CITRULLINATED AND MALONDIALDEHYDE-ACETALDEHYDE MODIFIED FIBRINOGEN ACTIVATES MACROPHAGES AND PROMOTES PROFIBROTIC RESPONSES IN HUMAN LUNG FIBROBLASTS.

Abstract

The objective of this study was to assess fibrinogen (FIB) co-modified 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 (SN) (Mϕ-SN [U-937-derived] or MϕP-SN [PBMC-derived]) or direct antigens were co-incubated 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. PDGF-BB and TGF- were measured in macrophage supernatants and inhibition studies performed using Su16f and SB431542, respectively. HLF gene expression of CD36, COL6A3, MMP-9, MMP-10, MMP-12 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 RA-ILD patients resulted in 4- to 30-fold increases in COL6A3 and MMP12 expression; up-regulation was greater in HLFs stimulated with MϕP-SN derived from RA-ILD vs. controls. HLF exposure to Mϕ-SN^FIB-MAA-CIT increased types I/VI collagen deposition vs. all other Mϕ-SN groups and was greater than FIB-MAA-CIT stimulation. PDGF-BB and TGF- 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- inhibitors, alone and in combination, significantly reduced HLF-mediated ECM deposition from Mϕ-SN stimulations. These results show that co-modified fibrinogen activates macrophages to produce PDGF-BB and TGF-β that promotes an aggressive HLF phenotype characterized 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.