Pam3CSK4 Enhances The Antibacterial Functions Of GM-CSF Induced Neutrophils To Methicillin-Resistant Staphylococcus Aureus

Objective:Pam3CSK4 is the synthetic Toll-like receptor 2(TLR2)ligand,which can upregulate neutrophil function through interacting TLR2.In this study,we investigated the effect of Pam3CSK4 on GM-CSF induced neutrophils,in term of anti-bactericidal function to the methicillin resistant Staphylococcus aureus(MRSA).We also dissected themechanism underlying the synergy between Pam3CSD4 and GM-CSF induced neutrophils.This study provide deeper insight into the prevention and treatment of MRSA using immunotherapy.Methods:1.Neutrophils were induced by GM-CSF and pretreated with Pam3CSK4 for 24 h and 48 h,respectively.The phagocytosis capacity was determined by the FITC labeled heat killed methicillin-resistant Staphylococcus aureus(HK-MRSA)or Escherichia coli(HK-E.coli),and was detected by flow cytometry.Agar plate counting method was used to detected the killing ability of neutrophils which were pretreated with Pam3CSK4 for 24 h and 48 h before being challenged by MRSA or E.coli.2.Transwell experiment was used to detected the neutrophils chemotaxis in GMCSF induced Pam3CSK4 after pretreatment for 24 h and 48 h.3.GM-CSF induced neutrophils were pretreated with Pam3CSK4 for 6h or 12 h before challenged by HK-MRSA.The q-PCR was used to detect the expression of inflammation related genes(IL-1?,IL-6,TNF-?,IL-10)and oxidative stress genes(COX2,iNOS),as well as antimicrobial peptides LL37.ELISA was used to detect the production of cytokines including IL-1?,IL-6,IL-10,TNF-?.4.The concentration of lactoferrin(LTF)in GM-CSF induced neutrophils and neutrophils respiratory burst was tested by ELISA and flow cytometry,after pretreatment of varying concentrations of Pam3CSK4.5.Western Blot was used to detect the phosphorylation levels of p38 MAPK,AKT,and ERK in neutrophils pretreated with Pam3CSK4.6.The p38 MAPK inhibitor(SB202190),the ERK inhibitor(PD98059),the JNK inhibitor(SP600125),and the PI3 K inhibitor(LY294002)were given to neutrophils before treatment with Pam3CSK4,and then the phagocytosis,respiratory and production of lactoferrin to HK-MRSA was detect by flow cytometry.Results:1.The phagocytosis and bacteriostasis to HK-MRSA of neutrophils were significantly increased after treatment with Pam3CSK4,whereas no significant difference was observed in Escherichia coli.2.Neutrophils pretreated with Pam3CSK4 enhanced the chemotaxis to NFormyl-Met-Leu-Phe(fMLP).3.Pam3CSK4 pretreated neutrophils upregulated the genes expression of IL-1?,IL-6,TNF-?,IL-10,iNOS,COX2 and LL37.In addition,the secretion of IL-6,IL-10,TNF-? was also enhanced after pretreatment with Pam3CSK4.4.The expression of lactoferrin and respiratory burst was increased by the pretreatment of Pam3CSK4 in a dose-dependent manner.5.Neutrophils pretreated with Pam3CSK4 increased the phosphorylation of p38 MAPK and AKT in a time and dose-dependent manner at the protein level as detected by Western Blot.However,there was no significantly difference in the phosphorylation level of ERK after pretreatment with Pam3CSK4.6.SB202190 and LY294002 significantly blocked the phagocytosis of neutrophils induced by Pam3CSK4 pretreatment in HK-MRSA,and the respiratory burst function was significantly blocked by SB202190,LY294002 and PD98059.All of the SB202190,LY294002,PD98059 and SP600125 inhibit the production of neutrophil lactoferrin mediated by Pam3CSK4.Conclusions:1.Pretreatment enhanced the killing function to MRSA by GM-CSF induced neutrophils.Pretreatment with with Pam3CSK4,also upregulated the phagocytosis,chemotaxis,increased release of lactoferrin,and enhanced the respiratory burst function to HK-MRSA.2.Pam3CSK4 enhancing anti-bactericidal function by GM-CSF induced neutrophil is associateed with the upregulation of the phosphorylation levels of p38 MAPK and AKT in neutrophils.