Total glucosides of paeony suppresses experimental autoimmune uveitis in association with inhibition of Th1 and Th2 cell function in mice.

Total glucosides of paeony (TGP) are active components extracted from the roots of Paeonia lactiflora Pall. In this study, we investigated the role and mechanisms of TGP in experimental autoimmune uveitis (EAU) model of mice. The C57BL/6 mice were randomly divided into three groups: sham group, EAU-control group, and EAU-TGP group. Clinical score of images of the eye fundus were taken on 7, 14, 21, and 28 days after induction of EAU. The concentrations of proinflammatory cytokines in intraocular fluid were measured at 14 days after EAU induction with the use of a multiplex assay system. Flow cytometry was used to analyze the frequency of CD4+, CD8+, interferon-gamma (IFN-γ), and CD4+/CD8+ ratio in spleen and lymph nodes. Western blotting was used to measure expressions of mitogen-activated protein kinase (MAPK) pathway-related proteins in retina. Clinical scores for uveitis were lower in TGP-treated EAU mice than those without TGP treatment. Importantly, the concentrations of cytokines induced by T-helper 1 (Th1) and T-helper 2 (Th2) cells in intraocular fluid were reduced in EAU mice treated with TGP. Furthermore, the frequency of CD4+, IFN-γ, and CD4+/CD8+ ratio was decreased and the frequency of CD8+ was increased in spleen and lymph nodes of mice treated with TGP. The anti-inflammatory effects of TGP were mediated by inhibiting the MAPK signaling pathways. Our results showed that TGP suppressed uveitis in mice via the inhibition of Th1 and Th2 cell function. Thus, TGP may be a promising therapeutic strategy for uveitis, as well as other ocular inflammatory diseases.

Introduction

Uveitis is a sight debilitating intraocular inflammatory disease caused by infection related to the immune responses or autoimmune responses.[1] Globally, uveitis is one of the main causes of severe vision impairment and even blindness in the working population.[2] Refractory uveitis is characterized by recurrence of inflammation that contributes to progressive destruction of affected tissues (such as neural retina, uveal tract, and adjacent tissues) and consequently leading to vision impairment and even blindness. Therefore, it still remains a great challenge for the treatment of refractory autoimmune uveitis. The conventional treatment of uveitis includes corticosteroids, immunosuppressive agents and biologics, which may be efficacious, but can be associated with serious systemic side effects including cataract, glaucoma and metabolic disorders.[3] Furthermore, many patients do not respond well to these treatments.[4] Therefore, development of effective and safe treatment strategies would be beneficial in patients with uveitis.

Experimental autoimmune uveitis (EAU) is a T cell-mediated autoimmune disease animal model, which has many clinical and histological characteristics in common with human autoimmune uveitis.[5] EAU is induced by injection of animals with S-antigen (S-Ag), human interphotoreceptor retinoid–binding protein (hIRBP), or adoptive transfer of retinal antigen-specific CD4+ T cells.[6-8] After immunization with these antigens for 2 or 3 weeks, animals would represent several pathological symptoms, such as bilateral uveitis, pinealitis, and retinitis. Histopathological lesions in EAU animals include serous retinal folding, retinal detachment, vasculitis, retinitis, photoreceptor damage, vitritis, choroiditis, and subretinal neovascularization.[9]

Earlier studies have demonstrated the involvement of T-helper (Th) lymphocyte in uveitis to be predominant.[10,11] The two subsets of Th lymphocytes, Th1 and Th2, are distinguished by the type of cytokines they secrete.[12,13] Th1 cells primarily secrete interleukin (IL)-2, IL-3, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), whereas Th2 cells secrete IL-4, IL-5, IL-6, and IL-13. Th1 control cells mediate functions such as the activation of macrophages, whereas Th2 cells stimulate humoral immunity.[14,15] It has been reported that Th1 cells play a pivotal role in the immunopathologic processes of EAU,[16,17] but the roles of Th1 and Th2 cells in human uveitis have not been fully explored yet.

Total glucosides of paeony (TGP) is a water/ethanol extract of the root of Paeonia lactiflora Pall, a traditional Chinese herb.[18] Paeoniflorin is the mainly active component of TGP and accounting for approximately 90% of the active components of TGP.[19] Studies have shown that TGP exerts effective role of analgesic,[20] anti-inflammatory,[21,22] immunomodulatory,[23,24] and antioxidant.[25] TGP has been approved as a disease modifying oral agent by the State Food and Drug Administration of China in the treatment of rheumatoid arthritis since 1998. TGP has been indicated to function as dual effects on the proliferation of lymphocytes, differentiation of Th lymphocytes, and production of proinflammatory cytokines and IgM-antibodies.[18] However, the role of TGP in uveitis has not been thoroughly studied. Therefore, in this study, we aimed to explore the effects of TGP in EAU mice and we also investigated the underlying molecular mechanism.

Materials and methods

EAU animal model

The animals used in this study were treated in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, and the study was approved by the Animal Care and Use Committee of Central South University. Six-week-old female C57BL/6 mice were obtained from Chiyoda Kaihatsu (Tokyo, Japan) and were fed normal diet. Mice were maintained in individual cages at 22°C–24°C, with a relative humidity of 50%–70% and with a 12 h light and 12 h dark cycle (lights on from 0700 to 1900 h). EAU was induced in mice at 8 weeks of age by subcutaneous injection of 50 μg of an NH2-terminal peptide fragment (residues 1–20, GPTHLFQPSLVLDMAKVLLD) of human IRBP (Scrum, Tokyo, Japan) emulsified with complete Freund’s adjuvant containing mycobacterium tuberculosis H37Ra at 6 mg/mL (Difco, Detroit, MI, USA). The emulsion (100 μL) was injected into a footpad and the inguinal region. Pertussis toxin (100 μg; Sigma, St. Louis, MO, USA) was also injected intraperitoneally at the same time. Mice were sacrificed by cervical dislocation at 21 or 28 days after induction of EAU.

For the analysis of the effect of TGP in EAU animal model, C57BL/6 mice were administrated with 100 mg/kg TGP. Briefly, for each experiment, nine female C57BL/6 mice were randomly allocated into three groups (n = 3 per group): (1) sham: did not receive any treatment; (2) EAU-control: EAU was performed and received single-dose intragastric 0.1 mL saline every day/time from the induction of EAU to sacrifice; and (3) EAU-TGP: EAU was performed and received 100 mg/kg TGP with intragastric administration every day/time from the induction of EAU to sacrifice.

Scoring of EAU

At 7, 14, 21, and 28 days after induction of EAU, mice were anesthetized by intraperitoneal injection of a mixture of ketamine (90 mg/kg; Daiichi Sankyo, Tokyo, Japan) and xylazine (10 mg/kg; Bayer Yakuhin, Osaka, Japan), and the pupils were dilated by topical instillation of 0.5% tropicamide and 0.5% phenylephrine hydrochloride ophthalmic solutions (Santen, Osaka, Japan). Eye gel (Scopisol; Senju, Osaka, Japan) was applied to the cornea for contact with a digital medical scope (VersaCam; NIDEK, Aichi, Japan), focus and illumination were adjusted, and images of the fundus were obtained. Clinical score for EAU was graded by retina specialists on the basis of the photographs in a blinded manner on a scale of 0 to 5: 0, no evidence of inflammation; 1, focal vasculitis and/or spotted soft exudate of less than 5 spots; 2, linear vasculitis and/or spotted soft exudate within half of the retina; 3, linear vasculitis and/or spotted soft exudate over half of the retina; 4, retinal hemorrhage along with severe vasculitis and/or spotted soft exudate; 5, exudative retinal detachment or subretinal hemorrhage.

Analysis of cytokines and chemokines in intraocular fluid

Mice were sacrificed and the eyeballs were removed 21 days after EAU induction. Each eyeball was trimmed of extraocular muscles and soft tissue and then crushed with the use of a BioMasher (Sarstedt, Germany). The crushed sample was centrifuged at 9000g for 30 s, and the resulting supernatant (intraocular fluid) was stored at −80°C. Assay of cytokine and chemokine concentrations was done using a Bio-Plex Pro Mouse Cytokine 23-Plex Panel and Bio-Plex Manager software version 4.1.1 (Bio-Rad, Hercules, CA, USA).

Isolation and culture of T cells

C57BL/6 mice were immunized with human IRBP peptide (Scrum) for EAU induction as described above. After 14 days, mice were sacrificed and T cells were isolated from spleen and lymph gland and cultured in six-well flat-bottom plates at a concentration of 1 × 107 cells/well with Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 2% penicillin–streptomycin (Gibco-BRL, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS; Gibco-BRL) in the absence or presence of 10 μg/mL human IRBP peptide.

Flow cytometry analysis

Flow cytometry was conducted to analyze the frequency of CD4+ cells, CD8+ cells, interferon γ (IFN-γ), and the ratio of CD4+/CD8+. Cells were stained with 10 µL of anti-porcine CD25 (AbD Serotec, MCA1736 clone K231.3B2), followed by 0.06 µg of Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG, H+L; Invitrogen, Molecular Probes, Carlsbad, CA, USA). The LYNX rapid antibody conjugation kit (AbD Serotec) was used to conjugate mouse anti-porcine CD4 with allophycocyanin (APC; clone 74-12-4; VMRD, Inc., Pullman, Wa, USA) and CD8 with RPECy7 (clone 76-2-11; VMRD, Inc.), and 0.05 µg of these antibodies were then added to the cell preparation. Forkhead box P3 (Foxp3) intracellular staining was performed with 0.03 µg of anti-rat/mouse Foxp3 (clone FJK-16s, with cross-reactivity with swine Foxp3; eBioscience, San Diego, CA, USA) and 0.03 µg of PE-conjugated rat IgG2a isotype control (clone eBR2a; eBioscience) using the Foxp3 Staining Buffer Set (Staining, Fixation/Permeabilization, and Permeabilization Buffers; eBioscience) to obtain the four-color stain CD4APCCD8RPECy7CD25Alexa488Foxp3PE. The frequency of regulatory T cells (Tregs) was evaluated by flow cytometry (BD FACS Canto II), and data were analyzed using BD FACS Diva 6.0 software.

Western blot analysis

The retinas were collected 14 days after immunization and lysed by Radioimmunoprecipitation Assay (RIPA) Lysis Buffer (Beyotime, Shanghai, China) including 1% proteases inhibitor (Beyotime). The lysate was centrifuged and the supernatant was collected. Protein concentration was determined by bicinchoninic acid (BCA) protein kit (Beyotime). All samples were diluted in sodium dodecyl sulfate (SDS) loading buffer (Beyotime) and boiled for 5 min. Equal amounts of protein (80 μg) were loaded to 10% polyacrylamide gel (PAG) for the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein was then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk or 5% bovine serum albumin (BSA) and incubated with specific primary antibodies over night at 4 °C, followed by the secondary antibody (1:3000; Abcam, Cambridge, MA, USA) at 37 °C for 1 h. The membranes were further developed by Western Bright™ ECL kit (Advansta, Menlo Park, CA, USA). Bands were analyzed using ImageJ software (Version1.43; Broken Symmetry Software, Bethesda, MD, USA). Analysis was normalized against a housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Statistical analysis

Quantitative data are presented as mean ± standard deviation (SD) and were analyzed with Prism version 6 (GraphPad Software, San Diego, CA, USA). EAU scores were compared among groups with nonparametric Mann–Whitney U test or Tukey–Kramer test. Other data were compared between groups with unpaired Student’s t test. A P value of <0.05 was considered statistically significant.

Results

Effects of TGP on clinical score

The images of the fundus were taken at 7, 14, 21, and 28 days after induction of EAU. The mice were anesthetized while taking the images. Examination of the fundus revealed that there was no significant difference in the clinical score of the mice fundus between the EAU-control group and EAU-TGP group on day 7 after human IRBP (1–20) injection (Figure 1). However, at 14, 21, and 28 days after EAU induction, the clinical scores in the EAU-control group were significantly higher than those in the EAU-TGP group (P < 0.05, P < 0.01, P < 0.001 or P < 0.0001; Figure 1). These results suggested that TGP suppressed the inflammation associated with EAU.

Figure 1.

Clinical score for EAU. EAU clinical scores were determined from fundus images of mice at 7, 14, 21, and 28 days after injection with hIRBP(1–20) and control or TGP. The sham mice were similarly examined. Data are represented as mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.

EAU: experimental autoimmune uveitis; hIRBP: human interstitial retinoid-binding protein; NS: not significant; SD: standard deviation; TGP: total glucosides of paeony.

Effects of TGP on inflammatory cytokines and chemokines in intraocular fluid

To investigate the mechanism by which TGP suppresses inflammation in EAU mice, we measured the concentrations of inflammatory mediators in intraocular fluid at 14 days after EAU induction with the use of a multiplex assay system. The concentrations of the proinflammatory cytokines IL-1β (P < 0.01), IL-6 (P < 0.05), and TNF-α (P < 0.001); the chemokines MCP-1 (monocyte chemoattractant protein–1; P < 0.01) and RANTES (regulated on activation, normal T expressed and secreted; P < 0.01); and the Th17 cytokine IL-17A (P < 0.05) were significantly reduced in the EAU-TGP group compared to the EAU-control group (Figure 2). These results indicated that TGP suppressed inflammation in EAU mice by decreasing the concentrations of inflammatory cytokines, chemokines, and Th17 cytokine IL-17A in intraocular fluid.

Figure 2.

Concentrations of inflammatory cytokines and chemokines in intraocular fluid of mice. The concentrations of (a) IL-1, (b) IL-6, (c) TNF-α, (d) MCP-1, (e) RANTES, and (f) IL-17A in intraocular fluid of EAU mice were determined with a multiplex assay at 14 days after injection of hIRBP(1–20) and control or TGP.

The sham mice were similarly examined. Data are represented as mean ± SD. ***P < 0.001, **P < 0.01, and *P < 0.05.

EAU: experimental autoimmune uveitis; hIRBP: human interstitial retinoid-binding protein; IL: interleukin; MCP-1: monocyte chemoattractant protein–1; RANTES: regulated on activation, normal T expressed and secreted; SD: standard deviation; TGP: total glucosides of paeony; TNF-α: tumor necrosis factor-alpha.

Effects of TGP on expression of CD4+, CD8+, CD4+/CD8+, and IFN-γ

Flow cytometry was used to analyze the frequency of CD4+ cells, CD8+ cells, IFN-γ, and CD4+/CD8+ ratio in spleen and lymph nodes. As shown in Figure 3, the frequency of CD4+, CD4+/CD8+, and IFN-γ was significantly higher in EAU-control group than that in the EAU-TGP group (P < 0.05). In contrast, the frequency of CD8+ was significantly lowered in the EAU-control group than that in the EAU-TGP group (P < 0.01). These findings suggested that TGP exerts immunomodulatory effects by decreasing the proportions of CD4+ and IFN-γ and increasing the proportions of CD8+ cells.

Figure 3.

Frequency of CD4+, CD8+, CD4+/CD8+, and IFN-γ. The frequencies of (a) CD4+, (b) CD8+, (c) CD4+/CD8+, and (d) IFN-γ populations were quantified by flow cytometry after injection of hIRBP(1–20) and control or TGP. The sham mice were similarly examined. Data are represented as mean ± SD. **P < 0.01 and *P < 0.05.

EAU: experimental autoimmune uveitis; hIRBP: human interstitial retinoid-binding protein; IFN-γ: interferon-gamma; SD: standard deviation; TGP: total glucosides of paeony.

Effects of TGP on MAPK pathway

To determine whether MAPK signaling pathway is involved in TGP-mediated anti-inflammatory effects, phosphorylation levels of p38, extracellular-activated kinase (ERK), and c-Jun N-terminal kinase (JNK) in the retina were measured by western blotting at 14 days after immunization. The phosphorylation levels of the three proteins demonstrated an increase in the EAU-control group, while significantly decreased in the EAU-TGP group (P < 0.05 or P < 0.01; Figure 4). These results showed that the anti-inflammatory effects of TGP in EAU eyes are mediated by inhibition of MAPK pathway.

Figure 4.

Involvement of the MAPK pathway in uveitis. The phosphorylation levels of (b) p38, (c) ERK, and (d) JNK in the sham, EAU-control, and EAU-TGP groups were determined by western blotting. (a) Western blot band intensities. The band intensities of p-p38, p-ERK, and p-JNK were normalized to p38, ERK, and JNK, respectively. Data are represented as mean ± SD. **P < 0.01 and *P < 0.05.

EAU: experimental autoimmune uveitis; ERK: extracellular regulated kinase; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; SD: standard deviation; TGP: total glucosides of paeony.

Discussion

In this study, we showed that TGP suppressed intraocular inflammation in mice with EAU. We demonstrated that the concentrations of proinflammatory cytokines and chemokines produced by Th1 and Th2 cells and IL-17A induced by Th17 cells were significantly increased in EAU mice. TGP treatment decreased the concentrations of these inflammatory cytokines and chemokines. We also showed that TGP reduced the proportions of CD4+, CD4+/CD8+ ratio, and IFN-γ and increased CD8+ in spleen and lymph nodes. Finally, we found that the anti-inflammatory effects of TGP in EAU might be mediated via inhibition of the MAPK signaling pathway.

Proinflammatory cytokines are mainly produced by activated macrophages and are responsible for upregulation of inflammation responses. The typical proinflammatory cytokines include IL-1β, TNF-α, and IL-6. IL-1β is secreted predominantly by monocytes and macrophages,[26] whereas TNF-α is secreted by Th1 cells and IL-6 is secreted by Th2.[14,15] IL-17 is produced mainly by Th17 cells.[19] IL-17 increases the production of TNF-α, IL-1, and IL-6 and enhances local inflammation.[27] MCP-1 and RANTES are proinflammatory cytokines that are members of the C-C class of beta chemokine supergene family and are mediators of acute and chronic inflammation.[28] Therefore, suppression of these proinflammatory cytokines leads to the alleviation of inflammation, which is a major symptom of uveitis.

The anti-inflammatory effect of TGP has been evaluated in many animal and clinical studies. A previous study has reported that TGP significantly suppressed the production of TNF-α and IL-1 and inhibited the proliferation of synoviocytes in rats with collagen-induced arthritis.[29] Xu et al.[30] showed that TGP inhibited the production of IL-1, TNF-α, IL-6, and prostaglandin E(2). In a clinical study that was conducted in diabetic patients, TGP significantly lowered the serum levels of MCP-1 and TNF-α.[31] Lin et al.[32] demonstrated that TGP significantly decreased the proportion of Th1 and Th17 (IL-17) cells in collagen-induced arthritis in mice. In agreement with these findings, we showed that TGP significantly reduced the concentrations of IL-1β, IL-6, TNF-α, MCP-1, RANTES, and IL-17A in intraocular fluid of EAU mice. These findings explain the anti-inflammatory effects of TGP in uveitis.

CD4+ T cells play vital role in the development of ocular autoimmunity. Activated CD4+ T cells differentiate into Th1 or Th2 phenotypes, which were characterized by the different cytokines they produce.[33] Autoreactive CD4+ T cells that exhibiting a Th1 phenotype play a crucial role in the pathogenesis of clinical uveitis and experimental uveitis.[9] Ohta et al.[34] reported that the increased concentrations of CD4 and CD8 lymphocytes were observed in the aqueous humor of patients with uveitis, when compared with peripheral blood of the patients and with aqueous humor of healthy subjects. In our study, we found that concentrations of CD4+ were increased and concentrations of CD8+ were decreased in EAU mice, which were reversed by TGP. In addition, we also investigated the concentration of IFN-γ. IFN-γ is a cytokine that is produced by Th1 cells,[15] which acts as a protective or proinflammatory cytokine, depending on the disease stage at which it is produced.[35] It has been shown that administration of IFN-γ when immunization induction exerts a protective effect in EAU mice.[36] In contrast to this finding, we found increased concentrations of IFN-γ in EAU mice, which were reduced by TGP. These findings revealed the immunomodulatory effects of TGP in autoimmune uveitis.

MAPK signaling pathway plays a pivotal role in the process of inflammation. Phosphorylation of this pathway-related core factors leads to the activation of macrophages and release of proinflammatory cytokines,[37,38] thus enhancing cell inflammation response. It has been suggested that TGP exhibited anti-inflammatory effects by modulating the activation of MAPKs. Zheng and Wei[22] showed that TGP inhibited the phosphorylation of p38, ERK, and JNK in rats with adjuvant arthritis. Similarly, in our study, we showed that TGP inhibited the EAU-induced phosphorylation of p38, ERK, and JNK, suggesting the role of the MAPK pathway in uveitis.

The immunomodulatory and anti-inflammatory effects of TGP provided the therapeutic basis in the treatment of autoimmune diseases. TGP has been tested in clinical trials for some autoimmune diseases, such as RA, systemic lupus erythematosus (SLE), Sjögren syndrome, ankylosing spondylitis, and chronic urticaria.[18,39-41] However, animal and clinical data evaluating the role of TGP in the treatment of uveitis are scarcely available. Therefore, this study could be the first to explore the role of TGP in EAU model of mice. We demonstrated that TGP could suppress the harmful effects of human IRBP in mice with EAU, as evidenced by ameliorating the clinical signs of uveitis. However, there still exists limitation in this study, and it would be better to use a positive control group for the comparison of TGP effects in EAU mice. We will apply to the positive control group as a comparison to better illustrate the effect of drug in our future study.

Conclusion

In summary, we demonstrated that treatment with TGP significantly reduced the clinical scores of EAU in mice, indicating protective effect of TGP in uveitis. Of significant interest, TGP reduced the concentrations of proinflammatory cytokines produced by Th1 and Th2 cells in intraocular fluid of EAU mice. In addition, we found that TGP acts as an immunomodulator by decreasing the frequency of CD4+, CD4+/CD8+ ratio, and IFN-γ and increasing the frequency of CD8+ in the spleen and lymph nodes. Finally, we suggested that TGP might exert the anti-inflammatory effect through its modulation on the activation of MAPK signaling pathway.