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. Author manuscript; available in PMC: 2025 Nov 25.
Published in final edited form as: J Hazard Mater. 2024 Jan 23;466:133590. doi: 10.1016/j.jhazmat.2024.133590

Nanoparticles with intermediate hydrophobicity polarize macrophages to plaque-specific Mox phenotype via Nrf2 and HO-1 activation

Shumei Zhai a,b,1,*, Xianzhi Zhang b,1, Mingdi Jiang b,1, Yujia Liu a, Guangbo Qu c, Xiaomiao Cui a, Cristina-Maria Hirschbiegel b, Yuanchang Liu b, Colby Alves b, Yi-Wei Lee b, Guibin Jiang c, Bing Yan d,*, Vincent M Rotello b,*
PMCID: PMC12643246  NIHMSID: NIHMS2118577  PMID: 38280324

Abstract

Mox macrophages were identified recently and are closely associated with atherosclerosis. Considering the potential health risks and the impact on macrophage modulation, this study investigated the Mox polarization of macrophages induced by nanoparticles (NPs) with adjustable hydrophobicity. One nanoparticle (C4NP) with intermediate hydrophobicity efficiently upregulated the mRNA expression of Mox-related genes including HO-1, Srxn1, Txnrd1, Gsr, Vegf and Cox-2 through increased accumulation of Nrf2 at a nontoxic concentration in both resting and LPS-challenged macrophages. Additionally, C4NP impaired phagocytic capacity by 20% and significantly increased the secretion of cytokines, including TNFα, IL-6 and IL-10. Mechanistic studies indicated that intracellular reactive oxygen species (ROS) were elevated by 1.5-fold and 2.6-fold in resting and LPS-challenged macrophages respectively. Phosphorylated p62 was increased by 2.5-fold in resting macrophages and maintained a high level in LPS-challenged ones, both of which partially accounted for the significant accumulation of Nrf2 and HO-1. Notably, C4NP depolarized mitochondrial membrane potential by more than 50% and switched macrophages from oxidative phosphorylation-based aerobic metabolism to glycolysis for energy supply. Overall, this study reveals a novel molecular mechanism potentially involving ROS-Nrf2-p62 signaling in mediating macrophage Mox polarization, holding promise in ensuring safer and more efficient use of nanomaterials.

Keywords: Nanoparticles, macrophages polarization, oxidative stress, atherosclerosis, p62

1. Introduction

Nanotechnology is progressively being integrated into all aspects of daily life, resulting in increased release of nanoparticles (NPs) into the environment and heightened human exposure.1,2 The released NPs contaminate soil, migrate through water and air, and eventually are gathered in the human body through bioaccumulation and bioconcentration.3, 4, 5 NPs can also infiltrate the human body through inhalation, ingestion, and skin contact.6 Due to their small size and surface functionalization, NPs can penetrate biological barriers and membranes, establishing contact and interactions with sub-cellular components.7, 8, 9 This capability potentially induces cyto-genotoxicity and mitochondrial dysfunction, ultimately leading to cardiac diseases, inflammation diseases or immune diseases.10,11,12,13,14

Studies have shown that NPs preferentially accumulate in macrophages following systemic administration, perturbing the polarization of macrophages.15, 16 The resulting subsets of polarized macrophages play critical roles in both progression and resolution of diseases.17, 18, 19, 20 The polarization of macrophages is traditionally divided into two phenotypes, classically activated macrophages (M1) and alternatively activated macrophages, which respectively exert pro-inflammatory/anti-tumor and anti-inflammatory/pro-tumor effects.21, 22 However, the phenotypic range of macrophages in pathological conditions such as atherosclerotic microenvironments is complicated, with new polarization phenotypes being discovered.23, 24 In recent studies, the Leitinger group identified a new plaque-specific ‘Mox’ macrophage phenotype induced by oxidized phospholipids, comprising approximately 30% of all macrophages in mouse atherosclerotic lesions.25, 26 Mox phenotype has been considered as the metabolic adaptation of macrophages to oxidative tissue damage, and investigation of their functions has been focused mainly on antioxidant and redox-regulatory activities.26 Although it is uncertain so far whether Mox is pro-atherosclerotic or anti-atherosclerotic, the imbalance of the ratio of macrophage subsets, including M1, M2, and Mox macrophages, could be a contributing factor for plaque formation, hindering the resolution of inflammation.27,28

Previous studies on the macrophage polarization effects in response to NP exposure have predominantly concentrated on the modulation of macrophage M1/M2 polarization.29, 30, 31 However, the induction to other phenotypes, including Mox, remains largely unexplored. Numerous studies have indicated that the unique physicochemical properties of NPs, such as size, hydrophobicity, and charge, are crucial to determining their interactions with the immune system.16,17 Particularly, it is well known that the hydrophobicity of NPs is correlated with oxidative stress and inflammatory response.32,33 Therefore, in this work, we fabricated a library of AuNPs with tuned surface hydrophobicity to investigate their potential to polarize macrophages to non-M1/M2 phenotypes. (Fig. 1). These NPs feature cationic surface elements with precisely tailored hydrophobicity, from hydrophilic (C1NP) to hydrophobic (C10NP). By screening macrophage polarization-related gene expression, we identified a specific particle (C4NP) that robustly promoted the Mox phenotype polarization alone or combined with lipopolysaccharide (LPS) through activating Nrf2 and HO-1 signal pathways. The generation of the Mox phenotype reveals a novel molecular mechanism involved in immune modulation by NPs, providing a cautionary tale for the risk of NP exposure.

Fig. 1. Gold nanoparticle (AuNP)-mediated macrophage M0-to-Mox phenotypic change.

Fig. 1.

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The R group of AuNPs was precisely tuned to dictate hydrophobicity, with only C4NP providing efficient generation of the Mox phenotype through activating Nrf2 and HO-1 signaling pathways. C4NP treatment inhibited mitochondrial respiration of macrophages while promoting their glycolytic pathway, inducing differentiation to the Mox phenotype. LogP represents the calculated hydrophobic values of the headgroup.

2. Materials and methods

2.1. NP fabrication

NPs were synthesized by following the previously reported procedures.34 The detailed synthesis and characterizations of NPs can be found in the Supporting Information.

2.2. Quantitative RT-PCR

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using a LightCycler480 System (Roche). All DNA primers were synthesized from Boshang Technologies (Shanghai, China). The sequences of primers used for PCR are shown in Table S2. Samples were first activated at 95 °C for 30 sec. Then denaturing occurred at 95 °C for 5 sec, followed by annealing at 60 °C for 30 sec. The denature/anneal process was repeated in 40 cycles. Relative gene expression was determined by comparing the Ct value of the gene of interest to that of β-actin, used as a housekeeping gene, by the 2ΔΔCt method. Three biological replicates were performed for each control group, and two technical replicates were used for each biological replicate.

2.3. Measurement of ROS production

Reactive oxygen species (ROS) were quantified by the DCFH-DA (Beyotime, China) assay. Briefly, after culturing the RAW 264.7 cells in a 24-well plate for 24 h, the medium was replaced with fresh high-glucose DMEM supplemented with 2% FBS. After 2 h of starvation, cells were incubated with AuNPs with/without LPS (100 ng/mL). After 20 h, cells were incubated with DCFH-DA for 30 min and washed with PBS (3 times), followed by digestion. Finally, the cells were suspended in 0.5 mL of PBS and analyzed by flow cytometry (ACEA Novo Cyte 3009, China).

2.4. Measurement of mitochondrial membrane potential

TMRE (tetramethylrhodamine, ethyl ester) (Beyotime, China) is used to label active mitochondria. TMRE is a cell-permeant, positively-charged, red-orange dye that readily accumulates in active mitochondria due to their relative negative charge. Depolarized or inactive mitochondria have decreased membrane potential and fail to sequester TMRE. RAW 264.7 cells were seeded, incubated, and treated as above. Then, cells were washed with warm PBS (3 times) and incubated with TMRE for 30 min at 37 °C. Cells were then washed with warm PBS (3 times) and analyzed with a flow cytometer using a 488 nm laser for excitation and at the emission of 575 nm. Fluorescence microscopy images were obtained via confocal laser scanning microscopy (CLSM, Lecia TCS SP8 STED 3X).

2.5. Measurement of oxygen consumption rate and glycolytic rate

RAW 264.7 cells were seeded into a 24-well plate (Seahorse Bioscience, North Billerica, MA) at a density of 2.5×104 cells/well for both the mitochondrial stress and glycolytic rate tests. The next day, the cells were treated with AuNPs with/without LPS for 24 h before the test. According to the user manual, the oxygen consumption rate (OCR) was determined for the mitochondrial stress test using the Seahorse XF-24 analyzer (Seahorse Bioscience). In brief, OCR was measured at baseline (Basal OCR) followed by the sequential addition of oligomycin (1.5 μM) to inhibit ATP synthase, FCCP (1.0 μM) to permeabilize the inner mitochondrial membrane and promote maximum electron flow, and rotenone/antimycin (0.5 μM) to inhibit complex I and complex III, respectively. ATP-linked OCR was calculated by subtracting the OCR after oligomycin treatment from the basal OCR. The spare respiratory capacity was calculated by subtracting the basal OCR from the maximal OCR after FCCP treatment. For the glycolytic rate assay, the glycolytic proton efflux rate (glycoPER) was determined by the Seahorse XF-24 analyzer. GlycoPER was measured at baseline, followed by the sequential addition of rotenone/antimycin (0.5 μM) to inhibit all electron transport and 2-DG (50 mM) to block glycolysis. GlycoPER, compensatory glycolysis, and non-glycolytic acidification were calculated.

2.6. Measurement of cytokine and nitric oxide (NO) secretion

Cells were treated with C4NP with or without LPS for 24 h; the supernatants were then collected and centrifuged at 12000 g for 10 min. TNFα, IL-6, and IL-10 secretion were measured by enzyme-linked immunosorbent assay (ELISA) using kits (BD Biosciences, CA, USA). Nitrite was determined by Invitrogen™ Griess Reagent Kit according to the manufacturer’s instructions.

2.7. Statistical analysis

Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA. All statistical analyses were done with GraphPad Prism.

3. Results and discussion

3.1. Synthesis of AuNPs with different hydrophobicity

A small library of AuNPs with a 2 nm core and functionalized with surface ligands were generated through ligand-exchange reactions using a 1-pentanethiol-coated gold core and the corresponding thiolate ligands. The surfaces of the AuNPs in the library feature a hydrophobic alkane chain to stabilize the NP in a biological environment, an oligo (ethylene glycol) spacer to enhance the solubility and biocompatibility,35 and a positively-charged terminal ligand with tunable hydrophobicity. This terminal group featured a hydrophobic alkyl tail ranging from one to ten carbons with logP values from 0.14 to 5.04 (Fig. 1), providing a systematic platform to investigate macrophage polarization. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) demonstrated that surface functionalization did not significantly alter the size and the morphology of AuNPs (Table S1 and Fig. S1). Zeta potential measurements were likewise unaffected by headgroup choice (Table S1).

3.2. Screening of AuNPs for Mox Modulation

Our initial studies explored the immune responses of macrophages induced by different NPs using RAW 264.7 cells in the absence and presence of lipopolysaccharide (LPS), mimicking the normal state and inflammatory environment found in a range of diseases including atherosclerosis.36 After 24 h treatment, AuNPs showed minimal toxicity on RAW 264.7 cells at concentrations below 200 nM (Fig. S2A). AuNPs with a surface area of 1.0 ×1015 nm2/mL (equals 136 nM) also did not affect cell viability in the presence of LPS (100 ng/mL) (Fig. S1B). In addition, LPS co-treatment did not alter the cell uptake of NPs (Fig. S2C) Therefore, 136 nM AuNPs and 100 ng/mL LPS were chosen for subsequent experiments to avoid cytotoxic response.

Previous works have reported genes closely associated with the Mox phenotype, including heme oxygenase, (HO)-1, sulfiredoxin (Srxn)1, thioredoxin reductase (Txnrd) 1, glutathione, reductase (Gsr), vascular endothelial growth (Vegf), and Cox-2.25 We, therefore, first assessed the mRNA levels of these genes using qRT-PCR. Oxidized phospholipid (OxPAPC), known to generate Mox macrophage, was used as a positive control. The results showed that RAW 264.7 cells’ exposure to NPs resulted in dramatically different gene expression profiles. Mox-specific gene expression was only observed with C4NP-treated cells, which significantly upregulated the mRNA expression of all Mox-identified genes by more than 1.5-fold (P<0.001) (Fig. 2A and Fig. S3A). When RAW 264.7 cells were co-treated with NPs and LPS, the mRNA expression of Mox-specific genes correlated with increasing hydrophobicity of the NPs. Notably, the expression of Mox-related genes after C4NP and LPS co-treatment was significantly higher than other NPs (Fig. 2B and Fig. S3B). These results demonstrate that C4NP induces macrophage gene expression patterns toward the Mox phenotype.

Fig. 2. Heatmap of Mox-related gene expression.

Fig. 2.

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(A) Gene expression of RAW 264.7 cells exposure only to NPs. (B) Gene expression of RAW 264.7 cells co-exposure to NPs and LPS. Oxidized phospholipids (OxPAPC, 50 μg/mL) were used as the positive control. The data were the average of three biological replicates. Fold changes in mRNA level were normalized to β-actin. The bar graph of gene expression is presented in Supplemental Fig. 3.

Since Nrf2 dominates the expression of Mox-specific genes,25 we next examined the presence and accumulation of both Nrf2 protein and its target HO-1 after treatment. Results showed that in resting macrophages, C1, C2 and C4 significantly induced Nrf2 expression compared with the negative control, with ~2.4 fold as the maximum induction from C4NP (Fig. 3A and C). In contrast, C6 and C10 slightly increased Nrf2 expression. Consistently, only C4NP treatment caused a marked increase of HO-1 (~2.1-fold higher than control, P<0.01). In LPS-challenged macrophages, both C2 and C4 treatment distinctly increased the accumulation of Nrf2 (~2.0-fold higher than LPS, P < 0.01 and ~3.5-fold higher than LPS, P < 0.0001, respectively), but only C4NP significantly induced the accumulation of HO-1 (~1.5-fold) compared with LPS treatment (Fig. 3B and D). These results suggested that C4NP can polarize macrophages to the Mox phenotype through the stabilization of Nrf2. The stabilization and accumulation of Nrf2 are presumably due to the oxidative stress after exposure to relatively hydrophobic nanoparticles (C1–C4). However, the much more hydrophobic C6 and C10 could induce exceedingly elevated oxidative stress, which could activate NFκB, leading to the suppression and less accumulation of Nrf2.37,38.

Fig. 3. Immunoblot of Nrf2 and HO-1.

Fig. 3.

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(A) RAW 264.7 cells exposed to NPs. (B) RAW 264.7 cells co-treated with AuNPs and LPS. The protein levels were normalized to β-actin and the basal level in the untreated sample was set at 1.0. Results were expressed as fold induction over control levels. (C) Quantitative data of the western blot when cells were exposed to different functionalized NPs (n-3). (D) Quantitative data of the western blot when cells were co-treated with AuNPs and LPS. Each data was presented as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA. *, **, ***, **** the value is significantly different from that of control (P < 0.05, P < 0.01, P <0.001 and P < 0.0001, respectively). ##, #### indicate a significant difference from that of LPS treatment (P < 0.01 and P < 0.0001).

Reduced phagocytosis is another key feature of Mox macrophages and is implicated in the reduced clearance of apoptotic cells in atherosclerotic plaques.39 We, therefore, quantified the phagocytic capacity of C4NP-treated macrophages for FITC-labeled beads (1.0 μm mean particle size) using flow cytometry. Macrophages treated with C4NP and LPS exhibited ~20% reduction in phagocytosis compared to those treated with LPS alone (Fig. S4). This reduced phagocytosis is consistent with the polarization of macrophages to the Mox phenotype.

3.3. Molecular mechanisms stabilizing Nrf2 after NP exposure

As a crucial transcription factor in regulating cellular redox homeostasis, intracellular Nrf2 is normally degraded quickly after binding to Keap1 (Kelch-like ECH-associated protein 1) for ubiquitination. Previous studies showed that the interactions of Keap1 with phosphorylated p62 or ROS could inhibit the binding of Nrf2 to Keap1, resulting in the stabilization of Nrf2.40 As a positive feedback loop, the activated Nrf2 could bind to an antioxidant response element (ARE) in the p62 promoter to induce p62 expression.41 We, therefore, measured p62 phosphorylation and ROS levels in macrophages. The results showed that NP exposure increased the level of p62 phosphorylation in resting macrophage, with the largest effects seen with C4NP (P < 0.01) (Fig. S5). In LPS-challenged macrophages, the high level of p62 phosphorylation induced by LPS was maintained by C4NP. The intracellular ROS of macrophages increased by ~1.5 fold after exposure to C4NP and ~2.6 fold after co-exposure to C4NP and LPS (Fig. 4) as quantified by flow cytometry. These results indicated that C4NP treatment integrates the upregulation of the positive feedback loop between Nrf2 and phosphorylated p62, and the induction of ROS, promoting the stabilization of Nrf-2.

Fig. 4. Determination of reactive oxygen species (ROS) using flow cytometry.

Fig. 4.

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(A) Intracellular ROS levels are determined by the fluorescence generation from activated 2’,7’-diacetyldichlorofluorescein diacetate (DCFH-DA). (B) Quantification of ROS levels. The bar graph represents the average fluorescence intensity calculated from three biological replicates. Statistical significance was determined by one-way ANOVA (**P < 0.01, ****P < 0.0001).

It is well known that the main sources of intracellular ROS in most cells including macrophages are NADPH oxidase and mitochondria.42 However, the increased expression of HO-1, just as in this study, has the potential to inhibit NADPH oxidase-dependent ROS but induce mitochondrial ROS (mtROS),43,44 which could damage mitochondria. Mitochondrial membrane potential (ΔΨm) was then measured to reflect mitochondrial activity. The results showed that the mitochondrial inhibitor carbonylcyanide-3-chlorophenylhydrazone (CCCP) and LPS treatment significantly reduced and elevated ΔΨm, respectively. In contrast, C4NP treatment dramatically reduced ΔΨm with the value decreased to 0.5-fold that of untreated cells. C4NP even prevented the LPS-induced elevation of ΔΨm, with the value in the C4NP plus LPS group decreased to 0.3-fold that of cells treated with LPS (Fig. 5 and Fig. S6). Thus, these results demonstrated that C4NP disturbs mitochondrial function and homeostasis.

Fig. 5. Fluorescence microscopy images showing changes in mitochondrial membrane potential.

Fig. 5.

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RAW 264.7 were treated with vehicle, C4 (136 nM), LPS (100 ng/mL), or C4 plus LPS C4 for 24 h. Cells were washed with PBS and incubated with fluorescent probe TMRE (tetramethylrhodamine, ethyl ester) for 30 min at 37 °C. Then, cells were washed with warm PBS (3 times). Fluorescence microscopy images were obtained via confocal laser scanning microscopy (CLSM, Lecia TCS SP8 STED 3X). LPS increased, but C4 decreased ΔΨM relative to the vehicle. CCCP (10 μM) is used as the positive control

3.4. NP-induced Mox macrophage transformation alters metabolic pathways

Given the obvious depolarization of the ΔΨm, we next measured the oxygen consumption rate (OCR) and glycolytic proton efflux rate (glycoPER) as measures of mitochondrial respiration and glycolysis. Our studies demonstrated that LPS or C4NP treatment significantly decreased basal OCR (P < 0.0001), ATP-linked OCR (P < 0.0001), and maximal OCR (P < 0.0001) compared with cells without treatment (control), and C4NP plus LPS dramatically decreased OCR (P < 0.0001) compared with LPS, highlighting the remarkable inhibition of respiratory capacity and ATP production (Fig. 6). These results were consistent with previous studies, which suggested that mitochondrial respiration was suppressed in Mox macrophages to support Nrf2-mediated redox homeostasis.26 On the other hand, in the case of mitochondrial disorders, lower ΔΨm and decreased activity of the respiratory chain have been associated with the simultaneous increase in mtROS production,45 which promotes the stabilization of Nrf2 resulting in Mox polarization.

Fig. 6. Mitochondrial respiration profile.

Fig. 6.

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(A) The real-time respiratory capacity of macrophages treated by C4NP with/without LPS. OCR =oxygen consumption rate. Oligomycin: the inhibitor of ATP synthesis (e.g., complex V); FCCP: the uncoupler of oxygen consumption from ATP production; Rotenone/antimycin: the inhibitor of complex I and III. (B-D) The average basal OCR, ATP-linked respiration, and maximal respiration are calculated from Fig. 6A, respectively. Data shown are mean ± SD (n=3). Statistical significance was determined by one-way ANOVA. **** The value statistically differs from the control’s (P < 0.0001). #### The value is significantly different from that of LPS treatment (P < 0.0001). OCR =oxygen consumption rate.

Glycolysis and oxidative phosphorylation (OXPHOS) are two major metabolic pathways that provide cell energy.46 The inhibition of OXPHOS should result in compensatory elevation of glycolysis to increase ATP generation for cell survival. Indeed, C4NP treatment significantly increased basal glycolysis (P < 0.0001), decreased post-2-DG acidification (P <0.001) and compensatory glycolysis (no significant difference) as compared to the control group. In contrast, LPS treatment markedly increased basal glycolysis (P < 0.0001), compensatory glycolysis (P<0.01) and post-2-DG acidification (P<0.001) as expected. Consistently, C4NP plus LPS increased basal glycolysis (P<0.05) compared with LPS treatment, but did not affect compensatory glycolysis and post-2-DG acidification obviously (Fig. 7). These results demonstrated that C4NP promotes glycolysis in macrophages consistent with a recent study, which demonstrated that Nrf2 activation promoted glycolysis in both resting and LPS-challenged macrophages.47 It is well known that the metabolic features of macrophages affect their functionality.48 In vitro, pro-inflammatory M1 macrophage has been associated with enhanced glycolytic metabolism, while increases in OXPHOS are associated with anti-inflammatory M2 macrophage. In addition, tumor-associated macrophage is highly dependent on glycolysis. All in all, our results suggested that C4NP promotes metabolic reprogramming and tunes the innate immune response in macrophages partially through Nrf2 activation.

Fig. 7. Glycolysis assessment.

Fig. 7.

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(A) The real-time glycolytic proton efflux rate (glycoPER) of macrophages under C4NP with/without LPS exposure. 2-deoxyglucose (2-DG): the inhibitor of glycolysis. (B-D) The average of basal glycolysis, compensatory glycolysis, and post 2-DG acidification is calculated from Fig 7A. Data shown are mean ± SD (n=3). Statistical significance was determined by one-way ANOVA. **, ***, **** the value is significantly different from that of control (P < 0.01, P <0.001 and P < 0.0001, respectively). # The value is significantly different from that of LPS treatment (P < 0.05).

3.5. C4NP regulates the production of cytokines and NO

Cytokines are pivotal immunoregulators to determine immune responses. The high level of Nrf2 protein accumulation and the metabolic reprogramming induced by C4NP and C4NP plus LPS triggered the exploration of cytokine expression and excretion, including tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and interleukin-10 (IL-10).49,50 As shown in Fig. 8A and B, C4NP significantly induced the mRNA expression of TNFα, IL-6, and IL-10 in both resting and LPS-challenged macrophages. Moreover, C4NP treatment induced the secretion of TNFα (P <0.0001) and IL-10 (P<0.05) but not IL-6 in resting (M0) macrophages, as compared to that of the control group (Fig. 8C). In contrast, C4NP treatment enhanced the secretion of TNFα (P<0.01), IL-6 (P <0.01) and IL-10 (P <0.001) in LPS-challenged macrophage (Fig. 8D). These results suggested that Mox might be a pro-atherosclerotic phenotype. In addition, C4NP treatment significantly inhibited the LPS-induced mRNA expression of inducible nitric oxide synthase (iNOS) and secretion of NO in macrophages (Fig. S7), indicating the potential inhibition of macrophage M1 polarization.

Fig. 8.

Fig. 8.

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(A-B) mRNA expression of inflammatory-related genes measured by qRT-PCR and (C-D) the cytokines secretion measured by ELISA in cells treated with LPS (100 ng/mL), C4NP (136 nM) or C4NP plus LPS (100 ng/mL) for 24 h. Cells without any treatment were used as the control group. The cell culture supernatants were collected to determine the secretion of TNF-α, IL-6, and IL-10 by ELISA. Cells were collected for RNA extraction and PCR. Data shown are mean ± SD (n=3). Statistical significance was determined by one-way ANOVA (*P<0.05, ** P <0.01, *** P <0.001, ****P <0.0001).

To reveal the mechanism underlying the regulation of cytokine secretion by C4NP, we determined the mRNA expression of TNF-α, IL-6, IL-10, and iNOS in LPS-stimulated macrophages pretreated with HO-1 inhibitor Zinc Protoporphyrin (ZnPP). The mRNA expression of those cytokines was regulated by ZnPP pretreatment, albeit in different patterns (Fig. S8), which indicates that C4NP partially promotes the secretion of these cytokines through the induction of HO-1. These results suggested that the induction of HO-1 plays a key role in the immune modulation of hydrophobic AuNPs.

4. Conclusion

In this work, we found that C4NP with intermediate hydrophobicity showed the most efficiency in inducing macrophage polarization to Mox phenotype. C4NP induced the accumulation of Nrf2 and HO-1 through the elevated level of intracellular ROS and phosphorylated p62, which constitute the canonical and non-canonical mechanisms of Nrf2 activation, respectively. It is worth noting that the inhibition of ΔΨm and the respiratory chain are considered to be associated with increased mtROS production, which together participated in the activation of the Nrf2 signaling pathway, resulting in enhanced Mox phenotype. Additionally, the metabolic shift from oxidative phosphorylation to glycolysis provided energy for cell survival under C4NP-induced oxidative stress and Nrf2 activation. Moreover, the positive feedback circuit between Nrf2 and phosphorylated p62, HO-1 and IL-10, as we found in this work, might also amplify the effects of C4NP in macrophage Mox polarization.

Supplementary Material

ESI

HIGHLIGHTS.

  • Intermediate hydrophobic gold nanoparticle polarizes macrophages to Mox phenotype.

  • The Mox phenotype is generated through the ROS-Nrf2-p62 signal pathway.

  • Polarized Mox macrophages switch from mitochondrial respiration to glycolysis.

Environment implication.

Nanoparticles (NPs) are now extensively used in food, agriculture, chemical, and medical industries. The ubiquitous use of NPs increases the environmental release and human exposure of NPs. The modulation of the immune system by NPs is central to understanding the environmental safety of nanomaterials due to the unique physicochemical properties of NPs.

Our study demonstrates the potential of hydrophobic NPs to polarize macrophages to the Mox phenotype, which plays a role in atherosclerosis. The findings emphasize the importance of rationally designing NPs to reduce the potential environmental impact and ensure the health safety of manufactured NPs in the future.

Acknowledgments

This work was supported by the NIH EB022641 (V.M.R.), the National Natural Science Foundation of China (21677090 to S.Z., and 22036002 to B.Y.), the National Key R&D Program of China (2023YFA0915103), the introduced innovative R&D team project under the “The Pearl River Talent Recruitment Program” of Guangdong Province (2019ZT08L387), and the State Scholarship Fund of the China Scholarship Council. C.-M.H. was partially supported by a fellowship from the University of Massachusetts as part of the Chemistry-Biology Interface Training Program (National Research Service Award T32 GM139789). This work was partly performed at the Analytical Center for Structural Constituent and Physical Property at Shandong University.

Appendix A. Supplementary data

Supplementary data associated with this article can be found in the online version.

Footnotes

CRediT authorship contribution statement

S.Z., X.Z., B.Y., and V.M.R. developed the idea. S.Z., X.Z., M.J., Y.L., X.C., C.-M.H., G.Q., Y.L., and Y.-W.L. performed experiments and analyzed the data. S.Z., X.Z., M.J., C.A., G.J., B.Y., and V.M.R. wrote and revised the manuscript.

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

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