Mechanism And Signal Pathway Of Pro-inflammatory Response And Phagocytic Vesicle Membrane Mediated Immune Evasion By Leptospira Interrogand Hemolysins

Leptospira interrogans is a global distribution zoontic diseases when flood outbreak, which is one of the major infectious diseases in China's key prevention and control. There are many genes that encode putative hemolysin Leptospira interrogans strain Lai genome, hemolysin may contribute to the pathogenesis of leptospirosis, but leptospira gene expression and secretion of hemolysin during infected with macrophages have not been reported.In this study, first, we constructed Leptospira hemolysin genes including sphl (LA1027), sph2(LA1029), sph3(LA4004), sph4(LA3050), hlpA (LA1650), hlyC (LA3937), hlyX(LA0378) and tlyA (LA0327) prokaryotic expression system by cloning, then the recombinant proteins were extracted and purified by Ni-NTA affinity chromatography, and hemolytic activity of recombinant proteins were measured by hemolytic test in sheep blood by spectrophotometry. Finally, real-time fluorescence quantitative PCR was applied to detect the change of hemolysin genes in mRNA levels and Western-blot was used to detect the secreted of hemolysin proteins during Leptospira interrogans infected THP-1and J774A.1cells.The results showed that eight recombinant proteins with varying degrees of hemolytic activity has been constructed with high-level expression, eight hemolysin genes mRNA levels upregulated after infected with macrophages, especaily for sph2and hlpA show more significant increase, rSphl, rSph2, rSph3, rH1pA and rTlyA can be secreted into the extracellular in THP-1and J774A.1cells.All these results lead us to have a conclusion that a prokaryotic expression system with high efficiency of L. interrogans hemolysin genes were constructed successfully and the hemolysin genes change in mRNA and protein levels during infected with macrophages, which lay a foundation for further study of pathogenic of leptospira hemolysin. Infection of pathogenic Leptospira species causes serious systemic inflammation in patients but the profile of proinflammatory cytokines has never been characterized. Although the presence of numerous hemolysin-encoding genes in the genomes of pathogenic L.interrogans strains suggests that the hemolysins may contribute to pathogenesis in leptospirosis, the ability of leptospiral hemolysins to induce proinflammatory cytokines and stimulate TLR-dependent signaling pathways has never been reported. In the present study, we measured the cytokine profile in sera from leptospirosis patients and leptospire-infected mice using cytokine protein microarrays. The infected mice provided less elevated serum cytokines with relatively lower levels compared to the patients. However, IL-1β, IL-6and TNF-a were the main proinflammatory cytokines in sera of both the patients and infected mice. The tested leptospiral recombinant sphingomyelinic hemolysins (rSphl to rSph4) and non-sphingomyelinic hemolysins (rHlpA, rHlyC, rHlyX and rTlyA) could induce the production of IL-1(3, IL-6and TNF-a in human macrophages, while the production of cytokines in mouse macrophages was relatively lower. Inhibition of TLR2and TLR4as well as the NF-kB and JNK pathways in the two species of macrophages significantly reduced the production of hemolysin-induced IL-1β,IL-6and TNF-a. Macrophages isolated from TLR2-, TLR4-or double TLR2-and4-deficient mice confirmed the TLR2-and TLR4-dependent regulation of proinflammatory cytokine production induced by leptospiral hemolysins. Taken together, we demonstrated that all eight leptospiral hemolysins have the ability to induce inflammatory cytokines via TLR2and TLR4through the JNK and NF-kB pathways. This study is aim to determine the modality of Leptospira interrogans invading human and murine mononuclear-macrophages and diversity of leptospiral phagocytotic vesicle formation. Transmission electron microscopy was applied to observe the invasion of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai into murine mononuclear-macrophage-like cell line J774A.1and PMA-activated human monocyte line THP-1and the formation of leptospiral phagocytotic vesicles. By using immunofluorescence plus either laser confocal microscopy or fluorescence spectrophotometry, the changes of intracellular leptospiral numbers in J774A.1and PMA-activated THP-1cells before and after block with endocytosis inhibitors monodansylcadaverin (MDC), phenylarsine oxide (PAO) and clathrin antibody were investigated.The leptospires in J774A.1cells were located in phagocytotic vesicles while the leptospires in THP-1cells had no package with phagocytotic vesicle membrane. Both MDC and PAO presented the effect inhibiting endocytosis of L. interrogans into J774A.1and THP-1cells in dose-dependent manner. The numbers of leptospires in J774A.1and THP-1cells that pre-blocked with10μmol/L or above MDC and1μmol/L or above PAO were significantly less than that in the two cells untreated with MDC and PAO (P＜0.05). After J774A.1and THP-1cells were blocked with clathrin antibody, the numbers of intracellular leptospires were also remarkbly decreased (P<0.05).Leptospira interrogans can invade into both human and murine mononuclear-macrophages through the way of clathrin-dependent endocytosis. There is an opposite diversity of leptospiral phagocytotic vesicle formations in human and murine mononuclear-macrophages, which may result in the difference of pathogenesis in humans and mice after infected with L. interrogans. Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira, but the symptoms of human and poultry infected with leptospira are different. Leptospira can enter the bloodstream through the skin and mucous membranes quickly, Phagocytic process is the most important innate immune protection mechanisms, so the monocytes and macrophages play a key role in removing invasive pathogen. However, Leptospira invaded human and mouse macrophages occured different phenomenons, most of leptospira limited in the phagocytic vacuole of mouse primary and passage macrophages and distributed in the cytoplasm of human primary and passage macrophages. Furthermore, phagocytic membrane containing leptospira fused with lysosomes and leptospira were killed in mouse, while fertile signs of leptospira within human macrophages.Cells of the innate immune system ingest and destroy invading microorganisms by initially engulfing them into a specialized vacuole, known as the phagosome. The nascent phagosome is not competent to kill and eliminate the ingested microorganisms. Phagosome maturation is the process by which internalized particles (such asbacteria and apoptotic cells) are trafficked into a series of increasingly acidified membrane-bound structures, leading to particle degradation. The basic evasion mechanism is to mediate the process of phagosome, resulting in the formation of the mature phagosome-lysosome could not be obtained to produce within the phagosome, such as Mycobacterium tuberculosis. However, the mechanisms of pathogenic leptospira in human macrophages escaped from the host immune system and reproducted in the organization, and whether the leptospira hemolysins involved in the phagosome maturation process are not reported.In this study, we found that THP-1and J774A.1cells formed phatocytic vesicle membrane at the beginning of infection, then the phatocytic vesicle membrane diappeared in THP-1cells while not in J774A.1cells using TEM. Then using laser confocal immunofluorescence, we found that the major hemolysin co-localizated with the early markers of phagocytic vesicle membrane (Rab5),less co-localizated with the late markers (Rab7and LAMP-1) in THP-1cells, hemolysins could co-localizated with different periods of surface markers of vesicle membrane in J774A.1cells.We can conclude preliminarily that leptospira hemolysins interfered with the process of vacuolar maturation and mediated the evasion of leptospira from human macrophages for pathogen survival which id different with mouse macrophages, which may result in the difference of pathogenesis in humans and mice after infected with L. interrogans...