Association between aqueous humor and vitreous fluid levels of Th17 cell-related cytokines in patients with proliferative diabetic retinopathy

Abstract

Inflammation is known to be involved in the progression of diabetic retinopathy. We have recently reported that vitreous levels of IL-4, IL-17A, IL-22, IL-31, and TNFα are higher than the respective serum levels in proliferative diabetic retinopathy (PDR) patients, and that vitreous levels of these cytokines are higher in PDR than in other non-inflammatory vitreoretinal diseases or uveitis associated with sarcoidosis. In the present study, we investigated inflammatory cytokines including Th17 cell-related cytokines in aqueous humor samples obtained from eyes with PDR, and analyzed the association between the aqueous humor and vitreous fluid levels of individual cytokines. The study group consisted of 31 consecutive type 2 diabetic patients with PDR who underwent cataract surgery and vitrectomy for vitreous hemorrhage and/or tractional retinal detachment. Undiluted aqueous humor was collected during cataract surgery, and then vitreous fluid was obtained using a 25G vitreous cutter inserted into the mid-vitreous cavity at the beginning of vitrectomy. IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-γ, soluble CD40 ligand (sCD40L), and TNFα levels in the aqueous humor and vitreous fluid were measured using a beads-array system. Although IL-17A was detected in the aqueous humor of eyes with PDR and the level correlated with IL-17A level in the vitreous fluid, both percent detectable and level of IL-17A in the aqueous humor were significantly lower than those in the vitreous fluid. Vitreous IL-17A level was related significantly to IL-10, IL-22, and TNFα levels in aqueous humor as well as in vitreous fluid, On the other hand, aqueous IL-17A level was not related significantly to aqueous or vitreous levels of IL-10, IL-22 or TNFα level. The present study demonstrated that IL-17A level and detectable rate in the aqueous humor of patients with PDR are markedly lower than those in the vitreous fluid and aqueous IL-17A does not correlate with vitreous levels of other cytokines, and hence should not be used as a surrogate for IL-17A in the vitreous fluid.

Fig 1. Comparison of cytokine levels in aqueous humor and vitreous fluid of patients with proliferative diabetic retinopathy.

Levels of (A) IL-6, (B) IL-10, (C) IL-17A, (D) IL-17F, (E) IL-22, (F) IL-25, (G) IL-31, (H) IFN-γ, (I) sCD40L, and (J) TNFα in aqueous humor (AC) and vitreous fluid (Vit) are shown as dot plots. * P < 0.05 by Wilcoxon signed-rank test.

Fig 2. Association of cytokine levels in aqueous humor and vitreous fluid of patients with proliferative diabetic retinopathy.

Correlation of aqueous level (AC, x-axis) vs. vitreous level (Vit, y-axis) for each cytokine was analyzed by Spearman’s correlation coefficient test. (A) IL-6: y = 0.45 + 0.08x, R² = 0.039, P = 0.284; (B) IL-10: y = 10.4 + 1.99x, R² = 0.165, P = 0.02; (C) IL-17A: y = 14.9 + 3.27x, R² = 0.287, P = 0.002; (D) IL-17F: y = 3.66 + 0.09x, R² = 0.042, P = 0.269; (E) IL-22: y = 10.8 + 0.03x, R² = 0.0001, P = 0.956; (F) IL-25: y = 0.24–0.001x, R² = 0.00001, P = 0.988; (G) IL-31: y = 4.08–0.04x, R² = 0.003, P = 0.775; (H) IFN-γ: y = 1.41–0.004x, R² = 0.0001, P = 0.9519; (I) sCD40L: y = 1.58 + 0.03x, R² = 0.009, P = 0.617; and (J) TNFα: y = 4.42 + 0.99x, R² = 0.059, P = 0.188.

Fig 3. Association of IL-17F level with levels of other cytokines in aqueous humor of patients with proliferative diabetic retinopathy.

Correlation of aqueous (AC) IL-17F level (x-axis) vs. aqueous levels of other cytokines (y-axis) was analyzed by Spearman’s correlation coefficient test. (A) IL-6: y = 306.1 + 57.7x, R² = 0.938, P < 0.0001; (B) IL-10: y = 1.74 + 0.010x, R² = 0.008, P = 0.6338; (C) IL-17A: y = 2.98–0.003x, R² = 0.0002, P = 0.9347; (D) IL-22: y = 1.84–0.008x, R² = 0.005, P = 0.6964; (E) IL-25: y = -0.32 + 0.06x, R² = 0.888, P < 0.0001; (F) IL-31: y = -0.82 + 0.30x, R² = 0.920, P < 0.0001; (G) IFN-γ: y = -1.60 + 0.38x, R² = 0.913, P < 0.0001; (H) sCD40L: y = -1.51 + 0.47x, R² = 0.952, P < 0.0001; and (I) TNFα: y = 1.10 + 0.002x, R² = 0.002, P = 0.8076.