Accumulation Of EBI3 Induced By Virulent Mycobacterium Tuberculosis Inhibits Apoptosis In Murine Macrophages

BackgroundTuberculosis is a major infectious disease that seriously threatens people's health.China has the third highest number of cases(about 920,000 new cases,9% of the global total).Therefore,it is urgent to conduct researches on mechanism of M.tb pathogensis and M.tb-mediated immune escape,contributing to TB prevention and treatment.EBI3,the common subunit of immune regulatory cytokines IL-27 and IL-35,has usually been explored as the secreted form and assessed in terms of its effects triggered by extracellular EBI3.Little is knowed about intracellular EBI3 function.ObjectiveThe current study aims to explore the effects of intracellular EBI3 on virulent M.tb-treated macrophages.MethodsThe expression of EBI3 in mononuclear cell of peripheral blood samples from the TB patients was determined by FCM.The levels of intracellular and secreted EBI3 in the murine macrophages upon stimulation with virulent M.tb and BCG at different time points were detected by ELISA and Western blot.The location of intracellular EBI3 was observed visually by confocal microscopy.The protein interacted with EBI3 was detected and validated by RNA interference,Western blot,Co-IP,LC-MS/MS.The intracellular EBI3 effects on macrophage apoptosis were assessed by FCM analysis.ResultsThere is a significantly increase in the EBI3 expression in peripheral blood mononuclear cells of the TB patients,compared with healthy people.The increasedlevels of intracellular EBI3 in the murine macrophages upon the stimulation with M.tb and BCG at different time points,and elevated levels of extracellular EBI3 were observed.The EBI3 secretion took longer time to reach its peak level in the virulent M.tb group than the BCG group,while the levels of intracellular EBI3 were rapidly reach its maxium level in virulent M.tb group.These results indicated that intracellular EBI3 were accumulated in virulent M.tb-treated macrophages.The result from RNA interference,Western blot,Co-IP,LC-MS/MS and FCM analysis demonstrated that protein eEF1A1 interacts with intracellular EBI3.The level of Lys48(K48)-linked ubiquitination on EBI3 mediated by eEF1A1 was inhibited by virulent M.tb,leading to the intracellular EBI3 accumulation in macrophages.The accumulated intracellular EBI3 were involved in inhibition of M.tb-induced and caspase-3-mediated macrophage apoptosis.ConclusionIn the present study,we show that eEF1A1 participates in the reduction of K48-linked ubiquitination of EBI3 upon stimulation with virulent M.tb.The accumulation of EBI3 hinders apoptosis in macrophages.We propose a model mechanism for intracellular EBI3 and its regulation of macrophage apoptosis in response to virulent M.tb.