www.sciencetranslationalmedicine.org/cgi/content/full/6/230/230ra44/DC1

Supplementary Materials for

IL-18 Attenuates Experimental Choroidal Neovascularization as a Potential Therapy for Wet Age-Related Macular Degeneration

Sarah L. Doyle,* Ema Ozaki, Kiva Brennan, Marian M. Humphries, Kelly Mulfaul, James Keaney, Paul F. Kenna, Arvydas Maminishkis, Anna-Sophia Kiang, Sean P.

Saunders, Emily Hams, Ed C. Lavelle, Clair Gardiner, Padraic G. Fallon, Peter Adamson, Peter Humphries, Matthew Campbell*

*Corresponding author. E-mail: matthew.campbell@tcd.ie (M.C.); sarah.doyle@tcd.ie (S.L.D.)

Published 2 April 2014, Sci. Transl. Med. 6, 230ra44 (2014) DOI: 10.1126/scitranslmed.3007616

The PDF file includes:

Materials and Methods Fig. S1. Analysis of human NK cells. Fig. S2. Effect of IL-18 on RPE cell integrity. Fig. S3. Viability of RPE cells after IL-18 treatment. Fig. S4. Differentially regulated NFκB-associated genes 6 hours after IL-18 treatment. Fig. S5. Differentially regulated NFκB-associated genes 24 hours after IL-18 treatment. Fig. S6. Decrease in angiogenesis-associated proteins and cytokines after IL-18 treatment. Fig. S7. Primary RPE cell characterization. Fig. S8. No apoptosis or necrosis of RPE cells after treatment with IL-18. Fig. S9. ZO-1 expression in RPE cells. Fig. S10. Western blot and densitometric quantification of ZO-1 in primary RPE cells. Fig. S11. Human RPE cell response to pro–IL-18 and pro–IL-1β. Fig. S12. Cell swelling after expression of pro–IL-18. Fig. S13. Pro–IL-18 expression in RPE for 3 months. Fig. S14. Necrosis gene array after transfection of RPE cells with pro–IL-18. Fig. S15. Necrosis gene array after transfection of RPE cells with pro–IL-1β. Fig. S16. Mouse ASC-cerulean oligomerization. Fig. S17. Autophagy prevents RPE cell swelling induced by pro–IL-18 expression. Fig. S18. Bone marrow chimera reconstitution.

Fig. S19. IL-18 injected 1 day before CNV has no effect on lesion volume.

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Spplementary Materials

METHODS

Primary human fetal RPE culture

Primary human fetal RPE cells were isolated from human donor eyes as previously described

and cultured in MEM-α containing 5 % FCS. Cells, provided by A. Maminishkis (National

Eye Institute, USA), were received at the Ocular Genetics Unit as a confluent monolayer of

P-0 cells and were designated with a unique reference number that related to each donor (fig.

S5). In this study, cells from 3 individual donors were used and all assays were conducted

using cells at P-1. When splitting cells from P-0, an aliquot of cells was taken for DNA

isolation and subsequent genotyping for the most common variants associated with AMD:

CFH, HTRA1, ARMS2, and PEDF variants.

ERG analysis of mice

Mice were dark-adapted overnight and prepared for electroretinography under dim red light.

Pupillary dilation was carried out by instillation of 1% cyclopentalate and 2.5%

phenylephrine. Animals were anesthetized by intraperitoneal (i.p) injection of ketamine (2.08

mg per 15 g body weight) and xylazine (0.21 mg per 15 g body weight). Standardized flashes

of light were presented to the mouse in a Ganzfeld bowl to ensure uniform retinal

illumination. The ERG responses were recorded simultaneously from both eyes by means of

gold wire electrodes (Roland Consulting Gmbh) using Vidisic (Dr Mann Pharma) as a

conducting agent and to maintain corneal hydration. Reference and ground electrodes were

positioned subcutaneously, approximately 1 mm from the temporal canthus and anterior to

the tail respectively. Body temperature was maintained at 37oC using a heating device

controlled by a rectal temperature probe. Responses were analysed using a RetiScan RetiPort

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electrophysiology unit (Roland Consulting Gmbh). The protocol was based on that approved

by the International Clinical Standards Committee for human electroretinography. Cone-

isolated responses were recorded using a white flash of intensity 3 candelas/m2/s presented

against a rod-suppressing background light of 30 candelas/m2 to which the previously dark

adapted animal has been exposed for 10 minutes prior to stimulation. The responses to 48

individual flashes, presented at a frequency of 0.5 Hz, were computer averaged. Following

the standard convention, a-waves were measured from the baseline to a-wave trough and b-

waves from the a-wave trough to the b-wave peak.

AAV production and sub-retinal inoculation

Mouse pro–IL-18 cDNA was cloned into pAAV-IRES-GFP expression vector (Cell Biolabs

Inc). AAV-2/9 was then generated using a triple transfection system in a stably transfected

HEK-293 cell line for the generation of high-titer viruses (Vector BioLabs). The eye was

prepared essentially as described below in the section outlining intra-vitreal injections,

however, single sub-retinal injections were performed on anaesthetized mice and 3 µl of

AAV at a dose of 1.5 × 1013, 0.75 × 1013, or 1.5 × 1011 GC/ml was injected. All experiments

were carried out in compliance with the ARVO statement for the use of animals in

ophthalmic and vision research.

Intra-vitreal injections of IL-18

Pupils of mice were dilated with 1% cyclopentalate and 2.5% phenylephrine and mice were

anaesthetized using a combination of ketamine and domitor. The eye was gently proptosed

and Vidisic was placed on the corneal surface followed by the placement of a supporting

polypropylene ring to allow for a flat contact lens to be placed over the entire eye and the

fundus to be easily visualized. A 30-G needle was used to penetrate the sclera, and a blunt

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ended needle tip (34 G) of a Hamilton syringe was inserted intravitreally under direct

ophthalmoscopic control. In our hands, 3 µl can be injected reliably into the vitreous with no

obvious signs of retinal inflammation or damage as assessed immediately after injection or

indeed histopathologically.

OCT and fundus fluorescein angiography

OCT was performed on mice using a Heidelberg Spectralis OCT (Heidelberg Engineering).

Briefly, pupils were dilated with instillation of 1% cyclopentalate and 2.5% phenylephrine

and mice were anaesthetized using a mixture of ketamine and domitor. OCT images were

captured with a 30-degree angle of view. Similarly, fundus fluorescein angiography was

obtained using the autofluorescent channel of the Spectralis HRA and mice were injected

intraperitoneally with a solution containing 2% fluorescein in PBS (200 µl). Early and late

stage images were taken post injection of fluorescein. Heidelberg eye explorer version 1.7.1.0

was used to capture images.

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SUPPLEMENTARY FIGURES

Fig. S1. Analysis of human NK cells. (A) Human IL-18 treated PBMCs (gated on CD56+CD3- NK cells). (B) Gating of NK and NKT cells from human PBMCs. (C) Percentage of cells expressing IFN-γ following stimulation of PBMCs with IL-12/IL-18 (RnD). Data are representative of n = 4 human donors. P-values determined by ANOVA with Dunnett's Multiple Comparison Test.

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Fig. S2: Effect of IL-18 on RPE cell integrity. (A) Phase contrast images of ARPE-19 cells treated with increasing doses of IL-18 (GSK) for 24 h. Scale bar, 100 µm. (B) Western blot analysis of the tight junction component occludin in ARPE-19 cells treated with increasing doses of IL-18 for 24 h.

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Fig. S3: Viability of RPE cells after IL-18 treatment. (A to C) Viability assay of ARPE-19 cells treated with IL-18 (10 ng/ml, 50 ng/ml, 100 ng/ml, and 1 µg/ml) for 48 h (A), 72 hours (B), and 1 week (C), with and without a media change. Left panels, MTS assay. Right panels, Trypan blue exclusion assay. Data are means ± SEM (n = 3 samples, with 3 replicates per sample)

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Fig. S4: Differentially regulated NFκB-associated genes 6 hours after IL-18 treatment. (A) Heat map of differentially regulated genes 6 h post treatment of ARPE-19 cells with IL-18 (GSK). (B) Clustergram analysis of NFκB-associated genes differentially regulated 6 h after treatment with IL-18 (GSK).

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Fig. S5: Differentially regulated NFκB-associated genes 24 hours after IL-18 treatment. (A) Heat map of differentially regulated genes 24 h post treatment of ARPE-19 cells with IL-18 (GSK). (B) Clustergram analysis of NFκB-associated genes differentially regulated 24 h after treatment with IL-18 (GSK).

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Fig. S6: Decrease in angiogenesis-associated proteins and cytokines after IL-18 treatment. Human angiogenesis proteome and cytokine arrays 0, 6, 12, and 24 h after treatment of ARPE-19 cells with IL-18 (40 ng/ml).

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Fig. S7: Primary RPE cell characterization. Genotype of donor RPE and ARPE-19 cells with respect to the CFH, HTRA1, ARMS2, and PEDF variants.

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Fig. S8: No apoptosis or necrosis of RPE cells after treatment with IL-18. (A) Phase contrast images of primary RPE cells treated with increasing doses of IL-18. Scale bar, 100 µm. (B) TUNEL staining to identify dead/dying cells in primary human RPE cells treated with increasing doses of IL-18. Positive control was DNAse-treated cells (red nuclei). Scale bar, 20 µm.

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Fig. S9: ZO-1 expression in RPE cells. Three-dimensional reconstruction of the apical localization of ZO-1 in primary human RPE cells treated with increasing doses of IL-18 (RnD). Images are representative of 2 individual donors with 2 technical repeats per donor.

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Fig. S10: Western blot and densitometric quantification of ZO-1 in primary RPE cells. Cells treated with increasing doses of IL-18 (10 ng/ml to 10 µg/ml). Data are representative of 3 biological repeats.

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Fig. S11: Human RPE cell response to pro–IL-18 and pro–IL-1 β. (A) Western blot analysis of NLRP3, pro–IL-1β, pro–IL-18, and β-actin following transfection of ARPE-19 cells with empty vector (EV), pro–IL-18 cDNA, or pro–IL-1β cDNA. (B) ARPE-19 cells transfected with EV, pro–IL-18, or pro–IL1β for 48 h. Scale bar, 100 µm. (C) Western blot analysis of NLRP3, pro–IL-1β, pro–IL-18, and β-actin following transfection of ARPE-19 cells with EV, pro–IL-18 cDNA, or pro–IL-1β cDNA. (D) ARPE-19 cells transfected with EV, pro–IL-18, or pro–IL1β for 48 h. Scale bar, 100 µm.

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Fig. S12: Cell swelling after expression of pro–IL-18. Pattern of cell swelling and AAV transduction in WT and Nlrp3-/- retinal sections. 4X objective, top panels; 10X objective, middle panels; 40X objective, bottom panels.

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Fig. S13: Pro–IL-18 expression in RPE for 3 months. (A) Rod isolated ERG after sub-retinal injection of different concentrations (genome copies per ml, GC/ml) of pro–IL-18 AAV. (B) RPE histology (H&E) 2 weeks after subretinal injection of different concentrations of pro–IL-18 AAV. Scale bar, 100 µm. (C) Depleted outer nuclear layer (20 µm) 3 months post-inoculation of pro–IL-18 AAV (1.5 X 1013 GC/ml). Left panel (phase contrast), middle panel (phase contrast with GFP/DAPI overlay), right panel (GFP/DAPI overlay). Scale bar, 50 µm.

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Fig. S14: Necrosis gene array after transfection of RPE cells with pro–IL-18. Heat map analysis of differentially regulated genes 24 hours after transfection of ARPE-19 cells with 100 ng/ml pro–IL-18 compared to transfection with 100 ng/ml empty vector. Data are representative of 3 biological repeats.

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Fig. S15: Necrosis gene array after transfection of RPE cells with pro–IL-1β. (A) Differential regulation of significantly increased or decreased genes associated with necrotic pathways 24 h after transfection of ARPE-19 cells with pro–IL-1β. P-values determined by Student's t-test. Data are means ± SEM (n = 3 samples with 3 biological repeats). (B) Clustergram analysis of necrosis-associated human genes differentially regulated 24 h after transfection of cells with pro–IL-1β.

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Fig. S16: Mouse ASC-cerulean oligomerization. Cells were left untreated, transfected with an empty vector, or pro–IL-18 AAV (n = 3 each). Positive control was ATP-treated cells (n = 2). Scale bars: (top) 40 µm, (bottom) 20 µm. Western blot of pro–IL-18 levels in ASC-cerulean cells following transient transfection with pro–IL-18 AAV.

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Fig S17: Autophagy prevents RPE cell swelling induced by pro–IL-18 expression. (A) LC3-1/LC3-II expression following over-expression of pro–IL-18 or pro–IL-1β in ARPE-19 cells. (B) LC3I/II expression post sub-retinal injection of pro–IL-18 expressing AAV. Scale bars: 100 µm (top), 20 µm (bottom). LC3I/II (red), DAPI (blue), pro–IL-18/eGFP (green). LC3A/B (red) rendered in middle images for GFP overlay. (C) ARPE-19 cells transfected with EV, pro–IL-18, or pro–IL-1β and pre-treated with either rapamycin (10 µM) or 3-MA (5 mM). Scale bar, 20 µm. Quantification of cell swelling as a percentage of total cells within the field of view (assessed by Hoechst stain). Data are means ± SEM (n = 3). P-values determined by Student's t-test. (D and E) ARPE-19 cell metabolism (D) and viability (E) following treatment with increasing doses of rapamycin (0, 0.01, 0.1, or 10 µM) or 3-MA (0.05, 0.5, or 5 mM). Data are means ± SEM (n = 3 biological repeats).

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Fig. S18: Bone marrow chimera reconstitution. (A) CD45.2+ cell expressed by wild type (WT) mice. (B) CD45.1+ cell expressed by Pepboy mice. (C) WT bone marrow chimeras expressing CD45.1+ cells.

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Fig. S19: IL-18 injected 1 day before CNV has no effect on lesion volume. Choroidal neovascularization volume after subcutaneous injection of mIL-18 (GSK) 1 day prior to CNV induction. Data are means ± SEM of individual lesion volumes from 10 mice per group. P-values determined by ANOVA with Tukey post-hoc test. Images below are representative lesion volumes from each experimental cohort.