Study On Mechanisms Of Leptospires Internalization On Different Cell-types

Background:Leptospirosis is a world-wild Zoonotic infectious disease caused by leptospires which is lethal to some mammanlian. Leptospires are able to penetrate mucosal tissues and skin of mammalian such as human and mouse, transported by blood stream results in a bacteremia, and target to organs such as livers, lungs and kidneys. Leptospirosis has been frequently reported in Asia, American, Europe. In mammalian, different cells play different functions when infected by leptospires. Macrophages play a crucial role in phagocytosis and killing pathogens by degradation them. Vascular endothelial cell is responsible for transport pathogens from blood stream to targeted organs, but the viability of pathogens with limited loss during transcytosis process. The purpose of this study is to discover the different leptospires internalization mechanisms of different cells, better understanding of leptospires infection will lay a solid foundation to therapy and drug development to leptospirosis.Methods:In this study, THP-l(The human monocytic cell line), J774A.1(the murine monocyte macrophage-like cell line) and HUVEC(human umbilical vein endothelial cell line) were used to investigated different internalization mechanisms infected by leptospires. In this experiment, ELISA test of adherence and adherence inhibition were used to evaluate the adhension of leptospires to ECM (FN, LN, COL1, COL2, COL3, COL4); Confocal microscopy and ELISA were used to detect expression of FN, LN, COL1, COL2, COL3å'ŒCOL4on THP-1, J774A.1and HUVEC; Detection of intracellular leptospires of THP-1, J774A.1and HUVEC by TEM; Identification of ITGB1, ITGB2, ITGB3and CAV-1on THP-1, J774A.1and HUVEC by flow cytometry and western-blot; Detection of ITGB1, ITGB2, ITGB3and CAV-1on leptospire-contained vesicals in THP-1, J774A.1and HUVEC; ITGB1,ITGB2,ITGB3were inhibited by antibody and siRNA, CAV-1were inhibited by filipin and siRNA in THP-1, J774A.1and HUVEC, after infected with leptospires, fluorescence intensity of confocal microscopy were used to detect the intracelluar leptospires of each treatment; Phosphorylation of FAK and PI3K in THP-1, J774A.1and HUVEC after infected by leptospires; THP-1, J774A.1and HUVEC were treated with inhibitor of FAK and PI3K respectively, after infected with leptospires, fluorescence intensity of confocal microscopy were used to detect the intracelluar leptospires of each treatment; Observation of cytoskeleton rearrange (microfilament and microtubule) in THP-1, J774A.1and HUVEC after infected with leptospires; THP-1, J774A.1and HUVEC were treated with cytochalasin D and colchicine, fluorescence intensity of confocal microscopy were used to detect the intracelluar leptospires of each treatment; Observation of leptospires in HUVEC by confocal microscopy3D reconstruction of cell Z axis; Antibody blockage of ITGB1, ITGB2and ITGB3, filipin inhibition to HUVEC, detection of by direction transcytosis of leptospires in HUVEC; Colocalization of leptospires and CAV-1in HUVEC; Colocalization of leptospires and lysosomes in THP-1, J774A.1and HUVEC; Viability of intracellualr of leptospires in THP-1, J774A.1and HUVEC.Results:Leptospires adhere to FN, LN, COL1, COL2, COL3and COL4; Confocal microscopy showed that only FN was expressed on THP-1and J774A.1, FN,LN,COL3and COL4were expressed on HUVEC, FN expressed on THP-1and J774A.1were4.2×107and3.8×107per cell respectively, FN, LN, COL3and COL4expressed on HUVEC were1.543×107,1.23×106,2.73×106and3.14×106per cell respectively; Transmission electron microscopy showed leptospires were internalized by THP-1, J774A and HUVEC; ITGB1, ITGB2and ITGB3were expressed on THP-1and J774A.1, while ITGB1, ITGB3and CAV-1were expressed on HUVEC; ITGB1was presented on the leptospire-contained vesicles of THP-1and J774A.1while ITGB1and CAV-1were presented on vesicles of HUVEC; Inhibition of ITGB1reduced the internalized leptospires in THP-1and J774A.1, while inhibition of ITGB1and CAV-1reduced the internalized leptospires in HUVEC; In THP-1and J774A.1, phosphorylation of FAK increased while phosphorylation of PI3K had no significant change, in HUVEC, after infected by leptospires phosphorylation of PI3K increased while phosphorylation of FAK had no significant change. Confocal microscopy indicated FAK inhibitor reduced the internalization of leptospires in THP-1and J774A.1, PI3K inhibitor did not, while in HUVEC PI3K inhibitor reduced the internalized leptospires, FAK inhibitor did not. Microfilament but not microtubule mediate internalization of leptospires in THP-1, J774A.1and HUVEC; Leptospires transmigrate integrity monolayer of HUVEC, blockage by ITGB1, ITGB2and ITGB3antibody, filipin treated HUVEC decreased the transmigration of leptospires; Leptospires fused with lysosomes in HUVEC but not in THP-1and J774A.1; Intracellular leptospire viability in HUVEC were higher than that in THP-1and J774A.1.Conclusion:Adherence tests and adherence inhibition tests indicated leptospires to all six ECM molecules used in this study, FN, LN, COL1, COL2, COL3and COL4. During infection of leptospires to THP-1and J774A.1, leptospires adhered to ECM of cells trigged the activation of ITGB1and FAK, microfilament rearranged for endocytosis leptospires. In HUVEC, transcytosis of leptospires happened when leptospires adhered to ECM, ITGB1, CAV-1, PI3K and microfilament mediated the transcytosis of leptospires, colocalization of leptospires and caveolae marker CAV-1indicated transcytosis in HUVEC was caveolae-dependent which avoid fushion with lysosomes, the viability of intracellular leptospires in HUVEC were higher than that in THP-1and J774A.1.