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. 2023 Aug;16(4):386-398.
doi: 10.1016/j.mucimm.2023.01.008. Epub 2023 Feb 14.

Rhinovirus C causes heterogeneous infection and gene expression in airway epithelial cell subsets

Sarmila Basnet  1 , Chitrasen Mohanty  2 , Yury A Bochkov  3 , Rebecca A Brockman-Schneider  3 , Christina Kendziorski  2 , James E Gern  3
Affiliations

Rhinovirus C causes heterogeneous infection and gene expression in airway epithelial cell subsets

Sarmila Basnet et al. Mucosal Immunol. 2023 Aug.

Abstract

Rhinoviruses infect ciliated airway epithelial cells, and rhinoviruses' nonstructural proteins quickly inhibit and divert cellular processes for viral replication. However, the epithelium can mount a robust innate antiviral immune response. Therefore, we hypothesized that uninfected cells contribute significantly to the antiviral immune response in the airway epithelium. Using single-cell RNA sequencing, we demonstrate that both infected and uninfected cells upregulate antiviral genes (e.g. MX1, IFIT2, IFIH1, and OAS3) with nearly identical kinetics, whereas uninfected non-ciliated cells are the primary source of proinflammatory chemokines. Furthermore, we identified a subset of highly infectable ciliated epithelial cells with minimal interferon responses and determined that interferon responses originate from distinct subsets of ciliated cells with moderate viral replication. These findings suggest that the composition of ciliated airway epithelial cells and coordinated responses of infected and uninfected cells could determine the risk of more severe viral respiratory illnesses in children with asthma, chronic obstructive pulmonary disease, and genetically susceptible individuals.

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Conflict of interest statement

DECLARATIONS OF COMPETING INTERESTS

The authors have no competing interests to declare.

Figures

Fig. 1
Fig. 1
RV-C infection of differentiated BEC induces changes in cellular morphology and composition. (A) Polymerase chain reaction quantification of RV-C15 counts (PFUe) detected in each of the three BEC donor cultures (donor 1, donor 2, donor 3) used in this experiment; (B) BEC cultures were infected with RV-C15 and imaged at time 0 (nv, no virus), 12, 24, and 42 hpi by H&E, Alcian Blue, and immunofluorescent staining (RV-C15 anti-VP2, green); (C) Percentage of Alcian blue-positive cells (mucin secreting cells) at each time point. Mean ± standard error, n = 3. *p < 0.05, **p < 0.01. BEC = bronchial epithelial cells; hpi = hours post infection; H&E = hematoxylin & eosin; nv = uninfected control; PFU = plaque forming units; RV = rhinovirus.
Fig. 2
Fig. 2
Identification of key biological processes and representative genes activated by RV-C from bulk RNA sequencing analysis. (A) Venn diagram illustrates all differentially expressed genes across 12, 24, and 42 hpi and genes that were differentially expressed at multiple time points; (B) charts show the significantly-induced biological processes occurring at 12, 24, and 42 hpi; (C) hierarchical clustering and expression profiles of representative genes involved in activated gene ontology pathways from the 12, 24, and 42 hpi time points; (D) BEC were infected with RV-C15 and cytokine secretion into the basal media was measured; (E) following infection with RV-C15, BEC were analyzed (bulk sequencing) for cytokine messenger RNA. Mean ± standard error, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. BEC = bronchial epithelial cells; hpi = hours post infection; miRNA = micro RNA; nv = uninfected control; RV = rhinovirus; RPKM = reads per kilobase per million.
Fig. 3
Fig. 3
RV-induced changes in cellular composition. (A) Mean expression of key genes identified by a multinomial naive Bayes classification model that are characteristic of each cell cluster (Log2); (B) mean expression of functional cell markers in each t-SNE cell cluster (Log2); (C) t-SNE cell cluster plots of the cellular ecosystem in BEC culture. Nine distinct clusters of cell populations were identified based on their transcript profile and are color-coded; (D) changes in cell composition in uninfected no virus (nv) cells and following RV-C infection (2, 8, 12, 16, 20, 24, 42 hpi) in donor 1. The ciliated cell groups (Ciliated 1, Ciliated 2, Ciliated 3, Ciliated CDHR3-, mucous Ciliated) and secretory cell groups (Progenitor, Secretory) were combined; (E) RV-induced changes in the composition of ciliated cell subpopulations. Mean ± standard error, n = 3. *p < 0.05, **p < 0.01. BEC = bronchial epithelial cells; CDHR3 = cadherin-related family member 3; hpi = hours post infection; nv = uninfected control; RV = rhinovirus.
Fig. 4
Fig. 4
RV-C abundance in clusters of airway epithelial cells over time. (A) Comparison of RV-C replication kinetics in differentiated BEC as measured by quantitative polymerase chain reaction (blue) and single-cell RNA-seq (red); (B) t-SNE plots colored by cell-associated RV-C RNA counts in uninfected (nv) cells and at each infection time point (2, 8, 12, 16, 20, 24, 42 hpi); (C) mean expression kinetics of RV-C and CDHR3 RNA in each cell subpopulation. Groups expressing ciliated cell markers are in solid lines and closed shapes. Non-ciliated cell types are in dotted lines and open shapes; Mean ± standard error, n = 3; (D) relationships between CDHR3 expression and RV-C RNA for each cell cluster; log mean counts; *p < 0.05, **p < 0.01. BEC = bronchial epithelial cells; CDHR3 = cadherin-related family member 3; hpi = hours post infection; nv = uninfected control; RV = rhinovirus.
Fig. 5
Fig. 5
Differential gene expression of Ciliated 1 cells. (A) Volcano plots of differentially expressed genes in Ciliated 1 (green) compared to Ciliated 2 or 3 (red). Vertical lines indicate 1 Log2 fold change for Ciliated 1 (green) and Ciliated 2 and 3 (red). Horizontal line (blue) indicates Y-axis threshold (FDR = 0.001); (B) DE genes in Ciliated 1 (green) group compared to Ciliated 2 and 3 (red) groups from uninfected (nv) cells as shown in volcano plots above; (C) 30 highest expressed genes in Ciliated 1 group from donor 1, donor 2, and donor 3 in uninfected (no virus, nv) culture. DE = differentially expressed; FDR = false discovery rate; hpi = hours post infection; nv = uninfected control.
Fig. 6
Fig. 6
Epithelial response to RV-C is cell type-specific. (A) t-SNE plots colored by RNA counts of RV-C, MX1, and CXCL10 genes in uninfected (nv) and at infection time points 12, 24, 42 hpi; (B) expression kinetics of RV-induced MX1 and CXCL10 messenger RNA for each cell type; (C) relationship between RV-C RNA and expression of MX1 and CXCL10 for each cell type and infection time point; log mean counts, donor 1. hpi = hours post infection; nv = uninfected control; RV = rhinovirus.
Fig. 7
Fig. 7
A subset of infected ciliated cells express interferon lambda. (A) t-SNE plots of IFN-λ3 gene expression in uninfected (nv) and in cells infected for 12, 24, and 42 hours. Arrow heads point to subsets of cells expressing relatively high IFN-λ3; (B) IFN-λ3 expression kinetics for each cell type and time point (nv, 2, 8, 12, 16, 20, 24, and 42 hpi); (C) percentage of IFN-λ3 expressing cells within a population of cadherin-related family member 3 positive ciliated cells that are associated with different levels of RV-C RNA (24 and 42 hpi). hpi = hours post infection; mRNA = messenger RNA; nv = uninfected control; RV = rhinovirus.
Fig. 8
Fig. 8
Expression of genes representing functional pathways and their correlation with RV-C RNA. (A) Expression of genes representing functional pathways in uninfected cells (nv) and in cells infected for 2, 8, 12, 16, 20, 24, and 42 hours. The values represent the normalized mean Log expression of individual cells; (B) Pearson correlations of the same set of genes with RV-C RNA under the same conditions as in (A). CDHR3 = cadherin-related family member 3; hpi = hours post infection; nv = uninfected control; RV = rhinovirus.
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