D2.138 - Type 3 cytokine - dependent E3 epithelial programming is driven by a novel transcriptional regulator that impacts airway barrier integrity in asthma

Airway epithelial - immune interactions are essential for maintaining mucosal barrier integrity and shaping asthma susceptibility. Recent paediatric nasal transcriptomic studies identified three epithelial phenotypes: E1 (type 1, IFN‑γ driven), E2 (type 2, IL‑4 driven), and E3 (type 3, IL‑17A-associated). The E3 signature, has been proposed as an independent risk factor for asthma, whereas E1 appears protective. We hypothesize that these signatures represent a distinct, inducible epithelial phenotypes with functional consequences for airway inflammation and barrier disruption.

Primary normal human bronchial epithelial (NHBE) cells were cultured as submerged monolayers (2D) or differentiated at air-liquid interface (ALI). Cells were stimulated with relevant cytokines and gene expression was assessed by qPCR and microarray. Secreted proteins were quantified using Mesoscale multiplex ELISA. ALI cultures underwent H&E staining to assess epithelial morphology and barrier structure. Upstream promoter analysis was performed to identify transcriptional regulators of the E3 program, followed by pharmacologic inhibition to test functional relevance. Barrier integrity was quantified using FITC‑dextran flux and transepithelial electrical resistance (TEER).

Distinct epithelial polarization states corresponding to E1, E2, and E3 were inducible by specific cytokine environments. IFN-γ and IL-4 robustly induced E1 and E2, respectively, while E3 polarization required combined IL-17A and IL-36γ stimulation. Inducibility was context-dependent: E2 emerged exclusively in ALI cultures, E3 was inducible in both 2D and ALI systems, and E1 showed mixed inducibility, suggesting involvement of distinct epithelial subsets. These phenotypes were confirmed at both mRNA and protein levels. Transcriptomic profiling revealed that E3 was enriched for genes associated with mucus production, barrier damage, and inflammation, whereas E1 preferentially upregulated protective and innate defence genes. Promoter analysis identified a transcription factor that promoted E3 while suppressing E1 and it ‘s pharmacologic inhibition dose-dependently suppressed E3 gene expression and restored baseline levels while upregulating E1 proteins. Histological examination of ALI cultures, together with FITC-dextran permeability and TEER measurements, demonstrated that E3 polarization impaired epithelial barrier integrity, an effect that was reversed by transcription factor inhibition.

The E3 epithelial state represents a cytokine-induced, type 3–biased polarization characterized by inflammation and compromised barrier function. Its regulation by a specific transcription factor reveals a mechanistic link between epithelial signalling and asthma pathology, offering a potential therapeutic target to stabilize epithelial homeostasis.