Man Wai Tang

Autonomic Dysfunction Precedes Development of Rheumatoid Arthritis: A Prospective Cohort Study

Background Heart rate variability (HRV) is a validated method to establish autonomic nervous system (ANS) activity. Rheumatoid arthritis (RA) is accompanied by ANS imbalance. We hypothesized that ANS dysfunction may precede the development of RA, which would suggest that it plays a role in its etiopathogenesis. Methods First, we assessed HRV parameters in supine (resting) and upright (active) position in healthy subjects (HS, n = 20), individuals at risk of developing arthritis (AR subjects, n = 50) and RA patients (RA, n = 20). Next, we measured resting heart rate (RHR), a parasympathetic HRV parameter, in an independent prospective cohort of AR subjects (n = 45). We also evaluated expression levels of the parasympathetic nicotinic acetylcholine receptor type 7 (α7nAChR) on circulating monocytes. Findings Both AR subjects (68 beats per minute (bpm), interquartile range (IQR) 68–73) and RA patients (68 bpm, IQR 62–76) had a significantly higher RHR compared to HS (60 bpm, IQR 56–63). RHR was significantly higher at baseline in individuals who subsequently developed arthritis. Expression levels of α7nAChR were lower in AR subjects with RHR ≥ 70 bpm compared to those with RHR < 70 bpm, consistent with reduced activity of the parasympathetic cholinergic anti-inflammatory pathway. Interpretation These data support the notion that autonomic dysfunction precedes the development of RA.

Btk inhibition suppresses agonist-induced human macrophage activation and inflammatory gene expression in RA synovial tissue explants

Objectives Brutons tyrosine kinase (Btk) is required for B lymphocyte and myeloid cell contributions to pathology in murine models of arthritis. Here, we examined the potential contributions of synovial Btk expression and activation to inflammation in rheumatoid arthritis (RA). Materials and methods Btk was detected by immunohistochemistry and digital image analysis in synovial tissue from biologically naive RA (n=16) and psoriatic arthritis (PsA) (n=12) patients. Cell populations expressing Btk were identified by immunofluorescent double labelling confocal microscopy, quantitative (q-) PCR and immunoblotting. The effects of a Btk-specific inhibitor, RN486, on gene expression in human macrophages and RA synovial tissue explants (n=8) were assessed by qPCR, ELISA and single-plex assays. Results Btk was expressed at equivalent levels in RA and PsA synovial tissue, restricted to B lymphocytes, monocytes, macrophages and mast cells. RN486 significantly inhibited macrophage IL-6 production induced by Fc receptor and CD40 ligation. RN486 also reduced mRNA expression of overlapping gene sets induced by IgG, CD40 ligand (CD40L) and RA synovial fluid, and significantly suppressed macrophage production of CD40L-induced IL-8, TNF, MMP-1 and MMP-10, LPS-induced MMP-1, MMP-7 and MMP-10 production, and spontaneous production of IL-6, PDGF, CXCL-9 and MMP-1 by RA synovial explants. Conclusions Btk is expressed equivalently in RA and PsA synovial tissue, primarily in macrophages. Btk activity is needed to drive macrophage activation in response to multiple agonists relevant to inflammatory arthritis, and promotes RA synovial tissue cytokine and MMP production. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA and other inflammatory diseases.

Synovial IL-21/TNF-producing CD4+ T cells induce joint destruction in rheumatoid arthritis by inducing matrix metalloproteinase production by fibroblast-like synoviocytes

Bone and cartilage destruction is one of the key manifestations of rheumatoid arthritis (RA). Although the role of T helper (Th)17 cells in these processes is clear, the role of IL‐21–producing cells T cells has been neglected. We sought to investigate the role of IL‐21 in RA by focusing on the functional characteristics of the main producers of this cytokine, synovial CD4+IL‐21+ T cells. We show that the frequency of both synovial fluid (SF) CD4+IL‐21+ or CD4+IL‐21+TNF+ T cells in patients with RA was significantly higher compared with patients with psoriatic arthritis (PsA). The frequency of peripheral blood (PB) IL‐21+CD4+ T cells in patients with RA positively correlated with disease activity score 28 (DAS28), serum anticyclic citrullinated peptide (anti‐CCP) antibodies and IgM‐rheumatoid factor (IgM‐RF). IL‐21 levels in RA SF were associated with matrix metalloproteinase (MMP)‐1 and MMP‐3. Related to this, IL‐21 induced significantly the secretion of MMP‐1 and MMP‐3 in RA synovial biopsies. Sorted SF CD4+IL‐21+ T cells significantly induced the release of MMP‐1 and MMP‐3 by fibroblast‐like synoviocytes (FLS) compared with medium or CD4+IL‐21− T cells in a coculture system. Neutralization of both IL‐21 and TNF resulted in significantly less production of MMP by FLS. The results of this study indicate a new role for synovial CD4+IL‐21+TNF+ T cells in promoting synovial inflammation/joint destruction in patients with RA. Importantly, IL‐21 blockade in combination with anti‐TNF might be an effective therapy in patients with RA by inhibiting MMP‐induced inflammation/joint destruction.

The prolactin receptor is expressed in rheumatoid arthritis and psoriatic arthritis synovial tissue and contributes to macrophage activation

Objectives. Prolactin (PRL) is a lactation-inducing hormone with immunomodulatory properties and is found at elevated levels in the serum of patients with RA and other rheumatic diseases. The PRL receptor (PRLR) has been shown to be expressed by macrophages in atherosclerotic plaques. The aim of this study was to examine PRLR expression by synovial macrophages and its role in the regulation of macrophage activation. Methods. Serum monomeric 23 kDa PRL levels were measured in 119 RA patients using a fluoroimmunometric assay. PRLR expression was assessed in synovial tissue of 91 RA, 15 PsA and 8 OA patients by immunohistochemistry and digital image analysis. Double IF was used to identify PRLR-expressing cells. The effects of PRL on monocyte-derived macrophage gene expression were examined by quantitative real-time PCR and ELISA. Results. Serum PRL levels were similar in female and male RA patients. Median (interquartile range) PRLR expression was significantly higher (P < 0.05) in RA and PsA synovial tissue compared with OA. PRLR colocalized with synovial CD68+ macrophages and von Willebrand factor+ endothelial cells. In vitro, PRLR was prominently expressed in IFN-γ-and IL-10-polarized macrophages compared with other polarizing conditions. PRL by itself had negligible effects on macrophage gene expression, but cooperated with CD40L and TNF to increase expression of pro-inflammatory genes including IL-6, IL-8 and IL-12β. Conclusions. Synovial PRLR expression is enhanced in patients with inflammatory arthritis compared with OA, and PRL cooperates with other pro-inflammatory stimuli to activate macrophages. These results identify PRL and PRLR as potential new therapeutic targets in inflammatory arthritis.

Hormone, metabolic peptide, and nutrient levels in the earliest phases of rheumatoid arthritis—contribution of free fatty acids to an increased cardiovascular risk during very early disease

Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with changes in several hormones and metabolic peptides. Crosstalk between these factors and the immune system may be important for homeostasis during inflammation. Here, we studied the levels of hormones, metabolic peptides, and nutrients in individuals at risk for developing RA (at risk). In total, 18 hormones, metabolic peptides, and nutrients were measured in fasting serum samples from 45 autoantibody-positive individuals at risk, 22 RA patients, and 16 healthy subjects. Triglyceride (TG) levels were also measured in an independent validation cohort of 32 individuals at risk, 20 early arthritis patients, and 20 healthy controls. We found an elevated TG level in individuals at risk and significantly higher TG levels in RA patients compared to healthy controls. These results were confirmed in the validation cohort. Similarly, free fatty acid (FFA) levels showed an increase in individuals at risk and were significantly higher in RA patients compared to healthy controls. In RA patients, FFA levels were positively correlated with disease activity. Pancreatic polypeptide (PP) and norepinephrine levels were highly significantly increased in individuals at risk and RA patients compared to healthy controls. TG and FFA levels are increased in RA patients and positively correlated with disease activity parameters. The results presented here suggest a role for FFAs in the pathogenesis of RA. Furthermore, PP and norepinephrine may be a biomarker that could assist in the identification of individuals at risk.

1.57 Prolactin is locally produced in the synovium of patients with inflammatory arthritic diseases and promotes macrophage activation

Background and Objectives The sex hormone prolactin (PRL) has immunomodulatory properties, can be produced by immune cells, and elevated PRL serum levels have been reported in rheumatoid arthritis (RA) patients. Here we examined synovial expression of PRL and PRL receptor (PRLR) in patients with inflammatory arthritis, their expression in polarised macrophages from patients and healthy donors, and the effects of PRL on macrophage activation. Materials and Methods PRL levels in paired synovial fluid (SF) and peripheral blood of RA (n = 19), psoriatic arthritis (PsA, n = 13) and gout (n = 11) patients were measured using an immunofluorescent metric assay. PRL mRNA expression was measured in synovial tissue (ST) of RA (n = 25), PsA (n = 11) and gout (n = 12) patients, and in macrophages differentiated in RA, PsA, spondyloarthritis (SpA) and gout SF by qPCR. PRLR protein expression was determined in ST of RA (n = 19), PsA (n = 15) and osteoarthritis (OA, n = 9) patients by immunohistochemistry and detected in specific cell types by immunofluorescence. IL-6 production by IFN-y and IL-10 -differentiated macrophages following stimulation with CD40L or TNF in the absence or presence of PRL was measured by ELISA. Results PRL protein levels were similar in serum and SF of RA, PsA and gout patients, as was mRNA expression in RA, PsA and gout ST. Of interest, PRL mRNA expression significantly correlated with clinical disease parameters in PsA (DAS28, R = 0.729, P = 0.017) and RA (ESR, R = 0.424, P = 0.049). PRL expression was also detected in monocyte-derived macrophages from RA patients, and significantly higher (P≤0.01) in healthy donor macrophages differentiated in pooled SF of RA and PsA compared to SpA and gout SF. In RA SF-differentiated macrophages PRL production was increased by CD40L or IgG stimulation but not LPS or TNFα. Median (IQR) PRLR expression was significantly higher (P<0.05) in RA [0.06 (0.00-0.33)] and PsA [0.18 (0.00-1.67)] ST compared to OA [0.00 (0.00-0.02)], and there was no significant difference in PRLR expression between (pre/postmenopausal) females and males, independently of disease. PRLR expression was mainly colocalised with CD68+ macrophages and vWF+ endothelial cells. In vitro, PRLR was prominently expressed in IFN-y and IL-10 polarised monocyte-derived macrophages compared to macrophages polarised in GM-CSF, M-CSF or RA SF. In these macrophages, PRL stimulation significantly enhanced IL-6 production in response to TNFα or CD40L. Conclusions Our results provide the first evidence that PRL is produced locally in the synovium of patients with inflammatory arthritis, and contributes to the activation of macrophages in the presence of other inflammatory stimuli.

Rheumatoid arthritis and psoriatic arthritis synovial fluids stimulate prolactin production by macrophages

Prolactin (PRL) is a neuroendocrine hormone that can promote inflammation. We examined the synovial tissue and fluid levels of PRL in patients with inflammatory arthritis, PRL expression in differentiated Mϕs from patients with arthritis and from healthy donors, and the effects of different stimuli on PRL production by Mϕs. PRL levels were measured in paired synovial fluid (SF) and peripheral blood of patients with rheumatoid arthritis (RA, n = 19), psoriatic arthritis (PsA, n = 11), and gout (n = 11). Synovial‐tissue PRL mRNA expression was measured by quantitative PCR in patients with RA (n = 25), PsA (n = 11), and gout (n = 12) and in Mϕs differentiated in SF of patients with RA, PsA, other subtypes of spondyloarthritis (SpA), and gout. Synovial‐tissue PRL mRNA expression correlated significantly with clinical disease parameters in patients with RA and PsA, including erythrocyte sedimentation rate (ESR, r = 0.424; P = 0.049) and disease activity score evaluated in 28 joints (DAS28, r = 0.729; P = 0.017). Synovial‐tissue PRL expression was similar in RA, PsA, and gout. PRL mRNA expression was detected in monocyte‐derived Mϕs from patients with RA and was significantly higher (P ≤ 0.01) in Mϕs differentiated in pooled SF from patients with RA and PsA compared with SpA or gout. PRL production by Mϕ differentiation in the SF from patients with RA was not further regulated by stimulation with CD40L, IgG, LPS, or TNF. PRL is produced locally in the synovium of patients with inflammatory arthritis. The production of PRL by Mϕs was increased by unknown components of RA and PsA SF, where it could contribute to disease progression.

Class 3 semaphorins modulate the invasive capacity of rheumatoid arthritis fibroblast-like synoviocytes

Objective Class 3 semaphorins regulate diverse cellular processes relevant to the pathology of RA, including immune modulation, angiogenesis, apoptosis and invasive cell migration. Therefore, we analysed the potential role of class 3 semaphorins in the pathology of RA. Methods Protein and mRNA expression in RA synovial tissue, SF and fibroblast-like synoviocytes (FLS) were determined by immunoblotting and quantitative PCR (qPCR). RA FLS migration and invasion were determined using wound closure and transwell invasion assays, respectively. PlexinA1, neuropilin-1 and neuropilin-2 expression was knocked down using small interfering RNA (siRNA). Activation of FLS intracellular signalling pathways was assessed by immunoblotting. Results mRNA expression of semaphorins (Sema)3B, Sema3C, Sema3F and Sema3G was significantly lower in the synovial tissue of early arthritis patients at baseline who developed persistent disease compared with patients with self-limiting disease after 2 years follow-up. Sema3B and Sema3F expression was significantly lower in arthritis patients fulfilling classification criteria for RA compared with those who did not. FLS expression of Sema3A was induced after stimulation with TNF, IL-1β or lipopolysaccharides (LPS), while Sema3B and Sema3F expression was downregulated. Exogenously applied Sema3A induced the migration and invasive capacity of FLS, while stimulation with Sema3B or Sema3F reduced spontaneous FLS migration, and platelet-derived growth factor induced cell invasion, effects associated with differential regulation of MMP expression and mediated by the PlexinA1 and neuropilin-1 and -2 receptors. Conclusion Our data suggest that modulation of class 3 semaphorin signaling could be a novel therapeutic strategy for modulating the invasive behaviour of FLS in RA.

FRI0006 Class 3 Semaphorins Modulate The Invasive Capacity of Rheumatoid Arthritis Fibroblast-like Synoviocytes

Background The semaphorin family is a large group of proteins initially described in axon guidance. However, semaphorins also play a role in other processes such as the regulation of immunity, angiogenesis, apoptosis and cell migration and invasion. Moreover, semaphorins have been related with the pathogenesis of multiple sclerosis, myocarditis, atherosclerosis and cancer. However, the potential role of class 3 semaphorins in rheumatoid arthritis (RA) remains unknown. Objectives The aim of this study was to analyze the role of class 3 semaphorins in the pathology of RA. Methods mRNA expression of class 3 semaphorins in the synovial tissue of early, DMARD-naive arthritis patients was analyzed by (q)uantitative PCR. RA FLS were stimulated for 4h with IL-1β, TNF or LPS. and mRNA and expression of class 3 semaphorins was analyzed by qPCR. RA FLS were stimulated with Sema3A, Sema3B or Sema3F in the presence or absence of PDGF. Cell migration and invasion were determined using wound closure motility and transwell invasion assays, respectively. Matrix metalloproteinase mRNA and protein expression was determined by qPCR array and luminex. PlexinA1 and neuropilin-1 expression were knocked-down by siRNA-mediated silencing. ERK and Rac1 activation were determined by immunoblot. Results mRNA expression of class 3 semaphorins in the synovial tissue of early arthritis patients negatively and significantly correlated with the mRNA expression of inflammatory mediators and the disease activity parameters of these patients. Moreover, Sema3B, Sema3C, Sema3F and Sema3G mRNA expression were significantly lower in early arthritis patients who developed persistent disease compared with patients with self-limiting disease after 2 years follow-up. Sema3F and Sema3B expression were significantly lower in patients that developed RA after 2 years of follow-up compared to those who remained as undifferentiated arthritis patients. FLS expression of Sema3A was significantly induced after IL-1, TNF or LPS stimulation, while the expression of Sema3B and Sema3F was down-regulated. Functional assays showed that Sema3A significantly induced the migration and invasion of FLS. In contrast, Sema3B and Sema3F reduced spontaneous FLS migration and PDGF-induced cell invasion. These effects were associated withthe differential regulation of MMP-1, MMP-3 and MMP-8 by these semaphorins, and were mediated by the receptor PlexinA1 and the co-receptor NRP-1. Finally, we observed that class 3 semaphorins reduced the PDGF-induced ERK and Rac1 activation. Conclusions Our data demonstrate that class 3 semaphorins are differentially expressed in the synovium of early patients depending on the severity and the progression of the disease and that Sema3A, Sema3B and Sema3F play an important role in the invasive capacity of RA FLS. Together, class 3 semaphorins and their receptors could be useful biomarkers and promising therapeutic targets for the treatment of RA. Disclosure of Interest None declared

A7.8 Autonomic dysfunction in the preclinical phase of rheumatoid arthritis

Background and objectives Rheumatoid arthritis (RA) is accompanied by changes in the autonomic nervous system, mainly lower activity of the parasympathetic nervous system. Autonomic changes can be detected by changes in heart rate variability (HRV). RA development starts years before the first presentation of arthritis; we hypothesised that autonomic changes may be present in individuals at risk of development of arthritis. Material and methods 10-minutes recordings of continuous blood pressure and heart rate were made in supine (resting) and upright (active) position in healthy subjects (HS, n = 20), individuals at risk of RA development (individuals at risk, n = 50) and RA patients (RA, n = 20). In addition, sympathetic proteins, (nor)epinephrine and the parasympathetic nicotinic acetylcholine receptor type 7 (α7nAChR) on monocytes were measured in venous peripheral blood samples. Resting heart rate (RHR) was also evaluated by a single, non-continuous measurement in an independent cohort of autoantibody positive individuals at risk of developing RA (n = 45). Results/Discussion Individuals at risk had a significantly higher RHR (68 beats per minute (bpm)) compared to HS (60 bpm, p = 0.006) and similar to RA patients (68 bpm, p = 0.38), indicating lower parasympathetic activity compared to HS. Other parasympathetic HRV parameters were lower compared to HS and norepinephrine levels were higher in individuals at risk compared to HS and RA. Dichotomizing RHR in < and ≥70 bpm in the group of individuals at risk, we could demonstrate that the majority of HRV parameters were significantly lower in individuals with RHR ≥ than <70 bpm. The parasympathetic receptor α7nAChR on peripheral CD14+ monocytes was lower in individuals at risk with RHR ≥ 70 bpm than <70 bpm. In the independent cohort RHR was higher at baseline in individuals who developed arthritis (n = 14) vs. those who did not (n = 31; 73.8 vs. 65.8 bpm, p = 0.018) after follow, and this was associated with RA development in the future (hazard ratio 1.098: 95% CI 1.012–1.191, p = 0.025). Conclusions Individuals at risk of RA development have lower parasympathetic activity compared to HS, and resemble RA patients. Increased RHR in individuals at risk is associated with RA development and a RHR ≥70 bpm measured by short-term HRV registration is accompanied by detrimental changes in all HRV parameters. These data support the notion that decreased vagus nerve tone may contribute to RA development.