Downregulation of SLC40A1 leads to iron accumulation in fibrotic lung fibroblasts.

Iron is an essential nutrient for almost all organisms. However, excess iron generates reactive oxygen species and causes tissue injuries. Iron is implicated in idiopathic pulmonary fibrosis (IPF). In this study, we examined iron accumulation in fibrotic lung fibroblasts and the underlying mechanisms. We hypothesize that the downregulation of Solute Carrier Family 40 Member 1 (SLC40A1) results in iron accumulation in lung fibroblasts of IPF patients. Using a Prussian Blue iron staining, we found that iron accumulated in the fibrotic region of the lungs from IPF patients and bleomycin- and asbestos-induced lung fibrosis mice. Iron was partially co-localized with the fibroblast marker vimentin in IPF lungs. By using publicly available single cell RNA sequencing datasets, we identified SLC40A1, the only known gene involved in iron export, as a downregulated gene in alveolar fibroblasts of IPF. The downregulation of SLC40A1 was confirmed in the lung fibroblasts isolated from IPF patients and bleomycin-treated mice. The treatment of human lung fibroblasts with transforming growth factor-β1 (TGF-β1), a major cytokine elevated in IPF, reduced SLC40A1 mRNA and protein expression. TGF-β1 downregulated SLC40A1 expression via SMAD3 as determined by chromatin immunoprecipitation and luciferase promoter reporter assays. Knockdown of SLC40A1 using lentiviral shRNAs or TGF-β1 treatment induced iron accumulation in human lung fibroblasts as determined by live cell ferrous dye staining using a SiRhoNox-1 probe. Knockdown of SLC40A1 enhanced the iron-induced lung fibroblast activation. In summary, we conclude that the downregulation of SLC40A1 caused by TGF-β1 induces iron accumulation in lung fibroblasts, resulting in fibroblast activation.