Interstitial lung diseases (ILDs) include a large number of diseases and causes with variable outcomes often associated with progressive fibrosis. Although each of the individual fibrosing ILDs are rare, collectively, they affect a considerable number of patients, representing a significant burden of disease. Idiopathic pulmonary fibrosis (IPF) is the typical chronic fibrosing ILD associated with progressive decline in lung. Other fibrosing ILDs are often associated with connective tissues diseases, including rheumatoid arthritis-ILD (RA-ILD) and systemic sclerosis-associated ILD (SSc-ILD), or environmental/drug exposure. Given the vast number of progressive fibrosing ILDs and the disparities in clinical patterns and disease features, the course of these diseases is heterogeneous and cannot accurately be predicted for an individual patient. As a consequence, the discovery of novel biomarkers for these types of diseases is a major clinical challenge. Heat shock proteins (HSPs) are molecular chaperons that have been extensively described to be involved in fibrogenesis. Their extracellular forms (eHSPs) have been recently and successfully used as therapeutic targets or circulating biomarkers in cancer. The current review will describe the role of eHSPs in fibrosing ILDs, highlighting the importance of these particular stress proteins to develop new therapeutic strategies and discover potential biomarkers in these diseases.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal form of interstitial lung disease, of unknown aetiology. We have shown that the small heat shock protein 27 (HSP27) is overexpressed in IPF patient lungs and promotes fibrogenesis. In cancer, HSP27 can also be secreted, notably within extracellular vesicles (EVs). During IPF, EVs accumulate in the BAL. Our aim is to characterize the role of extracellular HSP27 during lung fibrosis. Methods: Pulmonary fibrosis was induced in C57Bl/6J mice with bleomycin (BLM). EVs were isolated from BAL of mice exposed to BLM (or control) by differential ultracentrifugation and analyzed by Nanoparticle Tracking Analysis (NTA), flow cytometry and Western-blot. In vitro, A549 cells, murine Bone Marrow-Derived Macrophages (BMDM) and primary macrophages isolated from healthy donor blood were stimulated with recombinant HSP27 (rHSP27). Results: BLM induced a 10-fold increase of EVs in BAL compared to controls (NTA). These BAL-EVs were positive for EV-enriched proteins TSG101, ALIX and CD9 (Western blot). BALF-EVs from BLM mice had increased HSP27 levels (Western blot). In A549 cells, rHSP27 activated the NF-κB pathway (Western blot) and co-localized with TLR4 (immunofluorescence). Moreover, rHSP27 promoted IL1β and TNFα gene expression in human macrophages and in BMDM (qPCR). Conclusion: Upon BLM exposure, EV levels increase in BAL associated with elevated HSP27 compared to controls. Extracellular HSP27 seems to interact with TLR4 and to enhance pro-fibrotic cytokine expression in macrophages. Our hypothesis is that HSP27 is secreted during the fibrotic process by injured cells, and take part in macrophages activation by stimulating the production of a pro-fibrotic medium.