C70-37 Biophysical Forces Orchestrate Airway Mucosal Defense via Activating Epithelial GSDMD

Rationale

Airway mucus constitutes a fundamental mucosal barrier against inhaled pathogens and irritants, yet uncontrolled mucin hypersecretion drives the pathological progression of numerous chronic respiratory diseases. Uncovering non-canonical regulatory circuits that govern mucin exocytosis is therefore essential for advancing mechanistic understanding and developing innovative treatments for obstructive airway disorders. While Gasdermin D (GSDMD) is canonically defined as an executor of pyroptosis and inflammatory mediator release in immune cells, emerging evidence supports its broader, pyroptosis-independent functions in tissue homeostasis. Given its emerging multifunctionality, we hypothesized that GSDMD mediates a previously uncharacterized pathway governing airway mucin secretion and mucosal defense, with potential translational value for respiratory disease therapy.

Methods

Single-cell RNA sequencing datasets were analyzed via Seurat in R to explore Gsdmd-specific expression in pulmonary cells. Club cell-conditional GSDMD knockout (GsdmdΔ/CC) male mice with wild-type Gsdmdfl/fl littermates as controls were sacrificed after treatment with tamoxifen, H1N1, and HDM for sample collection. H&E staining, AB-PAS staining, and immunofluorescence staining were performed on clinical samples and murine lung tissues to investigate mucus secretion under physiological and pathological conditions, as well as airway mucus barrier and mucosal integrity. Molecular assays were performed in the Calu-3 cell line, as well as in vivo, to evaluate ROS production, NLRP1 inflammasome assembly, caspase-1 activation, GSDMD cleavage, ER localization, intracellular Ca²⁺ flux and mucin secretion efficiency.

Results

GSDMD was specifically expressed and constitutively cleaved in steady-state airway epithelial secretory cells of both human and mouse tissues. Conditional ablation of GSDMD in secretory cells disrupted the continuous airway mucus layer and increased host susceptibility to viral infection. Mechanistically, respiration-derived biophysical forces stimulated ROS accumulation, which activated the NLRP1 inflammasome and promoted caspase-1-mediated GSDMD cleavage. Cleaved GSDMD oligomerized on the ER membrane, increasing membrane permeability to induce robust Ca²⁺ release, which in turn facilitated efficient mucin secretion. Notably, both the co-expression level of GSDMD and MUC5AC and the number of double-positive club cells were significantly elevated in a subset of club cells from COPD patients, with immunofluorescence staining of bronchial brushings demonstrating a strong correlation between GSDMD-N and MUC5AC/MUC5B. Meanwhile, in HDM-induced asthmatic mice, GSDMD deficiency reduced airway mucus thickness and promoted intracellular mucin retention, thereby alleviating allergen-driven airway pathology.

Conclusion

We identify a biophysical force-driven signaling axis governing mucin secretion and mucosal defense through GSDMD. Targeting GSDMD suppresses pathological mucus hypersecretion in asthma and COPD, mitigating related clinical symptoms.

This abstract is funded by: None