Role of soluble epoxide hydrolase in the abnormal activation of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by enigmatic pathogenesis. Polyunsaturated fatty acids (PUFAs) are implicated in RA's development and progression, yet their exact mechanisms of influence are not fully understood. Soluble epoxide hydrolase (sEH) is an enzyme that metabolizes anti-inflammatory epoxy fatty acids (EpFAs), derivatives of PUFAs. In this study, we report elevated sEH expression in the joints of CIA (collagen-induced arthritis) rats, concomitant with diminished levels of two significant EpFAs. Additionally, increased sEH expression was detected in both the synovium of CIA rats and in the synovium and fibroblast-like synoviocytes (FLS) of RA patients. The sEH inhibitor TPPU attenuated the migration and invasion capabilities of FLS derived from RA patients and to reduce the secretion of inflammatory factors by these cells. Our findings indicate a pivotal role for sEH in RA pathogenesis and suggest that sEH inhibitors offer a promising new therapeutic strategy for managing RA.

Extracellular pyruvate kinase M2 promotes osteoclastogenesis and is associated with radiographic progression in early rheumatoid arthritis.

Extracellular PKM2 (exPKM2) levels have been reported to be increased in several cancers and inflammatory diseases, including rheumatoid arthritis (RA). This study aimed to investigate the association of circulating exPKM2 levels with radiographic progression in RA patients and the effect of exPKM2 on osteoclastogenesis. Plasma and synovial fluid exPKM2 levels were significantly elevated in RA patients. Plasma exPKM2 levels were correlated with RA disease activity and were an independent predictor for radiographic progression in RA patients with a disease duration of ≤ 12 months. CD14+ monocytes but not RA fibroblast-like synoviocytes secreted PKM2 upon stimulation with inflammatory mediators. Recombinant PKM2 (rPKM2) increased the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells and resorption pit in osteoclast precursors, dose-dependently, even in the absence of receptor activator of nuclear factor-kappa B ligand (RANKL). rPKM2 treatment upregulated the expression of dendrocyte-expressed seven transmembrane protein (DC-STAMP) and MMP-9 via the ERK pathway. Although rPKM2 did not directly bind to RAW264.7 cells, extracellular application of pyruvate, the end-product of PKM2, showed effects similar to those seen in rPKM2-induced osteoclastogenesis. These results suggest that exPKM2 is a potential regulator of RA-related joint damage and a novel biomarker for subsequent radiographic progression in patients with early-stage RA.

The Prominent Role of Hematopoietic Peptidyl Arginine Deiminase 4 in Arthritis: Collagen- and Granulocyte Colony-Stimulating Factor-Induced Arthritis Model in C57BL/6 Mice.

OBJECTIVE: Genome-wide association studies have connected PADI4, encoding peptidylarginine deiminase 4 (PAD4), with rheumatoid arthritis (RA). PAD4 promotes neutrophil extracellular trap (NET) formation. This study was undertaken to investigate the origin of PAD4 and the importance of NET formation in a C57BL/6 mouse model of arthritis. METHODS: To permit the effective use of C57BL/6 mice in the collagen-induced arthritis (CIA) model, we introduced the administration of granulocyte colony-stimulating factor (G-CSF) for 4 consecutive days in conjunction with the booster immunization on day 21. Mice with global Padi4 deficiency (Padi4-/- ) and mice with hematopoietic lineage-specific Padi4 deficiency (Padi4Vav1Cre/+ ) were evaluated in the model. RESULTS: G-CSF significantly increased the incidence and severity of CIA. G-CSF-treated mice showed elevated citrullinated histone H3 (Cit-H3) levels in plasma, while vehicle-treated mice did not. Immunofluorescence microscopy revealed deposition of Cit-H3 in synovial tissue in G-CSF-treated mice. Padi4-/- mice developed less severe arthritis and had lower levels of serum interleukin-6 and plasma Cit-H3, lower levels of Cit-H4 in synovial tissue, and less bone erosion on micro-computed tomography than Padi4+/+ mice in the G-CSF-modified CIA model. Similarly, Padi4Vav1Cre/+ mice developed less severe arthritis, compared with Padi4fl/fl mice, and presented the same phenotype as Padi4-/- mice. CONCLUSION: We succeeded in developing an arthritis model suitable for use in C57BL/6 mice that is fully compliant with high animal welfare standards. We observed a >90% incidence of arthritis in male mice and detectable NET markers. This model, with some features consistent with human RA, demonstrates that hematopoietic PAD4 is an important contributor to arthritis development and may prove useful in future RA research.

Factors affecting drug retention of Janus kinase inhibitors in patients with rheumatoid arthritis: the ANSWER cohort study.

This multi-center, retrospective study aimed to clarify the factors affecting drug retention of the Janus kinase inhibitors (JAKi) including baricitinib (BAR) and tofacitinib (TOF) in patients with RA. Patients were as follows; females, 80.6%; age, 60.5 years; DAS28-ESR, 4.3; treated with either BAR (n = 166) or TOF (n = 185); bDMARDs- or JAKi-switched cases (76.6%). The reasons for drug discontinuation were classified into four major categories. The drug retention was evaluated at 24 months using the Kaplan-Meier method and multivariate Cox proportional hazards modelling adjusted by confounders. Discontinuation rates for the corresponding reasons were as follows; ineffectiveness (22.3%), toxic adverse events (13.3%), non-toxic reasons (7.2%) and remission (0.0%). Prior history of anti-interleukin-6 receptor antibody (aIL-6R) ineffectiveness significantly increased the risk of treatment discontinuation due to ineffectiveness (p = 0.020). Aging (≥ 75 years) (p = 0.028), usage of PSL ≥ 5 mg/day (p = 0.017) and female sex (p = 0.041) significantly increased the risk of treatment discontinuation due to toxic adverse events. Factors not associated with treatment discontinuation were: number of prior bDMARDs or JAKi, concomitant MTX usage, difference of JAKi, and prior use of TNF inhibitor, CTLA4-Ig or other JAKi.

TNFα induces endothelial dysfunction in rheumatoid arthritis via LOX-1 and arginase 2: reversal by monoclonal TNFα antibodies.

AIMS: Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting joints and blood vessels. Despite low levels of low-density lipoprotein cholesterol (LDL-C), RA patients exhibit endothelial dysfunction and are at increased risk of death from cardiovascular complications, but the molecular mechanism of action is unknown. We aimed in the present study to identify the molecular mechanism of endothelial dysfunction in a mouse model of RA and in patients with RA. METHODS AND RESULTS: Endothelium-dependent relaxations to acetylcholine were reduced in aortae of two tumour necrosis factor alpha (TNFα) transgenic mouse lines with either mild (Tg3647) or severe (Tg197) forms of RA in a time- and severity-dependent fashion as assessed by organ chamber myograph. In Tg197, TNFα plasma levels were associated with severe endothelial dysfunction. LOX-1 receptor was markedly up-regulated leading to increased vascular oxLDL uptake and NFκB-mediated enhanced Arg2 expression via direct binding to its promoter resulting in reduced NO bioavailability and vascular cGMP levels as shown by ELISA and chromatin immunoprecipitation. Anti-TNFα treatment with infliximab normalized endothelial function together with LOX-1 and Arg2 serum levels in mice. In RA patients, soluble LOX-1 serum levels were also markedly increased and closely related to serum levels of C-reactive protein. Similarly, ARG2 serum levels were increased. Similarly, anti-TNFα treatment restored LOX-1 and ARG2 serum levels in RA patients. CONCLUSIONS: Increased TNFα levels not only contribute to RA, but also to endothelial dysfunction by increasing vascular oxLDL content and activation of the LOX-1/NFκB/Arg2 pathway leading to reduced NO bioavailability and decreased cGMP levels. Anti-TNFα treatment improved both articular symptoms and endothelial function by reducing LOX-1, vascular oxLDL, and Arg2 levels.

Semi-Mechanistic PK/PD Modeling and Simulation of Irreversible BTK Inhibition to Support Dose Selection of Tirabrutinib in Subjects with RA.

Tirabrutinib is an irreversible, small-molecule Bruton's tyrosine kinase (BTK) inhibitor, which was approved in Japan (VELEXBRU) to treat B-cell malignancies and is in clinical development for inflammatory diseases. As an application of model-informed drug development, a semimechanistic pharmacokinetic/pharmacodynamic (PK/PD) model for irreversible BTK inhibition of tirabrutinib was developed to support dose selection in clinical development, based on clinical PK and BTK occupancy data from two phase I studies with a wide range of PK exposures in healthy volunteers and in subjects with rheumatoid arthritis. The developed model adequately described and predicted the PK and PD data. Overall, the model-based simulation supported a total daily dose of at least 40 mg, either q.d. or b.i.d., with adequate BTK occupancy (> 90%) for further development in inflammatory diseases. Following the PK/PD modeling and simulation, the relationship between model-predicted BTK occupancy and preliminary clinical efficacy data was also explored and a positive trend was identified between the increasing time above adequate BTK occupancy and better efficacy in treatment for RA by linear regression.

Inhibition of SYK and cSrc kinases can protect bone and cartilage in preclinical models of osteoarthritis and rheumatoid arthritis.

The pathophysiology of osteoarthritis (OA) includes the destruction of subchondral bone tissue and inflammation of the synovium. Thus, an effective disease-modifying treatment should act on both of these pathogenetic components. It is known that cSrc kinase is involved in bone and cartilage remodeling, and SYK kinase is associated with the inflammatory component. Thus the aim of this study was to characterize the mechanism of action and efficacy of a small molecule multikinase inhibitor MT-SYK-03 targeting SYK and cSrc kinases among others in different in vitro and in vivo arthritis models. The selectivity of MT-SYK-03 kinase inhibition was assayed on a panel of 341 kinases. The compound was evaluated in a set of in vitro models of OA and in vivo OA and RA models: surgically-induced arthritis (SIA), monosodium iodoacetate-induced arthritis (MIA), collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA). MT-SYK-03 inhibited cSrc and SYK with IC50 of 14.2 and 23 nM respectively. Only five kinases were inhibited > 90% at 500 nM of MT-SYK-03. In in vitro OA models MT-SYK-03 reduced hypertrophic changes of chondrocytes, bone resorption, and inhibited SYK-mediated inflammatory signaling. MT-SYK-03 showed preferential distribution to joint and bone tissue (in rats) and revealed disease-modifying activity in vivo by halving the depth of cartilage erosion in rat SIA model, and increasing the pain threshold in rat MIA model. Chondroprotective and antiresorptive effects were shown in a monotherapy regime and in combination with methotrexate (MTX) in murine and rat CIA models; an immune-mediated inflammation in rat AIA model was decreased. The obtained preclinical data support inhibition of cSrc and SYK as a viable strategy for disease-modifying treatment of OA. A Phase 2 clinical study of MT-SYK-03 is to be started.

Glycolysis Rate-Limiting Enzymes: Novel Potential Regulators of Rheumatoid Arthritis Pathogenesis.

Rheumatoid arthritis (RA) is a classic autoimmune disease characterized by uncontrolled synovial proliferation, pannus formation, cartilage injury, and bone destruction. The specific pathogenesis of RA, a chronic inflammatory disease, remains unclear. However, both key glycolysis rate-limiting enzymes, hexokinase-II (HK-II), phosphofructokinase-1 (PFK-1), and pyruvate kinase M2 (PKM2), as well as indirect rate-limiting enzymes, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), are thought to participate in the pathogenesis of RA. In here, we review the latest literature on the pathogenesis of RA, introduce the pathophysiological characteristics of HK-II, PFK-1/PFKFB3, and PKM2 and their expression characteristics in this autoimmune disease, and systematically assess the association between the glycolytic rate-limiting enzymes and RA from a molecular level. Moreover, we highlight HK-II, PFK-1/PFKFB3, and PKM2 as potential targets for the clinical treatment of RA. There is great potential to develop new anti-rheumatic therapies through safe inhibition or overexpression of glycolysis rate-limiting enzymes.

Casein Kinase II exacerbates rheumatoid arthritis via promoting Th1 and Th17 cell inflammatory responses.

OBJECTIVES: Studies have demonstrated that CK2 is engaged in CD4+ T cell proliferation and activation. We investigated the potential involvement of CK2 in the pathogenesis of rheumatoid arthritis (RA). METHODS: Peripheral blood and synovial fluid mononuclear cells (PBMC and SFMC) of RA patients, as well as splenocytes of collagen-induced arthritis (CIA) mice were treated with different doses of CK2 inhibitor CX4945 in vitro. Then, the Th1, Th2, Th17, and Treg cell responses were analyzed. In addition, CIA mice were administrated with CX4945 via oral gavage. Accordingly, the arthritis scores, bone destruction, tissue damage, and the CD4+ T cell subsets were assessed. RESULTS: The expression of CK2 was upregulated in CD4+ T cells under RA circumstance. In vitro CX4945 treatment significantly inhibited the Th1 and Th17 cell responses, while promoted the Th2 cell responses in RA patient PBMC, SFMC and CIA mouse splenocytes, dampening IFN-γ and IL-17A production. Moreover, administration of CX4945 ameliorated the severity of arthritis in CIA mice, along with decreased Th1 and Th17 cells. However, CX4945 seemed to have minimal effect on RA Treg cells. CONCLUSION: CK2 serves as an important regulator of the Th1 and Th17 cell axes in RA, thus contributing to the disease aggravation.

Tofacitinib Suppresses Several JAK-STAT Pathways in Rheumatoid Arthritis In Vivo and Baseline Signaling Profile Associates With Treatment Response.

Objective: Current knowledge on the actions of tofacitinib on cytokine signaling pathways in rheumatoid arthritis (RA) is based on in vitro studies. Our study is the first to examine the effects of tofacitinib treatment on Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathways in vivo in patients with RA. Methods: Sixteen patients with active RA, despite treatment with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), received tofacitinib 5 mg twice daily for three months. Levels of constitutive and cytokine-induced phosphorylated STATs in peripheral blood monocytes, T cells and B cells were measured by flow cytometry at baseline and three-month visits. mRNA expression of JAKs, STATs and suppressors of cytokine signaling (SOCS) were measured from peripheral blood mononuclear cells (PBMCs) by quantitative PCR. Association of baseline signaling profile with treatment response was also investigated. Results: Tofacitinib, in csDMARDs background, decreased median disease activity score (DAS28) from 4.4 to 2.6 (p < 0.001). Tofacitinib treatment significantly decreased cytokine-induced phosphorylation of all JAK-STAT pathways studied. However, the magnitude of the inhibitory effect depended on the cytokine and cell type studied, varying from 10% to 73% inhibition following 3-month treatment with tofacitinib. In general, strongest inhibition by tofacitinib was observed with STAT phosphorylations induced by cytokines signaling through the common-γ-chain cytokine receptor in T cells, while lowest inhibition was demonstrated for IL-10 -induced STAT3 phosphorylation in monocytes. Constitutive STAT1, STAT3, STAT4 and STAT5 phosphorylation in monocytes and/or T cells was also downregulated by tofacitinib. Tofacitinib treatment downregulated the expression of several JAK-STAT pathway components in PBMCs, SOCSs showing the strongest downregulation. Baseline STAT phosphorylation levels in T cells and monocytes and SOCS3 expression in PBMCs correlated with treatment response. Conclusions: Tofacitinib suppresses multiple JAK-STAT pathways in cytokine and cell population specific manner in RA patients in vivo. Besides directly inhibiting JAK activation, tofacitinib downregulates the expression of JAK-STAT pathway components. This may modulate the effects of tofacitinib on JAK-STAT pathway activation in vivo and explain some of the differential findings between the current study and previous in vitro studies. Finally, baseline immunological markers associate with the treatment response to tofacitinib.

Genome-wide association study of liver enzyme elevation in rheumatoid arthritis patients starting methotrexate.

Aim: To identify novel genetic variants predisposing to elevation of Alanine aminotransferase (ALT) in rheumatoid arthritis (RA) patients after initiation of methotrexate (MTX) treatment. Patients & methods: We performed genome-wide association studies in 198 RA patients starting MTX. Outcomes were maximum level of ALT and ALT >1.5-times the upper level of normal within the first 6 months of treatment. Results:RAVER2 (rs72675408) was significantly associated with maximum level of ALT (p = 4.36 × 10-8). This variant is in linkage disequilibrium with rs72675451, which is associated with differential expression of JAK1 and RAVER2. Conclusion: We found an association between ALT elevation and genetic variants that may regulate the expression of JAK1 and RAVER2. JAK1 encodes a janus kinase involved in the pathogenesis of RA.

Longitudinal monitor of Jun N-terminal kinase pathway associated phosphatase reflects clinical efficacy to triple conventional disease-modifying anti-rheumatic drugs treatment in rheumatoid arthritis patients.

OBJECTIVE: This study aimed to investigate the correlation of Jun N-terminal kinase pathway associated phosphatase (JKAP) with inflammation, disease activity, and clinical efficacy to triple conventional disease-modifying anti-rheumatic drugs (cDMARDs) in rheumatoid arthritis (RA) patients. METHODS: A total of 119 active RA patients about to receive triple cDMARDs treatment were enrolled. Serum JKAP was detected by enzyme-linked immunosorbent assay at week0, week6, week12, and week24 (W24). According to clinical response status or remission status at W24, RA patients were classified as response patients and non-response patients, or remission patients and non-remission patients, respectively. RESULTS: JKAP was negatively correlated with erythrocyte sedimentation rate (ESR), C-reactive protein, and 28-joints disease activity score based on ESR (DAS28 score (ESR)), while JKAP was not correlated with disease duration, tender joint count, swollen joint count, health assessment questionnaire for rheumatoid arthritis or treatment history. Furthermore, during 24-week triple cDMARDs treatment, JKAP was increased overtime. Subgroup analyses showed that JKAP displayed a rising trend in response patients, remission patients, non-remission patients but not non-response patients, meanwhile its increment was more obvious in remission patients versus non-remission patients. Additionally, JKAP at W24 was higher in response patients compared with non-response patients, and JKAP at W12 and W24 was higher in remission patients compared with non-remission patients. CONCLUSION: Longitudinal monitor of JKAP might reflect clinical efficacy to the treatment of triple cDMARDs, which could improve outcomes in RA patients.

miR-483-3p Promotes IL-33 Production from Fibroblast-Like Synoviocytes by Regulating ERK Signaling in Rheumatoid Arthritis.

Our previous studies have identified miR-483-3p to be highly expressed in synoviocytes from patients with rheumatoid arhtirits (RA); however, its effects on inflammation of RA fibroblast-like synoviocytes (FLSs) have remained unclear. The expression of miR-483-3p and cytokines in RA FLSs was detected using quantitative real-time polymerase chain reaction. Enzyme-linked immunosorbent was conducted to determine interleukin (IL)-33 production from RA FLSs. Western blotting was employed to quantify the levels of p-ERK and total ERK. Overexpressed miR-483-3p significantly increased the mRNA and protein expression of IL-33, but not of IL-27 or IL-34, in RA FLSs, whereas miR-483-3p suppression showed the opposite effects. Furthermore, miR-483-3p upregulation activated the ERK signaling pathway. The ERK signaling inhibitor PD98059 partly reversed the elevation of IL-33 levels mediated by miR-483-3p overexpression. Our results reveal that miR-483-3p promotes IL-33 expression by regulating the ERK signaling pathway in RA FLSs. Thus, miR-483-3p may be a potential effective target for RA treatment.

Current jakinibs for the treatment of rheumatoid arthritis: a systematic review.

OBJECTIVE: One-third of patients with severe rheumatoid arthritis (RA) do not achieve remission or low disease activity, or they have side effects from cDMARD and bDMARD. They will need a new treatment option such as the small molecule JAK inhibitors. In this systematic review, we evaluate the efficacy and safety data of the current jakinibs: tofacitinib, peficitinib, decernotinib, upadacitinib, baricitinib and filgotinib in patients in whom treatment with conventional or biological disease-modifying antirheumatic drugs (cDMARD and/or bDMARD) failed. METHODS: We searched for randomized controlled trials comparing efficacy and safety of jakinibs for RA treatment using the Web of Science, Scopus, PubMed, and clinicaltrials.gov databases with the terms: "rheumatoid arthritis" OR "arthritis rheumatoid" OR "RA" AND "inhibitor" OR "jak inhibitor" AND "clinical trial" OR "treatment" OR "therapy". RESULTS: All jakinibs achieved good results in ACR 20, 50, 70 and with CRP-DAS28 for LDA and remission, upadacitinib showed better results compared to the others. In ESR-DAS28 for remission, tofacitinib achieved the best result. Regarding the safety of all jakinibs, peficitinib, baricitinib and filgotinib did not register deaths in their studies unlike tofacitinib that presented 11 deaths. Despite all benefits of jakinibs, the use in patients with severe liver and kidney disease should be avoided. CONCLUSIONS: Jakinibs in monotherapy or in combination with methotrexate can be considered a viable alternative in the treatment of moderate-to-severe RA. Even after failures with combination of cDMARDS and bDMARDS, jakinibs demonstrated efficacy.

Clinical pharmacology of filgotinib in the treatment of rheumatoid arthritis: current insights.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic autoimmune disease, whose natural course has been deeply modified thanks to the development of new therapeutic approaches. The Janus kinase inhibitors (Jakinibs) represent the newest class of drugs introduced for treating RA. Among these, Filgotinib (FIL) has been developed as Janus kinase1 (JAK1) selective inhibitor, specifically targeting key pro-inflammatory mediators in RA pathogenesis. AREAS COVERED: This narrative review provides an overview on FIL as new therapeutic approach for RA, with focus on its pharmacological properties, clinical efficacy, and safety profile. The following electronic databases were adopted for the study search: PubMed, Google Scholar, ClinicalTrials.gov and Abstract archive from the American College of Rheumatology and the European Alliance of Associations for Rheumatology. EXPERT OPINION: The phase II and phase III randomized controlled trials (RCTs) performed so far and their long-term extensions showed a comparable clinical efficacy of FIL to biologic treatments, with an acceptable safety profile. Thanks to these data, FIL was approved in Europe and Japan for the treatment of active RA, increasing the spectrum of therapeutic approaches and improving the possibility of a more tailored therapeutic strategy. Real-life data and head-to-head clinical trials will be needed to confirm its efficacy and safety.

Thromboembolic Adverse Drug Reactions in Janus Kinase (JAK) Inhibitors: Does the Inhibitor Specificity Play a Role?

Recent advances in immunology enabled the characterization of several signal transmitting pathways responsible for proper cytokine and chemokine signaling. Among them, Janus kinases (JAKs) are essential components of receptor activation systems. The discovery of JAK kinases enabled the synthesis of JAK kinase inhibitors (JAKi or Jakinibs), which have proven to be efficacious in the treatment of hematologic malignancies and several rheumatological disorders and continue to be investigated in many clinical indications. Blocking multiple cytokines belonging to several cytokine families with a single small molecule may, however, create a potential risk for the patients. Recently, a higher risk of thromboembolic complications, namely, deep vein thrombosis and pulmonary embolism, has been recognized as the main concern during treatment with Jakinibs. At present, it is not entirely clear whether this increased risk is related to direct cytokine blockade, the presence of concomitant diseases in treated patients or other unknown circumstances that work together to increase the risk of this side effect. In this review, we discuss data on the risk of thromboembolic side effects, with special emphasis on the mechanism that may be responsible for this increased risk. Many indirect data indicate that higher thromboembolic risk may be related to the specificity of JAK inhibitor action, such that preferentially blocking one signaling pathway upsets the balance between pro and anti-thrombotic activities.

Autocitrullination of PAD4 does not alter its enzymatic activity: In vitro and in silico studies.

BACKGROUND: Protein arginine deiminase 4 (PAD4) is an enzyme capable of converting arginine (positively charged residue) into citrulline (neutral residue). PAD4 is a promiscuous enzyme since it citrullinates various substrates, including small peptides, large proteins and itself. The effect of autocitrullination on PAD4 activity remains controversial and inconclusive. We hypothesized that PAD4 autocitrullination may influence the activity of PAD4 by indirectly altering its binding to substrate. METHODS: We employed mass spectrometry analysis to study the process of autocitrullination. The kinetics of citrullination of PAD4 and citrullinated PAD4 (citPAD4) towards substrates of different sizes (0.17-15.4 kDa), i.e. free arginine, a peptidyl substrate, and histone H3, were studied by colorimetric assay and Western blotting. Molecular dynamics (MD) simulations were performed to investigate structural dynamic and binding properties of PAD4/citPAD4 in the absence and presence of substrates. RESULTS: We observed that 23/27 arginine residues in PAD4 (85 %) can be citrullinated, including R372, R374 and R639 located near the substrate binding pocket. PAD4 and citPAD4 expressed comparable enzymatic activities towards different substrates. In agreement with experimental results, MD simulations indicated that autocitrullination does not change the shape of the substrate binding pocket and PAD4/citPAD4 exhibited comparable binding free energy with a H3-derived peptidyl substrate (6-TARKS-10). CONCLUSION: While the effect of autocitrullination on PAD4 activity thus far remained unclear and controversial, here we have demonstrated that autocitrullination does not affect the activity of PAD4. Thus, the regulation of PAD4 activity is probably not controlled by autocitrullination but likely by other mechanisms that need further investigation.

JNK pathway-associated phosphatase associates with rheumatoid arthritis risk, disease activity, and its longitudinal elevation relates to etanercept treatment response.

BACKGROUND: This study aimed to investigate the relationship of serum JNK pathway-associated phosphatase (JKAP) expression with rheumatoid arthritis (RA) risk and clinical features, also to explore the longitudinal change of JKAP during etanercept treatment and its relationship with etanercept treatment response in RA patients. METHODS: A total of 87 RA patients and 44 healthy controls (HCs) were enrolled; then, their JKAP expression in serum was determined by enzyme-linked immunosorbent assay (ELISA). Among 87 RA patients, 42 cases further received the 24-week etanercept treatment; then, their JKAP level in serum (detected by ELISA) and clinical response (evaluated by disease activity score in 28 joints (DAS28) score) were evaluated at week 4 (W4), week 12 (W12), and week 24 (W24) after initiation of etanercept treatment. RESULTS: JKAP expression was decreased in RA patients compared to HCs, which disclosed a good predictive value for RA risk. JKAP expression was negatively associated with tender joint count, swollen joint count, erythrocyte sedimentation rate, C-reactive protein, and DAS28 in RA patients, respectively. For RA patients who received 24-week etanercept treatment, their clinical response rate was 0.0%, 33.3%, 50.0%, and 69% at W0, W4, W12, and W24, respectively. Importantly, JKAP was gradually increased during etanercept treatment, whose longitudinal elevation positively related to etanercept treatment response in RA patients. CONCLUSION: Circulating JKAP links with decreased RA risk and mild disease activity, whose longitudinal elevation positively relates to etanercept treatment response.

Endophilin A2 deficiency protects rodents from autoimmune arthritis by modulating T cell activation.

The introduction of the CTLA-4 recombinant fusion protein has demonstrated therapeutic effects by selectively modulating T-cell activation in rheumatoid arthritis. Here we show, using a forward genetic approach, that a mutation in the SH3gl1 gene encoding the endocytic protein Endophilin A2 is associated with the development of arthritis in rodents. Defective expression of SH3gl1 affects T cell effector functions and alters the activation threshold of autoreactive T cells, thereby leading to complete protection from chronic autoimmune inflammatory disease in both mice and rats. We further show that SH3GL1 regulates human T cell signaling and T cell receptor internalization, and its expression is upregulated in rheumatoid arthritis patients. Collectively our data identify SH3GL1 as a key regulator of T cell activation, and as a potential target for treatment of autoimmune diseases.

Association of altered folylpolyglutamate synthetase pre-mRNA splicing with methotrexate unresponsiveness in early rheumatoid arthritis.

OBJECTIVES: An efficient pharmacological response to MTX treatment in RA patients relies on the retention and accumulation of intracellular MTX-polyglutamates catalysed by the enzyme folylpolyglutamate synthetase (FPGS). We recently identified a partial retention of FPGS intron 8 (8PR) as a prominent splice variant conferring FPGS dysfunction and decreased MTX polyglutamylation in acute lymphoblastic leukaemia. Here, we explored the association between FPGS 8PR levels and lack of MTX responsiveness in RA patients. METHODS: Thirty-six patients undergoing MTX treatment were enrolled from the Combinatie behandeling Reumatoide Artritis (COBRA)-light trial. RNA was isolated from blood samples at baseline, 13 weeks and 26 weeks of therapy, from patients in either COBRA-light (n = 21) or COBRA (n = 15) treatment arms. RT-qPCR analysis was used to assess RNA levels of FPGS 8PR over wild-type FPGS (8WT). RESULTS: In the COBRA-light treatment arm, higher baseline ratios of 8PR/8WT were significantly associated with higher 44-joint disease activity score (DAS44) at 13 and 26 weeks. Higher baseline ratios of 8PR/8WT also trended towards not obtaining low disease activity (DAS <1.6) and becoming a EULAR non-responder at 13 and 26 weeks. In the COBRA-treatment arm, a significant association was observed between high baseline 8PR/8WT ratios and higher DAS44 score at 26 weeks. Higher 8PR/8WT ratios were associated with non-response at week 26 based on both low disease activity and EULAR criteria. CONCLUSION: This study is the first to associate alterations in FPGS pre-mRNA splicing levels with reduced responsiveness to MTX treatment in RA patients. TRIAL REGISTRATION: ISRCTN55552928.