Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage

Abstract

Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1 + SiglecF + transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo . Human orthologues of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury.

Macrophages in wound healing: activation and plasticity

“…To this end, a study using single cell recognition, a reference‐based computational tool that enables unbiased annotation of SC‐RNA Seq, investigated macrophage heterogeneity in lung fibrosis . They characterized three distinct groups of macrophages, including alveolar macrophages (C1), interstitial macrophages (C3) and an intermediate cluster of cells (C2) in the lung.…”

A pathologic two‐way street: how innate immunity impacts lung fibrosis and fibrosis impacts lung immunity

“…However, during the fibrotic phase of bleomycin‐induced fibrosis, the numbers of alveolar macrophages increase, concomitant with the appearance of a population of alveolar macrophages expressing lower levels of Siglec F . Lineage‐tracing systems and transcriptome analyses have characterised these Siglec F lo macrophages as monocytic in origin, supporting the idea that bleomycin‐induced lung injury ablates TR‐AMs which are replaced by monocyte‐derived alveolar macrophages (Mo‐AMs) that migrate into the alveoli and upregulate Siglec F . Expansion of alveolar macrophages has also been observed in human lung tissue from patients with pulmonary fibrosis compared to healthy controls .…”

“…57 Following administration of bleomycin to induce pulmonary fibrosis in mice, a large portion of the alveolar macrophage population (distinguished as Siglec F + ) is lost. 58 However, during the fibrotic phase of bleomycin-induced fibrosis, the numbers of alveolar macrophages increase, concomitant with the appearance of a population of alveolar macrophages expressing lower levels of Siglec F. 58 Lineage-tracing systems and transcriptome analyses have characterised these Siglec F lo macrophages as monocytic in origin, supporting the idea that bleomycin-induced lung injury ablates TR-AMs which are replaced by monocytederived alveolar macrophages (Mo-AMs) that migrate into the alveoli and upregulate Siglec F. 55,56 Expansion of alveolar macrophages has also been observed in human lung tissue from patients with pulmonary fibrosis compared to healthy controls. 56 Similar to findings in mice, single-cell RNA sequencing revealed multiple distinct macrophage populations in lungs of patients with fibrosis.…”

“…For example, recruitment of Mo‐AMs was observed following diphtheria toxin depletion of CD11c + lung cells, liposomal clodronate administration and bleomycin treatment, but not in mice treated with LPS, indicating that mass ablation of TR‐AMs after severe lung injury may require differentiation of Mo‐AMs to replenish the alveolar macrophage population. Mo‐AMs localise to sites of fibroblast accumulation following bleomycin‐induced lung injury . Of note are recent findings identifying direct interactions between macrophages and myofibroblasts via cadherin‐11, which possibly mediate the Mo‐AM localisation to the profibrotic niche, and importantly, specific deletion of Mo‐AMs was found to ameliorate fibrosis .…”

“…54 More recently, reports have also highlighted the importance of a discrete subset of alveolar macrophages in driving fibrotic lung pathology. 55,56 In the na€ ıve lung, there exists a self-renewing population of macrophages that derive from foetal monocytes shortly after birth, referred to as tissue-resident alveolar macrophages (TR-AMs), that differ from macrophages that differentiate from circulating bone marrow-derived monocytes. 57 Following administration of bleomycin to induce pulmonary fibrosis in mice, a large portion of the alveolar macrophage population (distinguished as Siglec F + ) is lost.…”