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.

Single-cell gene expression analysis of cryopreserved equine bronchoalveolar cells

“…Previous scRNA-seq studies on BALF from other species ( 14 , 55 ) and on equine peripheral blood ( 53 ) used CD14 to identify monocytes. The expression of this canonical marker was barely detectable in our dataset, similar to previous observations in dog BALF ( 62 ). Yet, equine BALF monocytes have been described as CD14 + cells based on flow cytometric data ( 63 ).…”

“…Cluster Mo/Ma 4 stood out by the co-expression of lymphocyte and monocyte markers. Similar double positive cells were detected in three other BALF scRNA-seq studies ( 4 , 30 , 62 ). This was attributed to either ambient RNA contamination ( 30 , 62 ) or to doublet formation ( 4 ).…”

“…Similar double positive cells were detected in three other BALF scRNA-seq studies ( 4 , 30 , 62 ). This was attributed to either ambient RNA contamination ( 30 , 62 ) or to doublet formation ( 4 ). We considered several different hypotheses to explain this unusual expression pattern, including engulfing macrophages, a novel dual lineage cell population, technical multiplets (capture of several cells in a single droplet) and immune cell complexes.…”

Function of Macrophages in Disease: Current Understanding on Molecular Mechanisms

“…In pathogenic conditions, TRMs can be replaced and joined by recruited monocyte-derived macrophages to orchestrate an immune response ( 10 ). Current studies using single-cell RNA-sequencing (scRNA-seq) have revealed the presence of multiple macrophage subsets with distinct functions in various tissues ( 11 ). For example, Remmerie et al ( 12 ) reported that in fatty liver, hepatic resident KCs were destroyed and replaced by bone marrow-derived macrophages, and those recruited macrophages comprise two subsets resembling either KCs or lipid-associated macrophages expressing Osteopontin.…”

Diversity of Macrophages in Lung Homeostasis and Diseases

“…Thus, the circumstances under which pathogens induce long-term changes in TR-AMs or substantial replenishment of different functional Mo-AMs remain to be explored. 106) also used scRNA-seq to characterize macrophage/monocyte cell populations in the BALF from dogs with canine idiopathic pulmonary fibrosis (CIPF) (107) and found that monocytederived macrophages were enriched in profibrotic genes in CIPF. Misharin et al (6) found that during lung fibrosis, AMs are partially depleted and replaced by Mo-AMs.…”