Pam3Csk4预处理增强巨噬细胞对耐甲氧西林金黄色葡萄球菌杀菌作用

Increasing antimicrobial activity of macrophage to methicillin resistant staphylococcus aureus via TLR2 agonist-Pam3Csk4

目的:初步探讨TLR2激动剂Pam3Csk4预处理后,小鼠腹腔巨噬细胞对耐甲氧西林金葡菌(Methicillin-resistant S.aureus,MRSA)的免疫反应性。方法:1μg/ml Pam3Csk4作用于鼠腹腔巨噬细胞,12 h后以热灭活耐甲氧西林金葡菌刺激细胞,ELISA和荧光定量PCR(Q-PCR)法分别检测培养细胞中TNF-α、IL-6和IL-1及mRNA含量,流式检测小鼠腹腔巨噬细胞对热灭活金葡菌的吞噬能力,平板计数法检测Pam3Csk4预处理巨噬细胞对金葡菌杀菌能力;Q-PCR法检测Pam3Csk4预处理巨噬细胞6 h和12 h后吞噬相关受体与补体受体、一氧化氮诱导合成酶(i NOS)及抗菌肽LL37基因表达。结果:金葡菌刺激后,Pam3Csk4预处理组TNF-α、IL-6、IL-1蛋白和基因水平均显著低于未处理组(P<0.05),但Pam3Csk4预处理组细胞对金葡菌吞噬和杀菌能力均显著增强(P<0.05),对于MRSA菌株,增强的杀菌能力在补体和抗体参与。进一步Q-PCR结果显示Pam3Csk4预处理巨噬细胞6 h和12 h后调理吞噬相关受体FCγRⅠ/Ⅲ与补体受体CR1/3表达均显著增强,i NOS和LL37基因表达也显著增加。结论:Pam3Csk4预处理小鼠腹腔巨噬细胞能增强其对金黄色葡萄球菌甲氧西林敏感和耐药菌株杀菌或抑菌能力,并降低其相应炎症反应,该现象可能与Pam3Csk4激活巨噬细胞吞噬相关受体以及i NOS和抗菌肽表达有关。

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. Howerer, 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 opsonophag0cytosis in depended antibodies, complements manners. The results suggest Pam3Csk4 probably be a novel immunotherapy candidate against MRSA.