The Study On Toxic Mycobacterium Tuberculosis Induces EBI3 Production And Inhibits The Apoptosis Of Macrophages

Background:Tuberculosis(TB)is a chronic infectious disease caused by Mycobacterium tuberculosis(M.tb).According to the World Health Organization(WHO)2016 survey,10.4 million people fell ill with TB and 1.8 million died from the disease.Over 95%of TB deaths occur in low-and middle-income countries.China has an estimated about 1 million new cases of TB every year,more than any country except India and Indonesia.Therefore,it is of great significance to study the pathogenesis of M.tb and the mechanism of immune escape caused by the bacteria.Epstein-Barr virus induced 3(EBIB)is a common subunit of regulatory cytokines IL-3 5 and IL-27,EBI3 is currently reported to show a high level in serum from TB patients.The aim of this study was to investigate the effects of EBI3 on M.tb-induced immune escape.Objective:To assess the EBI3 production by macrophages induced by M.tb,and to explore the mechanism of EBI3 in macrophage apoptosis induced by virulent M.tb.Methods:The macrophage cell line Raw264.7 was stimulated with heat-inactivated M.tb H37Rv(iH37RV)and Bacillus Calmette-Guerin(BCG).The EBI3 production was detected by flow cytometry and the secreted extracellular EBI3 from cell culture supernatant was detected by ELISA.The intracellular distribution of EBI3 was observed by confocal microscope.IH37Rv/BCG-induced necrosis/apoptosis in macrophages was determined by Flow cytometry.The intracellular distribution of EBI3 was observed by confocal microscope.The EBI3 shRNA interference plasmid was constructed and transfected into Raw264.7 cells.EBI3-slienced Raw264.7 cells were subject to iH37Rv/BCG treatment and their necrosis/apoptosis was determined by flow cytometry.Results:Compared with BCG control group,virulent iH37Rv induced Raw264.7 cells to produce higher levels of EBI3.Results of immunofluorescence assay showed that EBI3 was trapped inside the cells upon stimulation with iH37Rv.The downregulation of EBI3 promoted the apoptosis of macrophages induced by iH37Rv.Conclusion:IH37Rv induced an increase of EBI3 production in the mouse macrophage.The increased EBI3 was trapped inside the macrophages upon iH37Rv stimulation.Downregulation of EBI3 promoted the apoptosis of macrophages induced by iH37Rv.These data indicate that accumulation of EBI3 inside the macrophage might inhibit the apoptosis of the macrophages,which helps to elucidate the immune escape mechanism of virulent M.tb H37Rv.