Due to their immunomodulatory functions, several herbal medicines have been used for the treatment of immunological disorders [1
]. Among them, Korean Red Ginseng (KRG) has been traditionally prescribed for various diseases, although the exact mechanisms by which KRG mitigates the severity of diseases remain unclear [2
]. Recent advances in understanding the pharmacological components of KRG shed light on identifying a variety of bioactive ingredients, such as ginsenosides, polysaccharides, phytosterols, peptides, polyacetylenic alcohols, and fatty acids, that prevent and eradicate diabetes, tumors, ulcers, aging, and depression [3
It has been suggested that ginsenosides are among the most potent biologically active constituents of KRG [7
]. More than 100 types of ginsenosides are present, stratified by the differential polarity attributed to their chemical backbones [8
]. Interestingly, numerous studies have reported that anti-inflammatory effects are common features of a myriad of ginsenosides [2
]. For example, enhanced Rg3, one of the major ginsenosides present in KRG, inhibits IFNγ-producing helper CD4+
T cells (Th1) that prevalently expand in the conditions of excessive inflammation [10
]. Rg3 also promotes immunosuppressive M2 macrophage polarization, which can resolve self-destructive immune responses [11
]. Moreover, increasing numbers of recent studies demonstrate that ginsenosides can ameliorate the disease severity in animal models of multiple sclerosis, Crohn’s disease, and rheumatoid arthritis, suggesting that ginsenosides modulate autoimmune responses in vivo [1
Autoimmune diseases are caused by unnecessarily aggressive autoreactive CD4+
T-cell responses that attack self-tissue and/or self-antigen. Among diverse CD4+
T-cell subsets, IL-17A-producing CD4+
T cells (Th17) provide protection against extracellular pathogens and fungi, but also exert detrimental roles in mediating tissue inflammation in autoimmune diseases [12
]. Indeed, antagonistic antibodies to IL-17A or IL-17RA have shown a clinical benefit in patients with psoriasis [14
]. The differentiation of Th17 cells is induced by IL-6 and TGFβ, which activate STAT3 and subsequently induce RORγt [15
]. Recently, two independent studies have demonstrated that KRG can inhibit Th17 cell differentiation by hampering STAT3 activation, leading to the amelioration of collagen-induced arthritis and alloantigen-induced inflammation [18
]. Our previous study also suggested a role of Rg3 in T-cell differentiation [10
], indicating a possible role of ginsenosides in T-cell-mediated immune disorders.
In the present study, we aimed to investigate whether ginsenoside Rg3 modulated Th17 differentiation and Th17-mediated experimental autoimmune encephalomyelitis (EAE), in an animal model for human multiple sclerosis. By using multiple in vitro T-cell culture systems, we found that Rg3 inhibited Th17 cell differentiation in a T-cell-intrinsic manner. Moreover, Rg3 alleviated the incidence and severity of EAE in vivo.
2. Materials and Methods
2.1. Ethics Statement
All animal experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University (IACUC protocol #: SNU-170120-1) and were conducted in accordance with guidelines of Seoul National University for the care and use of laboratory animals.
2.2. Mice and EAE Model
Six-week-old C57BL/6 female mice were purchased from Orient Bio (Gyeonggi, Korea). For T-cell-transfer EAE, mice were immunized with 300 μg myelin oligodendrocyte glycoprotein peptide (MOG35–55
) peptide emulsified in complete freund’s adjuvant (CFA) with heat-inactivated Mycobacterium tuberculosis
. Seven days after immunization, cells from draining lymph nodes were isolated and cultured with 20 μg/mL MOG35–55
and IL-23 (20 ng/mL) (PeproTech, Rocky Hill, NJ, USA). On day five post in vitro stimulation, cells were harvested and enriched with CD4+
T cells by using magnetic beads. The enriched CD4+
T cells (2 × 106
cells/mouse) were transferred into mice followed by immunization with MOG35–55
in CFA (s.c.) and subsequent pertussis toxin (PT) (i.p.) injection, and the clinical severity for EAE pathology was monitored daily as previously mentioned [20
2.3. Bone-Marrow-Derived Dendritic Cells (BMDCs) Generation
Bone marrow cells obtained from C57BL/6 mice were cultured in PRMI1640 supplemented with 10% FBS, 55 μM 2-mercaptoethanol, penicillin/streptomycin at 1 × 106 cells/mL (All from Gibco, Grand Island, NY, USA), and recombinant mouse GM-CSF (10 ng/mL) (PeproTech, Rocky Hill, NJ, USA). Twenty-four hours later, floating cells were transferred into new plates. Fresh medium was added every other day. On day seven, cells were recovered and CD11c+ cells were isolated using magnetic beads, and the sorted CD11c+ DCs were used in further studies.
2.4. In Vitro Th17 Cell Differentiation
Naïve CD4+ T cells (CD44lowCD62LhighCD25−) were isolated from wild-type mice using a cell sorter (BD BioScience, San Jose, CA, USA). For DC-mediated Th17 cell differentiation, BMDCs were co-cultured with naive CD4+ T cells in the presence of anti-CD3ε (0.3 μg/mL) antibody (145-2C11, BioXcell, NH, USA), TGFβ (1.5 ng/mL) (PeproTech, Rocky Hill, NJ, USA), and LPS (100 ng/mL) (Sigma, Seoul, Korea) for 96 h. For DC-free Th17 cell differentiation, anti-CD3ε (1 μg/mL) and anti-CD28 (1 μg/mL) (37.51, BioXcell, West Lebanon, NH, USA) were pre-coated in a 96-well flat-bottom plate overnight at 4 °C. After washing the plate with cold PBS three times, 1 × 105 naïve CD4+ T cells were stimulated with IL-6 (10 ng/mL) and TGFβ (1.5 ng/mL) (PeproTech, Rocky Hill, NJ, USA) for 96 h. For Rg3 (LKT Labs, Saint Paul, MN, USA; dissolved in DMSO) treatment, various concentrations (9.37, 18.75, and 37.5 μg/mL) were added at the beginning of in vitro culture.
IL-6, 12p40, IL-17A, and TNFα in the culture supernatants of LPS-stimulated BMDCs or T cells stimulated under Th17-skewing conditions were quantified by ELISA according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA).
2.6. Real-Time RT-PCR
Total RNA from cells was isolated by TRIzol reagent (Ambion, Austin, TX, USA) and cDNA was synthesized with a cDNA Synthesis kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Relative gene expression levels were evaluated using SYBR Green (Bio-Rad, Philadelphia, PA, USA) on ABI 7500 Fast Real-Time PCR Systems (Applied Biosystems, Singapore). Target genes were normalized to the Hprt level in each sample. Primer sets for genes were synthesized at Cosmogenetech (Seoul, Korea): Il6 (sense, 5′- TCG GAG GCT TAA TTA CAC ATG TTC T -3′, antisense, 5′- GCA TCA TCG TTG TTC ATA CAA TCA -3′), Il12b (sense, 5′- AAA CCA GAC CCG CCC AAG AAC -3′, antisense, 5′- AAA AAG CCA ACC AAG CAG AAG ACA G -3′), Il17a (sense, 5′- CTC CAG AAG GCC CTC AGA CTA C -3′, antisense, 5′- GGG TCT TCA TTG CGG TGG -3′), Il22 (sense, 5′- CAT GCA GGA GGT GGT ACC TT -3′, antisense, 5′- CAG ACG CAA GCA TTT CTC AG -3′), Rorc (sense, 5′-CCG CTG AGA GGG CTT CAC -3′, antisense, 5′- TGC AGG AGT AGG CCA CAT TAC A -3′),Tnfa (sense, 5′- ATG AGA AGT TCC CAA ATG GCC -3′, antisense, 5′- TCC ACT TGG TGG TTC GCT ACG -3′), Hprt (sense, 5′- GGT TAA GCA GTA CAG CCC CAA AAT -3′, antisense, 5′- ATA GGC ACA TAG TGC AAA TCA AAA GTC -3′).
2.7. Flow Cytometry Analysis
For intracellular cytokine staining, cells were incubated for 3 h with 100 ng/mL of PMA and 1 μM of ionomycin (all from Sigma-Aldrich, Saint Louis, MO, USA), brefeldin A, and monensin (all from eBioscience, San Diego, CA, USA). After washing cells with cold PBS containing 1.5% FBS, cells were stained with APC-Cy7-conjugated anti-CD45.2 mAb and PE/Cy7-conjugated anti-CD4 mAb (eBioscience, San Diego, CA, USA) for surface staining. Cells were then washed and stained with PerCp-Cy5.5-conjugated anti-IFNγ mAb, APC-conjugated anti-IL-17 mAb (all from BioLegend, San Diego, CA, USA) and PE-conjugated anti-RORγt mAb (eBioscience, San Diego, CA, USA) after incubation with fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) for 30 min at 4 °C. Cells were analyzed by LSR III flow cytometer (BD Bioscience, San Jose, CA, USA). Data were analyzed with FlowJo (TreeStar, Ashland, OR, USA).
2.8. Statistical Analysis
All experiments were performed more than three times. Statistical analysis was conducted with mean ± SEM by unpaired two-tailed Student’s t-test with GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA).
While the immunomodulatory effects of KRG extract (KRGE) are well documented, the immuno-pharmacological effects of each component within the extracts are less clear. In this regard, ginsenoside Rg3 has been proposed to be immuno-modulatory based on the observation that Rg3-enhanced KRGE ameliorates various inflammatory diseases in animal models, including T-cell-mediated autoimmune diseases [2
]. Among diverse helper T-cell subsets, Th17 cells appear to be critically pathogenic immune cells in autoimmune tissue inflammation, including psoriasis and multiple sclerosis. Therefore, in the present study, we aimed to investigate the role of Rg3 in Th17 cell differentiation and Th17-mediated autoimmune diseases by using animal models. Our in vitro studies revealed that Rg3 played only a minor role in the production of Th17-promoting cytokines IL-6, IL-12/23p40 and TNFα from DCs. Instead, Rg3 significantly inhibited the induction of RORγt expression in CD4+
T cells and therefore hampered the differentiation of Th17 cells from naïve precursors. Moreover, Rg3 appeared to inhibit the expansion of autoreactive MOG-specific Th17 cells during ex vivo restimulation with a cognate peptide, and thus also ameliorated the incidence and severity of EAE in the recipient mice. Thus, our findings suggest an anti-inflammatory mechanism by which Rg3 ameliorates autoimmune inflammation via inhibiting RORγt expression of CD4+
T cells during Th17 cell differentiation.
RORγt, a master transcription factor for Th17, is regulated by a complex regulatory circuit [26
], but whether Rg3 intervenes with the RORγt expression has not been identified. Activation of STAT3 upon IL-6 signaling with concomitant TGFβ signaling induces RORγt expression [17
]. Moreover, IL-23 further expands and stabilizes Th17 cells via STAT3. In this respect, it is noteworthy that Rg3 represses STAT3 phosphorylation in tumor cells by decreasing the hexokinase 2 level [27
]. We observed that the transcript level of Il21
in T cells stimulated with IL-6 and TGFβ was reduced by Rg3. IL-21 is known to be induced by IL-6 via STAT3 during Th17 cell differentiation [28
]. Thus, it is feasible to hypothesize that Rg3 inhibits RORγt expression by suppressing STAT3 activation in T cells upon IL-6 and IL-23 signaling. Alternatively, it is possible that STAT5, a negative regulator of RORγt, was involved in the Th17 reduction by Rg3, given that ginseng can up-regulate STAT5 phosphorylation in cyclosporine-treated splenocytes poised to Treg differentiation [18
]. It has also been delineated that natural ligands stemming from endogenous cholesterol metabolism can either promote or inhibit RORγt function by directly interacting with its binding sites [29
]. Ursolic acid is one of the most well-characterized RORγt antagonists, which ameliorates EAE and allergic asthma [30
]. Since Rg3 has a tetracyclic triterpenoid saponin structure [14
], a homologue of the ursolic acid, Rg3 might belong to the specific RORγt antagonists as well. Further studies are required to dissect the direct molecular target(s) of Rg3 in developing Th17 cells and to elucidate the molecular mechanisms by which Rg3 regulates Th17 cell differentiation and expansion.
Several studies including our own showed that KRGE can modulate the production of innate cytokines from DCs and macrophages [10
]. LPS stimulation induced the production of several Th17-promoting cytokines including IL-6, IL-23, TNFα, and IL-1β. The observed minor role of Rg3 in regulating the production of these cytokines from DCs suggests that other components within KRGE have DC-regulatory functions. The diminished RORγt expression by Rg3 treatment in T cells raises the possible role of Rg3 in regulating the functions of RORγt-expressing innate immune cells including type 3 innate lymphoid cells (ILC3) and LTi cells. Since these cells are known to play a crucial role in mucosal homeostasis and lymphoid tissue generation [32
], it will be important to determine the potential role of Rg3 in these innate RORγt-expressing cells. Antibodies against IL-17A or IL-17RA are proven to be effective in the treatment of psoriasis in humans [34
]. Thus, it will be of interest to investigate if Rg3 can ameliorate other autoimmune tissue inflammation, such as psoriasis.
In summary, the results of the present study indicate that Rg3 negatively regulates RORγt expression in CD4+ T cells, subsequently hindering Th17 cell differentiation and Th17-mediated neuro-inflammation. These findings are novel because they attest to the fact that ginsenoside Rg3 is the active constituent of KRG extract that can inhibit the differentiation of Th17 cells and Th17-cell-mediated autoimmunity. Our findings may provide a rationale for developing Rg3 as a potential candidate for the treatment of autoimmune disorders driven by Th17 cells.