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1.  IL-12p40 Homodimer Ameliorates Experimental Autoimmune Arthritis 
IL-23 is the key cytokine that induces the expansion of Th17 cells. It is composed of p19 and p40 subunits of IL-12. The p40 subunit binds competitively to the receptor of IL-23 and blocks its activity. Our aim was to assess the preventive and therapeutic effect of the IL-12p40 homodimer (p40)2 subunit in autoimmune arthritis animal models. In the current study, using IL-1R antagonist–knockout mice and a collagen-induced arthritis model, we investigated the suppressive effect of (p40)2 on inflammatory arthritis. We demonstrated that the recombinant adenovirus-expressing mouse (p40)2 model prevented the development of arthritis when given before the onset of arthritis. It also decreased the arthritis index and joint erosions in the mouse model if transferred after arthritis was established. (p40)2 inhibited the production of inflammatory cytokines and Ag-specific T cell proliferation. It also induced CD4+CD25+Foxp3 regulatory T (Treg) cells in vitro and in vivo, whereas the generation of retinoic acid receptor–related organ receptor γt and Th17 cells was suppressed. The induction of Treg cells and the suppression of Th17 cells were mediated via activated STAT5 and suppressed STAT3. Our data suggest that (p40)2 suppressed inflammatory arthritis successfully. This could be a useful therapeutic approach in autoimmune arthritis to regulate the Th17/Treg balance and IL-23 signaling.
PMCID: PMC4574520  PMID: 26324771
2.  Interleukin 17-expressing Innate Synovial Cells Drive K/Bxn Serum-induced Arthritis 
Immune Network  2016;16(6):366-372.
K/BxN serum can induce arthritis in normal mice because of abundant autoantibodies that trigger an innate inflammatory response in joints. To determine whether IL-17 is involved in the pathogenesis of serum-induced arthritis, we injected wild-type and IL-17−/− mice with K/BxN serum and evaluated them for signs of arthritis. Unlike wild-type mice, IL-17−/− mice did not show any signs of arthritis. IL-17 was produced predominantly by CD3− CD4− γδTCR− NK1.1− Sca1int Thy1hi cells residing in the inflamed synovial tissue. When synovial cells extracted from normal joints were stimulated with IL-23 or autoantibody-containing immune complexes, a substantial fraction of Sca1int Thy1hi cells produced IL-17. Thus, we have identified a novel population of IL-17-producing innate synovial cells that play a crucial role in the development of K/BxN serum-induced arthritis.
PMCID: PMC5195846  PMID: 28035212
IL-17; Synovial cells; Arthritis; Immune complex
3.  PTEN ameliorates autoimmune arthritis through down-regulating STAT3 activation with reciprocal balance of Th17 and Tregs 
Scientific Reports  2016;6:34617.
PTEN is a tyrosine phosphatase with significant function in inhibiting STAT3 activation. Recently, inactivation of STAT3 has been demonstrated as a therapeutic candidate for autoimmune arthritis. The expression of PTEN controlled by p53 regulates autoimmune arthritis through modulating the balance between Th17 and Treg. We hypothesized that PTEN regulated by p53 might reduce CIA severity and inflammatory response via inhibiting STAT3 activation. Our results revealed that PTEN could ameliorate experimental autoimmune arthritis by reducing STAT3 activity and Th17 differentiation. Systemic infusion of PTEN overexpression downregulated CIA severity. In addition, PTEN overexpression decreased the activation of T cells and modulated reciprocal differentiation of Th17 and Treg cells. We observed that PTEN expression downregulated by p53 deficiency induced the activation of STAT3. Loss of p53 exacerbated autoimmune arthritis and dysregulated the population of Th17 and Treg. These data suggest that induction of STAT3-modulatory activity of PTEN may be a therapeutic target for rheumatoid arthritis therapy.
PMCID: PMC5052580  PMID: 27708408
4.  Advances in Systems Biology Approaches for Autoimmune Diseases 
Immune Network  2014;14(2):73-80.
Because autoimmune diseases (AIDs) result from a complex combination of genetic and epigenetic factors, as well as an altered immune response to endogenous or exogenous antigens, systems biology approaches have been widely applied. The use of multi-omics approaches, including blood transcriptomics, genomics, epigenetics, proteomics, and metabolomics, not only allow for the discovery of a number of biomarkers but also will provide new directions for further translational AIDs applications. Systems biology approaches rely on high-throughput techniques with data analysis platforms that leverage the assessment of genes, proteins, metabolites, and network analysis of complex biologic or pathways implicated in specific AID conditions. To facilitate the discovery of validated and qualified biomarkers, better-coordinated multi-omics approaches and standardized translational research, in combination with the skills of biologists, clinicians, engineers, and bioinformaticians, are required.
PMCID: PMC4022781  PMID: 24851096
Autoimmune diseases; Systems biology; Multi-omics; Biomarker; Translational research; Network analysis
6.  Amelioration of autoimmune arthritis by adoptive transfer of Foxp3-expressing regulatory B cells is associated with the Treg/Th17 cell balance 
Foxp3 is a key regulator of the development and function of regulatory T cells (Tregs), and its expression is thought to be T cell-restricted. We found that B cells in mice can express Foxp3 and B cells expressing Foxp3 may play a role in preventing the development of collagen-induced arthritis (CIA) in DBA/1J mice.
Foxp3 expression was modulated in CD19+ B cells by transfection with shRNA or using an over-expression construct. In addition, Foxp3-transfected B cells were adoptively transferred to CIA mice. We found that LPS or anti-IgM stimulation induced Foxp3 expression in B cells. Foxp3-expressing B cells were found in the spleens of mice.
Over-expression of Foxp3 conferred a contact-dependent suppressive ability on proliferation of responder T cells. Down-regulation of Foxp3 by shRNA caused a profound induction in proliferation of responder T cells. Adoptive transfer of Foxp3+CD19+ B cells attenuated the clinical symptoms of CIA significantly with concomitant suppression of IL-17 production and enhancement of Foxp3 expression in CD4+ T cells from splenocytes.
Our data indicate that Foxp3 expression is not restricted to T cells. The expression of Foxp3 in B cells is critical for the immunoregulation of T cells and limits autoimmunity in a mouse model.
PMCID: PMC4924280  PMID: 27350539
Foxp3; Regulatory B cell; Th17; Arthritis
7.  TNFB Gene Polymorphism in Patients with Systemic Lupus Erythematosus in Korean 
To elucidate the gene frequency of TNFB Ncol polymorphism and its association with HLA class II antigen in patients with systemic lupus erythematosus (SLE) in Korea.
We investigated the gene frequency of the TNFB alleles using DNA obtained from peripheral mononuclear cells in 141 healthy controls and in 58 patients with SLE. The polymorphisms of TNFB gene (735 bp) were studied by Ncol PCR-RELP. A portion of TNFB gene(735 bp) was amplified by PCR and its products were digested with Ncol restriction enzyme. The digested samples of amplified DNA were analyzed by agarose gel electrophoresis. TNFB*1 and TNFB*2 alleles were identified according to polymorphic fragments on Ncol restriction site in the first intron of the TNFB gene. The generic types of HLA-DRBI were also determined by PCR with sequence specific primers (SSP) using genomic DNA from the same subjects.
The genotypic frequency of TNFB*2 homozygote was significantly increased in patients with SLE compared with controls (RR=2.36, P=0.011). The frequency of HLA-DRBI*15 was also significantly increased in patients (RR=2.27, P=0.029). However, the increased frequency of TNFB*2 homozygote was apparently increased in nephritis group (RR=2.79, P=0.035), whereas the significance of TNFB*2 homozygote was weakend in non-nephritis group.
Our results suggest that genetic predisposition of TNFB*2 homozygote is another risk factor in Korean SLE, especially in DR2 negative patients. In addition, TNFB*2 homozygote could have a tendency for the development of nephritis in patients with SLE.
PMCID: PMC4532044  PMID: 7495771
SLE; TNFB Gene; HLA-DR; Lupus nephritis; PCR-RFLP
8.  Green Tea Epigallocatechin-3-Gallate Suppresses Autoimmune Arthritis Through Indoleamine-2,3-Dioxygenase Expressing Dendritic Cells and the Nuclear Factor, Erythroid 2-Like 2 Antioxidant Pathway 
The activity of one of the major catechins in Green Tea, the polyphenol (−)-epigallocatechin-3-gallate (EGCG), has been shown to have a variety of health benefits. Recent studies suggest that EGCG can modulate both the innate and adaptive arms of the immune system. The goal of the current studies was to examine the immunomodulatory effects and mechanisms of action of EGCG on experimental arthritis in mice.
EGCG (10 mg/kg) was administered by oral gavage after CIA induction, while control mice were administered phosphate buffered saline (PBS). Disease mechanisms were studied in both groups of mice. Phenotypes were examined using repeated measure analysis of variance (ANOVA) and data from in vitro and ex vivo experiments were analyzed for significance using the Mann-Whitney U test.
EGCG treatment ameliorated clinical symptoms and reduced histological scores in arthritic mice. Serum type-II collagen-specific immunoglobulin (Ig) IgG2a antibodies were significantly lower in EGCG-fed mice compared to PBS-treated mice. EGCG significantly suppressed T cell proliferation and relative frequencies of CD4 T cells, CD8 T cells and B cell subsets including marginal zone B cells, T1 and T2 transitional B cells, while increasing the frequency of CD4+ Foxp3+ regulatory T cells (Tregs) and indoleamine‐2,3‐dioxygenase (IDO) expression by CD11b+ dendritic cells (DC). Splenic CD11b+ DC from EGCG fed mice induced an increased frequency of Tregs via an IDO-dependent mechanism in in vitro cultures. Importantly, joint homogenates from EGCG-fed mice exhibited significantly increased levels of Nuclear Factor, Erythroid 2-Like 2 (Nrf-2) and Heme oxygenase-1 (HO-1) compared with PBS-fed mice.
This is the first report of upregulation of the Nrf-2 antioxidant pathway in EGCG-mediated immunoregulation. EGCG ameliorated experimental arthritis in mice by eliciting IDO-producing DCs, increasing frequencies of T regs and inducing the activation of the Nrf-2 antioxidant pathway. It remains to be established whether EGCG is useful for the prevention and treatment of rheumatoid arthritis and other inflammatory disorders.
PMCID: PMC4570740  PMID: 26379475
Green Tea (−)-epigallocatechin-3-gallate (EGCG); Collagen-induced arthritis; Regulatory T cells; Nrf-2 signaling pathway
9.  A Plasmacytoid Dendritic Cells-Type I Interferon Axis Is Critically Implicated in the Pathogenesis of Systemic Lupus Erythematosus 
Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease that is characterized by the generation of immune responses to various nuclear components. Impaired clearance of apoptotic cells and loss of tolerance to self-antigens are involved both in the initiation and in the propagation of the disease. Dendritic cells (DCs) are key factors in the balance between autoimmunity and tolerance and play a role linking innate and adaptive immunity. DCs, particularly plasmacytoid DCs (pDCs), are the main source of type I interferon (IFN) cytokines, which contribute to the immunopathogenesis of SLE. There is accumulating evidence that pDCs and type I IFN cytokines take the leading part in the development of SLE. In this review, we discuss recent data regarding the role of pDCs and type I IFN cytokines in the pathogenesis of SLE and the potential for employing therapies targeting against aberrant regulation of the pDC-type I IFN axis for treating SLE.
PMCID: PMC4490545  PMID: 26110387
systemic lupus erythematosus; dendritic cells; type I interferon
10.  Kinetin modulates physio-hormonal attributes and isoflavone contents of Soybean grown under salinity stress 
Crop productivity continues to decline due to a wide array of biotic and abiotic stresses. Salinity is one of the worst abiotic stresses, as it causes huge losses to crop yield each year. Kinetin (Kn) has been reported as plant growth regulator since long, but its role in improving plant growth and food quality under saline conditions through mediating phytohormonal cross-talk is poorly studied. Current study was designed to evaluate the impact of exogenously applied Kn on growth, isoflovones and endogenous phytohormones of soybean grown under NaCl induced salt stress. Soybean plants were grown in perlite (semi hydroponic), and under controlled green-house conditions. Elevated levels of exogenous Kn significantly mitigated the adverse effect of NaCl and rescued plant growth attributes, i.e., plant height, fresh and dry biomass of soybean plants grown in all treatments. Higher diadzen, glycitin, and genistin contents were observed in plants treated with elevated Kn in the presence or absence of NaCl induce salt stress. The gibberellins (GAs) biosynthesis pathway was up-regulated by Kn as the bioactive GA1 and GA4 contents were significantly higher in Kn treated plants, as compared to control, while GAs level reduced in NaCl treated plants. Contrary to GAs, the abscisic acid contents declined with Kn but promoted in NaCl stressed soybean plants. The endogenous jasmonic acid and salicylic acid contents of soybean enhanced with elevated Kn application, but they showed an antagonistic response under salt stress. Current study supports the active role of Kn to ameliorate the adverse effects of salt stress on the growth and food quality of soybean. The favorable role of Kn toward soybean growth under salt stress may be attributed to its potential to modulate cross-talk between the various phytohormones involved in soybean growth and its resistance to salinity stress.
PMCID: PMC4450176  PMID: 26082785
kinetin; soybean; salt stress; gibberellins; abscisic acid; jasmonic acid; salicylic acid
11.  Red Ginseng Extract Ameliorates Autoimmune Arthritis via Regulation of STAT3 Pathway, Th17/Treg Balance, and Osteoclastogenesis in Mice and Human 
Mediators of Inflammation  2014;2014:351856.
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic joint inflammation. Red ginseng is a steamed and dried Panax ginseng C.A. Meyer, which has been used as alternative medicine for thousands of years. This study was undertaken to investigate the effects of red ginseng extracts (RGE) on autoimmune arthritis in mice and humans and to delineate the underlying mechanism. RGE was orally administered three times a week to mice with arthritis. Oral administration of RGE markedly ameliorated clinical arthritis score and histologically assessed joint inflammation in mice with CIA. A significant reduction in STAT3 phosphorylation and a decrease in the number of Th17 cells were observed with RGE treatment. There was also a marked reduction in RANKL-induced osteoclastogenesis with treatment of RGE. The inhibitory effect of RGE on Th17 differentiation and osteoclastogenesis observed in mice was also confirmed in the subsequent experiments performed using human peripheral blood mononuclear cells. Our findings provide the first evidence that RGE can regulate Th17 and reciprocally promote Treg cells by inhibiting the phosphorylation of STAT3. Therefore, RGE can ameliorate arthritis in mice with CIA by targeting pathogenic Th17 and osteoclast differentiation, suggesting a novel therapy for treatment of RA.
PMCID: PMC4132415  PMID: 25147435
12.  Prevalence of Systemic Lupus Erythematosus in South Korea: An Administrative Database Study 
Journal of Epidemiology  2014;24(4):295-303.
Systemic lupus erythematosus (SLE) is a rare autoimmune disease for which a population-based survey on the prevalence of the disease in South Korea has not yet been conducted. Our goal was to estimate the nationwide prevalence of SLE.
The International Classification of Diseases, Tenth Revision (ICD-10) code for SLE diagnosis—M32—was tentatively given when patients were suspected to have SLE before 2009. As such, the positive predictive value (PPV) of the M32 code shown in medical bills reflecting true SLE was uncertain. We attempted to estimate the prevalence of SLE in South Korea using national administrative database data from 2004–2006. We approximated the actual number of SLE patients by analyzing a list of SLE-coded patients provided by the National Health Insurance (NHI) and Health Insurance Review and Assessment Service. Prevalence was estimated by multiplying the PPV of the M32 diagnostic code by the number of patients receiving the code. The PPV was determined by three methods: direct investigation of the medical records of patients randomly selected from the SLE-coded patients list; assessment of all SLE patients treated at 56 selected hospitals in South Korea; and extrapolation from sub-groups at a single institute to the sub-groups of the national NHI data.
The estimated number of national SLE cases was between 9000 and 11 000, depending on the method of ascertainment, corresponding to a prevalence of 18.8–21.7 per 100 000 people.
This is the first report of a nationwide prevalence survey of SLE in South Korea. National databases may serve as a resource for epidemiologic studies of rare autoimmune diseases like SLE.
PMCID: PMC4074634  PMID: 24857955
systemic lupus erythematosus; prevalence; epidemiology
13.  Generation of disease-specific induced pluripotent stem cells from patients with rheumatoid arthritis and osteoarthritis 
Since the concept of reprogramming mature somatic cells to generate induced pluripotent stem cells (iPSCs) was demonstrated in 2006, iPSCs have become a potential substitute for embryonic stem cells (ESCs) given their pluripotency and “stemness” characteristics, which resemble those of ESCs. We investigated to reprogram fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) to generate iPSCs using a 4-in-1 lentiviral vector system.
A 4-in-1 lentiviral vector containing Oct4, Sox2, Klf4, and c-Myc was transduced into RA and OA FLSs isolated from the synovia of two RA patients and two OA patients. Immunohistochemical staining and real-time PCR studies were performed to demonstrate the pluripotency of iPSCs. Chromosomal abnormalities were determined based on the karyotype. SCID-beige mice were injected with iPSCs and sacrificed to test for teratoma formation.
After 14 days of transduction using the 4-in-1 lentiviral vector, RA FLSs and OA FLSs were transformed into spherical shapes that resembled embryonic stem cell colonies. Colonies were picked and cultivated on matrigel plates to produce iPSC lines. Real-time PCR of RA and OA iPSCs detected positive markers of pluripotency. Immunohistochemical staining tests with Nanog, Oct4, Sox2, Tra-1-80, Tra-1-60, and SSEA-4 were also positive. Teratomas that comprised three compartments of ectoderm, mesoderm, and endoderm were formed at the injection sites of iPSCs. Established iPSCs were shown to be compatible by karyotyping. Finally, we confirmed that the patient-derived iPSCs were able to differentiate into osteoblast, which was shown by an osteoimage mineralization assay.
FLSs derived from RA and OA could be cell resources for iPSC reprogramming. Disease- and patient-specific iPSCs have the potential to be applied in clinical settings as source materials for molecular diagnosis and regenerative therapy.
PMCID: PMC3978583  PMID: 24490617
14.  Gut-residing Microbes Alter the Host Susceptibility to Autoantibody-mediated Arthritis 
Immune Network  2014;14(1):38-44.
K/BxN serum can transfer arthritis to normal mice owing to the abundant autoantibodies it contains, which trigger innate inflammatory cascades in joints. Little is known about whether gut-residing microbes affect host susceptibility to autoantibody-mediated arthritis. To address this, we fed C57BL/6 mice with water containing a mixture of antibiotics (ampicillin, vancomycin, neomycin, and metronidazol) for 2 weeks and then injected them with K/BxN serum. Antibiotic treatment significantly reduced the amount of bacterial genomic DNA isolated from fecal samples, in particular a gene encoding 16S ribosomal RNA derived from segmented filamentous bacteria. Arthritic signs, as indicated by the arthritic index and ankle thickness, were significantly attenuated in antibiotic-treated mice compared with untreated controls. Peyer's patches and mesenteric lymph nodes from antibiotic-treated mice contained fewer IL-17-expressing cells than those from untreated mice. Antibiotic treatment reduced serum C3 deposition in vitro via the alternative complement pathway. IL-17-/- congenic C57BL/6 mice were less susceptible to K/BxN serum-transferred arthritis than their wild-type littermates, but were still responsive to treatment with antibiotics. These results suggest that gut-residing microbes, including segmented filamentous bacteria, induce IL-17 production in GALT and complement activation via the alternative complement pathway, which cause the host to be more susceptible to autoantibody-mediated arthritis.
PMCID: PMC3942506  PMID: 24605079
K/BxN serum-transferred arthritis; Gut-residing microbes; Antibiotics; IL-17; Segmented filamentous bacteria
15.  Grape Seed Proanthocyanidin Extract–Mediated Regulation of STAT3 Proteins Contributes to Treg Differentiation and Attenuates Inflammation in a Murine Model of Obesity-Associated Arthritis 
PLoS ONE  2013;8(11):e78843.
Grape seed proanthocyanidin extract (GSPE) is a natural flavonoid that exerts anti-inflammatory properties. Obesity is an inflammatory condition and inflammatory cells and their secretion of pro-inflammatory molecules contribute to the pathogenesis of obesity. Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by inflammation of joints lined by synovium. Previously, we demonstrated that obesity augmented arthritis severity in collagen induced arthritis (CIA), a murine model of human RA. Here, we investigated whether oral administration of GSPE showed antiobesity and anti-arthritic effects in high-fat diet-induced obese (DIO) mice and in obese CIA mice, respectively. The pathophysiologic mechanisms by which GSPE attenuates weight gain and arthritis severity in vivo were also investigated. In DIO mice, GSPE administration significantly inhibited weight gain, reduced fat infiltration in liver and improved serum lipid profiles. The antiobesity effect of GSPE was associated with increased populations of regulatory T (Treg) cells and those of decreased Th17 cells. Decrease of Th17 cells was associated with significant inhibition of their key transcriptional factors, pSTAT3Tyr705 and pSTAT3Ser727. On the contrary, GSPE-induced Treg induction was associated with enhanced pSTAT5 expression. To identify the anti-arthritis effects of GSPE, GSPE was given orally for 7 weeks after type II collagen immunization. GSPE treatment significantly attenuated the development of autoimmune arthritis in obese CIA model. In line with DIO mice, GSPE administration decreased Th17 cells and reciprocally increased Treg cells by regulating STAT proteins in autoimmune arthritis model. The expressions of pro-inflammatory cytokines and nitrotyrosine in synovium were significantly inhibited by GSPE treatment. Taken together, GSPE functions as a reciprocal regulator of T cell differentiation – suppression of Th17 cells and induction of Tregs in both DIO and obese CIA mice. GSPE may act as a therapeutic agent to treat immunologic diseases related with enhanced STAT3 activity such as metabolic disorders and autoimmune diseases.
PMCID: PMC3818494  PMID: 24223854
16.  In vivo action of IL-27: reciprocal regulation of Th17 and Treg cells in collagen-induced arthritis 
Interleukin (IL)-27 is a novel cytokine of the IL-6/IL-12 family that has been reported to be involved in the pathogenesis of autoimmune diseases and has a pivotal role as both a pro- and anti-inflammatory cytokine. We investigated the in vivo effects of IL-27 on arthritis severity in a murine collagen-induced arthritis (CIA) model and its mechanism of action regarding control of regulatory T (Tregs) and IL-17-producing T helper 17 (Th17) cells. IL-27-Fc-treated CIA mice showed a lower severity of arthritis. IL-17 expression in the spleens was significantly decreased in IL-27-Fc-treated CIA mice compared with that in the CIA model. The Th17 population was decreased in the spleens of IL-27-Fc-treated CIA mice, whereas the CD4+CD25+Foxp3+ Treg population increased. In vitro studies revealed that IL-27 inhibited IL-17 production in murine CD4+ T cells, and the effect was associated with retinoic acid-related orphan receptor γT and signal transducer and activator of transcription 3 inhibition. In contrast, fluorescein isothiocyanate-labeled forkhead box P3 (Foxp3) and IL-10 were profoundly augmented by IL-27 treatment. Regarding the suppressive capacity of Treg cells, the proportions of CTLA-4+ (cytotoxic T-lymphocyte antigen 4), PD-1+ (programmed cell death protein 1) and GITR+ (glucocorticoid-induced tumor necrosis factor receptor) Tregs increased in the spleens of IL-27-Fc-treated CIA mice. Furthermore, in vitro differentiated Treg cells with IL-27 exerted a more suppressive capacity on T-cell proliferation. We found that IL-27 acts as a reciprocal regulator of the Th17 and Treg populations in CD4+ cells isolated from healthy human peripheral blood mononuclear cells (PBMCs), as well as from humans with rheumatoid arthritis (RA) PBMCs. Our study suggests that IL-27 has the potential to ameliorate overwhelming inflammation in patients with RA through a reciprocal regulation of Th17 and Treg cells.
PMCID: PMC3809362  PMID: 24091748
collagen-induced arthritis; interleukin-27; interleukin-17-producing T cells; regulatory T cells; rheumatoid arthritis
17.  The Effects of Antihypertensive Drugs on Bone Mineral Density in Ovariectomized Mice 
Journal of Korean Medical Science  2013;28(8):1139-1144.
The effects of several antihypertensive drugs on bone mineral density (BMD) and micro-architectural changes in ovariectomized (OVX) mice were investigated. Eight-week-old female C57/BL6 mice were used for this study. Three days after ovariectomy, mice were treated intraperitoneally with nifedipine (15 mg/kg), telmisartan (5 mg/kg), enalapril (20 mg/kg), propranolol (1 mg/kg) or hydrochlorothiazide (12.5 mg/kg) for 35 consecutive days. Uterine atrophy of all mice was confirmed to evaluate estrogen deficiency state. BMD and micro-architectural analyses were performed on tibial proximal ends by micro-computed tomography (micro-CT). When OVX mice with uterine atrophy were compared with mice without atrophy, BMD decreased (P < 0.001). There were significant differences in BMD loss between different antihypertensive drugs (P = 0.005). Enalapril and propranolol increased BMD loss in mice with atrophied uteri compared with control mice. By contrast, thiazide increased BMD in mice with uterine atrophy compared with vehicle-treated mice (P = 0.048). Thiazide (P = 0.032) and telmisartan (P = 0.051) reduced bone loss and bone fraction in mice with uterine atrophy compared with the control. Thiazide affects BMD in OVX mice positively. The reduction in bone loss by thiazide and telmisartan suggest that these drugs may benefit menopausal women with hypertension and osteoporosis.
PMCID: PMC3744700  PMID: 23960439
Antihypertensive Agents; Osteoporosis; Ovariectomized Mice; Thiazides; Telmisartan
18.  Serum Level of Interleukin-33 and Soluble ST2 and Their Association with Disease Activity in Patients with Behcet's Disease 
Journal of Korean Medical Science  2013;28(8):1145-1153.
Interleukin (IL)-33 is an important mediator of innate immunity. Behcet's disease (BD) is an autoinflammatory disorder characterized by hyperactivity of the innate immune response. We measured serum levels of IL-33 and its receptor soluble ST2 (sST2) in patients with BD to investigate their association with disease activity. Serum levels of both IL-33 and sST2 were higher in patients with BD compared with those in normal controls (IL-33: 594.48±175.04 pg/mL in BD and 224.23±56.64 pg/mL in normal controls [P=0.048], sST2: 99.01±15.92 pg/mL in BD and 23.56±3.25 pg/mL in normal controls [P<0.001]). IL-33 and sST2 expression in skin tissue, as shown by immunohistochemistry, was higher in patients with BD compared with that in the normal controls. Serum sST2 level correlated significantly with the BD currently active form (BDCAF), Iranian BD dynamic activity measure (IBDDAM), erythrocyte sedimentation rate and C-reactive protein. Multiple linear regression showed that serum sST2 was an independent factor associated with IBBDAM (regression coefficient, 0.374; P=0.004), and BDCAF (regression coefficient, 0.236; P=0.047). These results demonstrate that IL-33 and sST2 are highly expressed in patients with BD and that serum sST2 is an independent factor associated with IBDDAM and BDCAF, suggesting a potential role for sST2 as a surrogate marker of disease activity in patients with BD.
PMCID: PMC3744701  PMID: 23960440
Interleukin-33; soluble ST2; Behcet Syndrome
19.  Coenzyme Q10 Ameliorates Pain and Cartilage Degradation in a Rat Model of Osteoarthritis by Regulating Nitric Oxide and Inflammatory Cytokines 
PLoS ONE  2013;8(7):e69362.
To investigate the effect of CoenzymeQ10 (CoQ10) on pain severity and cartilage degeneration in an experimental model of rat osteoarthritis (OA).
Materials and Methods
OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA) to the knee. Oral administration of CoQ10 was initiated on day 4 after MIA injection. Pain severity was assessed by measuring secondary tactile allodynia using the von Frey assessment test. The degree of cartilage degradation was determined by measuring cartilage thickness and the amount of proteoglycan. The mankin scoring system was also used. Expressions of matrix metalloproteinase-13 (MMP-13), interleukin-1β (IL-1β), IL-6, IL-15, inducible nitric oxide synthase (iNOS), nitrotyrosine and receptor for advanced glycation end products (RAGE) were analyzed using immunohistochemistry.
Treatment with CoQ10 demonstrated an antinociceptive effect in the OA animal model. The reduction in secondary tactile allodynia was shown by an increased pain withdrawal latency and pain withdrawal threshold. CoQ10 also attenuated cartilage degeneration in the osteoarthritic joints. MMP-13, IL-1β, IL-6, IL-15, iNOS, nitrotyrosine and RAGE expressions were upregulated in OA joints and significantly reduced with CoQ10 treatment.
CoQ10 exerts a therapeutic effect on OA via pain suppression and cartilage degeneration by inhibiting inflammatory mediators, which play a vital role in OA pathogenesis.
PMCID: PMC3718733  PMID: 23894457
20.  Curcumin Attenuates Acute Graft-versus-Host Disease Severity via In Vivo Regulations on Th1, Th17 and Regulatory T Cells 
PLoS ONE  2013;8(6):e67171.
In this study we examined the in vivo and in vitro effects and mechanisms of action of curcumin on the development of acute graft-versus-host disease (GVHD) using a murine model.
Methodology/Principal Findings
Mixed lymphocyte reactions were used to determine the in vitro effects of curcumin. Treatment with curcumin attenuated alloreactive T cell proliferation and inhibited the production of interferon (IFN)-γ and interleukin (IL)-17. In a murine acute GVHD model, transplantation of curcumin-treated allogeneic splenocytes into irradiated recipient mice significantly reduced the clinical severity scores of acute GVHD manifested in the liver, skin, colon and lung as compared with animals receiving vehicle-treated splenocytes. c-Fos and c-Jun expression levels in the skin and intestine, which are major target organs, were analyzed using immunohistochemical staining. Expression of both proteins was reduced in epithelial tissues of skin and intestine from curcumin-treated GVHD animals. The IFN-γ-expressing CD4+ splenocytes and IFN-γ-expressing lymph node cells were dramatically decreased in curcumin-treated mice. In contrast, CD4+Foxp3+ splenocytes were increased in the curcumin-treated acute GVHD animals. Flow cytometric analysis revealed that animals transplanted with curcumin-treated allogeneic splenocytes showed increased populations of CD4+ regulatory T cells (Tregs) as well as CD8+ Treg cells, compared to animals administered vehicle-treated splenocytes. Curcumin-treated acute GVHD animals could have a change in B cell subpopulations.
In the present study, we investigated the efficacy and mechanism of action of curcumin treatment against acute GVHD. The acute GVHD mice administered with curcumin-treated splenocytes showed significantly reduced severity of acute GVHD. Curcumin exerted in vivo preventive effects on acute GVHD by reciprocal regulation of T helper 1 (Th1) and Treg (both CD4+ and CD8+ Treg) cell lineages as well as B cell homeostasis.
PMCID: PMC3688629  PMID: 23840617
21.  Interferon Gamma Suppresses Collagen-Induced Arthritis by Regulation of Th17 through the Induction of Indoleamine-2,3-Deoxygenase 
PLoS ONE  2013;8(4):e60900.
C57BL/6 mice are known to be resistant to the development of collagen-induced arthritis (CIA). However, they show a severe arthritic phenotype when the Ifng gene is deleted. Although it has been proposed that IFN-γ suppresses inflammation in CIA via suppressing Th17 which is involved in the pathogenesis of CIA, the exact molecular mechanism of the Th17 regulation by IFN-γ is poorly understood. This study was conducted to 1) clarify that arthritogenic condition of IFN-γ knockout (KO) mice is dependent on the disinhibition of Th17 and 2) demonstrate that IFN-γ-induced indoleamine2,3dioxgenase (IDO) is engaged in the regulation of Th17. The results showed that the IFN-γ KO mice displayed increased levels of IL-17 producing T cells and the exacerbation of arthritis. Also, production of IL-17 by the splenocytes of the IFN-γ KO mice was increased when cultured with type II collagen. When Il17 was deleted from the IFN-γ KO mice, only mild arthritis developed without any progression of the arthritis score. The proportion of CD44highCD62Llow memory-like T cells were elevated in the spleen, draining lymph node and mesenteric lymph node of IFN-γ KO CIA mice. Meanwhile, CD44lowCD62Lhigh naïve T cells were increased in IFN-γ and IL-17 double KO CIA mice. When Th17 polarized CD4+ T cells of IFN-γ KO mice were co-cultured with their own antigen presenting cells (APCs), a greater increase in IL-17 production was observed than in co-culture of the cells from wild type mice. In contrast, when APCs from IFN-γ KO mice were pretreated with IFN-γ, there was a significant reduction in IL-17 in the co-culture system. Of note, pretreatment of 1-methyl-DL- tryptophan, a specific inhibitor of IDO, abolished the inhibitory effects of IFN-γ. Given that IFN-γ is a potent inducer of IDO in APCs, these results suggest that IDO is involved in the regulation of IL-17 by IFN-γ.
PMCID: PMC3628800  PMID: 23613752
22.  IL-17-mediated Bcl-2 expression regulates survival of fibroblast-like synoviocytes in rheumatoid arthritis through STAT3 activation 
Fibroblast-like synoviocytes (FLSs) are a major cell population of the pannus that invades adjacent cartilage and bone in rheumatoid arthritis (RA). The study was undertaken to determine the effect of interleukin-17 (IL-17) on the survival and/or proliferation of FLSs from RA patients and to investigate whether signal tranducer and activator of transcription 3 (STAT3) is implicated in this process.
Bcl-2 and Bax expression in FLSs was determined using the real-time PCR and western blot analysis. The expression of Bcl-2 and phosphoSTAT3 in synovial tissues was investigated by confocal microscope. Apoptosis of FLSs was detected by Annexin V/propidium iodide staining and/or phase contrast microscopy. The proliferation of FLSs was determined by CCK-8 ELISA assay.
The pro-apoptotic Bax is decreased and anti-apoptotic Bcl-2 is increased in FLSs from RA patients compared with those from patients with osteoarthritis (OA). IL-17 upregulated the expression of Bcl-2 in FLSs from RA patients, but not in FLSs from OA patients. STAT3 was found to mediate IL-17-induced Bcl-2 upregulation in FLSs from RA patients. Additionally, IL-17 promoted the survival and proliferation of FLSs from RA patients. Most importantly, treatment with STAT3 inhibitor reversed the protective effect of IL-17 on FLSs apoptosis induced by sodium nitroprusside (SNP).
Our data demonstrate that STAT3 is critical in IL-17-induced survival of FLS from RA patients. Therefore, therapeutic strategies that target the IL-17/STAT3 pathway might be strong candidates for RA treatment modalities.
PMCID: PMC3672783  PMID: 23421940
23.  Temporal differential effects of proinflammatory cytokines on osteoclastogenesis 
Bone destruction and inflammation are closely linked. Cytokines play an important role in inflammatory bone destruction by upregulating the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). The direct role of cytokines that act in a non-RANKL-dependent manner has yet to be elucidated. The aim of this study was to investigate the direct osteoclastogenic properties of inflammatory cytokines at different time-points of osteoclastogenesis. Mouse bone marrow macrophages were stimulated with the macrophage colony-stimulating factor (M-CSF) and various concentrations of RANKL. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-17 and IL-23, were added to the culture system of osteoclastogenesis. Two time-points of cytokine treatment were set. The ‘early’ effect of each cytokine was investigated at the time of first RANKL treatment, whereas the ‘late’ effect was investigated 48 h after the first RANKL challenge. Osteoclast differentiation and function were assessed using an osteoclast marker [tartrate-resistant acid phosphatase (TRAP)] and by visualization of pit formation. A permissive level of RANKL was required for cytokine-associated osteoclastogenesis in all experiments. In the M-CSF/RANKL monocellular culture system, IL-1β enhanced and IL-6 decreased osteoclast formation in a dose-dependent manner, regardless of temporal differences. Other cytokines showed various responses according to the phase of osteoclast maturation and the concentration of each cytokine and RANKL. Furthermore, luciferase assays showed that both IL-1β and RANKL activated the NF-κB signaling pathway. Collectively, our data revealed that targeting IL-1β may be a promising strategy to inhibit inflammation-associated bone destruction and osteoporosis.
PMCID: PMC3621814  PMID: 23403591
inflammation; osteoclast; receptor activator nuclear factor-κB ligand; interleukin-1β; interleukin-6; nuclear factor-κB
24.  Periarticular Osteoporosis Is a Prominent Feature in Early Rheumatoid Arthritis: Estimation Using Shaft to Periarticular Bone Mineral Density Ratio 
Journal of Korean Medical Science  2013;28(2):287-294.
We aimed to quantify periarticular osteoporosis and investigate its significance in 45 patients with rheumatoid arthritis (RA) and 106 controls. Dual-energy X-ray absorptiometry (DXA) was used to determine the ratio of shaft to periarticular bone mineral density (BMD) as an index of periarticular demineralization. Periarticular osteoporosis was measured by conventional radiography. The BMDs of shaft and periarticular regions in eight designated areas on proximal phalanges were quantified. Clinical variables were examined to identify risk factors for periarticular osteoporosis. The assessment of periarticular osteoporosis on X-ray images reached a moderate degree of interobserver agreement among four physicians (ĸ = 0.47). For BMD quantification, we designed three types of mathematical formulae: the ratio of shaft to periarticular BMD, the mean of the ratios, and the ratio of the sums. These ratios were significantly higher in the patients with early RA (disease duration ≤ 3 yr) than in controls (P < 0.01). The findings were not as distinctive in patients with established RA. Body mass index, cumulative dose of corticosteroid, and C-terminal telopeptide were correlated with BMD ratios. Conclusively, DXA-assisted localized quantification and BMD ratio calculations are feasible for assessing periarticular demineralization. Periarticular osteoporosis is a relatively distinctive feature of early RA.
PMCID: PMC3565142  PMID: 23399828
Bone Density; Arthrits; Rheumatoid; Periarticular Osteopenia
25.  Grape-Seed Proanthocyanidin Extract as Suppressors of Bone Destruction in Inflammatory Autoimmune Arthritis 
PLoS ONE  2012;7(12):e51377.
Chronic autoimmune inflammation, which is commonly observed in rheumatoid arthritis (RA), disrupts the delicate balance between bone resorption and formation causing thedestruction of the bone and joints. We undertook this study to verify the effects of natural grape-seed proanthocyanidin extract (GSPE), an antioxidant, on chronic inflammation and bone destruction. GSPE administration ameliorated the arthritic symptoms of collagen-induced arthritis (CIA), which are representative of cartilage and bone destruction. GSPE treatment reduced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and osteoclast activity and increased differentiation of mature osteoblasts. Receptor activator of NFκB ligand expression in fibroblasts from RA patients was abrogated with GSPE treatment. GSPE blocked human peripheral blood mononuclear cell-derived osteoclastogenesis and acted as an antioxidant. GSPE improved the arthritic manifestations of CIA mice by simultaneously suppressing osteoclast differentiation and promoting osteoblast differentiation. Our results suggest that GSPE may be beneficial for the treatment of inflammation-associated bone destruction.
PMCID: PMC3519627  PMID: 23251512

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