Department of Physiology, Immunology Laboratory, University of Calcutta, University Colleges of Science and Technology, 92 APC Road, Calcutta, 700009, West Bengal, India.
Department of Physiology, Immunology Laboratory, University of Calcutta, University Colleges of Science and Technology, 92 APC Road, Calcutta, 700009, West Bengal, India.
Microb Pathog. 2023 Oct;183:106294. doi: 10.1016/j.micpath.2023.106294. Epub 2023 Aug 9.
The CXCL8/CXCR1 axis in conjoint with the free radicals and anti-oxidants dictates the severity of inflammation caused by the bacteria, Staphylococcus aureus. S.aureus mediated inflammatory processes is regulated by NF-κB and its product, iNOS. The objective of this study was to examine the effects of inhibition of NF-κB and iNOS on CXCL8/CXCR1, alteration in M1/M2 polarization of macrophages and associated inflammatory responses during S.aureus infection in vitro. For this, the murine peritoneal macrophages were pretreated with NF-κB inhibitor, Pyrrolidine dithiocarbamate (PDTC) and iNOS inhibitor, L-N-monomethyl arginine (LNMMA), either alone or in combination, followed by time-dependent S.aureus infection. The chemotactic migrations of macrophages were determined by the agarose spot assay. The iNOS, NF-κB and CXCR1 protein expressions were evaluated. The ROS level (superoxide, HO, NO) and antioxidant activities (SOD, CAT, GSH, arginase) were measured. The intra-macrophage phagoctyic activity had been analyzed by confocal microscopy. S.aureus activated macrophages showed increased iNOS expression that symbolizes M1 characterization of macrophages. The results suggest that the combination treatment of LNMMA + PDTC was effective in diminution of CXCL8 production and CXCR1 expression through downregulation of NF-κB and iNOS signaling pathway. Consequently, there was decrement in macrophage migration, reduced ROS generation, elevated antioxidant enzyme activity as well as bacterial phagocytosis at 90 min post bacterial infection. The increased arginase activity further proves the switch from pro-inflammatory M1 to anti-inflammatory M2 polarization of macrophages. Concludingly, the combination of PDTC + LNMMA could resolve S.aureus mediated inflammation through mitigation of CXCL8/CXCR1 pathway switching from M1 to M2 polarization.
趋化因子 CXCL8/CXCR1 轴与自由基和抗氧化剂一起决定了细菌金黄色葡萄球菌引起的炎症严重程度。金黄色葡萄球菌介导的炎症过程受 NF-κB 及其产物 iNOS 调节。本研究旨在研究抑制 NF-κB 和 iNOS 对 CXCL8/CXCR1 的影响，以及金黄色葡萄球菌感染体外时巨噬细胞 M1/M2 极化的改变和相关炎症反应。为此，用 NF-κB 抑制剂吡咯烷二硫代氨基甲酸盐 (PDTC) 和 iNOS 抑制剂 L-N-单甲基精氨酸 (LNMMA) 预处理鼠腹腔巨噬细胞，单独或联合使用，然后进行金黄色葡萄球菌感染的时间依赖性实验。通过琼脂糖斑点测定法测定巨噬细胞的趋化迁移。评估 iNOS、NF-κB 和 CXCR1 蛋白表达。测量 ROS 水平（超氧化物、HO、NO）和抗氧化活性（SOD、CAT、GSH、精氨酸酶）。通过共聚焦显微镜分析巨噬细胞内吞噬活性。金黄色葡萄球菌激活的巨噬细胞显示 iNOS 表达增加，这标志着巨噬细胞的 M1 特征。结果表明，LNMMA+PDTC 联合治疗通过下调 NF-κB 和 iNOS 信号通路，有效减少 CXCL8 的产生和 CXCR1 的表达。因此，在细菌感染后 90 分钟，巨噬细胞迁移减少，ROS 生成减少，抗氧化酶活性升高，细菌吞噬作用增强。增加的精氨酸酶活性进一步证明了巨噬细胞从促炎 M1 向抗炎 M2 极化的转变。总之，PDTC+LNMMA 的联合治疗可以通过减轻 CXCL8/CXCR1 通路来解决金黄色葡萄球菌介导的炎症，从而从 M1 向 M2 极化转变。
