ORLL-NIH001 inhibits expansion of Th17 cells in a dose dependent manner.

<p>(A, B) Naïve CD4⁺ and CD8⁺ T cells were labeled with CFSE, stimulated for 4 days with anti-CD3/anti-CD28 under Th17 polarization condition in medium containing vehicle alone or different doses of ORLL-NIH001 (A). Numbers in the quadrants indicate percent of proliferating or non-proliferating CD4⁺ or CD8+ T cells expressing IL-17. (B) Proliferation of cells in cultures without CFSE was quantified by the Thymidine incorporation assay. Data presented are representative of at least 3 independent experiments.</p

ORLL-NIH001 inhibited proteins that mediate lymphocyte trafficking into retina.

<p>(A, B) LN cells from mice with EAU were re-stimulated with IRBP in medium containing ORLL-NIH001 or commercially available STAT3 inhibitors. Expression or activation of integrins and chemokine receptors were analyzed by FACS. Plots were gated on CD4⁺ T cells and numbers in quadrants indicate percent of CD4⁺ T cells expressing CD11a, CD29, CD44, CD49d, CXCR3, CCR6, α4β7α4β1 or Ly6c. Data presented are representative of 3 independent experiments.</p

ORLL-NIH001 inhibited the expansion of human Th17 cells.

<p>(A) CD4⁺ T cells isolated from PBMC of healthy human subjects were labeled with CFSE and then stimulated with anti-CD3/CD28 for 4 days in medium containing vehicle alone or different doses of ORLL-NIH001. Numbers in the quadrants indicate percent of proliferating or non-proliferating T cells expressing IL-17 or IFN-γ. (B) Human PBMC were activated with anti-CD3/CD28 abs for 4 days, stimulated with IL-6 in serum-free medium containing vehicle or ORLL-NIH001. The cells were then analyzed for the expression of STAT3, pSTAT1 or β-actin by Western blotting. Activated human (C) or mouse (D–H) CD4 T cells were stimulated with IL-2 or IL-6 and expression of pSTAT1, pSTAT3 and pSTAT5 was detected by Western blotting (C, D, E) or flow cytometry (F, G, H). (E) Western blots (B, C, D) were analyzed using NIH Image Quant program and each band was normalized to corresponding β-actin band and expressed in arbitrary relative protein expression units. Results are representative of 3 independent experiments.</p

STAT3-deficient T cells could not traffic into the retina nor induce EAU.

<p>(A) WT mice or mice with targeted deletion of STAT3 in T cells (CD4-STAT3KO) were immunized with IRBP in CFA and their eyes were enucleated 21 days post-immunization and histological sections through the retina were stained with H&E. Black arrows indicate presence of inflammatory cells in the vitreous (V) and blue arrows depict pathologic foci characterized by the presence of retinal folds and hemorrhage. Red arrow, choroiditis; White arrow, granuloma; green arrow, retinal vasculitis; OpN, optic nerve; V, vitreous; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (B) Freshly isolated cells from the draining lymph nodes or spleen of WT unimmunized WT control or EAU mice were analyzed by the intracellular cytokine assay. Plots were gated on CD4⁺ T cells and numbers in quadrants indicate percentage of CD4⁺ T cells expressing IL-17 and/or activated STAT3 (pSTAT3). (C) WT or CD4-STAT3KO mice were immunized with IRBP in CFA and T cells present in retina on day-21 post-immunization were detected and quantified by flow cytometry. Numbers in quadrants indicate percentage of CD3⁺ or CD4⁺ T cells or CD4⁺ T cells expressing IL-17 and/or IFN-γ. Data presented are representative of at least 3 independent experiments.</p

ORLL-NIH001 inhibited expansion of mouse uveitogenic Th17 cells.

<p>(A) Draining LN cells from vehicle-treated (control) or drug-treated mice (Protocol 1) were re-stimulated <i>in vitro</i> with IRBP for 3 days and effects of ORLL-NIH001 was assessed by Thymidine incorporation assay. Freshly isolated PBMC (B, C) was isolated from individual mice 4 days post-immunization. The levels of CD3⁺CD4⁺ T cells were quantified using FACS (B) and the number of cytokine-expressing T cells was assessed by intracellular cytokine assay (C). (D, E) Freshly isolated lymph node cells from vehicle or drug-treated mice were re-stimulated ex vivo with IRBP for 3 days and then analyzed by the intracellular cytokine assay. CFSE was added to some cultures (E). Plots were gated on CD3⁺ T cells and numbers in quadrants indicate percent of CD4⁺ T cells expressing IL-17 and/or IFN-γ. Data presented are representative of at least 3 independent experiments.</p

ORLL-NIH001 conferred protection from development of severe EAU.

<p>(A) Schematic depiction of immunization protocols used for EAU induction in B10.A mice. Mice treated under Protocol 1 received injections of ORLL-NIH001, starting 1 day before immunization (day −1) and on every other day thereafter until day-17 post-immunization. Protocol 2: mice began receiving the drug on day-5 post-immunization. Mouse eyes were harvested, fixed, embedded in paraffin and stained with H&E. (B, D) Histological analysis showing: inflammatory cells in the vitreous (black arrows), retinal folds (white arrows). OpN, optic nerve; V, vitreous; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (C, E) EAU scores were calculated for vehicle-treated (control) or ORLL-NIH001-treated mice based on and histology. Similar results were obtained in mice treated under Protocol 1 (B, C) or Protocol 2 (D, E) and data presented are representative of at least 3 independent experiments.</p

IL-27 induces expression of CFH through STAT1-mediated up-regulation of IRF-1 and IRF-8.

<p>(A–C) ARPE-19 cells were starved for 2 hours in serum free medium and cultures were then stimulated with IL-27 (10 ng/ml) for 30, 90 or 720 min. (B–D) In other cultures, cells were treated with CHX (35 ug/ml) for 30 min prior treatment with IL-27 for 30 min (B) or 24 h (C, D). Whole cell extracts were analyzed by Western blot assay and the antibodies used are indicated. (D) CFH mRNA transcripts were detected and quantified by qRT-PCR.</p

Expression of IRF-1, IRF-8 and IL-27 (IL27p28/EBi3) by retinal cells was up-regulated during experimental autoimmune uveitis (EAU).

<p>(A) Fundus images of the retina of un-immunized mouse and mouse with EAU. (B, C) Retinas were isolated from control or EAU mice and analyzed for IRF-1, IRF-8, CFH, IL27p28 or EBi3 expression by qRT-PCR.</p

Detection and localization of IRF-1 and IRF-8 proteins in the retina.

<p>Retinas were isolated from control or EAU mice and whole cell extracts were analyzed for IRF-1 (A) or IRF-8 expression by Western blot analysis. (C) Immunohistochemical localization of IRF-8 expression was detected in the normal mouse retina or retina of mice with EAU using an IRF-8-specific antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045801#s2" target="_blank">Methods</a> section. Retina pigmented epithelium (RPE); Photoreceptors (pho); outer nuclear layer (ONL); outer plexiform layer (OPL); inner plexiform layer (IPL).</p

IL-27 induces CFH expression in the retina through STAT1-dependent mechanism.

<p>(A) Characterization of retina from mice with specific deletion of STAT3 in the retina (Ret-STAT3^−/−). (B) Freshly isolated retinas from wild type (WT), STAT1 knockout (STAT1^−/−), (Ret-STAT3^−/−), or from mice with targeted over-expression of SOCS1 in the retina (SOCS1Tg) were analyzed by RT-PCR. (C, D) Freshly isolated retinal cells from WT or STAT1^−/− mice were cultured overnight, washed, starved for 2 hours in serum-free medium and then cultured for an additional 22 hours in medium containing IL-27 (10 ng/ml). We analyzed RNA and total cell extracts from the cells by RT-PCR (C) or Western blotting (D).</p