Identification of Pro-Fibrotic Macrophage Populations by Single-Cell Transcriptomic Analysis in West Highland White Terriers Affected With Canine Idiopathic Pulmonary Fibrosis

Abstract

Canine idiopathic pulmonary fibrosis (CIPF) affects old dogs from the West Highland white terrier (WHWT) breed and mimics idiopathic pulmonary fibrosis (IPF) in human. The disease results from deposition of fibrotic tissue in the lung parenchyma causing respiratory failure. Recent studies in IPF using single-cell RNA sequencing (scRNA-seq) revealed the presence of profibrotic macrophage populations in the lung, which could be targeted for therapeutic purpose. In dogs, scRNA-seq was recently validated for the detection of cell populations in bronchoalveolar lavage fluid (BALF) from healthy dogs. Here we used the scRNA-seq to characterize disease-related heterogeneity within cell populations of macrophages/monocytes (Ma/Mo) in the BALF from five WHWTs affected with CIPF in comparison with three healthy WHWTs. Gene set enrichment analysis was also used to assess pro-fibrotic capacities of Ma/Mo populations. Five clusters of Ma/Mo were identified. Gene set enrichment analyses revealed the presence of pro-fibrotic monocytes in higher proportion in CIPF WHWTs than in healthy WHWTs. In addition, monocyte-derived macrophages enriched in pro-fibrotic genes in CIPF compared with healthy WHWTs were also identified. These results suggest the implication of Ma/Mo clusters in CIPF processes, although, further research is needed to understand their role in disease pathogenesis. Overexpressed molecules associated with pulmonary fibrosis processes were also identified that could be used as biomarkers and/or therapeutic targets in the future.

Keywords: bronchoalveolar lavage fluid; canine idiopathic pulmonary fibrosis; dog; lung; macrophages; single-cell RNA-sequencing methods.

Figure 1

Single-cell RNA sequencing analysis identifies multiple cell populations in the canine bronchoalveolar lavage fluids (BALFs). The scRNA-seq analysis was performed on a single-cell suspension generated from eight BALFs obtained from three healthy West Highland white terriers (WHWTs) and five WHWTs affected with canine idiopathic pulmonary fibrosis (CIPF). Cells were visualized using t-distributed stochastic neighbor embedding (t-SNE) plots. (A) Cell clusters identified. (B) Cell populations identified. (C) Cells are colored according to the status of dogs either healthy or affected with CIPF. (D) Expression of differentially expressed genes representative of each cell population. Ma/Mo, macrophages/monocytes; DC, dendritic cell; MRC1, macrophage mannose receptor; MARCO, macrophage receptor with collagenous structure; CD163, scavenger receptor cysteine-rich type 1 protein M130; CD3E, T-cell surface glycoprotein CD3 epsilon chain; CD8b, T-cell surface glycoprotein CD8 beta chain; CCR7, C-C chemokine receptor type 7; CD83, CD83 molecule; SELL, selectin; TOP2A, DNA topoisomerase II alpha; FCRLA, Fc receptor like A; KRT19, cytokeratin 19; MS4A2, membrane spanning 4-domains A2.

Figure 2

Macrophages/monocytes (Ma/Mo) clusters identified. Cells identified as Ma/Mo after the annotation of scRNA-Seq data obtained from three healthy West Highland white terriers (WHWTs) and five WHWTs affected with canine idiopathic pulmonary fibrosis (CIPF) were selected and then clustered allowing the identification of five distinct clusters. (A) Clusters identified. Cells were visualized using a t-distributed stochastic neighbor embedding (t-SNE) plot. (B) t-SNE plot of Ma/Mo colored according to the disease status of the WHWTs either healthy or affected with CIPF. (C) Bar plot of the relative proportion in each disease status of each Ma/Mo cluster.

Figure 3

Differential genes expression analysis between macrophages/monocytes (Ma/Mo) clusters. Dot plot showing the expression of the principal gene markers used to characterize each Ma/Mo cluster. Dot size represents the percentage of cells expressing the genes, while the dot color represents the average expression of the indicated genes.

Figure 4

Enrichment in pulmonary fibrosis processes in M1 and M2 macrophages/monocytes clusters compared to others. (A, B) Gene set enrichment analyses between Comparative Toxicogenomics Database Pulmonary Fibrosis gene set and differentially expressed genes in M1 and M2 clusters, respectively, compared to others. (C) Dot plot showing the expression of genes involved in pulmonary fibrosis processes found to be upregulated in cluster M1 and M2 compared to others. Dot size represents the percentage of cells expressing the genes, while the dot color represents the average expression of the indicated genes.

Figure 5

M1 macrophages/monocytes cluster enrichment in pulmonary fibrosis processes in CIPF compared with healthy dogs. (A–C) Gene set enrichment analyses in M1 cluster between differentially expressed genes in West Highland white terriers (WHWTs) affected with canine idiopathic pulmonary fibrosis (CIPF) compared to healthy WHWTs and the Comparative Toxicogenomics Database Pulmonary fibrosis gene set and epithelial mesenchymal transition and angiogenesis Hallmark gene sets. (D–G) T-distributed stochastic neighbor embedding (t-SNE) plot of cluster M1 cells showing overexpressed genes in CIPF compared with healthy WHWTs, associated with pulmonary fibrosis according to the Comparative Toxicogenomics Database Pulmonary fibrosis gene set. Color represents the average expression of the indicated genes.