Molecular Mechanism Of Apoptosis Induced By Leptospira Interrogans And The Intracellular Survival And Replication Within Phagocytes

Partâ… :Leptospira interrogans Induced FasL/Fas Dependent and Toll like Receptor-2-p38 MAPK/JNK Regulated Apoptosis of Murine MacrophagesBackground and Objective:Leptospirosis is one of the most widespread zoonosis that causes an acute febrile and systemic illness in humans caused by pathogenic Leptospira species.The clinical syndromes include subclinical infection,self-limited anicteric febrile illness with or without meningitis,and severe and potentially fatal Weil's syndrome that manifests hemorrhage,jaundice,and renal failure.Although Leptospira virulence factors such as hemolysins,lipopolysaccharide,glycolipoprotein, peptidoglyean,heat shock proteins,flagellin,and others may contribute to the pathogenesis,their pathogenic mechanisms have not been clearly understood. Although it has been reported that Leptospira interrogans invades and induces apoptosis of murine monocyte macrophages-like cell line(J774A.1),the apoptotic mechanism remains unclear.Fas ligand(FasL)/Fas pathway is one of the most common pathway involved in cell apoptosis.This study is aimed to investigate the role of the FasL/Fas pathway on apoptosis induced by Leptospira inteerogans and the apoptotic regulation mechanism of associated signal transduction pathaws.Methods:The leptospiral lipopolysaccharide(L-LPS) and outer membrane protein (L-OMP) of L.interrogans serovar Lai strain Lai was prepared by the phenol-water method and Triton X-114 method,respectively.The apoptosis and apoptotic blocking effect of anti-mouse FasL neutralizing antibody in J774A.1 cells infected with leptospires and treated with L-LPS or L-OMP were quantified by flow cytomety.The J774A.1 cells infected with leptospires and treated with L-LPS or L-OMP were stained with PE-conjugated mouse FasL/Fas antibody,and then the alteration of FasL/Fas expression level was observed with fluorescence microscope or quantified by flow cytometry.The alteration of FasL/Fas mRNA expression level of J774A.1 cells infected with leptospires and treated with L-LPS or L-OMP was quantified with real time RT-PCR.The blocking effects of toll like receptor(TLR2 and TLR4) neutralizing antibodies and signal transduction pathway(p38-MAPK,JNK and ERK) inhibitors on FasL/Fas expression were also quantified by flow cytometry.Results:The results of flow cytometry showed that both leptospires and L-LPS induced apopotosis in J774A.1 cells and anti-mouse FasL neutralizing antibody large partly blocked the apoptosis,and the apoptosis blocking rates of eptospire-infected and L-LPS treated J774A.1 cells were 60.03%and 83.47%,respectively(P<0.05).The L-OMP also induced apoptosis in J774A.1 cells,but the anti-mouse FasL neutralizing antibody had no obvious blocking effect on L-OMP induced apoptosis(p>0.05). Immunofluorescence and flow cytometry detection results indicated that both the FasL and Fas expression rates of leptospire-infected and L-LPS treated-J774A.l cells, compared with the normal cells,increased in time-dependent manner(P<0.05),and approached to maximal expression rates at 12 h.The maximal expression rates of FasL and Fas expression were 71.57%and 93.57%for leptospire-infected J774A.1 cells,respectively,and 60.56%and 89.54%for L-LPS-treated J774A.1 cells,but no obvious alteration of FasL and Fas expression of L-OMP treated J774A.1 cells was detected(P>0.05).Besides,the real time RT-PCR detection results suggested that both the FasL and Fas mRNA level of leptospire-infected and L-LPS treated-J774A.1 cells were upregulated(P<0.05),but no alteration of FasL and Fas mRNA expression level of L-OMP treated J774A.1 cells was detected(P>0.05).Moreover,the results of flow cytometry indicated that TLR2 receptor neutralizing antibody large partly blocked the expression of FasL and Fas of J774A.1 cells infected with leptospires and treated with L-LPS for 12 h(P<0.05),and the blocking rates of TLR2 for FasL expression of leptospire-infected cells and L-LPS- treated J774A.1 cells were 84.74%and 87.92%. while the blocking rates of TLR2 for Fas expression of leptospire-infected cells and L-LPS- treated J774A.1 cells were 58.07%and 61.12%.The TLR4 neutralizing antibodies had no obvious blocking effect on the the up-regulated FasL and Fas expression of leptospire-infected and L-LPS-treated J774A.1 cells(P>0.05),and neither TLR2 nor TLR4 receptor neutralizing antibodies had blocking effect on the FasL and Fas expression of L-OMP treated J774A.1 cells(P>0.05).In addition,the results of flow cytometry indicated that both p38-MAPK inhibitors large partly suppressed the FasL and Fas expression of J774A.1 cells infected with leptospires and treated with L-LPS for 12 h(P<0.05),and JNK inhibitor small partly suppressed the expression(P<0.05).The blocking rates of p38MAPK inhibitor and JNK inhibitor for the FasL expression were 68.20%and 29.00%for leptospire-infected J774A.1 cells, and 70.04%and 42.63%for LPS-treated J774A.1 cells,while The blocking rates of p38MAPK inhibitor and JNK inhibitor for the Fas expression were 67.33%and 18.09%for leptospire-infected J774A.1 cells,and 60.93%and 28.00%for LPS-treated J774A.1 cells.The ERK inhibitor had no obvious blocking effect on the expression of FasL and Fas expression of leptospire-infected and L-LPS-treated J774A.1 cells (P>0.05),and all the three signal pathway inhibitors had no blocking effect on L-OMP treated J774A.1 cells(P>0.05).Conclusion:Induction of cell apoptosis is an important mechanism of L. interrogans strain Lai injuring J774A.1 cells.L.interrogans is able to up-regulate FasL/Fas expression levels of host cells and induce apoptosis of the cells via FasL/Fas pathway,and the apoptosis is regulated by TLR2 receptor and p38MAPK pathway and leptospiral LPS is the component of outer membrane of L.interrogans Besides, leptospiral OMP is also able to induce apoptosis of J774A.1 cells,but no alteration of FasL/Fas expression of L-OMP treated cells was detected,and the anti-mouse FasL neutralizing antibody had no apoptotic blocking effects on L-OMP induced cell apoptosis,which indicates FasL-like protein(s) may exsist in the outer membrane protein of Leptospira interrogans.Partâ…¡:Intracellular survival and replication of Leptospira interrogans within macrophagesBackground and objective:Leptospirosis is one of the most widespread zoonosis that caused by pathogenic Leptospira species.The carriers of leptospires are mainly wild or domestic animals,especially rodents,small marsupials,cattle,pigs,and dogs. The clinical manifestations of human leptospirosis range from mild illness to multiorgan failure,characterized by jaundice,pulmonary haemorrhage and renal failure.Pulmonary diffuse hemorrhage,a serious clinical form of leptospirosis,is fatal in about 25%of patients.On the contrary,most of the infected mammalian reservoir animals,such as rodents,only present mild chronic disease or are asymptomatic,and shed infectious organisms in the urine for their lifetime.The reasons for this diversity of infection remain unclear.Mononuclear macrophages are the most efficient phagocytes in humans and mammals,and can phagocytose large numbers of invading bacteria and other microbes.Many studies have demonstrated that leptospires are phagocytosed by macrophages in vivo and in vitro,but the intracellular fates of leptospires in macrophages is not well known.Therefor,this study is aimed to investigate the intracellular fates of leptospires within macrophages of humnan and murine origin.Methods:Fontana staining method was used to detect the adhesion of L. interrogans serovar Lai strain Lai and serovar Pomona strain Luo to primary and immortal(THP-1 and J774A.1) macrophages from human and mouse.Transmission electron microscopy(TEM) was used to observe the phagocytosed L.interrogans strain Lai in human and murine macrophages.Enumeration of colony-forming units (CFUs) and Quantitative fluorimetric assay were applied to determined the replication of L.interrogans strain Lai and Luo in macrophages.The Viability of intracellular L. interrogans strain Lai was assessed by staining with a Bacterial Viability Kit and quantified by flow cytometry.Confocal microscopy was carried out to determine the co-localization of phagocytosed L.interrogans strain Lai and Luo with the late-endosomal/lysosomal marker LAMP-1 in human and murine macrophages.The Cell Apoptosis Detection Kit was used to staining L.interrogans strain Lai infected human and murine macrophages and the apoptotic and necrotic cells were quantified by flow cytometry.Results:The Fontana staining method detection results showed that L.interrogans strain Lai and Luo had a similar ability to adhere to and enter primary and immortal (THP-1 and J774A.1) macrophages from human and murine.Transmission electron microscopy(TEM) revealed that the L.interrogans strain Lai resided within membrane-bound vacuoles in the murine macrophages,but occurred free in the cytosol of human macrophages,with no surrounding vesicular membrane.The results of CFU enumeration revealed increased numbers of L.interrogans strain Lai in human macrophages in a time-dependent manner,the number of both leptospires in murine macrophages gradually decreased as a function of incubation time(PO.05).The results of quantitative fluorimetric assay indicated that the fluorescence intensity of L. interrogans strain Lai increased in incubation time-dependent manner in human macrophages,while that decreased in incubation time-dependent manner in murine macrophages(P<0.05).The flow cytometry detection results of intracellular viability of L.interrogans strain Lai manifested that the percentages of live leptospires decreased slightly but continously in human macrophages during the whole infection process,compared to leptospires in EMJH medium,while the percentages of live leptospires in murine macrophages dropped sharply after 2 h infection and then progressively decreased during the remaining incubation time(P<0.05).Confocal microscopic images revealed that the percentage of colocalization of intracellular L. interrogans strain Lai and Luo with lysosomes in the murine macrophages increased progressively,whereas those in the human macrophages gradually decreased(P<0.05). The results of flow cytometry revealed that L.interrogans strain Lai induced higher maximal apoptotic ratios(57.3%for MPMs and 47.3%for J774A.1 cells after 4 h incubation) than the two human macrophages(32.9%for HPMs and 34.5%for THP-1 cells after 72 h incubation)(P<0.05).Moreover,late apoptotic/necrotic ratios in murine macrophages were also markedly higher than those in human macrophages, Furthermore,the dying cells showed typical morphologic changes of cell apoptosis such as cytoplasmic vacuolation,and chromatin condensation and margination.Conclusion:L.interrogans had a similar ability to adhere to primary and immortal macrophages of human and mouse origin,but its intracellular fate in human macrophages differed sharply from that in mouse.Most leptospires in murine macrophages resided within membrane-bound vacuoles and then were killed by lysosomal hydrolase,while most of the leptospires in human macrophages survived and replicated.These data strongly suggest that the outcome for intracellular leptospires depends on he differences among host macrophages,which may account for some of the differences in the severity of leptospirosis in humans and animals.