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

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.