Study Of RAGE-mediated Phagocytosis And Related Signaling Pathways

Phagocytosis is a process by which phagocytes internalize macromolecules(including pathogens and damaged cells)larger than 0.5 μm in diameter,which usually performed by professional phagocytes such as macrophages,however,non-professional phagocytes can also perform phagocytosis.Phagocytosis is an important pathway of immune response and maintaining body homeostasis.Phagocytes recognize specific ligands on target particles through phagocytosis receptors,then initiate intracellular phagocytosis signaling pathways.Receptor for advanced glycation end products(RAGE)is a multi-ligands receptor and its activation could induce various cellular responses such as phagocytosis and inflammation.Saccharomyces cerevisiae is a favorable model organism.Our previous study shown that RAGE can mediate the internalization of S.cerevisiae spores(3-4 μm in diameter)by non-professional phagocytes,but the detailed mechanism still unclear.Therefore,we explored the phagocytic signaling pathway of RAGE in non-professional phagocytic cell HEK293 T and macrophage RAW264.7,respectively.The results are as followed:(1)In professional phagocytes,Spleen tyrosine kinase(SYK)is a signaling molecule to mediate phagocytosis,but it is unknown whether SYK is required for phagocytosis in nonprofessional phagocytes.When HEK293 T cells were treated with SYK inhibitors,the phagocytic efficiency of S.cerevisiae spores obviously decreased;When knocked out or mutated the SYK gene in HEK293 T,phagocytic efficiency also significantly reduced.These results suggest that phagocytosis of S.cerevisiae spores by non-professional phagocyte is depend on SYK.SYK often binds directly to phagocytic receptors to transmit phagocytic signal,however,co-immunoprecipitation experiments shown that RAGE and SYK were not directly bound,but linked through some intermediate proteins to mediate phagocytosis.(2)Nucleic acid is one of the reported RAGE ligands,the phagocytic efficiency of macrophage RAW264.7 significantly reduced when S.cerevisiae spores surface RNA was removed.After knocked out the Rage gene in RAW264.7,internalized efficiency of S.cerevisiae spores and RAGE ligand-modified beads(2 μm in diameter)decreased obviously,it indicates that the phagocytosis of microparticles by macrophages is depend on RAGE.Further fluorescence localization analysis revealed that RAGE was recruited around target particles when macrophages initiate phagocytosis.In addition,both S.cerevisiae spores and RAGE ligands-modified beads could stimulate macrophage to secrete pro-inflammatory cytokines,and the level of cytokine secretion was regulated by RAGE.These results suggest that RAGE as a phagocytic receptor in macrophages,S.cerevisiae spores and macroparticles containing RAGE ligands can be internalized through the RAGE-mediated phagocytic pathway.Taken together,our study identified the mechanism of RAGE mediated phagocytosis in phagocytes to macroparticles by exploring the phagocytic signaling pathways,laid foundation for further research on the role of RAGE in immune system,provided ideas for the development of new micron-scale drug delivery systems.