Fig 2: The effect of IL-17A on pJAK1 expression in bEnd.3 cells and the WT mouse retina. JAK1 phosphorylation was examined in bEnd.3 cells by Western blot and immunostaining, and in mouse retinal sections 48 h after IL-17A i.v. administration. (A) Representative Western blot and corresponding densitometry of pJAK1 in control (0 min) and IL-17A-treated bEnd3 cells. (B) Quantification of pJAK1 expression detected by Western blotting. Mean ± SD; ** p ≤ 0.01, unpaired t-test, n ≥ 4. (C) Representative images of pJAK1 immunocytochemistry in bEnd.3 cells from n ≥ 3 independent experiments. (D) Immunostaining of pJAK1 expression (red) in the murine retina 48 h after IL-17A or PBS i.v. injection compared to control non-injected wild-type (WT) mice, n ≥ 4.

Fig 3: IL-17A may mediate release of IL-6, but not VEGFA, from bEnd.3 cells. Supernatants from control of IL-17A treated bEnd.3 cells were collected at relevant time points. The concentrations of IL-6 and VEGF were measured by ELISA. (A) The levels of IL-6 in bEnd.3 cell supernatant after 3 and 6 days of treatment with IL-17A. Mean ± SD, n = 2. (B) The levels of VEGFA in the supernatants from bEnd.3 cells after 3 days IL-17A treatment (black dots). Supernatants from bEnd.3 cells exposed to hypoxia (1% oxygen) for 8 days (red dots) were used as a positive control. Each point is representative of one technical replicate. Mean ± SD. UT, untreated; IL-17A, treated with 100 ng/mL of IL-17A.

Fig 4: The effect of IL-17A in BRB integrity in vivo. Retinal albumin expression and albumin leakage were assessed 48 h after IL-17A intravitreal (itv, A–C) or intravenous (i.v., D–F) administration in C57BL/6J mice. (A) Western blot analysis of total albumin expression in the retina from vehicle (Veh, PBS) or IL-17A ivt, and control mice (no injection). Corresponding densitometry of albumin normalized to β-Actin. n ≥ 16 retinas from n ≥ 8 mice per group. (B) Albumin immunostaining (green) in retinal sections from IL-17A or PBS ivt mice. Scale bar = 25 µm. Graph showing mean gray value of albumin in the neuroretina (n ≥ 2 animals per group). (C) Representative images showing FITC-Dextran leakage from retinal vessels 48 h after IL-17A or PBS control ivt. White arrows: FITC-Dextran leakage, n ≥ 4 eyes per group. (D) Western blot analysis of total albumin expression in the retina from vehicle (Veh, PBS) or IL-17A i.v. and control mice (no injection); n ≥ 6 retinas from n ≥ 6 animals per group. (E) Representative images of extravascular albumin (green) outside Isolectin B4+ blood vessels (red) in the retina from PBS or IL-17A i.v. injected mice. White arrows: albumin leakage. (F) Quantification of extravascular albumin in the retina from vehicle or IL-17A i.v. injected mice. Blue—DAPI; n ≥ 3 eyes per group. Scale bar = 20 µm. Mean ± SD; ** p < 0.001, * p < 0.05 with One-Way ANOVA followed by Dunnett’s multiple comparisons in A and D and unpaired t-test in (F).

Fig 5: pSTAT3 expression in ARPE-19 cells after IL-17A treatment. (A) Representative Western blot images of n = 4 independent experiments. Densitometry of (B) pSTAT3/β-Actin and (C) pSTAT3/STAT3. Mean ± SD; n = 4; * p < 0.05 by One-Way ANOVA with Dunnett’s post hoc tests, ns: no statistical significance.