Study On The Anti-MRSA Effect Of CP-PGN By MyD88-mediated Moderate Inflammatory Reaction

Objectives:In the previous study,the peptidoglycan of Corynebacterium Pyruviciproducens(CP-PGN),a TLR2 ligand,has been proven to be resistant to methicillin-resistant Staphylococcus aureus(MRSA)infection.This study focused on how CP-PGN exerts its anti-infective effect by regulating subsequent signaling of the TLR2 pathway.Methods:1.The model of blood infection and abdominal infection was established by injection of MRSA into the tail vein or intraperitoneal injection.Record the infection status of mice after pretreatment with CP-PGN.qRT-PCR detects changes of MyD88 mRNA level in the spleen,lung,liver and other organs.2.The mouse peritoneal primary macrophage was used made an infection model of MRSA in vitro.CP-PGN was used before and after infection of MRSA to simulate infection prevention model and infection treatment model.Western blot and ELISA method were used to detect MyD88 level and TNF-a,IL-6 level change after treatment with CP-PGN.3.Western blot and ELISA were used to detect the changes of MyD88 level and TNF-α and IL-6 levels in RAW264.7 after CP-PGN stimulation.The concentration and time dependence of CP-PGN on the MyD88 and cytokines were evaluated.At the same time,the CCK-8 method was used to detect changes in cell viability during the test.4.Western blot and ELISA method were used to detect the changes of MyD88 and cytokine TNF-a and IL-6 expression induced by CP-PGN before and after RAW264.7 MRSA infection.The prevention and treatment model of MRSA infection by CP-PGN was simulated at the cell level,explored the molecular mechanism and cytokine changes of TLR2-MyD88 induced by CP-PGN during anti-infective action.5.To explore the differences and associations between the action of CP-PGN and ST2825 during the regulation of MyD88,qRT-PCR was used to detect the changes of TNF-a and IL-6 mRNA levels induced by CP-PGN and MyD88 inhibitor ST2825 on RAW264.76.Western blot analysis of changes in TRIF levels induced by CP-PGN on RAW264.7,confirmed that whether CP-PGN anti-infective immune regulation mechanism involves TLR-non-MyD88-dependent pathway.7.To further clarify whether the basic level of MyD88 is involved in affecting the regulation of CP-PGN,after the expression of MyD88 in RAW264.7 was reduced by interfering RNA technology,the changes of MyD88 and IL-6 levels induced by CP-PGN were detected by Western blot and ELISA.Results:1.Pretreatment of M-PGN could aggravate MRSA infection,but CP-PGN greatly improved the survival in MRSA-infected mice no matter in blood infection model or abdominal infection model.The results of qRT-PCR showed that the level of MyD88 mRNA was significantly decreased in important organs such as spleen of slightly infected mice compared with severely infected mice,indicating that the moderate expression level of MyD88 affects the progress of infection,but whether this is related to CP-PGN Intervention related needs to be further determined.2.CP-PGN upregulated MyD88 expression in peritoneal macrophages in vitro,but the reverse order of CP-PGN and MRSA intervention unexpectedly downregulated the MyD88 protein level in peritoneal macrophages.3.In RAW264.7 cells,CP-PGN significantly down-regulated the expression of MyD88 following time lengthening and rising concentration of stimulation,but dose-and time-dependently promoted TNF-a and IL-6 secretion in RAW264.7 cells.Compared with other ligands of Toll like receptors(TLRs),MRSA,M-PGN,and TLR2 agonist Pam3CSK4,CP-PGN was most effective in inhibiting MyD88,and there was no significant difference in cell viability throughout the experiment.4.In the prevention and treatment model of CP-PGN anti-MRSA infection,Western blot results showed that CP-PGN can reduce the higher level of basal MyD88 expression in RAW264.7,and continue to maintain a low level after MRSA infection.CP-PGN acting after MRSA infection can reduce the level of MyD88 caused by infection and is accompanied by a fall in the level of TNF-a secretion.5.CP-PGN and MyD88 inhibitor ST2825 caused opposite changes of TNF-a and IL-6 mRNA levels in RAW264.7.ST2825 inhibited MyD88 homodimerization and could not completely reversed the elevated cytokine mRNA level caused by the CP-PGN,it is shown that the inhibition of MyD88 by CP-PGN is not through competitive binding domains.6.Western blot results showed that CP-PGN did not cause changes in TRIF levels in cell line RAW264.7,indicating that non-MyD88-dependent pathway was not involved in the immunomodulation of CP-PGN against MRSA infection.7.Interfering RNA reduced the basal expression level of MyD88 in RAW264.7,and the expression level of IL-6 was also decreased.The effect of CP-PGN did not cause significant changes in MyD88 protein levels,indicating that the basal expression level of MyD88 can directly affect the regulation of CP-PGN.Conclusion:We show that not all ligands of TLR2 could be used as anti-infective agents.Exactly,peptidoglycan derived from MRSA(M-PGN)aggravated serious MRSA infection.In addition,In the pro-infection prevention process before MRSA infection,CP-PGN inhibits the excessive elevation of MyD88 caused by subsequent MRSA infection by TLR2.During the treatment of infection after MRSA infection,CP-PGN was also able to reverse the hyper-adherence of MyD88 caused by MRSA infection by TLR2.In both models,CP-PGN was able to exhibit an excessive inflammatory response caused by infection.Furthermore,these data clearly demonstrate that such down-regulation of MyD88 is mainly dependent on the basal level of MyD88 at the CP-PGN time-dependently exposure,but more in-depth research is needed.Overall,this study found for the first time that the anti-infective effect of immunological adjuvants is closely related to MyD88-mediated moderate inflammatory response.