Fibroblast-derived extracellular vesicles contain SFRP1 and mediate pulmonary fibrosis
Olivier Burgy, Christoph H. Mayr, Déborah Schenesse, Efthymios Fousekis Papakonstantinou, Beatriz Ballester, Arunima Sengupta, Yixin She, Qianjiang Hu, Maria Camila Melo-Narvaéz, Eshita Jain, Jeanine C. Pestoni, Molly Mozurak, Adriana Estrada-Bernal, Ugochi Onwuka, Christina Coughlan, Tanyalak Parimon, Peter Chen, Thomas Heimerl, Gert Bange, Bernd T. Schmeck, Michael Lindner, Anne Hilgendorff, Clemens Ruppert, Andreas Güenther, Matthias Mann, Ali Önder Yildirim, Oliver Eickelberg, Anna Lena Jung, Herbert B. Schiller, Mareike Lehmann, Gerald Burgstaller, Melanie Königshoff
Olivier Burgy, Christoph H. Mayr, Déborah Schenesse, Efthymios Fousekis Papakonstantinou, Beatriz Ballester, Arunima Sengupta, Yixin She, Qianjiang Hu, Maria Camila Melo-Narvaéz, Eshita Jain, Jeanine C. Pestoni, Molly Mozurak, Adriana Estrada-Bernal, Ugochi Onwuka, Christina Coughlan, Tanyalak Parimon, Peter Chen, Thomas Heimerl, Gert Bange, Bernd T. Schmeck, Michael Lindner, Anne Hilgendorff, Clemens Ruppert, Andreas Güenther, Matthias Mann, Ali Önder Yildirim, Oliver Eickelberg, Anna Lena Jung, Herbert B. Schiller, Mareike Lehmann, Gerald Burgstaller, Melanie Königshoff
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Research Article Cell biology Pulmonology

Fibroblast-derived extracellular vesicles contain SFRP1 and mediate pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a lethal chronic lung disease characterized by aberrant intercellular communication, extracellular matrix deposition, and destruction of functional lung tissue. While extracellular vesicles (EVs) accumulate in the IPF lung, their cargo and biological effects remain unclear. We interrogated the proteome of EV and non-EV fractions during pulmonary fibrosis and characterized their contribution to fibrosis. EVs accumulated 14 days after bleomycin challenge, correlating with decreased lung function and initiated fibrogenesis in healthy precision-cut lung slices. Label-free proteomics of bronchoalveolar lavage fluid EVs (BALF-EVs) collected from mice challenged with bleomycin or control identified 107 proteins enriched in fibrotic vesicles. Multiomic analysis revealed fibroblasts as a major cellular source of BALF-EV cargo, which was enriched in secreted frizzled related protein 1 (SFRP1). Sfrp1 deficiency inhibited the activity of fibroblast-derived EVs to potentiate lung fibrosis in vivo. SFRP1 led to increased transitional cell markers, such as keratin 8, and WNT/β-catenin signaling in primary alveolar type 2 cells. SFRP1 was expressed within the IPF lung and localized at the surface of EVs from patient-derived fibroblasts and BALF. Our work reveals altered EV protein cargo in fibrotic EVs promoting fibrogenesis and identifies fibroblast-derived vesicular SFRP1 as a fibrotic mediator and potential therapeutic target for IPF.

Authors

Olivier Burgy, Christoph H. Mayr, Déborah Schenesse, Efthymios Fousekis Papakonstantinou, Beatriz Ballester, Arunima Sengupta, Yixin She, Qianjiang Hu, Maria Camila Melo-Narvaéz, Eshita Jain, Jeanine C. Pestoni, Molly Mozurak, Adriana Estrada-Bernal, Ugochi Onwuka, Christina Coughlan, Tanyalak Parimon, Peter Chen, Thomas Heimerl, Gert Bange, Bernd T. Schmeck, Michael Lindner, Anne Hilgendorff, Clemens Ruppert, Andreas Güenther, Matthias Mann, Ali Önder Yildirim, Oliver Eickelberg, Anna Lena Jung, Herbert B. Schiller, Mareike Lehmann, Gerald Burgstaller, Melanie Königshoff

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Figure 6

SFRP1 in overexpressed in IPF and is transported by fibroblast EVs.

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SFRP1 in overexpressed in IPF and is transported by fibroblast EVs. (A) ...
(A) Immunofluorescence staining for α-SMA (green) and SFRP1 (red) in FFPE lung sections from patients with IPF or donors. DAPI stains nuclei (blue). Scale bars = 200 μm. (B) Gene expression of SFRP1 in lung tissue from patients with IPF or controls. Data from GSE47460 and GSE68239. Box plots show the interquartile range, median (line), and minimum and maximum (whiskers). (C) SFRP1 expression by Western blot on lung tissue from patients with IPF (n = 5) or donors (n = 4). GAPDH serves as loading control. Densitometry over GAPDH is shown. (D) phLFs were cultured and EVs isolated from the cell culture SN. SFRP1 expression in cell lysate, EV-free fraction, and EV fractions. CD81 used as EV-enriched protein and Ponceau shows total protein amount. (E and F) phLF-EVs were isolated by SEC from conditioned media of control or IPF cells. Expected quantifications for EVs (blue) and proteins (yellow) are shown. Representative electron microscopy of pooled fractions 7–9 (E). Particle concentration in fractions 3–16 were quantified by nFCM (F). (G) Schematic presentation of ExoView analysis workflow for SFRP1 surface expression on EVs. ExoView chips have spotted capture antibodies targeting CD63, CD81, and CD9. EVs bound to the chip via the capture antibodies were visualized using a DyLink 550–conjugated α-SFRP1 antibody. (H) SFRP1+ EVs from phLFs (donor and IPF) were quantified using the ExoView system. Bound particles positive for SFRP1 at the different capture spots are presented as x-fold above mIgG background. SFRP1-positive EVs in green. Scale bar = 10 μm. (I) SFRP1+ EVs from human BALF (donor and IPF) were quantified using ExoView. Bound particles positive for SFRP1 at the different capture spots are presented as x-fold above murine IgG background. BALF from 5 donors each were pooled. Statistical analysis by parametric 2-tailed unpaired t test (C) or 2-way ANOVA (H and I). P values are indicated and *P < 0.05, **P < 0.01.