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Blanca Molins, Victor Llorens, Marina Mesquida, Laura Pelegrin, Alfredo Adan Civera; Regulatory T cell levels and cytokine production in active non-infectious uveitis: in vitro effect of anti-TNF-α, dexamethasone, and cyclosporine. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2029. doi: https://doi.org/.
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To evaluate circulating regulatory T-cell (Treg) levels and cytokine production by peripheral blood mononuclear cells (PBMC) in patients with active non-infectious uveitis and to evaluate the effect of in vitro treatment with infliximab, dexamethasone, and cyclosporine on Treg levels and cytokine production in PBMC from uveitis and healthy subjects.
We included 12 patients with active non-infectious uveitis from Hospital Clinic of Barcelona (Spain) and 11 age-matched healthy subjects. PBMC were obtained by Ficoll gradient and cultured for 24 h in the presence or absence of dexamethasone (0.1-1x10-6M), cyclosporine (100-200 ng/ml), and infliximab (5-20 μg/ml). Treg (CD3+CD4+Foxp3+CD25highCD127low) levels in PBMC were determined by flow cytometry using the following fluorochrome-labeled monoclonal antibodies: AF700-CD3, Fitc-CD4, APC-CD25, PeCy7-CD127, and PE-Foxp3 (eBiosciences). Supernatants of cultured PBMC were collected and IL-10 and TNF-α levels were measured by ELISA (R&D Duoset).
No significant differences were observed in Treg levels between uveitis patients and control subjects. However, PBMC from uveitis patients produced significantly higher amounts of TNF-α (20.3 ±8.9 pg/mL vs. 6.9 ± 3.4 pg/ml, P<0.05) and lower amounts of IL-10 (12.7± 3.9 pg/mL vs. 20.0±6.0 pg/mL, P<0.05). In vitro treatment with infliximab and cyclosporine did not modulate the levels of CD3+CD4+Foxp3+, CD3+CD4+CD25high, CD3+CD4+Foxp3+CD25high, and CD3+CD4+Foxp3CD25highCD127low in both groups. However, dexamethasone treatment significantly reduced CD25high in CD3+CD4+ cells from uveitis patients, decreasing Treg levels. Furthermore, dexamethasone also increased in a dose-dependent manner CD127 expression in CD3+CD4+ cells in both uveitis and control subjects, further reducing Treg levels. On the other hand, dexamethasone and infliximab treatment significantly reduced TNF-α production in PBMC from both groups without affecting IL-10 production.
Our results suggest that TNF-α and IL-10 production by PBMC rather than altered Treg levels are associated with active uveitis and that in vitro treatment with infliximab and dexamethasone lower the inflammatory response without increasing Treg levels.
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