THU0018 ANTI-GALECTIN-9 ANTIBODY AS A NOVEL TREATMENT OPTION IN RHEUMATOID ARTHRITIS TARGETING PATHOGENIC FIBROBLAST-LIKE SYNOVIOCYTES

Abstract

Background: Fibroblasts-like synoviocytes (FLS) present in the stromal environment are key effector cells in the persistence of synovial inflammation and joint damage in rheumatoid arthritis (RA). A particular subset of FLS characterized as Thy-1+Podoplanin+CD34- is significantly expanded in RA patients and has been described to be disease-associated1. Currently, no treatment targeting the stromal environment in RA is available2. Objectives: To identify a novel treatment option targeting the stromal environment in RA to modify the disease-associated subset of fibroblasts. Methods: An in vitro model was established with FLS derived from synovial fluid mononuclear cells (SFMC) from RA and osteoarthritis (OA) patients (n=6). FLS between passage 2-5 were used for further analyses. Untreated FLS in cultures were analyzed by flow cytometry for expression of the surface proteins CD34, CD45, Thy-1 and Podoplanin. The potential effect of cell detachment solutions on the expression of the surface proteins was examined by applying either trypsin or lidocaine. To evaluate on the inflammation status of the FLS, MCP-1 levels in supernatants from FLS in mono-cultures stimulated with either antibodies targeting galectin-9 (Gal-9), an anti-Gal-9 antibody-matched isotype control, LPS or steroid were analyzed by ELISA and compared to culture medium alone. Cell viability were examined using an MTT assay. Data are expressed as mean (95% CI) and analysed by a paired t-test. P-values Results: FLS derived from SFMC were characterized as CD34-CD45- and the majority co-expressed Thy-1 and Podoplanin [80.5%, (66.3-94.7%)] confirming the pathogenic phenotype of these cells. This phenotype was not altered by using different methods to detach the cells from the cell-culture plates. FLS receiving anti-Gal-9 antibody treatment showed a significant decreased fold change in MCP-1 secretion (0.84, [0.70;0.98]) compared with unstimulated cells (p=0.036). Treatment with an isotype control did not result in a significant decrease in MCP-1 secretion. This tendency was specific to FLS derived from RA patients as FLS derived from OA patients showed no significant decrease in MCP-1 secretion upon anti-Gal-9 antibody treatment. FLS derived from both RA or OA patients showed a significant increased fold change in secretion of MCP-1 upon LPS stimulation and significantly decreased levels of MCP-1 upon steroid treatment, consistent with the pathogenic phenotype of these cells. None of the different stimulations resulted in morphological changes of the FLS examined by light microscopy. Further, no significant changes in cell viability were detected after anti-Gal-9 antibody treatment. Conclusion: FLS cultures derived from RA patients at passage 2-5 consist mainly of disease-associated fibroblasts and secrete significantly lower amounts of MCP-1 when treated with an anti-Gal-9 antibody whitout affecting cell viability. Thus suggesting that Gal-9 neutralization may represent a novel treatment option targeting the stromal environment and inflammation in RA.