Airway epithelial cell response to RSV is mostly impaired in goblet and multiciliated cells in asthma

Abstract

Background: In patients with asthma, respiratory syncytial virus (RSV) infections can cause disease exacerbation by infecting the epithelial layer of the airways, inducing subsequent immune response. The type I interferon antiviral response of epithelial cells upon RSV infection is found to be reduced in asthma in most-but not all-studies. Moreover, the molecular mechanisms causing the differences in the asthmatic bronchial epithelium in response to viral infection are poorly understood.

Methods: Here, we investigated the transcriptional response to RSV infection of primary bronchial epithelial cells (pBECs) from patients with asthma (n=8) and healthy donors (n=8). The pBECs obtained from bronchial brushes were differentiated in air-liquid interface conditions and infected with RSV. After 3 days, cells were processed for single-cell RNA sequencing.

Results: A strong antiviral response to RSV was observed for all cell types, for all samples (p<1e-48). Most (1045) differentially regulated genes following RSV infection were found in cells transitioning to secretory cells. Goblet cells from patients with asthma showed lower expression of genes involved in the interferon response (false discovery rate <0.05), including OASL, ICAM1 and TNFAIP3. In multiciliated cells, an impairment of the signalling pathways involved in the response to RSV in asthma was observed.

Conclusion: Our results highlight that the response to RSV infection of the bronchial epithelium in asthma and healthy airways was largely similar. However, in asthma, the response of goblet and multiciliated cells is impaired, highlighting the need for studying airway epithelial cells at high resolution in the context of asthma exacerbation.

Keywords: Airway Epithelium; Asthma; Viral infection.

Figure 1

Experimental design and dataset overview. (A) Overview of the study design. BECs were obtained from healthy subjects and patients with asthma by bronchial brushes, and were differentiated in ALI cultures for 3 or 4 weeks before being treated with RSV. Cells were harvested and processed for scRNAseq. (B) UMAP representation of 30 604 single cells from all donors and conditions, clustered and annotated in 4 groups (left) and 10 subgroups (right), (C) coloured by disease (top) and treatment condition (bottom). (D) Proportions of cell types for each sample, calculated with scCODA. ALI, air-liquid interface; BECs, bronchial epithelial cells; RSV, respiratory syncytial virus; scRNAseq, single-cell RNA sequencing; UMAP, Uniform Manifold Approximation and Projection.

Figure 2

Similarities in cellular composition, barrier formation and transcriptional profiles between the ALI cultures derived from the patients with asthma and the ones derived from healthy subjects. (A) UMAP representation of untreated 7089 from untreated control and 6382 asthma cells, coloured by cell type (left), cell subtype (middle) and disease status (right). (B) Cellular frequencies of each cell subtype, coloured by disease status. Significance was determined by scCODA with an FDR-adjusted p<0.05. (C) TEER of the ALI cultures through time. Data are presented as mean±SD, comparisons between patient groups at a single time point were analysed using the Wilcoxon rank-sum test. Comparisons between two time points within the same group were done using two-way analysis of variance test. (D) Violin plots of the composite score of the genes involved in the epithelial barrier. ALI, air-liquid interface; FDR, false discovery rate; TEER, transepithelial electrical resistance; UMAP, Uniform Manifold Approximation and Projection.

Figure 3

RSV infection induces transcriptomic changes in ALI-cultured pBECs derived from healthy subjects. (A) UMAP representation of untreated 7089 from untreated and 9199 RSV-infected cells, coloured by cell subtype (left) and treatment (right). (B) Cellular frequencies of each cell subtype, coloured by treatment. Significance was determined by scCODA with an FDR-adjusted p<0.05. (C) UpSet plot depicting the unique and shared sets of DEGs with RSV infection among cell subtypes. An FDR of less than 0.05 was considered as statistically significant. (D) Venn diagram of the biological processes found to be significantly enriched by GO analysis of the genes differentially expressed in RSV compared with control, in basal, secretory and multiciliated cells. The 10 most enriched processes are indicated. ALI, air-liquid interface; DEGs, differentially expressed genes; FDR, false discovery rate; GO, Gene Ontology; pBECs, primary bronchial epithelial cells; RSV, respiratory syncytial virus; UMAP, Uniform Manifold Approximation and Projection.

Figure 4

RSV infection induces a different transcriptomic response and a shift in cellular communication in pBECs derived from patients with asthma compared with control. (A) UpSet plot depicting the unique and shared sets of DEGs with RSV infection among cell types in control (blue) and in asthma (red). An FDR of less than 0.05 was considered as statistically significant. (B) Change in gene expression induced by RSV, for goblet (left) and club (right) cells. Only genes found to be significantly different in RSV (FDR <0.05) in either the healthy-derived or the asthma-derived pBECs are displayed. DEGs found in both conditions are coloured in black, DEGs only found in healthy (resp. asthma) are coloured in blue (resp. red). Genes found significant (FDR <0.05) in the interaction analysis are coloured in purple. (C) Violin plots of the expression of OASL, ICAM1 and TNFAIP3 in goblet cells derived from patients with asthma (A) and healthy controls (C). (D) Differential number and strength of cell–cell interactions when comparing untreated with RSV-infected cells derived from healthy subjects (top) and from patients with asthma (bottom). Red (or blue) depicts an increase (or decrease) of these metrics in the RSV-infected cells compared with untreated cells. (E) Signalling changes of multiciliated cells in control (top) and in asthma (bottom) when comparing untreated and RSV-infected samples. DEGs, differentially expressed genes; FDR, false discovery rate; pBECs, primary bronchial epithelial cells; RSV, respiratory syncytial virus.