucus plugs were identifiable in multiple airways from patients with asthma and COPD (Figure 2, A–F). We first analyzed the relative proportions of MUC5AC and MUC5B (the principal gel-forming mucins in the airways) in these mucus plugs. We found that mucus plugs from patients with asthma were rich in MUC5AC (Figure 2, G and H), whereas those from patients with COPD were rich in MUC5B (Figure 2, I and J), and the ratio of MUC5AC to MUC5B was significantly higher in asthma than in COPD (Figure 2K). We noticed that mucus plugs from patients with both asthma and COPD were tethered to the surface of goblet cells by mucin strands that connected the edge of the mucus plug to the epithelium (Figure 2L and Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI186889DS1). Using a metric of mucus tethering (percentage tethering, Supplemental Figure 2A) to quantify the degree of continuity between the mucus plugs and the epithelium, we found that percentage of tethering correlated positively and significantly with MUC5AC immunostaining in asthma but not in COPD (Figure 2M and Supplemental Figure 1C). We also noticed that the epithelium around the plugs was frequently buckled, forming bronchial mucosal folds that penetrated into the airway lumen to decrease the lumen area. The mucosal folds were rich in goblet cells, and we noticed that mucus secreted by these goblet cells was attached to the luminal mucus on both sides of the fold (Figure 2N and Supplemental Figure 1, D and E), which could have an effect to maintain the folds. Using a measure of bronchial mucosal folding (percentage folding, Supplemental Figure 2B), we quantified the difference between the size of the airway lumen and the basement membrane perimeter. We found that the percentage of folding correlated positively with percentage of mucus tethering in both asthma and COPD (Figure 2O and Supplemental Figure 1F). Specifically, the percentage of folding was higher in mucus-plugged airways from patients with asthma and COPD than in unplugged airways in these patient groups or in unplugged airways from lung disease–free individuals acting as controls (Figure 2P and Supplemental Figure 1G), indicating that the airway lumen is thereby smaller in mucus-plugged airways relative to their basement membrane perimeter.

Selection of lung tissues from patients with asthma, patients with COPD, and lung disease–free individuals acting as controls. The flow diagram shows how tissues were selected from the James Hogg Lung Biobank at the University of British Columbia. It further illustrates the screening of lung tissue sections for mucus-plugged and unplugged airways and the number of samples analyzed by histology, immunofluorescence, and imaging mass cytometry.

MUC5AC is the principal mucin in asthma mucus plugs, and epithelial mucus plug tethering correlates with extent of mucosal folding. (A) Lung disease–free controlAsthma airway (participant ID 7018). (B) Unplugged asthma airway (participant ID 7239). (C) H&E-stained asthma mucus plug (participant ID 7016). (D) Lung disease–free controlCOPD airway (participant ID 7309). (E) Unplugged COPD airway (participant ID 7336). (F) H&E-stained COPD mucus plug (participant ID 7336). (G) Asthma mucus plugs stained for MUC5AC (green), MUC5B (magenta), and DNA (blue). (H) MUC5AC immunostaining in asthma mucus plugs is higher than in COPD mucus plugs. ***Significantly different from COPD, P < 0.001 (Mann-Whitney test). (I) COPD mucus stained for MUC5B (magenta), MUC5AC (green), and DNA (blue). (J) MUC5B immunostaining in asthma mucus plugs is lower than in COPD plugs. ****Significantly different from COPD, P < 0.0001 (Mann-Whitney test). (K) The MUC5AC/MUC5B ratio in asthma mucus plugs is higher than in COPD mucus plugs. ****Significantly different from COPD, P < 0.0001 (Mann-Whitney test). (L) Mucus strands (black arrowheads) connect the asthma mucus plug (MP) to the surface of goblet cells (GB) (participant ID 7187). (M) Mucus plug tethering percentage correlates with MUC5AC immunostaining in asthma mucus plugs (n = 61) (Spearman’s correlation). (N) Mucosal folds in mucus-plugged airways in asthma are rich in goblet cells (GB) and mucus is tethered to goblet cells at multiple points along the folds (black arrowheads) (participant ID 7237). (O) Mucus plug tethering percentage correlates with mucosal folding percentage in mucus plugs from patients with asthma (n = 61) (Spearman’s correlation). (P) Mucosal folding percentage is higher in mucus-plugged airways in asthma (asthma MP) than in unplugged airways in asthma (asthma UnP) or in lung disease–free controlAsthma airways. ****Significantly different from lung disease–free controlsAsthma, P < 0.0001; ####significantly different from unplugged asthma airways, P < 0.0001 (Kruskal-Wallis test with Dunn’s correction). Scale bars: 200 μm.

Spatial, single-cell proteomic characterization of mucus plugs and the airways in which they form. In a subset of 117 airways from individuals with asthma, airways from individuals with COPD, and lung disease–free control airways, we used spatial single-cell imaging mass cytometry (IMC) to analyze the interaction of mucins with structural and immune cells in the mucus plugs and the airway wall (Figure 3A). In selecting this subset of airways, our objective was to enhance our understanding of the cellular environment surrounding asthma mucus plugs. Therefore, we prioritized the number of asthma airways (n = 55) over the number of COPD airways (n = 17), which we mostly used as a disease control group for mucus plug composition. In total, 750,883 cells comprising immune, epithelial, endothelial, and smooth muscle cells were assessed (Figure 3B). The frequency of the cell types varied among participants (Figure 3C), and the relatively high frequency of unassigned cells was because markers for common lung cells such as fibroblasts and alveolar epithelial cells were not included in the cell segmentation panel. A total of 23 different cell types were identified by marker expression (Figure 3D and Supplemental Table 3), and the most abundant cells were granulocytes (eosinophils and neutrophils), macrophages, smooth muscle cells, and endothelial cells (Figure 3D). We extracted the mean cell area measurement for each cell type, and we categorized cells according to their location in the airway lumen, airway epithelium, or airway wall (Figure 3D). Final images could be visualized through direct immunostaining or single-cell segmentation (Figure 3E and Supplemental Figure 3). In this way, we confirmed the confocal imaging data for expression of MUC5AC and MUC5B in mucus plugs and that the cell markers for airway epithelial cells, smooth muscle cells, and endothelial cells generated the expected airway images (Figure 3E). Spatial visualization of the 23 cell types provided details about their distribution across the different airways (Supplemental Figure 3).

Spatial, single-cell characterization of mucus plugs and airways by imaging mass cytometry. (A) Schematic representation of the imaging mass cytometry (IMC) data generation and analysis pipeline. (B) Uniform manifold approximation and projection (UMAP) plot of all the cells identified in the IMC dataset, colored by cell classes. (C) Cell class frequency distribution among the different study participants: lung disease–free controlsAsthma, patients with asthma, lung disease–free controlsCOPD, and patients with COPD. (D) Heatmap of marker expression for all cell classes and cell types. An additional heatmap shows cell area (in pixel [1 pixel = 1 μm2]). The 2 bar graphs show cell spatial categorization and the number of cells of each type, respectively. (E) Representative images of mucin immunostaining and cell class distribution after cell segmentation in a lung disease–free control airway (participant ID 7272), an asthma unplugged airway (participant ID 7239), an asthma mucus-plugged airway (participant ID 7188), and a COPD mucus-plugged airway (participant ID 6971). Scale bars: 100 μm.

The airway epithelium of mucus-plugged airways in asthma is characterized by hyperplasia of basal cells and MUC5AC-positive goblet cells. To explore the pathology of the airway epithelium surrounding mucus plugs, single-cell IMC was used to assess the ciliated cells, basal cells, and goblet cells in individuals with asthma and lung disease–free individuals acting as controls (Figure 4, A–C). We found that the airway epithelium was composed of over 80% epithelial cells (Figure 4D), and the number of epithelial cells in asthma airways with mucus plugs was higher than in unplugged asthma airways or lung disease–free control airways (Figure 4E). While the number of ciliated cells was similar to that in lung disease–free individuals acting as controls (Figure 4F), the increase in total epithelial cell number in plugged airways was partially driven by an increase in the number of basal cells in these airways (Figure 4G). In addition, the basal cells in asthma airways with mucus plugs had higher proliferative activity, as evidenced by increased Ki-67 positivity (Figure 4H). Although the number of goblet cells expressing MUC5B was similar in asthma airways with mucus plugs, unplugged asthma airways, and lung disease–free control airways (Figure 4I), the number of goblet cells expressing MUC5AC was significantly higher in asthma airways occluded with mucus plugs (Figure 4J). The MUC5AC-expressing goblet cells were also larger in the mucus-plugged asthma airways (Figure 4K), indicating that MUC5AC-expressing goblet cells undergo both hyperplasia and hypertrophy. In COPD, the total number of epithelial cells in the airway epithelium of mucus-plugged airways was not significantly different than in unplugged COPD airways or in lung disease–free control airways (Supplemental Figure 4A).

The airway epithelium of mucus-plugged airways in asthma is characterized by hyperplasia of basal cells and MUC5AC-positive goblet cells. (A–C) Representative cell-segmented images of segmented epithelium and epithelial cell types in a lung disease–free controlAsthma airway (participant ID 7234) (A), an asthma unplugged airway (participant ID 7239) (B), and an asthma plugged airway (participant ID 7016) (C). Scale bars: 100 μm. (D) Pie chart showing the cell class diversity of cells in asthma and disease-free controlAsthma airway epithelium. (E) Epithelial cell numbers are increased in asthma mucus-plugged airways. (F) Ciliated cell numbers are similar in lung disease–free controlAsthma and asthma plugged airways. (G) Basal cell numbers are increased in asthma mucus-plugged airways. (H) Basal cell proliferation is increased in asthma mucus-plugged airways (violin plot shows the Ki-67 mean cellular expression of basal cells. and the red lines indicate the average for each subgroup). (I) Epithelial goblet cells expressing MUC5B are similar in all patient groups. (J) Epithelial goblet cells expressing MUC5AC are increased in asthma airways occluded with mucus. (K) Kernel density plot showing a higher area for epithelial goblet cells expressing MUC5AC in mucus-plugged asthma airways than that in unplugged asthma airways and in lung disease–free controlAsthma airways. *Significantly different from lung disease–free controlAsthma, P < 0.05; ***significantly different from lung disease–free controlAsthma, P < 0.001; ****significantly different from lung disease–free controlAsthma, P < 0.0001; #significantly different from asthma unplugged airways, P < 0.05; ####significantly different from asthma unplugged airways, P < 0.0001 (ordinary 1-way ANOVA with Tukey’s correction).

The airway wall of mucus-plugged airways in asthma is characterized by smooth muscle hyperplasia and ILC2 infiltration. To explore the pathology of the airway wall in which mucus plugs form in asthma, we applied IMC to characterize the structural cells and immune cells in the airway wall, excluding the airway epithelium (Figure 5, A–C). We found that most of the cells identified by IMC in the airway wall were immune cells, smooth muscle cells, and endothelial cells (Figure 5, A–D). Compared with lung disease–free control airways, endothelial cell numbers were increased in asthma airways with and without mucus plugs (Figure 5E), and smooth muscle cell numbers were specifically and significantly increased in asthma airways occluded with mucus plugs (Figure 5F). In addition, total immune cell number, comprising innate lymphoid type 2 cells (ILC2s), T cells, eosinophils, mast cells, B cells, macrophages, CD8+ T cells, and NK cells, was higher in asthma airways with and without mucus plugs than in lung disease–free control airways (Figure 5, G and H). Notably, ILC2 cells emerged from the IMC data as a cell type whose numbers were specifically increased in asthma airways occluded with mucus plugs (Figure 5I). In COPD, the number of endothelial cells, smooth muscle cells, or immune cells in the airway walls of mucus-plugged airways was not significantly different than in the airway walls of unplugged COPD airways or in lung disease–free control airways (Supplemental Figure 4, B–D).

Smooth muscle cell hyperplasia and ILC2 infiltration characterize asthma airways occluded with mucus. (A–C) Representative cell-segmented images of airway wall categorization and cell class identification of a lung disease–free controlAsthma airway (participant ID 7018) (A), an asthma unplugged airway (participant ID 7238) (B), and an asthma plugged airway (participant ID 7237) (C). Scale bars: 100 μm. (D) Pie chart showing the cell class diversity of cells in asthma and disease-free control airway walls. (E) Endothelial cell numbers in asthma airway walls (unplugged and plugged) are higher than in lung disease–free controlAsthma airways. (F) Smooth muscle cell numbers in the walls of asthma airways plugged with mucus are higher than in unplugged asthma airway walls or in lung disease–free controlAsthma airway walls. (G) Immune cell numbers in airway walls in asthma (unplugged and plugged) are higher than in lung disease–free controlAsthma airways. (H) Heatmap of the z scores values of the immune cell types that are significantly higher in number in the airway wall of asthma unplugged and plugged airways compared with lung disease–free controlAsthma airways. (I) ILC2 numbers in the walls of asthma airways occluded with mucus are higher than in unplugged asthma airways and in lung disease–free controlAsthma airways. ***Significantly different from lung disease–free controlAsthma, P < 0.001; ****significantly different from lung disease–free controlAsthma, P < 0.0001; #significantly different from asthma unplugged airways, P < 0.05 (ordinary 1-way ANOVA with Tukey’s correction).

Mucus plugs are infiltrated with immune cells that are dual positive for markers of eosinophils and neutrophils. To characterize the cellular profile of mucus plugs that occlude airways in asthma, we segmented mucus plugs to determine the number and type of immune cells that infiltrate them (Figure 6A). Among cells infiltrating asthma mucus plugs, immune cells predominated (92%), although there were some epithelial cells (6.3%) (Figure 6B), which were mainly goblet cells (Figure 6C). Among immune cells that infiltrate mucus plugs, cells double positive for eosinophil peroxidase (EPX) and neutrophil elastase (ELA2) were by far the most prevalent cells (Figure 6D). We used the “granulocyte” label for these EPX/ELA2 double-positive cells because of their dual expression of both eosinophil and neutrophil markers. We found that granulocytes were infrequent in some plugs and abundant in others, so we used the median split value for their number in plugs to categorize the plugs as either paucigranulocytic or granulocytic (Figure 6D). COPD mucus plugs, for their part, were also infiltrated by immune cells (Figure 6E) whose numbers were intermediate between those in asthma granulocytic and paucigranulocytic mucus plugs (Figure 6F). Similar to asthmatic plugs, the immune cells in COPD plugs were predominantly granulocytes (cells double positive for ELA2 and EPX, 79.1%), followed by macrophages (8.8%) and neutrophils (cells positive for ELA2 only, 5.4%) (Figure 6G and Supplemental Figure 4E).

Paucigranulocytic mucus plugs rich in MUC5AC mucin are frequent in fatal asthma whereas granulocytic mucus plugs comprising a mix of MUC5AC and MUC5B mucins are frequent in nonfatal asthma. (A) Mucin immunostaining and cell-segmented images of lumen categorization and cell identification of 2 asthma plugged airways (participant IDs 7188 and 7298). Scale bars: 100 μm. (B) Pie chart showing the cell class diversity of cells infiltrating asthma mucus plugs. (C) Pie chart showing the epithelial cell type diversity of epithelial cells infiltrating asthma mucus plugs. (D) Diversity and prevalence of immune cell types infiltrating asthma mucus plugs, with granulocytes identified as the predominant cells. The median of total immune cell number was used to categorize mucus plugs as paucigranulocytic or granulocytic. (E) Mucin immunostaining and cell-segmented images of immune cells in a COPD plugged airway (participant ID 6967). Scale bars: 100 μm. (F) Immune cell infiltration of COPD mucus plugs is intermediate to that of asthma mucus plugs. ***Significantly different from COPD mucus plugs, P < 0.001; ####significantly different from asthma granulocytic plugs, P < 0.0001 (ordinary 1-way ANOVA with Tukey’s correction). (G) Pie chart showing the immune cell type diversity of cells infiltrating COPD mucus plugs. (H) Relationship between DNA immunostaining and the total immune cell number infiltrating airway lumen of mucus plugs analyzed by confocal imaging and IMC (n = 25) (Spearman’s correlation). (I) The median of DNA percentage was used to categorize mucus plugs as paucigranulocytic or granulocytic. (J) Paucigranulocytic mucus plugs are more frequent in fatal asthma, whereas granulocytic mucus plugs are more frequent in nonfatal asthma (P < 0.01, Fisher’s exact test). (K) MUC5AC immunostaining in paucigranulocytic mucus plugs is higher than in granulocytic mucus plugs. (L) The MUC5AC/MUC5B ratio is higher in paucigranulocytic plugs than in granulocytic plugs. ****Significantly different from paucigranulocytic plugs, P < 0.0001 (Mann-Whitney test).

The frequency of paucigranulocytic and granulocytic mucus plugs differs in fatal and nonfatal cases of asthma. To determine if the frequency of asthma mucus plug subtypes differs in fatal and nonfatal cases, we used the higher number of asthma mucus plugs that had been analyzed by histology and confocal imaging than by IMC (62 vs. 32), which provided more statistical power for the analysis. To categorize the plugs in the larger histology dataset as granulocytic or paucigranulocytic, we used percentage DNA staining as a measure of immune cell infiltration of the mucus plugs. There was a high correlation between percentage DNA staining and immune cells numbers in the mucus plugs analyzed by IMC (r = 0.7080 and P < 0.0001, Figure 6H), allowing the median percentage of DNA staining data to segregate 62 asthma mucus plugs into paucigranulocytic and granulocytic plugs (Figure 6I). In this way, we found that paucigranulocytic mucus plugs occur frequently in fatal asthma and that granulocytic mucus plugs are more frequent in nonfatal asthma (Figure 6J). Analyzing mucus plug subtypes on an individual participant basis, we found that fatal asthma cases mostly had higher numbers of paucigranulocytic plugs and nonfatal asthma cases mostly had higher numbers of granulocytic plugs (Supplemental Figure 5A). In addition, we noticed that paucigranulocytic plugs were characterized by high MUC5AC mucin content, whereas granulocytic plugs had a more balanced mix of MUC5AC and MUC5B mucins (Figure 6, K and L). Furthermore, paucigranulocytic plugs were characterized by higher amounts of tethering and folding than granulocytic plugs (Supplemental Figure 5, B and C). Taken together, these results suggest that paucigranulocytic mucus plugs result from acute degranulation of MUC5AC-positive goblet cells, whereas granulocytic plugs have a more complicated pathogenesis to account for their infiltration with granulocytes and their more balanced mix of MUC5AC and MUC5B mucins.

Eosinophils interact with mucins secreted by IL-13–activated airway epithelial cells and undergo cytolysis. The IMC analyses identified that immune cells infiltrating mucus plugs from patients with asthma predominantly coexpressed EPX and ELA2 (Figure 7A). A minority of immune cells exclusively expressed ELA2 and very few cells expressed only EPX (Figure 7A). To validate the IMC data, we used immunofluorescence methods to immunostain the mucus plugs for EPX and ELA2. This analysis confirmed that the majority of granulocytes infiltrating mucus plugs were double positive for EPX and ELA2 (Figure 7B). These data indicate that both eosinophils and neutrophils infiltrate the plugs, but the cells are somehow interacting in the mucin-rich environment of the plug. We hypothesized that eosinophils undergo degranulation with mucin exposure and that the extracellular EPX is bound and internalized by phagocytic neutrophils, as it has been previously reported (16). We therefore set out to determine whether mucins could cause eosinophil degranulation, by culturing human eosinophils in mucus secreted by human airway epithelial cells (HAECs) stimulated with IL-13 to recapitulate an allergic asthma environment (Supplemental Figure 6A). We found that exposure of eosinophils to epithelial cell mucus formed primarily of MUC5AC caused eosinophils to undergo nonapoptotic cell death, as evidenced by FACS data showing high numbers of cells in the dead cell fraction and low expression of annexin-5 (Figure 7C). Notably, the percentage of eosinophils undergoing nonapoptotic cell death was significantly lower when eosinophils were overlaid on mucus-depleted epithelial cells (Figure 7, D and E). Although IL-13 stimulation is also known to change the basolateral secretions of epithelial cells (17), we found that basolateral secretions of IL-13–stimulated cells did not change eosinophil viability (Figure 7F and Supplemental Figure 6, B and C), indicating that the induction of nonapoptotic cell death is specific to apical secretions. Nonapoptotic death of eosinophils associated with degranulation in epithelial mucus could suggest that cytolysis has occurred. Eosinophil degranulation during cytolytic cell death is distinct from classical exocytosis where no cell death occurs or apoptosis where programmed cell death occurs without degranulation (18). To confirm that eosinophils degranulate and release EPX in MUC5AC-rich mucus, we used 3D renderings of whole-mount and apical washes of the epithelial and eosinophil cocultures. In this way, we showed that eosinophils degranulate and release EPX in the MUC5AC-rich mucus layer (Figure 7G, Supplemental Figure 6D, and Supplemental Video 1). In addition, we noted that eosinophils exposed to apical mucus are coated in MUC5AC (Supplemental Figure 6C), suggesting a direct, possibly contact-dependent, interaction between eosinophils and mucins.

MUC5AC rich mucus secreted by IL-13–activated airway epithelial cells (HAECs) causes cytolytic degranulation of eosinophils. (A) Granulocytes dual positive for eosinophil peroxidase (EPX) and neutrophil elastase (ELA2) infiltrate asthma mucus plugs (IMC), although ELA2 single-positive neutrophils also occur. (B) Confocal imaging confirms cells in mucus plugs are double positive for EPX (red) and ELA2 (yellow) (participant ID 7233). DNA is shown in blue. Scale bar: 200 μm. (C) Nearly half (44.4%) of the eosinophils are dead+/annexin5– (nonapoptotic dead cells) when overlaid on mucus layer of IL-13–activated human airway epithelial cells (HAECs). (D) Only 15.7% of eosinophils are dead+/annexin5– cells when overlaid on mucus-depleted HAECs. (E) Nonapoptotic dead cell percentage is lower when eosinophils are overlaid on mucus-depleted HAECs (n = 8). ***Significantly different from eosinophils overlaid on IL-13 mucus, P < 0.001 (paired t test). (F) No eosinophils incubated in basolateral media of HAECs underwent nonapoptotic death (n = 4). (G) Representative immunostaining and 3D rendering of whole-mount cocultures of HAECs and eosinophils. EPX (red), MUC5AC (green), and nuclei (blue). White arrows mark eosinophil degranulation. Scale bars: 20 μm. (H) Nonapoptotic dead cell percentage is higher when eosinophils are incubated with high-molecular-weight versus low-molecular-weight-fraction (n = 6). **Significantly different from eosinophils incubated with high-molecular-weight mucus, P < 0.01 (paired t test). (I) Incubation of eosinophils with anti-CD11b or pretreatment of high-molecular-weight mucus with periodate (NaIO4) decreased nonapoptotic dead cell percentage. Symbols represent independent experiments (n = 5). **Significantly different from eosinophils incubated with high-molecular-weight mucus alone, P < 0.01 (ordinary 1-way ANOVA with Tukey’s correction). ##Significantly different from eosinophils incubated with high-molecular-weight mucus and isotype control, P < 0.01 (Tukey’s correction). (J) MUC5AC-coated degranulating eosinophils (asterisks) are visible when eosinophils are incubated with high-molecular-weight mucus. Incubation of eosinophils with anti-CD11b or pretreatment of high-molecular-weight mucus with periodate decreased the frequency of these MUC5AC-coated degranulating eosinophils. Scale bars: 20 μm.

Mucins induce eosinophil cytolytic degranulation in a mechanism mediated by mucin glycans and eosinophil CD11b. To investigate the mechanism of interaction between mucus and eosinophils, we used ultrafiltration to separate the high- and low-molecular-weight fractions of apical secretions from IL-13–treated HAECs (Supplemental Figure 6E). We found that only the high-molecular-weight (mucin-rich) fraction induced eosinophil nonapoptotic cell death (Figure 7H and Supplemental Figure 6, F and G). In considering how mucins might cause eosinophil cytolysis, we noted prior research that shows that fibrinogen is a specific trigger for cytolytic eosinophil degranulation and that the mechanisms is integrin (CD11b) mediated (19). Other studies have also implicated CD11b in mechanisms of eosinophil adhesion to polymers and pathogens (20, 21). In addition, any mechanism of mucin-related cell activation needs to consider the role of mucin glycans, since mucins are heavily glycosylated and these glycans have important signaling functions (22). We therefore tested if inhibiting CD11b or removing the glycan coat of mucins would reduce the cytolytic effect of mucus on eosinophils. We found that inhibition of CD11b on eosinophils significantly decreased eosinophil nonapoptotic death when the eosinophils were exposed to the high-molecular-weight fraction of epithelial cell mucus (Figure 7I and Supplemental Figure 6, H and I). In addition, we found that eosinophils exposed to high-molecular-weight fraction of epithelial cell mucus that had been treated with periodate to remove glycans also showed significantly decreased eosinophil nonapoptotic death (Figure 7I and Supplemental Figure 6, H and K). Furthermore, using microscopy, we found that eosinophils incubated with high-molecular-weight mucus are heavily coated with MUC5AC and show a degranulating phenotype (Figure 7J). CD11b inhibition on eosinophils or periodate treatment of the high-molecular-weight fraction of the mucus decreased the number of these degranulating MUC5AC-coated eosinophils by 55% and 80%, respectively (P < 0.0001 for both reductions vs. control). Taken together, these findings demonstrate that eosinophils undergo cytolytic degranulation when in contact with MUC5AC-rich airway mucus in a glycan- and CD11b-dependent manner, making the released EPX available to be bound and internalized by neutrophils.

Granulocytic mucus plugs are infiltrated by extracellular traps. The high DNA levels and prominent infiltration of mucus plugs by granulocytes prompted us to explore if granulocytic mucus plugs are infiltrated with extracellular DNA traps. Using the IMC dataset, we found that mucus plugs from patients with COPD and granulocytic mucus plugs from patients with asthma showed extracellular histone H3 immunostaining, while the paucigranulocytic mucus plugs exhibited histone H3 staining exclusively within the cell boundaries (Figure 8A). The median intensity of histone H3 immunostaining in the airway lumen was significantly higher in COPD mucus plugs and asthma granulocytic mucus plugs than in asthma paucigranulocytic mucus plugs (Figure 8B). Asthma granulocytic mucus plugs are therefore characterized by high number of granulocytes that release extracellular traps and by a mucin profile that includes both MUC5AC and MUC5B mucins. In contrast, asthma paucigranulocytic plugs are characterized by low numbers of granulocytes and by a mucin profile dominated by MUC5AC.

Granulocytic mucus plugs have increased numbers of extracellular DNA traps. (A) COPD mucus plugs (participant ID 6968) and granulocytic asthma mucus plugs (participant ID 7239) show extracellular histone H3 immunostaining. Paucigranulocytic mucus plug (participant ID 7188) shows histone H3 immunostaining only within the cell boundaries. Scale bar: 100 μm. (B) Mean intensity of histone H3 immunostaining in COPD mucus plugs and asthma granulocytic mucus plugs is higher than in asthma paucigranulocytic mucus plugs. *Significantly different from COPD mucus plugs, P < 0.05 (Kruskal-Wallis test with Dunn’s correction). ####Significantly different from asthma granulocytic mucus plugs, P < 0.0001 (Kruskal-Wallis test with Dunn’s correction). (C) Paucigranulocytic mucus plugs are more common in acute asthma than in chronic asthma. (D) Schematic summary of findings for mucus plugs from patients with asthma. Asthma mucus plugs form in airways that are inflamed and remodeled, displaying features such as folding of the epithelium and hyperplasia of smooth muscle cells, basal cells, and goblet cells. Two subtypes of mucus plugs are identified in asthma: paucigranulocytic, which are high in MUC5AC and low in cellular infiltration, and granulocytic, which have a balanced mix of MUC5AC and MUC5B and high numbers of infiltrating granulocytes. Granulocytes in these plugs tend to degranulate and form DNA extracellular traps. Paucigranulocytic mucus plugs likely result from acute goblet cell degranulation while granulocytic mucus plugs may be a subset of acute mucus plugs that fail to resolve and become chronic.