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 1

EVs accumulate in lung fibrosis, initiate lung remodeling, and impair alveolar epithelial cell function.

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EVs accumulate in lung fibrosis, initiate lung remodeling, and impair al...
(A) C57BL/6J mice were exposed to orotracheal bleomycin or NaCl (control). Lung function was assessed and lung tissue and BALF were collected over the indicated time course. EVs were concentrated from BALF and characterized. BLM, bleomycin. (B) Quasistatic compliance and (C) hydroxyproline level of the corresponding experiments are shown. (D) BALF-EVs were observed by electron microscopy (scale bars indicate 600 nm) and (E) numbered by Nanosight (data expressed as number of particles per BALF). (F) EV quantification according to particle diameters (expressed in nm) at each time point after bleomycin exposure (mean ± SD). (G) Correlation between EV number and quasistatic compliance is depicted. (B–G) Each point corresponds to a mouse (n = 5–8 for NaCl groups, n = 13–20 for BLM groups). (H) BALF-EVs were isolated from mice with pulmonary fibrosis (14 days after bleomycin) or control mice and used for functional assays. (I and J) PCLS from normal C57BL/6J were cultured with the abovementioned BALF-EVs. After 7 days, the expression of fibrosis-related genes was assessed by qPCR. Data are representative of PCLS from individual mice exposed to control- (n = 4 PCLS) or fibrotic EVs (n = 6 PCLS). Gene expression was normalized to Hprt expression. (K) Murine EpCAM-positive cells and CCL-206 fibroblasts in Matrigel were exposed to BALF-EVs for 14 days. Representative images (left panel, scale bar = 1 mm or 500 μm for region of interest [ROI] zoom) and quantification (right panel, n = 4 control EVs, n = 4 fibrotic EVs) of the organoid formation efficiency. Statistical analysis by Kruskal-Wallis followed by Dunn’s multiple comparisons tests (B–D), Spearman’s correlation test (G), or nonparametric Mann-Whitney test (I–K). P values are indicated for each comparison.