Background/Purpose: The post translational modification of self-proteins with malondialdehyde-acetaldehyde (MAA) has been shown to alter protein function and antibodies to MAA are increased in both ACPA positive and negative RA patients.  Recently, we have shown that immunization of mice with MAA-modified proteins increases both ACPA and T-cell responses to MAA modified and citrullinated proteins.  However, few if any, MAA modified and/or citrullinated proteins have been identified in vivo.  One protein that is of interest and has been recently identified by our group is matrix Gla protein (MGP), one of the most powerful naturally occurring inhibitors of calcification. While much is known about this protein, there has been limited investigation examining whether this protein could be MAA-modified and/or citrullinated.  Therefore, it was the purpose of this study was to evaluate whether MGP could be “naturally” modified with MAA and/or citrulline. 

Methods: Human THP-1 monocytes were activated into macrophages with Phorbol 12-myristate 13-acetate (PMA).  To evaluate whether MGP could be modified in vitro, THP-1 activated  cells were incubated with 0.5mM MDA and 0.25mM AA (to form MAA modified proteins) ionomycin (a calcium ionophore) or media alone for 24 hours. Cell lysates were then assayed using a unique capture assay to confirm MGP could be MAA adducted and/or citrullinated.  In separate experiments, activated THP-1 cells were incubated as above and then loaded with a Fluo-4 calcium indicator and observed for fluorescence at 488nm to evaluate intracellular calcium level efflux and retention.

Results: THP-1 cell lysates assayed, using the antigen capture ELISA, confirmed that MGP was both MAA-adducted and citrullinated when incubated with MDA and AA (p< 0.0001) as compared to ionomycin incubated and media controls (Figure 1).  This was interesting as the cells were not exposed to peptidyl arginine deiminase (PAD) suggesting that citrullination was a secondary effect during the modification of cellular proteins MAA.  In previous experiments, it has been shown that exogenously added MAA modified proteins decrease calcium efflux which may contribute to the activation of endogenous PAD and the citrullination of proteins.  In this study, MDA and AA incubated THP-1 cells showed significantly increased (p< 0.01) levels of intracellular calcium as compared to both the ionomycin and media controls (Figure 2), suggesting that calcium was indeed retained inside the cell. 

Conclusion: Incubating THP-1 cells with the precursors (AA and MDA) of MAA modifies cellular proteins including MGP, which was also citrullinated in the absence of adding exogenous PAD.  PAD activity is calcium dependent and we were able to show that these THP-1 macrophages incubated with AA and MDA, retained more calcium than those incubated with with appropriate controls.  Mechanisms linking MAA formation to citrullination need to be identified, but it is possible that MGP function (binds calcium) is altered to help retain calcium and initiate this response. It is interesting to speculate that MGP is playing a role in the retention of calcium and the increased citrullination observed in these studies.

Figure 1

Figure 2

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/modification-of-thp-1-cells-with-malondialdehyde-acetaldehyde-increases-cellular-calcium-load-rendering-cells-susceptible-to-citrullination-of-self-proteins/