Fig 1: BCL3 controls cytokine production in PMCs without altering the quality of ET. BMMCs were left untreated or incubated with LPS (300 ng/ml) for 24 h to induce ET. Naive and tolerant BMMCs were then stimulated with LPS (1 µg/ml) for 90 min and Bcl3 gene expression was analysed (a). Data show mean ± SD from n = 3 independent experiments. One-sample t-Test was performed to calculate the p-values. Moreover, cells were stimulated with LPS (1 µg/ml) for 4 h and 6 h and lysates were analysed by Western Blotting with antibodies specific for BCL3 and GAPDH (loading control) (b). The Western blot is representative of three independent experiments with three biological samples. Next, BMMCs of WT and Bcl3−/− mice were pretreated with LPS (300 ng/ml) for 24 h to induce ET. Then the cells were stimulated with LPS (1 µg/ml) for 90 min to measure Il6 and Tnf mRNA expression (c), and for 4 h to detect IL-6 and TNF-α protein secretion (d). Also, WT and Bcl3−/− PMCs were incubated with LPS (300 ng/ml) for 24 h to induce ET, and then stimulated with LPS (1 µg/ml) for 4 h to analyse TNF-α protein production (e). Moreover, WT and Bcl3−/− PMCs were stimulated with LPS (1 µg/ml), FSL-1 (1 µg/ml), and IL-33 (2 ng/ml) for 4 h and IL-6 and TNF-α protein secretion was measured (f). Data show mean ± SD from three independent experiments. Student’s t-Test and one-sample t-Test were performed to calculate the p-values. Multiple testing p-values were corrected by FDR. *p < 0.05, **p < 0.01, ***p < 0.001.
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: IL‐33 treatment protects against RPE cell death and loss of metabolically important protein Hexokinase II in retina in Cfh+/−~HFD mice. A, Confocal images of ZO‐1/TUNEL stained RPE/choroid and DAPI/TUNEL stained retina flatmounts from Cfh+/−~HFD and C57BL/6 ~ HFD eyes treated with either vehicle or IL‐33 (1 ng/µL). Scale bar is 38 µm. B, Quantitative analysis of mean TUNEL + cells per field of view in RPE/choroid flatmounts. A significant reduction in cell death is observed in Cfh+/−~HFD IL‐33‐treated eyes (n = 9) compared to Cfh+/−~HFD vehicle‐treated eyes (n = 9; **P = .0037) and C57BL/6 ~ HFD IL‐33‐treated eyes (n = 5; *P = .040). C, Quantitative analysis of TUNEL + cells per field of view in retina. Cell death was significantly increased in Cfh+/−~HFD eyes (n = 7, 7) compared to C57BL/6 ~ HFD control (n = 4, 5), irrespective of treatment (*P = .032). D, Representative Western blot of Hexokinase II (HKII) protein expression in retina lysates, cropped lanes from the same blot. E, Densiometric analysis demonstrates HKII is significantly reduced in Cfh+/−~HFD vehicle‐treated eyes (n = 9) compared to Cfh+/−~HFD IL‐33 (1 ng/µL; n = 9), C57BL/6 ~ HFD veh (n = 5) and IL‐33 (1 ng/µL; n = 5)‐treated eyes (*P = .017, P = .010 and P = .039, respectively)
Fig 4: IL‐33 regulates the function of human choroidal fibroblasts. IL‐33 inhibited the ability of human choroidal fibroblasts to (A) migrate in a wound‐healing assay and (B) contract collagen gel (scale bar = 100 µm). (C) The MTT assay showed that IL‐33 treatment had no effect on their proliferation. (D) MMP2 and (E) MMP9 mRNA expression in choroidal fibroblasts upon treatment with IL‐33. (F) TIMP1 and TIMP2 expression in choroidal fibroblasts in response to IL‐33 (n = 3 per group). (G) Expression of uPA and its receptor uPAR in choroidal fibroblasts in response to IL‐33 (n = 3 per group). Data are shown as mean ± SEM. Data are representative of three independent experiments with similar results. *p < 0.05. Statistical analysis was performed with Student's t‐test.
Fig 5: Exogenous IL‐33 is non‐toxic in aged C57BL/6 ~ HFD and Cfh+/−~HFD mice. (A) Schematic to explain timeline of animal manipulations; TEM—transmission electron microscopy, IHC—immunohistochemistry, WB—Western blot (B) OCT scan of eyes from Cfh ± and C57BL/6 mice prior to high‐fat diet (HFD) and post‐HFD animals injected with either vehicle or IL‐33 (1 ng/µL). Scale bars are both 100 µm. Graph to show retinal thickness measured from OCT scans of Cfh± (n = 4) and C57BL/6 mice prior to high‐fat diet start (n = 6) (C) or Cfh+/−~HFD or C57BL/6 ~ HFD mice injected with either IL‐33 or vehicle control (n = 4‐11) (D). (E) Transmission electron micrograph images of sub‐RPE deposit in Cfh+/−~HFD mice treated with either IL‐33 (1 ng/µL) or vehicle. Arrowhead shows remnants of basal infoldings disrupted by deposit formation. In both IL‐33 and vehicle‐treated eyes, large numbers of vesicle‐like structures with an electron‐dense shell are observed within the deposits, as indicated by the asterix (*). BrM—Bruch's membrane. Scale bar is 2 µm. (F) Graph shows mean deposit height in eyes of Cfh+/−~HFD IL‐33 and vehicle‐treated eyes (n = 3 of each)
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