Amelogenin down-regulates MHC class II antigen presentation on macrophages.

Abstract: Enamel matrix derivatives (EMDs)-based regenerative therapy is suggested to promote better healing with minimal inflammatory response after periodontal surgery. We previously found stimulation of monocytes with amelogenin suppresses major histocompatibility complex class II (MHC II) gene expression using microarray analysis. However, the detailed molecular mechanisms for this process remain unclear. Here, we aimed to understand the underlying mechanism and its effects on immune system.THP-1 monocytic cells were stimulated with 50 µM PMA. The cells were pretreated with 10 µg/ml amelogenin for 24 hours, followed by the stimulation with 2.5 µg/ml IFNγ for 24 hours. The cell surface expression of antigens was evaluated by flow cytometry, and signal transduction pathway was analyzed by real-time PCR and western blotting. Cellular uptake of amelogenin was observed by using confocal laser microscopy. Histone modification of class II transactivator (CIITA) promoter region was analyzed by chromatin immunoprecipitation. T cell activation was evaluated by mixed leukocyte reaction (MLR). Amelogenin down-modulated IFNγ-induced cell surface expression of MHC II and this effect was widely conserved across species, as similar effect was confirmed in raw 264.7 murine macrophages. Ameloginin accumulated in the nucleus as early as 15 min following stimulation and inhibited subsequent CIITA expression. Reduced MHC II expression on macrophages pretreated with amelogenin down-regulated the expression of T cell activation markers CD25 and CD69, T cell proliferation, and IL-2 production by allogenic CD4+ T lymphocytes in MLR. H3K27ac and H3K4me3 in CIITA promoter IV region, which are essential for conversion to euchromatin, were markedly suppressed by amelogenin.Amelogenin suppresses MHC II expression by altering chromatin structure and inhibiting CIITA promoter IV transcription activity and attenuates subsequent T cell activation. Therefore, clinically observed better wound healing after the surgery may be, at least in part, explained by the mechanism elucidated in the current study.