Per- and Polyfluoroalkyl Substances (PFAS) Enhance Cholesterol Accumulation and Dysregulate Inflammatory Responses in Macrophages

Abstract

Epidemiological studies and in vivo animal models have shown that exposure to PFAS can lead to cardiovascular toxicity and promote atherosclerosis. In this study, we explored the effects of PFOA and PFOS exposure on lipid accumulation in macrophages and analyzed critical markers of foam cell formation, which are early precursors of atherosclerotic lesions. Our results demonstrate that PFOS and PFOA enhance lipid and cholesterol accumulation in human U937-derived macrophages, which is characteristic of foam cells. PFOS and PFOA induced the activity of the peroxisome proliferator-activated receptor gamma (PPARγ) and treatment with a PPARγ antagonist partly reversed the accumulation of lipids after PFAS exposure. Furthermore, the results show that PFOS and PFOA activate (NF)-erythroid-derived 2 (E2)-related factor 2 (Nrf2) and induce markers of oxidative stress. Gene expression analysis revealed that mRNA levels of interleukin-1β (IL-1β) and plasminogen activator inhibitor-2 (PAI-2) were upregulated in a time- and concentration-dependent manner in PFOS- and PFOA-treated macrophages. The expression of other key atherosclerosis-related enzymes, including cytochrome P450 8B1 (CYP8B1) and lanosterol synthase (LSS), was downregulated, whereas the expression of cyclooxygenase 2 (COX-2) and aldo-keto reductase family 1 member C3 (AKR1C3) was induced by PFOS and PFOA. Additionally, elevated levels of matrix metalloproteinases (MMP)-1 and MMP-12 were found in PFOS- and PFOA-treated cells, which were associated with increased cell migration. Furthermore, PFOS and PFOA enhanced the expression of IL-1β when macrophages were activated; however, elevated levels of IL-6 and COX-2 in activated macrophages were repressed by PFOS and PFOA. Together, the findings indicate that PFAS exposure modifies immune responses and promotes lipid accumulation in macrophages, potentially contributing to foam cell and plaque formation in atherosclerosis.

Keywords: Cardiovascular disease; Cytokines; Foam cells; Lipids; Macrophages; PFAS; PPAR; SARS-CoV-2.

Fig. 1

Cell viability of U937-derived macrophages after exposure to PFOS and PFOA. Percent nonviable cells were assessed by trypan blue exclusion test. Macrophages were treated with PFOS or PFOA at 5, 10, and 50 μM concentrations for (A) 24 h and (B) 5 days. Vehicle control cells received 0.1% DMSO only. Error bars represent mean ± SD of three independent experiments

Fig. 2

PFAS increases lipid accumulation and cholesterol in macrophages. (A) U937-derived human macrophages were treated with PFOS (10 µM) or PFOA (10 µM) for 5 d. Vehicle control cells received 0.1% DMSO. Lipid accumulation was detected by ORO staining at 520 nm and normalized in 2 × 10⁶ cells. (B) Cholesterol accumulated in U937 macrophages. Cells were treated for 5 days with 5, 10, or 50 μM PFOS or PFOA. Cells were co-treated with the PPARγ antagonist GW9662 or NAC in the presence of 50 μM PFOS or PFOA. Vehicle control cells received 0.1% DMSO. Total cholesterol was determined using a colorimetric method in the presence of cholesterol oxidase and cholesterol esterase. Values are given as mean ± SD of triplicates of three independent experiments. ^aSignificantly higher than vehicle control (p < 0.05). ^bSignificantly lower than cells treated with 50 μM PFOS or 50 μM PFOA (p < 0.05)

Fig. 3

PFOS and PFOA increase PPARγ and Nrf2 activity. U937-derived macrophages were transiently transfected with a (A) PPARγ or (B) Nrf2 luciferase reporter plasmid. After 16 h the cells were treated for 4 h with various concentrations of PFOS or PFOA (5, 10, and 50 μM). Additionally, PPARγ-transfected cells were treated with the PPARγ antagonist GW9662 (30 nM) in the presence of 50 μM PFOS or 50 μM PFOA. Nrf2-transfected cells were co-treated with the ROS scavenger NAC (1 mM) in the presence of 50 μM PFOS or 50 μM PFOA.^aSignificantly higher than control cells (p < 0.05). ^bSignificantly lower than PFOS- and PFOA-treated cells (p < 0.05)

Fig. 4

Time-course study of (A) IL-1β and (B) PAI-2 mRNA expression in U937-derived human macrophages. Macrophages were treated for 3, 6, 12, 24, and 48 h with 10 μM PFOS or 10 μM PFOA. Control cells of each time point received 0.1% DMSO. mRNA expression is shown as fold increase compared to the respective controls. *Significantly higher than control (p < 0.05)

Fig. 5

Concentration-dependent induction of (A) IL-1β and (B) PAI-2 mRNA. U937 macrophages were treated with 5, 10, and 50 μM PFOS or PFOA for 24 h, and mRNA was analyzed by real-time RT-PCR. Control cells received 0.1% DMSO used as vehicle. Results are normalized to β-actin and given as relative changes of the mRNA levels in PFAS-treated cells vs. control. *Significantly different from control (p < 0.05)

Fig. 6

PFOS and PFOA exposure stimulates chemotaxis of U937-derived macrophages. Cells were treated with 10 µM PFOS or 10 µM PFOA for 24, 48, or 72 h, after which they were placed in the upper wells of a chemotaxis chamber. The cells were allowed to migrate for 6 h, followed by an MTT assay. The plate was then read at 540 nm to determine the number of migrated cells. Results of three separate experiments are presented as means ± SD. *Indicates a significant increase compared to the 0-h time point (p < 0.05)

Fig. 7

Modulation of inflammatory markers in activated U937-derived macrophages by PFOS and PFOA. Cells were treated with 10 μM PFOS or 10 μM PFOA for 24 h in the absence or presence of spike S1 protein of SARS-CoV-2. (A) IL-1β mRNA, (B) IL-6 mRNA and (C) COX-2 mRNA were analyzed by real-time RT-PCR and normalized to the expression of β-actin. Results of three separate experiments are presented as means ± SD. ^aSignificantly different from non-activated control (p < 0.05). ^bSignificantly higher than non-activated control (p < 0.05). ^cSignificantly higher than cells treated with spike S1 only, (p < 0.05).^dSignificantly lower than cells treated with spike S1 only, (p < 0.05)