Peptidyl arginine deiminase expression and macrophage polarization following stimulation with citrullinated and malondialdehyde-acetaldehyde modified fibrinogen

•MAA and citrulline modifications induced modification-specific macrophage responses.•Citrullinated fibrinogen generated predominantly M1-like phenotype.•MAA and citrulline co-modification promoted a mix of M1 and M2-like phenotypes.•Macrophages upregulate PAD expression in response to MAA modified fibrinogen.•MAA modified proteins increase protein citrullination in macrophages. Post-translational modifications of extracellular matrix proteins such as fibrinogen may lead to tolerance loss and have been implicated in rheumatoid arthritis (RA) pathogenesis. The purpose of this study was to determine whether fibrinogen (FIB) modified with citrulline (CIT), malondialdehyde-acetaldehyde (MAA) or both leads to altered macrophage polarization, peptidyl arginine deiminase (PAD) expression, or production of citrullinated proteins. PMA-treated U-937 cells (M0 cells) were stimulated with MAA, CIT or MAA-CIT modified FIB. Macrophage (M1/M2) phenotypes were evaluated by flow cytometry, RT-PCR, and ELISA. PAD enzyme expression and protein citrullination was evaluated using RT-PCR and Western Blot. Flow cytometry revealed that M0 macrophages stimulated with FIB-MAA-CIT resulted in mixed M1/M2 phenotypes as demonstrated by cell surface expression and mRNA levels of CD14, CD192, CD163, and CD206 (p