BackgroundFibroblast-like synoviocytes (FLS) are pivotal mediators of rheumatoid synovitis expansive growth and invasiveness which respond insufficiently to disease-modifying antirheumatic drugs. Galectin-9 (Gal-9) is a lectin with well-conserved carbohydrate-recognition domains. Gal-9 has been reported to have both pro- and anti-inflammatory properties based on its ability to modulate RA FLS. Gal-9 levels are elevated in serum of rheumatoid arthritis (RA) patients, and high levels of Gal-9 have been identified in both the synovial fluid and in the inflamed synovial tissue of patients with RA.ObjectivesTo investigate the role of Gal-9 as a biomarker for disease severity in treatment naïve patients with early RA and to study aspects of Gal-9 effects on inflammatory RA FLS.MethodsSoluble plasma Gal-9 was measured in patients with newly diagnosed, treatment-naïve RA (n = 98) and in Healthy (HC) (n = 48) (Table 1). Over a 2-year period patients were randomized to either MTX alone or MTX and anti-TNF antibody treatments. Serial measurements of disease activity (DAS28CRP) were collected to evaluate the disease course. In another cohort of patients with established RA, plasma and synovial fluid samples were also examined for Gal-9 (n = 18) (Table 1). Synovial fluid mononuclear cells (SFMC) from established RA patients were used to harvest RA FLS (n=7). Osteoarthritis FLS were used as disease controls and obtained from patients with knee OA undergoing joint replacement surgery (n = 5). Monocultures of synovial fluid derived FLS (SF-FLS) (n= 6) and autologous co-cultures of SF-FLS and peripheral blood mononuclear cells (PBMC) were established (n=7) and subsequently analyzed by flow cytometry, MTT assay, and ELISA. In vitro, cultures were treated with a neutralizing anti-Gal-9 antibody.Table 1.Patient characteristicsEarly RA (n=98)Established RA (n=18)HC (n=48)Time after treatment initiation (months)032484 (12-288)Disease activityDAS28CRP (0-10)5.7 (5.1-6.4)2.1 (1.8-3.2)2.0 (1.8-2.7)5.0 (3.2-5.5)-Gal-9 levels (pg/ml)Plasma3315 (2683-4421)3217 (2706-4250)2904 (2639-3411)2190 (1652-2852)Synovial fluid21246 (13832-34727)Patient characteristics. Data are expressed as median with IQR range unless otherwise indicated.ResultsPatients with early and established RA had increased plasma levels of Gal-9 compared with HCs (P < 0.05) (Table 1) and levels of Gal-9 correlated positively with swollen joint counts at baseline (rho = 0.344, P < 0.05). The levels remained unaffected by treatment with MTX alone or by a combination of MTX and anti-TNF antibodies. Gal-9 levels were markedly elevated in the synovial fluid of chronic RA patients compared with the corresponding plasma samples (P < 0.05) (Table 1). In vitro, a neutralizing Gal-9 antibody mediated a 40% decrease in MCP-1 secretion (P < 0.05) and a 30% decrease in IL-6 secretion (P < 0.05) in RA FLS monocultures. In OA FLS, addition of anti-Gal-9 antibodies comparably decreased the production of both MCP-1 (P < 0.05) and IL-6 (P < 0.05). The changes in cytokine production were not attributable to reduction in the fraction of inflammatory FLS (CD34^-PDPN⁺THY1⁺), decreased viability or proliferation. We further investigated if the effect of neutralizing Gal-9 persisted in co-cultures between FLS and autologous PBMC. Also in this setting, neutralization of Gal-9 mediated a 40% reduction in both MCP-1 and IL-6 (P < 0.05, P < 0.05).ConclusionPretreatment plasma Gal-9 levels in patients with newly diagnosed RA were increased and correlated with baseline clinical disease activity but remained elevated during goal directed synovitis suppressive therapy. In addition, in vitro neutralization of Gal-9 decreased MCP-1 and IL-6 production in a subset of FLS linked to RA pathology. Collectively these findings indicate that Gal-9 overexpression is a co-player in the causation of acute and persistent RA synovitis by enhancing pro-inflammatory FLS pathways.AcknowledgementsWe thank Karin Skovgård Sørensen (Dept. of Biomedicine, Aarhus University) for technical assistance concerning the ELISA data and the FACS Core Facility (Aarhus University, Denmark) for technical assistant regarding Flow cytometry. We thank medical doctors and nurses at the Department of Rheumatology, Aarhus University Hospital for helping to collect the patient samples. We kindly acknowledge the generous grants from Aarhus University Research Foundation and the Danish Rheumatism Association.Disclosure of InterestsNone declared.