DataSheet_1_Macrophage metallothioneins participate in the antileishmanial activity of antimonials.docx

Host cell functions that participate in the pharmacokinetics and pharmacodynamics (PK/PD) of drugs against intracellular pathogen infections are critical for drug efficacy. In this study, we investigated whether macrophage mechanisms of xenobiotic detoxification contribute to the elimination of intracellular Leishmania upon exposure to pentavalent antimonials (SbV). Primary macrophages from patients with cutaneous leishmaniasis (CL) (n=6) were exposed ex vivo to L. V. panamensis infection and SbV, and transcriptomes were generated. Seven metallothionein (MT) genes, potent scavengers of heavy metals and central elements of the mammalian cell machinery for xenobiotic detoxification, were within the top 20 up-regulated genes. To functionally validate the participation of MTs in drug-mediated killing of intracellular Leishmania, tandem knockdown (KD) of MT2-A and MT1-E, MT1-F, and MT1-X was performed using a pan-MT shRNA approach in THP-1 cells. Parasite survival was unaffected in tandem-KD cells, as a consequence of strong transcriptional upregulation of MTs by infection and SbV, overcoming the KD effect. Gene silencing of the metal transcription factor-1 (MTF-1) abrogated expression of MT1 and MT2-A genes, but not ZnT-1. Upon exposure to SbV, intracellular survival of Leishmania in MTF-1KD cells was significantly enhanced. Results from this study highlight the participation of macrophage MTs in Sb-dependent parasite killing.

参与抗胞内病原体感染药物的药代动力学与药效动力学（pharmacokinetics and pharmacodynamics, PK/PD）的宿主细胞功能，对药物疗效具有关键作用。本研究旨在探讨巨噬细胞异生物质解毒机制在暴露于五价锑剂(SbV)时是否可促进胞内利什曼原虫的清除。研究团队从6例皮肤利什曼病(CL)患者体内分离原代巨噬细胞，体外开展巴拿马利什曼原虫（L. V. panamensis）感染模型及SbV处理实验，并完成转录组测序分析。转录组分析结果显示，7个金属硫蛋白(MT)基因——作为强效重金属清除剂、哺乳动物细胞异生物质解毒通路的核心组成部分——位列上调基因的前20位。为从功能层面验证MTs在药物介导的胞内利什曼原虫杀伤中的参与作用，研究团队在THP-1细胞中采用泛MT短发夹RNA(shRNA)策略，对MT2-A、MT1-E、MT1-F及MT1-X进行串联敲降(KD)。但由于感染与SbV处理可强烈上调MTs的转录水平，抵消了敲降的干预效果，因此串联敲降细胞内的寄生虫存活率未出现显著变化。对金属转录因子-1(MTF-1)实施基因沉默，可阻断MT1与MT2-A基因的表达，但不影响锌转运体-1(ZnT-1)的转录。在SbV暴露条件下，MTF-1敲降细胞内的利什曼原虫胞内存活率显著升高。本研究结果证实，巨噬细胞MTs参与了SbV依赖的寄生虫杀伤过程。