Alteration of bronchial epithelium is one of the main features of severe asthma. Bronchial epithelial cells promote and perpetuate airway inflammation and structural changes leading to persistent symptoms, bronchial obstruction and hyperresponsiveness. Bronchial epithelial cells are the first barriers in viral infections, which are the major contributor to asthma exacerbations. However, the consequences of the persisting long-term responses to viral infection of bronchial epithelium in severe asthma are not fully understood. Therefore, we investigated the in vitro response to rhinovirus of epithelial cells obtained from control donors or severe asthma patients and performed a long-term follow-up.

Primary human bronchial epithelial cells (HBECs) from control (C) (n=6) and severe asthmatics (SA) (n=6), cultured in air-liquid interface (ALI), were infected with Rhinovirus A16 (RV-A16). Differences in term of viral replication, epithelium cohesion (TEER measurement), antiviral and inflammatory responses (protein secretion) were assessed at the acute phase and at the late phase. Moreover, a single-cell RNA-sequencing analysis was performed at 3-, 7- and 14-days post-infection.

RV-A16 replication increased during the acute phase then decreased during the late phase, without any significative difference between C and SA. Epithelium cohesion was damaged by RV-A16 in SA at the late phase whereas it was strengthened in C. RV-A16 induced the secretion of various antiviral defence peptides (IFN type I and III) and inflammatory mediators (CXCL10, IL 33, TSLP) over the first week post-infection, with different kinetics and intensities between C and SA. For example, IL-33, TSLP, IFNλ2-3 and IFNα2 secretions were significantly more induced in SA than in C. By contrast, MUC5AC secretion was significantly less important in SA than in C. Single-cell RNA sequencing data revealed that SA epithelia displayed fewer genes up-regulated after RV-A16 infection than C epithelia for all cell types (max 202 genes upregulated in multiciliated cells vs max 1262 genes upregulated in goblet cells respectively at 3 dpi). NF-kB pathway (e.g: RIPK1, TNFAIP3, NFKBIA, MYD₈₈) was enriched only in C at 3 dpi, not in SA.

The antiviral response is different between control and severe asthmatic, both in the acute and late phases. The virus induces alteration of epithelium cohesion in severe asthmatics, suggesting a defect in cellular events normally induced by injury. Then, an altered response of HBECs in severe asthma, illustrated by the increased secretion of epithelial alarmins and the deficiency of NF-kB pathway, could contribute to increased disease susceptibility and an enhanced persistent inflammatory response to rhinovirus infection.