Dietary Influence on Lung Injury and Immune Modulation in Cadmium-Exposed Mice

Abstract

Cadmium (Cd), a toxic heavy metal found in air pollution, poses serious risks to lung health due to its efficient pulmonary absorption and prolonged biological half-life. This study examines how ad-libitum (AL), time-restricted feeding (TRF), and intermittent fasting (IF) influence Cd-induced lung injury and immune responses in mice. Adult male C57BL/6 mice were pre-acclimated to AL, TRF, or IF regimens for three weeks, followed by intratracheal exposure to cadmium chloride (CdCl₂, 0.5 mg/kg). Lung mechanics were assessed using flexiVent, bronchoalveolar lavage (BAL) fluid was analyzed for inflammation, and immune profiling was performed on spleens and mediastinal lymph nodes (MLNs) 14 days post-exposure. Cd exposure increased immune cell infiltration in BAL fluid. IF mice showed significantly elevated inflammatory cytokines, while TRF mice had modest increases. Histological analysis revealed greater lung inflammation in TRF mice, whereas lung mechanics were more impaired in IF mice, suggesting distinct injury profiles. Immune profiling showed that IF reduced activated and effector T-cell populations in the spleen but increased them in MLNs, indicating a shift in immune localization. Furthermore, compared to the AL, Cd-exposed IF mice had minimal changes in T-cell distribution but reduced effector CD4⁺ and CD8⁺ T-cells in the spleen and increased in MLNs. In contrast, TRF mice exhibited minimal changes in T-cell distribution. These findings suggest that dietary regimens modulate immune responses and lung injury following Cd exposure. Feeding patterns play a critical role in shaping susceptibility to environmental toxicants and should be considered in future toxicological and immunological studies.

New & Noteworthy:

This study reveals that time-restricted feeding (TRF) and intermittent fasting (IF) distinctly modulate cadmium-induced lung injury and immune responses in mice. TRF worsened lung inflammation, while IF impaired lung function and altered immune cell distribution, indicating divergent mechanisms. These findings highlight how feeding patterns influence pulmonary responses to environmental toxicants and suggest that metabolic rhythms may shape airway immunity, offering new insight into dietary modulation as a potential strategy in lung injury management.

Graphical Abstract

Introduction

Cadmium (Cd), a highly toxic heavy metal commonly found in particulate matter (PM2.5), is a significant contributor to air pollution-related diseases (1, 2). Cd exposure primarily occurs through inhalation of cigarette smoke, PM2.5, and emissions from coal-fired power plants, smelting industries, and natural events such as volcanic eruptions and forest fires (3–5). The lungs, being the primary site of exposure, absorb Cd efficiently (~50%) compared to ingestion (~5%), allowing it to reach the alveolar space and exacerbate both acute and chronic lung diseases (6–8). Cd’s long biological half-life (10–30 years) further amplifies its toxic effects, as its slow elimination increases the risk of respiratory illnesses such as chronic obstructive pulmonary disease (COPD), emphysema, and pulmonary fibrosis (9–12).

Emerging evidence suggests that dietary interventions hold therapeutic potential in modulating metabolism and inflammation, both of which contribute to respiratory illnesses, including those caused by heavy metal exposure (13–15). Intermittent fasting (IF), also known as intermittent calorie restriction, and time-restricted feeding (TRF) are considered fasting-mimicking diets and are widely used in weight loss programs (16). Although few studies have examined the impact of dietary interventions on Cd-induced injury, the role of IF or TRF in this context remains poorly defined. One study found that a 24-hour fasting period exacerbated Cd toxicity in rats, likely due to reduced hepatic glutathione levels, which compromised the liver’s detoxification capacity (17). In contrast, TRF has been shown to attenuate maternal high-fat diet (HFD)-induced fetal lung injury and improve outcomes related to circadian rhythms, hyperinsulinemia, hepatic steatosis, and inflammation in mouse models (18). Furthermore, IF has demonstrated protective effects against lung injury in several models, including the reduction of lipopolysaccharide (LPS)-induced acute lung injury and bleomycin-induced lung inflammation and fibrosis (19, 20). However, other studies suggest that fasting-induced depletion of lung glutathione may increase susceptibility to hyperoxic lung injury, underscoring the complex and context-dependent nature of dietary interventions on lung health (21).

Both obesity and dietary restriction have been linked to changes in immune cell frequencies, metabolism, and functions (22–26). Studies demonstrate that dietary interventions can significantly modulate the innate immune system (27). For instance, IF in mice has been shown to promote the beiging of white adipose tissue through IL-22 secretion by type 3 innate lymphoid cells (ILC3) (28). In another study, TRF altered innate immune responses to LPS challenge, as evidenced by changes in cytokine levels in the serum and spleen of mice (29). TRF also reduced renal innate immune cell populations, thereby improving renal health in a mouse model of hypertension (30). Additionally, short-term fasting and TRF have been shown to decrease circulating monocyte levels in both mice and humans (31, 32).

Diet and diet-induced metabolic changes significantly influence adaptive immunity, particularly CD4⁺ T-cell responses (33). In humans, TRF has been associated with a reduction in circulating Th2, Th17, and senescent CD4⁺ T-cells, along with an increase in regulatory T-cells (Tregs) and follicular helper T-cells (34). In multiple sclerosis patients, IF reduced effector memory and Th1 cells while increasing naïve CD4⁺ T-cells (35). Additional studies demonstrate that fasting can suppress CD4⁺ T-cell activation and differentiation, with prolonged fasting exerting stronger effects via FOXO4 induction and its downstream targets (36). Furthermore, IF has been shown to reduce inflammation in patients with rheumatoid arthritis and in overweight adults with moderate asthma, highlighting its broader immunoregulatory potential, particularly in CD4⁺ T-cells (37, 38).

Mouse studies have shown that TRF and IF exert diverse effects on T-cell responses, depending on the disease model and inflammatory context (39–43). In a psoriasis model, TRF reduced Th2 and Th17 cells in the spleen, increased Tregs in skin lesions, and decreased senescent CD4⁺ T-cells (39). In HFD-fed mice, TRF normalized elevated CD8⁺ T-cell levels in epididymal fat to levels observed in low-fat diet controls (40). In obese mice with allergic contact dermatitis, IF promoted TGF-β expression in M2 macrophages, suppressed CD4⁺ T-cell proliferation, and increased Tregs (41). Similarly, in experimental autoimmune encephalomyelitis (EAE), IF reduced IL-17 expression and enhanced Tregs in the small intestine lamina propria, indicating anti-inflammatory effects (42). In contrast, a study using a 4-day fasting-mimicking diet reported increased IL-17 levels in serum and colonic supernatants (43). These findings underscore the context-dependent immunomodulatory effects of dietary interventions and highlight the importance of further research to guide the development of tailored nutritional strategies for inflammatory diseases.

We and others have previously demonstrated that cadmium (Cd)-induced lung injury disrupts T-cell homeostasis in secondary lymphoid organs in both mice and humans (44–46). However, the potential of IF and TRF to modulate Cd-induced alterations in CD4⁺ and CD8⁺ T-cell populations has not been explored. In this study, we aim to investigate the direct impact of dietary interventions, ad-libitum [AL], TRF, and IF, on T-cell responses in the lungs and secondary lymphoid organs of Cd-exposed mice. Findings from this work may inform the development of targeted dietary strategies to mitigate immune dysregulation associated with cadmium exposure.

Materials and methods

Animals

Male C57BL/6J mice of wild-type genetic background were acquired from Jackson Laboratory (Bar Harbor, ME) at approximately 8–10 weeks of age. The animals were maintained in standard housing conditions with a 12-hour alternating light and dark cycle and unrestricted access to food and water in the Research Support Facility vivarium at the University of Kansas Medical Center. The mice were housed until they reached 12–14 weeks of age for experimental use. All procedures involving animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and followed the ARRIVE guidelines. The experimental protocols received approval from the Institutional Animal Care and Use Committee at the University of Kansas Medical Center (Protocol #2020–2575-1).

Ad-libitum, time-restricted feeding, and intermittent fasting dietary regimens

Male wild-type mice (12–14 weeks old, weighing 18–22 g) were randomly assigned to six experimental groups: PBS-AL, Cd-AL (ad-libitum), PBS-TRF, Cd-TRF (time-restricted feeding), PBS-IF, and Cd-IF (intermittent fasting). The ad-libitum (AL) groups, which received no food or water restrictions, were included in parallel experiments reported previously (44) and are referenced here to enable direct comparison with the current findings. All mice underwent a three-week acclimation period to their designated dietary regimens prior to cadmium (Cd) or PBS exposure. In the TRF groups, food access was limited to the 12-hour dark phase (6:00 PM to 6:00 AM), while the IF groups received food every other day for a continuous 24-hour period. All groups were maintained on a standard 12:12-hour light/dark cycle. These feeding schedules were strictly followed throughout the study duration.

Cadmium-induced lung injury

To examine cadmium-induced pulmonary damage, we utilized adult male C57BL/6 wild-type mice. Male mice were selected due to their higher susceptibility to cadmium-induced lung injury, as previously reported (44, 47, 48), and because they exhibit minimal hormonal fluctuations compared to females, which can introduce variability in immune and inflammatory responses. Our experimental design, including the selection of male mice approximately 2 months of age, the use of cadmium chloride (CdCl₂), the specific dosage, and the 14-day evaluation time point, was established based on findings from our previous research and other published studies (44, 49, 50). Following an adaptation period, mice received an intratracheal instillation of sterile 1X PBS or CdCl₂ (0.5 mg/kg body weight; 40 μl volume) at Zeitgeber time 6 (ZT6: 12:00 PM). We monitored the animals’ well-being and weight changes daily, with all experimental evaluations consistently conducted 14 days post-exposure at the same circadian timepoint (ZT6).

Lung mechanics (flexiVent)

Prior to measurements, we calibrated the flexiVent apparatus (SCIREQ Inc., Montreal, QC, Canada) and connected catheterized mice to the system through Y-tubing to assess respiratory function parameters using standardized perturbations as outlined in our previous work (51). We employed the single forced oscillation technique (FOT) to evaluate fundamental parameters, including dynamic compliance (Crs), elastance (Ers), and resistance (Rrs), considering the lung as a unified compartment. We further utilized broadband FOT to measure tissue-specific variables such as tissue elastance (H), tissue damping (G), and Newtonian resistance (Rn), conceptualizing the lung as an integrated system of central airways connected to peripheral alveolar structures. We determined inspiratory capacity (IC) through deep inflation procedures, while the deflation curve slope (K) and quasi-static compliance (Cst) were calculated from pressure-volume (PV) loop maneuvers to examine quasi-static mechanical lung properties. We analyzed PV-loop data and graphical outputs to evaluate elastic recoil characteristics of both chest wall and lung tissues during respiratory cycles across the experimental groups. All respiratory function metrics were extracted and processed from the system’s exported datasets.

Bronchoalveolar lavage fluid and differential cell counts

Fourteen days following PBS or Cd exposure, mice were anesthetized using an intraperitoneal injection combining ketamine (90 mg/kg) and xylazine (10 mg/kg). A small incision was made to insert a 22-gauge, 25-mm catheter into the trachea, which was then secured with a suture. Using this catheter, we first instilled and retrieved 0.6 ml of normal saline from the lungs, collecting this initial bronchoalveolar lavage (BAL) fluid sample for proinflammatory cytokine analysis. We repeated this procedure twice more with 0.6 ml of saline to obtain additional BAL fluid. All samples were centrifuged at 2,500 rpm for 10 minutes at 4°C. The resulting cell pellets were resuspended in 1 ml of saline for total cell count analysis. We mixed equal volumes of the cell suspension with acridine orange-propidium iodide (AO/PI) dye and performed counts using the Countess 3 cell counter (Thermo Fisher Scientific). For differential cell counts, we prepared duplicate cytospin slides containing 50,000 cells each using the Thermo Scientific Cytospin 4 system and stained them with Kwik-Diff™ (Thermo Fisher Scientific) according to manufacturer instructions. The total protein concentration in the BAL fluid was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) following the manufacturer’s protocol.

Histopathology and lung inflammation assessment

The larger left pulmonary lobe was inflated with 1% low-melting agarose under gravity, then rinsed with 1× PBS. The lung tissue was subsequently fixed in 10% neutral-buffered formalin for 48 hours at 4°C. Subsequently, the fixed pulmonary tissues underwent progressive dehydration through a gradient of ethanol solutions (30%, 50%, and 70%) before paraffin embedding. Tissue sections were thinly sliced and subjected to hematoxylin and eosin (H&E) staining. The staining protocol involved xylene deparaffinization (three 5-minute washes), followed by rehydration through descending ethanol concentrations (100% twice, 95%, and 70%, each for 3 minutes), and distilled water rinsing for 5–10 minutes. Sections were exposed to hematoxylin for 1 minute, rinsed in distilled water for 5 minutes, treated with ammonia water, and rinsed again. After 1 minute in 95% ethanol, sections received eosin counterstaining for 1 minute, underwent dehydration in 95% ethanol (twice), clearing in xylene (three washes), and were mounted for examination. Histological assessment was performed in a blinded manner, with the evaluator unaware of the sample groups. The evaluation focused on Cd-induced inflammatory changes in the peribronchiolar, perivascular, and alveolar regions. A five-point scale (0–4) was employed for scoring lung inflammation, as previously outlined (44, 52). A score of 0 represented normal pulmonary architecture without immune cell infiltration, while scores 1–4 indicated increasing inflammation severity, with 4 signifying extensive immune cell accumulation in affected areas. For statistical evaluation, mean inflammation scores were determined for both control PBS- and Cd-exposed groups. To ensure consistency, all images were captured using either a Nikon (USA) tiling microscope or an ECHO Revolve microscope (5× or 20× magnification), with identical imaging settings applied across all samples.

Proinflammatory cytokine analysis

Measurement of pro-inflammatory cytokines was performed on BAL fluid maintained at –80°C using a custom LEGENDplex panel (BioLegend) according to the manufacturer’s protocol. An Attune NxT flow cytometer was employed for data collection, with subsequent analysis conducted using the LEGENDplex Qognit software as previously reported (44, 52).

Isolation of lymphoid organs and cell preparation

Mice were subjected to either ad-libitum (AL), time-restricted feeding (TRF), or intermittent fasting (IF) regimens and exposed to PBS or cadmium (Cd). After 14 days, their spleens and mediastinal lymph nodes (MLNs) were collected for analysis. Spleen tissues underwent homogenization and washing with 1× PBS, followed by treatment with 1.66% ammonium chloride solution to lyse red blood cells. The isolated splenocytes were subsequently washed with 1× PBS, passed through a 70-μm cell strainer, and suspended in FACS buffer (1% FBS in PBS). After counting, cells were labeled with fluorophore-conjugated antibodies to analyze T-cell populations via flow cytometry. For MLNs, tissues were homogenized in 1× PBS containing 0.1 mM EDTA, filtered through a 70-μm cell strainer, and resuspended in FACS buffer. These cells were then enumerated and stained with fluorophore-conjugated antibodies for flow cytometric assessment of T-cell populations.

Flow cytometry analysis

For cell surface marker detection, approximately 1.0 × 10⁶ cells were suspended in 100 μL of staining buffer (FACS buffer). Fluorophore-conjugated anti-mouse antibodies employed in this analysis included CD4-APC-Cy7, TCR-β-Pacific Blue, CD8-V500 or CD8-PE, CD62L-APC, CD44-FITC, and CD25-PE-Dazzle. Single-cell preparations derived from spleens and mediastinal lymph nodes underwent antibody incubation for 30 minutes at 4°C. Following the staining procedure, cells were washed with FACS buffer and subsequently resuspended in 100 μL of this same buffer prior to analysis. Data acquisition was performed using a Cytek^™ Aurora flow cytometer, with subsequent analysis conducted using FlowJo software (version 10.8.2; Becton, Dickinson and Company). The list of antibodies, kits, reagents, their sources, and identifier (Cat# and Research Resource Identifier) information is summarized in the key resources Table S1.

Statistical Analysis

Statistical comparisons between PBS AL, TRF, and IF groups were conducted using one-way analysis of variance (ANOVA) with Tukey’s post hoc multiple comparison test. When analyzing more than two groups (PBS vs. Cd-exposed AL, TRF, and IF), we employed a two-way ANOVA mixed model with a subsequent Tukey’s post hoc test using GraphPad Prism 9 (La Jolla, CA). Each experimental group included 5–8 biological replicates to ensure adequate statistical power and scientific rigor, based on our prior experience with preclinical studies. Most analyses were conducted in a blinded manner, either through random sampling or by keeping the evaluator unaware of group classifications. Outliers were identified and removed using either Grubbs’ test or the extreme studentized deviation (ESD) method, as determined by GraphPad Prism’s outlier calculator. Data are expressed as means ± SD, with statistical significance defined as P < 0.05.

Results

AL, TRF, and IF conditions alter T-cell homeostasis in the spleen and mediastinal lymph nodes of PBS-treated mice

In this study, mice were acclimated to one of three feeding regimens: ad-libitum (AL), with unrestricted access to food and water (44); time-restricted feeding (TRF), with access to food only during the 12-hour dark phase (6:00 PM to 6:00 AM daily); or intermittent fasting (IF), with access to food every other day for 24 hours, all maintained on a 12:12 light-dark cycle (Fig. 1A). To determine whether dietary interventions influence T-cell homeostasis in secondary lymphoid organs, we harvested the spleens and mediastinal lymph nodes (MLNs) from PBS-treated mice and assessed T-cell frequencies by flow cytometry (Fig. S1A; Fig. 1B). In the spleen, the percentages of T-cells were significantly higher in both TRF and IF mice compared to AL control, with the highest percentage observed in the IF mice (Fig. 1C). However, no differences in total T-cell percentages were observed in the MLNs among the TRF and IF mice (Fig. 1D). Correspondingly, total T-cell numbers were significantly increased in the spleens of IF mice compared to both AL and TRF mice, while MLNs showed no significant differences (Fig. 1C–D).

Fig. 1. Ad-libitum (AL), time-restricted feeding (TRF), and intermittent fasting (IF) regimens modified T-lymphocyte distributions in splenic and mediastinal lymph node (MLN) tissues of PBS-administered mice.

(A) Wild-type C57BL/6 male mice (~2–3 months of age) received either PBS or CdCl₂ (Cd: 0.5 mg/kg body weight [BW], administered at ZT6; 12:00 PM). Before experimental procedures, mice were habituated to either ad-libitum (AL: unrestricted access to food and water), TRF protocol, with feeding limited to the 12-hour dark phase (6:00 PM to 6:00 AM daily), or IF protocol, with feeding occurring every alternate day for 24 hours within a standard 12:12 light-dark cycle. These dietary patterns were sustained throughout the experimental period. (B-D) Cellular components from splenic tissue and MLN were extracted, enumerated, and immunophenotyped using fluorochrome-labeled antibodies targeting TCRβ, CD4, CD8, CD44, and CD62L to identify effector CD4⁺ (TCRβ⁺CD4⁺CD44⁺CD62L⁻) and CD8⁺ (TCRβ⁺CD8⁺CD44⁺CD62L⁻) T-lymphocytes. Illustrative flow cytometry gating strategy demonstrating CD4⁺ and CD8⁺ T-cell proportions in splenic samples. Percentages and absolute numbers of total T-cells (TCRβ⁺ population), CD4⁺, and CD8⁺ T-cells in splenic tissue and MLN from PBS-administered mice under all the dietary conditions. (E-F) Representative flow cytometry gating approach for effector CD4⁺ (TCRβ⁺CD4⁺CD44⁺CD62L⁻) and CD8⁺ (TCRβ⁺CD8⁺CD44⁺CD62L⁻) T-cells in splenic samples, accompanied by summary graphs depicting percentages and absolute counts under TRF and IF regimens. Effector CD4⁺ and CD8⁺ T-cell proportions and quantities in MLN of PBS-administered mice under all the dietary protocols. Results are presented as mean ± SD (n=5–8 mice/group). Statistical comparisons were conducted using one-way ANOVA with the Tukey post-hoc test for multiple comparisons, which was employed for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, indicate significant differences between the AL versus TRF, AL versus IF, and TRF versus IF PBS groups.

Next, we examined the frequencies and absolute numbers of CD4⁺ and CD8⁺ T-cells to assess specific impacts on these subsets. In the spleen, the percentages of CD4⁺ T-cells were significantly lower in both TRF and IF mice compared to AL mice, whereas the percentage of CD8⁺ T-cells was significantly higher in these mice (Fig. 1C). Despite lower CD4⁺ T-cell percentages, the absolute number of CD4⁺ T-cells was higher in IF spleens than in TRF spleens, likely reflecting the overall increase in total T-cell numbers in IF mice. Additionally, the number of CD8⁺ T-cells was significantly elevated in IF mice compared to both AL and TRF mice (Fig. 1C). In the MLNs, a similar pattern was observed; the percentage of CD4⁺ T-cells was significantly lower in both TRF and IF mice compared to AL mice, whereas CD8⁺ T-cell percentages were significantly higher in the IF mice relative to AL and TRF mice (Fig. 1D).

Since dietary intervention-mediated immune modulation resulted in an overall increase in T-cell numbers, we anticipated a corresponding expansion of effector T-cell populations, characterized by a CD44⁺CD62L⁻ phenotype. Therefore, we compared effector T-cell subsets between the AL, IF, and TRF groups. (Fig. 1E–F). Consistent with our expectations, the IF mice exhibited significantly increased percentages and absolute numbers of splenic CD4⁺ and CD8⁺ effector T-cells compared to the AL mice. In the MLNs of IF mice, however, the percentages of CD8⁺ effector T-cells remain unaffected, while the percentages of CD4⁺ effector T-cells were significantly increased and absolute numbers reduced (Fig. 1E–F). In TRF mice, the percentages of CD4⁺ effector T-cells in both the spleen and MLNs were higher than in the AL mice but remained lower than those observed in the IF mice. Notably, despite the increased percentage, the absolute number of CD4⁺ effector T-cells in the MLNs of TRF mice was lower than in the AL mice, suggesting a relative rather than absolute expansion (Fig. 1E–F).

To assess T-cell activation status, we measured the expression of CD25⁺. In the spleen, the TRF mice showed significantly increased percentages and absolute numbers of activated CD25⁺CD4⁺ T-cells compared to both AL and IF mice. (Fig. S1B). In contrast, CD8⁺ T-cells exhibited an opposite pattern with significantly reduced frequencies and absolute numbers of CD25⁺CD8⁺ T-cells in spleen and MLNs of both TRF and IF mice relative to AL mice (Fig. S1B-C). In the MLNs, no significant differences in CD25⁺CD4⁺ T-cell percentages or numbers were observed between the AL, TRF, and IF groups, except for a modest increase in the percentage of CD25⁺CD4⁺ T-cells in TRF mice compared to IF mice (Fig. S1C). Overall, these findings indicate that dietary interventions differentially modulate T-cell homeostasis, activation, and effector function in both the spleen and MLNs, with IF favoring effector T-cell expansion and TRF influencing CD4⁺ T-cell activation in a compartment-specific manner.

Cadmium exposure affects body weight under AL, TRF, and IF conditions

We then assessed the impact of Cd exposure on body weight and found that the effects varied between the AL, TRF, and IF conditions. Body weight was not recorded in the PBS AL group, as it is generally unaffected. In contrast, Cd-exposed mice in the AL group exhibited transient weight loss during days 1–6 post-instillation, followed by recovery to baseline by days 10–11 (44). In TRF mice, Cd exposure initially led to a reduction in body weight, with significant weight loss observed between days 3 and 5 post-exposure. Gradual recovery began by day 7, with body weight returning to baseline levels by days 10–11. In IF mice, Cd exposure resulted in a marked reduction in body weight between days 3 and 6. Recovery was noted after day 7, with body weight returning close to baseline by days 13–15 (Fig. S2).

Cadmium-induced lung inflammatory responses under AL, TRF, and IF conditions

In this study, the lung inflammatory responses were assessed in mice maintained under AL, TRF, and IF conditions following Cd exposure, with the respective PBS-treated groups serving as controls (Fig. 1A). In the AL group, 14 days after Cd exposure, we observed a significant increase in both total and differential cell counts in bronchoalveolar lavage (BAL) fluid. Specifically, the Cd-exposed AL group showed a marked increase in total cells, macrophages, eosinophils, and neutrophils compared to the PBS-treated control group (Fig. 2A) (44). Cd exposure significantly increased the total cell count in the BAL fluid of both the TRF and IF groups compared to their respective PBS controls. Specifically, Cd exposure led to a significant increase in macrophages and neutrophils in the BAL fluid of both TRF and IF conditions (Fig. 2A). Additionally, Cd exposure significantly elevated eosinophil counts in the BAL fluid of the IF group, while the AL and TRF groups showed a trend toward increased eosinophil counts compared to the PBS control. Intergroup comparisons revealed that the Cd-exposed IF group exhibited significantly higher eosinophil counts compared to the Cd-exposed AL group (Fig. 2A).

Fig. 2. Dietary regimens influence cadmium-induced inflammatory responses in mouse lungs.

Wild-type male C57BL/6 mice (~2–3 months of age) received either PBS or CdCl₂ (0.5 mg/kg BW) administered at ZT6 (12:00 PM). Throughout the experiment, mice were maintained on one of three feeding protocols: ad-libitum (AL: unrestricted access to food and water), time-restricted feeding (TRF; food available from 6:00 PM to 6:00 AM daily), or intermittent fasting (IF; 24-hour feeding periods on alternate days). (A) BAL fluid cellular composition was evaluated using Diff-Quik staining 14 days after PBS or Cd administration in all the feeding groups. (B) BCA assay was employed to quantify total protein content in BAL fluid 14 days post-exposure in AL, TRF, and IF cohorts. (C) Inflammatory cytokines and chemokines (IL-6, TNFα, IP-10, and KC) in BAL fluid were quantified via custom-designed LegendPlex assay across all experimental groups. Results are presented as mean ± SD (n = 5–6 mice/group). A two-way ANOVA mixed model with Tukey post-hoc test was employed for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, indicate significant differences compared to respective PBS controls. ^# P < 0.05, denotes a significant difference between AL versus IF or TRF versus IF Cd groups.

While the macrophage percentage was only marginally reduced in the Cd-exposed AL group, it was significantly decreased in both the TRF and IF groups. Among the Cd-exposed mice, the TRF group showed a significant reduction in macrophage percentage compared to the AL group and a marginal reduction compared to the IF group (Fig. S3A). Conversely, the neutrophil and eosinophil percentages were marginally increased in the Cd-exposed AL group but showed significant increases in the TRF and IF groups compared to their respective PBS-treated controls. Among Cd-exposed groups, the TRF group exhibited a more pronounced increase in neutrophil percentage compared to the AL group (Fig. S3A). We also assessed total protein levels in the BAL fluid as a measure of lung barrier integrity following Cd exposure under both TRF and IF conditions. A significant increase in total protein was observed in the TRF group post-Cd exposure, while the IF group showed a trend toward increased protein levels compared to the PBS control (Fig. 2B).

Proinflammatory cytokines in BAL fluid of PBS- and Cd-exposed mice under the AL, TRF, and IF conditions were analyzed using the LEGENDplex assay (Fig. 2C). In the Cd-exposed AL group, proinflammatory cytokines and chemokines such as IL-6, TNFα, IP-10 (interferon-gamma inducible protein 10)/CXCL10, and KC (keratinocyte-derived chemokine)/CXCL1 showed an increasing trend compared to the PBS-treated control. In contrast, other cytokines and chemokines, IFNγ, IL-4, IL-5, IL-13, IL-17A, IL-17F, IL-22, and IL-10, remained unaffected in both PBS- and Cd-exposed AL groups (Fig. S3B). In the Cd-exposed TRF group, IL-6, IP-10, and KC levels were marginally increased, while TNFα levels decreased compared to the PBS-treated control. All other cytokines and chemokines remain unchanged in the TRF group. In the Cd-exposed IF group, IL-6 and KC levels were significantly increased compared to the PBS-treated control, while TNFα and IP-10 showed an upward trend (Fig. 2C). However, the IF group exhibited a significant increase in IL-4, IL-5, IL-17A, IL-17F, and IL-22, with IL-13 and IFNγ showing increasing trends, and IL-10 levels remaining unchanged (Fig. S3B).

Intergroup comparison revealed that the Cd-exposed IF group exhibited higher IL-6 levels compared to the Cd-exposed AL and TRF groups. Additionally, KC levels were significantly increased in the IF group compared to the Cd-exposed TRF group. Basal levels of most cytokines and chemokines remain unaffected across the AL, TRF, and IF groups, except for TNFα, which showed an increasing trend in the TRF group compared to the AL and IF groups (Fig. 2C). Conversely, IL-17A, IL-17F, and IL-22 levels were marginally reduced in the IF group compared to the AL and TRF groups (Fig. S3B). Collectively, these findings indicate that Cd exposure leads to increased immune cell infiltration in the lungs, accompanied by elevated proinflammatory cytokine and chemokine levels in the BAL fluid, particularly in mice under the IF regimen compared to those under AL and TRF.

Cadmium-induced lung inflammation/injury under AL, TRF, and IF conditions

Histological analysis of lung sections revealed significantly increased inflammation in the peribronchial, perivascular, and alveolar regions of Cd-exposed mice compared to their respective PBS-treated controls across the AL, TRF, and IF groups (Fig. 3; Fig. S4). At baseline, peribronchial inflammation was significantly higher in the PBS-treated IF group compared to the AL group and marginally higher than the TRF group. In the perivascular region, baseline inflammation was modestly higher in the IF group relative to the AL and TRF groups. In the alveolar region, baseline inflammation was significantly elevated in the IF control group compared to both the AL and TRF controls. Among Cd-exposed groups, the TRF group exhibited significantly greater inflammation in the peribronchial and alveolar regions compared to both the AL and IF groups. Perivascular inflammation was also significantly higher in the Cd-exposed TRF group compared to the IF group and marginally higher than in the AL group (Fig. 3; Fig. S4). Overall, histopathological analysis and inflammation scoring indicated increased immune cell infiltration and more pronounced lung inflammation and injury in the airway, vascular, and alveolar regions of Cd-exposed TRF mice compared to those in the AL and IF groups.

Fig. 3. Histological assessment of cadmium-induced lung injury in mice under AL versus TRF versus IF regimens.

Male C57BL/6 (WT) mice (~2–3 months of age) received either PBS or CdCl₂ (Cd: 0.5 mg/kg body weight [BW]) at ZT6 (12:00 PM). Mice were previously adapted to either ad-libitum (AL: unrestricted access to food and water), time-restricted feeding (TRF), consuming food only during the 12-hour dark phase (6:00 PM to 6:00 AM daily), or intermittent fasting (IF), with food provided every alternate day for 24 hours within a standard 12:12 light-dark cycle. This dietary schedule was continued throughout the experimental period. Characteristic hematoxylin and eosin (H&E)-stained pulmonary sections revealed varying inflammatory intensities in peribronchial (PB), perivascular (PV), and alveolar (AV) compartments at 14 days following PBS or Cd administration under TRF and IF conditions. The accompanying graph depicts blinded semi-quantitative inflammation scores as outlined in the methodological section. Results are presented as mean ± SD (n = 6–8 mice/group). A two-way ANOVA mixed model with Tukey post-hoc test was employed for statistical analysis. *** P < 0.001, indicate significant differences compared to respective PBS controls. ^# P < 0.05, ^{# #} P < 0.01, ^{# # #} P < 0.001, denotes a significant difference between AL versus TRF, AL versus IF, and TRF versus IF PBS or Cd groups.

Cadmium-induced changes in lung mechanics under AL, TRF, and IF conditions

Lung mechanics were assessed in mice maintained under AL, TRF, and IF conditions 14 days after PBS or Cd exposure. Cd exposure resulted in a slight decrease in dynamic compliance (Crs) and increases in elastance (Ers) and resistance (Rrs) across all three groups compared to their respective PBS-treated controls. Additionally, Cd exposure significantly reduced the slope of the deflation curve (K) in all groups. Newtonian resistance (Rn) did not significantly increase in the Cd-exposed AL and TRF groups but was significantly increased in the Cd-exposed IF group compared to its PBS control. Notably, the baseline K value was significantly lower in the IF group compared to the TRF group. Among Cd-exposed mice, Rn was also significantly increased in the IF group compared to the TRF group.

A downward shift in the pressure-volume (PV) loop was observed in all Cd-exposed groups (AL, TRF, and IF) relative to their PBS-treated controls (Fig. 4). Other lung function parameters, including tissue damping (G), tissue elastance (H), and static compliance (Cst), were also affected, though changes were not statistically significant. Specifically, G increased marginally in the Cd-exposed AL and IF groups but remained unchanged in the TRF group. H showed a modest increase across all Cd-exposed groups compared to their PBS controls. Cst decreased marginally in the AL and IF groups following Cd exposure, but the TRF group showed a significant reduction in Cst relative to its PBS control (Fig. S5). Overall, these findings indicate that Cd exposure impacts multiple lung function parameters across TRF and IF groups, with the most changes observed in the TRF and IF groups compared to their respective controls.

Fig. 4. Distinct effects of cadmium exposure on pulmonary mechanics in mice under TRF and IF regimens.

Male C57BL/6 (WT) mice (~2–3 months of age) received either PBS or CdCl₂ (Cd: 0.5 mg/kg body weight [BW]) at ZT6 (12:00 PM). Mice were previously adapted to either ad-libitum (AL: access to food and water always), time-restricted feeding (TRF; food access limited to the 12-hour dark phase from 6:00 PM to 6:00 AM daily), or intermittent fasting (IF; 24-hour feeding periods on alternate days) within a standard 12:12 light-dark cycle. These feeding protocols were continued throughout the experimental period. After 14 days, lung mechanical properties, including dynamic compliance (Crs), elastance (Ers), resistance (Rrs), and Newtonian resistance (Rn), were assessed using the forced oscillation technique (FOT). The coefficient of elasticity (K) was determined from pressure-volume (PV) curves using the Salazar–Knowles equation. The PV curve demonstrates alterations in pulmonary mechanics following Cd administration in mice under both TRF and IF conditions. Results are presented as mean ± SD (n = 5–8 mice/group). A two-way ANOVA mixed model with Tukey post-hoc test was employed for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, indicate significant differences compared to respective PBS controls. ^# P < 0.05, denotes a significant difference between TRF versus IF PBS or Cd comparison.

Cadmium exposure alters T-cell homeostasis in the spleen and mediastinal lymph nodes of mice under AL, TRF, and IF conditions

We next examined the impact of Cd exposure on the immune cell populations within the secondary lymphoid organs of the AL, TRF, and IF groups. Cd exposure led to a reduction in both the percentage and absolute number of total CD3⁺ T-cells in the spleens of TRF and IF mice, but not in the AL group, when compared to their respective PBS-treated controls (Fig. 5A). In contrast, Cd exposure significantly increased the percentage of T-cells in the MLNs of TRF mice, whereas no such increase was observed in the IF group (Fig. 5B). When directly comparing Cd-exposed groups, the percentages of T-cells did not differ significantly between the AL groups. However, the absolute number of T-cells was reduced in the spleens of both TRF and IF mice and in the MLNs of the IF mice compared to the AL group (Fig. 5A–B).

Fig. 5. Cadmium exposure modified T-cell proportions and numbers in both spleen and mediastinal lymph node (MLN) tissues under AL, TRF, and IF regimens.

Following isolation and enumeration, cells from spleens and MLNs were labeled with fluorophore-conjugated antibodies targeting TCRβ, CD4, and CD8. (A). The upper panel shows percentages, while the lower panel displays absolute counts of total T-cells (TCRβ⁺ cells), CD4⁺, and CD8⁺ T-cell subsets in the spleens of AL, TRF, and IF mice. (B). Corresponding percentages and absolute counts of total T-cells (TCRβ⁺ cells), CD4⁺, and CD8⁺ T-cell populations in the MLNs of mice under AL, TRF, and IF conditions. Results are presented as mean ± SD (n=5–8 mice/group). A two-way ANOVA mixed model with Tukey post-hoc test was employed for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, indicate significant differences compared to respective PBS controls. ^# P < 0.05, ^{# #} P < 0.01, ^{# # #} P < 0.001, denotes a significant difference between AL versus TRF, AL versus IF, and TRF versus IF Cd-exposed groups.

We further analyzed the CD4⁺ and CD8⁺ T-cell subsets in the spleen and MLNs. In AL and TRF mice, Cd exposure led to a decreased percentage of CD4⁺ T-cells and an increased percentage of CD8⁺ T-cells in both organs (Fig. 5A; upper panel). However, the absolute numbers of these subsets showed opposite trends; both CD4⁺ and CD8⁺ T-cell counts were reduced in the spleen, while they showed an increasing trend in the MLNs compared to PBS control (Fig. 5A; lower panel). In the IF group, Cd exposure did not alter the percentage of splenic CD4⁺ and CD8⁺ T-cells (Fig. 5A; upper panel), but their absolute numbers were significantly reduced relative to PBS-treated IF mice (Fig. 5A; lower panel). In the MLNs of Cd-exposed IF mice, only the percentage of CD4⁺ T-cells was increased, with no significant changes observed in the absolute numbers of CD4⁺ or CD8⁺ T-cells compared to PBS controls (Fig. 5B; Fig. S6).

Compared to the Cd-exposed AL spleens, the percentages of CD4⁺ and CD8⁺ T-cells were not significantly different in TRF and IF mice; however, their absolute numbers were significantly lower (Fig. 5A). These findings suggest that Cd exposure under dietary restriction (TRF and IF) disrupts T-cell homeostasis in the spleen. In the MLNs, no major differences in T-cell percentages or absolute numbers were observed in TRF mice compared to the Cd-exposed AL group. In contrast, Cd-exposed IF mice exhibited an increased percentage of CD4⁺ T-cells in the MLNs, but this was accompanied by a reduction in absolute numbers of total T-cells, as well as CD4⁺ and CD8⁺ T-cells, compared to Cd-exposed AL mice (Fig. 5B).

Direct comparison of Cd-exposed TRF and IF groups revealed that IF mice exhibited more pronounced alterations in lymphoid T-cell homeostasis. Specifically, Cd-exposed IF mice had significantly higher percentages of CD4⁺ T-cells in both the spleen and MLNs and lower percentages of CD8⁺ T-cells in the MLNs compared to their TRF counterparts (Fig. 5). Despite these proportional differences, the absolute numbers of both CD4⁺ and CD8⁺ T-cells were markedly reduced in the MLNs of Cd-exposed IF mice relative to TRF mice, likely reflecting an overall reduction in total T-cell numbers. This trend was not evident in the spleens (Fig. 5A–B; Fig. S6). These contrasting patterns highlight the differential influence of dietary regimens, AL vs. TRF vs. IF, on shaping T-cell dynamics in secondary lymphoid organs in the context of Cd-induced lung injury, which may have important implications for disease outcomes.

Cadmium exposure alters CD4⁺ and CD8⁺ effector T-cells in the spleen and mediastinal lymph nodes of mice under AL, TRF, and IF conditions

Since we observed alterations in CD4⁺ and CD8⁺ T-cell frequencies and counts in Cd-exposed mice under TRF and IF regimens, we next investigated whether these dietary interventions also modulate effector T-cell populations. Effector T-cells, which play key roles in immune responses by rapidly producing cytokines, exhibit a memory/activated phenotype characterized by high expression of CD44 and low expression of CD62L (53, 54). In the spleens of AL and TRF mice, Cd exposure led to a significant increase in the percentage, but not the absolute number of CD4⁺ effector (CD44⁺CD62L⁻) T-cells, compared to PBS-treated controls (Fig. 6A). CD8⁺ effector T-cell populations were not significantly altered. Conversely, in Cd-exposed IF mice, we observed a reduction in both the percentages and absolute numbers of splenic CD4⁺ and CD8⁺ effector T-cells compared to PBS-treated IF controls (Fig. 6A; Fig. S6). In the MLNs of TRF mice, Cd exposure induced an increased trend in CD4⁺ effector T-cell counts compared to PBS controls, while CD8⁺ effector T-cells remained unaffected (Fig. 6B; Fig. S6). In contrast, Cd-exposed IF mice exhibited increased percentages, but not counts, of both CD4⁺ and CD8⁺ effector T-cells in the MLNs relative to their PBS-treated counterparts (Fig. 6B).

Figure 6. Cadmium exposure modified effector T-cell populations in splenic and mediastinal lymph node (MLN) tissues under AL, TRF, and IF regimens.

Splenic and MLN cells were harvested, enumerated, and labeled with fluorescent antibodies against TCRβ, CD8, CD44, and CD62L to identify effector CD4⁺ (TCRβ⁺CD4⁺CD44⁺CD62L⁻) and CD8⁺ (TCRβ⁺CD8⁺CD44⁺CD62L⁻) T-lymphocytes. Panel A depicts the percentages (upper) and absolute numbers (lower) of effector CD4⁺ and CD8⁺ T-cells in spleen samples. Panel B illustrates the percentages (upper) and absolute numbers (lower) of effector CD4⁺ and CD8⁺ T-cells in MLN samples. Results are presented as mean ± SD (n=5–8 mice/group). A two-way ANOVA mixed model with Tukey post-hoc test was employed for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, indicate significant differences compared to respective PBS controls. ^# P < 0.05, ^{# #} P < 0.01, ^{# # #} P < 0.001, denotes a significant difference between AL versus TRF, AL versus IF, and TRF versus IF Cd-exposed groups.

To further assess the differential effects of dietary interventions on Cd-induced immune alterations, we directly compared Cd-exposed AL, TRF, and IF groups. Notably, we observed opposing trends in T-cell responses between the two lymphoid organs. A marked contrast was evident between the AL and IF groups, particularly in effector CD4⁺ and CD8⁺ T-cell populations, while the TRF group showed minimal differences (Fig. 6A–B). In the spleen, Cd-exposed IF mice exhibited a significant decrease in both the percentage and absolute number of effector CD4⁺ and CD8⁺ T-cells compared to AL and TRF mice (Fig. 6A). In contrast, in the MLNs, Cd exposure resulted in an increase in the percentages of both effector CD4⁺ and CD8⁺ T-cells, as well as an elevated absolute number of CD8⁺ effector T-cells in the IF group relative to the AL and TRF groups (Fig. 6B). Together, these findings underscore the capacity of distinct dietary regimens, particularly, IF to differentially modulate effector T-cell responses following Cd exposure. The divergent responses observed between the spleen and MLNs suggest that dietary interventions may have affected T-cell activation, proliferation, or trafficking in a tissue-specific manner.

Cadmium exposure differentially alters activation of CD4⁺ and CD8⁺ T-cells in the spleen and mediastinal lymph nodes (MLNs) of mice under AL, TRF, and IF conditions

To evaluate the impact of Cd exposure and dietary regimens on T-cell activation, we assessed the expression of CD25, a well-established marker of T-cell activation and the α-chain of the IL-2 receptor, as described previously (44). In the spleens of TRF mice, Cd exposure resulted in a significant increase in the percentage of activated (CD25⁺) CD4⁺ and CD8⁺ T-cells compared to PBS-treated TRF controls, although no significant changes were observed in the absolute numbers (Fig. 7A; Fig. S6). Conversely, in the spleens of IF mice, Cd exposure led to a significant reduction in the absolute number of activated CD4⁺ T-cells (Fig. 7A). In the MLNs of TRF mice, Cd exposure caused a significant decrease in the percentage of activated CD4⁺ T-cells, while the activated CD8⁺ T-cells remained unchanged compared to PBS controls (Fig. 7B; Fig. S6). In contrast, Cd-exposed IF mice showed significantly increased percentages and absolute numbers of activated CD4⁺ T-cells in the MLNs compared to PBS-treated IF mice (Fig. 7B).

Fig. 7. Alterations in activated CD4⁺ and CD8⁺ T-lymphocytes in splenic and mediastinal lymph node (MLN) tissues following cadmium exposure under AL, TRF, and IF regimens.

Splenic and MLN cells were harvested, enumerated, and immunophenotyped using fluorochrome-labeled antibodies specific for TCRβ, CD4, CD8, and CD25 to identify activated CD4⁺ (TCRβ⁺CD4⁺CD25⁺) and CD8⁺ (TCRβ⁺CD4⁺CD25⁺) T-lymphocyte populations. (A). Proportions (top panels) and absolute numbers (bottom panels) of activated CD4⁺ and CD8⁺ T-cells in splenic tissue from mice subjected to AL or TRF or IF conditions. (B) Top panels display the proportions, and bottom panels show the absolute numbers of activated CD4⁺ and CD8⁺ T-cells in MLN tissue from mice that were subjected to AL or TRF or IF conditions. Results are presented as mean ± SD (n=5–8 mice/group). A two-way ANOVA mixed model with Tukey post-hoc test was employed for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, indicate significant differences compared to respective PBS controls. ^# P < 0.05, ^{# #} P < 0.01, ^{# # #} P < 0.001, denotes a significant difference between AL versus TRF, AL versus IF, and TRF versus IF Cd-exposed groups.

Direct comparison of Cd-exposed groups revealed opposing effects on activated CD4⁺ and CD8⁺ T-cells between TRF and IF mice relative to the AL group. In the spleens, Cd-exposed TRF mice exhibited higher percentages of activated CD4⁺ and CD8⁺ T-cells compared to AL mice, whereas IF mice showed decreased percentages of activated CD4⁺ T-cells (Fig. 7A). A similar trend was observed for the absolute number of activated CD4⁺ T-cells, while no differences were observed in the absolute number of activated CD8⁺ T-cells in the spleen (Fig. 7A). In the MLNs, Cd-exposed IF mice had significantly higher percentages of activated CD4⁺ T-cells compared to both Cd-exposed AL and TRF groups (Fig. 7B). Compared to AL mice, the absolute number of activated CD4⁺ T-cells was reduced in TRF mice, whereas no difference in the absolute number of activated CD4⁺ and CD8⁺ T-cells was observed between Cd-exposed IF and either the AL or TRF groups (Fig. 7B). Together, these findings demonstrate that IF exerts a distinct and compartment-specific influence on T-cell activation under conditions of Cd-induced immune stress. While IF dampens T-cell activation in the spleen, it appears to enhance T-cell activation in the MLNs, highlighting a potentially important spatial regulation of immune responses mediated by dietary context during environmental toxicant exposure.

Discussion

Cadmium (Cd) is a well-documented environmental and occupational pollutant, widely recognized for its harmful effects on lung health (55, 56). Previous studies, including ours, have demonstrated that Cd exposure induces lung inflammation, promotes a profibrotic response, and disrupts immune homeostasis (44, 57–59). Recently, we showed that Cd exposure leads to lung injury in mice and alters immune homeostasis within secondary lymphoid organs, such as the mediastinal lymph nodes (MLNs) and spleen (44). However, the impact of dietary changes, particularly time-restricted feeding (TRF) and intermittent fasting (IF), on Cd-induced lung injury and immune homeostasis in secondary lymphoid organs remains poorly understood.

This study aimed to explore the effects of AL, TRF, and IF on Cd-induced lung injury, with a particular focus on the immune profiles of secondary lymphoid organs, including MLNs and spleen. Our findings revealed that the dietary regimens differentially impacted body weight changes in the AL, TRF, and IF groups. Mice in the AL group exhibited a 15% reduction in body weight by day 3, followed by gradual recovery, with body weight returning to baseline levels by day 14 (44). In contrast, mice in the TRF and IF groups showed a greater reduction in body weight, approximately 20%, after Cd exposure but similarly recovered to baseline body weight by day 14.

Previous studies showed that Cd exposure leads to an increase in total and differential cell counts, particularly macrophages in BAL fluid (44, 60). Similarly, Cd exposure significantly increased the total cells, and macrophage counts in the BAL fluid across all groups (AL, TRF, and IF). While neutrophil counts were only marginally increased in the AL group, they were significantly increased in the Cd-exposed TRF and IF groups compared to their respective PBS controls. Notably, eosinophil counts were significantly higher in the Cd-exposed IF group, whereas the AL and TRF groups showed only a trend toward increased eosinophils. Increased BAL fluid protein levels indicate alveolocapillary permeability due to lung injury and are a sign of epithelial barrier dysfunction in most chronic lung diseases (61).

Cd exposure significantly altered proinflammatory cytokines in BAL fluid, as demonstrated in our study and in previous reports (44, 56, 62). Recent research has also highlighted that fasting and feeding regimens can substantially impact inflammation (15). For instance, a TRF regimen has been shown to offer protective effects in mice fed with a high-fat diet (HFD) during their active phase (9:00 PM to 6:00 AM). This approach mitigated HFD-induced fetal lung injury by reducing Il1β, Il6, and Tnfα transcript levels and activated phospho-NF-κB protein levels in lung homogenates compared to ad-libitum HFD-fed mice (63). Based on these findings, we hypothesized that TRF could potentially reduce proinflammatory effects in vivo following Cd-induced lung injury.

Our data from the Cd-exposed AL, TRF, and IF groups showed marginal increases in proinflammatory cytokines (TNFα and KC) in BAL fluid; however, these cytokines were significantly elevated in the Cd-exposed IF group compared to PBS control. Interestingly, Cd exposure led to a modest reduction in TNFα levels in the TRF group, whereas TNFα levels were higher in both the AL and IF groups. Notably, basal TNFα levels were substantially lower in the PBS-treated AL and IF groups compared to the TRF group, suggesting that TNFα may be a key cytokine modulated by dietary regimens in the lungs, warranting further validation.

It is well established that nutrition and systemic metabolism influence immune function, and accumulating evidence suggests that dietary interventions can broadly impact autoimmunity by modulating metabolic pathways within immune cells (64, 65). Immune cell metabolism and function are tightly interconnected, and metabolic changes at both the systemic and cellular levels can enhance or suppress specific T-cell subsets (66). For instance, periodic 3-day cycles of a fasting-mimicking diet (FMD) have been shown to ameliorate demyelination and clinical symptoms in a murine experimental autoimmune encephalomyelitis (EAE) model. Furthermore, both FMD and chronic ketogenic diets have demonstrated safety, feasibility, and potential benefit in patients with relapsing-remitting multiple sclerosis (RRMS) (64). These findings support the idea that dietary interventions can shape immune responses by regulating immune cell metabolism.

In the present study, the heightened Th cytokine responses observed in Cd-exposed mice under IF may be a consequence of such metabolic-immune interactions. Specifically, the elevated levels of Th1, Th2, and Th17 cytokines in the Cd-exposed IF group suggest that IF induces broad and robust immune activation, amplifying the lung’s inflammatory response to environmental toxicants like cadmium. Additionally, the increased release of the anti-inflammatory cytokine IL-10 in this group likely reflects a compensatory mechanism in response to heightened inflammation. This also highlights the potential role of dietary regimens in promoting the polarization of IL-10–producing monocytes, macrophages, and/or regulatory T or B cells, which may contribute to dampening immune responses in inflammatory settings. Notably, Th17-related cytokines (IL-17A, IL-17F, and IL-22) were higher in the Cd-exposed IF compared to the Cd-exposed TRF group, indicating that IF may more strongly promote Th17-mediated responses.

The heightened Th17 cytokine production and broader T-helper activation observed in the IF group could reflect fasting-induced metabolic changes that promote T-helper cell differentiation, as previously reported in both multiple sclerosis patients (35, 67) and murine models (41). However, further mechanistic studies are necessary to delineate how Cd exposure under the IF regimen modulates effector functions of T-helper subsets. Importantly, we did not observe significant differences in basal cytokine levels between the TRF and IF groups under control conditions, suggesting that neither fasting regimen inherently alters the lung cytokine milieu. Instead, our data indicate that IF and TRF differentially shape the immune landscape only upon exposure to Cd, likely through distinct metabolic-immune regulatory pathways. This highlights the potential for specific dietary regimens to modulate susceptibility to environmental toxins by influencing immune system responsiveness.

Existing literature supports the protective benefits of IF against lung injury in various models (19, 20). It has been shown to mitigate lipopolysaccharide (LPS)-induced acute lung injury and bleomycin-induced lung inflammation and fibrosis by reducing immune cell populations, particularly regulatory T-cells and monocytes (19, 20). Histopathological analysis revealed more pronounced inflammation and greater lung injury in the Cd-exposed TRF group compared to the AL and IF groups. Interestingly, under basal conditions, the PBS-treated IF group exhibited higher levels of lung inflammation relative to the AL and TRF groups, suggesting increased immune cell infiltration in the lungs of IF mice even in the absence of Cd exposure. Overall, we propose that the pre-existing inflammatory state induced by the dietary regimen at baseline may have directly influenced the progression of Cd-induced lung inflammation and injury, as well as the observed changes in immune homeostasis in secondary lymphoid organs, such as the MLNs and spleen.

To date, no studies have directly compared the effects of fasting and feeding on lung function parameters under both healthy and injury conditions. In our study, Cd exposure led to distinct alterations in lung function across the AL, TRF, and IF groups when compared to their respective PBS controls. All three groups exhibited marginal reductions in dynamic compliance (Crs) and increases in both elastance (Ers) and resistance (Rrs), likely reflecting changes in extracellular matrix remodeling and collagen deposition in response to Cd-induced injury. However, a notable finding was the significant increase in Newtonian resistance (Rn) in the IF group, indicative of large conducting airway obstruction. This suggests that IF may exacerbate airway inflammation and remodeling following Cd exposure. Furthermore, Rn values were significantly higher in the Cd-exposed IF group compared to the Cd-exposed TRF group, highlighting the differential impact of these dietary regimens on airway function. All Cd-exposed groups showed a reduction in the slope of the deflation curve (K) and a downward shift in the pressure-volume (PV) loop, both indicative of increased lung stiffness (44). Interestingly, the IF group exhibited lower K values both at baseline and after Cd exposure compared to the TRF group, suggesting a more pronounced reduction in lung compliance and elastic recoil in the IF group. Tissue damping (G) and tissue elastance (H) were only marginally affected across all Cd-exposed groups relative to their PBS-treated controls. However, Cd exposure significantly decreased static compliance (Cst) in the TRF group, whereas this effect was less pronounced in the AL and IF groups. This may indicate greater structural damage and parenchymal remodeling in the TRF group. In contrast, the IF group appears to exhibit a more immune-driven inflammatory response rather than structural injury. Together, these findings suggest that IF and TRF differentially influence the lung’s physiological response to Cd exposure, with IF exacerbating airway inflammation and TRF contributing to more structural lung alterations.

Previously, we demonstrated that Cd exposure alters CD4⁺ and CD8⁺ T-cell frequencies, as well as their activation and effector phenotypes, in secondary lymphoid organs such as the spleen and MLNs (44). In the present study, we investigated how these T-cell profiles are further modulated by dietary interventions, specifically, TRF and IF, in the context of Cd exposure compared to AL-fed mice. Our earlier findings showed that Cd exposure in AL-fed mice diet had no significant impact on splenic T-cell counts but led to an increase in T-cell counts in the MLNs (44). In contrast, our current data revealed that both TRF and IF regimens resulted in a reduction of splenic T-cell counts following Cd exposure compared to AL mice. Interestingly, in the MLNs, T-cell counts remained elevated after Cd exposure only in TRF mice but were still lower than the Cd-exposed AL mice. Furthermore, previously we reported that Cd exposure in AL-fed mice reduced CD4⁺ T-cell percentages while increasing CD8⁺ T-cell percentages in both the spleen and MLNs (44). A similar pattern was observed in TRF mice. However, in IF mice, we observed an opposing trend in the MLNs, with increased CD4⁺ and decreased CD8⁺ T-cell percentages. Overall, compared to the AL group, both TRF and IF regimens significantly reduced the percentage of CD4⁺ T-cells while increasing the percentage of CD8⁺ T-cells in the spleen. Interestingly, these differences were abolished following Cd exposure, which led to a reduction in the absolute number of both CD4⁺ and CD8⁺ T-cells. Notably, only the IF group exhibited a similar trend and significant alterations in CD4⁺ and CD8⁺ T-cell populations within the MLNs, suggesting a more pronounced and compartment-specific impact of IF on T-cell homeostasis under both basal and Cd-exposed conditions. These results suggest that IF may uniquely alter T-cell distribution and dynamics in draining lymph nodes, potentially influencing immune surveillance and inflammatory responses observed in the lungs. In contrast, TRF had limited impact on T-cell distribution in the MLNs.

Previous studies have demonstrated that IF and TRF regimens modulate immune cell metabolism and function in humans and mouse models (23). However, the direct effects of these dietary interventions on CD4⁺ and CD8⁺ T-cell activation and effector status during Cd-induced injury have not been previously explored. We have previously shown that Cd exposure disrupts CD4⁺ and CD8⁺ T-cell activation and effector phenotypes in secondary lymphoid organs (44). Building on that work, the present study reveals that, compared to TRF, IF significantly reduced both the percentages and absolute numbers of activated and effector CD4⁺ and CD8⁺ T-cells in the spleens of Cd-exposed mice. In contrast, IF induced a marked increase in activated and effector CD4⁺ and CD8⁺ T-cell populations in the MLNs, relative to TRF. Compared to AL, the TRF regimen did not significantly affect the effector responses of CD4⁺ and CD8⁺ T-cells following Cd exposure but differentially influenced the activation of these T-cell subsets in the spleen and MLNs. In contrast, the IF regimen appeared to attenuate T-cell activation in the spleen while enhancing activation within the MLNs. This dichotomy highlights a spatially regulated immune response shaped by dietary context, suggesting that IF may differentially modulate systemic versus mucosal immunity in the setting of environmental toxicant exposure. These contrasting patterns suggest a potential redistribution of activated and effector T-cells from peripheral lymphoid organs, such as the spleen, to draining lymph nodes under IF conditions during Cd-induced injury. This may reflect enhanced T-cell trafficking or infiltration, consistent with the elevated Th cytokine levels observed in the BAL fluid of IF mice. Limitations: While this study did not directly examine T-cell populations in the lung parenchyma, future work should explore how dietary alterations influence the distribution and function of lung-resident T-cells during environmental toxin exposure. Together, these findings highlight the differential immunomodulatory effects of IF and TRF in the context of Cd-induced lung injury and point to the importance of dietary context in shaping immune responses.

These findings highlight the intricate relationship between diet, immune response, and environmental exposures. While TRF appears to exacerbate structural lung injury and tissue remodeling, IF amplifies inflammatory and immune responses, particularly through modulation of T-cell activation, effector function, and trafficking within secondary lymphoid organs in cadmium (Cd)-exposed mice. The differential effects observed in this study suggest that nutritional status and feeding behaviors could be important factors to consider when assessing susceptibility to environmental toxicants and the pathogenesis of immune-mediated lung diseases. Further studies are needed to explore these relationships in broader contexts. Moving forward, future studies should aim to elucidate the molecular mechanisms underlying these diet-immune-environment interactions and evaluate therapeutic strategies that leverage dietary interventions to mitigate Cd-induced lung injury.

Supplementary Material

Table S1 and Figures S1-S6.

10.6084/m9.figshare.29257208