Group A Streptococcus Suppress Inflammatory Response In Macrophages By Up-regulation Negative Regulatory Factor A20

Objective: Group A streptococcus(GAS), is a Gram-positive bacteriumthat is responsible for a wide range of human infections, ranging fromautoimmunity diseases and uncomplicated infections of the throat or skin tosevere and potentially life-threatening invasive infections of streptococcaltoxic shock syndrome (STSS) and necrotising fasciitis (NF). Macrophages,one of the most important innate immune cell, that recognize and combinebacterial pathogen associated molecular pattern (PAMP) by patternrecognition receptor (PRR). TLRs as an important pattern recognition receptor,which combine with adaptor MyD88to activate TRAF6and form a complexwith IRAK in macrophages, When infection occurred, GAS was recognizedby TLRs，then activate TRAF6by MyD88to affect NF-κB. The importanceof NF-κB in the innate immune system has been evidenced that it couldregulate the expression of inflammatory genes. A20is Zinc finger protein,utilizes dual ubiquitin enzyme function to facilitate purpose proteins degradedby proteasome and restrict ubiquitin-dependent signals. It regulate substratesinclude TRAF6, RIP1, IKK(NEMO), Caspase8. The previous work of our labhas showed compared GAS with Escherichia coli (E.coli), GAS coulddown-regulate secretion of cytokines and up-regulate negative regulatoryfactor A20.1. exploring the relationships between suppressed inflammatoryresponse and high level expression of A20: examining cytokine, NF-κB,TRAF6and A20in macrophages infected with GAS or E.coli at differenttimes.2. A20SiRNA were transiently transfected to RAW264.7cells in thepresence of lipo2000and then specifically degraded A20mRNA by formingRNA-induced silencing complex (RISC). SiRNA leads gene silencing aftertranscription reducing the level expression of A20.3. After GAS infectedRAW264.7cells transfected by A20SiRNA, NF-κB, TRAF6and cytokines (IL-6, IL-1β, TNF-α, IL-10) were detected in order to offer some evidencesfor macrophages, which generated weak inflammatory factor reaction, withGAS infection.4. To further detect GAS-induced A20expression whetherdepends on the MyD88direct pathway signaling in macrophages or not,BMDM obtained from wild-type(C57BL/6) and MyD88-/-mice werestimulated with GAS or E.coli to detect the level expression of NF-κB,TRAF6, A20.Methods:1Utilizing Real-time PCR and CBA to analyzes the expression of thecytokines(IL-6，IL-1β, TNF-α, IL-10) in RAW264.7cells infected withGAS or E.coli at the various periods.2TRAF6, A20and nuclear factor NF-κB expression in RAW264.7cellsand C57BL/6mice BMDM cells were analyzed after infection with GAS orE.coli by Western blot at different time points.3NF-κB, TRAF6, A20and cytokines (IL-6, IL-1β, TNF-α, IL-10) weredetected by Western blot, BCA and Real-time PCR, after RAW264.7cellswere transfected by A20SiRNA and then stimulated by GAS that specifically.4BMDM macrophages were obtained from wild-type(C57BL/6) andMyD88-/-mice were stimulated with GAS or E.coli, detecting NF-κB，TRAF6，A20by Western blot.Results:1The levels of the cytokines detected by Real-time PCR: compared withE.coli, RAW264.7cells infected with GAS, induced the less levels of thecytokines IL-1β, IL-6and TNF-α and even higher levels of IL-10within7hours. Meanwhile, the mRNA of IL-1β and TNF-α reached to peak at3-5hours and IL-10peaked after7hours.2RAW264.7cells infected with GAS always induced the less levels ofthe cytokines than E.coli by CBA. The trend of cytokines changes insupernatant supported with mRNA level above.3Analyzing the expression of NF-κB, TRAF6, and A20in RAW264.7cells infected with GAS or E.coli by Western blot. A20expression was enhanced after GAS infection within2hours and peaked at6-8hours, whereasthe peak levels of A20in E.coli-treated cells was delayed (elevated at4h andpeaked at6-8h). At the same time, the more of the A20expression were, theless of NF-κB, TRAF6comparied with E.coli-induced infection.In GAS-infected BMDMs, the trend of NF-κB, TRAF6, A20changes aresimilar with RAW264.7cells, furthermore, the A20expression was strongerthan RAW264.7.4RAW264.7cells pretreated by A20SiRNA, were infected by GAS andWestern blot analysis showed that A20expression were not detected anymore,while the expression of P-P65and TRAF6protein were increased accordingly.5To further assess the role of MyD88in GAS-induced A20expression,BMDM cells were obtained from wild-type(C57BL/6) and MyD88-/-miceand stimulated with GAS as indicated. As shown the level of A20wasabolished completely in MyD88-/-BMDMs compared with WT controls.Conclusions:1Compared with E.coli, GAS as a Gram-positive bacterium that leadmacrophages for milder and more retardant inflammation.2Negative regulatory factor A20collaborate with TRA6restrictingubiquitin-dependent signals to down-regulated NF-κB Mediated inflammation.3The up-regulation of A20in macrophages infected with GAS dependson the induction of MyD88.