Reference-based annotation of single cell transcriptomes identifies a profibrotic macrophage niche after tissue injury

Abstract

Myeloid cells localize to peripheral tissues in a wide range of pathologic contexts. However, appreciation of distinct myeloid subtypes has been limited by the signal averaging inherent to bulk sequencing approaches. Here we applied single-cell RNA sequencing (scRNA-seq) to map cellular heterogeneity in lung fibrosis induced by bleomycin injury in mice. We first developed a computational framework that enables unbiased, granular cell-type annotation of scRNA-seq. This approach identified a macrophage subpopulation that was specific to injured lung and notable for high expression of Cx3cr1+ and MHCII genes. We found that these macrophages, which bear a gene expression profile consistent with monocytic origin, progressively acquire alveolar macrophage identity and localize to sites of fibroblast accumulation. Probing their functional role, in vitro studies showed a trophic effect of these cells on fibroblast activation, and ablation of Cx3cr1-expressing cells suppressed fibrosis in vivo. We also found by gene set analysis and immunofluorescence that markers of these macrophages were upregulated in samples from patients with lung fibrosis compared with healthy controls. Taken together, our results uncover a specific pathologic subgroup of macrophages with markers that could enable their therapeutic targeting for fibrosis.

Transcriptomics and single‐cell RNA‐sequencing

“…), suggesting that the complexity of alveolar macrophage taxonomy has been underappreciated (unpublished data). Whilst these observations are preliminary, they are in line with similar reports by groups employing scRNA‐seq to describe macrophage taxonomy and differences in the alveolar macrophage phenotype in fibrosis . These early studies underpin the utility and importance of interrogating macrophage phenotypes in human lung disease using BAL, and provide a window to the practical application of scRNA‐seq.…”

“…These macrophages appeared to localize to sites of fibroblast accumulation and exert a trophic role on fibroblast activation. Macrophages with the same markers were also identified in the lungs of patients with IPF, and ablation of these cells suppressed bleomycin‐induced fibrosis in a murine model . Targeting CX3CR1 ‐expressing cells may represent a therapeutic opportunity for fibrotic lung diseases.…”

“…In preclinical pulmonary studies, scRNA‐seq has proven particularly helpful in dissecting cellular events during lung organogenesis; and, in disease models, in defining cellular heterogeneity amongst mesenchymal, epithelial and monocyte/macrophage populations. For example, Lee et al .…”

“…Macrophages with the same markers were also identified in the lungs of patients with IPF, and ablation of these cells suppressed bleomycin-induced fibrosis in a murine model. 27 Targeting CX3CR1-expressing cells may represent a therapeutic opportunity for fibrotic lung diseases. Using scRNA-seq to identify specific cellular subpopulations and their biomarkers is likely to yield further such discoveries.…”

“…In preclinical pulmonary studies, scRNA-seq has proven particularly helpful in dissecting cellular events during lung organogenesis 28,29 ; and, in disease models, in defining cellular heterogeneity amongst mesenchymal, 30,31 epithelial 32 and monocyte/macrophage 27 populations. For example, Lee et al used a variety of techniques including scRNA-seq to resolve mesenchymal populations in mouse lung which support epithelial growth.…”