Amelogenin Modulates Macrophage Phenotype During Inflammatory Responses

Objectives: Amelogenin, the major component of enamel matrix derivative (Straumann® Emdogain), is well recognized in periodontology. It is used in periodontal surgery to regenerate cementum, periodontal ligament, and alveolar bone. Together with the regenerative effects on periodontium, possible influences of amelogenin on wound healing and anti-inflammatory responses are also reported. However, the precise molecular mechanisms underlying these effects are still unclear. Macrophages plays central role in resolution of inflammation, and its phenotype can be characterized as proinflammatory (M1) or immunomodulatory and tissue remodeling (M2). In this study, we analysed the effects of amelogenin on inflammatory genes expression profiles, and investigated its effect on cellular responses in LPS-challenged macrophage.
Methods: Human monocyte U937 cells were PMA-differentiated into macrophages. In microarray analysis, the gene expression profiles of LPS stimulated cells with or without recombinant amelogenin (rM180) were examined by GO term and heatmap. Anti-inflammatory effects of rM180 in LPS stimulated U937 cells were validated by RT-RCP, western blots, and ELISA. To further evaluate whether rM180 could modulate macrophage plasticity and polarization, the expression levels of M1/M2 marker genes and morphological changes were observed by confocal microscopy. Levels of growth factors were also measured by ELISA.
Results: Microarray analysis demonstrated that rM180 enhanced anti-inflammatory responses of LPS-challenged macrophages in 24 hrs, while it temporary up-regulated inflammatory responses in 4 hrs. The expression of M2 macrophage markers (CD163, CD206) were increased in IL-4, rM180 and rM180+ LPS stimulated cells, whereas M1 macrophage markers (iNOS, CD86) were increased in LPS stimulated cells. Amelogenin-induced M2 polarization led to cellular elongation, and the level of VEGF was increased.
Conclusions: These results suggest that amelogenin could catalyze inflammatory response appropriately, which contributes to early termination of inflammation. Amelogenin stimulation skew macrophage function toward the M2 phenotype, thereby paving the way for periodontal tissue regeneration.