Ryan C. Murphy

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 with airway dysfunction and inflammation is incompletely understood. Objectives: Characterize 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 (FVC), 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.

Innate immune cell populations are critical in asthma with different functional characteristics based on tissue location, which has amplified the importance of characterizing the precise number and location of innate immune populations in murine models of asthma. In this study, we performed premortem intravascular (IV) labeling of leukocytes in mice in two models of asthma to differentiate innate immune cell populations within the IV compartment versus those residing in the lung tissue or airway lumen. We performed spectral flow cytometry analysis of the blood, suspensions of digested lung tissue, and bronchoalveolar lavage fluid. We discovered that IV labeled leukocytes do not contaminate analysis of bronchoalveolar lavage fluid but represent a significant proportion of cells in digested lung tissue. Exclusion of IV leukocytes significantly improved the accuracy of the assessments of myeloid cells in the lung tissue and provided important insights into ongoing trafficking in both eosinophilic and neutrophilic asthma models.

Rationale: Indirect airway hyperresponsiveness (AHR) is a highly specific feature of asthma, but the underlying mechanisms responsible for driving indirect AHR remain incompletely understood. Objectives: To identify differences in gene expression in epithelial brushings obtained from individuals with asthma who were characterized for indirect AHR in the form of exercise-induced bronchoconstriction (EIB). Methods: RNA-sequencing analysis was performed on epithelial brushings obtained from individuals with asthma with EIB (n = 11) and without EIB (n = 9). Differentially expressed genes (DEGs) between the groups were correlated with measures of airway physiology, sputum inflammatory markers, and airway wall immunopathology. On the basis of these relationships, we examined the effects of primary airway epithelial cells (AECs) and specific epithelial cell–derived cytokines on both mast cells (MCs) and eosinophils (EOS). Measurements and Main Results: We identified 120 DEGs in individuals with and without EIB. Network analyses suggested critical roles for IL-33–, IL-18–, and IFN-γ–related signaling among these DEGs. IL1RL1 expression was positively correlated with the density of MCs in the epithelial compartment, and IL1RL1, IL18R1, and IFNG were positively correlated with the density of intraepithelial EOS. Subsequent ex vivo modeling demonstrated that AECs promote sustained type 2 (T2) inflammation in MCs and enhance IL-33–induced T2 gene expression. Furthermore, EOS increase the expression of IFNG and IL13 in response to both IL-18 and IL-33 as well as exposure to AECs. Conclusions: Circuits involving epithelial interactions with MCs and EOS are closely associated with indirect AHR. Ex vivo modeling indicates that epithelial-dependent regulation of these innate cells may be critical in indirect AHR and modulating T2 and non-T2 inflammation in asthma.