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Bioactive Constituents, Metabolites, and Functions

Atractylodin suppresses dendritic cell maturation and ameliorates collagen-induced arthritis in a mouse model Cheng Hsuan Chuang, Yu Chieh Cheng, Shiming Li, Shih-Chao Lin, Caitlin W. Lehman, Der-Yuan Chen, Sen-Wei Tsai, and Chi-Chien Lin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01163 • Publication Date (Web): 02 Jun 2019 Downloaded from http://pubs.acs.org on June 4, 2019

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Journal of Agricultural and Food Chemistry

Atractylodin

suppresses

dendritic

cell

maturation

and

ameliorates collagen-induced arthritis in a mouse model Cheng Hsuan Chuang † #, Yu-Chieh Cheng † , ‡ #, Shih-Chao Lin § , Caitlin W. Lehman § , Shun-Ping Wang¶, Der-Yuan Chen∥, Sen-Wei Tsai⊥,▽, Chi-Chien Lin†,Ψ,ϥ † Institute

of Biomedical Science, National Chung-Hsing University, Taichung 402,

Taiwan ‡ Department

of Orthopaedics, Tungs' Taichung Metro Harbor Hospital, Taichung 433,

Taiwan § National

Center for Biodefense and Infectious Diseases, School of Systems Biology,

George Mason University, Manassas, VA 20110, USA ¶ Department

of Orthopaedics, Taichung Veterans General Hospital, Taichung 407,

Taiwan ∥

Division of Immunology and Rheumatology, Department of Internal Medicine,

China Medical University Hospital, Taichung 404, Taiwan. ⊥ Department

of Physical Medicine and Rehabilitation, Taichung Tzu Chi Hospital,

Buddhist Tzu Chi Medical Foundation, Taichung 427, Taiwan. ▽ Department

of Physical Medicine and Rehabilitation, School of Medicine, Tzu Chi

University, Hualien 970, Taiwan. ΨDepartment

of Medical Research, China Medical University Hospital, Taichung 404,

Taiwan ϥ

Department of Medical Research and Education, Taichung Veterans General

Hospital, Taichung 407, Taiwan

#Both

authors contributed equally to this work

Corresponding author: Dr. Chi-Chien Lin, Department of Life Sciences, National

ACS Paragon Plus Environment


Chung Hsing University, 145 Xingda Rd., South Dist., Taichung City 402, Taiwan (R.O.C.); E-mail: [email protected]

Abstract The aim of this study was to evaluate the immunomodulatory effects of atractylodin, a polyethylene alkyne, on the maturation of bone marrow-derived dendritic cells (BM-DC) as well as its anti-rheumatic effect on collagen-induced arthritis (CIA) in DBA/1 mice. Our results indicate that atractylodin effectively suppressed the secretion of pro-inflammatory cytokines, expression of costimulatory molecules, and p38 MAPK, ERK, and NF-κBp65 signaling pathways in LPS-incubated dendritic cells (DCs). Additionally, the proliferation and cytokine secretion (IFN-γ and IL-17A) of CD8+ and CD4+ T cells were reduced. In a murine CIA model, intraperitoneal injection of atractylodin significantly alleviated the severity of the disease progression, as indicated by reduced paw swelling, clinical arthritis scores, and pathological changes of joint tissues. In addition, the overall proliferation of T cells stimulated by type II collagen and the abundance of Th1 and Th17 in the spleens were also significantly decreased with atractylodin treatments. Furthermore, atractylodin significantly down-regulated the expression levels of CD40, CD80, and CD86 of DCs in the spleens. In conclusion, this study shows for the first time that atractylodin has potential to manipulate the maturation of BM-DCs and should be further explored as a therapeutic agent in the treatment of rheumatoid arthritis (RA).

Keywords: atractylodin, dendritic cells, collagen-induced arthritis, maturation, T cells

Introduction


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Rheumatoid arthritis (RA) is a chronic autoimmune disorder plagued with symptoms of joint stiffness, swelling, pain, and in severe cases, joint deformation. The pathogenesis of RA is mainly caused by inflammation in synovial tissue (synovitis) and erosion of cartilage or bone1. Currently, the clinical approaches in treating RA primarily include nonsteroidal anti-inflammatory drugs (NSAIDs) and cytokine receptor inhibitors. In addition to these drugs, recent studies seeking to suppress the maturation of dendritic cells (DCs) in RA have brought much attention to the drug development quest against RA. For example, gold sodium thiomalate and leflunomide derivatives have recently been shown to affect DC maturation and differentiation and potentially ease RA progression2-4. The pathogenic role played by DCs in arthritic patients is critical and the mechanisms behind DC-induced RA have been extensively studied. Under normal conditions, myeloid DCs are capable of migrating to the periphery tissues to identify and capture foreign antigens using Toll like receptors (TLRs)5 as well as fragmenting foreign antigens into small peptides inside the cells to be used for antigen display via the major histocompatibility complex II (MHC class II)6. DCs can also express co-stimulatory molecules, CD40, CD80, CD867, 8, leading to cytokine secretion and stimulation of the immune response. However, in RA patients, both immature and mature DCs were found to accumulate substantially in synovial joint tissues9, 10. In the typical arthritic animal model, collagen-induced arthritis (CIA) mice, the excessive production of pro-inflammatory cytokines or chemokines and continuous presentation of autoantigens by DCs are regarded as contributing factors to the pathogenesis and progression of murine arthritis11. As such, suppressing the upregulation of DCs is a potential strategy for regulating immune-mediated rheumatic diseases12-14. Natural compounds with immunomodulation properties have been used as approaches to combat DC-induced RA. Compounds derived from vegetables, fruits,



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and herbs have been used to induce anti-inflammatory activities and modulate DC functions. For example, naringenin, luteolin, and quercetin were shown to inhibit LPS-induced DC activation by blocking both NF-κB and MAPK pathways15-17. Among such natural compounds, we are particularly interested in atractylodin, an active polyethylene alkyne extract from atractylodis rhizoma (Cangzhu), due to its pharmacological

activities

against

rheumatic

diseases.

Traditionally,

dried

atractylodis rhizoma has been extensively used for regulating immune responses and treating limb illnesses caused by joint pain or swelling in Asian countries18. Recent and previous studies have attempted to clarify the anti-inflammatory mechanisms of atractylodin. Results indicated that atractylodin could regulate and equilibrate the inflammatory cytokine, interleukin-6 (IL-6), through blocking both phosphorylation of MAPKs and NPM-ALK signaling pathways in a human mast cell line, HMC-119, 20. Additionally, atractylodin was shown to relieve inflamed rat jejunal tissue in both constipation and diarrhea prominent rats21. Likewise, atractylodin attenuated lipopolysaccharide-induced acute lung injury in mice by inhibiting nucleotide-binding domain-(NOD-) like receptor protein 3 (NLRP3) inflammasome and toll like receptor 4 (TLR4) activation22. The mechanism of how atractylodin ameliorates arthritic inflammation and whether it involves regulating DCs is currently unknown. Therefore, we sought to investigate the therapeutic effects and underlying mechanisms exerted by atractylodin in treating arthritic disorders in vitro and in vivo. To this end, we exploited murine bone marrow-derived dendritic cells and collagen-induced arthritis mice as models to analyze the effects of atractylodin on DCs and evaluate its therapeutic potential in regards to RA.

Results


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Atractylodin

induced

non-significant

cytotoxicity

of

bone

marrow-derived DCs. Prior to evaluating atractylodin (ATL, Fig. 1A) and its effects on DCs, we first evaluated the cytotoxicity of ATL in mouse bone marrow-derived DCs. We measured the cell viability of BMDCs treated with different concentrations of ATL using a CCK-8 kit. The results in Fig. 1B show that the cell viability of BMDCs remained over 90% when treated from 12.5 to 100 μM of ATL, indicating ATL induced minimal cytotoxicity in BMDCs compared to our positive control, quercetin at 100 μM, which inflicted profound cell death16. Since the toxicity was non-significant among groups with various concentrations of ATL, we proceeded to investigate ATL and its potential as a functional modulator of BMDCs. Atractylodin inhibited the secretion of cytokines and nitric oxide (NO) by LPS-stimulated DCs. Since secretions of pro-inflammatory cytokines and NO are key features of DC activation and they could alternatively regulate one another23, we next sought to investigate whether atractylodin affected the secretions of cytokines and NO in LPS-stimulated DCs. We found that ATL dose-dependently reduced cytokine productions of TNF-α, IL-6, IL-1β, IL-23, IL-12, IFN-γ, and IL-10 in LPS-stimulated dendritic cells (Fig. 2A) and more surprisingly, NO production levels were comparable to the control with ATL treatment. We thus further examined the expression of iNOS and confirmed that iNOS expression in LPS-stimulated DCs was profoundly inhibited by ATL treatments, leading to the blockage of NO production (Fig. 2B). Atractylodin inhibited LPS-induced costimulatory molecules in DCs. To further understand the degree to which treatment with ATL can affect DCs, we examined the surface marker expressions of costimulatory molecules, CD40, CD80, and CD86, which indicate DC maturation and are key in stimulating downstream T cell differentiation 24. Flow cytometry data depicted in Fig. 3 revealed DCs expressed



higher CD40, CD80, and CD86 following LPS treatments whereas ATL inhibited the ability of LPS-stimulated dendritic cells to express these costimulatory molecules in a dose-dependent manner (Fig. 3 and Supplemental Fig. 1), suggesting that ATL potentially modulates DC maturation. Atractylodin inhibited the ability of LPS-stimulated DCs to activate antigen -specific T cells. Since the ultimate goal for a mature DC is to stimulate and activate antigen-specific T cell proliferation, we further analyzed the effects of ATL on DC-induced T cell proliferation. Immature DCs were pre-incubated with OVA257–264 (OVAP1) or OVA323−339 (OVAP2) peptide followed by treatment with LPS and ATL for a total of 18 hours. These ATL-treated and untreated DCs were then co-cultured with OT-I CD8+ or OT-II CD4+ T cells, which recognize this specific OVA peptide, for 96 hours. Our data in Fig. 4A shows that LPS-stimulated DCs without ATL treatment could successfully present the OVA peptide to CD8+ and CD4+ T cells and induce stronger cell proliferation than naïve DCs after co-culture. However, upon treatment with ATL (50µM) the antigen presenting ability of LPS-stimulated DCs was significantly suppressed and subsequently induced weaker T cell proliferation (Fig. 4A and Fig 4C). In addition, LPS-treated DCs could stimulate IFN-γ production from CD8+ T and CD4+ cells as well as IL-17A release from CD4+ cells whereas ATL treatment reversed this stimulatory effect. However, production of IL-10 was not affected by ATL treatment (Fig. 4B and 4C). These results suggest that ATL could reduce the capability of DCs to respond to LPS stimulation and downregulate induction of antigen-specific T cell proliferation and cytokine secretion. Atractylodin downregulated the MAPKs and NF-  B pathways in BMDCs activated by LPS. In addition to characterizing the immunomodulatory effects of ATL on DCs, we aimed to identify the possible molecular mechanisms between ATL and DCs. Since the activations of MAPKs and NF-κB are essential to


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dendritic cell maturation and in turn trigger the secretion of pro-inflammatory cytokines25, we examined the phosphorylation levels of extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPKs by Western blotting analysis and quantified the nuclear translocation of NF-κB by p65 binding assay to evaluate the activations of MAPK and NF-κB in LPS-stimulated BMDCs with or without ATL treatments. Our results suggest that ATL potentially inhibits the phosphorylation of p38 and ERK induced by LPS at 60 and 90 min respectively while the expressions of p-JNK appeared to remain unchanged regardless of ATL treatment (Fig. 5A). Conversely, the nuclear translocation of NF-κB p65 was elevated over time upon LPS stimulation but ATL treatment significantly reduced the translocation of NF-κB p65 (Fig. 5B). These results suggest that deactivation of MAPK and NF-κB could potentially be associated with the underlying mechanism of ATL’s influence on DCs. Atractylodin attenuated the severity of CIA in DBA/1 mice. In order to more comprehensively evaluate the potential use of ATL in treating inflammatory diseases, we selected to use the well-established collagen-induced arthritis (CIA) mouse model in order to induce disease and treat with ATL. CIA in DBA/1 mouse induced by type II collagen exhibits similar pathological features and tissue damages to those observed in human rheumatoid arthritis26. Thus, the CIA mouse model is an appropriate model to objectively assess whether an immunomodulatory compound, like ATL, can potentially alleviate disease severity and perhaps be suitable for future clinical trials. As described in the methods section, after initial collagen immunization both vehicle control and ATL experimental mice were dosed daily with vehicle and 40 mg/kg of ATL and clinical scores of arthritis in hind paws were evaluated. Mice with ATL treatments had significantly reduced joint swelling of arthritic hind paws (Fig. 6A), lower clinical scores (Fig. 6B), and maintained their body weights (Fig. 6C)



as compared to the normal and vehicle control groups. Additionally, we examined histological sections of ankle joints and found that ATL treated mice had significantly reduced synovial hyperplasia, inflammatory cell infiltration, and cartilage damage with corresponding lower histological scores than that in vehicle control mice (Fig. 7). These results suggest that ATL treatment can alleviate arthritic symptoms and degeneration of joint tissues in CIA mice. Atractylodin suppressed the productions of inflammatory mediators and splenic DC maturation in CIA mice. To further analyze the interaction between ATL and immune responses of CIA mice, we investigated the cytokine profile of CIA mice with or without ATL treatment. After mice were euthanized at the end of the experiment (day 42), we homogenized their hind-limbs to obtain supernatant for sandwich ELISA tests. The levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), Th17-related cytokines (IL-17A and IL-15), and Th1-related cytokine (IFN-γ) in ATL-treated CIA mice (40 mg/kg) were substantially reduced to similar levels as observed in normal mice. Notably, ATL treatment did not significantly change the production of anti-inflammantory cytokine, IL-10 (Fig. 8). Alongside, we sorted CD11c+ DCs from CIA mice and tested their activation in terms of costimulatory molecule expression of CD40, CD80, and CD86. Despite the elevation of the surface markers, 40 mg/kg of ATL treatment was sufficient to suppress the expression of these costimulatory molecules on the splenic DCs of CIA mice (Fig. 9 and Supplemental Fig. 2). Atractylodin treatment marginally reduced CII-specific IgG antibody in CIA. In addition to the cellular immune responses, the humoral response against type II collagen (CII) likely plays a critical role in CIA pathogenesis27. Therefore, we titered the specific serum antibody, anti-CII IgG1, IgG2a, and total IgG, in all mouse groups using ELISA. We observed that CII-specific IgG2a, but not total IgG or IgG1,


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was significantly reduced by ATL treatment. Otherwise, CII-specific antibodies did not reveal obvious differences, suggesting that humoral immune responses may be partially or have very limited involvement in the ATL-mediated therapeutic effects against CIA (Fig. 10). ATL suppressed the splenic Th1 and Th17 cells development in CIA mice. The activity of Th17, Th1 and regulatory T (Treg) populations have also been implicated in the pathogenesis of RA and CIA mice

28,

therefore we next evaluated

the levels of Th17, Th1 and Treg in CIA mice in order to further elucidate the mechanism underlying ATL’s ability to alleviate arthritis. We stained the splenocytes from CIA mice using indicated antibodies followed by quantifying the abundance of Th17, Th1, and Treg cells gated with CD4+ T cells using flow cytometry. The flow cytometry data show that CD4+IFN-γ+ Th1, CD4+ IL-17+ Th17 and CD4+Foxp3+ Treg cells increased significantly in spleens of vehicle-treated CIA mice compared with naïve mice. However, ATL treatment largely reduced the proliferation of Th1 and Th17, but not Treg, cellular populations (Fig. 11 and Supplemental Fig. 3), suggesting that ATL-mediated anti-arthritic activity could be associated with downregulating the populations of Th1 and Th17 cells.

Discussion Our findings in this study suggest that ATL is capable of either directly or indirectly downregulating the activation of bone marrow-derived DCs by reducing pro-inflammatory cytokines, NO, and costimulatory molecular markers. Treatment with ATL also influenced downstream cellular responses, such as the decrease observed in Th1 and Th17 cells. Thus, for arthritic diseases induced by over-activation of DCs by collagen II in synovial tissues, ATL treatment can alleviate the clinical symptoms and disease progression, as indicated by less swelling and



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cartilage tissue damage in ankle joints along with lower cytokines and cellular responses compared to vehicle control mice. Furthermore, reduced NO production and iNOS expressions of BMDCs as a result from ATL treatment could contribute to the T cell expansion in CIA mice since NO is essential for DC reprogramming and the release of NO from DCs negatively impacts T cell proliferation29. In our in vitro model, we attempted to investigate the underlying mechanisms of which ATL might exploit to suppress LPS-induced inflammation. Upon LPS treatment, MAPK and NF-κB are activated via TLR-4 in myeloid DCs30,

31.

We

confirmed that ATL attenuated the induction of phosphorylation in p38 and ERK, but not JNK, in MAPK pathways, which are somewhat in agreement with previous findings20, 22. However, the suppressed levels of phosphorylation by ATL were not continuously significant at every time point observed. Rather, the nuclear translocation activity of NF-κB was remarkably reduced after treatment with ATL (Fig. 5). As a result, we believe that the underlying molecular mechanism of ATL in suppression of DC functions is likely associated with the NF-κB cascade. A definitive answer as to the roles different DC subsets play in the pathogenesis of RA has yet to be identified and needs further investigation. The BMDCs used in our study were prepared using ex vivo culture with GM-CSF and were primarily conventional DC2 (cDC2)-like cells (CD11c+ CD11b+ CD103- B220-) as described in previous reported methods32, 33. Furthermore, plasmacytoid DCs (pDCs, CD11clow CD11b- B220+) were not included in our study because we gated CD11chigh cell population (Supplementary Fig. 1A and 2A). Previous analyses have revealed that cDC2 cells could be more efficient in regulating the activation of CD4+ T cells than other DC subsets34. Since we observed an anti-arthritic effect of ATL in cDC2 like cells, we sought to test its potential in vivo. Our in vivo results also indicate that ATL is capable of acting as a therapeutic agent in suppressing the maturation of CD11chigh


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DCs in the spleens (Supplementary Fig. 2). One point to consider with our CIA animal model is that ATL was administered via intraperitoneal injection rather than as a dietary supplement or oral formulation. Whether the routes of administration would alter the therapeutic outcomes requires further testing. Ultimately, oral delivery of atractylodin would provide clinical professionals more flexibility in treating RA patients and would also be more convenient for patients. ATLs ability to modulate cytokine profiles of DCs could be one of the essential mechanisms underlying its ability to alleviate symptoms associated with arthritic disease. In addition to well-known pro-inflammatory cytokines released from DCs, such as TNF-α, IL-6 and IL-1β, other cytokines could be more prone to induce T cell differentiations towards Th1 or Th17 development in response to IL-12, IFN-γ (Th1) and IL-23 (Th17). In this study, we observed lower levels of the aforementioned cytokines from BMDCs upon ATL treatment in vitro. Also, production of IFN-γ and IL-17A from OT-II T cells were reduced, suggesting that ATL may regulate the differentiation and activity of Th1 and Th17 cells (Fig. 2 and 4). To date, it has been widely acknowledged that RA is highly correlated to Th1 and Th17 responses in RA patients35-37 and as such RA is classified as a Th1-mediated autoimmune disorder. However, recent findings indicate that the infiltration of Th17 cells to synovial tissues and the upregulation of IL-15 and IL-17 are also contributing factors in RA pathogenesis38. Our findings are consistent with previous reports that Th1 and Th17 cells were found to be increased in the spleens of CIA mice39. Upon ATL treatment, both Th1 and Th17, but not Treg cells, were reduced as demonstrated by reduction of IFN-γ, IL-17A, and IL-15 in hind paw tissues (Fig 8). IL-10 plays a critical role in attenuating the severity of CIA mice potentially through regulating the differentiation of Treg cells and enhancing Treg cells to



suppress Th17 cells40-42. Despite the positive correlation of IL-10 and Treg cells, ATL did not induce a significant increase in IL-10 from BMDCs and OT-II CD4+ T cells nor did it result in elevated Treg populations in CIA mouse spleens (Fig. 2A, 4B and 11). Therefore, we believe that the DC-suppressing ability mediated by ATL appears to not be associated with IL-10 signaling or Treg cells. Also, we did not observe significant changes in total CII-specific IgG and IgG1 concentrations in CIA mice in the presence or absence of ATL treatment but only in IgG2a (Fig. 10), which is consistent with previous reports regarding the pathogenic anti-CII autoantibody in a DBA/1 arthritic mouse model43. Anti-CII IgG2a subclass antibody is shown to be a complex-fixing autoantibody elicited to promote Clq cross-linking and trigger the complement cascade subsequently resulting in further inflammation of joint tissues44. As a result, the lower CII-specific IgG2a antibody reduced directly or indirectly by ATL may be partially involved in the mechanism of the anti-arthritic efficacy observed with ATL treatment. Overall, our study provides a systematic inspection of ATL on controlling an arthritic disease mediated by BMDCs. To our knowledge, our data is the first indicating that ATL treatment could be utilized to regulate DC functions in RA. While our results are preliminary and further investigation of ATL is required, we believe our study can serve as a valuable resource for preclinical development of ATL or ATL-like anti-arthritic compounds.

MATERIAL and METHODS Animals. 8-week-old male C57BL/6 mice with body weights around 20-22 g were provided by National Laboratory Animal Center in Taiwan. Male DBA/1 and OT-I TCR transgenic mice at about 6-8 weeks old with similar weight range were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA). OT-II TCR


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transgenic mice were generously provided by Dr. Ching-Liang Chu at National Taiwan University, Taipei, Taiwan. These experimental animals were kept and maintained under specific pathogen-free (SPF) conditions with controlled temperature, humidity, and light/dark cycle (22 ± 2 °C, 60 ± 10%, and 12/12 h, respectively) and were supplied with unlimited regular rodent diet and water, following the regulations of IACUC of National Chung Hsing University. Related animal study protocols were approved by the committee and assigned a serial number (IACUC: 107076) prior to the study begun. Preparation of BMDCs. The femurs and tibia of C57BL/6 mice were collected to retrieve bone marrow cells followed by removal of red blood cells. Cells were subsequently cultured with RPMI 1640 growth medium, supplemented with L-glutamine, NEAA, sodium pyruvate, HEPES, 2-ME, penicillin/streptomycin, 10% of FBS and 10 ng/mL recombinant mouse GM-CSF and were cultured in a humidified 5% CO2 incubator at 37°C. Additional 2 mL of medium along with 10 ng/ml GM-CSF was supplied for BMDCs on day 3 and 5. By day 7, the non-adherent and loosely adherent cells were collected and regarded as immature BMDCs confirmed by Accuri 5 flow cytometer (BD Biosciences, San Jose, CA, USA) to ensure CD11c expression. Afterwards, immature BMDCs were further separated by CD11c microbeads (Miltenyi Biotec, Auburn, CA, USA) to obtain cell samples with over 90% of CD11c+-expressed BMDCs. Cell viability assay. 2 × 105 cells/mL of murine BMDCs were cultured with different concentrations of atractylodin, obtained from National Institute for Food and Drug control (Beijing, China), while vehicle control group was treated with 0.1% DMSO solvent (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Quercetin (100 μM) was purchased from Sigma-Aldrich which has been shown to exhibit high cytotoxicity and used as a positive control. Cell viability was determined based on the



concentrations of monosodium salt, WST-8, and determined with Cell Counting Kit-8 (CCK-8) reagent as per the manufacturer's protocol. Analysis of cytokines and nitric oxide (NO) secretion in vitro and hind paws ex vivo. 1×106 cells/mL of murine BDMCs from C57BL/6 were pretreated with atractylodin (25-100 μM) for 2 h prior to being stimulated with 100 ng/mL of Escherichia coli serotype O26:B6 LPS (Sigma-Aldrich) for additional 18 h. Cells treated with 0.1 % of DMSO was a vehicle control. Cytokine concentrations of TNF-α, IL-6, IL-1β, IL-23, IL-12 p40, IFN-γ, IL-10, and NO in the culture medium were analyzed by ELISA kits (eBioscience, San Diego, CA, USA) and modified Griess reagent (Sigma Aldrich), respectively. Griess reagent was added into the medium transferred to a new 96-well plate in 1:1 ratio, and the nitrite content was calculated according to a standard curve established from a set of various concentrations of sodium nitrite. To measure the cytokine levels in hind paw tissues, the paws of DBA/1 mice from each group were homogenized and lysed with tissue lysis buffer along with UltraCruz® Protease Inhibitor Cocktail (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and then centrifugation twice at 840 × g for 10 minutes at 4°C to obtain supernatant. ELISA kits (eBioscience) for measuring TNF-α, IL-6, IL-1β, IL-17A, IL-15, IFN-γ, and IL-10 levels were applied to evaluate each concentration in samples. Flow cytometry analysis of surface makers in BMDCs and maturation of DCs in vivo. 1 × 106 cells/mL of murine BMDCs from C57BL/6 were pretreated with concentration range of 25-100 μM of actractylodin for 2 h followed by stimulating with 100 ng/mL LPS for additional 18 h. After incubation, the cells were washed with 500 μL of staining buffer containing 2% FCS and 0.05% sodium azide in PBS and stained with FITC-labeled mouse anti-CD11c antibody,


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PE-anti- CD80 antibody, PE-CD86 antibody and PE/Cy5-labeled anti-MHC class II with isotype-matched control antibodies. Fluorescence intensity of CD40, CD80, and CD86 was determined by Accuri 5 flow cytometer following gating with forward side scatter (FSC) and CD11c+ expression. The mean fluorescence intensity (MFI) calculated by BD Accuri C6 system software was used to plot graphs. Spleens of DBA/1 mice were collected and then homogenized by sterile 50-mesh stainless mesh to obtain single-cell suspension. Splenocytes were further purified with use of RBC lysing buffer (Sigma-Aldrich) to remove excessive erythrocytes followed by washing with staining buffer. The primary antibodies aforementioned targeted cellular markers of DCs were diluted in 500 μL of staining buffer at 4oC overnight and cellular markers were analyzed by Accuri 5 flow cytometer under the gating of FSC, CD11c+ and MHC II+ to define splenic dendritic cells. Western blot analysis. Cell suspension from atractylodin-pretreated CD11c+ BMDCs were collected at 0, 30, 60, and 90 mins after stimulation of LPS to determine the phosphorylation of MAPK pathways (18 h for iNOS measurement), and RIPA cell lysis buffer containing protease inhibitor were used to lyse cells on ice for 30 min to obtain cell lysate followed by bicinchoninic acid (BCA) assay for determination of the protein concentration. 40 μg of protein lysate were boiled and loaded into 8-10% gradient SDS-PAGE gels and then electrotransferred to nitrocellulose membranes. Membranes were hybridized with primary antibodies that recognize phospho-p38 (Thr180/Tyr182), p38, phospho-ERK (Thr202/Tyr204), total ERK, phosphor-JNK, JNK and GAPDH at 4 °C for overnight. The secondary antibody was mouse anti-rabbit IgG-conjugated with HRP in 1:2000 dilution at 4°C, incubating for additional overnight prior to developing with ECL reagent (GE Healthcare Life Sciences, Piscataway, NJ, USA) and visualizing with the Hansor Luminescence Image System (Taichung, Taiwan). Densitometric analyses for target



bands were performed with ImageJ software (National Institute of Health, Bethesda, MD, USA) and normalized with the level of GAPDH in each lane. Preparation of nuclear extracts and measurement of NF-κB activity. NE-PER® (Thermo Fisher Scientific, Waltham, MA, USA), a nuclear and cytoplasmic extraction reagent, was exploited to generate nuclear fractions from BMDCs, and TransAM™ NF-κB p65 kit (Active Motif, Carlsbad, CA, USA), an ELISA-based kit, was used to measure NF-κB p65 subunit activation as instructed by each manufacturer’s manual. Assay of OT-I T cell activation in co-culture assays. 2 or 10 μg/mL of OVA257–264 (OT-I) and OVA323–339 (OT-II) peptides, synthesized by Echo Chemical Co. in Taiwan, were separately added to CD11c+ BDMCs for 6 h followed by addition of LPS alone (100 ng/mL), LPS plus 0.1% DMSO, or LPS along with atractylodin for additional 16 h. Ovalbumin-specific CD8+ or CD4+ T cells were positively purified from the splenocytes of OT-1 or OT-II mice by using either EasySep™ Mouse CD8+ or CD4+ T Cell Isolation Kit (Stem Cell, Grenoble, France). CD8+ or CD4+ T Cell were co-cultured with BMDCs in 1:5 ratio (DC: 5 × 104 cells/well; T cell: 2.5×105 cells/well). After 72 h incubation, T cell proliferation was quantified based on the incorporated amount of radiolabeled [3H] thymidine (1 μL Ci/well) in newly synthesized DNA of T cells. The radioactivity was measured by liquid scintillation counting (Beckman Instruments, Palo Alto, CA, USA). Additionally, supernatants from the co-culture system of BMDC and OT-I/OT-II T cells were collected to measure the IFN-γ, IL-17A, and IL-10 levels by ELISA (eBioscience) after 96 hours of incubation. CIA induction and assessment. A total of 15 male DBA/1 mice were randomly categorized into three groups (n = 5). Since males have a higher conversion rate after immunization with CIA, we chose to employ male DBA/1 mice45, 46 for our


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study. Bovine collagen type II (CII, Chondrex, Inc., WA, USA) was dissolved and diluted to 2 mg/mL in 10 nM of acetic acid and emulsified in 1:1 ratio with complete Freund's adjuvant (CFA) at 250 μg per mouse of heat-inactivated Mycobacterium tuberculosis H37Ra (Sigma-Aldrich, St. Louis, MO, USA). The mixture was subsequently injected intradermally into tail base of DBA/1 mice with volume of 200 μL/mouse. A booster dose of CII in incomplete Freund's adjuvant (IFA) was also administered intradermally to same mouse groups after 21 days of primary immunization. The arthritis severity was evaluated under double-blind setting and quantified with a scoring index. Each limb had an individual score from scale 0 to 4 based on the severity of redness and swelling (see Table 1)47, 48. Treatment. Mice were grouped into several groups, including healthy normal mice (normal control), CIA mice injected with 10% DMSO and 90% glyceryl trioctanoate (vehicle control), and mice received 40 mg/kg of atractylodin treatment. Single dose of atractylodin was given via intraperitoneal injection daily for 21 days since 3 weeks of the primary immunization. Histological Analysis. Ankle joints of control or CIA mice were examined for histological changes following paraffin sectioning. Joint tissue section with 5 μm of thickness was stained with hematoxylin and eosin (H&E) to reveal the tissue structure. Histopathological features in terms of severity of arthritis were scored with a scoring system as the previous study described49 by two individual investigators under double-blind setting. Analysis of anti-collagen Type II IgG antibodies. The antibody titers of anti-CII total IgG, IgG1, and IgG2a in mouse serum were determined by ELISA. Briefly, 100 μg of CII was coated to microplates at 4°C for overnight. Following PBS washings and blocking, serial diluted sera were added to the wells for overnight incubation at 4°C. Next day, plates were washed and incubated with 1:5000 dilution



of sheep anti-mouse IgG, goat anti-mouse IgG2a, or IgG1 antibody conjugated with HRP at 4°C for overnight. After removal of unbounded antibody, plates were developed with substrate ABTS (Roche Diagnostic Systems, Hong Kong, China), and terminated by adding H2SO4 solution before reading the optical density (OD) values at wavelength of 450 nm with a ELISA reader (Tecan Sunrise, San Jose, CA USA). Intracellular cytokine staining. The protocol of intracellular cytokine staining in this study was adapted from previous reports with necessary modifications50, 51. In short, 2 × 106 cell/mL of murine splenocytes were planted in 96-well plates with complete medium followed by culturing with 50 μg/mL of bovine CII for next 48 h at 37°C. 5 μg/mL of brefeldin A (Sigma-Aldrich) was added to each well during the last 2 hours of 48-hour culturing. PE-conjugated anti-mouse CD4 antibody was used to stain cellular CD4 marker before applying with BD Cytofix/CytopermTM Fixation/Permeabilization Solution Kit. FITC-labeled anti-IFN-γ, anti-IL-17A, and anti-Foxp3

primary antibodies were then added to stain intracellular target proteins

and the fluorescent intensity was then analyzed by using Accuri C5 cytometer. Statistical Analysis. The data in this study are presented as the means ± standard deviation in triplicate unless indicated elsewhere. Two-tailed Student’s t test was applied to compare two individual data sets. One-way or two-way ANOVA with a post-hoc Tukey HSD was used to compare multiple experimental groups with GraphPad Prism v5.0 software. P-value below 0.05 was regarded as statistically significant for all analyses

Disclosure statement The authors declared that there is no conflict of interest regarding the publication of this article.


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Funding This study was funded by the Animal Biotechnology Center from The Feature Areas Research Center Program of Taiwan Ministry of Education (MOE-107-S-0023-E) and by National Chung-Hsing University/ Tungs' Taichung Metroharbor Hospital (TTMHH-NCHULS07005).

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Table 1. Severity scoring system Severity score

Degree of inflammation

0 1 2 3

No evidence of erythema and swelling Erythema and mild swelling confined to the tarsals or ankle joint Erythema and mild swelling extending from the ankle to the tarsals Erythema and moderate swelling extending from the ankle to metatarsal joints Erythema and severe swelling encompassing the ankle, food and digits. Ankylosis of the limb might be present.

4



Figure Legend Fig. 1. Cell viability of BMDCs from 12.5 to 100 µM of ATL treatments. (A) Chemical structure of atractylodin. (B) Cell viability was examined by CCK-8 assay after 24 h of ATL or quercetin treatment. Data are expressed as the percentage of the control treated with DMSO. Values represent the means ± SD from triplicate samples for each treatment. ***p

Atractylodin suppresses dendritic cell maturation ... - ACS Publications

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