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.

Background: Interstitial lung diseases (ILDs) include a large number of diseases associated with progressive pulmonary fibrosis (PPF), including idiopathic pulmonary fibrosis (IPF). Despite the rarity of each of the fibrotic ILDs individually, they cumulatively affect a considerable number of patients. PPF is characterised by an excessive collagen deposition leading to functional decline.Objectives: Therapeutic options are limited to nintedanib and pirfenidone which are only able to reduce fibrosis progression. CD206-expressing M2 macrophages are involved in fibrosis progression, and whether they may be relevant therapeutic targets or biomarkers remains an open question.Results: In our study, CD206⁺lung macrophages were monitored in bleomycin-induced lung fibrosis in mice by combining flow cytometry, scRNAseq and in vivo molecular imaging using a single photon emission computed tomography (SPECT) radiopharmaceutical,^99mTc-tilmanocept. The antifibrotic effect of the inhibition of M2 macrophage polarisation with a JAK inhibitor, tofacitinib, was assessed in vivo. We demonstrate that CD206-targeted in vivo SPECT imaging with^99mTc-tilmanocept was able to accurately detect and quantify the increase in CD206⁺macrophages from early to advanced stages of experimental fibrosis and ex vivo in lung biopsies from patients with IPF. CD206-targeted imaging also specifically detected a decrease in CD206⁺lung macrophages on nintedanib and tofacitinib treatment. Importantly, early in vivo imaging of CD206⁺macrophages allowed the prediction of experimental lung fibrosis progression as well as nintedanib and tofacitinib efficacy.Conclusions: These findings indicate that M2 macrophages may be relevant theranostic targets for personalised medicine for patients with PPF.

Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-β gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-β1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-β1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-β1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-β1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-β1 signaling. Further investigations are needed to determine whether these results can be transposed to humans.