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.

Systemic sclerosis (scleroderma, SSc) is a devastating fibrotic disease with few treatment options. Fumaric acid esters, including dimethyl fumarate (DMF, Tecfidera; Biogen, Cambridge, MA), have shown therapeutic effects in several disease models, prompting us to determine whether DMF is effective as a treatment for SSc dermal fibrosis. We found that DMF blocks the profibrotic effects of transforming growth factor-β (TGFβ) in SSc skin fibroblasts. Mechanistically, we found that DMF treatment reduced nuclear localization of transcriptional coactivator with PDZ binding motif (TAZ) and Yes-associated protein (YAP) proteins via inhibition of the phosphatidylinositol 3 kinase/protein kinase B (Akt) pathway. In addition, DMF abrogated TGFβ/Akt1 mediated inhibitory phosphorylation of glycogen kinase 3β (GSK3β) and a subsequent β-transducin repeat-containing proteins (βTRCP) mediated proteasomal degradation of TAZ, as well as a corresponding decrease of TAZ/YAP transcriptional targets. Depletion of TAZ/YAP recapitulated the antifibrotic effects of DMF. We also confirmed the increase of TAZ/YAP in skin biopsies from patients with diffuse SSc. We further showed that DMF significantly diminished nuclear TAZ/YAP localization in fibroblasts cultured on a stiff surface. Importantly, DMF prevented bleomycin-induced skin fibrosis in mice. Together, our work demonstrates a mechanism of the antifibrotic effect of DMF via inhibition of Akt1/GSK3β/TAZ/YAP signaling and confirms a critical role of TAZ/YAP in mediating the profibrotic responses in dermal fibroblasts. This study supports the use of DMF as a treatment for SSc dermal fibrosis.

Endothelial cell (EC) activation underlies many vascular diseases, including pulmonary arterial hypertension (PAH). Several members of the E-twenty six (ETS) family of transcription factors are important regulators of the gene network governing endothelial homeostasis, and their aberrant expression is associated with pathological angiogenesis. The goal of this study was to determine whether deficiencies of the ETS family member, Friend leukemia integration 1 transcription factor (FLI1), and its closest homolog, ETS-related gene (ERG), are associated with PAH. We found that endothelial ERG was significantly reduced in the lung samples from patients with PAH, as well as in chronically hypoxic mice. Functional studies revealed that depletion of ERG or FLI1 in human pulmonary ECs led to increased expression of inflammatory genes, including IFN genes, whereas genes regulating endothelial homeostasis and cell-cell adhesion were down-regulated. Simultaneous knockdown of both ERG and FLI1 had synergistic or additive effects on the expression of these genes, suggesting that ERG and FLI1 coregulate at least a subset of their target genes. Functionally, knockdown of ERG and FLI1 induced cell monolayer permeability with a potency similar to that of vascular endothelial growth factor. Notably, stimulation of ECs with Toll-like receptor 3 ligand poly(I:C) suppressed ERG expression and induced ERG dissociation from the IFNB1 promoter, while promoting signal transducers and activators of transcription 1 (STAT1) recruitment. Consistent with the up-regulation of inflammatory genes seen in vitro, Erg and Fli1 double-heterozygote mice showed increased immune cell infiltration and expression of cytokines in the lung. In conclusion, loss of ERG and FLI1 might contribute to the pathogenesis of vascular lung complications through the induction of inflammation.

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