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Potent antagonists of ROR#t, cardenolides from Calotropis gigantea, exhibit discrepant effects on the differentiation of T lymphocyte subsets Juan Liu, Li-Ping Bai, Fen Yang, Xiaojun Yao, KAWAI LEI, Christopher Wai Kei Lam, QIBIAO WU, Yuxin Zhuang, Riping Xiao, Kangsheng Liao, Hioha Kuok, Ting Li, and Liang Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01063 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on December 31, 2018
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Molecular Pharmaceutics
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Potent antagonists of RORγt, cardenolides from Calotropis gigantea, exhibit
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discrepant effects on the differentiation of T lymphocyte subsets
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Juan Liu1, Li-Ping Bai1, Fen Yang1, Xiaojun Yao1, Kawai Lei1, Christopher Wai Kei
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Lam1, Qibiao Wu1, Yuxin Zhuang1, Riping Xiao1, Kangsheng Liao1, Hioha Kuok1,
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Ting Li1,*, Liang Liu1,*
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1. State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute
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for Applied Research in Medicine and Health, Macau University of Science and
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Technology, Macau, People's Republic of China.
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* Corresponding authors
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Address for correspondence: State Key Laboratory of Quality Research in Chinese
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Medicines/Macau Institute for Applied Research in Medicine and Health, Macau
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University of Science and Technology, Avenida Wai Long, Taipa, Macau, China
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E-mail addresses:
[email protected];
[email protected] 16
Tel: +853 8897-2401; +853 8897-2799
17
Fax: +853 2882-5886; +853 2882-7222
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Keywords:
Uscharin;
Calactin;
Calotropin;
T
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cell
subsets;
RORγt
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Molecular Pharmaceutics
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Abstract
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RORγt is the master transcription factor of IL-17 cytokine expression and Th17
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lymphocyte differentiation, which are responsible for the induction of many
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autoimmune diseases. Recently, RORγt has become an attractive target for drug
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development to treat these types of diseases, and the field of RORγt antagonist
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research is now extremely competitive. In our current study, molecular docking was
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applied to demonstrate that cardenolides, including uscharin, calactin and calotropin
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derived from Calotropis gigantea, probably directly bound to RORγt. Therefore, the
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inhibitory effect was further validated using a luciferase reporter assay. Because
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RORt is the key transcriptional factor for Th17 differentiation, the effects of these
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compounds on Th17 differentiation were studied by flow cytometry. The results
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showed that uscharin, calactin and calotropin inhibited Th17 differentiation from 100
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to 500 nM. Furthermore, uscharin had a better effect than digoxin, a well-known
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inverse agonist of RORt, in reducing Th17 polarization. Additionally, the effects of
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the cardenolides on the differentiation of other Th lineages, including Th1, Th2 and
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Treg, were investigated. Uscharin suppressed Th1, Th2 and Treg cell differentiation,
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while calactin suppressed the differentiation of Th1 cells and calotropin did not
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influence the other T cell subsets, indicating that calactin suppressed Th1 and Th17
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differentiation and calotropin selectively quenched Th17 polarization. Structural
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analysis of the three compounds showed that the selectivity of uscharin, calactin and
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calotropin on the suppression of the different subsets of T cells is correlated to the
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minor differences in their chemical structures. Collectively, calactin and calotropin
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have greater potential to be developed as lead compounds than uscharin to treat
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autoimmune diseases mediated by Th17 and/or Th1 cells.
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1. Introduction
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The expression of retinoic acid-related orphan receptors γt (RORγt, also termed
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RORC2) is predominantly found in immune organs, such as the thymus, immature
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double-positive thymocytes, and Th17 lymphocytes [1]. Th17 cells are absent in
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RORγt-deficient mice, and the differentiation of Th17 cells in vitro and
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Th17-mediated inflammatory disease in vivo require the induction of RORγt,
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indicating that RORγt is the key transcription factor for Th17 differentiation [2]. Th17
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cells are crucial effector cells by producing numerous cytokines, including IL-17 and
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IL-23, to orchestrate the pathology of various autoimmune diseases, including
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rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD)
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and systemic lupus erythematosus (SLE). Therefore, inhibition of Th17 cell
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differentiation through direct suppression of RORγt is a promising strategy to treat
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autoimmune diseases.
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According to the literature, digoxin and its derivatives can suppress Th17 cell
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differentiation by antagonizing RORt activity without affecting the differentiation of
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other T cell lineages [3], though its cardiotoxicity constrains its clinical application.
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Synthesized compounds, including T0901317 and its derivative SR1001, also act as
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inverse agonists of RORt and in turn inhibit the differentiation of Th17 cells in vitro
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and in vivo [4, 5]. However, the moderate efficacy of the two compounds does not
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fulfill the requirements of clinical application. Therefore, antagonists of RORγt
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remain unavailable and desirable.
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Cardenolides, including uscharin, calactin and calotropin, have been isolated
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from Calotropis gigantea, which is a common plant in Africa, Eastern Asia and
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Southeast Asia. This plant is a rich source of cardenolides [6, 7]. Flowers, latex, roots
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and leaves of this plant reportedly have biological activity against arthritis, as well as
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bacteria, fungi and influenza [8-11]. However, the effects of these compounds on
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Th17 cells and other subsets of the T lymphocyte population have not been
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investigated. 3
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Th1 cells are the quintessential cell type involved in inflammation. The signature
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cytokine of the Th1 subset, IFN-, is associated with the pathogenesis of several
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autoimmune diseases. According to several different observations in humans, Th2
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cells have a protective effect in autoimmune conditions via secretion of IL-4, the
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signature cytokine of Th2 lymphocytes. Another T cell subset, regulatory T cells
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(Treg), also mediates immune suppression through the expression of inhibitory
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receptors and secretion of anti-inflammatory cytokines.
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In the current study, we have for the first time demonstrated that uscharin,
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calactin and calotropin significantly inhibited Th17 cell differentiation by
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antagonizing RORγt activity. After determining the effects of these compounds on
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other subsets of T lymphocytes and analyzing their chemical structures, we found that
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the discrepant effects of these compounds on the different T lymphocyte subsets are
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most likely attributed to their differences in chemical structure.
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2. Material and methods
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2.1 Reagents and animals
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Anti-mouse CD3, CD28, IL-4, IFN-γ, recombinant mouse IL-6, IL-23, IL-2,
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IL-12, IL-4, recombinant human TGF-β1, APC anti-mouse IL-4, PE anti-mouse
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IL-17A, FITC anti-mouse IFN-γ, PerCP/Cy5.5 anti-mouse CD4, monensin solution,
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brefeldin A solution, and red blood cell lysis buffer were obtained from BioLegend
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(San Diego, CA, USA). A primary antibody against β-actin was obtained from Santa
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Cruz (Dallas, Texas, USA). APC anti-mouse Foxp3 and RORγt antibodies were
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obtained from eBioscience (San Diego, CA, USA). Phorbol-12-myristate-13-acetate
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(PMA) was obtained from Sigma (Santa Clara, CA, USA). Ionomycin was obtained
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from Millipore (Billerica, Massachusetts, USA). CytofixTM Fixation buffer and stain
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buffer were obtained from BD Bioscience (Franklin, NJ, USA). CD4+ T cell isolation
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kits were purchased from Miltenyi Biotec (Cologne, Germany). Lipofectamine™
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LTX Reagent with PLUS™ Reagent was obtained from Invitrogen (Carlsbad, CA, 4
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USA). RPMI-1640, DMEM, trypsin and fetal bovine serum (FBS) were provided by
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GIBCO (Grand Island, NY, USA).
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C57BL/6 mice (8-14 weeks old) were supplied by the Laboratory Animal
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Services Center, The Chinese University of Hong Kong (Hong Kong, China). Mice
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were separately housed under a 12:12 h light cycle in rooms maintained at 20 ± 0.5 °C.
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Animal care and experiments were conducted in accordance with Macau University
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of Science and Technology regulations.
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2.2 Compounds, plasmid and cell lines
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Uscharin, calactin, and calotropin (>98% purity, verified by HPLC) were
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isolated from Calotropis gigantea according to our previous report [12]. The purity of
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these compounds was validated by HPLC (Fig. 1A). These compounds were
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dissolved in DMSO at a concentration of 100 mM and stored at -80°C for future study.
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Digoxin was purchased from Sigma. Human ROR/RORC/NR1F3 luciferase stable
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reporter cell line and RORt plasmid was purchased from Novus Biologicals
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(Centennial, CO, USA). HEK293 cell line was provided by American Type Culture
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Collection (ATCC, Manassas, VA, USA).
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2.3 Molecular docking
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Molecular docking calculations were employed to study the interaction between
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the small molecules and RORγt through the Induced Fit Docking module in
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Schrodinger software (Schrodinger, Inc., New York, NY, USA, Year 2009). The 3D
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structures of uscharin, calactin and calotropin were constructed and optimized in the
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LigPrep module. The 3D structure of RORγt co-crystallized with digoxin was derived
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from the PDB database (PDB ID: 3B0W) and prepared using the Protein Preparation
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Wizard during the induced fit docking. The water molecules within 5 Angstrom from
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digoxin were kept, and the centroid of the digoxin was used to define the active site in
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the molecular docking calculations.
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2.4 Luciferase reporter assay
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ROR t LUCPorterTM Stable Reporter cells and LUCPorterTM Vector Control
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HEK293 cells were plated (5×104/well) in 96-well plates in triplicate in RPMI 1640
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medium with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100
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μg/ml) and puromycin (3 μg/ml) overnight. The cells were incubated with different
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concentrations of uscharin, calactin, calotropin or digoxin for an additional 16 h. One
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hundred microliters of assay solution was added to the wells. The mixture was
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incubated in the dark at room temperature for 30 min and transferred into a white
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96-well plate. Finally, the luciferase activity was assessed with a Sirius luminometer
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(Bertold Detection System GmbH, Pforzheim, Germany).
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2.5 Transfection
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The transfection assay was performed according to the manufacturer’s
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instruction for Lipofectamine LTX. HEK293 cells were seeded in DMEM with 10%
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FBS at 4×105 cells per well. Five hundred microliters of OPTI-MEM Reduced Serum
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Media containing 2 μg of DNA was added to the cells to be transfected, and then 2 μL
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of PLUS was added into the above diluted OPTI-MEM and DNA solution, followed
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by gentle mixing and incubation for an additional 5 min at room temperature.
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Subsequently, Lipofectamine LTX™ Reagent was added to the above solution and
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then mixed gently and incubated for 30 minutes at room temperature to form
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DNA-Lipofectamine
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DNA-Lipofectamine LTX Reagent complexes was added directly to each well
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containing the cells, mixed gently, and incubated at 37°C in a CO2 incubator for 24 h.
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2.6 Protein extraction and western blotting analysis
LTX
Reagent
complexes.
Finally,
500
μL
of
the
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HEK293 cells transfected with were transfected with 2g RORγt plasmid were
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incubated with the indicated compounds at different concentrations for 6 h. The cells
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were lysed with RIPA buffer containing 1× protease inhibitor mix to harvest total
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cellular proteins, which were measured using a bicinchoninic acid (BCA) assay. The
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total cellular extracts were then subjected to electrophoresis in 10% SDS/PAGE and
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transferred to nitrocellulose membranes. After blocking with 5% non-fat milk in a 6
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Tris-buffered saline-0.1% Tween 20 buffer, the membrane was subsequently
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incubated with specific primary antibodies and HRP-conjugated secondary antibodies.
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A chemiluminescence (ECL) detection system was used to detect the antibody-bound
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proteins on the membrane.
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2.7 Purification of CD4+ T cells and differentiation of T cell subsets
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CD4+ single T lymphocyte were sorted by magnetic bead (Miltenyi Biotec,
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Cologne, Germany). Briefly, the splenocytes were isolated from C57BL/6 mice, and
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the red blood cells were lysed using lysis buffer. The isolated cells were labeled using
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biotin-antibody cocktail and anti-biotin microbeads and sorted by magnetic separation
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in MS columns. The sorted CD4+ T cells were further utilized for differentiation into
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different T-helper cell subsets.
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Different T-helper cell subsets were polarized according to the following
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conditions. Briefly, 2 μg/ml anti-CD3 was coated in a plate and 5 μg/ml anti-CD28
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in the presence of 50 ng/ml IL-6, 1 ng/ml TGF-β1, 10 μg/ml anti-IL-4 and 5 ng/ml
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IL-23 were supplied to polarize Th17 cells. For the polarization of Th1 cells, 1 μg/ml
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anti-CD28 in the presence of 2 ng/ml IL-2 and 5 ng/ml IL-12 were provided. For the
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differentiation of Th2 cells, 1 μg/ml anti-CD28 in the presence of 2 ng/ml IL-2, 10
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ng/ml IL-4, and 10 μg/ml anti-IFN- were applied. For the differentiation of Treg
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cells, 1 μg/ml anti-CD28 in the presence of 5 ng/ml IL-2 and 5 ng/ml TGF-β1 were
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applied. The cells were collected and intracellular staining indicated antibodies for
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analysis of Treg. For Th17 polarization, cells were re-stimulated with 50 ng/ml
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phorbol-12-myristate-13-acetate (PMA), 1 μg/ml ionomycin and 1 μg/ml brefeldin A
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for 5 hours after three days. For Th1 and Th2 differentiation, cells were re-stimulated
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with 50ng/ml PMA, 1 μg/ml ionomycin for 1 h followed by incubation of 2 μM
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Monensin for additional 4 h before intracellular staining.
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2.8 Flow cytometric analysis
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For the analysis of the T-cell subsets with or without indicated treatments, the
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cells were stained with the indicated fluorophore-conjugated monoclonal antibodies. 7
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PerCP/Cy5.5 anti-CD4 was used to stain the cytokines on the membrane surface of
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cells. After the cells were permeabilized, FITC anti-IFN-, APC anti-IL-4, PE
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anti-IL-17A and APC anti-Foxp3 were used to stain intracellular cytokines. The
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expression levels of these cytokines were determined by BD FACSAriaTM III (BD
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Bioscience) and analyzed by FlowJo software.
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2.9 Statistical analysis
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Data are expressed as the mean ± S.E.M. Significant differences were analyzed
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by one-way analysis of variance (ANOVA) using IBM SPSS Statistics 20 software.
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Values of p < 0.05, p < 0.01 and p < 0.001 were considered statistically significant.
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3 Results
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3.1 Uscharin, calactin and calotropin have the potential to be RORt antagonists
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The three non-classical cardenolides, uscharin, calactin and calotropin, were
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isolated from Calotropis gigantea as previously described [12], and the purities of
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these compounds used in current study are approximately 98% according to the
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results of LC-MS (Fig. 1B). These cardenolides share a similar steroidal core with
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digoxin, a classical cardenolide, and digoxin can inhibit Th17 differentiation via
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directly targeting RORt. We therefore hypothesized that uscharin, calactin and
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calotropin have the potential to inhibit Th17 differentiation. Due to the key role of
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RORt in regulating Th17 differentiation, the effects of these compounds on RORt
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were studied by molecular docking. As shown in Table 1, the standard precision (SP)
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docking score of digoxin on RORt was -13.51 kcal/mol, and the scores of uscharin,
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calactin and calotropin were -14.13, -13.6 and -13.15 kcal/mol, respectively,
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suggesting that uscharin, calactin and calotropin have the potential to bind directly to
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RORt and uscharin most likely has better binding affinity on RORt than the other
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two compounds.
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We were then interested in determining whether the binding affinity discrepancy 8
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of uscharin and digoxin on RORt was attributable to the different binding pockets.
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Hence, the binding sites were further elucidated by a molecular docking assay. As
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shown in Fig. 2A, the binding pocket for digoxin was surrounded by a series of
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hydrophobic residues, including Trp317, Leu324, Phe377, Phe378, Phe388, Leu391
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and Ile400. Three water molecules were found to stabilize the pocket by forming a
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hydrogen network with the surrounding residues. One water molecule along with
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Phe377 formed hydrogen bonds with the hydroxyl group of digoxin. At the interior of
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the pocket, Arg367 forms a hydrogen bond with the carbonyl of 2(5H)-furanone. Fig.
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2B shows that the pocket for uscharin encompassed almost the same residues as
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digoxin while the polar interactions for uscharin were different. Two water molecules
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formed hydrogen bonds with a hydroxyl of uscharin; Leu287 and Arg367 both formed
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hydrogen bonds with the carbonyl of 2(5H)-furanone at the interior of the pocket.
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His479 formed an extra hydrogen bond with the other of uscharin outside the pocket.
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These differences can account for the better binding affinity of uscharin on RORt
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than digoxin.
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3.2 Uscharin, calactin and calotropin inhibit RORγt luciferase reporter activity
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and protein expression
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Because uscharin, calactin and calotropin showed the potential to bind RORγt by
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molecular docking assay, the RORγt luciferase reporter was employed to determine
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whether these compounds have inhibitory effects on RORγt. Consistent with the
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molecular docking results, uscharin, calactin and calotropin strongly inhibited RORγt
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luciferase activity from 100 to 250 nM (Fig. 3A-C), suggesting that these compounds
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suppress RORt luciferase activity most likely via directly binding to RORt.
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Because uscharin, calactin and calotropin could significantly inhibit the RORγt
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luciferase reporter, we were interested in investigating the effect of these compounds
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on the protein expression level of RORγt. RORγt plasmid was prepared and
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transfected into HEK293 cells to validate the effect of uscharin, calactin and
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calotropin on RORγt protein expression. Consistent with the inhibitory effect of these
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compounds on RORγt luciferase activity, all three compounds could significantly and 9
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dose-dependently inhibit RORγt expression. Of note, the effective concentrations of
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uscharin, calactin and calotropin were from 100 to 500 nM and the effective
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concentration of digoxin was 10 M (Fig. 3D-F), implying that uscharin, calactin and
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calotropin are more potent than digoxin in suppressing Th17 differentiation.
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3.4 Uscharin, calactin and calotropin inhibit Th17 differentiation
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Before determine the effect of uscharin, calactin and calotropin on Th17
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differentiation, we investigated the cytotoxicity of these compounds on CD4+ T
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lymphocytes. The CD4+ T lymphocytes were isolated and sorted from spleen and
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incubated with indicated compounds for 72h. The MTT results showed that the three
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compounds have not obvious cytotoxicity on CD4+ T lymphocytes up to 72 h (Fig.
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4A-C).
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As mentioned, Th17 lymphocytes have been implicated in the pathogenesis of
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most common autoimmune diseases, and the differentiation of Th17 is mainly
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regulated by RORt. Our results indicated that uscharin, calactin and calotropin
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directly bind to RORt and suppress the luciferase activity and protein expression
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level of RORt. We therefore hypothesized that uscharin, calactin and calotropin
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could inhibit Th17 differentiation. In our current study, CD4+ T cells were sorted and
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polarized under Th17 conditions with or without the cardenolide compounds. As
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shown in Fig. 4, Th17 was successfully differentiated in our current study. With the
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addition of 10 M digoxin, Th17 differentiation was significantly decreased, which is
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consistent with pervious literature reports. Consistent with the molecular docking
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prediction, uscharin, calactin and calotropin effectively and does-dependently
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inhibited Th17 differentiation from 100 to 500 nM (Fig. 4), and the IC50 of uscharin,
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calactin and calotropin are 267.5 nM, 191.5nM and 393.9nM, respectively.
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3.5 Uscharin and calactin rather than calotropin suppress Th1 differentiation
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In addition to Th17, Th1 lymphocytes also play a crucial role in autoimmune
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diseases and both subsets synergistically mediate disease progression [13-16].
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Therefore, we determined whether these cardenolides could affect Th1 differentiation. 10
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CD4+ T cells were isolated from the spleens of C57BL/6 mice and were cultured
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under Th1-polarizing conditions for 3 days with or without cardenolide treatments.
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The cells were then harvested and intracellular cytokine levels were detected using
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flow cytometry with intracellular staining for IFN-, the signature cytokine of Th1. As
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shown in Fig. 5, digoxin did not have a significant effect on Th1 differentiation.
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Interestingly, although uscharin, calactin and calotropin share a similar chemical
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structure, there was a discrepancy among these compounds in their inhibition of Th1
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differentiation. Uscharin and calactin decreased IFN- production, while calotropin
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showed no obvious effect in suppressing IFN- production (Fig. 5), implying that
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uscharin and calactin have the potential to reduce Th1 polarization.
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3.6 Uscharin rather than calactin or calotropin inhibits Th2 differentiation
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Th2 is another important subset of T lymphocytes. In autoimmune diseases, Th2
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cells were initially described as anti-inflammatory subsets based on their ability to
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suppress cell-mediated or Th1 models of disease [17-19]. Considering the critical role
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of Th2 in the progression of autoimmune diseases, we investigated the effects of these
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cardenolides on Th2 differentiation. Th2 was prepared from CD4+ T lymphocytes
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sorted from the splenocytes of C57BL/6 mice and differentiated in polarizing
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conditions for 3 days with or without cardenolides. As shown in Fig. 6, Th2 cells were
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successfully induced and digoxin could not prevent the differentiation of Th2.
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Uscharin dose-dependently decreased IL-4 production, indicating that uscharin
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reduced Th2 polarization. However, neither calactin nor calotropin reduced Th2
287
differentiation.
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3.7 Uscharin, calactin and calotropin inhibit Treg differentiation
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Treg lymphocytes were identified and designated in the mid-1990s and they have
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important immunoregulatory activity in many autoimmune disease conditions [20, 21].
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Increasing Treg numbers or enhancing their suppressive function is beneficial for
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treating autoimmune diseases [22-24]. We therefore investigated the effects of the 11
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cardenolides on Treg differentiation. As shown in Fig. 7, digoxin showed no obvious
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effect on Treg differentiation, while uscharin and calactin intensively inhibited Treg
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differentiation compared to calotropin at 500nM. Interestingly, all of these
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compounds even slightly increased Treg differentiation at 100 and 250 nM.
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Collectively, the results indicated that uscharin non-selectively inhibited the
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differentiation of T cell subsets, including Th1, Th2 and Th17 from 100 to 500nM.
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Calactin inhibited Th17 and Th1 differentiation and calotropin selectively suppressed
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Th17 differentiation.
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4 Discussion
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T helper (Th) lymphocytes play critical roles in orchestrating adaptive immune
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responses. The initial discovery of the existence of specialized Th effector populations
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demonstrated that differentiated CD4+ T cells can be classified into two groups,
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designated Th1 and Th2 cells, based on their cytokine production. With significant
306
technical advances, Th17 cells have been recognized as playing critical roles in the
307
pathogenesis of some autoimmune diseases [25-28]. According to literature reports,
308
digoxin can suppress Th17 differentiation and in turn ameliorate the symptoms of
309
EAE, indicating that compounds with a cardioglycoside chemical structure can have
310
an inhibitory effect on Th17 cells. In our previous study, uscharin, calactin and
311
calotropin, isolated from Calotropis gigantea, were found to have a steroidal
312
core similar to digoxin, a typical cardenolide. We therefore hypothesized that these
313
compounds could inhibit Th17. Accumulating studies have reported that RORγt is a
314
master regulator in Th17 differentiation and hence can be a promising therapeutic
315
target for autoimmune diseases [2, 4, 29]. Before our investigation on the effects of
316
cardenolide compounds on Th17 polarization, molecular docking assay was applied to
317
predict the affinity of these compounds on RORt.
318
According to previous reports, digoxin and its derivatives have been identified as
319
effective therapeutic RORt inhibitor candidates that attenuate autoimmune diseases
320
[3]. Therefore, digoxin was used as a reference compound in our current study. The 12
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results implied that the binding affinities of our cardenolides on RORt are similar to
322
that of digoxin. Furthermore, uscharin has greater potential than digoxin to inhibit
323
Th17 according to the molecular docking results. The binding sites were also explored
324
using the assay. Although the binding pocket of uscharin for RORt has almost the
325
same residues as digoxin, the polar interactions for uscharin were different, which
326
most likely accounted for the better binding affinity of uscharin for RORt than
327
digoxin.
328
To further identify the effects of the three compounds on RORt activity and
329
expression, we employed a luciferase assay and over-expressed RORt. We found that
330
all three compounds significantly inhibited RORt luciferase activity. Interestingly,
331
we found all of the three compounds could suppress RORt overexpression in a
332
dose-dependent manner. It was reported that transcription factors upstream
333
stimulatory factor 1 (USF-1) and USF-2 regulate the expression of RORγt in human
334
lymphocytes, and upregulation of USF-1 and USF-2 was found during the
335
differentiation of Th17 cells from naive CD4+cells[30]. Due to the indispensable role
336
of USF-1 and USF-2 for the transcription of RORγt in lymphocytes, uscharin, calactin
337
and calotropin suppressed RORγt expression probably resulting from regulation of
338
USF-1 and USF-2. Furthermore, the effective concentration of these compounds was
339
500 nM, lower than digoxin, indicating that uscharin, calactin and calotropin have
340
more potential than digoxin to prevent Th17 polarization. Consistent with these
341
results, the three compounds also exhibited significant inhibition on Th17 polarization
342
at 500 nM.
343
Abnormal activation of Th1 cells is thought to be the critical event in most
344
organ-specific autoimmune diseases [31, 32]. In contrast, Th2 cells play a protective
345
role or at least a neutral role [30, 33, 34]. Furthermore, it is thought that some
346
autoimmune diseases result from an imbalance between Treg cells and Th17 cells [20,
347
21]. Therefore, regulating the balance between Treg cells and Th17 cells may be
348
developed as a therapeutic strategy to treat autoimmune disorders. Hence, we
349
intensively investigated the effect of the three compounds on the different T cell
350
subpopulations. although uscharin had the best inhibitory effect on Th17 13
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Molecular Pharmaceutics
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differentiation among the three compounds, as it simultaneously suppressed the
352
differentiation of Th1, Th2 and Treg cells, suggesting that uscharin has no selective
353
effect in inhibiting T lymphocyte subsets.
354
Alternatively, calactin simultaneously inhibited Th1, Th17 and Treg cells without
355
an obvious effect on Th2 cells, indicating that calactin could be developed to treat
356
autoimmune diseases, more so than uscharin. Furthermore, calotropin selectively and
357
intensively suppressed Th17 differentiation rather than other T cell subsets.
358
Collectively, uscharin, calactin and calotropin are cardenolides with similar chemical
359
structures, and their discrepant effects on different T cell subsets are most likely due
360
to the subtle differences in chemical structure.
361
Uscharin, calactin and calotropin share an identical chemical structure except for
362
their functional groups at the C-3' position. A thiazoline ring plays a key role in
363
uscharin’s strongest inhibitory effect on Th17 differentiation but without selectivity
364
for the T cell subsets. A β-hydroxyl group contributes to calactin’s selectivity for
365
suppressing differentiation of both Th1 and Th17 cells while an α-oriented
366
counterpart in calotropin results in its specificity for Th17 cell differentiation. The
367
above structure-activity relationship suggests the importance of the C-3'
368
stereochemistry of nonclassical cardenolides for their inhibitory effects on the
369
differentiation of T cell subsets.
370
In summary, calotropin selectively inhibited Th17 differentiation rather than
371
other T cell subsets, and it should be preferred in treating autoimmune diseases
372
mediated by Th17, indicating that calotropin could be developed as therapeutic
373
compounds to treat autoimmune diseases. The differences in the chemical structures
374
of uscharin, calactin and calotropin are correlated with their selective inhibitory
375
effects on T cell subsets. This finding may lead to strategic modifications of chemical
376
structures in the future.
377 378
Conflict of interests
379
The authors declare that they have no conflicts of interest. 14
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381
Acknowledgments
382
This work was supported by Macau Science and Technology Development Fund
383
(project code 0017/2018/A1).
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469
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Figures
470
Fig. 1 Chemical structures and purity of uscharin, calactin and calotropin. (A) The
471
chemical structures of uscharin, calactin, calotropin and digoxin. (B) The purity of
472
uscharin, calactin and calotropin was analyzed by LC-MS.
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474 475 476
Table 1 Value of the SP docking score for uscharin, calactin, calotropin and digoxin on RORγt.
Ligand
Docking score (Kcal/mol)
Digoxin
-13.51
Uscharin
-14.13
Calactin
-13.61
Calotropin
-13.15
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478 479
A
B
480 481 482 483 484 485 486 487 488
Fig. 2 Identification of the interaction between the compounds and RORγt via molecular docking calculations. (A) Schematic diagram of the interaction mode between the RORγt binding site (yellow) and digoxin (green). The red spheres represent water. Hydrogen bonds are highlighted with a red dashed line. (B) Schematic diagram of the interaction mode between the RORγt binding site (yellow) and uscharin (cyan). The red spheres represent water. Hydrogen bonds are highlighted with a red dashed line. 20
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490 491 492 493 494 495 496 497 498 499 500 501 502
Fig. 3 Effect of uscharin, calactin and calotropin on RORγt luciferase reporter activity and protein expression level. (A-C) The effect of uscharin, calactin, calotropin and digoxin on RORγt luciferase reporter activity. The RORγt LUCPorter™ Stable Reporter Cells were incubated with uscharin, calactin, calotropin and digoxin at the indicated concentration for 16 h. The cell lysates were prepared and used for luciferase activity analysis. (D-F) The effect of uscharin, calactin, calotropin and digoxin on the protein expression of RORγt. The HEK293 cells transfected with RORγt plasmid were incubated with the indicated compounds at different concentrations for 6 h and then the total lysates were prepared and the expression of RORγt and β-actin were analyzed by western blotting. Data of three independent experiments are presented as mean ± SEM, *p