Fibroblast-derived extracellular vesicles contain SFRP1 and mediate pulmonary fibrosis

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 characterize their contribution to fibrosis. EVs accumulated 14 days post-bleomycin challenge, correlating with decreased lung function and initiated fibrogenesis in healthy precision-cut lung slices. Label-free proteomics of broncho-alveolar lavage fluid (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 Krt8, and WNT/β-catenin signaling in primary alveolar type 2 cells. SFRP1 is 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 fibrotic mediator and potential therapeutic target for IPF. EVs were concentrated from the conditioned media of Sfrp1-/- and Sfrp1+/+ primary mouse lung fibroblasts and instilled intratracheally into mice previously challenged with bleomycin at D0. EVs were administered repeatedly starting day 8 post-bleomycin (n=4 mice BLM + Sfrp1+/+pmLF-EVs, n=4 mice BLM + Sfrp1-/-pmLF-EVs). At D21, lung tissue samples were collected and flash frozen in liquid nitrogen before analysis. mRNA sequencing on whole lung extract was carried out to shed light in the mechanisms involved in vesicular SFRP1 effects during fibrosis. Total RNA was extracted using a Trizol standard protocol. Messenger RNAs were purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dTTP. It was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platform (NovaSeq PE150), according to effective library concentration and data amount (30M reads/sample).