The Mechanism Of Talaromyces Marneffei Influencing Macrophage RAW264.7 Polarization And Sterilization Ability Via Arginine Metabolism Pathway

Objective:To investigate the mechanism of Talaromyces marnefei(TM)influencing macrophage RAW264.7 polarization and sterilization ability via arginine metabolism pathway.Methods:(1)Construct the co-culture system of RAW264.7(Mφ)/LPS-activated RAW264.7(Mφ+LPS)and Talaromyces Marneffei conidia for 24h,48h and 72h.The relative mRNA expressions of iNOS,Argl,TNF-α,IL-10,CD301 and IL-1β were detected by Real-time PCR.The protein expressions of iNOS,Argl,CD86 and IL-4R were assayed by western blot method.The Arginase activity was measured by biochemical analysis.The production of NO was determined with microplate test.The flurescence intensities of CD86 and CD206 were determined by immunofluorescence.(2)Pretreat the LPS-activated RAW264.7 with arginase inhibitor Nω-hydroxy-nor-arginine(nor-NOHA)2h before co-cultured with TM conidia for 24h.The Arginase activity was assayed by biochemical analysis.The production of NO was determined with microplate test.The protein expressions of iNOS and Argl were measured by western blot method.The phagocytosis of macrophages was determined by phagocytic index.The sterilization ability of macrophages against TM condia was evaluated by plate colony-counting method.Results:(1)①Real-time PCR showed that the relative mRNA expressions of iNOS,Arg1,TNF-α,IL-10,CD301 and IL-1β in co-culture system were higher than Mφ/Mφ+LPS group,P<0.05.A certain trendency of expression changes with the co-culture period.②Western blot showed that the protein expressions of Argl and CD86 in co-culture system were lower compared with Mφ/Mφ+LPS group while the protein expressions of iNOS and IL-4R in co-culture system were higher,P<0.05.①Biochemical analysis exhibited that the arginase activity in co-culture system was higher than Mφ/Mφ+LPS group.The longer the co-culture period,the higher the arginase activity,P<0.05.④Microplate test exhibited that the production of NO in co-culture system was lower than Mφ/Mφ+LPS group,P<0.05.⑤The flurescence intensity of CD206 in co-culture system was brighter compared with Mφ/Mφ+LPS group while the flurescence intensity of CD86 showed no difference.(2)①Biochemical analysis demonstrated that the arginase activity in co-culture system with pretreatment of 20 μ M nor-NOHA was lower compared with the group without nor-NOHA,P<0.05.②Microplate test demonstrated that the production of NO in co-culture system with pretreatment of 20 μ M nor-NOHA was higher compared with the group without nor-NOHA,P<0.01.③Western blot showed that the protein expression of Argl in co-culture system with pretreatment of 20 p M nor-NOHA was lower compared with the group without nor-NOHA while the protein expression of iNOS in co-culture system with pretreatment of nor-NOHA was higher,p<0.01.④The phagocytic index in co-culture system with pretreatment of 20 p M nor-NOHA was higher compared with the group without nor-NOHA,P<0.05.⑤Plate colony-counting methods indicated that the sterilization ability of macrophages with pretreatment of 20 μM nor-NOHA was stronger than the co-culture system without nor-NOHA;The amount of TM colonies with pretreatment of 20 μ M nor-NOHA was less than that without nor-NOHA;The amount of TM colonies in co-culture system was more than that being cultured alone.Conclusion:(1)TM reduces the production of NO and promotes macrophages towards M2 polarization by increasling the arginase activity of macrophages.This mechanism protects TM from the oxidative stress in macrophages and promotes persistent infection in the host.(2)Arginase inhibitor nor-NOHA deereases the high arginase activity induced by TM,increases the production of NO and enhances the sterilization ability of macrophages.(3)Arginase inhibitor nor-NOHA inhibits the TM growth.