ABSTRACT
Objective:To evaluate immune response of murine peritoneal macrophage challenging by methicillin-resistant S.aureus(MRSA)after pretreatment with Pam3Csk4(TLR2 agonist).Methods: Murine peritoneal macrophage was pretreated with Pam3Csk4(1 μg/ml).Following pretreatment 12 h later,heat-killed MRSA( HK-MRSA) was added and incubated for another 2 or 6 hours.The protein and mRNA level of TNF-α, IL-6 and IL-1 were determined by ELISA and Q-PCR, respectively.To estimate phagocytosis of macrophage,HK-MRSA/MSSA labeled with FITC( FITC-HK-MRSA/MSSA) were added to well and incubated for 30 min.After washing 5 times with PBS,intracellular FITC-HK-MRSA was detected by flow cytometry.To estimate antimicrobal activity of macrophage,live MRSA and MSSA were added to well and incubated at indication time,the CFU of s.aureus was estimated via a 10-fold serial dilution on agar media.cDNA was further quantitative assessed using primers for mouse FCR-Ⅰ,FCR-Ⅲ,CR-1,CR-3,iNOS and LL37 by Q-PCR .Results: Compared with saline-pretreated cell, the protein and mRNA level of TNF-α, IL-6 and IL-1 were markely reduced, respectively.However, both the phagocytosis and antimicrobal activity to S.aureus were significantly increased in macrophages pretreated with Pam3Csk4.Further study found that the macrophages had higher FCR-Ⅰ,FCR-Ⅲ,CR-1,CR-3,iNOS and LL37 expression at 6 h and 12 h post-stimulation Pam3Csk4.Conclusion: The results suggest that Pam3Csk4 could activate murine antimicrobal activity of peritoneal macrophage challenging by methicillin-resistant Saureus via increasing opsonophagocytosis in depended antibodies, complements manners.The results suggest Pam3Csk4 probably be a novel immunotherapy candidate against MRSA.