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Funder: GlaxoSmithKline; doi: http://dx.doi.org/10.13039/100004330, BACKGROUND: Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of inflammation through cell death and proinflammatory cytokine production. This multicenter, randomized, double-blind (sponsor-unblinded), placebo-controlled, experimental medicine study evaluated the safety, pharmacokinetics (PK), and preliminary efficacy of GSK2982772, a RIPK1 inhibitor, in moderate to severe rheumatoid arthritis (RA). METHODS: Patients with moderate to severe RA who had received ≥12 weeks' stable-dose conventional synthetic disease-modifying antirheumatic drug (csDMARD) therapy were randomized (2:1) to GSK2982772 60 mg or placebo orally 2 or 3 times daily for 84 days. Safety, PK, disease activity, joint damage, and pharmacodynamic (PD) biomarkers were assessed at days 43 and 85. RESULTS: A total of 52 patients were randomized (placebo, 18; GSK2982772, 34). Adverse events (AEs) were reported in 13 (72%) in patients in the placebo group (n = 3 b.i.d; n = 10 t.i.d.) and 20 (61%) in the GSK2982772 group (n = 3 b.i.d; n = 17 t.i.d.). All treatment-related AEs were mild/moderate, except one severe case of alopecia areata at day 49 and retinal vein thrombosis at day 66 (which led to withdrawal from the study) in patients receiving GSK2982772 t.i.d. Disease Activity Score in 28 Joints-C-reactive protein (DAS28-CRP) scores, ACR20/50/70 response, and rates of low disease activity and remission were similar between placebo and GSK2982772 arms. CONCLUSIONS: These results suggest that inhibition of RIPK1 activity at the GSK2982772 exposure levels evaluated do not translate into meaningful clinical improvement of RA. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02858492 . Registered 8 August 2016.

Background CSF-1 or IL-34 stimulation of CSF1R promotes macrophage differentiation, activation and osteoclastogenesis, and pharmacological inhibition of CSF1R is beneficial in animal models of arthritis. The objective of this study was to determine the relative contributions of CSF-1 and IL-34 signaling to CSF1R in RA. Methods CSF-1 and IL-34 were detected by immunohistochemical and digital image analysis in synovial tissue from 15 biological-naïve rheumatoid arthritis (RA) , 15 psoriatic arthritis (PsA) and 7 osteoarthritis (OA) patients . Gene expression in CSF-1- and IL-34-differentiated human macrophages was assessed by FACS analysis and quantitative PCR. RA synovial explants were incubated with CSF-1, IL-34, control antibody (Ab), or neutralizing/blocking Abs targeting CSF-1, IL-34, or CSF1R. The effect of a CSF1R-blocking Ab was examined in murine collagen-induced arthritis (CIA). Results CSF-1 (also known as M-CSF) and IL-34 expression was similar in RA and PsA synovial tissue, but lower in controls (P

Background In rheumatoid arthritis (RA) synovial tissue (ST) angiogenesis can be observed already in the earliest phase of disease. The chemokine CXCL12, which is induced via the non-canonical nuclear factor-kappaB (NF-kB) pathway, plays an important role in angiogenesis, lymphocyte transendothelial migration, and the homing of endothelial progenitor cells. Therefore, the non-canonical pathway, with its key mediator NF-kB inducing kinase (NIK), may play an important role in pathological angiogenesis and the perpetuation of synovial inflammation in RA.

Background The Autoimmune Regulator (AIRE) is a transcription factor that is involved in the negative selection of self-reactive thymocytes in the thymus and is pivotal in the establishment of central tolerance. Recently, AIRE protein has also been detected in peripheral lymphoid organs, predominantly in dendritic cells (DC). In these peripheral sites, AIRE was found to regulate the expression of a group of tissue-specific antigens, suggesting that peripheral AIRE may play a complementary role in tolerance induction. It is currently unknown whether AIRE may play a role in inflamed tissues associated with ectopic lymphoid neogenesis, such as rheumatoid arthritis (RA) synovial tissue (ST).

Introduction: Synovial tissue macrophages play a key role in chronic inflammatory arthritis, but the contribution of different macrophage subsets in this process remains largely unknown. The main in vitro polarized macrophage subsets are classically (M1) and alternatively (M2) activated macrophages, the latter comprising interleukin (IL)-4 and IL-10 polarized cells. Here, we aimed to evaluate the polarization status of synovial macrophages in spondyloarthritis (SpA) and rheumatoid arthritis (RA). Methods: Expression of polarization markers on synovial macrophages, peripheral blood monocytes, and in vitro polarized monocyte-derived macrophages from SpA versus RA patients was assessed by immunohistochemistry and flow cytometry, respectively. The polarization status of the intimal lining layer and the synovial sublining macrophages was assessed by double immunofluorescence staining. Results: The expression of the IL-10 polarization marker cluster of differentiation 163 (CD163) was increased in SpA compared with RA intimal lining layer, but no differences were found in other M1 and M2 markers between the diseases. Furthermore, no significant phenotypic differences in monocytes and in vitro polarized monocyte-derived macrophages were seen between SpA, RA, and healthy controls, indicating that the differential CD163 expression does not reflect a preferential M2 polarization in SpA. More detailed analysis of intimal lining layer macrophages revealed a strong co-expression of the IL-10 polarization markers CD163 and cluster of differentiation 32 (CD32) but not any of the other markers in both SpA and RA. In contrast, synovial sublining macrophages had a more heterogeneous phenotype, with a majority of cells co-expressing M1 and M2 markers. Conclusions: The intimal lining layer but not synovial sublining macrophages display an IL-10 polarized-like phenotype, with increased CD163 expression in SpA versus RA synovitis. These differences in the distribution of the polarized macrophage subset may contribute to the outcome of chronic synovitis

Background Pathological angiogenesis can be observed in rheumatoid arthritis (RA) synovial tissue (ST) already in the earliest phase of disease and may be critical in the switch from acute to chronic inflammation. The chemokine CXCL12 is regulated by non-canonical NF- Bs ignaling and stimulates angiogenesis in endothelial cells (EC). Therefore, the non-canonical NF- Bp athway, with its key mediator NF- Bi nducing kinase (NIK), may play an important role in pathological angiogenesis leading to the perpetuation of synovial inflammation in RA.

Background and objectives CD55 (decay-accelerating factor) is a complement-regulatory protein, which is highly expressed by fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leucocytes. We recently showed that lack of either CD55 or CD97 ameliorates disease in murine collagen-induced and K/BxN serum transfer models of arthritis.1 Little is known regarding the regulation of CD55 expression in FLS. We therefore investigated the effect of toll-like receptors ligation and pro-inflammatory cytokines on CD55 expression. Materials and methods Synovial fibroblasts, obtained from biopsy samples of arthritis patients, were cultured and stimulated with cytokines (TNF, IFNγ, IL-1β, IL-6, IFNα) or TLR ligands (LTA, poly (I:C), LPS, imiquimod, CpG). Expression of CD55 was measured by flow cytometry using domain-specific monoclonal antibodies and recombinant CD97-loaded fluorescent beads. Chloroquine was used to inhibit TLR3 activity. Upregulation and functionality of dsRNA sensors in response to poly (I:C) or 59-triphosphate RNA was analysed by PCR. Apoptosis was measured by PI/annexinV staining and was blocked with the pan-caspase inhibitor Q-VD-OPH. Results Cultured synovial fibroblasts of patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis, and spondylarthritis express equal amount of CD55. Stimulation of RA-FLS with IL-1β (p=0.02) and poly (I:C) (p=0.001) induced a significant upregulation of CD55. Engagement of TLR3 by the dsRNA analog poly (I:C) was confirmed using chloroquine, an inhibitor of endosomal acidification that impairs TLR3 signaling. Synovial fibroblasts also expressed the cytoplasmic dsRNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). Stimulation of these receptors with either poly (I:C) or 59-triphosphate RNA induced CD55 expression, but, in case of MDA5, also induced significant cell death (p Conclusions We identify dsRNA as a potent inducer of CD55 upregulation on synovial fibroblasts. Our findings suggest that CD55 induction by viral dsRNA or dsRNA may facilitate the accumulation of CD97-expressing inflammatory immune cells in the synovial tissue.

Introduction: With the development of increasing numbers of potential therapeutic agents in inflammatory disease comes the need for effective biomarkers to help screen for drug efficacy and optimal dosing regimens early in the clinical trial process. This need has been recognized by the Outcome Measures in Rheumatology Clinical Trials (OMERACT) group, which has established guidelines for biomarker validation. To seek a candidate synovial biomarker of treatment response in psoriatic arthritis (PsA), we determined whether changes in immunohistochemical markers of synovial inflammation correlate with changes in disease activity scores assessing 28 joints (ΔDAS28) or magnetic resonance imaging synovitis scores (ΔMRI) in patients with PsA treated with a biologic agent. Methods: Twenty-five consecutive patients with PsA underwent arthroscopic synovial biopsies and MRI scans of an inflamed knee joint at baseline and 12 weeks after starting treatment with either anakinra (first 10 patients) or etanercept (subsequent 15 patients) in two sequential studies of identical design. DAS28 scores were measured at both time points. Immunohistochemical staining for CD3, CD68 and Factor VIII (FVIII) was performed on synovial samples and scored by digital image analysis (DIA). MRI scans performed at baseline and at 12 weeks were scored for synovitis semi-quantitatively. The ΔDAS28 of the European League Against Rheumatism good response definition (>1.2) was chosen to divide patients into responder and non-responder groups. Differences between groups (Mann Whitney U test) and correlations between ΔDAS28 with change in immunohistochemical and MRI synovitis scores (Spearman’s rho test) were calculated. Results: Paired synovial samples and MRI scans were available for 21 patients (8 anakinra, 13 etanercept) and 23 patients (8 anakinra, 15 etanercept) respectively. Change in CD3 (ΔCD3) and CD68 expression in the synovial sublining layer (ΔCD68sl) was significantly greater in the disease responders compared to non-responders following treatment (P = 0.005 and 0.013 respectively). ΔCD3, but not ΔCD68 or ΔFVIII, correlated with both ΔDAS28 (r = 0.49, P = 0.025) and ΔMRI (r = 0.58, P = 0.009). Conclusions: The correlation of ΔCD3 with ΔDAS28 and ΔMRI following biologic treatment in this cohort contributes to the validation of ΔCD3 as a synovial biomarker of disease response in PsA, and supports the further evaluation of ΔCD3 for predictive properties of future clinical outcomes.

Introduction: Preclinical work has suggested that IL-1 plays a critical role in the pathogenesis of rheumatoid arthritis (RA). The objective of the present study was to determine the effect of a long-acting IL-1 receptor inhibitor, AMG 108, in a double-blind, placebo-controlled, parallel-dosing study in patients with active RA who were receiving stable methotrexate (15 to 25 mg/week). Methods: Patients were randomized equally to receive placebo or 50, 125, or 250 mg AMG 108 subcutaneously every 4 weeks for 6 months. The primary efficacy endpoint was a 20% improvement in the American College of Rheumatology response (ACR20) at week 24; other efficacy endpoints included the ACR50, the ACR70, and the RA disease activity score (28-joint count Disease Activity Score) responses, patient-reported outcomes, and pharmacokinetic parameters. Safety endpoints included treatment-emergent adverse events (AEs), infectious AEs, serious AEs, serious infections, injection site reactions, laboratory abnormalities, and antibodies to AMG 108. Results: Of 813 patients enrolled in the study, 204 patients were randomized to the 50 mg group, 203 to the 125 mg group, 203 to the 250 mg group, and 203 to placebo. At week 24, 40.4% of the 250 mg group, 36% of the 125 mg group, 30.9% of the 50 mg group, and 29.1% of the placebo group achieved an ACR20 (P = 0.022, 250 mg vs. placebo). Of the individual ACR components, numerical dose-dependent improvements were only seen in tender joint counts, pain (visual analog scale), and the acute phase reactants, erythrocyte sedimentation rate and C-reactive protein. No dose-related increase was observed in the incidence of treatment-emergent AEs. No deaths were reported, and the incidence of AEs and infections, serious AEs and infections, and withdrawals from study for safety were similar in the AMG 108 and placebo groups. Conclusions: This large double-blind randomized trial with a long-acting IL-1 receptor blocker, AMG 108, is consistent with the experience of other IL-1 blockers, represents a definitive experiment showing that IL-1 inhibition provides only moderate symptomatic amelioration of arthritis activity in the majority of RA patients, and provides an answer to a question that has been discussed for many years in the rheumatologic community. Trial Registration: ClinicalTrials.gov NCT00293826.

Introduction: The Rho family GTPase Rac1 regulates cytoskeletal rearrangements crucial for the recruitment, extravasation and activation of leukocytes at sites of inflammation. Rac1 signaling also promotes the activation and survival of lymphocytes and osteoclasts. Therefore, we assessed the ability of a cell-permeable Rac1 carboxy-terminal inhibitory peptide to modulate disease in mice with collagen-induced arthritis (CIA).Methods: CIA was induced in DBA/1 mice, and in either early or chronic disease, mice were treated three times per week by intraperitoneal injection with control peptide or Rac1 inhibitory peptide. Effects on disease progression were assessed by measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. Serum levels of anti-collagen type II antibodies were measured by enzyme-linked immunosorbent assay. T-cell phenotypes and activation were assessed by fluorescence-activated cell sorting analysis. Results were analyzed using Mann-Whitney U and unpaired Student t tests.Results: Treatment of mice with Rac1 inhibitory peptide resulted in a decrease in paw swelling in early disease and to a lesser extent in more chronic arthritis. Of interest, while joint destruction was unaffected by Rac1 inhibitory peptide, anti-collagen type II antibody production was significantly diminished in treated mice, in both early and chronic arthritis. Ex vivo, Rac1 inhibitory peptide suppressed T-cell receptor/CD28-dependent production of tumor necrosis factor α, interferon γ and interleukin-17 by T cells from collagen-primed mice, and reduced induction of ICOS and CD154, T-cell costimulatory proteins important for B-cell help.Conclusions: The data suggest that targeting of Rac1 with the Rac1 carboxy-terminal inhibitory peptide may suppress T-cell activation and autoantibody production in autoimmune disease. Whether this could translate into clinically meaningful improvement remains to be shown.

Cellular activation, proliferation and survival in chronic inflammatory diseases is regulated not only by engagement of signal trans-duction pathways that modulate transcription factors required for these processes, but also by epigenetic regulation of transcription factor access to gene promoter regions. Histone acetyl trans-ferases coordinate the recruitment and activation of transcription factors with conformational changes in histones that allow gene promoter exposure. Histone deacetylases (HDACs) counteract histone acetyl transferase activity through the targeting of both histones as well as nonhistone signal transduction proteins important in inflammation. Numerous studies have indicated that depressed HDAC activity in patients with inflammatory airway diseases may contribute to local proinflammatory cytokine production and diminish patient responses to corticosteroid treatment. Recent observations that HDAC activity is depressed in rheumatoid arthritis patient synovial tissue have predicted that strategies restoring HDAC function may be therapeutic in this disease as well. Pharmacological inhibitors of HDAC activity, however, have demonstrated potent therapeutic effects in animal models of arthritis and other chronic inflammatory diseases. In the present review we assess and reconcile these outwardly paradoxical study results to provide a working model for how alterations in HDAC activity may contribute to pathology in rheumatoid arthritis, and highlight key questions to be answered in the preclinical evaluation of compounds modulating these enzymes.

BACKGROUND: Mannose-binding lectin (MBL) is an innate immune protein. The aim of our study was to determine whether genetically determined MBL deficiency is associated with susceptibility to juvenile rheumatoid arthritis (JRA) and whether MBL2 genotypes are associated with JRA severity. METHODS: In a retrospective cohort study of 218 patients with polyarthritis (n = 67) and oligoarthritis (n = 151), clinical and laboratory disease variables were obtained by clinical examination and chart reviews. Healthy Caucasian adults (n = 194) served as control individuals. MBL2 gene mutations were determined by Taqman analysis to identify genotypes with high, medium and low expression of MBL. Functional MBL plasma concentrations were measured using enzyme-linked immunosorbent assay. Associations between clinical and laboratory variables and MBL2 genotypes were determined by Kruskal-Wallis and chi2 tests. RESULTS: MBL2 genotype frequencies were similar in polyarthritis and oligoarthritis patients as compared with control individuals. MBL plasma concentrations were associated with the high, medium and low MBL genotype expression groups (P < 0.01). In polyarthritis patients, the presence of low-expressing (deficient) MBL2 genotypes was associated with early age at onset of disease (P = 0.03). In oligoarthritis patients, patients with low-expressing MBL2 genotypes were more often in remission (81%) than patients in the medium (54%) and high (56%) genotype groups (P = 0.02). The remaining clinical and laboratory variables, such as arthritis severity index, presence of radiographic erosions and antinuclear antibody positivity, were not associated with MBL2 genotypes. CONCLUSION: Genetically determined MBL deficiency does not increase susceptibility to JRA, but MBL deficiency is associated with a younger age at onset of juvenile polyarthritis. On the other hand, MBL-deficient children with juvenile oligoarthritis are more often in remission. Therefore, MBL appears to play a dual role in JRA