Resveratrol inhibits AhR/Notch axis and reverses Th17/Treg imbalance in purpura by activating Foxp3

Abstract

Background

Resveratrol has been reported to reverse the imbalance of T helper 17/regulatory T (Th17/Treg) by inhibiting the aryl hydrocarbon receptor pathway to treat immune thrombocytopenia. However, the regulation mechanism of the Notch signaling pathway by resveratrol has not been reported in purpura. This study is aimed to explore the mechanism of resveratrol ultrafine nanoemulsion (Res-mNE) in immune thrombocytopenia.

Methods

The immune thrombocytopenia mouse model was constructed to explore the effect of RES-mNE on immune thrombocytopenia. Cluster of differentiation 4 (CD4⁺) T cells were isolated and treated with different medications. CD4⁺ T cells were induced to differentiate into Th17 cells and Treg cells. Flow cytometry was used to detect the proportion of Th17 cells and Treg cells. The secretion was measured by the enzyme-linked immunosorbent assay (ELISA). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot were used to detect the mRNA and protein levels.

Results

Th17 cells, IL-17A and IL-22 increased in the immune thrombocytopenia mouse model, and the Treg cells and IL-10 decreased. Res-mNE promoted Treg cell differentiation and IL-10 secretion in CD4⁺ T cells while inhibiting Th17 cell differentiation and IL-17A and IL-22 levels. The AhR activator 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reversed the effect of Res-mNE. Notch inhibitors reduced the ratio of Th17/Treg differentiation. Res-mNE activated the expression of Foxp3 by mediating AhR/Notch signaling to reverse the imbalance of Th17/Treg differentiation in immune thrombocytopenia.

Conclusion

Taken together, our findings demonstrated that RES-mNE inhibited the AhR/Notch axis and reversed Th17/Treg imbalance by activating Foxp3.

Introduction

As an autoimmune disease, primary immune thrombocytopenia (ITP) is characterized by thrombocytopenia, with or without bruising of the skin and mucous membranes, epistaxis, and visceral bleeding.¹ The pathogenesis of ITP is mainly due to the excessive destruction of platelets mediated by humoral immunity and the mononuclear macrophage system and the inhibition of platelet production caused by cellular immune-mediated megakaryocyte damage.² Two subtypes of CD4⁺ T helper (Th) cells, including Treg cells and T helper 17 (Th17) cells, shared the same differentiation pathway and played contrary roles in immune tolerance and autoimmune diseases.³ Studies have shown that regulatory T (Treg) and Th17 jointly participated the pathogenesis of ITP and the imbalance between the two subtypes of Th cells played a vital role in the development of ITP.⁴ Immunoglobulin improved cellular immune function by adjusting the ratio of Th17/Treg. The Th17/Treg ratio was used as an indicator to evaluate the effect of ITP treatment.⁵ The aryl hydrocarbon receptor (AhR) works as a ligand-activated transcription factor and a highly conserved nuclear receptor, which has been paid more and more attention in immune and inflammatory diseases.⁶ Previous studies have shown that cinnamtannin D1 regulated Th17 and Treg differentiation to treat collagen-induced arthritis mice by inhibiting the expression of AHR.⁷ Studies have also shown that AhR signaling may affect the balance between Th17 cells and Treg cells.⁸ Therefore, we deduce that ITP may be treated by mediating the AhR pathway to regulate the imbalance between Th17 cells and Treg cells.

Resveratrol, as a natural polyphenol compound, has a wide range of beneficial properties, such as neuroprotective, cardioprotective, antioxidant, anticarcinogenic, and antiaging.^9,10 To improve the pharmacokinetic properties of the respiratory syncytial virus, various methods and different synthetic derivatives have been developed.¹¹ Resveratrol also revealed anti-inflammatory, anticatabolic, antiapoptotic, and antioxidant properties in various joint cell types (such as chondrocytes and synovial cells) and in T and B immunomodulatory specific lymphocytes.^12,13 Bakheet Saleh A et al. demonstrated that resveratrol could reduce the production and expression of the CCR and C-X-C motif chemokine receptor (CXCR) in CD4+ T cells, in the spleen and brain tissues.⁹ The mechanism of action of resveratrol suggested that resveratrol could regulate unbalanced endocrine, apoptosis, and oxidative stress. Resveratrol proved to be a strong AhR competitive antagonist (IC50 6 μM), which could inhibit the effects related to AhR activation.¹³

Resveratrol has been reported to participate in various signal pathways, such as TLRs, NF-κB, and the COX-2 signaling pathway. Resveratrol improved neuroimmune dysregulation by inhibiting neuronal Toll-like receptors and COX-2 signaling in BTBR T⁺ Itpr3^tf/J mice.¹⁵ Resveratrol ameliorated the dysregulation of Th1, Th2, Th17, and T regulatory cell-related transcription factor signaling in a BTBR T + tf/J mouse model.¹⁶ Previous studies have also shown that resveratrol could reverse the imbalance of Th17/Treg through the mechanism of inhibiting the AhR pathway to achieve the purpose of treating ITP.¹⁴ The Notch signal was involved in Th17 cell differentiation and function, and its inactivation could reverse Th17/Treg imbalance in ITP patients.^17,18 Notably, the effect of resveratrol on Notch1 signaling is conflicting, that is, resveratrol can enhance or inhibit Notch1 in a cell type-dependent manner.¹⁹ Studies have shown that the Notch signal could downregulate the expression of Foxp3, which inhibited Treg cell differentiation and promoted the occurrence and development of allergic rhinitis.²⁰ Moreover, proinflammatory mediators and Notch-1 signaling were proven to be associated with the imbalance of neuroimmune function in BTBR mice exposed to methylmercury chloride.²¹ However, the regulation mechanism of the Notch signaling pathway by resveratrol has not been reported in purpura.

Here, we speculate that resveratrol can reverse the Th17/Treg imbalance in purpura by inhibiting the AhR receptor and inhibiting the Notch signal to promote Foxp3 expression. Res-mNE was also prepared in our study, which showed better stability and solubility than pure resveratrol. The potential therapeutic benefits of resveratrol on the imbalance of Th17/Treg was explored using an ITP mouse model. Our findings would provide new ideas for the pathogenesis and treatment of ITP and contribute to the new drug discovery in the future.

Materials and methods

ITP mouse model construction

BALB/c mice (6 weeks, weight 18–22 g) were purchased. Our present study was permitted and approved by the second affiliated Hospital of Nanchang University Animal Ethics Committee (No. 2020117).

Preparation of antiplatelet serum (GP-APS)

First, BALB/c mice were anesthetized and anticoagulated whole blood was collected. We used gradient density centrifugation Ficoll to isolate platelets from whole blood. Ficoll was a synthetic sucrose polymer with high density, low osmotic pressure, and no toxicity. During separation, the stratified solution was first placed in the bottom of the test tube, and then the anticoagulant whole blood was diluted in phosphate-buffered saline (PBS) and superimposed on the stratified solution. After horizontal centrifugation, the platelets were suspended in the upper layer because of the low density, and the upper liquid was sucked out and washed three times with platelet washing solution to obtain purified platelets. The sample was washed with PBS and then resuspended, counted, and adjusted to a concentration of 2.5 × 10⁶/mL. Then, it was uniformly mixed with equal amounts of incomplete and complete Freund’s adjuvant as antigens. At week 0, the antigen containing complete Freund’s adjuvant was injected. In the first week, second week, and fourth week, the antigen containing incomplete Freund’s adjuvant was injected into the same area and the same spot in the same amount. Then, the nonanticoagulated whole blood from the mouse heart was obtained. After standing for a period of time, the whole blood sample was centrifuged to obtain GP-APS. To inactivate complement, GP-APS was placed in a 56°C water bath for 30 min. To prevent red blood cell adsorption, GP-APS was diluted to a concentration of 1:4 with saline.

ITP mouse model construction

All of the 18 mice were randomly divided into the sham group (n = 6), ITP model group (n = 6), and RES-mNE treatment group (n = 6) using a random number table. The mice of the ITP model group were injected intraperitoneally with GP-APS at a volume-to-mass ratio of 100 μL/20 g on 1, 3, 5, 7, 11, and 13 d. Thwenty-four hours after the first injection of GP-APS, every mouse was administered RES-mNE (250 μL, 3 mg/mL) by gavage for 15 consecutive days. The mice of the sham group were given the same volume of saline every time.

Model identification

On 0, 2, 4, 6, 8, 10, 12, and 14 d of the modeling, approximately 30 μL of EDTA-Na₂ anticoagulant blood was taken from the orbital vein in mice from the sham group and ITP model group using a capillary glass tube. An automatic hematology analyzer was used to load samples to determine the number of peripheral blood platelets in mice.

Preparation and pharmacokinetic test of RES-mNE

The titration method was used to optimize the formulation of RES-mNE. Ethyl oleate, RH40, and anhydrous ethanol were used as the oil phase, emulsifier, and coemulsifier, respectively. According to a certain proportion, a milky-white state was obtained for follow-up experimental research. The nano-laser particle size analyzer (JB6 100-B, Kewang) was used to detect the particle size of RES-mNE. The results showed that the average particle size of RES-mNE was 80 nm and evenly distributed, indicating its good physical stability.

Isolation and culture of CD4⁺ T cells

The collected mouse spleen tissue samples were cut into pieces, and placed in new 60 mm petri dishes containing 3 ml of PBS. Then, sterile nylon mesh was put on the tissue. After pipetting, filtering, and centrifugation, the supernatant was obtained. Then, the supernatant was added to EZ.-SepTM Mouse l X Lymphocyte Separation Solution for centrifugation. After removing the lymphocyte layer and centrifugation again, the supernatant was discarded, the cells were resuspended in PBS, CD4⁺ labeled magnetic beads were added, 10 μL per 1 × 10⁷ cells were added, and avoided at 4°C. After light incubation for 15 min and centrifugation for 10 min, the supernatant was discarded, 0.5 mL of buffer was added to resuspend the cells. The LS separation column was placed in the Midi MACS sorter and washed with 1 ml of buffer, and the cell suspension was added to the separation column. The column was washed three times, with 3 mL of the buffer solution each time. The LS separation column was removed from the magnetic field, CD4⁺ T cells retained in the LS separation column were slowly flushed out, PBS was added to the wash and centrifuged for 10 min 300 × g, and the supernatant was discarded. The cell pellet was frozen and stored in liquid ammonia for later use. CD4⁺T cells were cultured in 24-well culture plates precoated with the anti-CD3 antibody (5 μg/mL) together with soluble anti-CD28 antibody (1 μg/mL) in the RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 50 μg/mL streptomycin.

CD4⁺T cells induced the differentiation of Th17/Treg cells in vitro

Every cell suspension (1 mL) in each well (1 × 10⁶ cells/well) was seeded in 24-well plates coated with anti-CD3 and soluble anti-CD28. Different medications treated and grouped were as follows: uninduced differentiation group (control), induced differentiation group (induction), induction + vehicle, induction + Res-mNE, induction + Res-mNE+2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, AhR agonist), induction + Res-mNE + TCDD + LY3039478 (Notch inhibition agent), induction + Res-mNE + valproic acid (VPA) Notch activator, induction + Res-mNE + siNC, and induction + Res-mNE + siFoxp3. In our study, 50 μM Res-mNE, 10 nM TCDD, 1 mM LY3039478, and 1 mM VPA were correspondingly used to treat CD4⁺ T cells in the corresponding groups for 48 h. The small interfering RNA negative control (siNC) and Foxp3 (siFoxp3) synthesis were purchased from Thermo Fisher. The used concentrations of RES, TCDD (the AhR agonist was added to reverse the effect of RES on AHR), LY3039478, VPA, and siFoxP3 were specified in the reference instructions.

The appropriate amounts of cytokines were added to the culture medium for induction, iTreg, 30 U/mL hIL-2, 15 ng/mL hTGFβ, 5,000 ng/mL IFN-g (clone number XMG1.2), 5,000 ng/mL IL-4 (clone number 11B11). 3 ng/mL hTGFβ, 20 ng/mL rmIL-6, 5,000 ng/mL IFN-g (clone number XMG1.2), and 5,000 ng/mL IL-4 were added to induce Th17. The plates were incubated with 5% CO₂, 37°C for 4–5 days. Cytokines were removed from the original plate to avoid a continuous stimulation of CD3 and CD28, adjusted to 1 × 10⁶ cells/mL with a fresh medium, added 10 U/mL IL-2 to the iTreg new medium, and, in new wells (uncoated), continued to culture and boosted proliferation and final yield.

Flow cytometry detection

Based on the International Society for Hematotherapy and Graft Engineering (ISHAGE) Gating Strategy,^22,23 CD4⁺ T cells were stained with fluorescein isothiocyanate (FITC)–labeled antihuman CD4 antibody for 30 min at 4°C to detect the proportion of Th17/Treg cells. CD4⁺ T cells were treated with fixed/permeabilized buffer (Invitrogen, Cat: 00–8333–56) and stained with APC/cy7-labeled anti-human IL-17A antibody in the dark at room temperature for 30 min. CD4⁺ T cells were simultaneously labeled by an FITC-labeled antihuman CD4 antibody and a PE-labeled antihuman CD25 antibody and stained with an Alexa 647-labeled antihuman Foxp3 antibody in the dark at room temperature. All staining procedures use isotype-matched control antibodies. The LSRII flow cytometer was used for flow cytometry (Becton Dickinson, USA). The data were analyzed by FlowJo.

ELISA

Serum samples from each group were collected and centrifuged, and the culture supernatant was obtained. According to the manufacturer's instructions, the ELISA kit (Hangzhou Cancer probe Biotech Company, Zhejiang, China) was used to detect the concentration of IL-17A (abs520009-96 T), IL-22 (abs552808), and IL-10 (abs520005). Cytokine levels in the serum of ITP patients and healthy patients were directly determined using the aforementioned mouse ELISA kit. The experiment was repeated three times and analyzed with a microplate reader.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted with the TRIzol® reagent (Takara, Tokyo, Japan). The purity of RNA was measured by a NanoDrop 1000 spectrophotometer. RNA was reversely transcribed into cDNA with PrimeScript reverse transcription (RT) kit. Following the manufacturer’s instructions, qRT-PCR was performed using TransStart Tip Green qPCR SuperMix (Transgen Biotech, Beijing, China) on the ABI 7500 system (Applied Biosystems, Carlsbad, CA, USA). Relative expression levels were according to the 2^-ΔΔCT method. The specific primers used in real-time PCR were as follows: AhR forward: 5’-CAAATCCTTCCAAGCGGCATA-3′; AhR reverse: 5’-CGCTGAGCCTAAGAACTGAAAG-3′; Foxp3 forward: 5’-GTGGCCCGGATGTGAGAAG-3′; Foxp3 reverse: 5’-GGAGCCCTTGTCGGATGATG-3′; Notch1 forward: 5′- TCTGTGTGGATGAGGGAGATAA-3′; Notch1 reverse: 5’-AGCCGCCGAGATAGTCAGT-3′; GAPDH forward: 5’-AGCCACATCGCTCAGACAC-3′; GAPDH reverse: 5’-GCCCAATACGACCAAATCC-3′.

Western blot assay

Total protein was extracted out from cultured cells, separated by dodecyl sulfate, and measured by the BCA protein assay (Beyotime). The following primary antibodies were used: anti-AhR antibody (1:1,000 dilution, Invitrogen, cat. no. MA1–513), anti-Notch1 antibody (1:1,000 dilution, Abcam, cat. no. Ab52627), anti-IL-17A antibody (1:1,000 dilution, Abcam, cat. no. Ab79056), anti-IL-22 antibody (1:1,000 dilution, Invitrogen, cat. no. PA5–115408), anti-IL-10 antibody (1:1,000 dilution, Abcam, cat. no. Ab9969), anti-Foxp3 antibody (1:1,000 dilution, Abcam, cat. no. Ab22510), anti-GAPDH antibody (Invitrogen, cat. no. PA1–987), and horseradish peroxidase–conjugated (HRP) secondary antibodies (1:5,000, Abcam, cat. no. ab7090). The protein expression was analyzed with biometrics digital image software and recorded as integrated density (ID). The ratio of ID (each protein) to ID (GAPDH) was the final result of protein expression.

Statistical analysis

All continuous data are presented as the means ± SD of the mean of at least three experiments. Before analysis, normal distribution was carried out. The comparisons of multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by LSD (GraphPad version 5.0; Inc, La Jolla, CA, USA). The difference was statistically significant at P < 0.05.

Results

Res-mNE improved Th17 and Treg cell imbalance in ITP mouse model

CD4⁺ T cells were isolated from the ITP mouse model, and Th17/Treg-specific markers were used to stain on the surface or in the cells. Compared with the sham group, the ITP group showed an increase in Th17 cells, which were then significantly reduced after Res-mNE treatment (P < 0.05, Fig. 1a). Compared with the sham group, the Treg cells in the ITP group were decreased, which was reversed with the Res-mNE treatment (P < 0.05, Fig. 1b). The Th17/Treg differentiation ratio in the ITP group was increased but significantly reduced after Res-mNE treatment (P < 0.05, Fig. 1c). The IL-10 levels in the ITP group were decreased, and the IL-17A and IL-22 levels were increased, compared with the sham group. Res-mNE treatment significantly reversed the levels of IL-17A, IL-22, and IL-10 in ITP (P < 0.05, Fig. 1d). The expression level of AhR was increased in the ITP group and significantly inhibited by Res-mNE treatment (P < 0.05, Fig. 1e). Our results indicated that the Th17/Treg ratio in ITP mice was unbalanced, and Res-mNE improved the Th17/Treg ratio and decreased the levels of related inflammatory factors in ITP by regulating AhR expression.

Fig. 1.

Res-mNE improved Th17 and Treg cell imbalance in ITP mouse model. Gating strategy for flow cytometric analysis of the proportion of Th17 cells a, c) and Treg cells b, c) in the CD4⁺ T-cell population isolated from the blood of ITP mouse models and ITP mice treated with res-mNE; d) the inflammatory factor expression levels of IL-17A, IL-22, and IL-10 was detected by ELISA; and e) the expression level of AhR was detected using western blot. All continuous data are presented as mean ± SD. Comparison among three groups using one-way ANOVA, followed by LSD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Res-mNE inhibited the differentiation of Th17 and promoted the differentiation of Treg by inhibiting AhR

In order to further explore whether Res-mNE regulates Th17/Treg differentiation through AhR, we used AhR agonist TCDD to treat cells and induced the differentiation of CD4⁺ T cells. The results showed that Th17 was successfully induced to increase the number of differentiated cells. Res-mNE treatment inhibited Th17 cell differentiation. After adding TCDD, the inhibitory effect of Res-mNE on differentiation was weakened (P < 0.05, Fig. 2a). Treg cells were successfully induced to differentiate, and the number was increased. After Res-mNE treatment, the differentiation of Treg cells was promoted. When adding TCDD, the differentiation promotion effect of Res-mNE was diminished (P < 0.05, Fig. 2b). After the induction of differentiation, the Th17/Treg differentiation ratio was decreased. After Res-mNE treatment, the differentiation ratio was further inhibited. Meanwhile, the inhibitory effect of Res-mNE was weakened by TCDD treatment, and the Th17/Treg differentiation ratio was increased after TCDD treatment (P < 0.05, Fig. 2c). After induced differentiation, the expression of IL-17A and IL-22 was increased, and the expression of IL-10 was decreased (P < 0.05). Res-mNE treatment inhibited the expression of IL-17A and IL-22 and promoted the expression of IL-10 (P < 0.05). After adding TCDD, the treatment of Res-mNE effect was reversed (Fig. 2D and E). The expression of AhR was significantly increased after inducing differentiation and decreased after Res-mNE treatment (P < 0.05). Adding the AhR agonist TCDD promoted AhR expression (P < 0.05, Fig. 2F and G). Our results indicated that Res-mNE regulated the differentiation of Th17/Treg via inhibiting the expression of AhR.

Fig. 2.

Res-mNE inhibited the differentiation of Th17 cells and promoted the differentiation of Treg cells by inhibiting AhR. We treated Th17/Treg cells with the AhR agonist TCDD. Gating strategy for the flow cytometric analysis of the number of Th17 a) and Treg b) cells induced by CD4⁺ T cells t; the ratio of Th17/Treg differentiation after CD4⁺ T-cell differentiation, c) ELISA (D) and western blot e) was used to detect the inflammatory factor expression levels of IL-17A, IL-22, and IL-10; qRT-PCR f) and western blot g) was used to detect the expression of AhR mRNA and protein. All continuous data are presented as mean ± SD. Comparison among the five groups using one-way ANOVA, followed by LSD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

AhR activation inhibited the therapeutic effect of Res-mNE by the Notch pathway

In order to explore whether AhR affects the differentiation of CD4⁺ T cells into Th17/Treg through the Notch pathway, Notch inhibitor LY3039478 was used to treat Th17/Treg differentiation after adding Res-mNE. The results showed that, after the addition of LY3039478, the number of differentiated Th17 cells (Fig. 3a) and Treg cells (Fig. 3b) reversed the inhibitory effect after Res-mNE treatment (P < 0.05), as well as the promoting effect after TCDD and Res-mNE treatment. Meanwhile, the Th17/Treg differentiation ratio was downregulated after LY3039478 treatment (P < 0.05), and the result was basically consistent with the Th17/Treg differentiation ratio during the Res-mNE treatment period (P < 0.05, Fig. 3c). The addition of LY3039478 reversed the inhibitory effect of TCDD on Res-mNE treatment and restored the inflammatory factor level (Fig. 3d and e). Notch inhibitor LY3039478 reversed the promotion of TCDD on AhR and Notch, returning to the treatment level of Res-mNE (P < .05, Fig. 3f and g). Our results revealed the activation of AhR might inhibit the effect of Res-mNE through the Notch pathway.

Fig. 3.

AhR activation inhibited the therapeutic effect of res-mNE by the Notch pathway. We used Notch inhibitor LY3039478 to treat the addition of res-mNE treatment and AhR activator TCDD, gating strategy for the flow cytometric analysis of the number of Th17 cells a) and Treg cells b) differentiated and the change of Th17/Treg differentiation ratio c); ELISA d) and western blot e) detected the inflammatory factor expression levels of IL-17A, IL-22, and IL-10; qRT-PCR f, g); and western blot h) detected the expression of AhR and Notch in mRNA and protein levels. All continuous data are presented as mean ± SD. Comparison among the five groups using one-way ANOVA, followed by LSD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Res-mNE promoted Foxp3 regulation of Th17/Treg cell differentiation by inhibiting Notch signaling pathway

In order to further explore the changes of Notch downstream signaling molecule Foxp3 after Res-mNE treatment, we used Notch activator VPA to treat Res-mNE-treated cells. The addition of VPA reversed the inhibitory effect of Res-mNE on Th17 differentiation (P < 0.05, Fig. 4a) and inhibited Treg differentiation (P < 0.05, Fig. 4b). The Th17/Treg differentiation ratio was increased after adding VPA (P < 0.05, Fig. 4c). Compared with Res-mNE treatment, addition of VPA promoted the expression of IL-17A, IL-22 ELISA and inhibited the expression of IL-10 after VPA treatment (P < 0.05, Fig. 4d and e). Res-mNE treatment reduced the expression of Notch and promoted the expression of Foxp3. VPA reversed the effect of Res-mNE, significantly promoted the expression of Notch and inhibited the expression of Foxp3 (P < 0.05, Fig. 4f and g). Our results revealed that the therapeutic effect of Res-mNE promoted the expression of Foxp3 by inhibiting the Notch signaling pathway, thereby regulating Th17/Treg cell differentiation.

Fig. 4.

Res-mNE promoted the Foxp3 regulation of Th17/Treg cell differentiation by inhibiting the Notch signaling pathway. We used Notch activator VPA to treat the addition of res-mNE treatment and the AhR activator TCDD, gating strategy for flow cytometric analysis of the number of Th17 cells a) and Treg cells b) differentiated and the change of Th17/Treg differentiation ratio c); ELISA d) and western blot e) detected the inflammatory factor expression levels of IL-17A, IL-22, and IL-10; qRT-PCR f, g) and western blot h) detected the expression of Notch and Foxp3 in mRNA and protein levels. All continuous data are presented as mean ± SD. Comparison among the four groups using one-way ANOVA, followed by LSD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Res-mNE inhibited AhR/Notch signaling pathway and activated Foxp3 to reverse Th17/Treg imbalance

Finally, we interfered with the expression of the key factor Foxp3 to determine whether the therapeutic effect of Res-mNE was to mediate the AhR/Notch signal and activate Foxp3 to regulate Th17/Treg differentiation. The transfection of siFoxp3 reversed the inhibitory effect of Res-mNE, which promoted the number of Th17 cell differentiation and inhibited Treg cell differentiation (P < 0.05, Fig. 5a and b). The Th17/Treg differentiation ratio was increased after induction or Res-mNE treatment, but the effect was reversed by siFoxp3 (P < 0.05, Fig. 5c). The expression of IL-17A and IL-22 was promoted, and the expression of IL-10 was inhibited after siFoxp3 transfection (P < 0.05, Fig. 5d and e). The analysis of expression of AhR, Notch, and Foxp3 showed that siFoxp3 transfection had little effect on the expression of AhR and Notch but significantly reduced Foxp3 expression (P < 0.05, Fig. 5f–i). Our results revealed that the therapeutic effect of Res-mNE was to reverse the imbalance of Th17/Treg differentiation by mediating AhR/Notch signaling to activate the expression of Foxp3.

Fig. 5.

Res-mNE inhibits the AhR/Notch signaling pathway and activates Foxp3 to reverse Th17/Treg imbalance. We transfected siFoxp3 in the res-mNE treatment group, gating strategy for flow cytometric analysis of the number of Th17 cells a) and Treg cells b) differentiated and the change of Th17/Treg differentiation ratio c), ELISA, d) and western blot e) detected the inflammatory factor expression levels of IL-17A, IL-22, and IL-10; qRT-PCR f–h) and western blot i) detected the expression of AhR, Notch, and Foxp3 in mRNA and protein levels. All continuous data are presented as mean ± SD. Comparison among the five groups using one-way ANOVA, followed by LSD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Discussion

In the current study, we explore the mechanism of resveratrol in participating the imbalance between Th17 cells and Treg cells using an ITP mouse model. For the first time, our findings demonstrated that Res-mNE, as an inhibitor of targeting AhR, activated the expression of Foxp3 by mediating AhR/Notch signaling to reverse the imbalance of Th17/Treg differentiation in ITP. Th17 cells, IL-17A and IL-22 increased in CD4⁺ T cells, while Treg cells and IL-10 decreased, which could be reversed by Res-m NE. The AhR activator reversed the effect of Res-mNE. Notch inhibitors showed the same therapeutic effect on Th17/Treg differentiation, thereby reducing the ratio of Th17/Treg differentiation.

ITP was characterized by increased platelet clearance and impaired platelet production, which could result in decreased platelet production.^24,25 ITP could be considered a disease mediated by B cells. In order to produce an antiplatelet immune response, B cells needed the help of autoreactive CD4⁺ T cells.²⁶ The imbalance of Treg/Th17 was confirmed in several autoimmune diseases.^17,27 Studies have shown that the ratio of Treg/Th17 may be related to the clinical diversity of ITP patients.⁴ The current clinical treatment of ITP further proved the importance of T-cell homeostasis in weakening autoimmunity. Both clinical and experimental studies have shown that treatment methods such as the intravenous injection of platelet production mimics exerted their therapeutic effects by regulating Treg activation and expansion and/or inhibiting the survival and proliferation of effector T cells.²⁸ In Dou Z et al.’s study, resveratrol could promote the Th1/Th2 balance in the lamina propria of the small intestine to tilt toward Th2 polarization and the Treg/Th17 balance to tilt toward Treg and reduce the expression of intestinal proinflammatory cytokines by regulating the intestinal flora.²⁹ In our study, Res-mNE was used to treat ITP mice for the first time, and it was found that resveratrol significantly improved the balance of Th17/Treg in ITP mice.

Immune responses can be regulated by cytokines through proinflammatory or anti-inflammatory pathways. IL-1 family cytokines could differentiate primary ITP from systemic lupus erythematosus–associated thrombocytopenia.³⁰ IL-17A, IL-22, and IL-23 have been proven as the markers of psoriasis activity, which were significantly elevated in psoriasis patients.³¹ In BTBR T+ Itpr3tf/J mice, the expression of cytokines associated with immunologic dysfunction also can be regulated by the activation of adenosine A2A receptor signaling.³² The expression of IL-2⁺, IL-6⁺, IL-9⁺, IFN-γ⁺, and TNF-α⁺ were all significantly decreased, and the expression of TGF-β⁺ was significantly increased in splenic CD4⁺ T cells in the spleen tissues of A2AR agonist mice. Resveratrol proved to attenuate proinflammatory cytokines and the activation of JAK1-STAT3 in BTBR T+ Itpr3tf/J autistic mice.³³ Resveratrol (20 and 40 mg/kg) could significantly reduce the expression of IL-6+, TNF-α+, IFN-γ+, and STAT3+ in CD4+ spleen cells and the expression IL-6, IFN-γ, TNF-α, pJAK1, and pSTAT3 (Tyr705) in the brain tissue. These aforementioned reports support our findings that Res-mNE treatment could significantly promote the expression of IL-10 and inhibit the expression of Th17 cells, IL-17A, and IL-22 from CD4⁺ T cells.

Previous studies have demonstrated that the ratio of AhR in Th17 cells to AhR in Treg cells could predict skin damage in systemic lupus erythematosus.³⁴ Resveratrol interfered with the metabolic degradation of 6-formylindolo[3,2-b] carbazole (FICZ), which was the endogenous AHR ligand and could indirectly activate AHR.³⁵ Our results indicated that Res-mNE may regulate the differentiation of Th17/Treg by inhibiting the expression of AhR. The result was consistent with a previous study on the relationship between the AhR pathway and Th17/Treg balance.¹⁴ Meanwhile, it also revealed that resveratrol was a natural AHR antagonist and an ROS scavenger.³⁶ This was consistent with our findings that resveratrol regulated Th17/Treg differentiation by inhibiting the expression of AhR.

It has been reported that AhR could induce the transcription factor Notch to establish Notch signaling as a key pathway downstream of AhR in innate lymphogenesis.^37,38 AhR regulated the cell cycle, cell proliferation, and cell differentiation by the regulation of the interaction of the Notch signaling pathway.³⁹ In humans with chronic aplastic anemia, bone marrow mesenchymal stromal cells could regulate Treg/Th17 balance by affecting the Notch/RBP-J/FOXP3/RORγt pathway.⁴⁰ The intracellular marker Foxp3 can be expressed by Treg cells, which could effectively suppress the excessive immune responses and exerted an immune effect on regulating inflammatory processes.⁴¹ In human autoimmune diseases, the deficiency of Treg cell function could lead to tolerance failure.⁴² Histamine 4 receptor agonist JNJ upregulated Foxp3 gene expression, which was correlated with increased numbers of CD4⁺CD25⁺Foxp3⁺ Treg cells.⁴³ In our study, Res-mNE treatment reduced the expression of Notch and promoted the expression of Foxp3. Moreover, siFoxp3 transfection could reverse Res-mNE treatment, reduce the Th17/Treg differentiation ratio and the expression of IL-10, and promote the expression of IL-17A, IL-22. Our findings demonstrated that the treatment of ITP with Res-mNE activated the expression of Foxp3 by mediating the AhR/Notch signaling pathway, thereby reversing the imbalance of Th17/Treg differentiation.

Studies have identified the differential effects of resveratrol on signaling and transcriptional pathways that affected the expression of Tregs, Th17, and inflammatory cytokines. AHR can regulate Tregs and Th17 cell differentiation in a ligand-specific manner.⁴⁴ Different AhR ligands lead to different outcomes, which is a limitation of this study and will be further explored in future studies. It is also likely that resveratrol can establish tolerance through its estrogen receptor agonist activity as other reported AhR antagonists. Thus, although the antithrombotic activities of RES have pharmacological promise, its estrogenic properties may produce undesirable side effects and limit the circumstances under which it can be used safely. As evidence, the finding that resveratrol could stimulate the growth of human breast cancer cells is problematic. In this regard, structure function studies could lead to the development of more selective receptor (i.e. AHR and ER) agonists and antagonists, which could be useful as therapeutic agents.

There are also some limitations in this study. There are many cell models at present used in basic research. Here, we explored the mechanism of resveratrol in ITP using an ITP mouse model. Animal-level verification may be not perfect. The ITP mouse model functions on the basis of passive transfer of antiplatelet antibodies, which may only present the innate response phase of human ITP disease. Whether the experimental ITP model could accurately mirror the human condition is still questionable due to its potential shortcomings. Human ITP is an acquired autoimmune disease, which could be influenced by many factors such as the dysfunction of immune system components, the nature of the autoantigen, and the inflammatory response. More studies are still needed.

In summary, the current study for the first time revealed that resveratrol could inhibit the AhR/Notch axis and reverse Th17/Treg imbalance in purpura by activating Foxp3. Our findings provide new evidence for the pathogenesis and treatment of ITP, which contributes to the new drug discovery in the future.

Author contributions

Jing Cheng (conceptualization, methodology, validation, visualization, and supervision), Sheng Wang (formal analysis and investigation), Shi-Qin Lv (resources and data curation), Yuan Song (writing—original draft), and Ning-Hong Guo (writing—review and editing, project administration, and funding acquisition).

Funding

This work was supported by Key research and development projects of Jiangxi Provincial Department of Science and Technology (No. 20202BBGL73031).

Conflict of interest statement: None declared.

Data availability

The data underlying this article are available in [repository name, eg, the GenBank Nucleotide Database] at [URL], and can be accessed with [unique identifier, eg, accession number, deposition number].

Ethics approval and consent to participate

Our present study was permitted and approved by the second affiliated Hospital of Nanchang University Animal Ethics Committee of No. 2020117.

Consent for publication

Informed consent was obtained from study participants.