IL-17A Damages the Blood-Retinal Barrier through Activating the Janus Kinase 1 Pathway

Abstract

Blood-retinal barrier (BRB) dysfunction underlies macular oedema in many sight-threatening conditions, including diabetic macular oedema, neovascular age-related macular degeneration and uveoretinitis. Inflammation plays an important role in BRB dysfunction. This study aimed to understand the role of the inflammatory cytokine IL-17A in BRB dysfunction and the mechanism involved. Human retinal pigment epithelial (RPE) cell line ARPE19 and murine brain endothelial line bEnd.3 were cultured on transwell membranes to model the outer BRB and inner BRB, respectively. IL-17A treatment (3 days in bEnd.3 cells and 6 days in ARPE19 cells) disrupted the distribution of claudin-5 in bEnd.3 cells and ZO-1 in ARPE19 cells, reduced the transepithelial/transendothelial electrical resistance (TEER) and increased permeability to FITC-tracers in vitro. Intravitreal (20 ng/1 μL/eye) or intravenous (20 ng/g) injection of recombinant IL-17A induced retinal albumin leakage within 48 h in C57BL/6J mice. Mechanistically, IL-17A induced Janus kinase 1 (JAK1) phosphorylation in bEnd.3 but not ARPE19 cells. Blocking JAK1 with Tofacitinib prevented IL-17A-mediated claudin-5 dysmorphia in bEnd.3 cells and reduced albumin leakage in IL-17A-treated mice. Our results suggest that IL-17A can damage the BRB through the activating the JAK1 signaling pathway, and targeting this pathway may be a novel approach to treat inflammation-induced macular oedema.

Keywords: JAK/STAT signaling; Tofacitinib Citrate; blood–retinal barrier; inflammation; interleukin-17; macular oedema; retina; retinopathy.

Figure A1

Study design: effect of Tofacitinib Citrate on IL-17A-mediated BRB leakage in WT mice.

Figure A2

Array of JAK/STAT phosphorylation in ARPE19 cells in response to IL-17A treatment. Human ARPE-19 cells were treated with 50 ng/mL IL-17A for 30 min. Protein extracted from n = 4 independent experiments were pooled for array analyses, with one membrane per time point. (A) Array blot images from control and IL-17A treated group and blot identities were shown in the table. (B) Graphs showing the expression levels of the phosphorylated form of JAK/STAT family members.

Figure A3

IL-17A does not significantly alter pJAK1 expression in ARPE-19 cells. Western blot (A) and corresponding (B–D) densitometry analysis of pJAK1, JAK1 and β-actin. (E) Representative immunostaining for pJAK1 (red) in control (UT) and IL-17A-treated ARPE19 cells, and (F) corresponding quantification. UT: untreated control.

Figure A4

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.

Figure A5

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.

Figure A6

The effect of Tofacitinib Citrate on the viability of ARPE19 and bEnd.3 cells. The ARPE19 or bEnd.3 cells were treated with different concentrations of Tofacitinib for 7 days. Cell viability was evaluated by using the Alamar blue assay. (A) Viability fold change in ARPE-19 cells. (B) Viability fold change in bEnd.3 cells. *** p ≤ 0.0005, **** p ≤ 0.0001. One-Way ANOVA and Dunnett’s multiple comparisons test.

Figure 1

The effect of IL-17A on bEnd.3 cell and ARPE19 cell tight junctions and barrier function. Tight junction morphology and barrier function in bEnd.3 and ARPE19 cells were assessed after IL-17A treatment. (A) Immunostaining for claudin-5 (green) and DAPI (blue) in bEnd.3 cells after treatment with IL-17A (100 ng/mL) for 72 h. Scale bar = 20 µm. (B) TEER and (C) FITC-Na leakage of bEnd.3 cells cultured in transwell membrane after 3 days of treatment with IL-17A (100 ng/mL). (D) Immunostaining for ZO-1 (red) and DAPI (blue) in ARPE-19 cells after 6 days of treatment with IL-17A (50 ng/mL). Scale bar = 25 µm. (E) TEER and (F) FITC-Dextran permeability of ARPE-19 cells cultured in transwell membrane after 6 days of treatment with IL-17A (50 ng/mL). Mean ± SD; * p < 0.05 by unpaired t-test.

Figure 2

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).

Figure 3

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.

Figure 4

The effect of Tofacitinib Citrate on IL-17A-induced barrier dysfunction and claudin-5 junction dysmorphia in bEnd.3 cells. The bEnd.3 cells were pretreated with Tofacitinib Citrate (4.955 µM) for 30 min, followed by IL-17A (100 ng/mL) incubation for 30 min. TEER and claudin-5 expression in bEnd.3 cells was examined 72 h later. (A) Scheme of experimental design. (B) TEER in bEnd.3 cells from different groups. UT, untreated; Mean ± SD; *** p < 0.001, **** p ≤ 0.0001 by One-Way ANOVA with Tukey’s multiple comparisons test. (C) Immunostaining for claudin-5 (red), DAPI (blue). Scale bar = 25 µm.

Figure 5

The effect of Tofacitinib Citrate on IL-17A-induced retinal pJAK1 expression. C57BL/6J mice received IL-17A or vehicle (PBS) i.v. injection were treated with or without Tofacitinib Citrate twice. Retinae were collected 48 h later and processed for immunostaining of pJAK1. (A) Schematic of experimental design. (B) Representative images of pJAK1 expression in retinas of mice that received PBS, IL-17A, IL-17A + Tofacitinib Citrate or IL-17A + vehicle control; n ≥ 5 animals per group. (C) Quantification of pJAK1 integrated density. Two or three images from the central retina were quantified per animal. Values were normalized to PBS control on the same slide. Mean ± SD. ROUT outliers test 2% was used to remove outliers. Cleaned data were compared by using One-Way ANOVA with Tukey’s multiple comparisons test, * p < 0.05; ns: no statistical significance.

Figure 6

Tofacitinib Citrate ameliorates IL-17A-induced leakage in wild-type mice. C57BL/6J mice were treated with or without Tofacitinib Citrate immediately after IL-17A or vehicle (PBS) intravenous (iv) injection and were treated with Tofacitinib/vehicle again 24 h later. Retinae were collected 48 h later and processed for immunostaining of albumin and isolectin B4. (A) Schematic of experimental design. (B) Representative images showing albumin (green) and isolectin B4 (red, marker of vascular endothelial cells). Albumin outside the isolectin B4⁺ area was considered indicative of vascular leakage. White arrows: albumin leakage. (C) Quantification of albumin extravasation. Two or three images from the central retina were quantified per animal. Values were normalized to PBS control on the same slide. Mean ± SD; n ≥ 5 animals per group; ROUT outliers test 2% was used to remove outliers. Cleaned data were compared by using One-Way ANOVA with Tukey’s multiple comparisons test, * p < 0.05; ns: no statistical significance.