The Role Of Epithelial Cell-derived Exosome In Immune Regulation Of Macrophages In Response To BCG Infection

Tuberculosis is a chronic infectious disease caused by the infection of Mycobacterium tuberculosis(Mtb).Macrophages are the main target cells of Mtb,which can be cleared by cell autophagy,apoptosis and other pathways,In contrast,Mtb can also escape the killing of host cells through a variety of mechanisms.Exosomes play an important role in cellular communication.Exosome-derived factors can directly affect gene transcription and translation.They can alter protein modification by affecting signal transmission,regulate protein localization and key enzyme-catalyzed reactions,and then regulate the physiological activities of receptor cells.In order to investigate the regulatory role and mechanism of epithelial-derived exosomes on the immune response of infected macrophages to BCG infections.In this study,exosomes derived from BCG-infected or uninfected epithelial cells was isolated and identified,and then their roles of immune regulation in macrophages in response to BCG infection were examined.Western blotting,immunofluorescence,flow cytometry and other technologies were employed for testing the inflammation,apoptosis and autophagy of macrophages from the morphological to molecular levels.In order to reveal the regulatory role of exosomes in macrophage infection of BCG,Rapamycin was used to further investigate whether the regulation of autophagy by exosomes depends on the mTOR pathway.Finding from our experiments are described as follows:1.The isolated exosomes are disc-shaped under electron microscopy and with a size ranging from 30 nm to 150nm;Surface molecular markers CD81,CD63,HSP70 of exosomes were expressed;DIR red dye marks of exosome could be phagocytosis into the macrophages;exosomes from BCG-infected or uninfected epithelial cells with a concentration of 50ug/ml can significantly upregulated the expression of TLR signal ligands TLR2,TLR4,TLR6(p<0.05),promoted the cellular inflammatory factors IL-6,IL-1? secretion and upregulated expression of mediator MyD88,effectors NF-?B and TRAF6 proteins.This indictes that exosomes derived from epithelial cells can promote BCG-induced macrophage inflammation.2.Exosomes derived from BCG-infected and uninfected epithelial cells can significantly increase the expression of macrophage apoptosis-related proteins Bax and Caspase3(p<0.01),increase the apoptosis rate and reduce survival rate;After macrophages infected by BCG,compared with normal exosomes,BCG-infected exosomes can down-regulate the expression of apoptosis-related proteins Bax and Caspase3,and reduce the apoptosis rate of macrophages.3.The expression of Beclin1,Atg7 and LC3 protein was the highest at 12 hours after BCG infection in macrophages.The expressions of autophagy-related proteins Beclinl,Atg7 and LC3 were increased after exosomes from the two sources stimulated BCG infected macrophages.However,the upregulation effect of BCG infected exosomes on autophagy-related proteins was weaker than that of normal exosomes and inhibited the formation of autophagy flow.Meanwhile,exosomes could up-regulate the expression of mTOR pathway related proteins p-Akt,AKT,P-MTOR,mTOR and PI3K,indicating that both exosomes from the two sources could promote BCG-induced autophagy of macrophages,while the enhancement effect of BCG-infected exosomes from epithelial cells on BCG-induced autophagy of macrophages was less than that of normal exosomes.4.Compared with normal exosomes,the addition of rapamycin can up-regulate the expression of Atg5,Atg7,and LC3 proteins in BCG-infected epithelial cells derived exosomes group,indicating that rapamycin can reverse inhibitory effect of exosomes derived from epithelial cells on autophagy-related proteins.Meanwhile,rapamycin down-regulates the expression of P-AKT,P-mTOR and mTOR proteins,reversing the promoting effect of BCG-infected exosomes on mTOR related proteins.This indicates that exosomes derived from epithelial cells can participate in the regulation of autophagy in BCG-induced macrophages through the mTOR pathway.In summary,exosomes can promote the inflammatory response of macrophages,after BCG infected macrophages,exosomes derived from normal epithelial cells can upregulate apoptosis and autophagy in macrophages,the effect of exosomes derived from BCG-infected epithelial cells were weaker than normal exosomes,and the regulation of autophagy by the two different exosomes depends on the mTOR signaling pathway.This indicates that exosomes derived from epithelial cells can participate in the regulation of the immune response of BCG-infected macrophages by regulating the inflammation,apoptosis and autophagy of macrophages.