Immunotoxicity Studies of trans-Resveratrol in Male B₆C₃F₁/N Mice

Abstract

Resveratrol is a naturally-occurring polyphenol that is being investigated to treat and prevent various diseases, both experimentally and in the clinic. Despite increased use and interest in resveratrol due to its immunomodulatory properties, there is a lack of studies evaluating potential toxicities, particularly immunotoxicity, associated with resveratrol use. A previous 2-wk study found decreasing thymus weight in male B₆C₃F₁/N mice with increasing exposure to trans-resveratrol. This study is a follow-up on those findings by evaluating immune function. Male adult B6C3F1/N mice were given trans-resveratrol (0, 156, 312, 625, 1250, 2500 mg/kg/day) via oral gavage for 28 d and functional immune tests and histopathology were evaluated. There were no treatment-related effects on body weight during the study. Humoral, cell-mediated, and innate immune function were not altered after 28 d of trans-resveratrol treatment. There were also no changes in organ weight or microscopic alterations in immune organs. Overall, under the conditions of this study, there was no evidence of immunotoxicity or improvements in immune function associated with oral exposure to trans-resveratrol in male mice. Importantly, the immunomodulatory benefits of resveratrol may require a pre-requisite level of inflammatory activity and may not be observable in healthy individuals.

Introduction

Resveratrol is a naturally-occurring polyphenol that is found in grapes, peanuts, and various berries. Despite challenges with its bioavailability, resveratrol (typically trans-resveratrol) continues to be widely studied for its anti-inflammatory properties both experiment-ally and in clinical trials (Baur and Sinclair 2006; Ramirez-Garza et al. 2018). Numerous in vitro studies support the anti-inflammatory properties of resveratrol that could be harnessed for treatment of diseases that involve inflammation, such as cancer or cardiometabolic diseases (Bonnefont-Rousselot 2016; Oliveira et al. 2017; Springer and Moco 2019; Meng et al. 2020). Resveratrol appears to inhibit the production of inflammatory factors through activation of a regulatory deacetylase, sirtuin 1 (Sirt1). Activation of SIRT1 reduces expression of NF-κB-regulated genes responsible for leukocyte activation and inflammatory cytokine signaling, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and metalloproteases (Manna et. al. 2000; Singh et al. 2010). Other mechanisms of resveratrol include the reduction of oxidative stress through Nrf2 activation and inhibition of cyclooxygenase 2 (reviewed in Malaguarnera 2019).

Benefits of resveratrol have generally been shown in animal models of disease, such as in autoimmune disease models for systemic lupus erythmatosis, type 1 diabetes, diet-induced obesity, multiple sclerosis, colitis, and cancer (Jang et al. 1997; Yang et al. 2008; Wang et al. 2013, 2014; Jhou et al. 2017; Gandy et al. 2019; Alrafas et al. 2020). For example, after 13 wk of treatment with 250 mg resveratrol/kg/day, non-obese diabetic mice exhibited improved hyper-glycemia, decreased insulitis, and impaired trafficking of inflammatory T-cells into the pancreas (Lee et al. 2011). Resveratrol has also shown to be protective when administered with known toxicants (Singh [N] et al. 2011; Banu et al. 2016; Singh [I] et al. 2017; Ourique et al. 2016). Additionally, a number of clinical trials with trans-resveratrol have been or are being conducted (Park and Pexxuto 2015; Sawda et al. 2017; Pezzuto 2019). Many trials evaluate its use either alone or in conjunction with other therapies for reducing cancer risk (Hofseth et al. 2010; Patel et al. 2010; Howells et al. 2011; van Die et al. 2017). Resveratrol has also been tested for use in the management of chronic inflammatory diseases such as neurodegenerative disorders and diabetes (Moussa et al. 2017; Ramirez-Garza et al. 2018; Springer and Moco 2019). The benefits of resveratrol for prevention of disease in humans has yet to be conclusively identified in clinical trials (Smoliga et al. 2013). Research and development of ways to improve the low bioavailability of resveratrol associated with its rapid metabolism and clearance in both humans and rodents, increase bioefficacy, and investigate alternate routes of administration (e.g., dermally) of resveratrol further indicate that use of resveratrol will continue, if not increase (Ndiaye et al. 2011; Amri et al. 2012; Biasutto et al. 2017; Ratz-Lyko and Arct 2019).

Despite these advances in research, there are limited data on the long-term effects of resveratrol exposure, particularly at the higher doses being investigated for therapeutic use. Additionally, given the distribution of resveratrol as a dietary supplement as opposed to a pharmaceutical, there is a lack of adequate data available to evaluate potential toxicities associated with long-term resveratrol use. Resveratrol is generally well-tolerated in short-term clinical trials but it is unknown if there could be adverse effects associated with continuous resveratrol treatment. In particular, immuno- toxicity may exist given the immunomodulatory effects of resveratrol. In 14-day studies conducted by the National Toxicology Program (NTP), B₆C₃F₁/N mice given trans-resveratrol via gavage for 14 d exhibited a statistically significant trend of decreasing thymus weight with increasing exposure only in male mice, suggesting a potential effect on the immune system (NTP Toxicity Report 102, Table PA06 at https://tools.niehs.nih.gov/cebs3/views/?action= main.dataReview&bin_id=14563). The present paper describes results of a follow-up study to evaluate if trans-resveratrol affects immune function. To ensure sufficient exposure to resveratrol given its low bioavailability, the doses used in this study have been previously shown to produce internal doses in mice that are within 10-fold of reported internal doses in humans (Mutlu et al. 2020).

Materials and Methods

Chemicals and dose formulation

Trans-resveratrol (CAS #501–36-0; herein referred to as resveratrol) was obtained from Bayville Chemical Supply Co. Inc (lot #231AD; Deer Park, NY) and stored at −20°C. The identity of resveratrol was confirmed using infrared spectrometry and nuclear magnetic resonance spectrometry. Purity of the test article was estimated, via liquid chromatography, to be > 99%. Resveratrol doses were prepared in 0.5% methylcellulose and stored in the dark at −20°C in sealed, glass bottles. Resveratrol in formulation was determined to be stabile for up to 42 d; thus, formulations were prepared and used within 42 d. Positive controls used to assess assay performance were cyclophosphamide (CAS #6055–19-2; Sigma, St. Louis, MO), maleic vinyl ether, and anti-asialo GM1 rabbit antibody (Wako Bioproducts, Richmond, VA), which were diluted in phosphate-buffered saline (PBS).

Animals

Male B₆C₃F₁/N mice were obtained from Taconic Biosciences Inc. (Germantown, NY) at 4–8 wk-of-age. After 5 d of quarantine, mice were randomized using an Apple computer-generated randomization procedure to treatment groups. Mice were housed 1 animal/cage with hardwood bedding and maintained on the NTP-2000 diet and tap water ad libitum (City of Richmond). Animals were housed in a facility with a temperature between 64.4–78.8°F, relative humidity between 30–70%, and a 12-hr light/dark cycle. Studies were conducted under a protocol approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee in an AAALAC-accredited facility.

Study design

Mice were assigned to seven studies; endpoints evaluated in each study are listed in Table 1. In each study, mice were administered 0, 156, 312, 625, 1250, 2500 mg resveratrol/kg/day via oral gavage (0.1 ml/10 g body weight) for 28 d (N = 8/group). These doses are the same doses used in preliminary 14-day studies at the NTP where decreases in thymus weight were observed. Positive controls varied by study and included cyclophosphamide (CPS; 50 mg/kg/day via daily intraperitoneal (IP) injection on last 4 d of exposures), anti-asialo GM1 rabbit antibody (AA; 0.2 ml of a 1:10 dilution, via intravenous (IV) injection 24 hr prior to NK cell evaluation), and maleic vinyl ether (MVE; 50 mg/kg given IV ~24 hr prior to MPS evaluation).

Table 1.

Study design.

Study	Body Weight	Organ Weighta	Positive Control	Assays
1	X	-	CPS	Antibody-forming cell response to sRBC, serum IgM response to sRBC
2	X	-	CPS	Cytotoxic T-lymphocyte response
3	X	-	CPS	Serum IgM response to KLH
4	X	X	CPS, AAb	NK cell activity, anti-CD3 response, mixed leukocyte response
5	X	-	CPS	Antibody-forming cell response to sRBC, serum IgM response to sRBC
6	X	X	MVE	Histopathology
7	X	-	CPS	Mononuclear phagocytic system activity

CPS = cyclophosphamide; sRBC = sheep red blood cell; IgM = immunoglobulin mu; KLH = keyhole limpet hemocyanin; AA = anti-asialo GM 1 rabbit antibody; NK = natural killer; MVE = maleic vinyl ether.

X indicates the endpoint was evaluated.

^aOrgans weighed: liver, spleen, lung, thymus.

^bPositive control for the natural killer cell assay only

Body weight was measured on Day 1, 8, 15, 22, and 29 of the study. Animals were observed at the time of dosing in all cohorts. On Day 29, one day after the last dosing, animals were euthanized by CO₂ anesthesia and examined for gross pathology (e.g., spleen, thymus, liver, lungs, kidneys, adrenals). Organ weight (liver, spleen, lung, thymus, lung) was evaluated in Studies 4 and 6. Liver, kidneys, lung, spleen, bone marrow, proximal and distal lymph nodes, and thymus were collected from Study 6 animals, fixed in neutral buffered 10% formalin, embedded in paraffin, sectioned to 5 μm, and stained with hematoxylin and eosin (H&E) for histopathological examination.

Histopathology

At necropsy, the liver, spleen, lungs and bronchial-associated lymphoid tissue, thymus, kidneys, adrenals, bone marrow (femur), gastrointestinal tract with Peyer’s patches, and mesen-teric, submandibular, and popliteal lymph nodes were collected, fixed in 10% neutral buffered formalin, sectioned to 4–6 μm, and stained with H&E for histopathological evaluation, Lymphoid organs were evaluated using enhanced histopathology guidelines (Elmore 2006a-e); non-lymphoid organs were evaluated by traditional histopathology. All evaluations were conducted in accordance with the NTP Immunotoxicity Study Pathology Specifications (https://ntptools.niehs.nih.gov/policiesAndProcedures/NTP%20Immunotoxicity%20Specification%20Document%202016.pdf).

Immunological assays

T-cell dependent antibody-forming cell response to sheep erythrocytes (sRBC)

The primary IgM response to sheep erythrocytes (sRBC) was enumerated using a modi-fied hemolytic plaque assay (White et al., 2010). Mice in Studies 1 and 5 were immunized IV with 7.5 × 10⁷ sRBC (Colorado Serum Company, Denver, CO) on Day 25 of exposure. On Day 29, one day after the final treatment with resveratrol, animals were euthanized, and spleen cells were prepared. An aliquot of cells was added to a test tube containing guinea pig complement (Accurate Chemical Company, Westbury, NY), sheep erythrocytes, and warm agar. After thoroughly mixing, the test tube mixture was plated in a petri dish, covered with a microscope cover slip, and incubated at 37°C for 3 hr. Cell counts were performed and the number of cells/spleen, antibody-forming cells (AFC)/spleen and AFC/10⁶ spleen cells determined. The plaques that developed were counted using a Bellco plaque viewer (Bellco Glass Inc., Vineland, NJ). Data were expressed as specific activity (AFC/10⁶ spleen cells) and total spleen activity (AFC per spleen).

Serum IgM response to sRBC

An ELISA assay system (Temple et al. 1993) was used to determine the serum titers of the primary IgM response to sRBC in Study 1 and 5 mice. Briefly, sRBC membrane antigen was prepared at a 1:100 dilution of sRBC membrane preparation in PBS and incubated with Immulon 2 (Thermo-Fisher Scientific, Greenville, NC) microtiter plates at 4°C overnight. Serum samples diluted with the assay buffer were added to wells of the appropriate plates and probed with the secondary antibody (affinity-purified horseradish peroxidase-conjugated goat anti-mouse IgM antibody diluted 1:500 in PBS with 0.05% Tween 20). Plates were subsequently washed and incubated for 45 min with peroxidase substrate (2,2’-azino-bis[3-ethyl-benzthiazoline-6-sulfonic acid] (Thermo-Fisher Scientific, Waltham, MA). The color in each well was read at 405 nm on a Molecular Devices plate reader (Sunnyvale, CA). Results were obtained using SoftMax (v. 2.32, Molecular Devices) where the titer for each sample was determined using multi-point analysis.

Serum IgM response to keyhole limpet hemocyanin (KLH)

Mice in Study 3 were sensitized 5 d prior to study termination with 2 mg KLH (Thermo Fisher Scientific) per animal (IV). An ELISA was used to determine the antigen-specific serum concentration of the primary IgM response to KLH. In brief, 100 μl of a 5 μg/ml concentration of KLH were applied to Immulon-2 microtiter plates (Thermo-Fisher Scientific) and incubated at 4°C overnight. Plates were blocked with PBS-milk for 1 hr to prevent non-specific binding. After blocking, serum from each animal was diluted 1:32 with PBS-milk and added to wells of the Immulon-2 plates. After a 1-hr incubation at room temperature, horseradish peroxidase-conjugated goat anti-mouse IgM antibody was added at a 1:1000 dilution and allowed to incubate for 1 hr. Thereafter, peroxidase substrate (2,2-azino-bis[3-ethylbenzthia-zoline-6-sulfonic acid]; Sigma) was added; after 45 min, absorbance at 405 nm in each well was read on a Thermomax microplate reader (Molecular Devices). Results were obtained using Softmax. The sample absorbances were quantified against an anti-KLH IgM standard (purified mouse IgM anti KLH antibody, BD Pharmingen, San Jose, CA) and expressed as mean IgM (μg/ml).

Cytotoxic T Lymphocyte response

One day after the final resveratrol dose (Day 29), mice in Study 2 were euthanized and single-cell suspensions of splenocytes from control and treated mice were prepared. The cell concentration for each isolate was adjusted to 4 × 10⁷ total cells in 19.5 ml of medium (Eagle’s Minimal Essential Medium [E-MEM] supplemented with 10% fetal bovine serum [FBS], 25 mM HEPES, 1 mM L-glutamine, 50 μg gentamicin/ml and 10 μM 2-mercaptoethanol; all culture materials were purchased from GibcoBRL [Grand Island, NY]) and the total volume was then placed in a 25-cm² tissue culture flask (Corning, Corning, NY). Mitomycin C (50 μg mitomycin C/2 × 10⁷ cells)-treated P815 mastocytoma cells (ATCC; Manassas VA) were then added to each flask to yield a final responder-target cell ratio of 50:1, and the cultures then incubated in an upright position for 5 d at 37°C in 5% CO₂.

Following the incubation, cultured spleen cells were harvested and re-suspended in E-MEM media for determination of cytotoxic T-cell activity. For this, P815 cells cultivated in Dulbeccco’s Modified Minimal Essential Media (with 10% FBS) were labeled for 60 min at 37°C with 500 μCi ⁵¹Cr/2 × 10⁷ cells (as sodium chromate; Perkin Elmer, Wellesley, MA). Labeled target cells (2 × 10⁴ cells/well) were then co-cultured in duplicate with splenic effector cells in “U” bottom microtiter Costar culture plates to yield serial half dilutions of effector:target ratios from 25:1 to 0.75:1. After a 4-hr incubation at 37°C and 5% CO₂, the plates were centrifuged for 10 min at 300 × g. Radioactivity in the supernatant was measured in a Wizard®3 gamma counter (Perkin Elmer). The labeled target cells in the presence of E-MEM media or 0.1% Triton X-100 served as the spontaneous and maximum ⁵¹Cr release controls, respectively.

Mixed-leukocyte response (MLR) to DBA/2 mouse spleen cells

One day after the final resveratrol dose (Day 29), mice in Study 4 were euthanized and spleen cells prepared under aseptic conditions. Spleen cells in complete RPMI-1640 media supplemented with 10% FBS were added into wells of a U-bottom microtiter plate at a concentration of 10⁵ cells/well. DBA/2 mouse spleen cells were used as the allogeneic cell (stimulator cell) for the B₆C₃F₁ (responder) mice. Stimulator cells were treated with mitomycin C to render them unable to proliferate; the ratio of stimulators to responders had previously been optimized at 4:1. The cells were cultured for 5 d, the last 18 hr in the presence of 1 μCi [³H]-thymidine (Perkin Elmer). The cells were then collected with a cell harvester and counted in a liquid scintillation counter (LKB Instruments, Mount Waverly, Victoria, Australia). Incorpora-tion of [³H]-thymidine into the proliferating cells was measured as a surrogate for DNA synthesis and was expressed as counts per minute (cpm)/10⁵ cells.

Anti-CD3 cell response

Splenocytes isolated from mice in Study 4 were incubated in flat-bottom microtiter plates at a concentration of 2 × 10⁶ cells/ml in RPMI 1640 supplemented with 10% FBS and 50 μM 2-mercaptoethanol. The splenocytes were cultured in either control (non-treated) or T-cell activation plates (BD Biosciences, San Jose, CA) for 3 d. At 18–24 hr prior to harvest, the cells were pulsed with [³H]-thymidine and the total cpm/2 × 10⁵ spleen cells was then measured (see above).

Mononuclear phagocytic system (MPS) activity

One day after the final treatment, mice in Study 7 were injected IV with [⁵¹Cr]-labeled sRBC ([⁵¹Cr]-sRBC) at 10 μl/gram of body weight. The concentration of [⁵¹Cr]-sRBC was adjusted to a 10% hematocrit, i.e., equal to ≈ 5 × 10⁹ sheep erythrocytes/ml. Clearance of [⁵¹Cr]-sRBC from the blood was determined over the first 30 min (i.e., 3, 6, 9, 12, 15, and 30 min) by taking 5-μl blood samples via the tail vein from each animal after [⁵¹Cr]-sRBC injection. Due to the delayed clearance produced by MVE, the positive control, timepoints of longer duration (i.e., 5, 10, 15, 20, 30, and 60 min) were utilized for the MVE hosts. Radioactivity in the blood was used to determine the vascular half-life of the [⁵¹Cr]-sRBC. After 60 min, the animals were euthanized by decapitation and exsanguinated. Specific organs were then removed, weighed, and counted using the Wizard3 gamma counter to determine distribution of the [⁵¹Cr]-sRBC to the major organs of the MPS. Uptake into the liver, spleen, lungs, thymus, and kidneys was deter-mined and the data are expressed as percent uptake of the total counts injected in the total organ and cpm per mg tissue.

Natural killer (NK) cell activity

Splenocytes isolated from mice in Study 4 were placed in RPMI 1640 supplemented with 10% FBS at six concentrations, i.e., 2 × 10⁷, 1 × 10⁷, 5 × 10⁶, 2.5 × 10⁶, 1.25 × 10⁶, and 0.625 × 10⁶ cells/ml. The target cells, YAC-1 cells (10⁷ cells/ml), were labeled by incubation with 200 μCi of [⁵¹Cr, as sodium chromate; see above] for 90 min at 37°C with frequent agitation. The cells were then washed in RPMI with 10% FBS, counted, and adjusted to 10⁵ nucleated cells/ml. Equal volumes of target cells and isolated splenocytes (effector cells) were combined to obtain effector-to-target ratios of 200:1, 100:1, 50:1, 25:1, 12.5:1, and 6.25:1 (four replicates each). The maximum (total) release from the cells was determined by treating 10⁴ YAC-1 cells (0.1 ml volume) with 0.1 ml 0.1% Triton X-100. Spontaneous release was determined by adding 0.1 ml of medium to 10⁴ YAC-1 cells (0.1 ml volume) only. The plates were incubated for 4 hr at 37°C at 5% CO₂. At the end of the incubation, the plates were centrifuged at 250 × g for 10 min, 0.1 ml supernatant was removed from each well, and total [⁵¹Cr] present determined using the Wizard3 system. The mean (± SE) percent cytotoxicity at each effector concentration was deter-mined for each exposure group and compared to the comparable values for the vehicle mice. The results were expressed as: % cytotoxicity = 100 × (CPM_exp - CPM_spon)/(CPM_total - CPM_spon), where CPM_exp = counts in experimental wells, CPM_spon = total spontaneous release, and CPM_total = total release due to addition of Triton X. When treatment with the test compound or positive control resulted in cytotoxicity percentages ≤ 1.0, the response was considered 100% suppressed.

Statistical analysis

Data were analyzed in Provantis (Instem, Staffordshire, UK). Statistical analysis performed were a Jonckheere (trend) test and then a pair-wise test. Williams/Dunnett pairwise tests were used for organ weights, Shirley/Dunn pair-wise tests were used for all other endpoints. Statistical analysis for the positive control group compared to the vehicle control group was performed using the Kruskal-Wallis test. Significance was defined at a p-value ≤ 0.05.

Results

All study data, including that for each endpoint described below, are available in the NTP Chemical Effects in Biological Systems (CEBS) database: https://doi.org/10.22427/NTP-DATA-002–02772-0033–0000-6.

Mortality and body weights:

There were two deaths in the 1250 mg/kg/day groups and seven deths in the 2500 mg/kg/day groups that appeared to be due to the impaction of test article in the intestine (CEBS I01). There were no significant differences in body weight or body weight gain or any adverse clinical observations during any of the studies (CEBS I04).

Organ weight and histopathology:

At necropsy, one day after the final dosing (Day 29), there were no gross lesions noted in any of the animals. Differences in organ weight potentially due to trans-resveratrol were observed in the liver. Absolute and relative liver weights were signifi-cantly higher (~ 20% higher than control) in the 2500 mg/kg/day group compared to controls in Study 6 (CEBS PA06). In Study 4, relative liver weight in the 2500 mg/kg/day group was significantly higher by 8% compared to controls (CEBS PA06). Histological evaluation was done on tissues collected in Study 6. No treatment-related lesions were observed in the thymus, spleen, mesenteric lymph node, popliteal lymph node, mandibular lymph node, bronchial associated lymphoid tissue, gut associated lymphoid tissue, bone marrow, liver, kidneys, or lung (CEBS P09).

Humoral immunity:

Humoral responses in the mice were assessed using two T-cell dependent antigens, i.e., sRBC and KLH. There was not a consistent response to sRBC in the AFC assay: specific activity was elevated in resveratrol-treated mice at most doses in Study 1 but there was no significant change in any treated groups in Study 5 (Figure 1). IgM titers from resveratrol-treated animals were not significantly different from control in both Study 1 and 5. After KLH exposure, there were no significant differences in anti-KLH antibody concentration from control in any resveratrol-treated groups (CEBS M09). For both the sRBC and KLH assays, the vehicle controls were within historical ranges and CPS significantly decreased AFC response and antibody titers/concentrations, indicating that the assays performed as expected.

Figure 1. T-cell dependent antibody response to sRBC in male B₆C₃F₁/N mice after trans-resveratrol exposure for 28 d.

Mice treated with trans-resveratrol for 28 d were injected with sRBC 4 d prior to isolation of splenocytes. Positive control group mice were injected daily with 50 mg cyclophosphamide (CPS)/kg for 4 d prior to sacrifice. After Study 1 was completed, Study 5 was conducted to confirm findings. Specific activity (A, B) and total activity (C, D) are reported as means ± SEM; N = 6–8. *p ≤ 0.05 and **p < 0.01 as compared to vehicle control group.

Cell-mediated immunity:

Resveratrol treatment did not affect spleen cell proliferation after stimulation with anti-CD3 or allogenic leukocytes in the MLR (CEBS M11, M20V). There were no significant differences in the cytotoxic T lymphocyte response (Figure 2).

Figure 2. Cytotoxic T-lymphocyte response in mice after 28-day trans-resveratrol exposure.

Splenocytes (effector) isolated from exposed male mice were sensitized to P815 mastocytoma cells prior to incubation with [⁵¹Cr]-labeled target P815 cells. Cytotoxicity in assays using varying effector:target ratios is shown. Maleic vinyl ether (MVE) was used as the positive control in this assay. Data are shown as means ± SEM, N = 7–8. **p < 0.01 vs. control group.

Innate immunity:

In the mononuclear phagocytic system assay, resveratrol treatment did not modify the vascular half-life or uptake of [⁵¹Cr]-labeled sRBC into the liver, spleen, lungs, or thymus (Figure 3; CEBS M14V). Furthermore, resveratrol did not affect NK cell activity (Figure 4).

Figure 3. Functional activity of mononuclear phagocytic system in mice exposed to trans-resveratrol for 28 days.

Mice were injected with [⁵¹Cr]-labeled sRBC. (A) Clearance (vascular half-life) and uptake of radioactivity in the (B) liver and (C) spleen, shown as percent of total counts, over the subsequent 30 min were evaluated. A single dose of 50 mg maleic vinyl ether (MVE)/kg was used as the positive control here. Data are presented as means ± SEM, N = 5–8. **p < 0.01 vs. control.

Figure 4. Natural killer cell activity after 28-day exposure of mice to trans-resveratrol.

Splenocytes were isolated from mice (after 28-d exposure to trans-resveratrol via oral gavage) and incubated with [⁵¹Cr]-labeled YAC-1 target cells for 4 hr. After incubation, the splenocytes (effector) were combined with YAC-1 (target) cells at varying ratios and cytotoxicity was monitored by evaluating supernatant radioactivity. Anti-asialo GM1 rabbit antibody (AA; 1:10 dilution) was used as a positive control. Data shown are means ± SEM, N = 6–8. **p < 0.01 compared to control within respective effector:target ratio experiments.

Discussion

Growing interest in using resveratrol to treat disease necessitates a better understanding of its potential long-term toxicity. Here, we examined the immunotoxicity of resveratrol using various functional immune tests. Due to a previous study showing decreases in thymus weight only in male B₆C₃F₁/N mice, male mice were evaluated in this study. Notably, the internal doses of resveratrol in mice from the current study were similar to that seen in humans during clinical trials evaluating the therapeutic use of resveratrol. Specifically, the study in humans evaluated kinetics after oral administration of either 2.5 or 5.0 g of resveratrol (Boocock et al. 2007). Comparing systemic exposure data (i.e., C_max and AUC) from studies in mice and humans, the internal dose in mice given the doses in this study are 3- to 8-fold higher than that seen in humans (Mutlu et al. 2020). In addition, in B6C3F1/N mice given the same doses, the plasma concentrations of resveratrol in mice (4–7 μM; Mutlu et al. 2020) are within the range of concentrations where immune effects of resveratrol are seen in vitro (1 nM to 400 μM; Meng et al. 2020).

Resveratrol was well-tolerated by male mice throughout the 28-d study, with minimal changes in body weight and no adverse clinical observations. Unlike the 14-d study, there was no indication of a decrease in thymus weight. This discrepancy suggests that the decreasing trend in thymus weight observed previously in the 14-d study may be a sporadic finding or due to adaptations in the animal during the 28-d exposure. There were some increases in liver weight with no histopathological correlates, which may be due to the induction of hepatic enzymes involved in metabolism and/or anti-oxidant pathways that have been observed following resveratrol treatment (Baur and Sinclair 2006). Moreover, there were no treatment-related histo-logical lesions in any of the lymphoid or non-lymphoid tissues evaluated.

After 28 d of resveratrol treatment, there were no significant effects on humoral, cell-mediated, or innate immune function in male B₆C₃F₁/N mice. The discordant responses in the two T-cell dependent antibody response to sRBC assays are likely due to the lower, although still within historical range, AFC response in controls of Study 1. Overall, these data suggest that there was no toxicity, nor improvement of immune cell function, of resveratrol in a healthy, functioning immune system. These findings are in contrast to in vitro studies showing that resveratrol can modulate or inhibit T- and B-cell activity, suppress inflammatory responses in macrophages, and promote NK cell cytotoxic activity (Malaguarnera 2019). However, many of the immune effects have largely been observed in the presence of inflammation, either through disease (e.g., cancer, autoimmunity) or stimulation via microbial agents, high-fat diet, exercise, or chemicals (Meng et al. 2020).

The beneficial effect of resveratrol may lie in modulating the response of the immune system to a stimulant or a priori inflammation. For instance, in vitro incubation of human peripheral blood mononuclear cells with clinically-relevant concentrations of resveratrol (1–10 μM) augmented the proliferation of regulatory T-cells and γδT-cells only when cells were stimulated (Espinoza et al. 2017). Similarly, resveratrol did not increase TNFα or nitric oxide in unstimulated monocytes but did in lipopolysaccharide-stimulated monocytes (Gualdoni et al. 2014; Ma et al. 2015). While resveratrol was shown to improve energy metabolism for obese individuals (Timmers et al. 2011) and rodents (Bauer et al. 2006; Lagouge et al. 2006) where inflammation and oxidative stress are known to be present, there were no significant changes in non-obese women with normal glucose tolerance (Yoshino et al. 2012) and normal-weight rodents (Turrens et al. 1997; Jeon et al. 2012). Interestingly, resveratrol supplementation did not improve glucose tolerance or modify inflammatory markers in obese but metabolically-healthy men, suggesting that a certain inflammatory threshold may need to be present before resveratrol is effective (Poulsen et al. 2013).

Overall, these studies suggest that trans-resveratrol does not induce immunotoxicity in male B₆C₃F₁ mice administered doses of up to 2500 mg/kg/day. Resveratrol did not affect the adaptive or innate immune response in these mice at clinically-relevant doses. The immunomodulatory benefits of resveratrol may require a pre-requisite level of inflammatory activity and may not be observable in healthy individuals.