Treponema Pallidum Delays The Apoptosis Of Human Polymorphonuclear Neutrophils Through The Intrinsic And Extrinsic Pathways

Objective: Treponema pallidum is a “stealth pathogen” responsible for infectious sexually transmitted diseases.Although neutrophils are usually present in skin lesions of early syphilis,the role of these cells in T.pallidum infection has received almost no attention.Neutrophils are short-lived cells that undergo spontaneous apoptosis,and phagocytosis usually accelerates this process.Taking human polymorphonuclear neutrophils(hPMNs)as the research object,this study preliminarily investigated the interaction between neutrophils and T.pallidum in vitro,exploring cell phagocytosis and apoptosis.Methods: 1.T.pallidum was injected into the rabbit’s testicles.The bacteria were extracted after they proliferated in intratesticular.The morphology of T.pallidum was examined by silver staining or dark-field microscopy.2.Human neutrophils were isolated from the whole blood of healthy adult volunteers.hPMNs were purified by Neutrophil Isolation Kit.The purity of hPMNs was assessed by using fluorescent dyes.Using a Neubauer-improved counting chamber to determine the number of cells.The viability of cells was assessed by trypan blue dye membrane exclusion method.3.After Neutrophils were incubated with Tp for different time points,immunofluorescence was used to observe the phagocytosis of T.pallidum by hPMNs.4.hPMNs were incubated with Tp for the indicated time and the apoptotic cells were quantified by flow cytometry.hPMNs were stained with Giemsa staining solution and the apoptotic change in nuclear morphology was quantified using light microscopy.The nuclear DNA fragmentation was detected by modified TUNEL assay.5.After T.pallidum stimulated hPMNs,the expression of cleaved caspase-3,cleaved caspase-8 and cleaved caspase-9 at the translation level were analyzed by western blotting,the caspase-Glo 3/7,8,and 9 luminescent assay kits were responsible for detecting the enzyme activities of caspase.6.After T.pallidum pre-infection with hPMNs,extrinsic apoptotic stimulation anti-Fas Ig M or intrinsic apoptotic pathway stimulation Staurosporine(STS)was added respectively.Apoptosis was assessed by flow cytometry.Levels of cleaved caspase-3protein were evaluated by western blotting.The enzyme activity detection kit analyzed the activity of caspase-3,caspase-8 and caspase-9.7.The cells were stimulated with heat-killed T.pallidum to determine the effect of dead bacteria on hPMNs apoptosis by flow cytometry.8.hPMNs were infected with T.pallidum and the supernatants were harvested.The IL-8concentration in supernatants was determined by ELISA.In some tests,hPMNs were cultured in the supernatant of T.pallidum-hPMNs.The apoptosis rate was analyzed by flow cytometry.Results: 1.By fluorescence microscopy,it was observed that hPMNs were able to phagocytose T.pallidum in vitro after hPMNs were infected with T.pallidum.Autologous human serum opsonization promoted hPMNs to take up T.pallidum.2.T.pallidum inhibited hPMNs apoptosis in a dose-and time-dependent manner,and the cells were unaffected by opsonization.3.TUNEL assay results showed that the number of TUNEL-positive cells in the T.pallidum-stimulated group decreased significantly compared with the control group.The results of Giemsa staining showed that the number of cells with apoptotic nuclei decreased after challenge by T.pallidum.4.Levels of cleaved caspase-3,cleaved caspase-8 and cleaved caspase-9 were reduced in hPMNs infected with T.pallidum.Consistent with the western blotting results,the activities of caspase-3,caspase-8 and caspase-9 were reduced significantly.5.After hPMNs were treated with anti-Fas immunoglobulin(Ig)M or staurosporine(STS),the protein expressions of cleaved caspase-8 and cleaved caspase-9 were up-regulated respectively,and the activities of caspase-8 and caspase-9 were significantly increased.Moreover,The protein expression of cleaved caspase-3 protein expression and the enzyme activity of caspase-3 were up-regulated by both apoptotic stimulations,which in turn promoted apoptosis.In contrast,T.pallidum-infected hPMNs were protected from STS-or anti-Fas-induced apoptosis.6.Spontaneous apoptosis of hPMNs is inhibited by heat-killed T.pallidum.7.This research measured the amount of IL-8 produced by hPMNs after T.pallidum infection.T.pallidum induced IL-8 secretion from hPMNs in a time-and dose-dependent manner,and it is independent of opsonization and bacterial viability.8.The apoptosis rate of the hPMNs treated with the supernatant of T.pallidum-hPMNs was significantly decreased,while the depletion of IL-8 from the supernatant restored the hPMNs apoptosis rate.Conclusions: 1.Serum opsonization promoted hPMNs to take up T.pallidum.2.T.pallidum could inhibit hPMNs apoptosis through intrinsic and extrinsic pathways.3.T.pallidum induced production of IL-8 by hPMNs,which is the potential mechanism for prolonging the lifespan of hPMNs.