Death Of Human Mononuclear Cells And Secretion Of Interleukin-8 Induced By Outer Membrane Protein Tp92 Of Treponema Pallidum

Background and objective The innate immune response system plays an important role in the early anti-Treponema pallidum infection,but syphilis patients often have no obvious symptoms at the beginning of infection,suggesting that Treponema pallidum may have some mechanisms for immune escape.The traditional view has always believed that Treponema pallidum mainly relies on mutations in the bacterial surface antigens to evade the innate immune response system of the host.Tp92 is the first outer membrane protein which has been screened out.Some researchers compared the genome of the pathogenic Treponema pallidum strains with non-pathogenic strains,suggesting that the gene of the protein may relate to the pathogenesis of Treponema pallidum,but we know little about its function.In previous experiments,Tp92 was found to have the ability to induce the death of innate immune cells(monocytes).The aim of this study was to further explore the effect of Tp92 on monocyte death and cytokine secretion,and the main signal pathways involved in transduction.Methods and results1.Tp92 protein induces the death of THP-1 cells.After Tp92 protein stimulate the THP-1 cell.The AO/EB staining was performed respectively and the cells membrane were observed,and the nuclei were stained with Hoechst33342 dye and observed.CCK-8 assay was used to determine the total death rate of THP-1 cells.The results showed that damages of cell membrane and nucleus,even the total cell death rate,were all intensified along following the ncreases of concentration and treatment duration of Tp92.2.Tp92 protein induces atypical pyroptosis of THP-1 cells via pro-caspase-1 pathway.After stimulating the THP-1 cell with Tp92 protein,Caspase-1 activity was inspected by caspase-1 kit;Caspase-1,pro-caspase-1and GSDMD protein expression were determined by Western blotting;The concentrations of IL-1? and IL-18 were measured by ELISA.Pretreat the cells with caspase-1 specific inhibitor VX-765 or caspase inhibitor Z-VAD-FMK,then detect release rate of LDH,total cell death rate and caspase-1 activity.The results showed that Tp9 up-regulated the expression of pyroptotic molecules,but Tp92 could not up-regulate the levels of IL-1? and IL-18,and caspase-1 inhibitors inhibited pyroptosis.3.Tp92 protein results in apoptosis of THP-1 cells via RIPK1 / caspase-8 /caspase-3 pathway.Effect of Tp92 protein on apoptosis of THP-1 cells was determined by flow cytometry.Effect of Tp92 protein on activities of caspase-3,8 and 9.Cells were pretreated with caspase-3 inhibitor or caspase-8 inhibitor.Treated with Tp92 before measuring apoptotic rate,JC-1,reactive oxygen species(ROS)and the activities of caspase-3 and 8.Effect of RIPK1 inhibitor Necrostatin-1 on apoptosis and caspase activity.The results showed that Tp92 induced apoptosis of THP-1.Tp92 up-regulated the activity of apoptosis-related molecules.Caspase-3 and caspase-8 inhibited the apoptosis of THP-1 cells,and Tp92 regulated the apoptosis of THP-1 cells by RIPK1.Tp92 can regulate the apoptosis of THP-1 through mitochondrial pathway.Tp92 up-regulates the level of ROS in THP-1 cells.4.Tp92 mediates THP-1 cell death by recognizing CD14 and / or TLR2 on cell surface.THP-1 cells were pretreated with TNFR1,Fas L,TLR4,CD14 or TLR2 neutralizing antibodies.The cells were treated with Tp92,and CCK-8assay was performed to determine total cell death rate.Then THP-1 cells were pretreated with interfering plasmids of TLR2 or TLR4,the cells were treated with Tp92,measured total cell death rate with CCK-8 assay,inspected expressions of TLR2 and TLR4 proteins by Western blotting.The results showed that CD14 and TLR2 neutralizing antibody and TLR2 interference plasmid can effectively reduce the cell death rate.5.Tp92 induces IL-8 secretion from THP-1 cells via NF-?B.Effect on the secretion of cytokines was measured by ELISA kit after THP-1 cells was treated by Tp92.Concentration of secreted IL-8 was tested by ELISA and NF-?B protein expression was determined by Western blotting after the cells was pretreated by NF-?B inhibitor QNZ.The results showed that Tp92 up-regulated the level of IL-8 and NF-?B protein level,NF-?B inhibitor can down-regulate the level of IL-8 and NF-?B protein levels.6.Tp92 protein mediates the secretion of IL-8 by recognizing CD14 and / or TLR2 on the surface of THP-1 cells.Measure concentration of secreted IL-8through ELISA after the cells were treated by neutralizing antibodies or pretreated with CD14,TLR2 or TLR4 interfering plasmids.The results showed that only CD14 and TLR2 neutralizing antibody and TLR2 interference plasmid can effectively reduce the secretion of IL-8.7.Tp92 reduces the number of monocytes in PBMCs,and increases IL-8secretion by PBMCs.The PBMCs were treated with Tp92,and then incubated with FITC-conjugated anti-CD14,CD3 or HLA-DR antibody before flow cytometry.The cells were treated with Tp92,and then incubated with FITC-conjugated anti-CD14 antibody before flow cytometry.Concentration of secreted IL-8 by PBMCs determined by ELISA.The results showed that Tp92 only induced mononuclear cell death in PBMCs and induced IL-8 secretion of PBMCs.Conclusion1.Tp92 induced cell death through CD14 and / or TLR2 on the surface of monocytes.The death were mainly composed of pyroptosis mediated by pro-caspase-1 pathway and apoptosis by RIPK1 / caspase-8 / caspase-3 pathway mediated.2.Tp92 induces cells secreting IL-8 by the NF-?B pathway via CD14 and / or TLR2 on the surface of monocytes.