Study On The Pattern Recognition And Response Effect Of Mast Cell To B.suisin Vitro

Objective:To investigate the pattern recognition and immune response of mast cell to brucella,thus explain mast cell recognition molecular mechanism to brucella, and explore the newway and new method of the treatment to brucellosis and vaccine development.Methods:1.Mast cells were infected by B.suis S2, B.suis WboA-S2and escherichia coli according toMOI50:1.The supernatants were collected at1h,12h and24h post-infection, and the contentsof IL-6, TNF-α, LT, IL-1β, histamine, tryptase, IL-12, IFN-γ were detected by ELISA withnomal mast cells as the control group.2. The mRNAexpressions of TLR2,48,9of mast cells were detected by RT-PCR.3. Mast cells were infected by B.suis S2, B.suis WboA-S2and escherichia coli accordingto MOI50:1. Mast cells were collected at1h,12h and24h post-infection, and the mRNAexpressions of TLR4,8,9were detected by RT-PCR.4. TLR4was inhibibited by RNAi technology with no inhibition group as the controlgroup, and inhibition effect was detected by RT-PCR and flow cytometry.5. TLR4RNAi groups and TLR4groups were infected by B.suis S2, B.suis WboA-S2andescherichia coli according to MOI50:1.The culture supernatants were collected at1h,12h and24h post-infection, and the contents of IL-6, TNF-α, LT,histamine were detected by ELISA.Results:1. The supernatants of IL-6was obviously higher than the nomal groups(P<0.05) at1h,12h and24h post-infection by B.suis S2, B.suis WboA-S2and escherichia coli,and thesupernatants of IL-6in the same infection groups step by step increased. The supernatants ofIL-6of B.suis S2groups had no statistical significance with the supernatants of IL-6of B.suisWboA-S2groups(P﹥0.05).2. The supernatants of TNF-α had no statistical significance with the nomal groups(P﹥0.05) at1h post-infection by B.suis S2, B.suis WboA-S2and escherichia coli.The supernatantsof TNF-α were higher than the nomal group(P<0.05) at12h,24h post-infection.The supernatants of TNF-α of B.suis S2groups had no statistical significance with thesupernatants of TNF-α of B.suis WboA-S2groups(P﹥0.05).3. The supernatants of LT were higher than nomal groups (P<0.05) at1h,12h and24hpost-infection by B.suis S2, B.suis WboA-S2and escherichia coli. The supernatants of LT inthe same infection groups at different times had no change(P﹥0.05).The supernatants of LTof B.suis S2groups had no statistical significance with the supernatants of LT of B.suisWboA-S2groups(P﹥0.05).4. The supernatants of histamine were higher than the nomal groups(P<0.05) at1h,12hand24h post-infection by B.suis S2, B.suis WboA-S2and escherichia coli.The supernatants ofhistamine rised,then reduced,again rised after mast cells were infected by B.suis S2,escherichia coli.The supernatants of histamine did not obviously change at1h,12,24hpost-infection by B.suis WboA-S2.The supernatants of histamine of B.suis S2groups werelower than the supernatants of histamine of B.suis WboA-S2groups (P﹤0.05),5. IL-1β,tryptase,IL-12and IFN-γwere not detected at all infection times by B.suis S2,B.suis WboA-S2and escherichia coli.6. The high mRNA expressions of TLR4,TLR9and the low mRNA expression of TLR8were detected in mast cells.It is noteworthy that the mRNA expressions of TLR2were notdetected in mast cells P815.7. The mRNA expression of TLR4changed at12h,24h post-infection by B.suis S2, B.suisWboA-S2and escherichia coli. The mRNA expression of TLR4step by step enhanced aftermast cells were infected by B.suis S2. The mRNA expression of TLR4weakened at12hpost-infection,but enhanced at24h post-infection by B.suis WboA-S2. The mRNA expressionof TLR4obviously enhanced at12h post-infection,but weakened at24h post-infection byescherichia coli.8. The mRNA expression of TLR9changed at12h,24h post-infection by B.suis S2,B.suis WboA-S2and escherichia coli. The mRNA expression of TLR9enhanced at12hpost-infection, but weakened at24h post-infection by B.suis S2.The mRNA expression ofTLR9did not change at12h post-infection,and enhanced at24h post-infection by B.suisWboA-S2.The mRNA expression of TLR9obviously weakened at12h post-infection,butobviously enhanced at24h post-infection by escherichia coli. 9. The mRNA expression of TLR8did not change at12h,24h post-infection by B.suis S2,B.suis WboA-S2and escherichia coli.10. TLR4RNAi condition was decided and optimized: per well had1μl transfectionreagent,6pmol TLR4siRNA,1×105mast cells in24well-plate.In this condition,the inhibitionefficiency of TLR4at the mRNAand protein level was highest,it could reach50%.11. The supernatants of IL-6of TLR4RNAi groups were obviously higher than thesupernatants of IL-6of correspondly TLR4groups(P<0.05) after mast cells were infected byB.suis S2, B.suis WboA-S2and escherichia coli.The supernatants of both TNF-α and LT had nostatistical significance with the supernatants of TNF-α ang LT of correspondly TLR4groups(P﹥0.05).12. The supernatants of histamine of TLR4RNAi groups had statistical significance withthe supernatants of histamine of correspondly TLR4groups(P<0.05).The supernatants ofhistamine of TLR4RNAi groups were lower than the supernatants of histamine ofcorrespondly TLR4groups at1h post-infection, but higher than the supernatants of histamineof correspondly TLR4groups at12h post-infection,then again lower than the supernatants ofhistamine of correspondly TLR4groups at24h post-infection by B.suis S2,escherichia coli.The supernatants of histamine of TLR4RNAi groups were lower than the supernatants ofhistamine of correspondly TLR4groups at1h post-infection, but higher than the supernatantsof histamine of correspondly TLR4groups at12h,24h post-infection by B.suis WboA-S2.Conclusion：1. The infection of B.suis S2, B.suis WboA-S2and escherichia coli to mast cells couldstimulate mast cells release IL-6,TNF-α, LT and histamine, but did not releasetryptase, IL-1β,IL-12and IFN-γ;2. The production of WboA gene was the main pattern ligand of stimulating mast cellsrelease histamine;3. The high mRNA expressions of TLR4,TLR9and the low mRNA expression of TLR8were detected in mast cells.The mRNA expression of TLR2was not detected in mastcells P815;4. TLR4and TLR9participated in the recognisition of mast cells to B.suis S2, B.suisWboA-S2,escherichia coli,but TLR8had no obvious function in recognizingBrucella; 5. TLR4was the main pattern recognition receptor of mast cells in the release of IL-6and histamine infected by brucella.The release processes of IL-6of mast cellsinfected by B.suis WboA-S2and B.suis S2were similer,thus TLR4did not onlyrecognize LPS0f brucella,but also could recognize else bacterial proteins tostimulate mast cells release IL-6. However, the release process of TNF-α and LT ofmast cells infected by brucella had nothing to do with pattern recognition process ofTLR4.