Long-term exposure to nanoparticles alters senescence-associated markers and immune responses in human monocyte-derived macrophages

Abstract

Background: Macrophages are essential in maintaining tissue homeostasis. However, their functionality and phenotype can be impaired by senescence, which impacts immune competence and inflammatory responses. Despite the growing use of engineered nanoparticles (NPs) in biomedical applications or chronic exposure to environmental NPs, their impact on macrophage senescence and immune function remains poorly understood, particularly in the context of prolonged NP exposure. Results: Here, we first confirm that human monocyte-derived macrophages (MDMs) undergo senescence in vitro, as indicated by a panel of senescence-associated markers that increased within 10 days of culture. Then, we investigate how gold (AuNPs), silica (SiO₂ NPs), and polyethylene terephthalate (PET NPs) influence senescence-associated markers, and immunocompetence in MDMs. By examining key markers such as senescence-associated beta-galactosidase (SA-β-gal), CDKN2A (p16), and senescence-associated secretory phenotype (SASP) cytokines, e.g., interleukin (IL)-6 and IL-8, over extended exposure periods up to 10 days, we revealed material-specific effects: AuNPs induce a strong pro-inflammatory response, SiO₂ NPs demonstrate low inflammatory potential, and PET NPs modulate the gene expression level of CDKN2A involved in cell cycle regulation. Conclusion: Our findings underscore the need to characterize long-term NP behavior in biological systems and reveal material-specific effects on macrophage senescence-associated traits and immune function. Rather than assessing NP-induced senescence, this study defines how prolonged NP exposure modulates selected senescence-associated signatures in non-proliferative macrophages, offering valuable insights into the functional consequences of chronic NP accumulation.

Fig. 1. Characterization of senescence in macrophages without NPs. (A) Qualitative brightfield observation (upper row) of primary human macrophage morphology and detection of the senescence-associated beta-galactosidase (SA-β-gal; lower row) by laser scanning microscopy (LSM) at cultivation time points of day 0 (control), day 5, and day 10. Scale bars: 100 μm and 50 μm in inset. (B) Magnified image of SA-β-gal-stained macrophages after 10 days of cultivation. Scale bar: 20 μm (C) RT-qPCR results showing gene expression levels of CDKN2A, which encodes the cell cycle regulation protein p16, for the control (dashed line) and at 5 and 10 days. Statistical significance was determined by a paired t-test (n = 3); * p ≤ 0.05.

Fig. 2. Characterization of the NPs used in this study and dosimetry. Representative TEM micrographs of AuNPs (A), SiO₂ NPs (B), and PET NPs (C). Size-distribution histograms derived from TEM analysis are shown for AuNPs (D), SiO₂ NPs (E), and PET NPs (F). SEM images of PET NPs at lower (G) and higher (H) magnification illustrate their polydisperse morphology. The dosimetry estimation curves (I) display the predicted fraction of AuNPs (black), SiO₂ NPs (red), and PET NPs (blue) deposited onto the bottom of the culture wells over time. The vertical line indicates the time points at which 50% of each NP population is estimated to have sedimented.

Fig. 3. Assessment of the interaction between AuNPs, SiO₂ NPs, and PET NPs with human MDMs after 10 days. The first column presents the visualization of NPs interacting with macrophages using LSM. Scale bars 100 μm and 50 μm in the inset. The middle column displays cross-sections of the macrophages captured by FIB-SEM. White arrows denote NPs, red arrows indicate lipid droplets, and yellow arrows highlight the nucleus. Scale bar: 5 µm. Quantitative uptake was measured using ICP-OES for AuNPs and flow cytometry for SiO₂ and PET NPs after 5 and 10 days of exposure, under both acute (24 h NPs exposure followed by medium replacement) and continuous (no medium change) conditions.

Fig. 4. Gene expression levels of CDKN2A, IL-8, and IL-6 in human monocyte-derived macrophages after exposure to Au, SiO₂, and PET NPs (20 μg mL⁻¹). Macrophages were exposed to Au, SiO₂, and PET NPs for 5 days (A–C) and 10 days (D–F) under two conditions: acute exposure (24 h NPs exposure followed by medium replacement) and continuous exposure (no medium change). Gene expression was assessed using RT-qPCR, and values plotted are expressed as 2^−ΔΔCT. Data are presented as individual values with mean ± SEM (n ≤ 3). Statistical analysis was performed using the ΔCT values, with a paired t-test comparing NPs-exposed cells to the control group (dotted line); * p ≤ 0.05, ** p ≤ 0.01.

Fig. 5. Immunocompetence of human MDMs after exposure to Au, SiO₂, and PET NPs (20 μg mL⁻¹). Macrophages were treated with AuNPs (A and D), SiO₂ NPs (B and E), or PET NPs (C and F) for 4 days (A–C) and 9 days (D–F) under two exposure conditions: acute exposure, where the medium was replaced after 24 hours of NPs exposure, and continuous exposure, where the cells remained in NPs containing medium without replacement throughout the exposure period. Following these exposures, cells were stimulated with LPS (2 ng mL⁻¹) for 24 hours. IL-8 and IL-6 levels in the supernatant were quantified by ELISA. Control samples correspond to untreated cells incubated for 4 or 9 days followed by 24 hours of LPS stimulation, depending on the experimental condition. LPS control samples of freshly differentiated MDMs (not shown) exhibited significantly higher cytokine secretion levels for both IL-6 and IL-8 than NPs-treated and untreated controls. Data are presented as mean ± SEM (n ≤ 3), and statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparison tests against the control (untreated cells); * p ≤ 0.05.