Relationships between basement membrane zone thickness and airway physiology, epithelial gene expression and innate immune cell infiltration in asthma

Abstract

Rationale

Thickening of the basement membrane zone (BMZ) has often been identified in airway biopsies of individuals with asthma, but the relationship of BMZ thickness to airway dysfunction and inflammation is incompletely understood. The objectives of the present study were to characterise the relationship between BMZ thickness and airway physiology, immune cell infiltration and airway gene expression.

Methods

We used design-based stereology to measure BMZ thickness in endobronchial biopsy samples obtained from 10 healthy control subjects and 31 individuals with asthma not receiving asthma-directed anti-inflammatory therapies (only using as-needed albuterol for the study period). BMZ thickness was correlated with measures of airway physiology, sputum inflammatory markers, airway wall immunopathology and gene expression from airway epithelial brushings.

Results

Individuals with asthma had a significantly thicker BMZ relative to healthy controls. BMZ thickness was negatively correlated with age and positively correlated with higher baseline lung function (forced vital capacity), mast cell (MC) infiltration of the airway epithelial compartment and expression of type 2 (T2) inflammatory genes in the airway. Thickening of the BMZ was not significantly associated with the bronchodilator response, direct airway hyperresponsiveness (AHR) to methacholine, indirect AHR in the form of exercise-induced bronchoconstriction, T1 or T3 inflammation, or airway eosinophilia.

Conclusions

MC infiltration of the airway epithelial compartment and T2 inflammation are significantly associated with a thickened BMZ in asthma.

Shareable abstract

Basement membrane thickening is significantly associated with mast cell infiltration of the airway epithelial compartment and type 2 inflammation https://bit.ly/4xlx8Gz

Introduction

The specific role and associated histological features of airway wall remodelling in asthma pathobiology remains incompletely understood, particularly its relationship with asthma inflammatory endotypes and airway physiology. One of the most cited features of airway remodelling in asthma is thickening of the basement membrane zone (BMZ) beneath the airway epithelium, which represents an increased density of extracellular matrix components like collagen, fibronectin, tenascin and proteoglycans in the lamina reticularis [1, 2]. Prior studies have identified increased BMZ thickness in individuals with severe asthma relative to milder severity asthma and smoking-related COPD [3], and also demonstrated associations between thicker BMZ and eosinophilic [4] or type 2 (T2)-predominant airway inflammation [5]. However, the pathological role of BMZ thickening in asthma is still unclear, including its role in modulating airway physiology [5–8], relationship to key cell populations implicated in asthma pathogenesis, association with airway remodelling gene expression programmes in the airways and response to asthma-directed therapies [9].

A recent study used design-based stereology to measure BMZ thickness in individuals from the Severe Asthma Research Program-3 (SARP-3) [10], which provided some novel insights into associations between features of asthma and BMZ thickening among individuals with severe asthma. These included significant positive correlations between BMZ thickening and improvement in lung function following albuterol use (bronchodilator responsiveness (BDR)), allergic sensitisation to pet dander, and airway gene expression of T2 inflammatory genes and mast cell (MC)-specific genes (TPSAB1, CPA3). However, the study was unable to address the relationship between BMZ thickness and other key physiological features of asthma, such as airway hyperresponsiveness (AHR) or direct assessments of critical inflammatory cell populations in the airway wall. Further, the study analysed individuals with severe asthma who were extensively treated with asthma-directed anti-inflammatory therapies (oral corticosteroids, inhaled corticosteroids (ICS), leukotriene receptor antagonists (LTRAs)), making it difficult to uncouple their findings from exposure to asthma treatments.

Here we measured BMZ thickness in endobronchial biopsies obtained from individuals with mild–moderate asthma that were not exposed to asthma-directed anti-inflammatory therapies (only utilising albuterol on as needed basis for the study period) using the same design-based stereology technique. This study population underwent detailed characterisation of airway physiology relevant for asthma, including direct AHR to methacholine and indirect AHR to dry air exercise challenge. Using this same cohort, we have previously described relationships between airway physiology in asthma with the quantity and location of MCs [11, 12] and eosinophils (EOS) [13] in the airway wall using design-based stereology as well as airway gene expression patterns from endobronchial brushings [14]. Here we analysed relationships between BMZ thickness and baseline lung function, BDR, AHR, MC and EOS infiltration of the airway wall, and airway expression of genes implicated in indirect AHR.

Materials and methods

Study population and procedures

We conducted a cross-sectional study of individuals with and without asthma who have previously been described [11–14]. All participants provided informed consent, and the study protocol was approved by the University of Washington Institutional Review Board. Additional details regarding our study population, inclusion and exclusion criteria, and study procedures are available in the supplementary material.

Briefly, we recruited healthy adults and adults with mild–moderate asthma. Patients with asthma were eligible for the study if they used either an inhaled short-acting β₂-agonist alone or in combination with a single daily therapy such as a LTRA, low-dose ICS or low-dose ICS combined with a long-acting β₂-agonist and had a forced expiratory volume in 1 s (FEV₁) >80% of predicted. “Low-dose ICS” was defined as outlined in the Global Initiative for Asthma (GINA) 2025 guidelines for ICS potency [15]. All participants underwent methacholine challenge testing and dry air exercise challenge. Participants without asthma had an FEV₁≥80% predicted, negative methacholine challenge (PC₂₀ >8 mg·mL⁻¹) and negative dry air exercise challenge test (<7% fall in FEV₁ following exercise). Participants with asthma were required to have a positive methacholine challenge test (PC₂₀ <8 mg·mL⁻¹) and were determined to have indirect AHR in the form of exercise-induced bronchoconstriction (EIB) by a ≥10% decrease in FEV₁ following exercise challenge. Six participants with asthma were using a daily asthma-directed anti-inflammatory therapy prior to study enrolment and were monitored off these treatments for 2 weeks prior to methacholine challenge testing, 3 weeks prior to exercise challenge testing and 5 weeks prior to research bronchoscopy (supplementary figure S1). The remaining subjects did not use any daily controller therapy for their asthma, and none of the subjects were treated with daily controller therapies during the study.

Study participants underwent induced sputum testing for sputum cytology analysis. To assess airway T2 gene expression in this cohort, the expression levels of interleukin (IL)-4, IL-5 and IL-13 in induced sputum cells were centred, scaled and combined into the summary T2 gene mean (T2GM) metric as previously described [11, 16].

Finally, participants underwent research bronchoscopy, which included collection of epithelial brushings and endobronchial biopsies. RNA isolated from epithelial brushings underwent RNA-sequencing (RNA-seq) and differential gene expression analysis as previously described [14]. Our prior analysis identified 120 DEGs between individuals with asthma with EIB versus without EIB (47 genes with lower expression and 73 genes with significantly higher expression in individuals with EIB). Gene signatures for T1, T2 and T3 immune responses were generated as previously described [10].

Design-based stereology of the airway BMZ

Endobronchial biopsy specimens were obtained from the 3rd to 5th generation airways as previously described [17]. Briefly, immediately after fixation, each biopsy was embedded individually in a 4-mm isector mold (Stereology Research Laboratory, Aarhus, Denmark) to impose isotropic-uniform-random (IUR) orientation and eliminate bias from tangential sectioning. From each spherical section, three serial sections spaced 3 µm apart were mounted per slide and stained with haematoxylin & eosin (H&E). This workflow follows the original orthogonal-intercept protocol of Ferrando et al. [18] and its subsequent optimisation by Leung et al. [10]. The H&E stained slides were converted to a digital format using a Hamamatsu NanoZoomer (Brightfield at 20× magnification). These images were subsequently imported into Visiopharm (Visiopharm A/S, Hørsholm, Denmark) for stereological analysis. The Microimager module and a 60× virtual lens was used for image processing. To prepare each slide for stereological analysis, each digital image was randomly sectioned into a series of high-resolution fields using a meander sampling scheme with a 50% sampling fraction. Tissue was considered valid if it contained ≥1 layer of intact epithelial cells directly contacting an intact submucosal layer. All 41 participants that completed the research bronchoscopy and met the airway physiology criteria for inclusion had adequate biopsy samples for measurement of BMZ thickness. We measured the orthogonal intercept with 10 equidistant, fixed-orientation line probes that were superimposed on every high-resolution field. At each intercept between a line probe and the apical surface of the basal lamina, the length of a probe drawn orthogonal to the basal lamina and spanning the full BMZ was recorded using the Visiopharm measurement tool (supplementary figure S1). On average, 59±45 (controls), 100±69 (EIB − asthma) and 104±81 (EIB + asthma) intercepts were collected per participant. We estimated the mean BMZ thickness using the harmonic mean of all orthogonal-intercept lengths $(\mathrm{L})$ per participant was converted to an unbiased thickness estimate $(\mathrm{T})$ using the following formula:

$$T={\displaystyle \frac{8}{3\pi }\cdot L}$$

Multiplication by ${\displaystyle \frac{8}{3\pi }}$ corrects for overestimations of BMZ thickness caused by tangential sectioning. Arithmetic means were not used because they overweight large intercepts and inflate thickness estimates. The coefficient of error (CE) for each estimate was calculated from the squared mean of within-biopsy variances. Mean±sd CE values were 0.056 (controls), 0.036 (EIB⁻) and 0.037 (EIB⁺). Collection of BMZ thickness measurements was performed by analysts (M. Liu, A. Mazzotta and J. Lee) who were blinded to all clinical data.

Correlation with existing data repository

We correlated BMZ thickness measurements from each study participant with previously published data from this cohort (table 1), including the quantity and location of MCs [11] and EOS [13] in the epithelium and subepithelial space of endobronchial tissue in this study population.

TABLE 1.

Number of study participants with individual data points included in the analysis, including those with asthma further subset by the presence or absence of exercise-induced bronchoconstriction (EIB)

	Asthma	EIB+	EIB−	Control
Basement membrane zone stereology	31	19	12	10
Mast cell stereology	30	19	11	10
Eosinophil stereology	30	18	12	10
Sputum T2 gene mean (T2GM)	21	13	8	7
RNA-seq of endobronchial brushings	20	11	9	0

Statistics

Statistical methods are explicitly discussed within individual figure legends and detailed in the supplementary material. Relationships between mean BMZ thickness and continuous measures of airway physiology, induced sputum inflammatory markers and lipid mediators, and densities of MCs and EOS in the airway wall were assessed using unadjusted and adjusted linear regression models. Measures of baseline lung function (FEV₁, forced vital capacity (FVC) and FEV₁/FVC) were adjusted for age, sex and body mass index (BMI). All other regression analyses were adjusted for age, sex, BMI and baseline FEV₁. We reported the main effects as β coefficients and 95% confidence intervals. Unadjusted linear regression analyses were used to assess relationships between mean BMZ thickness with airway gene expression (T1, T2 and T3 gene signatures; individual genes of interest previously described as being significantly correlated with indirect AHR). Statistical analyses were performed in R version 4.5.3 and Prism version 10 (GraphPad). A p-value <0.05 was considered statistically significant.

Results

BMZ is thicker in individuals with asthma but does not significantly correlate with measures of airway physiology associated with asthma

We obtained adequate endobronchial biopsy samples for design-based stereology analysis of BMZ thickness from 31 individuals with asthma (19 EIB⁺, 12 EIB⁻) and 10 individuals without asthma (healthy controls). Study participant characteristics are presented in table 2. Individuals with asthma had a significantly thicker BMZ relative to healthy controls (median 5.52 μm (IQR 4.50–6.10) versus 1.93 μm (IQR 1.86–2.84); figure 1a). 30 out of 31 individuals with asthma had a BMZ thickness above the maximum measured value for healthy individuals (>2.98 μm). There was no significant difference in BMZ thickness between individuals with asthma and indirect AHR and individuals with asthma without indirect AHR (figure 1b).

TABLE 2.

Baseline characteristics of the study groups

Characteristic		Individuals with asthma		p-value
	Control	EIB−	EIB+	3 groups	EIB+ versus EIB−
Individuals, n	10	12	19
General#
Age, years	30.4±12.7	24.8±5.0	26.8±8.6	0.35	0.56
Sex – male	2 (20)	3 (25)	6 (32)	0.80	0.70
Ethnicity – white	7 (70)	11 (92)	16 (84)	0.42	0.60
Body mass index, kg·m−2	22.0±2.4	22.4±2.7	24.1±3.7	0.19	0.16
Airway physiology#
FEV1 % predicted	96.5±11.3	90.7±9.7	89.2±10.9	0.22	0.71
FVC % predicted	95.2±12.8	96.2±8.9	103.3±9.6	0.08	0.07
FEV1/FVC ratio	0.86±0.06	0.80±0.09	0.74±0.09	0 .002	0 .04
FEV1 albuterol response %	0.5±0.6	6.5±3.1	13.4±6.7	<0.001	0 .002
Methacholine PC20, mg·mL−1	>8±0	1.8±1.3	0.6±1.5	<0.001	0 .01
Dry air exercise challenge
Max fall in FEV1 %	2.1±2.1	2.6±2.6	27.7±9.5	<0.001	<0.001
Severity of EIB (AUC30)	19.3±58.1	23.1±69.5	624.7±295.7	<0.001	<0.001
Induced sputum markers¶
Type 2 gene mean	−0.34 (−0.52 to −0.16)	−0.80 (−1.16 to −0.04)	0.24 (−0.11 to 0.79)	0 .01	0 .005
Sputum eosinophils ×103·mL−1	8.8 (4.5 to 28.5)	8.6 (3.5 to 18.0)	11.2 (5.1 to 24.0)	0.49	0.23
Sputum eosinophil %	1.9 (0.5 to 4.4)	1.8 (0.4 to 3.0)	1.6 (0.5 to 2.8)	0.93	0.76
Induced sputum lipid mediators¶
Cysteinyl leukotrienes, pg·mL−1	46.7 (27.6 to 62.3)	60.7 (45.7 to 89.0)	62.6 (57.8 to 79.2)	0.07	0.80
Prostaglandin D2, pg·mL−1	38.3 (33.5 to 78.1)	25.2 (20.2 to 43.0)	41.9 (20.6 to 74.0)	0.16	0.09
Prostaglandin E2, pg·mL−1	63.2 (27.3 to 223.8)	56.1 (40.7 to 101.0)	59.6 (40.7 to 131.0)	0.88	0.64
CysLT/PGE2 ratio	0.64 (0.27 to 0.86)	1.17 (0.56 to 1.52)	0.96 (0.52 to 1.56)	0.09	0.66
Airway wall morphometry¶
Epithelial MCs ×103·mm−3	3.4 (0.0 to 6.7)	2.1 (0.0 to 12.2)	10.3 (7.7 to 22.8)	0 .002	0 .006
Subepithelial MCs ×103·mm−3	5.9 (4.2 to 7.3)	4.0 (2.1 to 5.1)	3.6 (2.6 to 7.5)	0.25	0.91
Epithelial EOS ×103·mm−3	0.0 (0.0 to 0.0)	0.0 (0.0 to 2.2)	1.7 (0.0 to 3.4)	0 .009	0.20
Subepithelial EOS ×103·mm−3	4.1 (2.3 to 9.4)	6.3 (1.9 to 19.7)	15.1 (6.8 to 30.8)	0.06	0.08
Total airway EOS ×103·mm−3	4.1 (2.3 to 9.4)	7.4 (2.0 to 21.4)	18.0 (7.2 to 31.2)	0.07	0.13
Allergen skin prick testing¶
Number of positive allergens	0.5 (0 to 2.25)	3.0 (1.5 to 9.5)	8.5 (4.8 to 10.0)	<0.001	0.21

Statistical differences between study groups were assessed for continuous normally distributed variables using ordinary 1-way analysis of variance (ANOVA) with correction for multiple comparisons using two-stage step-up method of Benjamini, Krieger and Yekutieli. Statistical differences between study groups were assessed for continuous non-normally distributed variables using a Kruskal––Wallis test with correction for multiple comparisons using two-stage step-up method of Benjamini, Krieger and Yekutieli. Bold for p-values denotes statistical significance. EIB: exercise-induced bronchoconstriction; FEV₁: forced expiratory volume in 1 s; FVC: forced vital capacity; FEV₁/FVC: ratio of forced expiratory volume in the first second of exhalation divided by the FVC; FEV₁ albuterol response: absolute change in FEV₁ post-albuterol versus pre-albuterol expressed as a percentage of pre-albuterol FEV₁; PC₂₀: provocative concentration of methacholine required to reduce FEV₁ by 20%; AUC30: area under the curve of FEV₁ versus time curve during exercise challenge; MCs: mast cells; EOS: eosinophils. ^#: data are presented as number of subjects (n), mean±sd or n (%); ^¶: data are presented as median (interquartile range).

FIGURE 1.

Basement membrane zone (BMZ) thickness is elevated in asthma. a) BMZ is thicker in individuals with asthma relative to healthy controls. Median values are shown with error bars representing the interquartile range. p-values represent the result of a Mann–Whitney test. b) BMZ is nonsignificantly thicker in individuals with asthma and indirect airway hyperresponsiveness to dry air exercise challenge (EIB⁺) relative to individuals with asthma that are EIB⁻. Median values are shown with error bars representing the interquartile range. p-values represent the result of a Kruskal–Wallis test with repeated measures performed and adjusted for multiple comparisons using the two-stage step-up method of Benjamini, Kriegerand Yekutieli. ***: p <0.001; ****: p <0.0001.

Next, we assessed for relationships between BMZ thickness and measures of airway physiology in individuals with asthma (table 3). Unadjusted linear regression analyses identified a significant negative correlation between BMZ thickness and age (β=−3.33, p=0.002) as well as positive correlations with baseline FEV₁ (β=3.26, p=0.04) and FVC (β=3.33, p=0.02). We subsequently revised our model to adjust for age, sex and BMI, which weakened the correlation between BMZ thickness and FEV₁ (β=2.75, p=0.11) but still identified a significant correlation between a BMZ thickness and baseline FVC (β=4.51, p=0.02). Given the anticipated effect of baseline FEV₁ on dynamic changes in lung function with either albuterol exposure or bronchoprovocation, our aim to keep the linear modelling consistent for the remainder of analyses, and similar effect size as FVC, all subsequent adjusted linear regression analyses included baseline FEV₁ as a covariate. We did not identify any significant correlation between BMZ thickness with changes in FEV₁ following albuterol exposure (BDR), methacholine PC₂₀ or markers of severity of EIB (figure 2a–c). However, it is notable that the maximum decline in FEV₁ following dry air exercise challenge had a stronger correlation with BMZ thickness (β=4.51, p=0.06) relative to bronchodilator response to albuterol (β=1.77, p=0.13) and direct AHR to methacholine (β=−0.20, p=0.51).

TABLE 3.

Relationship between basement membrane zone (BMZ) thickness and age, airway physiology, markers of airway inflammation and immune cell infiltration of the airway wall in individuals with asthma

	BMZ thickness#
	Unadjusted			Adjusted
	β	95% CI	p-value	β	95% CI	p-value
Demographics
Age	−3.33	−5.22 to −1.45	0.002
Airway physiology
FEV1 % predicted	3.26	0.35 to 6.17	0.04	2.75	−0.56 to 6.05	0.11
FVC % predicted	3.33	0.50 to 6.16	0.02	4.51	1.12 to 7.90	0.02
FEV1/FVC ratio	−0.002	−0.03 to 0.03	0.88	−0.01	−0.05 to 0.02	0.50
FEV1 albuterol response %	−0.47	−2.52 to 1.58	0.64	1.77	−0.57 to 4.11	0.13
Methacholine PC20, mg·mL−1	0.06	−0.40 to 0.51	0.81	−0.20	−0.81 to 0.41	0.51
Dry air exercise challenge
Maximum decline in FEV1 %	3.19	−1.25 to 7.62	0.15	5.66	−0.31 to 11.63	0.06
Severity of EIB (AUC30)	76.7	−37.6 to 190.7	0.19	122.9	−34.7 to 280.5	0.12
Induced sputum markers
Type 2 gene mean	0.30	−0.09 to 0.69	0.13	0.39	−0.04 to 0.82	0.08
Sputum eosinophils ×103·mL−1	2.42	−5.02 to 9.86	0.57	6.23	−5.89 to 18.4	0.31
Sputum eosinophil %	−0.08	−0.68 to 0.51	0.78	0.06	−0.81 to 0.92	0.90
Induced sputum lipid mediators
Cysteinyl leukotrienes, pg·mL−1	19.7	−14.7 to 54.1	0.25	22.1	−25.4 to 69.7	0.35
Prostaglandin D2, pg·mL−1	8.3	−6.4 to 23.1	0.26	14.1	−5.9 to 34.0	0.16
Prostaglandin E2, pg·mL−1	15.8	−7.0 to 38.6	0.18	23.4	−5.35 to 52.2	0.11
CysLT/PGE2 ratio	114.2	−198.2 to 426.6	0.47	225.1	−170.2 to 620.3	0.27
Airway wall morphometry
Epithelial MCs ×103·mm−3	5.15	1.47 to 8.82	0.008	7.70	2.44 to 12.96	0.006
Subepithelial MCs ×103·mm−3	1.11	−0.03 to 2.26	0.06	1.43	−0.25 to 3.11	0.09
Epithelial EOS ×103·mm−3	0.23	−0.45 to 0.90	0.49	−0.08	−1.08 to 0.93	0.88
Subepithelial EOS ×103·mm−3	0.22	−6.39 to 6.83	0.94	0.96	−8.52 to 10.44	0.84
Total airway EOS ×103·mm−3	0.46	−5.94 to 6.86	0.88	1.01	−8.62 to 10.64	0.83

β coefficients, 95% confidence intervals and p-value result from unadjusted linear regression analyses comparing BMZ thickness with the predictive variable. Adjusted values for FEV₁ and FVC result from a linear regression model adjusted for age, sex and body mass index (BMI). All other adjusted values result from a linear regression model adjusted for age, sex, BMI and baseline FEV₁. FEV₁: forced expiratory volume in 1 s; FVC: forced vital capacity; FEV₁ albuterol response: absolute change in FEV₁ post-albuterol versus pre-albuterol expressed as a percentage of pre-albuterol FEV₁; PC₂₀: provocative concentration to reduce FEV₁ by 20%; EIB: exercise-induced bronchoconstriction; AUC30: area under the curve of FEV₁ versus time curve during exercise challenge; MCs: mast cells; EOS: eosinophils. ^#: expressed in micrometres.

FIGURE 2.

Basement membrane zone (BMZ) thickness is not significantly correlated with airway dysfunction in individuals with asthma. β coefficients, 95% confidence intervals (CI) and p-value result from adjusted linear regression analyses (adjusting for age, sex, body mass index and baseline forced expiratory volume in 1 s (FEV₁)) comparing BMZ thickness with a) the maximum FEV₁ albuterol response, b) the provocative concentration of methacholine to reduce FEV₁ by 20% (PC₂₀) and c) the maximum decline in FEV₁ in the 30-min interval following dry air exercise challenge. Linear regression lines are shown with the 95% CI as dotted lines. AHR: airway hyperresponsiveness.

BMZ thickness is positively correlated with MC infiltration of the airways but not airway eosinophilia

We also assessed for correlations between BMZ thickness and asthma endotype, key cell populations and inflammatory mediators that have been associated with asthma (table 3). Although this study did not include assessment of either blood EOS or fraction of exhaled nitric oxide, we did directly measure airway T2 gene expression and characterised EOS in induced sputum samples. There was a nonstatistically significant positive correlation between the sputum T2 gene mean with BMZ thickness (β=0.39, p=0.08; figure 3a) but no correlation with sputum EOS (β=0.06, p=0.90; figure 3b). We also did not identify any significant correlations between BMZ thickness and measurements of lipid mediators from induced sputum samples (table 3).

FIGURE 3.

Basement membrane zone (BMZ) thickness is positively correlated with the density of intraepithelial mast cells (MC_IE) but not airway eosinophils (EOS). β coefficients, 95% confidence intervals (CI) and p-value result from adjusted linear regression analyses (adjusting for age, sex, body mass index and baseline FEV₁) comparing BMZ thickness with a) type 2 gene mean measured from induced sputum samples, b) EOS in induced sputum samples as a percentage of total leukocytes identified by sputum cytology, c) the density of intraepithelial mast cells (MC_IE) and d) the density of total EOS in the airway wall (sum of intraepithelial and subepithelial EOS). Linear regression lines are shown with the 95% CI as dotted lines.

We previously performed design-based stereology of endobronchial biopsies from this same cohort of patients to characterise the density of both MCs and EOS in the epithelial and subepithelial compartment of the airway wall. There was a significant positive correlation between BMZ thickness and the density of MCs infiltrating the epithelial compartment (MC_IE; β=7.70, p=0.006; figure 3c). However, there was no association between BMZ thickness with EOS infiltration of the airway wall (β=1.01, p=0.83; figure 3d).

BMZ thickness is correlated with airway expression of type 2 inflammatory genes

We have previously performed bulk RNA-sequencing analysis of endobronchial brushings obtained from a subset of individuals with asthma in this study population [14], which allowed for examination of relationships between airway gene expression patterns and BMZ thickness. Using previously defined gene sets specific for T1, T2 and T3 inflammation derived from airway epithelial cells in ex vivo culture, we found a significant positive correlation between BMZ thickness and the T2 inflammation gene set (figure 4b). However, there was no significant association between T1 or T3 gene sets with BMZ thickness (figure 4a, 4c).

FIGURE 4.

Basement membrane zone (BMZ) thickness is positively correlated with airway type 2 (T2) gene expression. β coefficients, 95% confidence intervals (CI) and p-value result from unadjusted linear regression analyses comparing BMZ thickness with the average normalised gene expression of a) a type 1 (T1) gene set, b) type 2 (T2) gene set or c) type 3 (T3) gene set using bulk RNA-sequencing data obtained from epithelial brushings of matched study participants. Linear regression lines are shown with the 95% CI as dotted lines.

We also evaluated for associations between BMZ thickness and airway expression of genes that we recently identified as having significant positive or negative correlations with indirect AHR in the form of EIB (table 4). Three genes that were previously shown to have significantly decreased expression in individuals with asthma and indirect AHR in the form of EIB were also negatively correlated with BMZ thickness. Thus, lower expression of ARMC12 (armadillo repeat containing 12 protein), GPT (glutamic-pyruvic transaminase) and CRIP1 (cysteine rich protein 1) correlated with a thicker BMZ. In contrast, 11 genes previously shown to have significantly higher expression in individuals with asthma and indirect AHR in the form of EIB were positively correlated with BMZ thickness. This notably includes genes classically associated with T2 airway inflammation (POSTN, SERPINB2, CLCA1 and NOS2) and eicosanoid synthesis (PTGS1, encoding for cyclooxygenase-1). There were also significant positive correlations with BMZ thickness and other genes that have less well-defined roles in the airways and asthma. SEMA7A (encoding for semaphorin 7A) has been shown to modulate TGF-β1-mediated epithelial–mesenchymal transition [19], can be expressed by EOS following stimulation with IL-3, IL-5 and granulocyte–macrophage colony-stimulating factor [20], and modulated IgE-mediated neutrophilic airway inflammation [21]. IGF2BP3, which encodes for insulin-like growth factor II mRNA binding protein III, is elevated in the airways of individuals with T2-high asthma and may regulate IL-13-mediated signalling in airway epithelial cells [22]. Finally, SLC7A5 encodes for L-type amino acid transporter 1 (LAT1), and LAT1 inhibitors have been shown to attenuate AHR in mice [23] and have been hypothesised as a potential therapeutic for steroid-refractory asthma [24].

TABLE 4.

Correlation of basement membrane zone (BMZ) thickening and epithelial brushing RNA-sequencing (RNA-seq) results

	BMZ thickness# Unadjusted linear regression
Airway epithelial brushings	β	95% CI	p-value
ARMC12	−0.11	−0.21 to −0.01	0.03
GPT	−0.18	−0.34 to −0.02	0.03
CRIP1	−0.22	−0.40 to −0.05	0.01
POSTN	0.56	0.12 to 0.99	0.01
SERPINB2	0.27	0.05 to 0.50	0.02
CLCA1	0.88	0.06 to 1.71	0.04
NOS2	0.30	0.04 to 0.57	0.03
PTGS1	0.25	0.01 to 0.50	0.04
SEMA7A	0.31	0.08 to 0.54	0.01
ANLN	0.20	0.02 to 0.37	0.03
CHST2	0.13	0.01 to 0.26	0.04
IGF2BP3	0.17	0.01 to 0.33	0.04
FAM177B	0.17	0.01 to 0.34	<0.05
SLC7A5	0.36	0.08 to 0.63	0.01

β coefficients, 95% confidence intervals and p-value result from unadjusted linear regression analyses comparing BMZ thickness with the expression of previously characterised differentially expressed genes associated with indirect airway hyperresponsiveness in the form of exercise-induced bronchoconstriction. ^#: expressed in micrometres.

Discussion

We used design-based stereology to precisely quantify BMZ thickness in endobronchial biopsies obtained from a well-characterised population of healthy individuals and individuals with mild–moderate asthma only utilising albuterol on an as needed basis for the study period, and were able to make several novel observations. A thickened BMZ was found in nearly all individuals with asthma relative to healthy individuals, confirming that BMZ thickening is a feature of mild–moderate asthma. We also demonstrate that the BMZ is thinner in older individuals and thicker in individuals with higher baseline lung function. Notably, BMZ thickness did not have significant correlations with classical airway physiology seen in asthma and does not correlate with airway eosinophilia. However, BMZ thickness is significantly associated with T2 airway inflammation and MC infiltration of the airway epithelium. Despite the consistent associations between BMZ thickness and asthma, our study suggests that BMZ thickness has at most a modest effect on airway physiology features seen in asthma and may instead be a more reliable indicator of MC-driven T2 airway inflammation.

BMZ thickness has previously been associated with airway T2 gene expression in multiple asthma populations, including those with milder disease not receiving asthma controller therapies [5] and those with severe disease [10]. Here we again found increased T2 gene expression in the airways of individuals with asthma that had a thicker BMZ in our study population and confirmed that there was no relationship with either T1 or T3 inflammatory gene expression. We also specifically identify positive correlations with individual classical T2 genes including POSTN, SERPINB2, CLCA1 and NOS2, the latter being closely associated with IL-13-mediated airway inflammation [25, 26] and mucus hypersecretion [27, 28]. No study to date has quantified BMZ thickness in individuals with asthma before and after initiating corticosteroids or biologic therapies [29] targeting specific end products of T2 inflammation, and thus it is unclear whether asthma-directed anti-inflammatory therapies that adequately sustain attenuation of T2 airway inflammation affects BMZ thickness. However, given that other studies of severe asthma populations have identified associations between thicker BMZ [29] and persistent T2 airway inflammation despite treatment with either high-dose inhaled corticosteroids and/or oral corticosteroids [30], BMZ thickening may be a histological feature of treatment-resistant T2 inflammation.

Despite this consistent identification of a relationship between T2 airway inflammation and BMZ thickness across study populations, there has been less clarity on the key cell populations contributing to T2 inflammation in this context and their precise location in the airway wall. An earlier study found that BMZ thickness was positively associated with airway EOS infiltration in a study population that included healthy controls and individuals with mild, moderate and severe oral corticosteroid-dependent asthma [4]. More recently, Leung and colleagues [10] identified increased expression of MC-specific genes (TPSAB1, encoding for tryptase; CPA3, encoding for carboxypeptidase 3) in individuals with asthma and a thickened BMZ. Here we further refine the relationships between the precise location of these critical innate immune cells with BMZ thickening. Specifically, we found that BMZ thickness was positively correlated with MC_IE but was not associated with EOS in induced sputum or EOS infiltration of the airway wall. These findings are notable given that the recent study by Leung and colleagues found a significant positive association between blood EOS counts and BMZ thickness. Although we did not measure blood EOS in our study, our findings suggest that BMZ thickening associated with T2 inflammation is more closely related to MC infiltration of the epithelial compartment than airway eosinophilia. Given that Leung and colleagues similarly identified higher MC gene expression in individuals with severe asthma receiving corticosteroids with thicker BMZ and more airway T2 gene expression, it is possible that MCs are a key source of steroid-resistant T2 inflammation and contribute to airway remodelling.

A key strength of our study is the detailed characterisation of airway physiology relevant to asthma in a population not receiving asthma-directed anti-inflammatory therapies, which allowed us to provide further resolution into the relationship between BMZ thickness and airway dysfunction seen in asthma. BMZ thickness was not significantly associated with BDR to albuterol treatment, direct AHR to methacholine, or indirect AHR in the form of EIB. However, there were nonsignificant positive correlations between BMZ thickness and both BDR to albuterol as well as maximum decline in FEV₁ following dry air exercise challenge, suggesting that our study may have been underpowered to detect these differences and/or that the effect of BMZ thickness on airway reactivity is modest. Importantly, indirect AHR is a highly specific feature of asthma as it requires endogenous alterations in cell populations in the airway wall [11–13] and inflammatory mediators in the airway lumen [31–33] prior to exposure to a stimulus to generate bronchoconstriction. In contrast, direct AHR involves exposure to a stimulus that directly acts on airway smooth muscle cells to generate bronchoconstriction and is highly dependent on baseline lung function and airway geometry, which reduces its specificity for asthma as other chronic airway diseases also result in positive methacholine challenge tests. We have previously demonstrated in this same study population that indirect AHR, but not direct AHR, is tightly correlated with epithelial MC density, which we suspect may be the explanation for associations between BMZ thickness and indirect AHR. Interestingly, our findings are not in alignment with Leung and colleagues, who did identify higher BDR in individuals with asthma who have a thicker BMZ. This is almost certainly related to differences in our study populations, most importantly that our population was not receiving asthma-directed anti-inflammatory therapies at the time of assessment and nearly our entire asthma population had a thickened BMZ relative to our healthy control population (while only 38 of 109 individuals with asthma in the study by Leung and colleagues had a BMZ thickness greater than the 90th percentile of the healthy control population). It is notable that prior studies have demonstrated poor correlations between BDR and AHR, including indirect AHR in the form of eucapnic voluntary hyperpnoea [34] and direct AHR to methacholine [35–37]. However, most of these studies were completed in individuals not receiving asthma-directed anti-inflammatory therapies, such as inhaled corticosteroids.

Finally, our study raises still further questions about the pathophysiological significance of a thickened BMZ in the context of asthma. Although BMZ thickening is a consistent feature of airway histopathology in asthma, BMZ thickening is not specific for asthma as studies have identified similar amounts of BMZ thickening in patients with allergic sinus disease [7, 38] and COPD [39]. Similarly, studies have not consistently identified differences in BMZ thickness with asthma severity [40] or airway physiology features of asthma [38, 41]. We submit that the published literature support that the BMZ is often thickened in asthma where it is identified in individuals that tend to be younger, have biomarkers of T2 inflammation, have positive markers of allergic sensitisation and are more likely to have MCs infiltrating their airway mucosa. However, the published literature on the relationship between a thickened BMZ and airway physiology vary between studies. Here we report that higher baseline lung function (FVC) is positively correlated with a thicker BMZ, which has not previously been shown. We also show that there are likely positive correlations between BMZ thickness and BDR or indirect AHR but these associations are not strong. One potential explanation for the heterogeneity of the published literature in this area is that a thickened BMZ may have counteracting effects on airway function. A prior study hypothesised that a thickened BMZ could contribute to less bronchoconstriction by increasing the load on airway smooth muscle cells, which results in reduced shortening during contraction and overall less bronchoconstriction [7]. However, another study has proposed that airway smooth muscle contraction causes the airway mucosa to fold and bulge into the airway lumen resulting in a narrower airway diameter (“mucosal buckling”), which would be accentuated by a thicker BMZ [42]. Thus, these may be competing mechanisms that vary between patient populations due to differences in ongoing degree of T2 airway inflammation or MC infiltration of the epithelium as well as the uniformity of BMZ thickness throughout the airways. Unfortunately, our study is not designed to answer whether either or both phenomena are the predominant effect of a thickened BMZ on airway physiology in asthma.

In summary, we confirm the close relationship between BMZ thickness and T2 gene expression in the airways, and our data indicate that this is related to MC infiltration of epithelial compartment rather than airway eosinophilia. Gene expression analysis of the airway epithelium identified genes that are associated with BMZ thickening, implicating known regulators of T2 inflammation in addition to novel regulators of airway dysfunction. The two most notable limitations of our study are the moderate sample size and lack of collection of key peripheral biomarkers used to identify individuals with T2-high asthma, including blood EOS and fraction of exhaled nitric oxide. Although we make several important observations regarding BMZ thickness, airway inflammation and airway physiology relevant to asthma, several critical questions remain unanswered, including how standard asthma therapies and biologic therapies that specifically target T2 airway inflammation modify BMZ thickness and other features of airway remodelling in asthma.

Supplementary material

Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.

Supplementary material

00510-2026.SUPPLEMENT