| BackgroundTalaromyces marneffei(TM)infection can cause opportunistic infection,mainly spreading within immunodeficiency population such as HIV-patients.With increased use of immunosuppressants,the number of individuals with TM infection is on the rise.TM infection has nonspecific clinical signs,including fever,splenomegaly,skin damage and abnormal hemogram.For immunodeficient patients,TM also can cause lethal disseminated infections.Therefore,it is especially urgent to reveal the pathogenetic mechanisms of TM infection and provide the scientific basis for clinical diagnosis and treatment.There were also several reports on some virulence factors of TM such as HSP,antioxidant enzymes,MP1p and nutritional metabolism-related enzymes.However,the specific pathogenesis of TM has not been fully figured out and it was needed to be investigated from a new perspective.Extracellular vesicles(EVs),a kind of nano-sized lipid bilayer membrane structures,could act as a carriage with a lipid bilayer-enclosed structure to encapsulate numerous active molecules biologically.A large body of literature confirms that showed that EVs could be secreted from fungi and mediated inflammatory responses,however,whether TM could release EVs and involved in immune reactions have not been proven yet.Therefore,our research mainly focused on the specific pathogenic effect of TM from a new perspective of EVs.MethodsIn this study,the TM-EVs were isolated by differential centrifugation and further purified through OptiPrep density gradient ultracentrifugation.To investigate the morphology characteristic and size distribution of the TM-EVs,the TEM and NTA were utilized.A co-culture model of RAW264.7 macrophage cells with DiI labeled EVs was built,and then the function of TM-EVs in cellular inflammatory response was explored.The change in immune stimulation of TM-EVs was observed after protein,DNA and RNA being hydrolyzed respectively for investigating the main functional components of TM-EVs.Next TM-EVs were further analyzed by LC-MS/MS and classified for working out their functions and cellular localization.Finally,LC-MS/MS was performed in order to investigate the proinflammatory mechanism of TM-EVs.ResultsOur experiment indicated that purified TM-EVs were mainly localized in the 20%fraction.The NTA and TEM showed that the mean diameter of EVs was 170 nm and their structures were typical of cup-shaped vesicles.The moment of TM releasing EVs has also been visualized by TEM.Immunofluorescence experiments showed that macrophage could intake TM-EVs,but could not with pretreatment of actin polymerization inhibitors cytochalasin D,which confirmed that vesicle internalization of macrophage was an active process but not a passive movement.Our study revealed that TM-EVs could significantly promote NO and ROS secretion,and enhance the secretion of IL-1β,IL-6 and TNF-α in RAW264.7 macrophage cells.In addition,TM-EVs could enhance antigen presentation capacities of RAW264.7 macrophage cells.Different enzymatic experiment showed that protein could play a significant role in the proinflammatory of TM-EVs,and some virulence factors were also identified in the proteomics of TM-EVs.LC-MS/MS data showed that upregulated proteins were mainly clustered in the NF-kB pathway.The NF-κB mRNA and protein expression were also increased significantly in qPCR and WB,NF-κB activation inhibitor BAY 11-7082 could suppress inflammation response significantly.RNA interference experiments also showed that pro-inflammatory of TM-EVs was partially dependent on TLRs.ConclusionTaken together,our results indicated that the TM-derived EVs could mediate inflammatory response in RAW 264.7 macrophage cells.In addition,WB and PCR showed that TM-EVs may promote the inflammation of RAW264.7 macrophage cells through TLRs and NF-kB pathway. |
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