Study On Regulation And Mechanism Of Exosomes Dervied From Toxoplasma Gondii On Macrophages

Objective:To isolate and identify exosomes derived from T.gondii RH strain,and to analyze specific protein components from T.gondii.To investigate the regulatory effect of T.gondii exosomes on macrophage derived from human acute monocytic leukemia cell line(THP-1)in vitro,and to further explore the role of MAPK signal pathway and NF-κB signal pathway in the regulation of macrophages by T.gondii exosomes.Methods:The exosomes of T.gondii were separated by differential centrifugation,ultrafiltration and kit extraction,and the structures and characteristics of isolated vesicles were identified by electron microscopy,nanoparticle tracking analysis(NTA)and Western blotting.THP-1 cells were induced by phorbol 12-myristate 13-acetate(PMA)for 48 h,from the suspended monocytes to the adherent macrophages,and treated with different concentrations of T.gondii exosomes at different time points in vitro.The tracking of T.gondii exosomes into the cells was observed by immunofluorescence technology.The expression of cytokines was detected by real-time quantitative PCR and ELISA,and the production of NO was detected by Flow cytometry with the fluorescence probe.To show activation of MAPK signaling pathway and NF-κB signaling pathway,the expression and distribution of related proteins was detected by Western blotting and immunofluorescence respectively.Results:The exosomes derived from T.gondii were successfully isolated and identified.The results of electron microscopy showed that the structure of the membrane was round,and the diameter range was about 30-150 nm.The results of NTA showed that the distribution of particle size ranged from 30 to 150 nm,with an average size of 119.8 nm,which was consistent with characteristics of exosomes.The results of Western blotting confirmed that exosomes were successfully detected for HSP70,CD63,SAG1,ROP16,ROP18 and GRA1 proteins.T.gondii exosomes boosted THP-1 macrophages to secrete pro-inflammatorycytokines and NO,and accelerated the inflammatory response of macrophages to promote polarization of macrophages to M1 type.The exosomes derived from T.gondii promoted the activation of MAPK/JNK signaling pathway,and induced a large number of phosphorylated JNK protein into the nucleus.T.gondii exosomes also activated NF-κB dimer by promoting the phosphorylation of IκBα to promote the activation of NF-κB signaling pathway.Conclusions:The exosomes derived from T.gondii RH strain tachyzoites were successfully isolated and identified.T.gondii exosomes positively regulated THP-1 macrophages and promoted the activation of MAPK/JNK signaling pathway and NF-κB signaling pathway.This study provides a basis for further elucidating the biological function of T.gondii exosomes and its role in the relationship between hosts and parasites.