Journal: PLoS ONE

Article Title: Interleukin-17 Retinotoxicity Is Prevented by Gene Transfer of a Soluble Interleukin-17 Receptor Acting as a Cytokine Blocker: Implications for Age-Related Macular Degeneration

doi: 10.1371/journal.pone.0095900

Figure Lengend Snippet: IL17A mRNA expression in paraffin-embedded (A) sub-macular choroid button and (B) macula. (C) IL17RC mRNA expression in paraffin-embedded macula. (D) IL17RC mRNA expression in macula vs. periphery of 3 donors. (E, F) Macular IL17A and IL17RC mRNA were verified in paraformaldehyde-fixed fresh frozen tissue. (G) Immunohistochemical detection of IL17A and IL17RC in paraffin-embedded macular sections. Isotype controls lacked primary antibodies. (H) Comparison of macular and peripheral IL17A immunostains. For box plots: center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; width of the boxes is proportional to the square root of the sample size; data points are plotted as open circles; sample numbers are indicated beneath each column. GA = geographic atrophy; nAMD = neovascular AMD; GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer; IS/OS = inner/outer segment; RPE = retinal pigment epithelium. *: P<0.05; **: P<0.005; ***:P<0.0001.

Article Snippet: Retinas were treated with 50 ng/ml recombinant mouse Il17a (R&D Systems) for 0, 5, or 15 min.

Techniques: Expressing, Immunohistochemical staining, Comparison, Software

Journal: PLoS ONE

Article Title: Interleukin-17 Retinotoxicity Is Prevented by Gene Transfer of a Soluble Interleukin-17 Receptor Acting as a Cytokine Blocker: Implications for Age-Related Macular Degeneration

doi: 10.1371/journal.pone.0095900

Figure Lengend Snippet: (A) Confocal microscopy of ARPE-19 treated 48h with IL17A. Nuclear signal of cleaved Caspase-3 (arrowheads). Blue = DAPI. Scale bar = 50 µm. Graphical representation of confocal data presented to the right of the panel. (B) ARPE-19 ultrastructure cultured without (i–ii) and with (iii–vi) 10 ng/ml IL17A for 48h. Indicated are healthy mitochondria (i, white arrow), lipid deposits (iii, iv, blue arrowheads), autophagosomes (iv, green arrowhead), mitochondrial damage (iii, iv, vi, yellow arrowheads), pyknotic nuclear clumps (v, black arrowhead), and cytoplasmic necrosis (vi, red arrowhead). (C) ARPE-19 vs. COS-7 viability after 48h treatment with IL17A. (D) Relative expression of IL17 receptors in ARPE-19 vs. COS-7. Data are presented as mean ± SEM. *: P<0.05, **: P<0.01, ***: P<0.001; ****: P<0.0001.

Article Snippet: Retinas were treated with 50 ng/ml recombinant mouse Il17a (R&D Systems) for 0, 5, or 15 min.

Techniques: Confocal Microscopy, Cell Culture, Expressing

Journal: PLoS ONE

Article Title: Interleukin-17 Retinotoxicity Is Prevented by Gene Transfer of a Soluble Interleukin-17 Receptor Acting as a Cytokine Blocker: Implications for Age-Related Macular Degeneration

doi: 10.1371/journal.pone.0095900

Figure Lengend Snippet: (A) IL17RC knockdown by 0.04 µM siRNA. IL17RC was measured by qPCR at 24h, 48h, and 72h post-transfection relative to a universal mRNA standard. (B) Relative viability of ctrl or IL17RC siRNA-transfected ARPE-19 with 100 ng/ml IL17A. (C) Confocal microscopy of ctrl or IL17RC siRNA-transfected ARPE-19 treated with or without IL17A. Nuclear localization of Caspase-3 (arrowheads). Graphical representation of the Caspase:DAPI ratio are indicated to the right of the images. Blue = DAPI. Scale bar = 50 µm. *: P<0.05; **: P<0.005; ****: P<0.0001. Data are presented as mean ± SEM.

Article Snippet: Retinas were treated with 50 ng/ml recombinant mouse Il17a (R&D Systems) for 0, 5, or 15 min.

Techniques: Knockdown, Transfection, Confocal Microscopy

Journal: PLoS ONE

Article Title: Interleukin-17 Retinotoxicity Is Prevented by Gene Transfer of a Soluble Interleukin-17 Receptor Acting as a Cytokine Blocker: Implications for Age-Related Macular Degeneration

doi: 10.1371/journal.pone.0095900

Figure Lengend Snippet: (A) Retinal Il17a transcripts as a function of animal age. (B) Fundoscopic results 2 months post-intervention. (C) A2E quantification. For box plots: center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; width of the boxes is proportional to the square root of the sample size; data points are plotted as open circles. (D) Histopathological findings: sIL17R preserved photoreceptor IS/OS thickness compared to EV (asterisks) and maintained thickness of the ONL. EV retinas showed RPE degeneration (red arrow). Dystrophic rd8 lesions are indicated (black arrows). (E) Abundant lipofuscin in EV but not in sIL17R RPE (yellow arrowheads, upper left and right). Mitochondria (m) are damaged and disorganized within EV RPE (lower left) but are viable and organized in sIL17R RPE (lower right). EV RPE show extensive vacuolization, undigested OS and poor basal infoldings (lower left). (F) Western blot of downstream Il17 signaling. EV and sIL17R neuroretinas were treated with 50 ng/ml Il17a for 0, 5, or 15 min ex vivo . Relative Signal Intensities are presented of phosphorylated proteins relative to total protein to the right of the blots. NFL = nerve fiber layer; GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer; IS/OS = inner/outer segment; RPE = retinal pigment epithelium *: P<0.05; **: P<0.005; ****: P<0.00001.

Article Snippet: Retinas were treated with 50 ng/ml recombinant mouse Il17a (R&D Systems) for 0, 5, or 15 min.

Techniques: Software, Western Blot, Ex Vivo