Fathalla Ahmed M, Chow Seong H, Naderer Thomas, Zhou Qi Tony, Velkov Tony, Azad Mohammad A K, Li Jian
Biomedicine Discovery Institute, Infection and Immunity Program and Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Biomedicine Discovery Institute, Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Antimicrob Agents Chemother. 2020 Aug 20;64(9). doi: 10.1128/AAC.00013-20.
Innate immunity is crucial for the host to defend against infections, and understanding the effect of polymyxins on innate immunity is important for optimizing their clinical use. In this study, we investigated the potential toxicity of polymyxins on human macrophage-like THP-1 and neutrophil-like HL-60 cells. Differentiated THP-1 human macrophages (THP-1-dMs) and HL-60 human neutrophils (HL-60-dNs) were employed. Flow cytometry was used to measure the concentration-dependent effects (100 to 2,500 μM for THP-1-dMs and 5 to 2,500 μM for HL-60-dNs) and time-dependent effects (1,000 μM for THP-1-dMs and 300 μM for HL-60-dNs) of polymyxin B over 24 h. Effects of polymyxin B on mitochondrial activity, activation of caspase-3, caspase-8, and caspase-9, and Fas ligand (FasL) expression in both cell lines were examined using fluorescence imaging, colorimetric, and fluorometric assays. In both cell lines, polymyxin B induced concentration- and time-dependent loss of viability at 24 h with 50% effective concentration (EC) values of 751.8 μM (95% confidence interval [CI], 692.1 to 816.6 μM; Hill slope, 3.09 to 5.64) for THP-1-dM cells and 175.4 μM (95% CI, 154.8 to 198.7 μM; Hill slope, 1.42 to 2.21) for HL-60-dN cells. A concentration-dependent loss of mitochondrial membrane potential and generation of mitochondrial superoxide was also observed. Polymyxin B-induced apoptosis was associated with concentration-dependent activation of all three tested caspases. The death receptor apoptotic pathway activation was demonstrated by a concentration-dependent increase of FasL expression. For the first time, our results reveal that polymyxin B induced concentration- and time-dependent cell death in human macrophage-like THP-1 and neutrophil-like HL-60 cells associated with mitochondrial and death receptor apoptotic pathways.
固有免疫对于宿主抵御感染至关重要，了解多粘菌素对固有免疫的影响对于优化其临床应用具有重要意义。在本研究中，我们调查了多粘菌素对人巨噬细胞样THP-1和中性粒细胞样HL-60细胞的潜在毒性。使用了分化的THP-1人巨噬细胞（THP-1-dMs）和HL-60人中性粒细胞（HL-60-dNs）。采用流式细胞术测量多粘菌素B在24小时内的浓度依赖性效应（THP-1-dMs为100至2500μM，HL-60-dNs为5至2500μM）和时间依赖性效应（THP-1-dMs为1000μM，HL-60-dNs为300μM）。使用荧光成像、比色法和荧光法检测多粘菌素B对两种细胞系中线粒体活性、半胱天冬酶-3、半胱天冬酶-8和半胱天冬酶-9的激活以及Fas配体（FasL）表达的影响。在两种细胞系中，多粘菌素B在24小时时诱导浓度和时间依赖性的活力丧失，THP-1-dM细胞的50%有效浓度（EC）值为751.8μM（95%置信区间[CI]，692.1至816.6μM；希尔斜率，3.09至5.64），HL-60-dN细胞的50%有效浓度（EC）值为175.4μM（95%CI，154.8至198.7μM；希尔斜率，1.42至2.21）。还观察到线粒体膜电位的浓度依赖性丧失和线粒体超氧化物的产生。多粘菌素B诱导的细胞凋亡与所有三种测试半胱天冬酶的浓度依赖性激活相关。死亡受体凋亡途径的激活通过FasL表达的浓度依赖性增加得到证实。我们的结果首次揭示，多粘菌素B在人巨噬细胞样THP-1和中性粒细胞样HL-60细胞中诱导浓度和时间依赖性细胞死亡，这与线粒体和死亡受体凋亡途径有关。
