Citrullinated and malondialdehyde-acetaldehyde-modified fibrinogen activates macrophages and promotes profibrotic responses in human lung fibroblasts

The objective of this study was to assess fibrinogen (FIB) comodified with citrulline (CIT) and/or malondialdehyde-acetaldehyde (MAA) initiates macrophage-fibroblast interactions, leading to extracellular matrix (ECM) deposition that characterizes rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Macrophages (Mϕ) were stimulated with native-FIB, FIB-CIT, FIB-MAA, or FIB-MAA-CIT. Supernatants (SNs) [Mϕ-SN (U-937-derived) or MϕP-SN (PBMC-derived)] or direct antigens were coincubated with human lung fibroblasts (HLFs). Gene expression was examined using RT-PCR. ECM deposition was quantified using immunohistochemistry and Western blot; cell signaling mechanisms were delineated. Platelet-derived growth factor (PDGF)-BB and TGF-β were measured in macrophage supernatants, and inhibition studies were performed using Su16f and SB431542, respectively. HLF gene expression of <i>CD36</i>, <i>COL6A3</i>, <i>MMP-9</i>, <i>MMP-10</i>, and <i>MMP-12</i> was increased following stimulations with Mϕ-SN generated from modified FIB but not from direct antigens. HLF stimulated with MϕP-SN^FIB-MAA-CIT derived from patients with RA-ILD resulted in 4- to 30-fold increases in <i>COL6A3</i> and <i>MMP12</i> expression; upregulation was greater in HLFs stimulated with MϕP-SN derived from RA-ILD versus controls. HLF exposure to Mϕ-SN^FIB-MAA-CIT increased types I/VI collagen deposition versus all other Mϕ-SN groups and was greater than FIB-MAA-CIT stimulation. PDGF-BB and TGF-β signaling had the highest concentrations identified in Mϕ-SN^FIB-MAA-CIT and MϕP-SN^FIB-MAA-CIT, particularly from RA-ILD-derived cells. PDGF-BB and TGF-β inhibitors, alone and in combination, significantly reduced HLF-mediated ECM deposition from Mϕ-SN stimulations. These results show that comodified fibrinogen activates macrophages to produce PDGF-BB and TGF-β that promotes an aggressive HLF phenotype characterized by increased ECM deposition. These results suggest that targeting CIT and/or MAA modifications or downstream cellular signals could represent novel approaches to RA-ILD treatment.<b>NEW & NOTEWORTHY</b> This report demonstrates that fibrinogen simultaneously harboring two common posttranslational modifications activates macrophages to secrete platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-β. Resulting cross talk between activated macrophages and human lung fibroblasts leads to marked increases in extracellular matrix deposition. These protein modifications are abundant and colocalize in lung tissues from patients with rheumatoid arthritis-associated interstitial lung disease (RA-ILD), and the results suggest that agents targeting citrullination and/or malondialdehyde-acetaldehyde (MAA) adduct formation could represent novel therapeutic strategies.