Search

Background:Chronic rheumatological manifestations similar to those of rheumatoid arthritis (RA) have been described after chikungunya virus infection. However, the clinical significance of the symptoms and disease severity in the two conditions has not been directly compared.Objectives:To compare, using identical measures of disease severity and patient outcomes, the impact of disease severity measures and symptoms on outcomes in RA and chronic chikungunya disease.Methods:Forty patients with chronic chikungunya arthralgia two years post-infection and 40 matched patients with RA were enrolled in Roraima, Brazil. Twenty-eight joints were assessed for tenderness and swelling, a pain intensity visual analogue scale, musculoskeletal stiffness questionnaire, modified Health Assessment Questionnaire and the EuroQol EQ5D-5L quality of life assessment were completed. The importance of the various measures of disease severity were analysed using Spearman’s rank correlation and regression analysis.Results:Tender and swollen joint counts, pain and stiffness were all predictive of the HAQ disability index in RA, but only stiffness was significantly associated with disability in chikungunya patients (Table 1). Tender and swollen joint counts, pain and stiffness were predictive for all EQ5D quality of life domains (except anxiety/depression) in RA patients. In contrast, in chikungunya disease, tender joint counts were predictive only of usual daily activities; pain was predictive of impaired mobility, self-care and discomfort, while stiffness was predictive for the mobility and anxiety/depression domains (Figure 1). Swollen joint counts were not associated with any of the patient outcomes in chikungunya disease. Linear regression analysis confirmed (p=0.003) that the effect of swollen joint count on the HAQ disability index depends on the underlying disease.Table 1.Association of disease severity with HAQ disability index in rheumatoid and CHIKV+ arthritisSeverity measureRheumatoid arthritisCHIKV+ arthritisr (p)r (p)Tender joint count0.56 (0.0002)0.24 (0.14)Swollen joint count0.60 (0.002 (0.99)Joint pain (VAS)0.55 (0.0002)0.29 (0.07)Stiffness severity0.57 (0.0001)0.38 (0.02)Figure 1.Association of disease severity with quality of life domains in rheumatoid and CHIKV+ arthritisConclusion:The value of all the disease severity measures tested in RA were confirmed, but tender joint counts may have more limited value in the assessment of chronic chikungunya disease. Joint swelling appears to have little impact for chikungunya patients, while stiffness appears to be an important metric to quantify chikungunya arthritis disease severity.Disclosure of Interests:Hugh Watson Shareholder of: Sanofi, Employee of: Sanofi, Ramão Luciano Nogueira-Hayd: None declared, Maony Rodrigues-Moreno: None declared, Felipe Naveca: None declared, Giulia Calusi: None declared, Richard Amdur: None declared, Karol Suchowiecki: None declared, Gary S. Firestein: None declared, Gary Simon: None declared, Aileen Chang: None declared

Background:The “Targeting Immune Responses for Prevention of RA” (TIP-RA) collaboration studies individuals at high risk for developing RA because of serum anti-citrullinated protein antibody positivity in absence of arthritis, and is focused on defining how they transition from at-risk to classifiable disease. One potential mechanism is through alterations in epigenetics patterns in adaptive immune cells.Objectives:Previous studies showed that DNA methylation patterns of early RA (ERA) synoviocytes differ from long-standing RA, suggesting that abnormal methylation occurs early in synovium and evolves over time. To extend these observations, we performed a cross-sectional analysis in TIP-RA of DNA methylation signatures in peripheral blood cells in ERA, at-risk anti-CCP3+ individuals and demographically matched CCP- controls.Methods:Genomic DNA was isolated from two independent cohorts of CCP- (cohorts 1 and 2, respectively: B cell: n = 17/34; memory T cell: n = 21/34; and naïve T cell: n = 21/33), CCP3+ (B cell: n = 18/37; memory T cell: n = 20/36; and naïve T cell: n = 20/35), and CCP3+ ERA (B cell: n = 4/18; memory T cell: n = 5/18; and naïve T cell: n = 5/18) after separating PBMCs using antibodies and magnetic beads. Methylation was measured by Illumina Infinium MethylationEPIC chip. Differentially methylated loci (DMLs) were identified using Welch’s t-test and mapped to gene promoter regions to define DM genes (DMGs). Principal component analysis (PCA) was used to represent relationship among groups. Pathway analysis was applied by Reactome.Results:For the initial cohort, 1494, 1097 and 1330 DMLs were identified among CCP+, CCP- and ERA in B cells, memory T cells and naïve T cells, respectively. For the confirmatory cohort, 523, 793 and 548 DMLs were found in corresponding cell populations. The DML overlap between the 2 cohorts was highly significant (p= 2.48E-77). The DMLs were combined for both groups and corresponded to 411, 412, and 351 DMGs in B cells, memory T cells and naïve T cells. Of these, we found 246, 198 and 195 DMGs between CCP3+ and ERA in each peripheral blood cell population, respectively. PCA showed separation of CCP+, CCP- and ERA in each of the three blood cell types by DMLs (Fig. 1). DMGs were mapped to biological pathways to identify DM pathways. Although most were not significant, there were several highly significant differences comparing CCP+, ERA and CCP- in memory T cells involving pathways, including “Interferon gamma signaling” (FDR 7.48E-14), “PD-1 signaling” (FDR 8.71E-10), “Translocation of ZAP-70 to Immunological synapse” (FDR 4.75E-10), and “Phosphorylation of CD3 and TCR zeta chains” (FDR 8.71E-10).Figure 1.PCA shows the separation of CCP+, CCP- and ERA patients in memory T cells in confirmatory cohort.Conclusion:We identified reproducible methylation signatures of CCP-, CCP+, and ERA in peripheral blood B cells, memory T cells and naïve T cells in initial and confirmatory cohorts. The methylome of ERA also demonstrated a distinctive pattern from CCP+, indicating that progression to RA is accompanied by epigenetic remodeling, especially in T cell signaling and interferon responses. These signatures identify critical pathways in CCP positivity and classifiable RA and could provide the basis of novel interventions to prevent disease.Disclosure of Interests:Rizi Ai: None declared, David Boyle: None declared, Deepa Hammaker: None declared, Kevin Deane Grant/research support from: Janssen, Consultant of: Inova, ThermoFisher, Janseen, BMS and Microdrop, V. Michael Holers Grant/research support from: Janssen, Celgene, and BMS, Andre Matti: None declared, William Robinson: None declared, Jane Buckner Grant/research support from: Bristol-Myers Squibb, Janssen, Navin Rao Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Frederic Baribaud Shareholder of: Janssen Research & Development, LLC, Employee of: Janssen Research & Development, LLC, Alyssa Johnsen Employee of: Janssen, Sunil Nagpal Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Wei Wang: None declared, Gary Firestein Grant/research support from: Lilly, Janssen, Abbvie

Background:The Targeting Immune Responses for Prevention of RA (TIP-RA) Collaborative prospectively studies individuals at high risk for developing RA because of serum ACPA positivity in absence of baseline inflammatory arthritis (IA).Objectives:The objective of the analyses presented herein is to evaluate the role of baseline and changing levels of ACPA and rheumatoid factor (RF) in relationship to incident IA/RA.Methods:ACPA+ subjects and ACPA- controls were identified who did not have baseline historical or examination evidence of IA. ACPA+ was defined by serum elevation of anti-CCP3 ≥20 units (Inova). Subjects were evaluated annually or sooner if they had changes in joint symptoms. Factors including RFIgM and RFIgA (Inova) were also assessed, and relationships between autoantibody levels at baseline and over time and incident IA/RA were evaluated using t-tests, with paired testing where applicable.Results:Baseline characteristics of ACPA+ and ACPA- subjects are in Table 1. Sixteen of the 94 (17%) ACPA+ subjects developed IA/RA a mean of 518 days from the baseline visit; 14 of these met 2010 ACR/EULAR criteria for RA at the time of detection of IA. There was a trend for ACPA+ subjects who later developed IA/RA to have higher baseline levels of anti-CCP3 compared to those who did not develop IA/RA (Table 2). In addition, those who developed IA/RA had significantly higher mean levels of RFIgM and RFIgA compared to those who did not. While not statistically significant, in longitudinal analyses in the ACPA+ subjects with incident IA/RA, anti-CCP3 levels increased from baseline to identification of IA (mean [SD] of 119 [102] to 126 [100], p=0.42). Furthermore, RFIgM levels increased from 36 [49] at baseline to 43 [51] at the time of IA (p=0.31), and RFIgA levels increased from 16 [29] to 21 [31] (p=0.10). In contrast, in ACPA+ subjects who did not develop IA/RA, anti-CCP3 levels increased only slightly over follow-up of a mean of 712 days: 75 [75] to 80 [76], p=0.70 while the levels of RFIgM and RFIgA decreased slightly during the same follow-up: for RFIgM mean [SD] levels went from 9 [22] to 8 [19], p=0.74; for RFIgA, 5 [16] to 3 [12], p=0.67.Table 1.Baseline characteristics of ACPA+/- subjectsACPA-(n=162)ACPA+(n=94)p-valueAge, mean58580.90% Female69680.67% Ever smoker33340.87RF-IgM, mean (SD)3.2 (10.0)13.5 (30.2)RF-IgA, mean (SD)0.3 (0.6)6.5 (19.1)Table 2.Baseline characteristics of 16 ACPA+ subjects who developed incident IA/RA vs. 78 ACPA+ who did notDid not develop IA/RA (n=78)Developed IA/RA (n=16)p-valueDays from baseline to IA/RA or follow-up, mean (SD)712 (124)518 (295)–% Meeting 2010 criteria at time of IA-88–CCP3, mean (SD)74.5 (75.3)119.1 (102.1)0.05RFIgM, mean (SD)9 (22)36 (49)RFIgA, mean (SD)4 (16)16 (29)0.03Conclusion:In this prospectively followed cohort of ACPA+ subjects, higher levels of RFIgM and RFIgA at baseline were significantly associated with development of IA/RA within the follow-up period. Furthermore, there was a trend for rising levels of anti-CCP3 and RFIgM and A to be associated with development of IA/RA. These finding support the use of higher and/or rising levels of autoantibodies as additional features to predict imminent onset of IA/RA in ACPA+ individuals as well as potentially to use as outcomes of success of preventive interventions. Furthermore, the trend of increasing levels of RFIgM and RFIgA over time in individuals who developed IA/RA suggests that targeting pathways of RF development may lead to preventive interventions in a subset of RA.References:NoneDisclosure of Interests:Kevin Deane Grant/research support from: Janssen, Consultant of: Inova, ThermoFisher, Janseen, BMS and Microdrop, Gary Firestein Grant/research support from: Lilly, Janssen, Abbvie, David Boyle: None declared, Jane Buckner Grant/research support from: Bristol-Myers Squibb, Janssen, Eddie A. James Grant/research support from: Janssen, Pfizer, Sanofi, Novartis, Sylvia Posso Grant/research support from: Janssen, William Robinson Grant/research support from: Janssen, Laurie K. Moss Grant/research support from: Janssen, Jennifer Seifert Grant/research support from: Janssen, Roger Gilmore Grant/research support from: Janssen, Saman Barzideh Grant/research support from: Janssen, Navin Rao Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Frederic Baribaud Shareholder of: Janssen Research & Development, LLC, Employee of: Janssen Research & Development, LLC, Sunil Nagpal Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Alyssa Johnsen Employee of: Janssen, V. Michael Holers Grant/research support from: Janssen, Celgene, and BMS

ObjectivesRecent studies indicate that glucose metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Hexokinases (HKs) catalyse the first step in glucose metabolism, and HK2 constitutes the principal HK inducible isoform. We hypothesise that HK2 contributes to the synovial lining hypertrophy and plays a critical role in bone and cartilage damage.MethodsHK1 and HK2 expression were determined in RA and osteoarthritis (OA) synovial tissue by immunohistochemistry. RA FLS were transfected with either HK1 or HK2 siRNA, or infected with either adenovirus (ad)-GFP, ad-HK1 or ad-HK2. FLS migration and invasion were assessed. To study the role of HK2 in vivo, 108 particles of ad-HK2 or ad-GFP were injected into the knee of wild-type mice. K/BxN serum transfer arthritis was induced in HK2F/F mice harbouring Col1a1-Cre (HK2Col1), to delete HK2 in non-haematopoietic cells.ResultsHK2 is particular of RA histopathology (9/9 RA; 1/8 OA) and colocalises with FLS markers. Silencing HK2 in RA FLS resulted in a less invasive and migratory phenotype. Consistently, overexpression of HK2 resulted in an increased ability to migrate and invade. It also increased extracellular lactate production. Intra-articular injection of ad-HK2 in normal knees dramatically increased synovial lining thickness, FLS activation and proliferation. HK2 was highly expressed in the synovial lining after K/BxN serum transfer arthritis. HK2Col1 mice significantly showed decreased arthritis severity, bone and cartilage damage.ConclusionHK2 is specifically expressed in RA synovial lining and regulates FLS aggressive functions. HK2 might be an attractive selective metabolic target safer than global glycolysis for RA treatment.

Objective In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) β-target gene, PTPRK , promotes RA FLS invasiveness. Methods Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The RPTPκ substrate was identified by pull-down assays. Results PTPRK expression was higher in FLS from patients with RA versus patients with osteoarthritis, resulting from increased TGFB1 expression in RA FLS. RPTPκ knockdown impaired RA FLS spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumour necrosis factor and interleukin 1 stimulation. Furthermore, RPTPκ deficiency impaired the in vivo invasiveness of RA FLS. Molecular analysis revealed that RPTPκ promoted RA FLS migration by dephosphorylation of the inhibitory residue Y527 of SRC. Conclusions By regulating phosphorylation of SRC, RPTPκ promotes the pathogenic action of RA FLS, mediating cross-activation of growth factor and inflammatory cytokine signalling by TGFβ in RA FLS.

Objectives Interferon beta (IFN beta) therapy is effective in multiple sclerosis and murine models of arthritis. Surprisingly, systemic IFN beta treatment induces only minimal improvement in rheumatoid arthritis (RA). To explain this paradox, the authors evaluated the mechanism of IFN beta benefit in passive K/BxN arthritis and the effect of IFN beta treatment on RA synovium. Methods Interleukin 10 (IL-10) null, IL-1 receptor antagonist (IL-1Ra) null, IL-1Ra transgenic and wildtype mice were administered K/BxN serum and in some cases treated with IFN beta or normal saline. Clinical response and histological scores were assessed. Gene expression was measured by quantitative PCR. Serum IL-1Ra and IL-6 were measured by ELISA. Paired synovial biopsy specimens from RA patients pre-IFN beta and post-IFN beta treatment (purified natural fibroblast IFN beta (Frone) subcutaneously three times weekly 6 million IU, 12 million IU or 18 million IU) were immunostained for IL-1Ra and IL-10. Results Il1rn transgenic mice had an attenuated course of arthritis, whereas Il1rn(-/-) and Il10(-/-) mice had more severe serum transfer arthritis than wild-type mice. Daily IFN beta treatment significantly decreased arthritis severity in Il10(-/-) but not Il1rn(-/-) mice. IFN beta treatment did not reduce the histological scores in Il1rn(-/-) mice or gene expression of articular cytokines and chemokines. Paired synovial biopsy specimens from RA patients treated with IFN beta demonstrated a trend towards increased IL-1Ra and reduced IL-10 expression on day 85 levels compared with pretreatment specimens. Conclusions The anti-inflammatory effects of IFN beta in passive K/BxN arthritis are dependent on IL-1Ra, but not IL-10. Systemic IFN beta treatment in RA increases synovial IL-1Ra production, but also decreases IL-10 production

Background Rheumatoid arthritis is an inflammatory disease marked by intra-articular decreases in pH, aberrant hyaluronan regulation and destruction of bone and cartilage. Acid-sensing ion channels (ASICs) are the primary acid sensors in the nervous system, particularly in sensory neurons and are important in nociception. ASIC3 was recently discovered in synoviocytes, non-neuronal joint cells critical to the inflammatory process. Objectives To investigate the role of ASIC3 in joint tissue, specifically the relationship between ASIC3 and hyaluronan and the response to decreased pH. Methods Histochemical methods were used to compare morphology, hyaluronan expression and ASIC3 expression in ASIC3+/+ and ASIC3−/− mouse knee joints. Isolated fibroblast-like synoviocytes (FLS) were used to examine hyaluronan release and intracellular calcium in response to decreases in pH. Results In tissue sections from ASIC3+/+ mice, ASIC3 localised to articular cartilage, growth plate, meniscus and type B synoviocytes. In cultured FLS, ASIC3 mRNA and protein was also expressed. In FLS cultures, pH 5.5 increased hyaluronan release in ASIC3+/+ FLS, but not ASIC3−/− FLS. In FLS from ASIC3+/+ mice, approximately 50% of cells (25/53) increased intracellular calcium while only 24% (14/59) showed an increase in ASIC3−/− FLS. Of the cells that responded to pH 5.5, there was significantly less intracellular calcium increases in ASIC3−/− FLS compared to ASIC3+/+ FLS. Conclusion ASIC3 may serve as a pH sensor in synoviocytes and be important for modulation of expression of hyaluronan within joint tissue.

Background Animal models of arthritis are frequently used to evaluate novel therapeutic agents. However, their ability to predict responses in humans is variable. Objective To examine the time course of signalling molecule and gene expression in two models of arthritis to assist with selection of the model and timing of drug administration. Methods The passive K/BxN serum transfer and collagen-induced arthritis (CIA) models were studied. Activation of MAP kinase and interferon (IFN)-response pathways was evaluated by quantitative PCR and western blot analysis of ankle joints at various time points during the models. Results The kinetics of gene expression and kinase phosphorylation were strikingly different in passive K/BxN and CIA. All three MAP kinases (ERK, JNK and p38) and upstream kinases were activated within days in passive K/BxN and declined as arthritis severity decreased. Surprisingly, IFN-regulated genes, including IRF7, were not induced in the model. In CIA, activation of ERK and JNK was surprisingly low and p38 phosphorylation mainly peaked late in the disease. IFN-response genes were activated during CIA, with especially prominent peaks at the onset of clinical arthritis. Conclusions Timing of treatment and selection of CIA or passive K/BxN might have an important impact on therapeutic response. p38, in particular, increases during the late stages of CIA. ERK and JNK patterns are similar in passive K/BxN and rheumatoid arthritis (RA), while IFN-response genes in CIA and RA are similar. The dichotomy between RA and animal models could help explain the poor correlation between efficacy in RA and preclinical studies.

Objectives:The IκB kinase (IKK)-related kinase IKKϵ regulates type I interferon expression and responses as well as proinflammatory mediator production. We examined the role of IKKϵ in arthritis and its ability to enhance the therapeutic response to systemic interferon (IFN) β therapy in passive murine K/BxN arthritis.Methods:IKKϵ–/–, IFNα∼βR–/– and wild type mice were given K/BxN serum and treated with polyinosinic polycytidylic acid (poly(I:C)), IFNβ, or normal saline. Clinical response and histological scores were assessed. Gene expression in the paws was measured by quantitative PCR. Serum interleukin 1a receptor agonist (IL1Ra) and IL10 were measured by ELISA and multiplex bead array.Results:Arthritis was almost completely blocked in wild type mice if arthritogenic K/BxN serum and the Toll-like receptor (TLR)3 ligand, poly(I:C), were coadministered at the onset of the model, but not in established disease. Mice deficient in IFNα∼βR had an accelerated course of arthritis, and did not respond to poly(I:C). IKKϵ null mice had a modest decrease in clinical arthritis compared with heterozygous mice. Low doses of IFNβ that were ineffective in wild type mice significantly decreased clinical arthritis in IKKϵ null mice. Articular chemokine gene expression was reduced in the IKKϵ–/– mice with arthritis and secreted IL1Ra (sIL1Ra) mRNA was significantly increased. Serum levels of IL1Ra were increased in low dose IFNβ-treated IKKϵ–/– mice.Conclusions:Subtherapeutic doses of IFNβ enhance the anti-inflammatory effects of IKKϵ deficiency, possibly by increasing production of IL1Ra and unmasking the antichemokine effects. Combination therapy with low dose IFNβ and an IKKϵ inhibitor might improve efficacy of either agent alone and offers a novel approach to RA.

Chronic inflammatory processes are based on a sustained and tightly regulated communication network among different cells types. This network comprises extracellular mediators such as cytokines, chemokines and matrix-degrading proteases, which orchestrate the participation of cells in the chronic inflammatory process. The mirrors of this outside communication world are intracellular transcription factor pathways, which shuttle information about inflammatory stimuli to the cell nucleus. This review examines the function of one key signal transduction pathway of inflammation--the p38 mitogen-activated protein kinases (p38MAPK). The signalling pathway is considered as crucial for the induction and maintenance of chronic inflammation, and its components thus emerge as interesting molecular targets of small molecule inhibitors for controlling inflammation. This review not only summarises the current knowledge of activation, regulation and function of the p38MAPK pathway but also examines the role of this pathway in clinical disease. It gives an overview of current evidence of p38MAPK activation in inflammatory arthritis and elaborates the key molecular determinants which contribute to p38MAPK activation in joint disease.

Oxidative stress in RA synovial tissue can cause DNA damage and suppress the DNA mismatch repair (MMR) system in cultured synoviocytes. This mechanism includes two enzyme complexes, hMutSalpha (hMSH2/hMSH6) and hMutSbeta (hMSH2/hMSH3).To examine the expression and distribution of MMR enzymes in synovial tissues from patients with arthritis and from normal subjects.Synovial tissues from patients with RA, osteoarthritis (OA), or normal subjects were analysed by immunohistochemistry using monoclonal antibodies to hMSH2, hMSH3, and hMSH6. MMR protein expression was evaluated by computer assisted digital image analysis.hMSH2, hMSH3, and hMSH6 were found in most synovial tissues evaluated, with greater levels in the intimal lining than sublining regions. In RA and OA, sublining perivascular staining for hMSH6 and hMSH3 was also prominent. Significantly higher sublining expression of hMSH2, hMSH3, and hMSH6 was seen in RA and OA than in normal synovium. Double label immunohistochemistry demonstrated that the main cells expressing MMR enzymes were CD68(+) and CD68(-) cells in the intimal lining.DNA MMR enzyme expression is greatest in the synovial intimal lining layer, where maximal oxidative stress in RA occurs. Although MMR enzyme expression is greater in RA than in normal tissue, this compensatory response cannot overcome the genotoxic environment, and DNA damage accumulates.

ObjectivesLittle is known about targeting the metabolome in non-cancer conditions. Choline kinase (ChoKα), an essential enzyme for phosphatidylcholine biosynthesis, is required for cell proliferation and has been implicated in cancer invasiveness. Aggressive behaviour of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) led us to evaluate whether this metabolic pathway could play a role in RA FLS function and joint damage.MethodsCholine metabolic profile of FLS cells was determined by 1H magnetic resonance spectroscopy (1HMRS) under conditions of ChoKα inhibition. FLS function was evaluated using the ChoKα inhibitor MN58b (IC50=4.2 μM). For arthritis experiments, mice were injected with K/BxN sera. MN58b (3 mg/kg) was injected daily intraperitoneal beginning on day 0 or day 4 after serum administration.ResultsThe enzyme is expressed in synovial tissue and in cultured RA FLS. Tumour necrosis factor (TNF) and platelet-derived growth factor (PDGF) stimulation increased ChoKα expression and levels of phosphocholine in FLS measured by Western Blot (WB) and metabolomic studies of choline-containing compounds in cultured RA FLS extracts respectively, suggesting activation of this pathway in RA synovial environment. A ChoKα inhibitor also suppressed the behaviour of cultured FLS, including cell migration and resistance to apoptosis, which might contribute to cartilage destruction in RA. In a passive K/BxN arthritis model, pharmacologic ChoKα inhibition significantly decreased arthritis in pretreatment protocols as well as in established disease.ConclusionsThese data suggest that ChoKα inhibition could be an effective strategy in inflammatory arthritis. It also suggests that targeting the metabolome can be a new treatment strategy in non-cancer conditions.

Synovial biomarkers are increasingly important in the development of novel therapeutic agents for the treatment of rheumatoid arthritis (RA). To identify biomarkers correlating with changes in clinical disease activity, real-time quantitative PCR (Q-PCR) was used to evaluate changes in synovial gene expression after treatment with corticosteroids. Patients with active RA received either oral prednisolone (n=10, 60 mg daily for the first week and 40 mg daily for the second week) or placebo (n=11) for 14 days. Real-time Q-PCR was used to quantify gene expression of tumour necrosis factor (TNF)alpha, IL1beta, IL8 and matrix metalloproteinase (MMP) 1 in synovial tissue samples obtained through an arthroscopic procedure before and after treatment. mRNA levels were reported as relative expression units compared with a cell-based standard. Statistical analysis was performed using an analysis of covariance model. Prednisolone markedly decreased IL8 and MMP1 expression compared with placebo, and the CIs excluded the likelihood of no effect. A trend towards reduction was seen in IL1beta and TNFalpha mRNA expression in the prednisolone group, although CIs included the value for no effect. These data suggest that Q-PCR can be used to measure synovial mRNA expression of mediators implicated in the pathogenesis of RA in small proof-of-concept trials.

Objectives Epigenetics can influence disease susceptibility and severity. While DNA methylation of individual genes has been explored in autoimmunity, no unbiased systematic analyses have been reported. Therefore, a genome-wide evaluation of DNA methylation loci in fibroblast-like synoviocytes (FLS) isolated from the site of disease in rheumatoid arthritis (RA) was performed. Methods Genomic DNA was isolated from six RA and five osteoarthritis (OA) FLS lines and evaluated using the Illumina HumanMethylation450 chip. Cluster analysis of data was performed and corrected using Benjamini– Hochberg adjustment for multiple comparisons. Methylation was confirmed by pyrosequencing and gene expression was determined by qPCR. Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes. Results RA and control FLS segregated based on DNA methylation, with 1859 differentially methylated loci. Hypomethylated loci were identified in key genes relevant to RA, such as CHI3L1, CASP1, STAT3, MAP3K5, MEFV and WISP3 .H ypermethylation was also observed, including TGFBR2 and FOXO1. Hypomethylation of individual genes was associated with increased gene expression. Grouped analysis identified 207 hypermethylated or hypomethylated genes with multiple differentially methylated loci, including COL1A1, MEFV and TNF. Hypomethylation was increased in multiple pathways related to cell migration, including focal adhesion, cell adhesion, transendothelial migration and extracellular matrix interactions. Confirmatory studies with OA and normal FLS also demonstrated segregation of RA from control FLS based on methylation pattern. Conclusions Differentially methylated genes could alter FLS gene expression and contribute to the pathogenesis of RA. DNA methylation of critical genes suggests that RA FLS are imprinted and implicate epigenetic contributions to inflammatory arthritis.

ObjectivesThe objective of this study was to investigate the effect of the novel Janus kinase inhibitor CP-690,550 in fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA).MethodsRA FLSs were isolated from tissue obtained by arthroplasty, cultured and serum-starved 48 h prior to stimulation. Messenger RNA and protein levels were determined by quantitative PCR and ELISA or multiplex bead assay, respectively. Phosphorylation of STAT (signal transducers and activators of transcription) proteins was determined by western blot.ResultsInterleukin-6-induced phosphorylation of STAT1 and STAT3 was inhibited by CP-690,550 with IC50 values of 23 and 77 nM, respectively. Unexpectedly, although tumour necrosis factor (TNF) did not induce immediate phosphorylation of either STAT, CP-690,550 inhibited TNF-induced expression of several chemokines (IP-10, RANTES and MCP1) at the messenger RNA and protein levels. Chemokine expression was inhibited by cycloheximide, implying a need for de novo protein synthesis, and cycloheximide abolished the effect of CP-690,550 (tofacitinib). TNF induced early interferon (IFN) β expression and STAT1 phosphorylation beginning at 3 h, which was blocked by CP-690,550. The dependence of TNF-induced chemokine expression on type I IFN was confirmed in FLSs from mice lacking type I IFN receptors (IFNARs) and in RA FLSs using an IFNAR blocking antibody.ConclusionsThe Janus kinase/STAT pathway in FLS is indirectly activated by TNF through autocrine expression of type I IFN, resulting in IFNAR engagement and production of T cell chemokines. These findings illuminate a novel role of CP-690,550 in the treatment of RA: the reduction of chemokine synthesis by FLS, thereby limiting recruitment of T cells and other infiltrating leucocytes.

Despite the success of biological therapies in rheumatoid arthritis (RA), orally active small-molecule drugs are desirable. Signal transduction inhibitors have been the focus of intense efforts, with some recent notable successes and failures. p38alpha is a signalling molecule that regulates proinflammatory cytokines, which makes it a logical target for RA. Unfortunately, selective p38alpha inhibitors have limited efficacy. An attempt is made here to put these studies into perspective and offer possible explanations for the failure of p38alpha blockers. Alternative strategies, such as targeting kinases higher in the signalling cascade or using less selective compounds, might be more successful as suggested by the efficacy seen with Syk and JAK inhibitors.

Orally bioavailable compounds that target key intracellular signalling molecules are receiving increasing attention for the treatment of rheumatic diseases. The mitogen activated protein (MAP) kinases are especially attractive because they regulate both cytokine production and cytokine action. The MAP kinases are expressed and activated in rheumatoid arthritis (RA) synovium. Preclinical studies using MAP kinase inhibitors are very effective in animal models of arthritis, supporting their potential utility in human disease. Although the available data suggest a rationale for MAP kinase blockade, development of drugs has been hampered by toxicity and limited efficacy. Alternative strategies, such as targeting other kinases in the cascade or development of allosteric inhibitors have been proposed. These approaches might permit effective use of MAP kinase inhibitors for the treatment of rheumatic and immune-mediated diseases.

Chronic pain is a major health problem affecting approximately 20% of the population resulting in marked reduced quality of life for the individual, as well as extensive costs for society. A large-scale survey undertaken in 15 European countries showed that 40% of the people who reported persistent pain (>6 months) had joint pain and that osteoarthritis (OA) and rheumatoid arthritis (RA) combined was the most common cause of pain (42%). 1 Preclinical studies of pain mechanisms in RA have …

Background/Objectives Cartilage destruction mediated by invasive fibroblast-like synoviocytes (FLS) plays a central role in pathogenesis of RA. Increased cell migration and degradation of extracellular matrix are fundamental to these processes. The Class I phosphoinositide 3-kinases (PI3K) control cell survival, proliferation and migration, which might be involved with cartilage damage in RA. PI3Kdelta isoform expression was recently identified as a key regulator of FLS growth and survival, suggesting that it could contribute to synoviocyte aggressive behaviour. Therefore, we assessed the role of PI3Kdelta in synoviocyte invasion and matrix degradation using isoform selective PI3K inhibitors. Methods FLS were cultured in Matrigel coated transwells. PI3K inhibitors or vehicle were added to the upper chamber and PDGF was used as a chemoattractant in the lower chamber. The invading cells were quantified by staining the philtres with 1% crystal violet. F-actin was visualised with Rhodamin phalloidin and analysed with fluorescent microscopy. Rac1 activation was measured using PAK1/PBD GST pull down and quantified by Western blot analysis. PI3K inhibitors: pan (GDC-0941), PI3Kalpha (A66), PI3Kbeta (TGX-221), PI3Kgamma (AS-252424), PI3Kdelta (INK007 and CAL-101) and PI3K-delta/gamma (IPI-145). Results PDGF-directed invasion was completely inhibited by the pan PI3K inhibitor (1 uM). To define the role of the individual isoforms, we tested the effect of the isoform selective PI3K inhibitors. PI3Kdelta inhibition (INK007) significantly decreased the number of invading cells, with 60 ± 5% inhibition at 1 uM (p Conclusions PI3Kdelta is a major regulator of FLS migration and invasion and functions by inhibiting Rac1 activation and modulating F actin cytoskeleton rearrangement. These observations, together with previous findings that PI3Kdelta regulates FLS growth and survival, suggest that PI3Kdelta inhibition could be chondroprotective in RA by modulating synoviocyte growth, migration and invasion.