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 5

SFRP1-EVs promote the accumulation of keratin 8–positive expressing AT2 cells.

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SFRP1-EVs promote the accumulation of keratin 8–positive expressing AT2 ...
(A) Heatmap of differentially expressed genes (P < 0.05) between WT EV and KO EV groups (n = 4 samples/group). Genes are ranked by averaged decreasing expression in the WT EV group. Krt8, keratin 8. (B and C) Correlation of WT EV signature (top 10) versus the ADI gene signature (B) or Sfrp1 expression versus ADI gene signature (C). Data from GSE40151 (D14 to D28 after bleomycin, n = 24). (D) Immunofluorescence analysis of lung tissue sections from bleomycin-treated mice at day 14 after injury (BLM D14) displaying the appearance of SFRP1+ cells (red) surrounding Krt8+ expressing ADI cells (green). Arrowheads in the magnified inset point out single Krt8+ ADI cells. Nuclei stained with DAPI (blue). Scale bars = 20 μm. (E) Immunofluorescence analysis of lung tissue sections from bleomycin-treated mice at day 14 after injury (BLM) compared with healthy controls (NaCl). BLM-treated mice were additionally treated with/without SFRP1-containing EVs. Podoplanin (PDPN) in red, Krt8 in green, and nuclei stained with DAPI in blue. Scale bars = 1,000 μm and 200 μm (ROI). (F and G) pmAT2 cells were cultured with or without recombinant (r) SFRP1. After 6 days, cells were analyzed for the expression of Krt8, Sprr1a, or Itgb6 by immunofluorescence (F) or qPCR (G). Scale bars = 50 μm. Representative data from 3 independent experiments. Box plots show the interquartile range, median (line), and minimum and maximum (whiskers). (H) Immunofluorescence analysis of Sprr1a (red) in organoids treated with WT or SFRP1–/– containing EVs or SFRP1–/– containing EVs supplemented with rSFRP1. Nuclei stained with DAPI (blue). Single points represent MFI from 4 single organoids for each biological replicate (n = 3–4). (I) Real-time qPCR to determine Sprr1a gene expression of organoids treated with WT or SFRP1–/– containing EVs or SFRP1–/– containing EVs supplemented with rSFRP1. Single points represent biological replicates (n = 3–4). Statistical analysis by nonparametric Mann-Whitney or Pearson’s correlation testing. P values and correlation coefficient indicated in corresponding panels.