| Background:Syphilis is a chronic and systemic sexually transmitted disease,caused by treponema pallidum.As the initiator and executor of the innate immune system,macrophage is the most important cell for Tp clearance.Both of autophagy and phagocytosis are highly conserved functions of cell,which plays an essential role in pathogen infections and immunity.However,the immune mechanism of autophagy and phagocytosis remains unclear in Tp infections.Therefore,this study aims to clarify the effect of Tp on macrophage autophagy and phagocytosis,analyze the effects of autophagy on macrophage phagocytosis and killing Tp,and further investigate the regulatory role of the NLRP3 inflammasome on macrophage autophagy and phagocytosis.Methods:Different concentrations(MOIs of 10:1、20:1、50:1 or 100:1)of Tp were co-incubated with macrophages for 12 h.Then,RT-PCR and western blot was used to detect the mRNA and protein levels of autophagy gene beclinl and LC3B,and mRNA levels of phagocytic receptors,FR and SR,distributed on the surface of cells.Also,indirect immunofluorescence assay(IFA)was used to detect the percentage of Tp positive macrophages and the mean fluorescence intensity of internalized Tp,explicating that the phagocytosis of macrophages.Cells treated with PBS were set as the control group.Then,different time periods(0、1、2、4、6、8、12 and 24 h)of Tp were co-incubated with macrophages,and the above indicators were analyzed.Next,macrophages were transfected with si-Beclin1 to inhibit cells autophagy,the levels of phagocytic receptors and the phagocytosis of macrophages was determined.The untransfected macrophages were set as the control group.Macrophages transfected with normal control was set as si-NC group.Meanwhile,the clearance of Tp in macrophages was further verified by animal experiments.Finally,cells were transfected with siNLRP3,and the role of NLRP3 inflammasome on macrophage autophagy and phagocytosis were determined.Results:Tp promoted macrophage autophagy in a dose-and time-dependent manner.The mRNA and protein levels of autophagy related gene beclin1 and LC3B were significantly increased at a MOI of 20:1(vs PBS,P<0.05),and peaked at a MOI of 100:1(vs PBS,P<0.05).And the mRNA and protein levels of beclin1 and LC3B increased over time and reached its maximal at 12 h(vs 0 h,P<0.05).Also,Tp enhanced the levels of macrophage phagocytic receptors,FR and SR.In addition,the percentage of Tp-positive macrophages and the mean fluorescence intensity(MFI)of internalized spirochetes increased in a dose-and time-dependent manner.Both indicators were increased at a MOI of 10:1(vs PBS,P<0.001),and peaked at a MOI of 100:1(vs PBS,P<0.001).Also,these indicators started to increase at 1 h(vs PBS,P<0.001),and reached maximal at 12 h and 24 h,respectively(vs PBS,P<0.001).Inhibition of autophagy,the levels of FR and SR were reduced to 40.19%and 43.60%,respectively(vs Control,P<0.001).The percentage of Tp-positive macrophages(70.93%vs 22.34%,P<0.001)and the MFI of internalized spirochetes(20.33 vs 9.62,P<0.001)were also decreased.Moreover,animal experiment results showed that inhibition of autophagy,the clearance of Tp in macrophage was decreased,proved by the 3.00 days in advance of lesions occurrence(14.55+/-1.99 days and 17.55+/-3.73 days),and the increased number of lesions(66.7%(20/30)and 36.7%(11/30),χ2=5.406,P=0.020).Furthermore,inhibition of the gene of NLRP3,the mRNA and protein levels of autophagy-related genes beclin1 and LC3B were decreased by 49.86%,43.02%and 22.31%,24.24%,respectively.Also,the percentage of Tp-positive macrophages(30.29%vs.70.53%,P<0.001)and MFI of internalized spirochetes(9.82 vs 19.33,P<0.001)were significantly decreased.Conclusions:Tp enhanced macrophages autophagy,increased the levels of phagocytic receptors,and promoted the phagocytosis of macrophages.Inhibition of autophagy decreased the phagocytosis and killing of Tp in macrophage.The levels of NLRP3 inflammasome regulated autophagy and phagocytosis of Tp in macrophage. |
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