Epithelial Cell Membrane Perforation Induces Allergic Airway Inflammation

Allergens that induce allergic airway inflammation are highly diverse, yet they commonly activate type 2 immune responses1,2. Airway epithelial cells are crucial in the immune sensing of allergens3-5. However, what shared features among diverse allergens lead to their similar innate immune sensing, and how epithelial cells detect these features, remain poorly defined1,2,6-9. Here, we show that pore-forming proteins represent one of the common stimuli of allergic airway inflammation and elucidate their immune activation mechanisms. Using the prevalent mold allergen Alternaria alternata (A. alternata) as a model, we established a unique in vitro system to investigate type 2 innate immune sensing. Through a six-step biochemical fractionation, we identified its core immune-stimulatory components as Aeg-S and Aeg-L. Biochemical reconstitution and cryo-electron microscopy demonstrate that these proteins form 16-20-mer transmembrane pore complexes. Their cooperative perforation acts as a bona fide type 2 immune adjuvant to support antigen-specific TH2 and IgE responses. Genetically engineered A. alternata strains lacking such pore-forming activity fail to induce allergic responses in mice. Moreover, pore-forming proteins from various species, despite structural and membrane target differences, are sufficient to trigger respiratory allergies. Perforations in airway epithelial cells initiate allergic responses through two mechanisms: one triggers IL-33 release; another involves Ca2+ influx, which induces MAPK pathway activation and type 2 inflammatory gene expression. These findings provide insight into how type 2 immune responses detect common perturbations caused by structurally diverse stimuli. Targeting downstream signaling of epithelial cell perforation may open new avenues for treating respiratory allergies. Overall design: To assess early airway epithelial cell responses induced by pore-forming proteins, mice were intranasally administered one of the following treatments: mock, A. alternata extract, or pore-forming proteins. Lung epithelial cells were isolated 6 hours post-treatment, with each treatment including at least two biological replicates. • To investigate the transcriptomic response induced by pore-forming proteins in vitro, NCI-H1437 and NCI-H292 cells were treated with A. alternata extract or pore-forming proteins, with at least two biological replicates for each condition. Total RNA was extracted after 4 hours of stimulation for RNA sequencing. Differential gene expression analysis was then performed using RNA-seq data, comparing the different treatments to the mock control.