Myelin basic protein charge isomers change macrophage polarization

Abstract

Elene Tsitsilashvili,1 Maia Sepashvili,1,2 Marika Chikviladze,1 Lali Shanshiashvili,1,2,* David Mikeladze1,2,* 1Institute of Chemical Biology, Ilia State University, Tbilisi, Georgia; 2Department of Biochemistry, I. Beritashvili Center of Experimental Biomedicine, Tbilisi, Georgia *These authors contributed equally to this work Purpose: During a neuronal injury, a variety of immune cells infiltrate into the local microenvironment at the demyelination site. After the destruction of the intact myelin sheath, its major constituent myelin basic protein (MBP) dissociates from the plasma membrane and acts as a free ligand on the infiltrated immune cells. MBP exhibits charge microheterogeneity as a result of post-translational modifications, but the effect of various isomers of MBP on the activity of macrophages is not known.Materials and methods: MBP was isolated and purified from bovine brain white matter. RAW 264.7 macrophages were cultured in DMEM supplemented with heat-inactivated fetal bovine serum. For evaluation of macrophage polarization following treatment of RAW 264.7 cells with MBP charge isomers, inducible nitric oxide synthase (iNOS) expression (M1 phenotype marker) and arginase-1 expression (M2 phenotype marker) were determined in cell lysates by ELISA. To assess Rac activity, G-LISA Rac Activation Assay system was used. The expression of receptor for advanced glycation end-products (RAGE) and high mobility group box 1 (HMGB1) protein were assayed by Western blot analysis.Results: Our results have shown that minimally modified C1 component of MBP increases the expression of arginase-1 in cells, decreases the expression of iNOS, does not change the secretion of HMGB1 protein, but significantly elevates surface expression of RAGE, and in parallel, increases the activity of small GTPase Rac. On the other hand, highly modified deiminated isomer C8-MBP increases the secretion of HMGB1 protein but does not change the expression of arginase-1 or the content of RAGE.Conclusion: These data indicate that deiminated C8 isomer of MBP tends to polarize RAW macrophages into M1 phenotypes, whereas C1 enhances the activity of M2 phenotype markers. Keywords: Arginase-1, iNOS, HMGB1, RAG