Fra-2–expressing macrophages promote lung fibrosis
Alvaro C. Ucero, Latifa Bakiri, Ben Roediger, Masakatsu Suzuki, Maria Jimenez, Pratyusha Mandal, Paola Braghetta, Paolo Bonaldo, Luis Paz-Ares, Coral Fustero-Torre, Pilar Ximenez-Embun, Ana Isabel Hernandez, Diego Megias, Erwin F. Wagner
Alvaro C. Ucero, Latifa Bakiri, Ben Roediger, Masakatsu Suzuki, Maria Jimenez, Pratyusha Mandal, Paola Braghetta, Paolo Bonaldo, Luis Paz-Ares, Coral Fustero-Torre, Pilar Ximenez-Embun, Ana Isabel Hernandez, Diego Megias, Erwin F. Wagner
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Research Article Inflammation Pulmonology

Fra-2–expressing macrophages promote lung fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a deadly disease with limited therapies. Tissue fibrosis is associated with type 2 immune response, although the causal contribution of immune cells is not defined. The AP-1 transcription factor Fra-2 is upregulated in IPF lung sections, and Fra-2 transgenic mice (Fra-2Tg) exhibit spontaneous lung fibrosis. Here, we show that bleomycin-induced lung fibrosis is attenuated upon myeloid inactivation of Fra-2 and aggravated in Fra-2Tg bone marrow chimeras. Type VI collagen (ColVI), a Fra-2 transcriptional target, is upregulated in 3 lung fibrosis models, and macrophages promote myofibroblast activation in vitro in a ColVI- and Fra-2–dependent manner. Fra-2 or ColVI inactivation does not affect macrophage recruitment and alternative activation, suggesting that Fra-2/ColVI specifically controls the paracrine profibrotic activity of macrophages. Importantly, ColVI-KO mice and ColVI-KO bone marrow chimeras are protected from bleomycin-induced lung fibrosis. Therapeutic administration of a Fra-2/AP-1 inhibitor reduces ColVI expression and ameliorates fibrosis in Fra-2Tg mice and in the bleomycin model. Finally, Fra-2 and ColVI positively correlate in IPF patient samples and colocalize in lung macrophages. Therefore, the Fra-2/ColVI profibrotic axis is a promising biomarker and therapeutic target for lung fibrosis and possibly other fibrotic diseases.

Authors

Alvaro C. Ucero, Latifa Bakiri, Ben Roediger, Masakatsu Suzuki, Maria Jimenez, Pratyusha Mandal, Paola Braghetta, Paolo Bonaldo, Luis Paz-Ares, Coral Fustero-Torre, Pilar Ximenez-Embun, Ana Isabel Hernandez, Diego Megias, Erwin F. Wagner

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Figure 1

Alternative activation of macrophages in Fra-2Tg fibrotic lungs.

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Alternative activation of macrophages in Fra-2Tg fibrotic lungs. (A) Con...
(A) Confocal microscopy images of IF for Fra-2 (green) and Vimentin (Vim), Csf-1r, or SPC (red). Arrows point to double-positive cells. Nuclei are counterstained with DAPI (blue). Scale bars: 25 μm. (B) GSEA on ECM- and type 2 immunity-related pathways in Fra-2Tg lungs compared with WT littermates (6 weeks, RNA-Seq, n = 3/3 from 1 experiment). Normalized enrichment score (NES), nominal (NOM) P values, FDR, and Q values are indicated. (C) Flow cytometry analysis on GFP expression in epithelial (CD45–EpCAM+), endothelial (CD45–EpCAM–CD31+), and mesenchymal (CD45–EpCAM–CD31–CD140a+) cells and in myeloid cell subpopulations in WT (black) and Fra-2Tg (green) mouse lungs at 12 weeks of age. Percentages of GFP-positive cells in the plots are shown. (D) Relative myeloid subpopulation cell numbers. Experiment was repeated twice. Mean, maximum, and minimum are plotted with individual values. *P < 0.05; ***P < 0.001, 1-way ANOVA; Bonferroni’s post test. Group comparisons in each cell type are shown. macs, macrophages. (E) Expression of surface markers on nonalveolar (Siglec-F–CD11bhiF4/80+; left) and alveolar (Siglec-F+CD11blo; right) macrophages in the lungs of 12-week-old WT (black) and Fra-2Tg (purple) mice. Median fluorescence intensity (MFI) is plotted. (F) qRT-PCR analysis of lung macrophages isolated by FACS (n = 3; biological replicates from 1 experiment). Relative expression in WT is set to 1. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired 2-tailed t test. (G) Left, confocal microscopy images of Fra-2 (green), Csf-1r (white), and Ym1 (red) costaining. Nuclei are counterstained with DAPI (blue). Arrows point to triple-positive cells. Scale bars: 50 μm. Right, computational quantification of double Fra-2– and Ym1-positive cell numbers (n = 2; biological replicates from 1 experiment).