Extracellular vesicles with HSP90 promote pulmonary fibrosis.

<bold>Background and aims:</bold> Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition. Cellular miscommunication is a major hallmark of the disease. Extracellular vesicles (EVs) accumulate in the IPF lung and carry active molecules including Heat Shock Proteins (HSP). We studied HSPs linked to fibrotic EVs and whether these contribute to pulmonary fibrosis. <bold>Methods:</bold> Broncho-alveolar lavage fluid (BALF) from mice with bleomycin-induced pulmonary fibrosis and cell culture media were used to purify EVs by ultracentrifugation. EVs were characterized with western blot and flow cytometry. Their fate in vivo was followed by flow cytometry. EV functions were tested on precision cut lung slices (PCLS) using qPCR and microscopy. Extracellular HSP90 was monitored by in vivo SPECT-CT imaging. <bold>Results:</bold> Increased HSP90 was observed at the surface of BALF-EVs from mice exposed to bleomycin compared to controls while undetected in non-vesicular fraction (western blot). These HSP90+EVs were mainly secreted by fibroblasts in vitro and taken up in vivo by macrophages (55+/-6.3%, n=6). PCLS exposed to Hsp90-/-fibroblast-EVs had decreased collagen accumulation (second harmonic signal, p<0.001) and Col3a1 mRNA compared to WT fibroblast-EVs. PCLS exposed to BALF-EVs and incubated with HSP90 blocking antibody exhibited less collagen accumulation compared to PCLS exposed to EVs only (sirius red). SPECT-CT analysis revealed increased extracellular HSP90 in the lung of mice exposed to bleomycin compared to control. <bold>Conclusions:</bold> EVs transport HSP90 at their surface during pulmonary fibrosis. HSP90+ vesicles seem to mediate a fibroblasts to macrophages crosstalk. The inhibition of HSP90 decreases the activity of fibrotic EVs to promote fibrosis.