Inflammatory Cytokines in Psoriatic Arthritis: Understanding Pathogenesis and Implications for Treatment.

Psoriatic arthritis (PsA) is a persistent, inflammatory disease that affects individuals with psoriasis, arthritis, and enthesitis. Research has demonstrated that inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-23 (IL-23), and interleukin-17 (IL-17) play a pivotal role in both the onset and progression of PsA. These cytokines are generated by activated immune cells and stimulate the attraction of inflammatory cells to the synovium and joint tissues, resulting in the deterioration of cartilage and bone. The blocking of these cytokines has become a successful treatment strategy for PsA, as biological drugs that inhibit TNF-α, IL-23, and IL-17 have demonstrated notable clinical benefits. The association between PsA and other types of inflammatory cytokines or chemokines, excluding TNF-α, IL-23, and IL-17, has been extensively investigated in numerous studies. These findings may provide a chance for the discovery of novel therapeutic agents targeting other molecules, distinct from the currently approved biologics and targeted synthetic disease-modifying anti-rheumatic drugs. In this review, we discuss the current understanding of the role of inflammatory cytokines in PsA pathogenesis and clinical implications of targeting these cytokines for PsA treatment.

Prevalence, incidence, and risk factors of malignancy in patients with rheumatoid arthritis: a nationwide cohort study from Korea.

BACKGROUND/AIMS: This study aims to evaluate the incidence of malignancy in patients with rheumatoid arthritis (RA) and to investigate risk factors for such in a nationwide, population-based cohort. METHODS: In a large, prospective, observational cohort study, 5,077 patients with RA were enrolled from July 2009 to December 2011 and followed until February 2017. Standardized incidence ratios (SIRs) for malignancy were calculated using age- and sex-specific cancer rates in the Korean general population. Poisson regression was used to identify the risk of incident malignancy. RESULTS: The cohort included 5,023 participants with RA contributing 16,689 person-years of follow-up. A total of 148 malignancies were recorded. The risks of stomach cancer (SIR, 0.41; 95% confidence interval [CI], 0.21 to 0.74), colon cancer (SIR, 0.13; 95% CI, 0.03 to 0.37), and lung cancer (SIR, 0.35; 95% CI, 0.14 to 0.72) were lower in RA patients than in the general population. Poisson regression modeling demonstrated that the malignancy risk was more than two-fold greater in patients with thyroid disease than in those without thyroid disease. Hydroxychloroquine therapy was associated with a reduced risk (relative risk, 0.39; 95% CI, 0.189 to 0.801) of malignancy development. CONCLUSION: The overall risk of malignancy in patients with RA is decreased relative to in the general population. In particular, stomach, colon, and lung cancers in Korean RA patients are less common, while brain and central nervous system cancers in male RA patients are more frequent. The patients with thyroid disease and longer RA disease duration were at increased risk for developing malignancy, while hydroxychloroquine users were at lower risk.

The role of retinoid-related orphan receptor-α in cigarette smoke-induced autophagic response.

BACKGROUND: Retinoid-related orphan receptor-α (RORα) and autophagy dysregulation are involved in the pathophysiology of chronic obstructive pulmonary disease (COPD), but little is known regarding their association. We investigated the role of RORα in COPD-related autophagy. METHODS: The lung tissues and cells from a mouse model were analyzed for autophagy markers by using western blot analysis and transmission electron microscopy. RESULTS: Cigarette smoke increased the LC3-II level and decreased the p62 level in whole lung homogenates of a chronic cigarette smoking mouse model. Although cigarette smoke did not affect the levels of p62 in Staggerer mutant mice (RORαsg/sg), the baseline expression levels of p62 were significantly higher than those in wild type (WT) mice. Autophagy was induced by cigarette smoke extract (CSE) in Beas-2B cells and in primary fibroblasts from WT mice. In contrast, fibroblasts from RORαsg/sg mice failed to show CSE-induced autophagy and exhibited fewer autophagosomes, lower LC3-II levels, and higher p62 levels than fibroblasts from WT mice. Damage-regulated autophagy modulator (DRAM), a p53-induced modulator of autophagy, was expressed at significantly lower levels in the fibroblasts from RORαsg/sg mice than in those from WT mice. DRAM knockdown using siRNA in Beas-2B cells inhibited CSE-induced autophagy and cell death. Furthermore, RORα co-immunoprecipitated with p53 and the interaction increased p53 reporter gene activity. CONCLUSIONS: Our findings suggest that RORα promotes autophagy and contributes to COPD pathogenesis via regulation of the RORα-p53-DRAM pathway.

Chronic kidney disease in Korean patients with lupus nephritis: over a 35-year period at a single center.

INTRODUCTION: Chronic kidney disease (CKD) is a major risk factor for overall morbidity and mortality even in lupus nephritis (LN) patients. However, less attention has been paid to the development of CKD in patients with LN. The objective of this study was to identify predictors for CKD with 35-year experience depending on newly revised guidelines for patients with LN. METHODS: We conducted a retrospective cohort study for 401 patients who visited Seoul St. Mary's Hospital between January 1985 and December 2019. We analyzed clinical and laboratory indices, treatment response, the final renal function, and biopsy findings. The timing and cumulative risk of developing CKD were identified by Kaplan-Meier methods. Independent risk factors for developing CKD were examined by Cox proportional hazard regression analyses. RESULTS: The median follow-up time after the diagnosis of LN was 131 months. CKD occurred in 15.5% of patients within 10 years after the diagnosis of LN. The development of CKD was associated with delayed-onset LN, acute renal dysfunction at onset of LN, and failure to reach complete response (CR) at 6 or 12 months rather than histopathological findings or the severity of proteinuria at onset of LN. Cumulative incidence of progression to CKD was significantly higher in patients with the three predictors mentioned above. CONCLUSION: Ten-year cumulative incidence of CKD was about 15%. Our results showed that delayed-onset LN, acute renal dysfunction at the onset of LN, and inadequate treatment response assessed at 6 or 12 months after treatment were predictors for the development of CKD in LN. Key Points • CKD is a major risk factor for overall morbidity and mortality in LN patients. • Ten-year cumulative incidence of CKD was about 15% • Delayed-onset LN, acute renal dysfunction at the onset of LN, and inadequate treatment response assessed at 6 or 12 months after treatment were predictors for the development of CKD in LN.

The arachidonic acid metabolite 11,12-epoxyeicosatrienoic acid alleviates pulmonary fibrosis.

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid that are rapidly metabolized into diols by soluble epoxide hydrolase (sEH). sEH inhibition has been shown to increase the biological activity of EETs, which are known to have anti-inflammatory properties. However, the role of EETs in pulmonary fibrosis remains unexplored. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to analyze EETs in the lung tissues of patients with idiopathic pulmonary fibrosis (IPF, n = 29) and controls (n = 15), and the function of 11,12-EET was evaluated in in vitro and in vivo in pulmonary fibrosis models. EET levels in IPF lung tissues, including those of 8,9-EET, 11,12-EET, and 14,15-EET, were significantly lower than those in control tissues. The 11,12-EET/11,12-DHET ratio in human lung tissues also differentiated IPF from control tissues. 11,12-EET significantly decreased transforming growth factor (TGF)-ß1-induced expression of α-smooth muscle actin (SMA) and collagen type-I in MRC-5 cells and primary fibroblasts from IPF patients. sEH-specific siRNA and 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU; sEH inhibitor) also decreased TGF-ß1-induced expression of α-SMA and collagen type-I in fibroblasts. Moreover, 11,12-EET and TPPU decreased TGF-ß1-induced p-Smad2/3 and extracellular-signal-regulated kinase (ERK) expression in primary fibroblasts from patients with IPF and fibronectin expression in Beas-2B cells. TPPU decreased the levels of hydroxyproline in the lungs of bleomycin-induced mice. 11,12-EET or sEH inhibitors could inhibit pulmonary fibrosis by regulating TGF-ß1-induced profibrotic signaling, suggesting that 11,12-EET and the regulation of EETs could serve as potential therapeutic targets for IPF treatment.

The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of both B cell differentiation and the production of inflammatory cytokines.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an important transcription factor that plays a pivotal role in cellular defense against oxidative injury. Nrf2 signaling is involved in attenuating autoimmune disorders such as rheumatoid arthritis (RA). B cells play several roles in the pathogenesis of RA, such as in autoantibody production, antigen presentation, and T-cell activation. We investigated the anti-arthritic mechanisms of sulforaphane, an activator of Nrf2, in terms of its effect on B cells. To investigate the effect of sulforaphane on collagen-induced arthritis (CIA), sulforaphane was administered intraperitoneally after CIA induction. Hematoxylin and eosin-stained sections were scored for inflammation, pannus invasion, and bone and cartilage damage. We assessed the expression levels of inflammation-related factors by real-time PCR and the levels of various IgG subclasses by enzyme-linked immunosorbent assay. Sulforaphane treatment reduced the arthritis score and the severity of histologic inflammation in CIA mice. The joints from sulforaphane-treated CIA mice showed decreased expression of interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α, receptor activator of NF-κB ligand, and tartrate-resistant acid phosphatase. Sulforaphane-treated mice showed lower circulating levels of type-II-collagen-specific IgG, IgG1, and IgG2a. In vitro, sulforaphane treatment significantly reduced the differentiation of lipopolysaccharide-stimulated murine splenocytes into plasma B cells and germinal-center B cells. Finally, sulforaphane significantly inhibited the production of IL-6, TNF-α, and IL-17 by human peripheral blood mononuclear cells stimulated with an anti-CD3 monoclonal antibody in a dose-dependent manner. Inhibition of differentiation into plasma B and Germinal Center B cells may be the mechanism underlying the anti-arthritic effect of sulforaphane.

Network-based modeling of drug effects on disease module in systemic sclerosis.

The network-based proximity between drug targets and disease genes can provide novel insights regarding the repercussions, interplay, and repositioning of drugs in the context of disease. Current understanding and treatment for reversing of the fibrotic process is limited in systemic sclerosis (SSc). We have developed a network-based analysis for drug effects that takes into account the human interactome network, proximity measures between drug targets and disease-associated genes, genome-wide gene expression and disease modules that emerge through pertinent analysis. Currently used and potential drugs showed a wide variation in proximity to SSc-associated genes and distinctive proximity to the SSc-relevant pathways, depending on their class and targets. Tyrosine kinase inhibitors (TyKIs) approach disease gene through multiple pathways, including both inflammatory and fibrosing processes. The SSc disease module includes the emerging molecular targets and is in better accord with the current knowledge of the pathophysiology of the disease. In the disease-module network, the greatest perturbing activity was shown by nintedanib, followed by imatinib, dasatinib, and acetylcysteine. Suppression of the SSc-relevant pathways and alleviation of the skin fibrosis was remarkable in the inflammatory subsets of the SSc patients receiving TyKI therapy. Our results show that network-based drug-disease proximity offers a novel perspective into a drug's therapeutic effect in the SSc disease module. This could be applied to drug combinations or drug repositioning, and be helpful guiding clinical trial design and subgroup analysis.

The Practice Guideline for Vaccinating Korean Patients with Autoimmune Inflammatory Rheumatic Disease.

To develop a clinical practice guideline for vaccination in patients with autoimmune inflammatory rheumatic disease (AIIRD), the Korean College of Rheumatology and the Korean Society of Infectious Diseases developed a clinical practice guideline according to the clinical practice guideline development manual. Since vaccination is unlikely to cause AIIRD or worsen disease activities, required vaccinations are recommended. Once patients are diagnosed with AIIRD, treatment strategies should be established and, at the same time, monitor their vaccination history. It is recommended to administer vaccines when the disease enters the stabilized stage. Administering live attenuated vaccines in patients with AIIRD who are taking immunosuppressants should be avoided. Vaccination should be considered in patients with AIIRD, prior to initiating immunosuppressants. It is recommended to administer influenza, Streptococcus pneumoniae, hepatitis A, hepatitis B, herpes zoster, measles-mumps-rubella virus, human papillomavirus, and tetanus-diphtheria-pertussis vaccines in patients with AIIRD; such patients who planned to travel are generally recommended to be vaccinated at the recommended vaccine level of healthy adults. Those who live in a household with patients with AIIRD and their caregivers should also be vaccinated at levels that are generally recommended for healthy adults.

Direct Binding to NLRP3 Pyrin Domain as a Novel Strategy to Prevent NLRP3-Driven Inflammation and Gouty Arthritis.

OBJECTIVE: The NLRP3 inflammasome is closely linked to the pathophysiology of a wide range of inflammatory diseases. This study was undertaken to identify small molecules that directly bind to NLRP3 in order to develop pharmacologic interventions for NLRP3-related diseases. METHODS: A structure-based virtual screening analysis was performed with ~62,800 compounds to select efficient NLRP3 inhibitors. The production of caspase 1-p10 and interleukin-1ß (IL-1ß) was measured by immunoblotting and enzyme-linked immunosorbent assay to examine NLRP3 inflammasome activation. Two gouty arthritis models and an air pouch inflammation model induced by monosodium urate monohydrate (MSU) crystal injection were used for in vivo experiments. Primary synovial fluid cells from gout patients were used to determine the relevance of NLRP3 inflammasome inhibition in human gout. RESULTS: Beta-carotene (provitamin A) suppressed the NLRP3 inflammasome activation induced by various activators, including MSU crystals, in mouse bone marrow-derived primary macrophages (P < 0.05). Surface plasmon resonance analysis demonstrated the direct binding of ß-carotene to the pyrin domain (PYD) of NLRP3 (KD = 3.41 × 10-6 ). Molecular modeling and mutation assays revealed the interaction mode between ß-carotene and the NLRP3 PYD. Inflammatory symptoms induced by MSU crystals were attenuated by oral administration of ß-carotene in gouty arthritis mouse models (P < 0.05), correlating with its suppressive effects on the NLRP3 inflammasome in inflamed tissues. Furthermore, ß-carotene reduced IL-1ß secretion from human synovial fluid cells isolated from gout patients (P < 0.05), showing its inhibitory efficacy in human gout. CONCLUSION: Our results present ß-carotene as a selective and direct inhibitor of NLRP3, and the binding of ß-carotene to NLRP3 PYD as a novel pharmacologic strategy to combat NLRP3 inflammasome-driven diseases, including gouty arthritis.

Daurinol Attenuates Autoimmune Arthritis via Stabilization of Nrp1-PTEN-Foxp3 Signaling in Regulatory T Cells.

Optimizing Treg function and improving Treg stability are attractive treatment strategies for treating autoimmune rheumatoid arthritis (RA). However, the limited number of circulating Tregs and questions about the functional stability of in vitro-expanded Tregs are potential limitations of Treg-based cell therapy. The aim of this study was to analyze the regulatory effect of daurinol, a catalytic inhibitor of topoisomerase IIα, on Th cell differentiation and to evaluate their therapeutic potential in a preclinical experimental model of RA. We investigated the effect of daurinol on T cell differentiation by flow cytometry. Foxp3 stability and methylation were analyzed by suppression assays and bisulfite pyrosequencing. Daurinol was treated in the collagen-induced arthritis (CIA) model, and the effects in vivo were determined. We found that daurinol can promote Treg differentiation and reciprocally inhibit Th17 differentiation. This Treg-inducing property of daurinol was associated with decreased activity of Akt-mTOR and reciprocally increased activity of neuropilin-1 (Nrp1)-PTEN. Daurinol treatment inhibited aerobic glycolysis in Th17 conditions, indicating the metabolic changes by daurinol. We found that the daurinol increase the Treg stability was achieved by Foxp3 hypomethylation. In vivo daurinol treatment in CIA mice reduced the clinical arthritis severity and histological inflammation. The Treg population frequency increased and the Th17 cells decreased in the spleens of arthritis mice treated with daurinol. These results showed the anti-arthritic and immunoregulating properties of daurinol is achieved by increased differentiation and stabilization of Tregs. Our study provides first evidence for daurinol as a treatment for RA.

Metformin Augments Anti-Inflammatory and Chondroprotective Properties of Mesenchymal Stem Cells in Experimental Osteoarthritis.

Mesenchymal stem cells (MSCs) can protect against cartilage breakdown in osteoarthritis (OA) via their immunomodulatory capacities. However, the optimization strategy for using MSCs remains challenging. This study's objective was to identify the in vivo effects of metformin-stimulated adipose tissue-derived human MSCs (Ad-hMSCs) in OA. An animal model of OA was established by intra-articular injection of monosodium iodoacetate into rats. OA rats were divided into a control group and two therapy groups (treated with Ad-hMSCs or metformin-stimulated Ad-hMSCs). Limb nociception was assessed by measuring the paw withdrawal latency and threshold. Our data show that metformin increased IL-10 and IDO expression in Ad-hMSCs and decreased high-mobility group box 1 protein, IL-1ß, and IL-6 expression. Metformin increased the migration capacity of Ad-hMSCs with upregulation of chemokine expression. In cocultures, metformin-stimulated Ad-hMSCs inhibited the mRNA expression of RUNX2, COL X, VEGF, MMP1, MMP3, and MMP13 in IL-1ß-stimulated OA chondrocytes and increased the expression of TIMP1 and TIMP3. The antinociceptive activity and chondroprotective effects were greater in OA rats treated with metformin-stimulated Ad-hMSCs than in those treated with unstimulated Ad-hMSCs. TGF-ß expression in subchondral bone of OA joints was attenuated more in OA rats treated with metformin-stimulated Ad-hMSCs. Our findings suggest that metformin offers a promising option for the clinical application of Ad-hMSCs as a cell therapy for OA.

Notoginseng Radix and Rehmanniae Radix Preparata Extract Combination (YH23537) Reduces Pain and Cartilage Degeneration in Rats with Monosodium Iodoacetate-Induced Osteoarthritis.

Notoginseng Radix and Rehmanniae Radix Preparata have been widely used traditionally for treating inflammatory diseases. This research studies the therapeutic effects of YH23537, the extracts of Notoginseng Radix and Rehmanniae Radix Preparata, on pain and cartilage degeneration in an experimental osteoarthritis (OA) model. Male Wistar rats were inoculated intra-articularly with 3 mg of monosodium iodoacetate (MIA) in the right intra-articular. Four days later, the animals were administrated orally with YH23537 daily for 24 days. Tactile allodynia and weight bearing were measured. Macroscopic and microscopic observations for articular cartilage were performed at the end of the experiment. Protein expression in the joint was determined by immunohistochemistry. The effects of YH23537 on mRNA levels in chondrocytes stimulated with interleukin (IL)-1ß were analyzed using random polymerase chain reaction. OA induction was confirmed by significant decrease of paw withdrawal latency, paw withdrawal threshold, and weight bearing compared with the normal group at 3 days after MIA injection. The YH23537-treated groups displayed significant increases in pain thresholds and weight bearing throughout the observation period. The damage to articular cartilage was significantly lessened visually and histopathologically by YH23537 treatment. YH23537 suppressed the expression of metalloproteinase-3, nitrotyrosine, IL-1ß and IL-6 increased in OA joints. YH23537 upregulated tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-3 in IL-1ß-stimulated human OA chondrocytes. The protein levels of the NF-κBp65 and HIF-2α in the joint tissues were reduced by YH23537. YH23537 exerted antinociceptive effects and cartilage protective effects in experimental OA rats by suppressing oxidative injury, inflammatory mediators, and inducing anabolic factors. We suggest that YH23537 may have efficacy for treating OA in humans.

Differences in quality of life determinants according to the presence of fibromyalgia in middle-aged female patients with systemic lupus erythematosus: a multicenter, cross-sectional, single-ethnicity cohort.

OBJECTIVE: The purpose of this study was to identify whether determinants of health-related quality of life (HRQoL) in middle-aged female patients with systemic lupus erythematosus (SLE) differed according to the presence or absence of fibromyalgia. METHODS: One hundred and fifty-two patients with SLE and 139 healthy controls (HCs) completed the Medical Outcomes Study 36-Item Short Form (SF-36) and EuroQol EQ-5D questionnaires about HRQoL. Disease activity and cumulative disease damage were assessed with standard indices. Sleep quality was assessed using the Korean version of the Pittsburgh Sleep Quality Index (K-PSQI). RESULT: The mean EQ-5D and physical and mental components of SF-36 were lower in SLE patients with fibromyalgia (n = 41) than in those without fibromyalgia (n = 111) and HCs. The scores in all eight domains of the SF-36 were lower in SLE patients with fibromyalgia than in patients without fibromyalgia and HCs. Poor sleep (defined as a K-PSQI > 5) was reported by 85% of SLE patients with fibromyalgia, by 51% of patients without fibromyalgia, and by 33% of HCs. Multivariate logistic regression analysis showed that lower educational level, cumulative organ damage severity and poor sleep quality were independent determinants of HRQoL in SLE patients with fibromyalgia, whereas disease activity, sleep quality and depressive mood were independent determinants of HRQoL in those without fibromyalgia. CONCLUSION: Poor sleep quality is the common independent risk factor for poor HRQoL in both middle-aged SLE patients with fibromyalgia and without fibromyalgia. Sleep quality improvement may improve HRQoL in female SLE patients, even in those without fibromyalgia.

Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the synovial joints. Although involvement of the fibroblast growth factor (FGF) signaling pathway has been suggested as an important modulator in RA development, no clear evidence has been provided. In this study, we found that synovial fluid basic FGF (bFGF) concentration was significantly higher in RA than in osteoarthritis (OA) patients. bFGF stimulates proliferation and migration of human fibroblast-like synoviocytes (FLSs) by activation of the bFGF-FGF receptor 3 (FGFR3)-ribosomal S6 kinase 2 (RSK2) signaling axis. Moreover, a molecular docking study revealed that kaempferol inhibited FGFR3 activity by binding to the active pocket of the FGFR3 kinase domain. Kaempferol forms hydrogen bonds with the FGFR3 backbone oxygen of Glu555 and Ala558 and the side chain of Lys508. Notably, the inhibition of bFGF-FGFR3-RSK2 signaling by kaempferol suppresses the proliferation and migration of RA FLSs and the release of activated T-cell-mediated inflammatory cytokines, such as IL-17, IL-21, and TNF-α. We further found that activated phospho-FGFR3 and -RSK2 were more highly observed in RA than in OA synovium. The hyperplastic lining and sublining lymphoid aggregate layers of RA synovium showed p-RSK2-expressing CD68+ macrophages with high frequency, while pRSK2-expressing CD4+ T-cells was observed at a lower frequency. Notably, kaempferol administration in collagen-induced arthritis mice relieved the frequency and severity of arthritis. Kaempferol reduced osteoclast differentiation in vitro and in vivo relative to the controls and was associated with the inhibition of osteoclast markers, such as tartrate-resistant acid phosphatase, integrin ß3, and MMP9. Conclusively, our data suggest that bFGF-induced FGFR3-RSK2 signaling may play a critical role during the initiation and progression of RA in terms of FLS proliferation and enhanced osteoclastogenesis, and that kaempferol may be effective as a new treatment for RA.

Therapeutic Effect of Exogenous Truncated IK Protein in Inflammatory Arthritis.

Inhibitor K562 (IK) protein was first isolated from the culture medium of K562, a leukemia cell line. It is known to be an inhibitory regulator of interferon-γ-induced major histocompatibility complex class (MHC) II expression. Previously, we found that transgenic (Tg) mice constitutively expressing truncated IK (tIK) showed reduced numbers of pathogenic Th1 and Th17 cells, which are known to be involved in the development of rheumatoid arthritis (RA). Here, we investigated whether exogenous tIK protein has a therapeutic effect in arthritis in disease models and analyzed its mechanism. Exogenous tIK protein was produced in an insect expression system and applied to the collagen antibody-induced arthritis (CAIA) mouse disease model. Injection of tIK protein alleviated the symptoms of arthritis in the CAIA model and reduced Th1 and Th17 cell populations. In addition, treatment of cultured T cells with tIK protein induced expression of A20, a negative regulator of nuclear factor-κB (NFκB)-induced inflammation, and reduced expression of several transcription factors related to T cell activation. We conclude that exogenous tIK protein has the potential to act as a new therapeutic agent for RA patients, because it has a different mode of action to biopharmaceutical agents, such as tumor necrosis factor antagonists, that are currently used to treat RA.

The active metabolite of leflunomide, A77 1726, attenuates inflammatory arthritis in mice with spontaneous arthritis via induction of heme oxygenase-1.

BACKGROUND: Leflunomide is a low-molecular-weight compound that is widely used in the treatment of rheumatoid arthritis. Although leflunomide is thought to act through the inhibition of the de novo pyrimidine synthesis, the molecular mechanism of the drug remains largely unknown. We investigated the antiarthritis effects and mechanisms of action of the active metabolite of leflunomide, A77 1726, in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice. METHODS: 14- to 15-week-old male IL-1Ra-KO mice were treated with 10 or 30 mg/kg A77 1726 via intraperitoneal injection three times per week for 6 weeks. The effects of A77 1726 on arthritis severities were assessed by clinical scoring and histological analysis. The serum concentrations of IL-1ß, tumor necrosis factor-α (TNF-α), and malondialdehyde were measured by enzyme-linked immunosorbent assay. Histologic analysis of the joints was performed using Safranin O, and immunohistochemical staining. The frequencies of interleukin-17-producing CD4+ T (Th17) cells were analyzed by flow cytometry. Heme oxygenase-1 (HO-1) expression in splenic CD4+ T cells isolated from A77 1726-treated arthritis mice were assessed by western blotting. RESULTS: A77 1726 treatment induced heme oxygenase-1 (HO-1) in Jurkat cells and primary mouse T cells. Interestingly, A77 1726 inhibited Th17 cell differentiation. In vivo, A77 1726 reduced the clinical arthritis severity of histological inflammation and cartilage destruction. The joints isolated from A77 1726-treated mice showed decreased expression of inducible nitric oxide synthase, nitrotyrosine, TNF-α, and IL-1ß. The serum levels of TNF-α, IL-1ß, and malondialdehyde were also decreased in A77 1726-treated mice. Whereas the number of Th17 cells in spleens was decreased in A77 1726-treated arthritis mice, a significant increase in the number of Treg cells in spleens was observed. Interestingly, HO-1 expression was significantly higher in splenic CD4+ T cells isolated from A77 1726-treated mice compared with those from vehicle-treated mice, whereas HO-1 expression of splenic non-CD4+ T cells did not differ between groups. CONCLUSION: The inhibitory effects of A77 1726 on joint inflammation and oxidative stress in autoimmune arthritis may be associated with HO-1 induction in CD4+ T cells.

IL-17-mediated mitochondrial dysfunction impairs apoptosis in rheumatoid arthritis synovial fibroblasts through activation of autophagy.

Fibroblast-like synoviocytes (FLSs) are a major cell population of the pannus that invades cartilage and bone in rheumatoid arthritis (RA). FLS resistance to apoptosis is a major characteristic of RA. The aims of this study were to investigate the effects of interleukin-17 (IL-17) and IL-17-producing T helper (Th17) cells on resistance to apoptosis in FLSs from RA patients (RA FLSs) and their roles in mitochondrial dysfunction and autophagy. Mitochondrial function was assessed in RA FLSs and FLSs from osteoarthritis patients (OA FLSs). FLSs were treated with IL-17 and their morphological features, respiratory level and mitochondrial gene expression were measured. The effects of IL-17 and Th17 cells on the relationship between autophagy and apoptosis were evaluated by measuring the expression of apoptosis-related genes using sodium nitroprusside or 3-methyladenine. The mitochondria of FLSs isolated from RA and osteoarthritis patients displayed different morphological and physiological features. RA FLSs exhibited greater autophagosome formation and greater dysfunction of mitochondrial respiration compared with OA FLSs. IL-17 induced mitochondrial dysfunction and autophagosome formation in RA FLSs, suggesting that they were resistant to apoptosis. Autophagy-related antiapoptosis induced by IL-17 was restored by inhibition of autophagy, suggesting a relationship between mitochondrial dysfunction and cell survival in RA FLSs. Th17 cells and IL-17 increased autophagy of RA FLSs by causing mitochondrial dysfunction. Our findings suggest that, in RA, interactions between RA FLSs and Th17 cells may be involved in the tumorous growth of FLSs and the formation of pannus in joints.

IK acts as an immunoregulator of inflammatory arthritis by suppressing TH17 cell differentiation and macrophage activation.

Pathogenic T helper cells (TH) and macrophages have been implicated in the development of rheumatoid arthritis (RA), which can lead to severe synovial inflammation and bone destruction. A range of therapies have been widely used for RA, including specific monoclonal antibodies and chemical inhibitors against inflammatory cytokines produced by these cells. However, these have not been sufficient to meet the medical need. Here, we show that in transgenic mice expressing truncated IK (tIK) cytokine, inflammatory arthritis symptoms were ameliorated as the result of suppression of the differentiation of TH1 and TH17 cells and of macrophage activation. During inflammatory responses, tIK cytokine systemically regulated macrophage functions and TH17 cell differentiation through inactivation of the MAPK and NF-κB pathways. Interestingly, the level of tIK cytokine was higher in synovial fluid of RA patients compared with that in osteoarthritis (OA) patients. Our observations suggest that tIK cytokine can counterbalance the induction of inflammatory cells related to RA and thus could be a new therapeutic agent for the treatment of RA.

Overexpression of soluble RAGE in mesenchymal stem cells enhances their immunoregulatory potential for cellular therapy in autoimmune arthritis.

Mesenchymal stem cells (MSCs) are attractive agents for cellular therapy in rheumatoid arthritis (RA). The receptor for advanced glycation end products (RAGE) serves as a pattern recognition receptor for endogenous inflammatory ligands. Soluble RAGE (sRAGE) is a truncated form of RAGE that functions as a decoy and acts as an anti-inflammatory molecule. The aim of this study was to determine whether sRAGE has therapeutic effects and the mechanisms active in sRAGE-overexpressing MSCs (sRAGE-MSCs) in an experimental model of RA. sRAGE-MSCs were generated by DNA transfection of human adipose tissue-derived MSCs (Ad-hMSCs). MSCs showed increased expression of VEGF, IL-1ß, IL-6, and HMGB-1 under inflammatory conditions. However, sRAGE-MSCs showed significantly lower production of these proinflammatory molecules. Expression of immunomodulatory molecules such as IL-10, TGF-ß, and indoleamine 2, 3-dioxygenase was higher in sRAGE-MSCs than in mock-MSCs. sRAGE-MSCs showed enhanced migration potential. Transplantation of sRAGE-MSCs into arthritic IL-1Ra-knockout mice markedly suppressed inflammatory arthritis, decreased Th17 cells, and reciprocally increased regulatory T cells. The differentiation of IFN-γ+CD4+ and IL-17+CD4+ cells was inhibited by incubation with sRAGE-MSCs compared with mock-MSCs. These findings suggest that sRAGE overexpression in Ad-hMSCs optimizes their immunoregulatory properties, which may be useful as a novel cellular therapy for RA.

Simultaneous determination of plant growth regulator and pesticides in bean sprouts by liquid chromatography-tandem mass spectrometry.

A simple and sensitive analytical method based on QuEChERS approach using liquid chromatography tandem mass spectrometry was developed and validated for the determination of 6-benzylaminopurine, carbendazim and thiabendazole in bean sprouts. Sodium chloride and sodium acetate were used for salting-out step and magnesium sulfate for clean-up. The validation of optimized method was satisfactory with recoveries, between 89.5% and 103.2% for the three compounds, and relative standard deviation (RSD) values less than 3.3% at 20 and 40ng/g fortification levels (n=5). Limit of detection (LOD) and limit of quantification (LOQ) was 2.1-3.7ng/g and 6.3-11.1ng/g, respectively. Monitoring of 126 bean sprout samples collected from local markets was performed to verify the adaptability in real samples. No pesticides were detected but 6-benzylaminopurine was found in 3 samples at the level of 15-20ng/g. The optimized method should be applicable for monitoring of 6-benzylaminopurine, carbendazim and thiabendazole in bean sprouts in short time.