Abstract Rationale Rhinovirus-induced asthma exacerbations are common in children with allergic asthma, yet the molecular mechanisms underlying the synergy between viral infection and type-2 (T2) inflammation remain poorly characterized. Methods We generated single-cell RNA-seq data using the 10x Genomics Fixed RNA platform from organotypic air-liquid interface bronchial epithelial cultures derived from well-characterized children with asthma (n = 14) and healthy controls (n = 5) across four experimental conditions: Uninfected, RV16-infected (MOI = 0.5), IL-13-stimulated (10 ng/mL), and IL-13-stimulated followed by RV16 infection. Following quality control filtering, 834,932 cells were analyzed. Unsupervised clustering with manual annotation identified 12 distinct epithelial cell populations: basal cells, mitotic basal cells, suprabasal cells, secretory cells, goblet cells, club cells, mucous secretory cells, mucous ciliated cells, ciliated cells, deuterosomal cells, ionocytes, and tuft cells. Identified the unique transcriptional signatures for dual IL-13 stimulation plus RV16 by comparing them with the individual conditions IL-13 and RV16 conditions. Results All cell types were captured across all donors and conditions. Goblet cells and mucous secretory cells were more abundant in the IL-13 and IL-13 plus RV16 conditions compared to the Uninfected or RV16 conditions. In contrast, club cells, deuterosomal cells and suprabasal cells were more abundant in the uninfected and RV16 conditions compared to the IL-13 or IL-13 plus RV16 conditions. Rhinovirus infection alone induced robust interferon signaling and antiviral response genes, while IL-13 stimulation alone activated pathways characteristic of type 2 inflammation including JAK-STAT signaling, endoplasmic reticulum stress, and mucus glycosylation across many cell types. Critically, dual condition IL-13 stimulation plus rhinovirus infection revealed unique synergistic transcriptional signatures. In goblet cells, we identified 155 synergy-specific genes characterized by amplified interferon responses (IFI6, IFI27, IFI35, IFITM1, IFITM3, ISG15, OAS1, OAS2) and innate immunity genes (TLR3, NOD2, TICAM1), enhanced cytokine signaling (IL32, IL15RA, CXCL16, CXCL17), and endoplasmic reticulum stress with upregulation of XBP1 (a master regulator of the unfolded protein response). Goblet cells also exhibited upregulation of proteasome genes (PSMB8, PSMB9, PSMA2, PSMA4) and epithelial barrier dysfunction markers (GJB2, SCEL, SPRR3, SPINK2). Conclusions These findings suggest that T2 cytokine exposure primes the bronchial epithelium for exaggerated antiviral responses, creating a pathological synergy that drives epithelial barrier dysfunction and may explain the heightened severity of virus-induced asthma exacerbations in children with T2-high asthma. This abstract is funded by: NIH
Cruz et al. (Fri,) studied this question.