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Abstract or Description

Background. Peptide citrullination and adduction with malondialdehyde-acetaldehyde adduct (MAA) are involved in the pathogenesis of rheumatoid arthritis (RA). Anti-cyclic citrullinated peptide antibodies are >90% specific for the diagnosis of RA, and MAA co-localizes with citrullinated proteins in RA synovial fluid. In addition, macrophages demonstrate increased expression of peptidyl arginine deiminase-2 (PAD2), an isozyme of PAD, in response to these MAA-adducted proteins. PAD catalyzes protein citrullination and may mediate the immunogenic transformation of synovial proteins and subsequent auto-antibody formation in RA. Here, we determine whether inhibition of PAD effects inflammatory and fibrotic markers in macrophages and RA human fibroblast-like synoviocytes (HFLS-RA) in response to stimulation with MAA and citrulline (CIT)-modified fibrinogen (FIB).

Methods. U-937 cells were differentiated into activated macrophages and subsequently stimulated with unmodified FIB, FIB-MAA, FIB-CIT, or FIB-MAA-CIT in the presence (treatment group) and absence (control group) of BB-CLA for 48 hours. Supernatants collected from the media were assessed by ELISA for interleukin-1b (IL-1b), interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). HFLS from RA patients were stimulated with treatment and control supernatants from the macrophage cultures and assessed via immunofluorescent staining for the fibrotic markers vimentin and type II collagen.

Results. The effect of PAD inhibition on inflammatory cytokine levels (Figure 1) was pronounced with FIB-CIT and FIB-MAA-CIT stimulations and mild with FIB and FIB-MAA stimulations. If fold decrease could not be calculated due to cytokine levels reaching 0, it is denoted as *0. Macrophages stimulated with FIB, FIB-MAA, FIB-CIT, and FIB-MAA-CIT, respectively, showed decreases in IL-1b (*0, *0, 4-fold, 6-fold, respectively), IL-6 (*0, *0, *0, 3-fold), IL-8 (*0, 1.6-fold, 8-fold, 14-fold), and MCP-1 (1.3-fold, 2-fold, 15-fold, 17-fold). In the absence of PAD inhibition, control group cytokine levels generally followed the trend of FIB-MAA-CIT > FIB-CIT > FIB-MAA > FIB. A similar pattern of cellular response was observed (Figure 2) in HFLS-RA cells stimulated with macrophage supernatants from the treatment group, where vimentin (1.3-fold for FIB, 2-fold for FIB-MAA, 2-fold for FIB-CIT, 4-fold for FIB-MAA-CIT) and type II collagen (9-fold, 11-fold, 18-fold, 21-fold) expression were diminished compared with the control group. In the absence of PAD inhibition, mean pixel density levels followed FIB-MAA-CIT > FIB-CIT > FIB-MAA > FIB for both anti-vimentin and anti-type II collagen staining. Conclusions. This study provides insight into the degree to which inflammatory and fibrotic responses from macrophages and HFLS to MAA and CIT modified fibrinogen may be PAD-mediated. MAA and CIT modification of fibrinogen increased inflammatory cytokines from macrophages and fibrotic proteins from HFLS-RA. BB-CLA markedly diminishes these responses. These observations further the understanding of the roles of MAA and CIT modified antigen in the joint synovium of RA patients, and the therapeutic use of PAD inhibitors in RA treatment.

Publication Date

3-20-2025

Disciplines

Medical Education

The Peptidyl Arginine Deiminase Inhibitor BB-CLA Decreases the Inflammatory and Fibrotic Responses in Macrophages and Rheumatoid Arthritis Synovial Fibroblasts Exposed to Fibrinogen Modified with Malondialdehyde-Acetaldehyde Adduct and Citrulline

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