Fig 2: Intravitreal administration of IL-33 attenuates CNV in an ST2-dependent manner. (A) Representative OCT images showing choroidal neovascular lesions in control and IL-33-treated eyes. INL = inner nuclear layer; ONL = outer nuclear layer; RPE = retina pigment epithelium. The white asterisk denotes the sub-retinal fluid. Scale bar = 200 µm. (B) Fluorescence IB4 staining of choroidal neovascular (CNV) lesions in all treatment groups. Images are representative of CNV volume in each experimental group. Scale bar = 75 µm. (C) Quantitative analysis of the volume of CNV lesions showed that the two lower doses of IL-33 significantly attenuated CNV development. IL-33 did not affect the integrity and thickness of the retina, as shown in histological sections (D) and OCT analysis (E) of eyes that had only intravitreal injection of IL-33. ONH = optic nerve head. Data are representative of two measurements per retina with four eyes per group. (F) Il33-/- mice had more pronounced lesions but not to a statistically significant level. (G) IL-33 treatment did not affect the severity of CNV development in St2-/- mice (n = 10 eyes per group). Data are shown as mean ± SEM. Data are representative of at least three independent experiments with similar results. *p < 0.05. Statistical analysis was performed with ANOVA with post-hoc t-test.

Fig 3: HH signaling synergizes with IL-33 in EHBD hyperplasia. (A) EHBDs from WT mice treated with either Veh or IL-33 (1 µg/mouse/day for 4 days; necropsy on day 5) were analyzed by immunohistochemistry for ST2 expression (representative images of three samples). (B,C) EHBDs from WT, pCMV-Shh, and Gli1lacZ/lacZ mice treated with either Veh or mitogen IL-33 intraperitoneally, as shown in (B) schema, were (C) examined macroscopically (representative photographs; ruler scale, 1 mm) and (D) histologically with H&E (scale bars, 20 µm; asterisks, BD lumen). (D) BD wall thickness from immunohistochemistry images was measured using ImageJ software and compared among WT, pCMV-Shh, and Gli1lacZ/lacZ mice treated with either Veh or IL-33 (n = 7-11 animals per group). Results are expressed as mean ± SEM; one-way ANOVA; *P < 0.05, ***P < 0.001. Abbreviations: i.p., intraperitoneally; ns, not significant.

Fig 4: IL-33-induced BDO cell proliferation in vitro. (A) Total RNA was isolated from the WT mouse-derived organoids and analyzed for mRNA expression of St2 and Gli1 (n = 3 organoid lines). (B,C) BDOs were treated with Veh or recombinant IL-33 (100 ng/mL) for 72 hours (day 3) starting 24 hours after passage of established BDOs and analyzed for growth (n = 6 technical replicates per group). (D,E) In a separate experiment, BDOs were treated with either Veh or pretreated for 1 hour with the NF-?B inhibitor QNZ (1 µM) and then treated with recombinant IL-33 (100 ng/mL) for 72 hours (day 3) after passaging of established BDOs. BDOs were analyzed for proliferation (n = 3 technical replicates per group). (D) The organoids were incubated with EdU for 9 hours to label proliferating cells (green) among all BDO cells (cell nuclei, DAPI [blue]; original magnification ×600). (E) ImageJ software was used to enumerate proliferating cells. Results are expressed as mean ± SEM; one-way ANOVA (C,E); ***P < 0.001, ****P < 0.0001. Abbreviation: D0/D3, day 0/day 3.

Fig 5: IL-33 is expressed in murine and human retinas, and is induced in RPE. Expression of IL-33 (A) and its receptor, ST2L (B), mRNA expression in retinal pigment epithelium (RPE) treated with TLR agonists [LPS, poly(I:C)] as indicated for 24 h (n = 4 per group). (C) Western blot analysis of IL-33 and ST2 in mouse (B6RPE-07) and human (ARPE-19) RPE cells upon activation with TLR agonists. (D) Immunofluorescence staining showing expression of IL-33 in adult murine retina. GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer. Scale bar = 100 µm. (E) ST2 expression in choroidal mast cells (arrows), choroidal fibroblasts (arrowheads), and RPE. (F) Immunofluorescence staining showing expression of IL-1 receptor accessory protein (IL1RAP) in murine retina. Scale bar =100 µm. (G) Representative images of immunofluorescence staining showing expression of IL-33, ST2, and IL1RAP in sections of human retina. Scale bar = 100 µm. Data are shown as mean ± SEM. Data are representative of at least three independent experiments with similar results. *p < 0.05. Statistical analysis was performed with Student's t-test.