Study On Leptospira Interrogans-Induced Apoptosis In Murine Macrophages And Its Signal Transduction Mechanisms

Leptospirosis,caused by pathogenic Leptospira interrogans(L.interrogans),is a worldwide zooanthroponotic disease and has emerged as one of the key epidemic preventing and surveillancing infectious disease in natural disasters such as flood and earthquake.It has been reported that L.interrogans has the capability to enter host blood by penetrating skin and mucous membranes,and then rapidly disseminate to other tissues, where they survive,grow and cause pathological changes.The typical clinical manifestations of human leptospirosis are jaundice,pulmonary hemorrhage and renal failure. So far,exotoxin is not found in L.interrogans.And the reported cytotoxic factors and hypotoxic endotoxin-like substance can not explain host pathological changes during L. interrogans infection.Thus,mechanisms of pathogenesis in Leptospirosis remain poorly understood.Apoptosis is an active and highly ordered process in which cell death is executed.A series of genes and proteins,such as caspase and MAPK family members,are involved in this process and play important roles in apoptotic signal transduction.There is increasing evidence that a wide range of microbial pathogens can trigger host cell apoptosis and thereby influence the progression of the disease.It has been found that L.interrogans induces apoptosis in mono-macrophage like cells in vitro and in hepatocytes of guinea pigs. However,the underlying mechanisms responsible for L.interrogans-induced apoptosis remain uncharacterized.Taken into consideration that host cell apoptosis maybe not only relate to L.interrogans virulence,but also involve in immune evasion and survival in vivo. Thus,a deep understanding of apoptotic mechanisms induced by L.interrogans is valuable to elucidate the pathogenesis of Leptospirosis.Objective:The present study was undertaken to investigate the effects of L.interrogans infection on different host cells and the mechanisms of apoptotic signal transduction in murine macropahges,so that to establish the experimental foundation for the elucidation of the pathogenesis of Leptospirosis.Methods:The cytotoxic effects of Leptospira infection on different host cells were measured by LDH release assays.The percentage of apoptosis and necrosis induced by L. interrogans in different cells was evaluated by Annexin V/PI double staining using flow cytometry.The morphological changes in L.interrogans-infected J774A.1 and murine peritoneal macrophages were observed by transmission electron microscopy(TEM).By using caspase fluorometric substrates and flurometer,the enzymatic activities of caspases were measured in L.interrgans-infected J774A.1 and murine peritoneal macrophages. Western blot was employed to examine the effect of L.interrgans infection on the cleavage of PARP and Lamin A/C and the expression of FADD in J774A.1 and murine peritoneal macrophages.ELISA was performed to test the phosphorylation levels of JNK and P38MAPK in L.interrogans-infected J774A.1 cells.Caspase broad-spectrum and specific inhibitors were chosen to detect their inhibitory effect on L.interrogans-induced apoptosis and caspase signal transduction system,and JNK and p38MAPK specific inhibitors were used to determine the roles of JNK and p38MAPK in L.interrogans-induced J774A.1 apoptosis,by using flow cytometry,flurometer and western blot,respectively.Results:(1) L.interrogans reduced the viability of J774A.1,A549 and murine peritoneal macrophage in concentration- and time-dependent manners.The viability of human umbilical vein endothelial cells was not affected by L.interrogans infection.(2) L. interrogans induced concentration- and time-dependent apoptosis in J774A.1 and murine peritoneal macrophages.And primary murine macrophages were more susceptible to induction of apoptosis by L.interrogans than J774A.1 cells.In addition,L.interrogans induced time-dependent necrosis in A549 cells,whereas there was no measurable apoptosis or necrosis in human umbilical vein endothelial cells(EVC304 and EVC-EC-C).(3) Caspase broad-spectrum inhibitor reduced L.interrogans-induced apoptosisin J774A.1 and murine peritoneal macrophages in a dose-dependent manner.(4) L.interrogans-induced J774A.1 and murine peritoneal macrophage apoptosis was associated with the activation of caspases(caspase-3,-6,-8 and -9),the cleavage of caspase substrates PARP and Lamin A/C, and the increased expression of FADD.(5) Caspase-8 inhibitor,but not caspase-9 inhibitor, impaired L.interrogans-induced caspase-3 and -6 activation,as well as PARP and Lamin A/C cleavage and apoptosis,suggesting that apoptosis is initiated via caspase-8 activation, caspase-9 is activated through caspase-3 feedback amplification loop and mitochondrial cytochrome c-caspase-9-dependent signaling is not involved in this apoptotic process.(6) Caspase-3,served as upstream factor,mediated the activation of caspase-6 in J774A.1 and murine peritoneal macrophage infected by L.interrogans.(7) The phosphorylation level of JNK and p38MAPK was significantly increased in J774A.1 cells infected by L.interrogans. (8) JNK and p38MAPK inhibitors impaired L.interrogans-induced caspase-3 and -6 activation,as well as PARP and Lamin A/C cleavage and apoptosis.Conclusion:(1) The cellular effects of L.interrogans infection occurred in organ- and cell-type-specific contexts.L.interrogans induced concentration- and time- dependent apoptosis in J774A.1 and murine primary macrophages,and time- dependent necrosis in A549 cells.However,apoptosis or necrosis was not induced in human umbilical vein endothelial cells(EVC304 and HUV-EC-C) infected by L.interrogans.(2) L. interrogans-induced apoptosis in J774A.1 and murine primary macrophages was mediated by caspase-3 and -6 activation through a FADD-caspase-8-dependent pathway, independently of mitochondrial cytochrome c-caspase-9-dependent signaling.(3) JNK and p38MAPK signal transduction system was activated by L.interrogans in J774A.1 cells. JNK and p38MAPK played an important role in apoptosis by mediating the activation of caspase-8 and -3 and the increase of FADD expression.All these results provide new idea for understanding cellular and molecular mechanism of L.interrogans-induced apoptosis.