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Midwest Biomedical Research Conference

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Background: Post-translational modifications of self-proteins have been implicated in the pathogenesis of Rheumatoid Arthritis (RA). One of these protein modifications termed malondialdehyde-acetaldehyde-adduct (MAA) has recently gained interest for its involvement in RA. This protein adduct contributes to inflammation by inducing immune cells to generate pro-inflammatory cytokines, T-cell specific responses, responses and circulating autoantibodies. Recently, novel reactive aldehyde species (RASP) inhibitors (ADX-629 and ADX-246) became available that have been shown to prevent the formation of MAA adducts by covalently binding and sequestering MDA and AA in a mouse model of alcoholic liver disease. These inhibitors prevented the release of key inflammatory cytokines and protected animals from progressive liver damage. While these experiments demonstrated the binding of RASP inhibitors to MDA and AA prior to MAA formation, they did not determine the capacity of these agents to scavenge pre-formed MAA protein adducts. Therefore, the purpose of this study was to determine if RASP inhibitors sequester MAA-adducts and block the subsequent cellular release of pro-inflammatory cytokines.

Significance of the problem: MAA modified self-proteins may render harmful effects in patients with RA. Preventing inflammation and inflammation leading to fibrosis by inhibiting cellular binding of could represent significant advancement in the treatment of RA.

Hypothesis: ADX-629 and -246 will prevent MAA protein adducts from binding to macrophage receptors and, as a result, prevent the release of pro-inflammatory cytokines.

Experimental Design: Human monocytic cells (U-937 cell line) were activated to professional macrophages using phorbol 12-myristate 13-acetate (PMA) for 48 hours. Following pre-treatment with decreasing doses of ADX-629 or ADX-246 for 30 minutes, macrophages were incubated with 25µg/mL of fibrinogen (FIB) or MAA-modified fibrinogen (FIB-MAA) for 24 hours. Supernatants were collected for measurement of IL-6 and MCP-1 using commercially available kits. Cells were collected and tested for membrane integrity using a lactate dehydrogenase (LDH) assay.

Results: In the absence of RASP inhibition, FIB-MAA stimulation of cells significantly increased the release IL-6 with mean concentration of approximately 30pg/mL compared to FIB alone 6-fold increase. (p<0.0001). IL-6 release was significantly reduced with only 1 µM of ADX-629 and fell to native FIB levels with drug concentrations exceeding 10 µM (Figure 1A). ADX-246 demonstrated similar results (Figure 1B). Likewise, similar patterns were observed for the release of MCP-1 (data not shown). LDH assays showed no evidence of cellular toxicity regardless of ADX dose.

Conclusions: In addition to confirming the capacity of RASP inhibitors to sequester MAA, results of this study demonstrate that both ADX-629 and ADX-246 attenuate or prevent MAA-modified proteins from initiating the macrophage-mediated release of pro-inflammatory cytokines implicated in RA pathogenesis. These findings support the need for additional in vivo investigations of these RASP inhibitors as novel therapies in in the management of RA and possibly in other conditions wherein MAA adducts mediate tissue damage.

Reactive Aldehyde Species (RASP) Inhibitors Sequester MAA-Adducts and Reduce Pro-Inflammatory Cytokines