| BackgroundBrucellosis is an zoonotic infectious disease caused by Brucella.Brucella is a gram-negative and intracellular pathogenic bacteria,which causes serious systemic infection in livestock,and damage to human health.After entering chronic infection,Brucella often causes multiple organs and systems damage.Lipopolysaccharide(LPS)as a classic virulence factor of gram-negative bacteria,however,there has reported shows that the LPS of Brucella is different from other gram negative bacteria such as Escherichia coli.It is not the main virulence factor for Brucella.A few reports shows that outer membrane proteins may have the effect of virulence factor,such as lipoprotein causing host inflammation.Our previous studies confirmed that lipoprotein L19 or L16 co-incubation with THP-1 cells induces the secretion of inflammatory factors,such as TNF-?,IL-1? and IL-6,but the secretion of IL-18 was only detection by L19 group.The inflammatory response induced by L19 and L16 showed significant difference.Therefore,based on the important findings of previous studies,this study will continue to focus on the virulence and molecular mechanism of outer membrane lipoproteins OMP19 and OMP16,so as to provide theoretical basis for the discovery and identification of new virulence factors of Brucella.ObjectiveTo study the interaction mechanism between lipoproteins 19 or 16 and bacteria susceptible host cells,reveal the molecular mechanism of L19 and L16 differentially induced IL-18 secretion and the correlation with the occurrence and development of Brucella infectious inflammation.To reveal the effect mechanism of lipoprotein L16 induced cell pyroptosis,to analyze the value of Brucella lipoprotein as a virulence factor,to provide molecular modification target for the development of brucellosis vaccine,and provide a new research idea for the pathogenesis of multiple organ injury caused by Brucella infection.MethodsIn this study,activated THP-1 cells was induced by Brucella outer membrane lipoproteins L19,L16 or their un-lipidated U19 and U16 of 5 5 ?g/ml,respectively.Various experiments were detected to the inflammatory cytokine secretion,programmed cell death and other indicators,to identify the pathway and key molecules interacting with lipoprotein.The main experiments are as follows:1.Cytotoxicity test.THP-1 cells which activated by PMA and induced by recombinant proteins L19,L16 or LPS,observe and record the morphological changes of the cells through microscope,detect the cells viability through the CCK-8 assay,lactate dehydrogenase(LDH)release rate analysis cell death rate,detect of related indicators after different proteins stimulated,and then analysis the toxicity of lipoprotein to THP-1 cells.2.L19 and L16 differentially induced IL-18 secretion.L19 and L16 were incubated with PMA-activated THP-1 cells,respectively,and the relative light unit(RLU)was measured to detect caspase-1/3/8 activity.On the other hand,caspase-3 inhibitors were used to detect the expression of IL-18 in cells lysate and culture supernatant by Western blot(WB).To further identify the difference IL-18 secretion induced by L19 or L16 duo to the caspase-3 activity.3.The molecular mechanism of lipoprotein induced pyroptosis.Annexin V and 7-AAD double staining flow cytometry detects the mode of cell death,WB and real-time fluorescent quantitative PCR(RT-qPCR)were used to determine gene transcription,translation,and phosphorylation levels.To observe the executive protein expression in pyroptosis and inflammatory pathway,and to further identify the key role of protein by gene silence.Results1.Either L19,L16 or both(5 ?g/mL each)induced significantly higher secretion levels of TNF-?,IL-6 and IL-1? in cell cultures.L19 stimulated high secretion of IL-18 in the supernatant of THP-1 cells,while low secretion of IL-18 was also detected from L19 and L16 co-induction2.L19 and L16 induced pro-inflammatory cytokines secretion mainly through P38 MAPK signaling pathway3.L16 induced cells pyroptosis:Annexin V and 7-AAD double staining flow cytometry results showed that THP-1 cells was induced by L16 for 24 h.Annexin V and 7-AAD double-positive cells is as high as 33%,and the release rate of LDH was as high as 69%which is significantly higher than negative control(NC)and L19 control group(P<0.01).4.L16 down-regulates the expression of inflammasome NLRP3:After activated THP-1 cells were incubated with L19 or L16 for 4 h,the expression of NLRP3 and activated Caspase-1(P20)were down-regulated in L16 stimulated group,but could up-regulates in L19 stimulated group.5.L16 induces the activation of caspase-8/3 and cleaved GSDME:L19 or L16 were incubated with activated THP-1 cells for 4 h,24 h and 48 h,respectively.RLU assay was used to detect the activity of caspase-1/3/8.The results showed that L16 could significantly activate caspase-8 after 4 h,and could significantly activate caspase-3 after 24 h,and WB could detect the activated fragment of caspase-8/3 in cells lysates of the L16-stimulated group,P18 and P17,respectively.RT-qPCR results showed that the level of GSDME mRNA was significantly increased in L16 stimulated group(P<0.01)and the expression of GSDME-N could be detected in the L16 stimulated group.L16 was added after caspase-3 siRNA was transferred into THP-1 cells 72 h and further stimulated by L16 for 24 h.WB shows that activated caspase-3(P17)and GSDME-N were significantly reduced.6.L16 negatively regulates IL-18 through activated caspase-3:Compared with L16 stimulation group,the activity of caspase-3 was significantly down-regulated in L16 and AC-DEVD-CHO co-induced group(.P<0.001)and delayed pro-IL-18 cutting.WB results showed that after added inhibitors,the 16 kD and 9-6 kD fragments produced by pro-IL-18 cleavage were only present in the supernatant and no form of pro-IL-18 was found in cells lysate.7.L19 up-regulates the phosphorylation of inhibitor of apoptosis protein(XIAP):Activated THP-1 cells was induced by L19 or L16 for 24 h5 phosphorylated XIAP(P-XIAP)was only detected in L19 stimulated group.THP-1 cells were transfected with XIAP siRNA and then stimulated by L19 for 24 h.WB test results showed that P-XIAP was not detected,but the expression of activated caspase-3(P17)could be detected.8.Co-incubation of L19 and L16 could affect each other's virulence;When THP-1 cells were incubated with both L19 and L16,activated IL-18 secretion could be detected like L19 and pyroptosis in THP-1 cell was leaded like L16.9.Lipoprotein has a toxic effect on PBMCs:PBMCs incubated with L19,L16 or both of them showed similar responses in THP-1 cells.Conclusion1.The difference between L16 and L19 induced host inflammatory response in a caspase-3-dependent manner.L16 induces host inflammatory response by caspase-3 activation rather than classical inflammatory NLRP3/caspase-1.L16 can down-regulate NLRP3 expression to inhibit caspase-1 activity,while activated caspase-8 and induced caspase-3 activated.On the other hand,L16 down-regulates XIAP phosphorylation and promotes caspase-3 self-cleavage and activation,and production of a large number of inactive IL-18(15,16 kD)from activated IL-18 or pro-IL-18.In contrast,L19 promotes caspase-1 activation by up-regulation of NLRP3 expression,leading to an inflammatory response that relies on the classical inflammatory NLRP3/caspase-1,during which pro-IL-18 is cleavaged into IL-18(18 kD)by activated caspase-1.Moreover,p-XIAP antagonized the cleavage activity of caspase-3 and activated IL-18 was secreted.Therefore,L16 negatively regulates IL-18 secretion by activating caspase-3.2.L16 induces host cells executed pyroptosis mediated by caspase-3/GSDME.L16 activated caspase-8 and further activated caspase-3 and cleavaged GSDME,resulting in GSDME-N(the activated product of GSDME cleavage),which performs cell pyroptosis and causes inflammation. |
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