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. 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.