Per- and polyfluoroalkyl substances (PFAS) suppress macrophage alternative activation to disrupt hepatic lipid metabolism [RNA-seq II]

Per- and polyfluoroalkyl substances (PFAS) are pervasive environmental pollutants with diverse toxic effects, including endocrine disruption and hepatotoxicity. As a key class of immune cells, macrophages are critical target for endocrine disrupting chemicals (EDCs); however, their role in PFAS-induced toxicity and the underlying mechanisms remain poorly understood. In this study, we developed a high-content cell model by utilizing the activation and differentiation of human THP-1 monocytes into macrophages, enabling rapid quantitative screening of various PFAS chemicals. Our results identified that PFOA, PFUnDA, and HFPODA suppressed macrophage alternative activation to varying extents by disrupting the PPAR signaling pathway. In vivo, oral exposure to PFOA and PFUnDA in wild-type C57BL/6J mice significantly impaired macrophage alternative activation in the liver, resulting in hepatocyte hypertrophy, liver dysfunction, and systemic lipid metabolism disorders. Furthermore, macrophage-specific PPAR? knockout exacerbated PFAS-induced hepatotoxicity. These findings highlight the immunometabolic regulatory role of macrophage activation in PFAS-induced liver toxicity and provide novel insights into the health risks posed by PFAS exposure in humans. Overall design: In the study of in vivo toxic effects, wild-type (WT) and macrophage PPAR? knockout (Mac-ko) mice were randomly divided into 4 groups, each receiving an oral gavage of PFOA, PFUnDA, and HFPODA at a dose of 1 mg/kg bw/day, along with 0.1% DMSO, for 35 days (n=6-8). This dose was based on the no observed adverse effect level (NOAEL) for oral administration of PFOA and PFUnDA in mice, which is 1 mg/kg bw/day (ATSDR, 2021). HFPODA was used as a substitute for PFOA, maintaining the same gavage dosage. A 0.1% DMSO group served as the control (CT). Mouse body weight was recorded weekly, and at the end of the experiment, blood samples were collected via retro-orbital venipuncture. Subsequently, cervical dislocation was performed for euthanasia, and the liver was dissected and weighed. The liver was divided into three parts: the first part was fixed in 10% neutral buffered formalin for histopathological examination and immunohistochemical analysis; the second part was quickly frozen in liquid nitrogen and stored at -80°C for liver tissue RNA sequencing; the third part was washed with PBS and used for flow cytometry analysis.In this experiment, PPRE-eGFP THP-1 cells were seeded at a density of 70,000 cells per well in black-walled 96-well plates (model: 960096, n=3) to induce their differentiation into M2 macrophages. During the differentiation induction period, the cells were exposed to perfluorinated compounds. After 48 hours of treatment, they were incubated in the dark at room temperature for 30 minutes with fluorescently conjugated antibodies CD36 (1:100) and CD209 (1:100), along with the nuclear dye Hoechst (1:100). After washing with PBS, FluoroBrite DMEM (Thermo Scientific, A1896701) was added, and fluorescent images of the cells were captured using a high-content high-throughput imaging system (version 6.5, Molecular Devices). The analysis was conducted using CellReporterXpress imaging and analysis software (version 6.5, Molecular Devices). In the analysis, the average stained area and the percentage of positively stained cells were calculated by normalizing the total fluorescent-stained cell area and count to the total cell number in the same image.

全氟和多氟烷基物质（Per- and polyfluoroalkyl substances, PFAS）是一类广泛分布的环境污染物，具有多种毒性效应，包括内分泌干扰与肝毒性。作为一类关键免疫细胞，巨噬细胞是内分泌干扰物（endocrine disrupting chemicals, EDCs）的重要作用靶点；然而其在PFAS诱导毒性中的作用及潜在机制仍有待阐明。本研究通过将人THP-1单核细胞活化并分化为巨噬细胞，构建了高内涵细胞模型，可用于多种PFAS类化合物的快速定量筛选。研究结果显示，全氟辛酸（PFOA）、全氟十一烷酸（PFUnDA）与六氟环氧丙烷二聚酸（HFPODA）可通过干扰过氧化物酶体增殖物激活受体γ（PPARγ）信号通路，在不同程度上抑制巨噬细胞的替代性活化。在动物实验中，野生型C57BL/6J小鼠经口暴露PFOA与PFUnDA后，肝脏内巨噬细胞替代性活化功能显著受损，进而导致肝细胞肥大、肝功能异常与全身性脂质代谢紊乱。此外，巨噬细胞特异性PPARγ敲除会加剧PFAS诱导的肝毒性。上述研究结果揭示了巨噬细胞活化的免疫代谢调控在PFAS诱导肝毒性中的作用，为人类PFAS暴露相关健康风险提供了全新的研究视角。

总体实验设计：在体内毒性效应研究中，将野生型（WT）与巨噬细胞PPARγ敲除（Mac-ko）小鼠随机分为4组，每组分别经口灌胃给予PFOA、PFUnDA、HFPODA（剂量为1 mg/kg体重/天），以及0.1%二甲基亚砜（DMSO），连续染毒35天（每组n=6~8）。该剂量参考了PFOA与PFUnDA经口暴露于小鼠的未观察到有害作用水平（no observed adverse effect level, NOAEL），即1 mg/kg体重/天（ATSDR, 2021）。HFPODA作为PFOA的替代化合物，灌胃剂量保持一致。0.1% DMSO组作为空白对照（CT）。每周记录小鼠体重，实验结束时通过眶后静脉丛采血收集血液样本。随后采用颈椎脱臼法处死小鼠，解剖并称量肝脏。将肝脏分为三部分：第一部分用10%中性缓冲福尔马林固定，用于组织病理学检查与免疫组化分析；第二部分迅速置于液氮中冷冻，于-80℃保存，用于肝脏组织RNA测序；第三部分用磷酸盐缓冲液（PBS）洗涤后，用于流式细胞术分析。

本实验采用PPRE-eGFP标记的THP-1细胞，以70,000个/孔的密度接种于黑壁96孔板（型号：960096，n=3），诱导其分化为M2型巨噬细胞。在分化诱导期间，对细胞进行全氟化合物暴露处理。染毒48小时后，将细胞与荧光偶联抗体CD36（1:100稀释）、CD209（1:100稀释），以及核染料Hoechst（1:100稀释）于室温避光孵育30分钟。经PBS洗涤后，加入FluoroBrite DMEM培养基（赛默飞世尔科技，Thermo Scientific，货号：A1896701），使用高内涵高通量成像系统（版本6.5，美谷分子仪器，Molecular Devices）采集细胞荧光图像。采用CellReporterXpress成像与分析软件（版本6.5，美谷分子仪器，Molecular Devices）进行数据分析：将每张图像中总荧光染色细胞面积与细胞计数归一化至总细胞数，以此计算平均染色面积与阳性染色细胞百分比。