Nano-sized zinc oxide and silver, but not titanium dioxide, induce innate and adaptive immunity and antiviral response in differentiated THP-1 cells

Nano-sized metal oxides are currently the most manufactured nanomaterials (NMs), and are increasingly used in consumer products. Recent exposure data reveal a genuine potential for adverse health outcomes for a vast array of NMs, however the underlying mechanisms are not fully understood. To elucidate size-related molecular effects, differentiated THP-1 cells were exposed to nano-sized materials (n-TiO_2, n-ZnO and n-Ag), or their bulk-sized (b-ZnO and b-TiO₂) or ionic (i-Ag) counterparts, and genome-wide gene expression changes were studied at low-toxic concentrations (<15% cytotoxicity). TiO₂ materials were nontoxic in MTT assay, inducing only minor transcriptional changes. ZnO and Ag elicited dose-dependent cytotoxicity, wherein ionic and particulate effects were synergistic with respect to n-ZnO-induced cytotoxicity. In gene expression analyzes, 6 h and 24 h samples formed two separate hierarchical clusters. N-ZnO and n-Ag shared only 3.1% and 24.6% differentially expressed genes (DEGs) when compared to corresponding control. All particles, except TiO₂, activated various metallothioneins. At 6 h, n-Zn, b-Zn and n-Ag induced various immunity related genes associating to pattern recognition (including toll-like receptor), macrophage maturation, inflammatory response (TNF and IL-1beta), chemotaxis (CXCL8) and leucocyte migration (CXCL2-3 and CXCL14). After 24 h exposure, especially n-Ag induced the expression of genes related to virus recognition and type I interferon responses. These results strongly suggest that in addition to ionic effects mediated by metallothioneins, n-Zn and n-Ag induce expression of genes involved in several innate and adaptive immunity associated pathways, which are known to play crucial role in immuno-regulation. This raises the concern of safe use of metal oxide and metal nanoparticle products, and their biological effects.

纳米金属氧化物是目前全球量产规模最高的纳米材料（nanomaterials, NMs），且在消费品领域的应用愈发广泛。现有暴露相关研究数据表明，种类繁多的纳米材料确实存在引发不良健康结局的潜在风险，但其背后的分子调控机制尚未完全阐明。为阐明与材料尺寸相关的分子效应，本研究将分化型THP-1细胞暴露于纳米级材料（n-TiO₂、n-ZnO及n-Ag），或其对应的块体材料（b-ZnO与b-TiO₂）及离子型对应物（i-Ag），并在细胞毒性低于15%的低毒性浓度条件下，检测全基因组水平的基因表达变化。在MTT（噻唑蓝）细胞活性实验中，二氧化钛材料未表现出明显细胞毒性，仅引发轻微的转录组学变化。氧化锌与银基材料则呈现出剂量依赖性的细胞毒性，其中离子型效应与颗粒型效应在纳米氧化锌诱导的细胞毒性过程中具有协同作用。基因表达谱分析结果显示，6小时与24小时的受试样本各自形成独立的层级聚类簇。相较于各自对应的对照组，纳米氧化锌与纳米银之间仅分别共享3.1%与24.6%的差异表达基因（differentially expressed genes, DEGs）。除二氧化钛外，所有受试颗粒均能激活多种金属硫蛋白（metallothioneins）。暴露6小时后，纳米氧化锌、块体氧化锌与纳米银可诱导多种免疫相关基因的表达，这些基因涉及模式识别（包括toll样受体（toll-like receptor））、巨噬细胞成熟、炎症反应（肿瘤坏死因子（tumor necrosis factor, TNF）与白细胞介素1β（interleukin-1β, IL-1β））、趋化作用（CXCL8）以及白细胞迁移（CXCL2-3与CXCL14）。暴露24小时后，尤其是纳米银，可诱导与病毒识别及I型干扰素（type I interferon）应答相关的基因表达。上述研究结果强烈表明，除金属硫蛋白介导的离子效应外，纳米氧化锌与纳米银还可诱导参与多种先天免疫（innate immunity）与适应性免疫（adaptive immunity）相关通路的基因表达，而这些通路在免疫调控（immuno-regulation）中发挥着关键作用。该研究结果也引发了学界对金属氧化物与金属纳米颗粒产品安全使用及其生物学效应的广泛担忧。