Chromium(VI) Modulates Macrophage Polarization and Metabolic Reprogramming to Impair Immune Function

Abstract

Hexavalent chromium (Cr(VI)) is a recognized environmental and occupational hazard with significant implications for immune function. However, the cell-intrinsic mechanisms by which Cr(VI) coordinately reshapes macrophage polarization together with immunometabolic and mitochondrial alterations remain incompletely characterized. This study investigated how Cr(VI) exposure influences macrophage morphology, polarization, energy metabolism, and mitochondrial integrity using an in vitro model. Macrophages exposed to Cr(VI) exhibited morphological changes, including pseudopod growth and fusiform shapes, alongside a shift toward M1-type polarization. Key M1 associated biomarkers, including TNF-α, CD36, and CD80, increased 24 h after Cr(VI) exposure, whereas the M2 associated VEGFb decreased. Cr(VI) exposure also impaired energy metabolism, reducing ATP production and shifting metabolism towards glycolysis, despite increased glucose uptake. Mitochondrial damage, membrane potential collapse, and elevated oxidative stress further highlighted the immunotoxic effects of Cr(VI). Cr(VI) exposure may drive a metabolic shift in macrophages toward less efficient energy production pathways, such as glycolysis. These findings provide critical insights into Cr(VI)-induced macrophage dysfunction and emphasize the environmental risks associated with Cr(VI) pollution, underscoring the need for further mechanistic research and mitigation strategies to safeguard public health.

1. Introduction

Hexavalent chromium (Cr(VI)) is widely utilized in industries such as electroplating, leather tanning, and stainless steel production and is known to be toxic and carcinogenic [1]. In recent years, the demand for chromium, particularly hexavalent chromium, has increased significantly, leading to its release into the air, water, soil, and food through production and usage emissions [2]. This widespread distribution means that millions of workers worldwide may now be exposed to Cr(VI) and its oxides, posing significant occupational health risks [3,4]. Moreover, these pollutants also present various health hazards to nearby residents and ecosystems [3]. Contamination of drinking water and soil by Cr(VI), a hazardous heavy metal, has thus emerged as a critical issue that demands attention.

Human exposure to Cr(VI) can occur through inhalation, ingestion, and dermal contact, particularly in industrial settings [4]. Once chromium enters the bloodstream, it is distributed to almost all tissues, such as heart, kidneys, liver, and gastrointestinal tract [5]. This distribution can lead to various health problems, including asthma, cancer, allergic reactions, neurological and cardiovascular diseases, and organ failure [6,7]. Among the affected systems, the immune system stands out as the first line of defense against external damage and invasion while also maintaining internal balance [8]. The immune system produces responses when exposed to harmful environmental factors. We previously found that immune inflammatory indicators in the peripheral blood of workers exposed to chromate initially increased and then decreased, and they may contribute to the development of genetic damage [9,10]. Additionally, a study involving 106 chromate workers and 50 matched controls indicated that urinary chromium levels negatively correlated with IgA and IgG levels in peripheral blood, suggesting that chromate may cause immune system responses similar to those induced by haptens [11]. However, the specific cells of the immune system involved in the health damage caused by chromate exposure are not fully understood.

Macrophages are essential cells within the innate immune system, distinguished by their long lifespan and robust phagocytic capabilities [12,13]. These cells not only initiate immune and inflammatory responses against external damage but also play a pivotal role in maintaining tissue homeostasis and facilitating repair [13,14]. In response to various environmental signals, macrophages can polarize into two distinct states, M1 and M2, which are associated with pro-inflammatory and anti-inflammatory processes [15]. Recent advances in immunometabolism have demonstrated that macrophage polarization was tightly coupled with distinct metabolic programs [16]. Especially, much of the Cr(VI) related metabolic reprogramming evidence has been derived from epithelial or transformed cell models [17,18]. However, immunometabolic remodeling in macrophage populations remains comparatively underexplored. Classically activated M1 macrophages rely primarily on aerobic glycolysis for rapid ATP generation, even in the presence of oxygen [19]. This metabolic shift is accompanied by two points of interruption in the tricarboxylic acid (TCA) cycle, leading to the accumulation of succinate and itaconate. Succinate stabilizes hypoxia-inducible factor 1α (HIF-1α), thereby enhancing the transcription of glycolytic and pro-inflammatory genes such as IL-1β, while itaconate contributes to antimicrobial activity. In contrast, alternatively activated M2 macrophages depend on oxidative phosphorylation (OXPHOS) and fatty acid oxidation, utilizing an intact TCA cycle to fuel anti-inflammatory and tissue-repair functions. Moreover, M2 macrophages also engage the pentose phosphate pathway (PPP) to support redox homeostasis and anabolic demands [20,21]. However, these distinct metabolic programs were not merely byproducts of polarization but can actively shape macrophage functional states [22]. Our previous study revealed that serum levels of IL-1β, IL-23, IFN-γ, and suPAR increased with rising blood chromium levels in chromate-exposed workers, with each one-unit increase in ln-transformed blood Cr associated with increases of 7.22%, 8.5%, 3.14%, and 9.31%, respectively. This suggests a notable mediating role in club cell secretory (CC16) elevation [23]. Additionally, we observed significant alterations in the polarization status of macrophages within the lung tissue of C57BL/6J mice exposed to Cr(VI) aerosol (150 μg Cr(VI)/m³) for 13 weeks [24].

Although previous studies have reported macrophage activation, inflammatory signaling, and lung immune alterations following Cr(VI) exposure, how Cr(VI) perturbs macrophage polarization in parallel with immunometabolic remodeling and mitochondrial dysfunction at the cellular level remains insufficiently defined. This study aimed to delineate changes in macrophage functional state after Cr(VI) exposure by jointly profiling polarization associated markers, glucose handling through glucose uptake and glucose transporter expression, cellular ATP status, oxidative stress, mitochondrial integrity, and by relating these phenotypes to transcriptome wide pathway signatures.

2. Materials and Methods

2.1. Chemicals

Dulbecco’s modified Eagle medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA; 6124225) and fetal bovine serum (FBS; Procell, Wuhan, China; 164210-50) were used for routine cell culture. Phosphate-buffered saline (PBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA; C10010500BT). Potassium dichromate (K₂Cr₂O₇; Nanjing Reagent, Nanjing, China; purity ≥ 90.0%) was used as the Cr(VI) source and dissolved in ultrapure water to prepare stock solutions.

Cell viability was assessed using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan; CK04) and live/dead staining using calcein-AM/PI (Beyotime, Shanghai, China; C015M). For RNA experiments, TransZol reagent (Transgen, Beijing, China; ET101-01-V2) was used for RNA extraction, and Top Green qPCR SuperMix (TransGen, Beijing, China) was used for RT-qPCR. Secreted cytokines were measured using ELISA kits for mouse TNF-α (EK282) and mouse IL-10 (EK210) (Lianke Bio, Hangzhou, China).

For Western blotting, RIPA and SDS-PAGE reagents were obtained from EpiZyme (China). Protein concentrations were determined using a BCA protein assay (Beyotime, Shanghai, China; P0012). PVDF membranes and related consumables were obtained from Beyotime (Shanghai, China; FFP24). Chemiluminescence detection was performed using ECL substrate (GLPBIO, Montclair, CA, USA; GK10008). Intracellular ATP was quantified using an ATP assay kit (Beyotime, Shanghai, China; S0026). Glucose uptake was measured using 2-NBDG (Aladdin, Shanghai, China, N121715). Oxidative stress and mitochondrial function were assessed using DCFH-DA (Beyotime, Shanghai, China; S0033M) for ROS detection and JC-1 mitochondrial membrane potential assay kit (Beyotime, Shanghai, China; C2006), with CCCP provided in the kit as a positive control. Lipid peroxidation was quantified using an MDA assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China; A003-1-2).

2.2. Cell Culture and Cr(VI) Treatment

The mouse macrophage cell line RAW264.7 was purchased from Sunncell Biotech (Wuhan, China; ATCC-derived) and maintained in DMEM supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO₂. Potassium dichromate (K₂Cr₂O₇; Nanjing Reagent, Nanjing, China) was dissolved in ultrapure water to prepare a 3 mM stock solution, aliquoted, and stored at −20 °C. Working solutions were freshly prepared in DMEM immediately before experiments. Unless otherwise specified, cells were exposed to Cr(VI) at 0.63, 1.25, 2.50, 5.00, 10.00, and 20.00 μmol/L for 24 h.

2.3. Cell Viability

Cell viability was assessed using CCK-8 (Dojindo Laboratories, Kumamoto, Japan; CK04). Cells were seeded in 96-well plates (technical triplicates per condition), and experiments were independently repeated four times. After 24 h of exposure, 20 μL of CCK-8 reagent was added to the cells in each well, and absorbance at 450 nm was measured using a multifunctional microplate reader (CLARIOstar, BMG LABTECH, Ortenberg, Germany). Viability was evaluated by live/dead staining using calcein-AM/PI (Beyotime, Shanghai, China; C015M) according to the manufacturer’s protocol, and images were acquired using an inverted fluorescence microscope (Ts2R, Nikon, Tokyo, Japan).

2.4. RT-qPCR

Total RNA was extracted from cells that had been exposed for 24 h to different Cr(VI) doses using TransZol reagent. RNA concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 700 ng RNA, and quantitative PCR was performed using Top Green qPCR SuperMix (Transgen, Beijing, China) with 45 cycles of 94 °C for 5 s and 60 °C for 30 s. Relative mRNA expression was calculated using the 2^−ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Primer sequences are listed in Table S1.

2.5. ELISA

Culture supernatants were collected from four independent biological replicates per group and assayed in technical triplicates. Secreted TNF-α and IL-10 were quantified using ELISA kits (Mouse TNF-α, EK282; Mouse IL-10, EK210; Lianke Bio, Hangzhou, China). Absorbance was measured at 450 nm using a microplate reader (CLARIOstar, BMG LABTECH, Ortenberg, Germany).

2.6. Western Blotting

Cells were washed with PBS and lysed in ice-cold Radio Immunoprecipitation Assay (RIPA) buffer (EpiZyme, Shanghai, China, PC101). After washing, 150 μL of RIPA buffer was added to each well of a 6-well plate, followed by incubation on ice for 30 min. Cells were then scraped, and the lysates were transferred to sterile 1.5 mL microcentrifuge tubes. Lysates were centrifuged at 12,000× g for 10 min at 4 °C. Protein concentration was determined by BCA assay. Equal amounts of protein were separated by SDS-PAGE (EpiZyme, Shanghai, China, PG112) and transferred to PVDF membranes (Beyotime, Shanghai, China, FFP24, 0.2 μm). Membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with primary antibodies overnight at 4 °C (see Table S2 for antibody information, including vendor and catalog numbers); secondary antibodies were followed by diluted in TBST (EpiZyme, Shanghai, China, TF103) and incubated for 1 h at 37 °C. Bands were visualized using Enhanced Chemiluminescence (ECL; GLPBIO, Montclair, CA, USA; GK10008) and quantified with ImageJ (version 1.54p; National Institutes of Health, Bethesda, MD, USA), normalized to β-actin.

2.7. Bioenergetic and Glucose Uptake Assays

RAW264.7 cells were seeded in 6-well plates (5 × 10⁵ cells/well) in DMEM supplemented with 10% FBS and 1% penicillin streptomycin. After 12 h, the medium was replaced with DMEM (control; n = 3) or DMEM containing 2.50 μmol/L Cr(VI) (n = 3) for 24 h. Total RNA was extracted and quality-checked prior to library construction. Libraries were sequenced on an Illumina platform (SBS). Clean reads were aligned to the mouse reference genome, and gene counts were obtained using feature Counts; expression levels were reported as FPKM. Differential expression was analyzed using DESeq2 with Benjamini Hochberg FDR correction; genes with padj ≤ 0.05 and |log₂FC| ≥ 1 were defined as DEGs. GO/KEGG enrichment (clusterProfiler) and PPI analysis (STRING) were performed.

2.8. Transcriptomic Analysis

ATP levels were measured in cell lysates using the ATP assay kit (Beyotime, Shanghai, China, S0026) from four independent biological replicates per group, assayed in technical triplicates. Luminescence was recorded using a plate reader (CLARIOstar, BMG LABTECH, Ortenberg, Germany). Glucose uptake was assessed using 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Aladdin, Shanghai, China). 3 mM 2-NBDG stock solution was prepared in ultrapure water and stored at −20 °C. Cells were incubated with 100 μmol/L 2-NBDG for 1 h, and fluorescence was measured at Ex/Em = 485/535 nm using a plate reader (CLARIOstar, BMG LABTECH, Ortenberg, Germany).

2.9. Oxidative Stress and Mitochondrial Assessments

Intracellular ROS was assessed by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining (10 μmol/L) and analyzed by flow cytometry (Gallios 10-color, Beckman Coulter, Brea, CA, USA) using the FITC channel (Ex/Em = 485/525 nm). Mitochondrial membrane potential was assessed using JC-1 (Beyotime, Shanghai, China; C2006). CCCP provided in the kit was used as a positive control (10 μmol/L). Fluorescence images were acquired using a laser confocal microscope (DMi8, Leica, Wetzlar, Germany); JC-1 monomers were detected at Ex/Em = 514/529 nm and aggregates at Ex/Em = 585/590 nm. The red/green fluorescence ratio was quantified using ImageJ. For mitochondrial ultrastructure, cells were fixed with 2.5% glutaraldehyde at 4 °C for 2 h and examined using TEM (JEM-F200, JEOL, Tokyo, Japan). Lipid peroxidation was evaluated by measuring MDA using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with three independent biological replicates per group, assayed in technical triplicates. Absorbance was measured at 532 nm and normalized to total protein concentration.

2.10. Statistical Analysis

Data were analyzed using GraphPad Prism 9.0. One-way ANOVA with Tukey’s post hoc test was used for multiple-group comparisons. Data are presented as mean ± SD, and p < 0.05 was considered statistically significant.

3. Results

3.1. Evaluation of Macrophage Proliferative Viability

As shown in Figure 1a, Calcein/PI staining of RAW264.7 cells revealed a progressive increase in PI-positive (dead) cells with increasing Cr(VI) concentrations. Consistently, Figure 1b demonstrates a concentration dependent elevation in cell death following Cr(VI) exposure. In parallel, CCK-8 results (Figure 1c) were normalized to the control group (set as 100%) to calculate relative cell viability. Cell viability remained above 90% at 0.63 and 1.25 μmol/L Cr(VI), indicating minimal cytotoxicity at these lower concentrations despite statistically significant differences compared with the control. At 2.50 μmol/L, viability decreased to approximately 80%, suggesting the onset of measurable intolerance to Cr(VI)-induced toxicity. At 5.00 μmol/L, viability declined to 50%, indicating pronounced cytotoxicity. As shown in Figure 1d, flow cytometric analysis revealed a concentration-dependent increase in intracellular ROS levels following Cr(VI) exposure, indicating enhanced oxidative stress.

3.2. Effects of Different Concentrations of Cr(VI) on the Expression of Polarization Biomarkers in Macrophages

RT-qPCR analysis of RAW264.7 macrophages exposed to Cr(VI) for 24 h showed concentration dependent changes in polarization-associated gene expression (Figure 2a–f). The mRNA levels of the M1-associated biomarkers TNF-α, CD36, and CD80 increased progressively with increasing Cr(VI) concentration (Figure 2a–c). By contrast, the M2 associated biomarkers displayed divergent patterns, Arg-1 showed no significant differences among groups, IL-10 increased with Cr(VI) concentration, and VEGFb decreased steadily (Figure 2d–f). ELISA measurements of TNF-α and IL-10 in culture supernatants (Figure 2g,h) showed an increase in both cytokines, with TNF-α levels consistently higher than IL-10 across conditions. Western blotting further showed a concentration-dependent increase in TNF-α protein abundance (Figure 2i,j).

3.3. Transcriptomic Analysis of Macrophages Exposed to Different Concentrations of Cr(VI)

As shown in Figure 3a, 310 genes were uniquely detected in the control group, whereas 273 genes were uniquely detected after exposure to 2.50 μmol/L Cr(VI). 10,532 genes were shared between the two conditions. Differential expression analysis is summarized in the volcano plot (Figure 3b). Using a nominal threshold of p < 0.05, 642 genes showed differential expression in response to Cr(VI), including 307 upregulated and 335 downregulated genes. KEGG enrichment analysis of the differentially expressed genes (Figure 3c) revealed prominent enrichment in inflammation and immune related signaling, including chemokine signaling, NF-κB signaling, Toll-like receptor signaling, and NOD-like receptor signaling, as well as pathways linked to cellular stress and metabolism, including cellular senescence, HIF-1 signaling, oxidative phosphorylation, mTOR signaling, and carbon metabolism.

3.4. Effects of Different Concentrations of Cr(VI) on the Energy Metabolism of Macrophages

As shown in Figure 4a, increasing concentrations of Cr(VI) result in a significant decrease in cellular ATP content, with the RLU (relative light unit) value of the 5.00 μmol/L group being about one-third of the control group, indicating a substantial decline in the cell’s ATP production capacity. To investigate glucose uptake in macrophages, the uptake of the fluorescent glucose analog 2-NBDG was measured, alongside a semi-quantitative analysis of the gene expression of glucose transporter Slc2a1 using RT-qPCR, which reflects the cell’s glucose uptake capability. As shown in Figure 4b,c, cells in all groups transported nearly equal amounts of 2-NBDG within the same time frame, while the expression of Slc2a1 increased with higher concentrations of Cr(VI). Notably, the expression level in the 5.00 μmol/L group was approximately three times that of the control group. Although ATP production decreases following Cr(VI) exposure in macrophages, glucose uptake capacity was actually enhanced.

3.5. Effect of Varying Cr(VI) Concentrations on Mitochondrial Membrane Potential, Oxidative, and Morphology in Macrophages

As shown in Figure 5a, JC-1 staining revealed a dose-dependent loss of mitochondrial membrane potential following 24 h Cr(VI) exposure. Control group cells showed predominantly red fluorescence, whereas Cr(VI) treated cells exhibited progressively increased green fluorescence, indicating mitochondrial depolarization. Figure 5b confirmed a significant, concentration-dependent decrease in membrane potential. In Figure 5c,d, Mitochondrial ultrastructure was further examined by transmission electron microscopy (TEM). In control cells (0.00 μmol/L), mitochondria displayed intact double membranes and well-defined cristae. By contrast, mitochondria in cells treated with 2.50 μmol/L Cr(VI) showed pronounced swelling, disrupted cristae, and blurred membrane boundaries, as indicated by the arrows. As shown in Figure 5e, Consistent with these structural alterations, malondialdehyde (MDA) levels increased with rising Cr(VI) concentrations, supporting enhanced lipid peroxidation.

4. Discussion

Cr(VI) is a potent environmental toxin that primarily targets the respiratory system upon inhalation. Once deposited in the lung tissue, Cr(VI) can readily penetrate alveolar macrophages, the immune cells responsible for engulfing and eliminating foreign particles [25,26]. The internalization of Cr(VI) by macrophages leads to its reduction to Cr(III) within the cells, generating reactive intermediates and free radicals. These reactive species trigger oxidative stress, DNA damage, and inflammatory signaling cascades, contributing to cellular dysfunction and immune activation [27,28]. Transcriptomic profiling in the present study revealed upregulation of genes enriched in pro-inflammatory pathways, including the NF-κB, TNF-α, and chemokine signaling pathways. KEGG enrichment further highlighted chemokine signaling pathways, supporting an inflammatory chemotactic response to Cr(VI) exposure. In contrast, pathways typically associated with M2 differentiation and repair programs showed weaker enrichment, aligning with limited changes in Arg-1 expression in both RNA-seq and RT-qPCR validation [29,30]. Together, these data suggest that Cr(VI) disrupts macrophage function through coordinated inflammatory activation, oxidative mitochondrial stress, and metabolic alterations.

Based on the current results, it can be concluded that Cr(VI) evoked activation does not conform to a strict M1 or M2 switch. The results are more consistent with an incomplete macrophage polarization phenotype [31]. M1-associated readouts (TNF-α, CD36, and CD80) increased with Cr(VI), indicating a shift toward a pro-inflammatory activation state. However, M2-associated biomarkers did not show concordant changes. Arg-1 remained largely unchanged, IL-10 increased, whereas VEGFb decreased. The discordant regulation of IL-10, VEGFb, and Arg-1 indicates that Cr(VI) does not elicit a uniform M2 program. Overall, the marker pattern supports a mixed activation phenotype with predominant M1 characteristics, rather than a single uniformly polarized state. At the same time, Cr(VI) reduced cellular ATP levels while increasing Slc2a1 (GLUT1) expression and maintaining or enhancing glucose uptake, suggesting a compensatory shift toward glycolytic metabolism under energetic stress. This pattern is compatible with a Warburg-like metabolic adaptation reported in inflammatory macrophage activation [32,33]. At the same time, Cr(VI) increased ROS, reduced mitochondrial membrane potential, altered mitochondrial ultrastructure, and elevated MDA, collectively indicating oxidative damage and mitochondrial dysfunction. This mitochondrial damage provides a plausible upstream constraint on oxidative phosphorylation, thereby favoring glycolytic compensation. The supplementary morphology and phalloidin staining data (Figure S1) provide a supportive context for the functional readouts. Cr(VI) exposure induced visible changes in cell shape and cytoskeletal organization, which are commonly associated with macrophage activation, adhesive and migratory behavior, and cellular stress responses.

Several limitations should be considered when interpreting these findings. First, the study relied on an immortalized RAW264.7 macrophage cell line. Although widely used and reproducible, it may not fully recapitulate primary macrophage biology. Second, the polarization assessment was based on a limited marker set, with most readouts measured at the mRNA level and only selected cytokines validated at the protein level; broader protein-level phenotyping and single-cell approaches could better resolve mixed activation states. Third, the experiments did not distinguish macrophage subtypes relevant in vivo (e.g., resident alveolar macrophages versus recruited monocyte-derived macrophages), nor did they model tissue microenvironmental factors such as epithelial immune crosstalk, extracellular matrix cues, or cytokine gradients [34]. Finally, while the data support concurrent polarization-associated changes and metabolic/mitochondrial disruption, direct causal links and upstream regulators were not interrogated.

5. Conclusions

This study sheds light on the complex effects of hexavalent chromium on macrophage functionality, emphasizing the dual impact on polarization and energy metabolism. The findings underscore the potential of Cr(VI) to compromise immune responses through metabolic reprogramming and mitochondrial damage. Further research is needed to explore the long-term implications of Cr(VI) exposure on immune health and to develop strategies for mitigating its harmful effects.