THU0342 Anti-TNF Treatment Reduces Serum Serotonin Levels in Patients with Ankylosing Spondylitis and Downregulates Serotonin Signaling in an Experimental Cell Line Model

Background The mechanisms involved in new bone formation in ankylosing spondylitis (AS) are incompletely understood. Evidence suggests that gut-derived serotonin is an inhibitor of bone formation; serotonin binds to the serotonin receptor Htr1b on osteoblasts, inhibits Creb phosphorylation and decreases osteoblast proliferation. Objectives To assess i) serum serotonin levels in patients with AS, a prototype bone-forming disease, compared to patients with osteoporosis (OP) and healthy subjects ii) the effect of anti-TNFa treatment on serum serotonin levels in patients with AS and iii) the effect of serum of AS patients on serotonin signaling, in an experimental model. Methods Serum serotonin levels were measured in 37 patients with AS (19 on anti-TNFa treatment), 8 patients with OP and 29 healthy subjects by RIA methodology. The effect of serum on serotonin signaling was assessed using an experimental model based on osteoblast-like, Saos2 cells. On day 0, 2X106 Saos2 cells were cultured in EMEM/10%FBS; on day 2, 10% of serum was added and on day 3 cells were lysed, protein was extracted and expression of phospho-Creb and GAPDH were assessed by Western blot. We assessed the effect of serum from 11 patients with AS (5 on anti-TNFa), 6 patients with rheumatoid arthritis (RA) (2 on anti-TNFa) and 9 healthy subjects. Proliferation of Saos2 cells was evaluated using MTT assay. Results Serum serotonin levels were similar in patients with AS, OP and healthy subjects (mean±SEM ng/ml: 106.3±11.65, 99.19±31.77, 115.6±17.48, respectively, p=ns for all comparisons). However, serotonin levels were significantly lower in patients with AS on anti-TNFa treatment compared to patients with AS not on such treatment (mean±SEM: 140.1±17.11 vs 71.16±10.66 for patients, respectively, p=0.002). In order to explore if these differences in serotonin levels have a functional effect on serotonin signaling, we assessed phospho-CREB expression in Saos2 cells. We found that phospho-CREB expression in Saos2 cells treated with serum from AS patients on anti-TNF treatment was significantly higher compared to AS patients not on such treatment (mean±SEM of densitometric intensity ratio pCREB/GAPDH:0.80±0.14 vs 0.34±0.13, respectively, p=0.04). We next assessed the effect of serotonin on Saos2 proliferation; we found that serotonin at 75ng/ml (a concentration relevant to that of human serum) suppresses Saos2 proliferation (mean±SEM:0.23±0.01 vs 0.18±0.01, for cells not treated and treated, resp