Troy Noordenbos

Interleukin-17–positive mast cells contribute to synovial inflammation in spondylarthritis

OBJECTIVE Studies comparing spondylarthritis (SpA) to rheumatoid arthritis (RA) synovitis suggest that innate immune cells may play a predominant role in the pathogenesis of SpA. Recent observations have indicated a marked synovial mast cell infiltration in psoriatic SpA. We therefore undertook the present study to investigate the potential contribution of mast cells to synovial inflammation in SpA. METHODS Synovial tissue and fluid were obtained from patients with either nonpsoriatic or psoriatic SpA (n=82) and patients with RA (n=50). Synovial biopsy tissue was analyzed by immunostaining and used in ex vivo cultures. Synovial fluid was analyzed by enzyme-linked immunosorbent assay. RESULTS We observed a strong and specific increase of c-Kit-positive mast cells in the synovium from patients with SpA compared to the synovium from patients with RA synovitis, which was independent of disease subtype (nonpsoriatic versus psoriatic), disease duration, and treatment. Staining of mast cell granules, analysis of synovial fluid, and results in ex vivo tissue culture did not indicate increased degranulation in SpA synovitis. However, mast cells expressed significantly more interleukin-17 (IL-17) in SpA than in RA synovitis, and mast cells constituted the major IL-17-expressing cell population in the SpA synovium. Ex vivo targeting of synovial mast cells with the c-Kit inhibitor imatinib mesylate significantly decreased the production of IL-17 as well as other proinflammatory cytokines in synovial tissue cultures. Analysis of paired pre- and posttreatment synovial tissue samples indicated that the mast cell/IL-17 axis in SpA was not modulated by effective tumor necrosis factor (TNF) blockade. CONCLUSION The specific and TNF-independent increase in IL-17-expressing mast cells may contribute to the progression of synovial inflammation in peripheral SpA.

Absence of a classically activated macrophage cytokine signature in peripheral spondylarthritis, including psoriatic arthritis.

OBJECTIVE Peripheral spondylarthritis (SpA) is characterized by macrophages that express CD163, a marker of alternative activation (M2). The purpose of this study was to assess whether this differential infiltration with macrophage subsets was associated with a different local inflammatory milieu in SpA as compared with rheumatoid arthritis (RA). METHODS The effect of SpA and RA synovial fluid (SF) on macrophage polarization was tested in vitro on normal peripheral blood monocytes. SF levels of classically activated macrophage (M1)-derived and alternatively activated macrophage (M2)-derived mediators were analyzed by enzyme-linked immunosorbent assay and multiparameter Luminex bead assay in 47 patients with non-psoriatic SpA, 55 with RA, and 15 with psoriatic arthritis (PsA). Paired synovial biopsy samples were analyzed histologically. RESULTS SF from SpA patients promoted preferential expression of the M2 markers CD163 and CD200R in vitro, even if SF levels of the prototypical M2-polarizing factors (interleukin-4 [IL-4], IL-13, and IL-10) were not increased as compared with those in RA SF. Despite a similar degree of overall joint inflammation in SpA and RA, SpA synovitis displayed strongly reduced SF levels of M1-derived, but not M2-derived, mediators, such as tumor necrosis factor alpha (TNFalpha), IL-1beta, IL-12p70, and interferon-gamma-inducible protein 10. SF levels of M1-derived mediators correlated well with peripheral joint inflammation in RA, but neither these mediators nor IL-1alpha and IL-17 did so in SpA. Of interest, the SF cytokine profile in PsA, a more destructive subtype of SpA, was similar to that in non-psoriatic SpA. CONCLUSION The local inflammatory milieu is clearly different in SpA as compared with RA peripheral arthritis. Synovitis in SpA, including that in PsA, is characterized by a selective decrease in M1-derived proinflammatory mediators, such as TNFalpha and IL-1beta.

Inflammatory pathways in spondyloarthritis

Spondyloarthritis is the second most common form of chronic inflammatory arthritis and a unique hallmark of the disease is pathologic new bone formation. Several cytokine pathways have been genetically associated with ankylosing spondylitis (AS), the prototypic subtype of SpA, and additional evidence from human and animal studies support a role of these pathways in the disease. TNF has a key role in SpA as blockade significantly reduces inflammation and destruction, however the treatment does not halt new bone formation. New insights into the TNF pathway were recently obtained from an animal model specifically overexpressing the transmembrane form of TNF. This model leads to axial and peripheral new bone formation which is not seen in soluble TNF overexpression models, indicating different pathogenic roles of soluble and transmembrane TNF in arthritis development. Besides TNF, the IL-23/IL-17 axis is emerging as an important inflammatory pathway in SpA, as a SNP in the IL-23R locus has been associated with developing AS, mice overexpressing IL-23 develop SpA-like features and IL-17 blockade has been shown to be efficacious for AS patients in a phase II trial. In this review, we focus on the cytokine pathways that have recently been genetically associated with SpA, i.e. TNF, IL-1, IL-6 and IL-23/IL-17. We review the current genetic, experimental and human in vivo data available and discuss how these different pathways are involved in the pathophysiology of SpA. Additionally, we discuss how these pathways relate to the pathogenic new bone formation in SpA.

The abundant synovial expression of the RANK/RANKL/Osteoprotegerin system in peripheral spondylarthritis is partially disconnected from inflammation

OBJECTIVE Spondylarthritis (SpA) and rheumatoid arthritis (RA) have different patterns of bone damage, with more pronounced bone erosions in RA. The RANK/RANKL/osteoprotegerin (OPG) system plays a central role in bone resorption by promoting the maturation and activation of osteoclasts. To assess the potential role of this system in the distinct bone phenotype, we studied the synovial expression of these mediators in SpA and RA peripheral synovitis. METHODS Synovial biopsy specimens were obtained from the actively inflamed peripheral joints of 35 patients with SpA and 19 patients with RA. Paired synovial biopsy samples were obtained from 24 patients with SpA after tumor necrosis factor alpha (TNFalpha) blockade. Synovial tissue sections were immunostained for RANKL, OPG, RANK, and TRAP and assessed by semiquantitative scoring and digital image analysis. RESULTS After extensive validation of the reactivity and specificity of the antibodies, we demonstrated the abundant expression of RANKL and OPG in SpA synovitis. RANKL was expressed by both fibroblast-like synoviocytes and sublining T lymphocytes. RANK-positive osteoclast precursors but no mature TRAP-positive osteoclasts were present in the inflamed tissue. The expression of these mediators was not different between patients with nonpsoriatic SpA, patients with psoriatic SpA, and patients with RA, was not related to the degree of systemic or local inflammation, and was not significantly modulated by highly effective treatment with TNFalpha blockers. Only the subset of patients with the best systemic response to TNFalpha blockade had decreased RANKL expression in the intimal lining layer. CONCLUSION The relative protection against bone erosions in SpA cannot be explained by qualitative or quantitative differences in the synovial expression of RANKL, OPG, and RANK. The abundant expression of these factors in SpA peripheral synovitis is largely disconnected from systemic and local inflammation.

Intimal lining layer macrophages but not synovial sublining macrophages display an IL-10 polarized-like phenotype in chronic synovitis.

IntroductionSynovial 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).MethodsExpression 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.ResultsThe 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.ConclusionsThe 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.

Distinct synovial immunopathology in Behçet disease and psoriatic arthritis.

IntroductionThe aim of the study was to investigate synovial immunopathology differences between early Behçet disease (BD) and psoriatic arthritis (PsA).MethodsNeedle arthroscopy of an inflamed knee joint was performed in patients with early untreated BD (n = 8) and PsA (n = 9). Synovial fluid (SF) was collected for cytokines, perforin, and granzyme analysis. Eight synovial biopsies per patient were obtained for immunohistochemical analysis of the cellular infiltrate (T cells, natural killer cells, macrophages, B cells, plasma cells, mast cells, and neutrophils), blood vessels as well as expression of perforin and granzyme. The stained slides were evaluated by digital image analysis.ResultsThe global degree of synovial inflammation was similar in the two types of arthritis. In the analysis of the innate immune cell infiltration, there was a striking neutrophilic inflammation in BD synovitis whereas PsA displayed significantly higher numbers of cells positive for c-kit, a marker of mast cells. As for lymphocytes, CD3+ T cells, but neither CD20+ B cells nor CD138+ plasma cells, were significantly increased in BD versus PsA. Further analysis of the T-lymphocyte population showed no clear shift in CD4/CD8 ratio or Th1/Th2/Th17 profile. The SF levels of perforin, an effector molecule of cytotoxic cells, displayed a significant four- to fivefold increase in BD.ConclusionsThis systematic comparative analysis of early untreated synovitis identifies neutrophils and T lymphocytes as important infiltrating cell populations in BD. Increased levels of perforin in BD suggest the relevance of cytotoxicity in this disease.

Tumor necrosis factor α drives cadherin 11 expression in rheumatoid inflammation

OBJECTIVE Cadherin 11 expressed on fibroblast-like synoviocytes (FLS) plays a key role in normal synovial architecture. The purpose of this study was to examine the expression of cadherin 11 in human synovitis. METHODS Cadherin 11 expression in synovial biopsy samples from patients with various types of arthritis and in lung biopsy samples from patients with interstitial pneumonitis (IP) was examined by immunostaining. The regulation of cadherin 11 expression in human FLS was assessed by quantitative reverse transcription-polymerase chain reaction analysis and Western blotting. Therapeutic modulation of synovial cadherin 11 was assessed before and after effective antiinflammatory therapy. RESULTS Abundant staining for cadherin 11 was seen in the intimal lining layer and the synovial sublining in inflamed tissues, with discrete staining in noninflammatory osteoarthritic (OA) tissues. The pattern and degree of immunostaining were similar in tissues from patients with rheumatoid arthritis (RA), nonpsoriatic spondylarthritis (SpA), psoriatic arthritis (PsA), and inflammatory OA. Clear staining for cadherin 11 was also observed in lung tissues from RA-associated IP and idiopathic IP patients, but was very limited in normal lung tissue. Cadherin 11 staining correlated strongly with the degree of inflammatory infiltration of the tissue, as well as with the C-reactive protein level and the erythrocyte sedimentation rate in RA patients. In vitro, cadherin 11 expression by FLS was consistently up-regulated by tumor necrosis factor alpha (TNFalpha) at the protein, but not the messenger RNA, level. Cadherin 11 staining in vivo was strongly down-regulated by prednisone treatment in RA patients and by TNFalpha blockade in SpA patients. CONCLUSION Cadherin 11 expression is regulated by mediators of inflammation, such as TNFalpha. Since cadherin 11 plays an important role in cartilage destruction in experimental arthritis, down-modulation of cadherin 11 by potent antiinflammatory therapies in humans with arthritis may contribute to halting cartilage damage.

Disease-specific and inflammation-independent stromal alterations in spondylarthritis synovitis

OBJECTIVE The molecular processes driving the distinct patterns of synovial inflammation and tissue remodeling in spondylarthritis (SpA) as compared to rheumatoid arthritis (RA) remain largely unknown. Therefore, we aimed to identify novel and unsuspected disease-specific pathways in SpA by a systematic and unbiased synovial gene expression analysis. METHODS Differentially expressed genes were identified by pan-genomic microarray and confirmed by quantitative polymerase chain reaction and immunohistochemical analyses of synovial tissue biopsy samples from patients with SpA (n=63), RA (n=28), and gout (n=9). The effect of inflammation on gene expression was assessed by stimulating fibroblast-like synoviocytes (FLS) with synovial fluid and by analysis of synovial tissue samples at weeks 0 and 12 of etanercept treatment. RESULTS Using very stringent statistical thresholds, microarray analysis identified 64 up-regulated transcripts in patients with SpA synovitis as compared to those with RA synovitis. Pathway analysis revealed a robust myogene signature in this gene set. The myogene signature was technically and biologically reproducible, was specific for SpA, and was independent of disease duration, treatment, and SpA subtype (nonpsoriatic versus psoriatic). Synovial tissue staining identified the myogene expressing cells as vimentin-positive, prolyl 4-hydroxylase β-positive, CD90+, and CD146+ mesenchymal cells that were significantly overrepresented in the intimal lining layer and synovial sublining of inflamed SpA synovium. Neither in vitro exposure to synovial fluid from inflamed SpA joints nor in vivo blockade of tumor necrosis factor modulated the SpA-specific myogene signature. CONCLUSION These data identify a novel and disease-specific myogene signature in SpA synovitis. The fact that this stromal alteration appeared not to be downstream of local inflammation warrants further analysis of its functional role in the pathogenesis of the disease.

Human mast cells capture, store, and release bioactive, exogenous IL-17A

IL‐17A, a major proinflammatory cytokine, can be produced by a variety of leukocytes, but its exact cellular source in human inflammatory diseases remains incompletely understood. IL‐17A protein is abundantly found in mast cells in human tissues, such as inflamed synovium, but surprisingly, mechanistic murine studies failed to demonstrate IL‐17A production by mast cells. Here, we demonstrate that primary human tissue mast cells do not produce IL‐17A themselves but actively capture exogenous IL‐17A through receptor‐mediated endocytosis. The exogenous IL‐17A is stored in intracellular granules and can subsequently be released in a bioactive form. This novel mechanism confers to mast cells the capacity to steer IL‐17A‐mediated tissue inflammation by the rapid release of preformed cytokine.

OP0156 Targeting Synovial Mast Cells in Spondyloarthritis: A Proof-Of-Concept Study with Nilotinib A Tyrosine Kinase Inhibitor

Background Immunopathological studies on synovitis recently identified the mast cell as potential novel therapeutic target in spondyloarthritis (SpA).[1] Mast cells can be targeted by inhibiting the signalling of c-Kit, which is one of the targets of the tyrosine kinase inhibitor nilotinib. Objectives To evaluate the immunomodulating and clinical effects of nilotinib in the treatment of SpA. Methods 28 patients with active peripheral and/or axial SpA were included in a randomized, double-blind, placebo-controlled clinical trial. Patients were treated 1:1 with nilotinib or placebo for 12 weeks, followed by an open label extension for another 12 weeks. Paired synovial tissue biopsies, serum sampling and assessment of clinical symptoms were performed serially. Results In peripheral SpA (n=13) synovial inflammation was markedly reduced after 12 weeks of nilotinib treatment as evidenced by histopathology (decrease in number of infiltrating CD68+ and CD163+ macrophages and mast cells). Compared to placebo the mRNA expression of c-Kit as mast cell marker (p=0.037) and of pro-inflammatory cytokines such as IL-6 (p=0.024) were reduced. The improvement of synovial inflammation was paralleled by a decrease in serum biomarkers of inflammation such as C-reactive protein (CRP) from 9.2 (IQR 1.7-33.1) to 5.2 (IQR 1.7-25.1) mg/L (p=0.024) and calprotectin from 359.9 (IQR 183.3-484.9) to 287.9 (IQR 116.7-457.1) ng/mL (p=0.055). Also clinical parameters such as patients global assessment of disease activity (week 0: 52 (IQR 43-65) vs week 12: 21 (IQR 0-51) mm; p=0.031) and Ankylosing Spondylitis Disease Activity Score (ASDAS) (week 0: 2.2 (IQR 1.2-3.0) vs week 12: 1.1 (IQR 0.7-2.4); p=0.031) showed improvement upon 12 weeks of nilotinib but not placebo treatment, and this improvement was further augmented at week 24. In sharp contrast to peripheral SpA, neither serum biomarkers of inflammation nor clinical parameters improved upon nilotinib treatment in axial SpA. During the trial one serious adverse event occurred, which was considered unrelated to the study drug. There were no unexpected safety signals in comparison with published large scale data on nilotinib in chronic myeloid leukemia (CML). Conclusions This small proof-of-concept study supports the concept that mast cells can contribute to synovial inflammation in SpA and that tyrosine kinase inhibition targeting these cells has a biological and clinical immunomodulatory effect in peripheral but not axial SpA. These results support further clinical evaluation of nilotinib in larger clinical trials in pure peripheral SpA, as well as evaluation of other drugs targeting mast cells in SpA. References Noordenbos T, et al. Interleukin-17-positive mast cells contribute to synovial inflammation in spondylarthritis. Arthritis Rheum 2012;64:99-109. Acknowledgements We thank Novartis for the supply of the study medication for this investigator initiated and independent study. Disclosure of Interest J. Paramarta: None declared, M. Turina: None declared, T. Noordenbos: None declared, T. Heijda: None declared, I. Blijdorp: None declared, N. Yeremenko: None declared, D. Baeten Grant/research support: AbbVie, Centocor, Janssen-Cilag, MSD, Novartis, and Pfizer, Consultant for: AbbVie, BMS, Boehringer Ingelheim, Centocor, Janssen, MSD, Novartis, Pfizer, and UCB DOI 10.1136/annrheumdis-2014-eular.2823