Journal of Ethnopharmacology 155 (2014) 1306–1314

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Clematichinenoside AR induces immunosuppression involving Treg cells in Peyer's patches of rats with adjuvant induced arthritis Ying Xiong a, Yan Ma a, Wang Han a, Nandani Darshika Kodithuwakku a, Li-Fang Liu b, Feng-Wen Li c, Wei-Rong Fang a,n, Yun-Man Li a,nn a

State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, # 24 TongJiaXiang, Nanjing 210009, PR China Department of Pharmacognosy, the Key Laboratory of Modern Chinese Medicines (Ministry of Education), China Pharmaceutical University, # 24 TongJiaXiang, Nanjing 210009, PR China c Department of Traditional Chinese Pharmacy, China Pharmaceutical University, # 24 TongJiaXiang, Nanjing 210009, PR China b

art ic l e i nf o

a b s t r a c t

Article history: Received 28 December 2013 Received in revised form 23 May 2014 Accepted 15 July 2014 Available online 23 July 2014

Ethnopharmacological relevance: Clematichinenoside AR (AR) has been defined as a major active ingredient of triterpenoid saponins extracted from Clematidis Radix et Rhizoma, which is a traditional Chinese herbal medicine that has long been used in the treatment of rheumatoid arthritis (RA). To further explore the mechanism of AR in the treatment of RA, we investigated whether its immunomodulatory effects are related to Treg-mediated suppression derived from Peyer's patches (PPs) in adjuvant induced arthritis (AIA) rat model. Materials and methods: AR (8, 16, 32 mg/kg) was orally administered daily from Day 18 to Day 31 after immunization. The effect of AR on AIA rats was evaluated by hind paw swelling and histopathological examination. Percentages of CD4 þ CD25 þ Foxp3 þ T regulatory cells were determined by flow cytometry. Levels of IL-10, TGF-β1, IL-17A and TNF-α were measured by ELISA. Expressions of Foxp3 and RORγ in synovium were detected using immunohistochemical analysis. Results: AR treatment significantly reduced paw swelling of AIA rats, and histopathological analysis confirmed it could suppress severity of established arthritis. AR treatment upregulated the percentages of CD4 þ CD25 þ Foxp3 þ Treg cells among CD4þ T cells in PPs lymphocytes, and increased the levels of IL10 and TGF-β1 secreted from ConA-activated PPs lymphocytes, whereas decreased the levels of IL-17 A and TNF-α. Similar tendency of circulating CD4 þ CD25 þ Foxp3 þ Treg cells percentages and serum cytokine levels were observed. Moreover, AR decreased the expression levels of Foxp3 and RORγ in joint synovial membrane. Conclusions: In conclusion, these results suggested AR has a potent protective effect on the progression of AIA, probably by augmenting CD4 þ CD25 þ Foxp3 þ Treg cells in PPs to induce immunosuppression, and modulating the balance between Treg cells and Th17 cells systemically. These findings may help to develop AR as a potent immunosuppressive agent for the treatment of RA. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Clematichinenoside AR Rheumatoid arthritis Adjuvant induced arthritis Peyer's patch CD4þ CD25 þFoxp3þ Treg cell Immunosuppression

1. Introduction Clematis chinensis Osbeck, a member of Ranunculaceae family, is a semi-evergreen perennial deciduous vine and mainly distributed in People's Republic of China. The dried roots and rhizomes of

Abbreviations: AIA, adjuvant-induced arthritis; AR, clematichinenoside AR; Foxp3, forkhead box P3; GALT, gut-associated lymphoid tissues; IL-10, interleukin-10; IL-17 A, interleukin-17 A; PPs, Peyer's patches; RA, rheumatoid arthritis; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; Treg cells, regulatory T cells n Corresponding author. Tel./fax: þ 86 25 83271173. nn Corresponding author. E-mail addresses: [email protected] (W.-R. Fang), [email protected] (Y.-M. Li). http://dx.doi.org/10.1016/j.jep.2014.07.028 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

Clematis chinensis Osbeck, Clematis hexapetala Pall., and C. mandshurica Rupr., collectively termed Radix Clematidis et Rhizoma (Weilingxian, clematis root) with the effects of dispelling wind, eliminating dampness and activating meridians to stop pain, have long been used in traditional Chinese herbal medicine for the treatment of rheumatic arthralgia, limbs numbness, tendons spasm, discomforts of flexion and extension. According to the traditional Chinese medicinal view, the nature and flavor of Radix Clematidis are pungent, salty and warm, and channel tropism shows that Radix Clematidis acts on the urinary bladder meridian (State Pharmacopoeia Committee of People's Republic of China, 2010). Accumulative evidence has demonstrated that saponins extracted from Clematidis Radix possess anti-inflamatory (Fu et al., 2010; Shi et al., 2006) and chondroprotective (Hsieh et al., 2011;

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Wu et al., 2013, 2010) properties, and clematichinenoside AR (AR) has been isolated and identified as the major active component of triterpenoid saponins (Liu et al., 2009; Ma et al.,2009; Zhou et al., 2012). Our previous studies indicated that intragastric administration of AR markedly improved the hind paw swollen ratio and histopathological changes in collagen-induced arthritis (CIA) rats, decreased the levels of IL-1β and TNF-α in serum, and downregulated the expression levels of NF-κB p65 subunits, TNF-α and COX-2 in synovial membrane tissues (Peng et al., 2012). Moreover, AR treatment significantly reduced the expression levels of TNF-α, PI3K and p-Akt in synovium of the CIA rats (Han et al., 2013). These studies demonstrated that AR is beneficial in preventing experimental arthritis, due to its anti-inflammatory and immunosuppressive properties. In addition, AR has been shown to have a favorable membrane permeability within the whole segment of small intestine, and be able to diffuse passively into the intestinal mucosa (Wang et al., 2012). AR was mainly metabolized by intestinal microflora, eight metabolites of which have been identified and might contribute synergisticly to the total pharmacological activities of AR (Li et al., 2013). Therefore, we hypothesized that the mechanism underlying the anti-arthritis effect of AR may be related to the regulation of enteric mucosal immune system when orally administrated. Peyer's Patches (PPs), located within the small intestinal epithelium, are highly organized lymphoid follicles of the gut-associated lymphoid tissues (GALT), which is a well-developed immune network. PPs serve as essential physiological inductive sites of oral tolerance, mucosal immunity and systemic immunity. Moreover, PPs are hotbeds for Regulatory T cells (Treg cells) generation (Ahlers and Belyakov, 2010; Nagatani et al., 2004). Treg cells are the kind of cells that acquire their regulatory potential during differentiation in the thymus and the population induced from naive T cells in the periphery under various tolerogenic conditions, which are termed as naturally occurring Tregs and inducible Tregs, respectively. Although the phrase ‘Treg cells' encompasses different cell subtypes, those expressing the specific transcription factor forkhead box P3 (Foxp3) named CD4 þ CD25 þ Foxp3 þ Treg cells have received considerable attention due to their prominent immunosuppressive properties. CD4 þ CD25 þ Foxp3 þ Treg cells are responsible for the induction of mucosal immune suppression, maintaining immune system homeostasis and negative regulation of systemic immunity. The expansion of naturally occurring and inducible CD4 þ CD25 þ Foxp3 þ Treg cells considerably induced therapeutic benifits in experimental polyarthritis models and RA patients (Huang et al., 2012; Kong et al., 2012; Moon et al., 2013). It has been established that de novo induction of CD4 þ CD25 þ Foxp3 þ Treg cells in peripheral sites outside the thymus preferentially occurs in GALT (Ahlers and Belyakov, 2010), and PPs have been demonstrated to be the key site for the generation of interleukin-10 (IL-10) producing CD4 þ CD25 þ Foxp3 þ Treg cells and transforming growth factor-β (TGF-β) þ Treg cells (Takayama et al., 2007; Zhong et al., 2010). There is a constant migration of lymphocytes from PPs or other mucosal inductive sites to the mucosal regulatory network, and to the peripheral circulation where they control the systemic immune homeostasis (Park et al., 2009; Simecka, 1998; Wang et al., 2009). Once the function of GALT lymphocytes was induced to change, the function of systemic immune cells might alter subsequently (Biondo et al., 2008; Wieten et al., 2010). Therefore, modulation of PPs lymphocytes activity might influence the host's ability against immunologic responses, and upregulation of Treg cells derived from PPs has been confirmed to be beneficial for controlling the severity of experimental arthritis (Park et al., 2008; Zhou et al., 2006). Based on the characteristics of intestinal absorption and metabolism, the anti-arthritis effect of AR was supposed to be involved in the enteric mucosal immune system,

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and this hypothesis was tested in this study using the adjuvantinduced arthritis (AIA) rat model. RA is characterized by T cells imbalance, among which Treg and Th17 subsets play the antagonistic roles in the initiation and development of autoimmune arthritis, and modulation of the balance between Treg and Th17 subsets is crucial for the treatment of RA (Moon et al., 2013; Seki et al., 2008). So far, the effects of AR on mucosal and peripheral immunity associated with Treg cells have not been investigated. This study would emphasize on the effects of AR via oral administration on PPs-associated and systemic immune function based on the regulation of CD4 þ CD25 þ Foxp3 þ Treg cells. In addition, the function of Th17 cells was also studied on account of the immunomodulatory effect of AR. Taken together, the aim of the study is to determine the potential of AR as a therapeutic agent for RA treatment.

2. Materials and methods 2.1. Drugs and reagents AR was provided by Jiangsu Chia-tai Tianqing Pharmaceutical Co., Ltd. (Nanjing, China), and was suspended in distilled water at required concentrations. Tripterygium glycosides (TG) used as a positive control was purchased from Jiangsu Meitong Pharmaceutical Co., Ltd. (Taizhou, China), and was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) solution. The reagents included complete Freund's adjuvant (CFA) (Chondrex, USA), Rat organs and tissues lymphoprep (Hao Yang Biological Manufacture, CN), RPMI-1640 medium (Gibco, USA), Concanavalin A (ConA) (Sigma-Aldrich, USA), enzyme-linked immunosorbent assay (ELISA) kits for TGF-β1 and IL-10 (ExCell Biology, CN), ELISA kits for TNF-α and IL-17 A, anti-Mouse/Rat Foxp3 Staining Set APC, anti-Rat CD4 FITC, anti-Rat CD25 PE, flow cytometry staining buffer and erythrocyte lysis buffer (eBioscience, USA), anti-Foxp3 antibody and anti-ROR gamma antibody (Abcam, USA). Other chemicals and reagents used in these experiments were analytical grade from commercial sources. 2.2. Animals Male Sprague-Dawley rats weighing 110–130 g were purchased from Shanghai Super-B&K Laboratory Animal Co., Ltd. (Shanghai, China). Rats were adapted for a week and housed under standard laboratory condition of constant temperature (23 72 1C) with a 12-h alternate light/dark cycle. Rats had free access to commercial standard diet and tap water ad libitum. The experimental use of animals and procedures were performed in accordance with current ethical regulations for animal care and use at China Pharmaceutical University. 2.3. Induction, treatment and evaluation of AIA SD rats were immunized by intradermal inoculation into the plantar surfaces of right hind paws with 0.1 ml of CFA containing 10 mg/ml of heat-killed M. tuberculosis H37 RA. The day of inoculation was regarded as day 0. On day 18 after immunization, rats were randomly allocated into six groups, among which AR (8, 16, 32 mg/kg) and TG (18 mg/kg) were administered intragastrically once daily for 2 weeks. Normal and AIA model group rats were given distilled water in parallel. The volumes of non-inoculated hind paws were measured with a YLS-7C plethysmometer (Jinan Yiyan Technology Development Co., Ltd., China). Paw volume (ml) was measured on days 0, 18, 21, 25, 28, and 32 after immunization. Results were expressed as the volume of increase with respect to day 0 volume.

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2.4. Preparation of Blood samples

2.8. Histopathological examination

After 14 days of AR administration, peripheral blood was collected under anesthesia and the rats were sacrificed. A portion of collected blood was allowed to clot, centrifuged at 3000 rpm for 10 min, and then separated serum was collected and stored at 20 1C until assay. Another portion of the blood was immediately transferred into a blood collection tube containing EDTA for flow cytometric analysis.

On day 32 after immunization, the rats were sacrificed and the rear paw from each rat was removed, fixed in 10% neutral buffered formalin, and decalcified in EDTA solution for 3 weeks at 4 1C. The paws then were embedded in paraffin, sectioned at 5 μ m and stained with hematoxylin-eosin (HE) for histological examination microscopically. The pathological evaluation was performed by a pathologist blind to the groups of specimens.

2.5. Isolation and culture of PPs lymphocytes, and induction of Treg- and Th17-related cytokine production The whole length of small intestine was placed in cold D-Hanks' balanced salt solution containing 100 IU/ml penicillin and 100 μg/ml streptomycin. Visible PPs were carefully excised, mechanically dispersed by grinding, and filtrated through a 300mesh nylon gauze to yield single cell suspensions. Then the cells were resuspended in D-Hanks' solution containing 10% FBS, and carefully overlaid onto the surface of lymphoprep. After centrifugation at 2000 rpm for 15 min at room temperature, the white cloudy band at the interface was collected and washed twice with washing solution. Then the cells were suspended in complete RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 2 mM glutamine, 25 mM HEPES, 50 μM β-mercaptoethanol, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cell viability was assessed by trypan blue dye exclusion showing that more than 95% of the cells were viable. Isolated lymphocytes were seeded (1  106 cells/well) in 24well plates and stimulated with 5 μg/ml ConA, a polyclonal T-cell mitogen, in a humidified incubator at 37 1C containing 5% CO2. After 48 h of incubation, the supernatants were harvested for measurement of the levels of different cytokines including IL-10, IL-17A and TNF-α. In addition, cells cultured with ConA for 8 h were washed and resuspended in serum-free RPMI-1640 medium, then incubated for 72 h at 37 1C with 5% CO2. Culture supernatants were then collected for determination of TGF-β1 level. 2.6. Measurement of Treg- and Th17-related cytokines by ELISA Serum samples and culture supernatants of ConA-stimulated PPs lymphocytes were stored at  20 1C until assays. The levels of Treg-related cytokines (IL-10, TGF-β1) and Th17-related cytokines (IL-17 A, TNF-α) were measured by ELISA kits following the protocols recommended by the manufacturers. Acid treatment of the samples was used to activate latent TGF-β1 in serum and supernatants. Absorbance at 450 nm was measured and the results were calculated using standard curves.

2.9. Immunohistochemical staining of Foxp3 and RORγ The hind paw tissue specimens were prepared as described above, and sections of 5 μm thickness were cut, dewaxed using xylene, and hydrated in gradients of alcohols. After microwave heating mediated antigen retrieval, endogenous peroxidase activity was blocked using 3% H2O2. Slides were incubated in sheep non-immune serum for 10 min to block nonspecific binding. Each section was incubated with 20 μg/ml anti-Foxp3 antibody or 10 μg/ml anti-RORγ antibody for an hour at room temperature, followed by incubation with peroxidase linked goat anti-mouse/rabbit IgG secondary antibody for 10 min. Immune complexes were stained with diaminobenzidine tetrahydrochloride (DAB), and nuclei were counterstained with hematoxylin. Control sections were treated in parallel with PBS substituting primary Abs and no staining. After dehydration in gradients of alcohols and transparentization using xylene, the sections were mounted with neutral gum. Samples were photographed with an Olympus photomicroscope (Japan). For immunohistochemical analysis, the numbers of positively stained cells in five high power (400  ) fields of vision were counted and averaged in each section.

2.10. Statistical analysis Values were presented as mean7standard deviation (mean7SD). Multiple comparisons among different treatment groups were performed by one-way ANOVA, followed by SNK test (equal variances assumed) or Dunnett's T3 test (equal variances not assumed) as posthoc analysis. P values less than 0.05 were considered statistically significant.

3. Results 3.1. AR suppressed the progression in AIA rats

2.7. Flow cytometric analysis of CD4 þ CD25 þ Foxp3 þ Treg cells After erythrocytes were lysed, cells of the whole blood samples were collected, washed twice, and suspended in PBS at a concentration of 1  106 cells/ml. The freshly isolated PPs lymphocytes were suspended in PBS as above. After blocking nonspecific binding sites, cells were resuspended in staining buffer, and stained using FITC-conjugated anti-CD4 and PE-conjugated antiCD25 mAbs as cell surface markers at 4 1C for 30 min. Then cells were stained with APC-conjugated anti-Foxp3 mAb for intracellular staining after fixation and permeabilization using a Foxp3 staining set, according to the manufacturer's recommendations. Isotype controls were given to enable correct compensation and confirm antibody specificity. Stained cells were then resuspended in staining buffer for analysis by FACSCalibur flow cytometer (BD Bioscience).

As shown in Fig. 1A, left hind paw swelling was significantly increased in the AIA model rats compared with the normal rats. Treatment with AR and TG apparently diminished the severity of non-injected hind paw swelling during the course of the experiment. As shown in Fig. 1B, no inflammation or joint destruction was observed in the normal rats. Whereas in the AIA model rats, the joint architectures exhibited signs of severe arthritis such as synovial hyperplasia, a large amount of inflammatory cells infiltration into the synovial lining layer, pannus formation, bone and cartilage destruction partially. The pathological changes in rats with AIA were attenuated by the treatment of AR and TG for 14 days. Synovial hyperplasia and inflammatory cell infiltration were reduced, pannus formation was inhibited, and the articular cartilage destruction was alleviated, indicating that treatment with AR and TG could effectively attenuate the inflammatory response.

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Fig. 1. AR induced a protective effect against AIA. (A) The severity of arthritis evaluated by the paw swelling during the course of AIA. (n¼ 8 per group). ##Po 0.01 vs. normal group; nPo 0.05, nnPo 0.01 vs. model group (B) Effects of AR on histopathological examinations of ankle joints by HE staining under a light microscope. (original magnification, 100  ). (n ¼8 per group).

3.2. AR upregulated Treg-related cytokine production in ConA-activated PPs lymphocytes and serum To investigate the effects of AR on Treg cells function, we analyzed the concentrations of IL-10 and TGF-β1 in serum and culture supernatants of ConA-activated PPs lymphocytes. As shown in Fig. 2A, concentrations of IL-10 and TGF-β1 secreted into the culture supernatants were reduced significantly in the model group compared with the normal group. The concentrations of IL10 and TGF-β1 in supernatants of ConA-activated PPs lymphocytes were significantly elevated by the treatment with AR for 14 days. Moreover, AR and TG led to increased levels of IL-10 and TGF-β1 in serum (Fig. 2B), indicating that their beneficial effects might be derived from upregulation of Treg-related cytokine production. 3.3. AR downregulated Th17-related cytokine production in ConA-activated PPs lymphocytes and serum As shown in Fig. 3A, treatment of PPs lymphocytes with ConA for 48 h resulted in significant elevation of Th17-related cytokine production in the model group, compared with the normal group.

Concentrations of IL-17 A and TNF-α in culture supernatants were decreased significantly due to the treatment of AR and TG for 14 days. Similar tendency of the Th17-related cytokine concentrations was also observed in serum (Fig. 3B). These results indicated that AR and TG were able to decrease the production of Th17-related cytokines, which may inflect their inhibitory effect on Th17 cells function. 3.4. AR increased the CD4 þ CD25 þ Foxp3 þ regulatory T cells in PPs lymphocytes and peripheral blood The Treg-related cytokine level changes were likely due to the proportions of lymphocyte populations influenced by effects of AR and TG, thus the number of CD4 þ CD25 þ Foxp3 þ Treg cells in PPs and peripheral blood were determined by flow cytometry. As shown in Fig. 4A and B, the percentage of CD4 þ CD25 þ Foxp3 þ cells among CD4 þ T cells of PPs lymphocytes decreased significantly in the model group. Treatment of AR and TG led to increased percentages of CD4 þ CD25 þ Foxp3 þ Treg cells among CD4 þ T cells. Similar tendency was observed for the percentages of circulating CD4 þ CD25 þ Foxp3 þ Treg cells (Fig. 4C and D).

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Fig. 2. Effects of AR on Treg-related cytokine levels in AIA rats. (A) Treg-related cytokine concentrations in culture supernatants of ConA-activated lymphocytes isolated from PPs. (n ¼5 per group) (B) Treg-related cytokine concentrations in serum. (n¼ 8 per group) ##Po 0.01 vs. normal group; nP o0.05, nnP o 0.01 vs. model group.

Fig. 3. Effects of AR on Th17-related cytokine levels in AIA rats. (A) Th17-related cytokine concentrations in culture supernatants of ConA-activated lymphocytes isolated from PPs. (n ¼5 per group) (B) Th17-related cytokine concentrations in serum. (n ¼8 per group) #P o0.05, ##P o0.01 vs. normal group; nPo 0.05, nnP o0.01 vs. model group.

3.5. AR decreased the expression levels of Foxp3 and RORγ in synovial membrane As shown in Fig. 5, an increased population of Foxp3-expressing cells was detected in the model group, and the expression levels of Foxp3 were obviously decreased in the groups treated with AR and TG (Fig. 5A and B). In addition, it showed the increased number of RORγ-expressing cells within the flamed synovial tissues in the model group, and the expression levels of RORγ were reduced markedly by the treatment of AR and TG (Fig. 5C and D).

4. Discussion RA is a chronic and systemic autoimmune disease involving T lymphocytes, and is characterized by hyperplastic synovitis which is responsible for cartilage and bone destruction. Increasing evidence supports the notion that, in addition to Th1/Th2 cells, Treg/Th17 cells participate in the etiology and pathogenesis of RA. Experimental arthritis model challenged with heat-killed Mycobacterium tuberculosis H37Ra (Mtb) in adjuvant has been used extensively to investigate potential therapeutic targets, and the pathogenetic mechanism involved in this model is confirmed to be mainly mediated by T cells. In the present study, we demonstrated that AR ameliorated the severity of arthritis and inhibited the progression of AIA in rats.

The experiments in vivo confirmed that AR significantly reduced paw swelling. Histological evaluation showed that oral administration of AR lead to the reduction of chronic synovium hyperplasia, inflammatory cell infiltration, pannus formation, cartilage destruction and bone erosion, contributing to the protective effect of AR against AIA. It should be noted that T cells are present in PPs and their activation is pivotal in both regulatory and effector activities of immune system (Simecka, 1998). Among suppressive T cell subtypes, CD4 þ CD25 þ Foxp3 þ Treg cells are essential for suppression of the immune system activation, thereby preventing autoimmune responses and limiting chronic inflammatory reactions. The transcription factor Foxp3 represents a specific marker and serves as a master regulator of the development and function of CD4 þ CD25 þ Treg cells (Fontenot et al., 2003). CD4 þ CD25 þ Foxp3 þ Treg cells inhibit the proliferation and cytokine production of responding T cells, and target other cell types including natural killer T cells, antigen presenting cells such as dendritic cells, macrophages, and B cells as well. Treg cells utilize multiple means to mediate suppression, such as in a direct cell-contact dependent manner, as well as via secreting soluble factors mainly including TGF-β1 and IL-10 with marked anti-inflammatory and immunosuppressive properties. It has been shown that IL-10 suppressed Th17 cells and promoted Treg cells in the CD4 þ T cell population of RA patients (Heo et al., 2010). Moreover, TGF-β1 is essential for the peripheral generation and expansion of Foxp3 þ Tregs (Chen

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Fig. 4. Effects of AR on the percentages of CD4 þ CD25 þ Foxp3 þ Treg cells in AIA rats. Lymphocytes isolated from PPs and peripheral blood were prepared after AR administration for 14 days, stained with fluorescent antibodies for CD4, CD25 and Foxp3, and then analyzed by flow cytometry. (A) Representative images of CD25 þ Foxp3 þ cells gated on CD4 þ cells in Lymphocytes isolated from PPs. (B) Quantification of the percentages of CD4 þ CD25 þ Foxp3 þ Treg cells among CD4 þ T cells in PPs lymphocytes. (C) Representative images of CD25 þ Foxp3 þ cells gated on CD4 þ cells in peripheral blood. (D) Quantification of the percentages of CD4 þ CD25 þ Foxp3 þ Treg cells among CD4 þ T cells in peripheral blood. (n¼ 3 per group) ##P o 0.01 vs. normal group; nPo 0.05, nnP o 0.01 vs. model group.

et al., 2003; Lan et al., 2007). A recent study demonstrated that significant and persistent increases in TGFβRII expression on CD4 þ T cells occur in the PPs immediately after tolerizing events, and when challenged these PPs CD4 þ TGFβRII þ cells upregulate Foxp3 expression (Gilbert et al., 2011). Accumulated evidence suggested that CD4 þ CD25 þ Foxp3 þ Treg cells play a critical role in the prevention of autoimmune arthritis (Lu et al., 2012; Luo et al., 2011). Our experimental results demonstrated the decreased Tregrelated cytokine levels following ConA-activated lymphocytes derived from PPs in the AIA rats, which were likely due to the proportions of lymphocyte populations. Correspondingly, it was observed that CD4 þ CD25 þ Foxp3 þ Treg cells population among PPs CD4 þ T lymphocytes were diminished, suggesting that both the functions and percentages of Treg cells might be inhibited in the AIA rats. Whereas oral administration of AR promoted the expansion of CD4 þ CD25 þ foxp3 þ Treg cell population in PPs CD4 þ T lymphocytes, and these Treg cells might migrate into

systemic circulation to exert their inhibitory activities on the immune response. Consistently, the levels of IL-10 and TGF-β1 secreted from ConA-activated PPs lymphocytes were observed to be increased after AR administration, indicating the immunosuppressive effect of AR could be mediated by Treg-related cytokines. Moreover, peripheral CD4 þ CD25 þ foxp3 þ Treg cells and Tregrelated cytokines in serum were also upregulated, which suggested the systemic immune suppression might be induced by AR treatment. A delicate balance between suppressive Treg cells and inflammatory Th17 cells is maintained under homeostatic conditions and is shifted towards Th17 cells predominance in autoimmune arthritis. Th17 cells are effector T cells that characteristically express retinoic acid-related orphan nuclear receptor-γt (RORγt) as the specific transcription factor (Ivanov et al., 2006), and secrete a set of proinflammatory cytokines mainly including IL-17 A, IL-17 F, and TNF-α, which antagonize Treg cells functions and promote

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Fig. 5. Effects of AR on Foxp3 and RORγ expressions in synovial membrane of joint tissues in AIA rats. The expression levels of Foxp3 and RORγ were examined by immunohistochemical techniques. Positive cells expressing the marker of interest are stained brown. (original magnification, 400  ). (A) Representative immunohistochemical staining for Foxp3 protein. (B) The number of Foxp3-expressing cells per high power field. (C) Representative immunohistochemical staining for RORγ protein. (D) The number of RORγ-expressing cells per high power field. (n¼ 6 per group) ##Po 0.01 vs. normal group; nP o0.05, nnPo 0.01 vs. model group.

autoimmune diseases. The specific cytokine IL-17 A synergized with TNF-α had potent abilities to stimulate the proliferation of T cells (Zhang et al., 2013), promote the production of IL-6, IL-8, CCL20, and MMP-3 by RA synoviocytes (Zrioual et al., 2008), induce the migration and invasion of synoviocytes (Hot et al., 2012), and drive the degradation of matrix and cartilage (Moran et al., 2009). Our studies indicated that the levels of Th17-related proinflammatory cytokines secreted from ConA-stimulated PPs lymphocytes as well as in the periphery blood in AIA model rats were elevated significantly, showing the hyperfuntion of Th17 cells in response to arthritic inflammation. Whereas by AR oral treatment, the levels of IL-17A and TNF-α were decreased effectively, indicating that AR may exert its immunosuppressive effects on the enteric mucosal immunity and systemic immune responses relevant to hyperactivated Th17 cells. RA is characterized as a progressive proliferative synovitis following the influx of various inflammatory cells, mainly activated CD4 þ T cells. It has been well established that many chemokines and adhesion molecules, expressed in nonlymphoid tissues and inflammatory sites, may attract Foxp3 þ T cells to display sufficient suppression of excessive inflammation (Kim, 2006). The presence of Treg cells in RA inflamed synovial tissue has been demonstrated in several studies, which reported a restricted distribution of Foxp3þ T cells among lymphocytes. Furthermore, the presence of Foxp3 þ cells was mainly restricted to the T-cell-rich areas (Behrens et al., 2007; Raghavan et al., 2009). The upregulatory capacity of AR on Treg cells of PPs derived lymphocytes and peripheral blood led us to search thoroughly for the presence of

Treg cells in joint tissues locally. Herein, we found the expression levels of Foxp3 in synovium was higher in those AIA model rats when comparing with the normal controls, suggesting that Foxp3 þ cells migrated towards inflamed joint tissues, which might be needed to suppress activation of autoreactive lymphocytes (Kim, 2006). However, accompanied with the articular inflammation being well controlled, AR treatment led to the reduction of Foxp3 þ cells infiltration. Substantial evidence supported that pathogenic Th17 cells, which were expanded and activated in rheumatoid synovium, play the critical role for the inflammatory response and tissue damage in RA affected joints (Dong and Zhu, 2012; Gullick et al., 2010). Based on the data that AR treatment could modulate Treg/Th17 cytokines balance in PPs and peripheral blood, we were curious to know whether the reciprocal differentiation pattern of Th17 cells at the inflammatory site of joints could be altered or not. According to our experiment, RORγ-expressing cells were present at significantly higher numbers within the inflamed synovium in AIA rats, indicating that an increased population of Th17 cells infiltrated into the synovium, which might exacerbate the inflammatory phase of arthritis. AR treatment caused a significant reduction of RORγ-expressing cells in synovium, demonstrating that the infiltration of Th17 cells was inhibited, in parallel with the attenuation of local articular inflammation. In summary, our results demonstrated that induction of PPsassociated Treg cells accompanied by suppressive Treg-related cytokines production from ConA-activated PPs lymphocytes might be key initiators contributing to the immunosuppressive activities

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of AR. By the suppressive effects on Th17 cells function, AR was able to regulate the imbalance between Treg cells and Th17 cells, thereby shifting the pathological immune responses towards immunosuppression in experimental arthritis. Taken together, the present information added a novel facet to the underlying mechanisms conncerning the antiarthritic properties of AR, which may offer a possible option for RA treatment.

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