Resveratrol Possesses Protective Effects in a Pristane-Induced Lupus Mouse Model

Background Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease characterized by the production of autoantibodies. To date, no therapy has been found to satisfactorily treat SLE. SIRT1 deficiency results in the development of an autoimmune syndrome in mice, including a high titer of anti-nuclear antibody in serum, immunoglobulin deposition in the kidney, and immune complex glomerulonephritis. Resveratrol is an activator of SIRT1 and possesses anti-inflammation and immune-regulatory properties. Objective To evaluate the preventative effects of resveratrol on a pristane-induced lupus animal model and assess its putative immune modulation effects. Methods BALB/c mice received a single intraperitoneal injection of 0.5 ml of pristane on day 1 and then various doses of resveratrol were given to the mice daily starting on day 2 and continuing for seven months. The autoantibodies in serum and supernatants were measured. Single cells isolated from spleen, isolated CD4+ T cells, and CD19+ B cells were cultured with or without resveratrol in vitro and assessed by flow cytometry. Results Resveratrol attenuated proteinuria, immunoglobuin depositon in kidney, and glomerulonephritis as well as IgG1 and IgG2a in serum in pristane-induced lupus mice. Resveratrol also suppressed CD69 and CD71 expression on CD4+ T cells as well as CD4+ T cell proliferation, induced CD4+ T cell apoptosis, and decreased CD4 IFNγ+ Th1 cells and the ratio of Th1/Th2 cells in vitro. In vitro antibody production and proliferation of B cells were also inhibited. Conclusion Resveratrol possesses protective effects in pristane-induced lupus mice and may represent a novel approach for the management of SLE.


Introduction
Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease characterized by autoantibodies to components of the cell nucleus. Pathogenic autoantibodies are the primary cause of tissue damage in patients with SLE. Development of the disease is thought to arise due to genetic factors together with environmental triggers [1,2,3]. In addition, renal damage is the most important predictor of mortality [4]. However, despite intensive research, no therapy to date has been found to cure SLE, and those that adequately treat the disease may have severe and unfavorable side effects.
Several SLE animal models have been established and play an important role in investigating the mechanisms of the disease. One model is the pristane-induced lupus mouse model, which develops several autoantibodies and immune-complex glomerulonephritis [5,6]. Studies have shown that these mice have disparate T cell requirements of two subsets of lupus-specific autoantibodies as well as the toll-like receptor 7 (TLR7)-dependent and FccR-independent production of type I interferon [7,8]. TLR7 is required for the production of autoantibodies and the development of murine lupus nephritis [9].
Several different elements of the immune system are potential targets for therapeutic intervention in patients with SLE [1,10,11]. Current therapeutics used to treat SLE, including glucocorticoids and cyclophosphamide (CTX), are directed at suppressing humoral immunity and the production of autoantibodies as well as helper T cells (Th) and B lymphocytes. In addition, a new generation of biological agents is currently under development; however, the long-term beneficial and adverse effects of such agents remain unknown [10,11]. Therefore, more effective drugs with a favorable safety profile are urgently needed. Many natural compounds possess immune-modulatory effects and have the potential for treating autoimmune diseases, such as SLE. In this study, we assessed the efficacy of resveratrol for treating SLE.
Resveratrol (3,5,4-trihydroxystilbene) is a natural antimicrobial compound found in various plants and fruits [12,13]. It has attracted great attention since the discovery of its cardioprotective properties several years ago [13,14], and the compound has been shown to also possess anti-inflammatory, immuneregulatory, antioxidant, and blood fat-regulatory properties [13,15]. Moreover, studies have shown that resveratrol can inhibit several experimental autoimmune diseases, including collagen-induced arthritis, encephalomyelitis, colitis, and diabetes, though the mechanisms are not fully understood [16,17,18,19,20].
Resveratrol is an activator of silent mating type information regulation 2 homolog 1 (SIRT1), which is a class III histone deacetylase [13]. SIRT1 deficiency results in the development of an autoimmune syndrome in mice that manifests as a high titer of anti-nuclear antibody in serum, immunoglobulin deposition in the kidney, and immune complex glomerulonephritis [21,22].
However, to date, the effects of resveratrol on SLE and pristane-induced lupus have not been explored. Therefore, in this study we evaluated whether resveratrol can prevent the development of pristane-induced lupus in a mouse model.

Mice
Female BALB/c mice that were 9-10-weeks-old were obtained from Weitonglihua, Ltd. (Beijing, China). All mice were housed at Peking University on a diurnal 12 h light/dark cycle. All experimental protocols described in this study were approved by the Ethical Committee for Animal Experimentation of Peking Union Medical College Hospital.

Induction and treatment of pristane-induced lupus in mice
Forty female BALB/c mice were randomly divided into the following four groups: (1) Resveratrol A group: 10 BALB/c mice received a single intraperitoneal (i.p.) injection of 0.5 ml of pristane (Sigma-Aldrich, USA) on day 1 and were fed with resveratrol (Sigma-Aldrich, USA) (50 mg/kg/d) daily starting on day 2 for seven months; (2) Resveratrol B group: 10 BALB/c mice received a single i.p. injection of 0.5 ml of pristane on day 1 and were fed with resveratrol (75 mg/kg/d) daily starting on day 2 for seven months; (3) Model control group: 10 BALB/c mice received a single i.p. injection of 0.5 ml of pristane on day 1 but were not given resveratrol; and (4) Normal control group: 10 BALB/c mice not injected with pristane and not fed with resveratrol.

ELISA
Antibody concentrations were determined by ELISA. The anti-nuclear antibody (ANA), anti-ds-DNA, and anti-RNP/Sm antibodies were diluted 1:100 in murine serum. (ANA ELISA, Anti-ds-DNA ELISA, and Anti-nRNP/Sm ELISA, respectively; Euroimmun Medizinische Labordiagnosika AG, Germany). For ANA, the secondary antibodies were goat polyclonal anti-mouse IgG-Fc conjugated to horseradish peroxidase (HRP) (Abcam, USA) and goat polyclonal anti-mouse IgM-mu chain conjugated to HRP (Abcam, USA). For the anti-ds-DNA and anti-RNP/Sm antibodies, the secondary antibody was a goat polyclonal anti-mouse IgG-Fc conjugated to HRP (Abcam, USA). Assessment of supernatants and the determination of serum Ig isotypes were performed using the Mouse Immunoglobulin Isotyping ELISA Kit (BD Biosciences, USA). IFN-a were determined using the mouse IFN-alpha ELISA Kit (eBioscience, USA).

Proliferation assay
Freshly islated SMCs, CD4+ T lymphocytes, and CD19+ B lymphocytes were labeled with Carboxyfluorescein Succinimidyl Ester (CFSE) (CFSE cell proliferation kit, Invitrogen, USA) and cultured under various conditions. Four days later, the cells were analyzed by fow cytometry.

Apoptosis assay
Freshly isolated CD4+ T cells from the SMC fraction were cultured with or without resveratrol for 36 h in vitro. The cells were then stained with Annexin V/ propidium iodide (PI) (Alexa Fluor 488 Annexin V and PI kit For Flow Cytometry, respectively; Invitrogen, USA) to evaluate CD4+ T cell apoptosis by flow cytometry.

Histology
One kidney from each animal was fixed in 10% neutral-buffered formalin, cut into 5 mm-thick sections, and then stained with hematoxylin and eosin (H&E). Glomerular cellularity was evaluated by counting the number of nuclei per glomerular cross-section(30-40 glomerular cross-sections per mouse) after staining with hematoxylin and eosin. The other kidney from each animal was embedded in ornithine carbamoyltransferase and cut into 5-mm-thick sections for detecting IgG and IgM by direct immunofluorescence.

Statistical analysis
Clinical scores were analyzed using the non-parametric Mann-Whitney U test and histological scores were assessed using the one-way analysis of variance (ANOVA) test. Other data were compared using the Student's t-test or one-way ANOVA. The statistical significance of the various tests was examined by 2-sided hypothesis testing. P values ,0.05 were considered statistically significant. Statistical analysis was performed using SPSS version 17.0 (SPSS, Chicago, IL, USA).

Proteinuria
At the end of observation period, urinalysis revealed that the proteinuria of the normal control group was 0 or 1+, and 1+ to 3+ for the model control mice. The resveratrol A and resveratrol B groups had a lower proteinuria level compared to the model control group (Z52.013; P50.044; and Z52.071; P50.038, respectively; S1 Table). Therefore, resveratrol can prevent the development of proteinuria in this pristane-induced lupus model.

Histological pathogenesis
At the end of observation period, light microscopy of kidney sections revealed that 6 mice developed segmental or diffuse proliferative glomerular lesions with mononuclear cell infiltration among 10 model control mice (Fig. 1B): 3 mice among 10 resveratrol A group mice and 10 resveratrol B group mice, respectively. No mouse developed glomerular lesions among the normal control group mice. The mice from the resveratrol A group had a marked reduction in pathological lesions of the glomerulus compared to mice of the model control group (Fig. 1C) and slightly less lymphocyte infiltration was also observed. Similar results were detected in mice from the resveratrol B group as well (Fig. 1D).
The number of nuclei per glomerular cross-section, which is a measure of glomerular cellularity, was 39

Immunoglobuin G (IgG) and IgM deposition in kidney
At the end of the observation period, IgG deposition in kidney of normal control mice was negative, 022+ in model control mice, and 021+ in mice from the resveratrol A and B groups ( Fig. 2A-D, S2 Table). Moreover, the extent of IgG deposition in the kidney of resveratrol A and B mice was significantly lower than that in the model control mice (Z52.294; p50.022; and Z52.294; p50.022, respectively; S2 Table). However, the IgG deposition in mice from the resveratrol A and B groups was not significantly different (P.0.05). Similar results were observed for IgM deposition (Fig. 2E-H; S3 Table). Taken together, these results indicate that resveratrol attenuates pristane-induced nephritis.

Resveratrol inhibits IgG1 and IgG2a in pristane-induced lupus
To assess the immune mechanisms responsible for the resveratrol-mediated prevention of ongoing pristane-induced lupus, we assessed its role in the regulation of humoral immunity by measuring total immunoglobulin levels and ANA as well as anti-ds-DNA and anti-RNP/Sm antibody levels in serum. At the end of the observation period, the IgG1 levels in mice from the resveratrol A group were significantly lower than those in the model control group (2.08¡0.04 vs. 1.93¡0.04, respectively; P,0.01; Fig. 3A). Similar findings were observed for IgG2a levels between the two groups (Fig. 3B), but no differences were observed in IgG2b, IgG3, IgM, and IgA levels between the treatment and the model control groups (data not shown). Similar results were also detected in mice from the resveratrol B group (data not shown).
Throughout the observation period, ANA levels in mice from the resveratrol A and resveratrol B groups were lower than those in mice from the model control group, but the difference was not statistically significant (0.74¡0.32 vs.

IFN-a levels in serum
In light of the role of IFN-a in SLE, we analyse IFN-a in serum. At the end of observation period, IFN-a levels in mice from the resveratrol A and resveratrol B groups were lower than those in mice from the model control group, but the difference was not statistically significant (0.

Discussion
SLE is an autoimmune disease that responds poorly to current therapy options in many cases. Our study showed for the first time that resveratrol can attenuate proteinuria, decrease IgG and IgM deposition in kidney, and reduce kidney histological lesions, suggesting that resveratrol can effectively prevent pristaneinduced lupus in an animal model. CD4+ T cells and B cells are intimately associated with SLE and pristaneinduced lupus [1,7,23]. In humoral immunity, helper T cells interact with B lymphocytes and stimulate their proliferation and differentiation, resulting in the production of antibodies to components of the cell nucleus. Our results suggest that resveratrol has a profound inhibitory effect on CD4+ T cells and B cells. Moreover, we showed that resveratrol inhibits the activation of CD4+ T cells in vitro, similar to a previous report [24]. Our results also indicate that resveratrol inhibits the proliferation of CD4+ T cells and induces cellular apoptosis in vitro.
More than half of the T cells were already apoptotic (Anexin V+PI2) in the absence of treatment with resveratrol in our study, which is related to the culture duration of up to 36 h in vitro. Indeed, it is known that the viability of T cells  decreases as culture duration is prolonged [25,26]. In addition, resveratrol can inhibit the antibody production and proliferation of B cells in vitro, which is consistent with previous reports [27].
Taken together, our results suggest that resveratrol has protective properties in a pristane-induced lupus animal model that appear to be attributed to its inhibitory effect on CD4+ T cells and B cells.
The detailed mechanisms responsible for resveratrol-mediated effects remain unclear, though it is postulated that the compound may inhibit CD4+ T cells through effects on SIRT1. Resveratrol is an activator of SIRT1, which is essential for maintaining T cell tolerance through AP-1 in mice [21,28]. Resveratrolinduced apoptosis may be mediated by Fas, Bcl-2, Bax, p53, or by depolarizing mitochondrial membranes and activating Caspase 9 [29,30,31,32]. Resveratrol induces growth arrest through activation of FOXO transcription factors in prostate cancer cells [33] and suppresses the immune response through CD28/ CTLA-4 and CD80 co-stimulatory pathways [34].
Antigen-presenting cells (APCs) present antigens to naive T cells during the recognition phase of immune responses to initiate these responses, and some APCs present antigens to differentiated T cells during the effector phase to trigger elimination of the antigens. Resveratrol can affect dendritic cell maturation and the antigen presentation capacity in vitro [35]. However, our results do not rule out a regulatory role of resveratrol in dendritic cells.
Inflammation that is induced by the deposition of antigen-Ab complexes in kidney is an important mechanism for the development of glomerular lesions. Resveratrol inhibits COX expression by suppressing NF-kappa B activation [36] and also inhibits TNF-a-induced inflammation in fibroblasts by activating SIRT1 [37]. The resveratrol-mediated suppression of inflammation may also be benefical for reducing pristane-induced lupus glomerulonephritis.
Imbalance towards Th1 predominance is associated with acceleration of lupuslike autoimmune syndrome in MRL mice [38]. Recent findings in lupus glomerulonephritis show that Th1and Th17 cells have an essential role in the development of diffuse proliferative lupus nephritis and Th2 cytokines have an essential role membranous lupus nephritis [39]. Our study found that CD4 IFNc + Th1 cells, the ratio of Th1/Th2 cells, B cell, and Th1 cytokine promoting Ig (IgG2a, IgG3) decreased after treatment with resveratrol in a dose-dependent manner. Our findings suggest resveratrol may have effects for alleviating diffuse proliferative lupus nephritis. Resveratrol may also have effects for alleviating membranous lupus nephritis for it supressed Th2 cytokine promoting immunoglobulin (IgG1).
At the end of the observation period, ANA, anti-ds-DNA antibody and IFN-a levels in mice from the resveratrol treatment groups were lower than those in mice from the Model control group, but the differences were not statistically significant. The mechanism for this result remains unclear and will require further investigation through a well-designed study (including larger sample numbers) in the future.