| Objectives: Group A streptococcus (GAS), the most common bacterialpathogen of Gram-positive bacteria, can cause serious invasive infection,including mild infections such as pharyngitis, tonsillitis, etc. as well aspost-infection autoimmune diseases and highly lethal toxic disease such asrheumatic fever, rheumatic heart disease, autoimmune encephalopathy,psoriasis, glomerulonephritis, toxic shock syndrome, and scarlet fever, whichare a serious threat to human health. The body tries to remove bacteria andblock infection, while due to the strong ability of GAS immune escape, GASinfection manifests itself by severe and lingering disease and repeatedinfections. At present, some researches have shown that GAS escapes immuneattack in various ways. On the one hand, it depends on its virulence factor,including M protein, Fba and SpeB, etc. On the other hand, it relies on theinteraction with host cells to weaken or inhibit immune defense function forimmune escape. Innate immunity is the body’s first line of defense againstbacteria and the basis of specific immune. Macrophages, a kind of the mostimportant inherent immune cell, recognize and combine with bacteriapathogen associated molecular patterns by its pattern recognition receptors,were activated by signaling pathway for killing and clearing bacteria with avariety of mechanisms. GAS must challenge inherent immune cells in theprocess of immune escape to resist the immune clearance by various strategies.Negative regulators up-regulated in host cells is one of the important meansfor GAS immune escape. The NF-κB pathway is important to activatemacrophages, in which there are many immune negative regulatory factors,such as MyD88s, SHIP1, RP105, IRAK-M Tollip, A20, TRAILR, SOCS-1,NOD2, C5a, Stlr, SIGIRR and RIP3, etc. Their molecular regulatory mechanisms are not identical, but they all can negatively regulate the NF-κBsignal pathway. The previous work of our lab has confirmed that GAS caninduce high level immune negative regulatiory factor A20in macrophages, sothis research focused on components of GAS raising immune inhibitory factorA20and its mechanisms.A20is Zinc finger protein, also known as tumor necrosis factor alphainduced protein3(TNFAIP3) or tumor necrosis factor alpha induced proteinA20(TNFAIPA20). As a negative regulatory factor, A20can induceexpression in multiple tissue cells by a wide variety of stimuli including B cellsurface receptor CD40, TNF, IL-1, phorbol esters, hydrogen peroxide, bacteriaand its LPS, virus and its EBV-LMP1, etc.The adjustment function of A20shows to inhabit excessive release of TNF alpha, IL-1, IL-6, IL-8, intercellularadhesion molecules and other pro-inflammatory factors, and to control cellapoptosis and necrosis.A20makes use of its dual ubiquitin enzyme function to regulatesignaling pathway. In the cytoplasm, a variety of key proteins are modified byubiquitin via A20, then identified and degraded by proteasome. As a result,signaling pathway is suppressed, followed by decreased expression ofpro-inflammatory factors and increased expression of anti-inflammatoryfactors. Targeting the key substrates TRAF6, RIP1in the upstream, A20makes them degradation via Ubiquitylation, resulting in inhibition ofinflammatory pathways. The previous work of our lab has confirmed thatGAS can induce high level immune negative regulatory factor A20inmacrophages, however we don’t know clearly how A20is changing after theinteraction of GAS and macrophage. We also don’t know specificity therelated components of GAS that induce A20expression. This research willtarget to explore these problems.Methods:1. Detection of A20expression characteristics produced by RAW264.7cellsfollowing infection with GAS：The comparative kinetics of A20produced byRAW264.7cells following infection with GAS (MOI=10:1,50:1,100:1) were identified at0.5h,2h,4h,6h,8h,12h by Western blot.2. To study expression mechanisms of A20produced by RAW264.7cellsfollowing infection with GAS2.1The expression changes of TNF-α mRNA and protein produced byRAW264.7cells following infection with GAS at different times wereidentified by Real-time PCR and immunohistochemical assay.2.2After RAW264.7cells blocked by cycloheximide, the expression changesof TNF-α and A20in RAW264.7cells following infection with GAS weremeasured respectively by immunohistochemical assay or Western blot.2.3After RAW264.7cells blocked by anti-TNFR1, the expression changes ofA20in RAW264.7cells following infection with GAS were measured byWestern blot.2.4In gene knockout mouse MyD88-/BMDM cells following infection withGAS, A20expression changes were identified by Western blot.3. The gene mutant strains of GAS, i.e., SpeB-/GAS and M-/GAS wereconstructed. The homologous fragment suicide plasmids pFW5/△S peBandpFW5/△M were constructed, transformed, and then identified.4. In RAW264.7cells, comparisons of A20and its related molecule in NF-κBsignal pathway, between the stimulated groups of gene mutant strains and thestimulated group of wild type strain：4.1In RAW264.7cells following infection with wt GAS, SpeBˉ/-GAS, andM-/GAS, the expression changes of A20mRNA and protein，TRAF6and P65proteins in NF-κ B signal pathway were identified by Real-time PCR orWestern blot.4.2In RAW264.7cells following infection with wt GAS, SpeBˉ/-GAS, andM-/GAS, the expression changes of the pro-inflammatory cytokines (such asTNFα, IL-1β, IL-6) were identified by Real-time PCR.5. In the mouse BMDM cells following infection with wt GAS, SpeBˉ/-GAS,and M-/GAS, the expression changes of A20and the NF-κ B pathwaymolecules (TRAF6and P65) were identified by Western blot. Results:1.The expression characteristics of A20produced by RAW264.7cellsafter infection with GAS：The expression of A20maintained a high level from2h to12h after RAW264.7cells infected with low density GAS (MOI=10:1).With the cells infected with medium density GAS (MOI=50:1), A20also hada peak expression at6h, but maintained a shorter period than that in cellsinfected with low density GAS (MOI=10:1), while A20expression quantityincreased within2h in those infected with high density GAS（MOI=100:1）,it’s lower than that in those infected with the low-dose GAS.2. The relationship between A20expression and the TNF-α pathway: Theexpression levels of TNF-α produced by RAW264.7cells following infectionwith GAS were identified by Real-time PCR and immunohistochemicalanalysis. The results showed that inflammatory degree gradually increasedwith time passing (p<0.05). After RAW264.7cells blocked by cycloheximide,the TNF-α was detected by immunohistochemistry in the cells followinginfection with GAS. The results testified that TNF-α was indeed effectivelyblocked. The A20expression was decreased significantly in RAW264.7cellsfollowing infection with GAS after the cells blocked by10μg/mlcycloheximide, which showed that the expression of A20depended on theinflammatory factors. After RAW264.7cells blocked further by anti-TNFR1and then infected with GAS, the protein expression of A20in cells wassignificantly lower than that in those of positive control group, but higher thanthat in those of normal control group, all of which implied that A20waspartially induced through TNFR1signal pathway.3. Tested by Western blot, A20was no stimulating expression inMyD88-/BMDM cells from gene knockout mouse after infected with GAS,which suggested that the expression of A20depended on MyD88molecules.4. PFW5/△SpeBand pFW5/△Mwere successfully constructed, andthen, SpeBˉ/-GAS and Mˉ/-GAS were also constructed by the plasmidsstransformed into GAS through electroporation and identified. Theexpressions of A20mRNA and protein produced by RAW264.7cellsfollowing infection with wt GAS, SpeBˉ/-GAS and Mˉ/-GAS were identified respectively by Real-time PCR and Western blot. The results showed that theexpressions of A20mRNA and protein were both lower in Mˉ/-GASstimulated group than those in wt GAS stimulated group (p<0.01), but theywere higher in SpeBˉ/-GAS stimulated group than those in wt GAS stimulatedgroup (p<0.01).5.The signaling molecules expression changes of A20regulating pathwayin the process of the A20changes produced by RAW264.7cells followinginfection with wt GAS, Mˉ/-GAS, and SpeBˉ/-GAS: This research focused onthe expression differences of TRAF6(the substrate of A20), and P65(a NF-κB subunit). The kinetic analyses of TRAF6and P65in RAW264.7cellsfollowing infection with wt GAS, Mˉ/-GAS, SpeBˉ/-GAS at1h,2h,4h,6hand8h were identified by Western blot, the results of which were increasedover time in every group. These showed that inflammatory responses werestrengthened in all groups whether the components of GAS were missing ornot. Compared with wt GAS stimulated group, TRAF6and P65were higherin Mˉ/-GAS stimulated group, but they were lower in SpeBˉ/-GAS stimulatedgroup, the results were consistent with the changes of A20induced by mutantstrains.6.The expression changes of pro-inflammatory cytokines mRNAproduced by RAW264.7cells following infection with wt GAS, SpeBˉ/-GAS,and Mˉ/-GAS were identified by Real-time PCR. The mRNA levels of IL-1βand IL-6in Mˉ/-GAS stimulated group were significantly higher than that inwt GAS stimulated group (p<0.01), the comparison results had remarkabledifferences, but TNF-α mRNA level was lower than that in wt GASstimulation group. The levels of IL-1β, IL-6, and TNF-α mRNA inSpeBˉ/-GAS stimulation group were close to those in wt GAS stimulatedgroup. The cytokine changes were consistent with A20expression in the samegroup, showed a negative correlation.7. The expression changes of A20, TRAF6and P65produced by mouseBMDM cells following infection with wt GAS, Mˉ/-GAS, and SpeBˉ/-GASwere identified by Western blot, and the kinetic changes of A20, TRAF6, and P65were analyzed at2h,4h, and6h. The expression features and tendenciesof A20, TRAF6, and P65in BMDM cells were consistent with those inRAW264.7cells.Conclusions:1. The tested results of macrophages blocked by cycloheximide testifiedthat the expression of A20depended on cytokines, and another experimentresult of the cells blocked by anti-TNFR1proved the expression of A20depended partly on TNFR1pathway.2. The expression of A20depended partly on MyD88molecules.3. M protein could improve the expression of A20and contribute to GASimmune escape, the SpeB protein did not however. All results supportedabove conclusions proved by testing TRAF6, P65and cytokines in RAW264.7cells and in BMDM cells infected with GAS. |
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