Berberine Suppresses Th17 and Dendritic Cell Responses

Abstract

Purpose.: Berberine (BBR) has been shown to exert immunosuppressive and anti-inflammatory effects in several autoimmune diseases. This study was designed to investigate the possible effect of BBR on Th17 and dendritic cell (DC) responses.

Methods.: Twelve patients with active VKH disease and 20 healthy individuals were enrolled in this study. CD4⁺ T cells and CD14⁺ monocytes were isolated from peripheral blood mononuclear cells using magnetic-activated cell sorting. Monocyte-derived DCs were generated by culturing monocytes with GM-CSF and IL-4. Proinflammatory cytokines secreted by CD4⁺ T cells and LPS induced DCs when exposed to BBR or only vehicle were detected by ELISA. The frequency of IL-17–producing CD4⁺ T cells and the surface markers of BBR-treated DCs were measured by flow cytometry.

Results.: Activation of CD4⁺ T cells using anti-CD3 and anti-CD28 showed a higher Th17 response in active VKH patients. BBR showed a direct suppression of the Th17 response both in active VKH patients and healthy donors. It also suppressed the Th17 response indirectly by influencing DC maturation. On the one hand, BBR downregulated the expression of costimulatory molecules (CD40, CD80, and CD86) and, on the other hand, it inhibited IL-6, IL-1β, and IL-23 secretion by DCs.

Conclusions.: These findings suggest that the inhibitory effect of BBR on the Th17 response was mediated by a direct action on T cells as well as an indirect effect via DCs. This study provides new evidence that the natural compound BBR is of great value in the search for novel therapeutic agents in the treatment of T-cell–mediated autoimmune diseases.

Introduction

Many autoimmune diseases are caused by an aberrant autoreactivation of CD4⁺ T-cell responses. ^1–3 Recently, Th17 effector cells were shown to be involved in the disease pathogenesis of experimental autoimmune uveitis (EAU), an animal model for human uveitis, as well as in clinical uveitis. ^4–6 Further evidence demonstrated that neutralization of IL-17 or inhibition of Th17 cells led to an amelioration of EAU. ^4,7 Immunosuppressive agents, such as cyclosporin A and corticosteroids, which are often used to treat clinical uveitis, were shown to exert their immunosuppressive role in part by downregulating Th17 cells. ⁸  

It is well established that antigen-presenting cells (APCs) are crucial for the full activation of T cells, whereas dendritic cells (DCs) are regarded as the most powerful professional APCs due to their unique characteristics. The costimulatory molecules, expressed on DCs, interact with corresponding ligands on T cells to provide a complete signal, which is required for full activation of T cells. ⁹ In addition, proinflammatory cytokines, such as IL-6, IL-1β, and IL-23, secreted by APCs, are crucial for differentiation of Th17 cells. ^10–13  

Berberine (BBR) is an isoquinoline alkaloid that is present in numerous plants of the genera Berberis and Coptis . ¹⁴ These herbs have traditionally been used for centuries in China for the treatment of various conditions, such as diarrhea and other gastrointestinal disorders. One of the main active ingredients of these herbs, BBR also exhibits antitumor, antimicrobial, and even antidiabetic effects. ^15–18 Recent reports have suggested that BBR may suppress inflammation in collagen-induced arthritis, an animal model for human rheumatoid arthritis, ¹⁹ experimental type I diabetes, ²⁰ and experimental autoimmune encephalomyelitis, an animal model for human multiple sclerosis. ^21,22 These findings raised the question of whether BBR could possibly also be applied in the treatment for clinical autoimmune uveitis. As yet, no detailed reports have been published on the immunosuppressive action of BBR in humans and especially on Th17 cells and this was therefore the subject of our study. 

We chose Vogt-Koyanagi-Harada (VKH) as a clinical entity to perform our research because it is a relatively well-defined autoimmune disease that is frequently encountered in our uveitis clinic ²³ and in view of the fact that active VKH patients express enhanced Th17 cell responses. ⁵ VKH disease affects many organs and systems, including pigmented tissues in the eye, auditory system, integumentary system, and central nervous system. It manifests as a bilateral granulomatous panuveitis. If not treated properly, VKH disease can lead to severely decreased vision or even blindness. ^23–25 Although its pathogenesis is still not completely understood, a number of studies have indicated that aberrant autoreactive Th17 cells play an important role in the pathogenesis of this disease.^5,26  

The study presented here was undertaken to investigate the effect of BBR on the Th17 response, which, as already mentioned, is thought to be critical in the pathogenesis of VKH disease. Our results showed that BBR was able to inhibit the Th17 response directly and indirectly through impairing DC function. 

Materials and Methods

Berberine

BBR was obtained from Sigma-Aldrich (catalog number: 14050; Sigma-Aldrich, St. Louis, MO) and was dissolved in dimethyl sulfoxide (DMSO) at 50 mM as stock solution stored at −20°C. The final concentration of DMSO was kept at less than 0.05% (vol/vol) in the culture media. 

Patients and Healthy Donors

Twelve patients with active VKH disease (7 men and 5 women), with an average age of 37.5 years, and 20 healthy individuals (11 men and 9 women), with an average age of 38.6 years, were included in the study. Patients were seen in our clinic between March 2012 and August 2012. The diagnosis of VKH disease was made according to the diagnostic criteria revised for VKH disease by an international committee on nomenclature. ²⁷ The patients had active uveitis, as evidenced by diffuse bilateral choroiditis in association with exudative retinal detachment in the first uveitis attack or by mutton fat keratic precipitates, cells in the anterior chamber, and sunset glow fundus in the VKH patients with recurrent episodes. The systemic findings included headache (75.0%), tinnitus (58.0%), dysacusis (33.3%), poliosis (25.0%), alopecia (42.0%), and vitiligo (16.7%). This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. All procedures complied with the Declaration of Helsinki, and informed consent was obtained from all patients with VKH disease and controls. 

Cell Isolation and Culture

Peripheral blood mononuclear cells (PBMCs) were isolated from anticoagulant whole blood using density gradient centrifugation (Ficoll-Hypaque; TBDScience, Tianjin, China). CD4⁺ T cells and CD14⁺ monocytes were purified using microbead isolation kits followed by magnetic-activated cell sorting (MACS) according to the manufacturer's instructions (Miltenyi Biotec, Palo Alto, CA). The purity of isolated cells, identified by flow cytometry (FCM), was more than 93%. Isolated cells (PBMCs, CD4⁺ T cells, and monocytes) were resuspended at a concentration of 1 × 10⁶ cells/mL in RPMI 1640 medium (Gibco-Invitrogen, Carlsbad, CA) containing L-glutamine (2 mM), penicillin/streptomycin (100 U/mL), and 10% fetal calf serum (Gibco-Invitrogen). 

Immature DCs (iDCs) were generated from monocytes in the presence of recombinant human granulocyte macrophage–colony-stimulating factor (GM-CSF) (100 ng/mL) and IL-4 (50 ng/mL) (PeproTech, London, UK) for 6 days. On day 3, half of the culture medium, including GM-CSF and IL-4, was refreshed. For DC maturation, 100 ng/mL lipopolysaccharide (LPS) (Sigma-Aldrich) with BBR (5 μM) or the vehicle DMSO was added to the cells at day 6 and cultured for 24 hours. 

CD4⁺ T cells were cultured with BBR (5 μM) or the vehicle DMSO in the presence of anti-CD3 (OKT3, 0.5 μg/mL) and anti-CD28 antibodies (15E8, 0.1 μg/mL; Miltenyi Biotec) for 3 days. 

BBR- or DMSO-treated DCs were washed three times, and then cocultured with CD4⁺ T cells at a ratio of 1:5 for 5 days. 

Cell Viability and Proliferation

For assessment of BBR cytotoxicity, PBMCs, activated by anti-CD3 and anti-CD28 antibodies, were cultured with different concentrations of BBR for 3 days. The effect of BBR on DCs was tested as follows. Monocytes were cultured in the presence of recombinant human GM-CSF and IL-4 for 6 days and then 100 ng/mL LPS was added either in the presence of BBR (5 μM) or without and cells were cultured for another 24 hours. To assess cell viability, 10% (vol/vol) water-soluble tetrazolium salt (WST-8; Cell Counting Kit 8; Dojindo, Tokyo, Japan) was added to the culture for the last 4 hours. WST-8 is reduced by dehydrogenase activities in cells to give a yellow-color formazan dye and the amount of the formazan dye generated is a measure of the number of living cells. The absorbance was determined at 450 nm with an ELISA plate reader (SpectraMax M2e; Molecular Devices, Sunnyvale, CA). Viability was calculated by the following equation: cell viability (%) = 100 (OD treatment/OD control). 

For DC and T-cell coculture assays, BBR- or DMSO-treated DCs were washed three times, then cocultured with CD4⁺ T cells at the indicated ratio for 5 days. During the final 4 hours, the proliferation was determined by cell counting (Cell Counting Kit 8; Dojindo). Ten percent (vol/vol) WST-8 was added into each well and incubated for 4 hours and the absorbance was determined at 450 nm with an ELISA plate reader (SpectraMax M2e; Molecular Devices). 

Flow Cytometry

To detect intracellular expression of IL-17 in CD4⁺ T cells, cells were stimulated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL ionomycin (Sigma-Aldrich) for 5 hours. During the final 4 hours, 10 μg/mL Brefeldin A (Sigma-Aldrich) was added to the cultured CD4⁺ T cells. The stimulated cells were washed, fixed, permeabilized, and subsequently stained with anti–IL-17 antibody or its isotype (eBioscience, San Diego, CA). 

For surface-marker staining of DCs, cells were incubated for 30 minutes at 4°C with FITC-conjugated antihuman CD80, CD86, or phycoerythrin-conjugated antihuman HLA-DR, CD40, or appropriate isotypes (eBioscience). 

FCM analysis was performed on a FACS Aria cytometer (BD Biosciences, San Diego, CA) and analyzed with FlowJo software (Tree Star, Inc., San Carlos, CA). Mean fluorescent intensity was expressed as the geometric mean channel fluorescence minus the appropriate isotype control. 

Cytokine Measurement

Supernatants from CD4⁺ T cells, LPS-induced DCs, or DC-CD4⁺ T-cell cocultures were collected and stored at −80°C until the cytokine measurement. The levels of IL-17, IL-6, and IL-1β were measured using Duoset ELISA development kits (R&D Systems, Minneapolis, MN). IL-23 was measured using an ELISA kit (eBioscience) according to the manufacturer's instructions. 

Statistical Analysis

Data are expressed as mean ± SD. The analysis was performed using SPSS 13.0 (IBM SPSS, Chicago, IL). One-way ANOVA, Student's t-test, Wilcoxon, and Mann-Whitney U tests were applied. P less than 0.05 was considered statistically significant. 

Results

Effect of BBR on Cell Viability of PBMCs and DCs

The chemical form of BBR used in this study was BBR hydrochloride (Fig. 1A). PBMCs separated from healthy donors were cultured with different concentrations of BBR (0∼50 μM) in the presence of anti-CD3 and anti-CD28 antibodies for 3 days to detect its possible cytotoxicity. BBR displayed no effect on the viability of the PBMCs up to a concentration of 5 μM, whereas higher concentrations resulted in cell death (Fig. 1B). At the concentration of 5 μM, BBR did not affect the viability on DCs either (data not shown). A concentration of 5 μM was therefore used in the following experiments. 

Figure 1. 

 

Berberine. (A) Chemical structure. (B) Cell viability as measured by cell-counting kit in PBMCs from healthy donors stimulated by anti-CD3 and anti-CD28 and cultured for 3 days in the presence of increasing concentrations of BBR. Statistical analyses were performed using one-way ANOVA with Bonferroni post hoc analysis (representative experiment of n = 6).

Effect of BBR on the Th17 Response in VKH Patients and Healthy Donors

CD4⁺ T cells from active VKH patients and healthy donors were cultured with anti-CD3 and anti-CD28 antibodies for 3 days. Culture supernatants were collected to detect the expression of IL-17 and cells were harvested to analyze the frequency of Th17 cells. The results showed that the concentration of IL-17 in cell culture supernatants was significantly higher in active VKH patients (644.7 ± 133.8 pg/mL) as compared with healthy donors (253.3 ± 77.2 pg/mL, P = 0.001). Exposure to BBR (5 μM) significantly inhibited the production of IL-17 by CD4⁺ T cells in both active VKH patients (from 644.7 ± 133.8 pg/mL to 392.5 ± 158.1 pg/mL, P < 0.001) and healthy donors (from 253.3 ± 77.2 pg/mL to 155.1 ± 44.6 pg/mL, P = 0.001) (Fig. 2A). Consistent with the ELISA data, intracellular cytokine analysis revealed that a significantly higher frequency of IL-17–producing CD4⁺ T cells was observed in VKH patients as compared with healthy controls (2.25% ± 0.57% vs. 1.2% ± 0.45%, P = 0.005 ). Addition of BBR also significantly inhibited the frequency of IL-17–producing CD4⁺ T cells both in active VKH patients (from 2.25% ± 0.57% to 1.47% ± 0.42%, P < 0.001) and healthy donors (from 1.2% ± 0.45% to 0.8% ± 0.32%, P = 0.001) (Figs. 2B, 2C). 

Figure 2. 

 

Effect of BBR on Th17 response. CD4⁺ T cells from active VKH patients (n = 6) and healthy donors (n = 6) were cultured with BBR or the vehicle DMSO in the presence of anti-CD3 and anti-CD28 antibodies for 3 days. (A) IL-17 in the supernatants was measured by ELISA. Statistical analyses were performed using paired-samples t-test for healthy donors and Wilcoxon test for active VKH patients (DMSO versus BBR). For active VKH patients versus healthy donors (DMSO group), independent-samples t-test was performed. (B) Percentages of CD4⁺IL-17⁺ cells among the CD4⁺ T cells. Statistical analyses were performed using paired-samples t-test for DMSO versus BBR. For active VKH patients versus healthy donors (DMSO group), independent-samples t-test was performed. (C) A representative patient and healthy donor with data near the mean of each group in (B). Numbers indicate percentages of positive cells in that gate.

Effect of BBR-Treated DCs on the Th17 Response in VKH Patients and Healthy Donors

The aforementioned experiment revealed a direct inhibitory effect of BBR on the Th17 response. As DCs function as a bridge between innate and adaptive immunity, ^28,29 a further study was performed to test whether BBR could also exert its effects on the Th17 response via an indirect action on DCs. Immature DCs generated from monocytes stimulated with GM-CSF and IL-4 were treated with LPS, along with BBR or the vehicle DMSO for 24 hours and then cocultured with CD4⁺ T cells. IL-17 was subsequently measured by ELISA in the cell culture supernatants and the frequency of CD4⁺ IL-17⁺ T cells was assessed by FCM. The results showed that the production of IL-17 was significantly decreased in the supernatants of CD4⁺ T cells cocultured with BBR-treated DCs compared with coculture with DMSO-treated DCs (Fig. 3A). Flow cytometry showed that the frequency of CD4⁺ IL-17⁺ T cells was also decreased in the CD4⁺ T cells cocultured with BBR-treated DCs as compared with those cultured with control DCs (Figs. 3B, 3C). Furthermore, T-cell proliferation was significantly decreased when CD4⁺ T cells were cocultured with BBR-treated DCs (Fig. 4). 

Figure 3. 

 

Effect of BBR-treated DCs on the Th17 response. Allogeneic CD4⁺ T cells, purified from active VKH patients (n = 6) and healthy donors (n = 6), were cocultured with BBR- or DMSO-treated DCs for 5 days. (A) IL-17 in the supernatants was measured by ELISA. (B) The results represent the percentages of IL-17⁺ cells among the CD4⁺ T cells. Statistical analyses in B and C were performed using paired-samples t-test for DMSO-DCs versus BBR-DCs. For active VKH patients versus healthy donors (DMSO-DCs group), Mann-Whitney U test was performed. (C) A representative patient and healthy donor with data near the mean of each group in (B). Numbers indicate percentages of positive cells in that gate.

Figure 4. 

 

Effects of BBR on DC-mediated T-cell activation. Immature DCs were stimulated with LPS in the presence of BBR or vehicle DMSO for 24 hours. Varying ratios of DCs and T cells (1:5, 1:10, 1:20, 1:50, 1:100) were subsequently cocultured for 5 days and T-cell proliferation was measured by a cell-counting kit. Results showed decreasing induction of T-cell proliferation in DCs treated with BBR (1:5 ratio: P = 0.033, 1:10 ratio: P = 0.032, 1:20 ratio: P = 0.036, 1:50 ratio: P = 0.033, 1:100 ratio: P = 0.015). Data are shown as mean ± SD of triplicate values and representative of three separate experiments. Statistical analyses were performed using paired-samples t-test except DCs-T cells (1:50) (using Wilcoxon test) (representative experiment of n = 4).

Influence of BBR on DC Markers and Cytokine Production

To investigate the influence of BBR on DCs, immature DCs were stimulated following LPS with BBR or DMSO for 24 hours, and the expression of surface markers was investigated by FCM. BBR inhibited the expression of costimulatory molecules, including CD80, CD86, and CD40 (Fig. 5). 

Figure 5. 

 

Effect of BBR on surface marker expression in DCs. Immature DCs from healthy donors were stimulated with 100 ng/mL LPS, in the presence of BBR or the vehicle DMSO, for 24 hours. DCs were then stained with specific antibody against CD40, CD80, CD86, and HLA-DR, and analyzed by FCM. (A) Histograms with overlays are from a representative experiment. (B) Fluorescence intensity of surface marker expression on LPS-induced DCs. Statistical analyses were performed using paired-samples t-test. Similar results were obtained in eight independent experiments.

In a following set of experiments, we stimulated immature DCs with LPS in the presence of BBR or DMSO for 24 hours to evaluate its influence on the production of IL-6, IL-1β, and IL-23, cytokines that have been shown to be critical for Th17 differentiation. ^10–11,13 The results showed that BBR significantly inhibited IL-6, IL-1β, and IL-23 production induced by LPS (Fig. 6). 

Figure 6. 

 

Effect of BBR on proinflammatory cytokine secretion by DCs. Immature DCs from healthy donors were stimulated with 100 ng/mL LPS, in the presence of BBR or the vehicle DMSO, for 24 hours. The supernatants were collected and IL-6 (A), IL-1β (B), and IL-23 (C) were measured by ELISA. Statistical analyses were performed using paired-samples t-test (representative experiment of n = 6).

Discussion

In this study, we demonstrated that BBR could directly inhibit IL-17 production by CD4⁺ T cells and the DC-mediated differentiation of CD4⁺ IL-17⁺ T cells from both VKH patients and healthy donors. Moreover, it could inhibit the expression of costimulatory molecules on DCs, and inhibited proinflammatory cytokine production by these cells. These data provide a rationale for investigating the possibilities of using this drug in the treatment of clinical uveitis entities mediated by a Th17 response. 

BBR is an isoquinoline alkaloid that can be isolated from certain herbs, such as Berberis , Hydrastis canadensis , and Coptidis rhizoma . ¹⁴ The anti-inflammatory role of BBR has already been reported in several autoimmune diseases, but not yet in clinical uveitis. ^20–22,30 The only related study on the role of BBR in uveitis comes from an animal model for human uveitis, whereby Berberis aristata , whose main components include BBR, showed a potent anti-inflammatory activity against endotoxin-induced uveitis in rabbits. ³¹  

In a recent study from our laboratory, we showed that BBR significantly inhibited the TNF-α–induced expression of IL-6, IL-8, and monocyte chemoattractant protein 1 by ARPE-19 cells at both the protein and mRNA level and that it downregulated phosphorylation of p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase. ³² All these studies support a role for BBR as an immunosuppressive and anti-inflammatory agent. As Th17 responses are critically involved in the pathogenesis of several autoimmune diseases, ^1,4,33 we focused our attention to the role of BBR on the Th17 response, as this could offer a functional support for the use of this agent in the treatment of these diseases. In animal models, BBR has been shown to suppress the Th17 effector response.^20,21 Our study is, as far as we know, the first to study the effect of BBR on the human Th17 response. 

DCs play an important role in T-cell differentiation and activation and suppression of DC function may very efficiently control the specific immune response. The aforementioned results showed that BBR directly inhibited the Th17 response but it was not clear whether BBR could also exert its effect on Th17 cells in humans via an interaction with DCs. Our experiments revealed that CD4⁺ T cells cocultured with BBR-treated DCs became hyporesponsive. Analysis of the Th17 population and IL-17 secretion showed that in the responder T cells primed by BBR-treated DCs, there was a decrease of CD4⁺IL-17⁺ cells and a downregulated IL-17 production. To further examine the mechanisms that might be involved, we performed a further analysis on the effect of BBR on DC function. The results showed that BBR treatment significantly inhibited the LPS-induced production of proinflammatory cytokines, including IL-6, IL-1β, and IL-23, which are critical for the commitment of the Th17 subset. This result is, by and large, in agreement with that reported by Qin et al. ²¹ They found that BBR could inhibit the production of IL-6 and IL-23 in CD11b⁺ APCs from mice. This finding is also consistent with our observation that BBR inhibited maturation-associated IL-6, IL-1β, and IL-23 secretion by LPS-induced DCs. BBR also suppressed the expression of costimulatory molecules in DCs, which is in agreement with earlier findings in animals, which showed that BBR was able to downregulate the expression of CD80 and CD86 in mouse DCs stimulated by LPS. ¹⁹ A downregulated expression of costimulatory molecules on DCs may partially contribute to the observed impaired T-cell reactivity. Based on the study presented here and previous studies, it becomes evident that BBR can act as a negative modulator of the Th17 cell response via an effect on APCs, especially DCs. Extracts obtained from the roots of Berberidaceae species have been shown to have an effect on complement activation. ³⁴ Whether BBR also has an effect on the complement system in VKH is not yet known and deserves further study. 

The levels of inflammatory parameters in BBR-treated cells obtained from active VKH patients decreased, but not to the same low level as observed in the healthy donor group, which suggests that BBR treatment alone will be insufficient to control the disease. We speculate that it may be used as a steroid-sparing agent. Many agents can have an effect on inflammatory disease, but differences between the efficiency of BBR with other immunosuppressive agents was not the main interest of our study at this time and may be subject of future investigations. 

BBR is currently used in China for treating gastrointestinal diseases, including acute gastroenteritis and bacillary dysentery, and as yet no unexpected safety concerns have been noted. A recent randomized, placebo-controlled, double-blind trial concerning the effects of BBR gelatin on recurrent aphthous stomatitis, has shown that the use of BBR gelatin was safe and effective. ³⁵ However, the safety and efficacy of BBR treatment for ocular autoimmune diseases should be verified by clinical research. Our findings, together with previous animal studies, suggest that BBR, in combination with the currently used immunosuppressive agents, may potentially modulate an aberrant immune response and is likely to be an additive therapeutic approach for clinical uveitis entities, such as VKH and other autoimmune diseases mediated by an abnormal Th17 immune response. 

Acknowledgments

We thank all patients and healthy donors enrolled in the present study. 

Supported by Key Project of Natural Science Foundation Grant (81130019), National Basic Research Program of China (973 Program) (2011CB510200), National Natural Science Foundation Project Grant (81070722), Clinical Key Project of Ministry of Health, Basic Research Program of Chongqing, the Fund for PAR-EU Scholars Program, and Key Project of Health Bureau of Chongqing (2012-1-003), Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003). 

Disclosure: Y. Yang, None; J. Qi, None; Q. Wang, None; L. Du, None; Y. Zhou, None; H. Yu, None; A. Kijlstra, None; P. Yang, None 

Footnotes

 YY and JQ contributed equally to the work presented and should therefore be regarded as equivalent authors.