A C-terminal peptide from type I interferon protects the retina in a mouse model of autoimmune uveitis

Abstract

Experimental autoimmune uveitis (EAU) in rodents recapitulates many features of the disease in humans and has served as a useful tool for the development of therapeutics. A peptide from C-terminus of interferon α1, conjugated to palmitoyl-lysine for cell penetration, denoted as IFNα-C, was tested for its anti-inflammatory properties in ARPE-19 cells, followed by testing in a mouse model of EAU. Treatment with IFNα-C and evaluation by RT-qPCR showed the induction of anti-inflammatory cytokines and chemokine. Inflammatory markers induced by treatment with TNFα were suppressed when IFNα-C was simultaneously present. TNF-α mediated induction of NF-κB and signaling by IL-17A were attenuated by IFNα-C. Differentiated ARPE-19 cells were treated with TNFα in the presence or absence IFNα-C and analyzed by immmunhistochemistry. IFNα-C protected against the disruption integrity of tight junction proteins. Similarly, loss of transepithelial resistance caused by TNFα was prevented by IFNα-C. B10.RIII mice were immunized with a peptide from interphotoreceptor binding protein (IRBP) and treated by gavage with IFNα-C. Development of uveitis was monitored by histology, fundoscopy, SD-OCT, and ERG. Treatment with IFNα-C prevented uveitis in mice immunized with the IRBP peptide. Splenocytes isolated from mice with ongoing EAU exhibited antigen-specific T cell proliferation that was inhibited in the presence of IFNα-C. IFNα-C peptide exhibits anti-inflammatory properties and protects mice against damage to retinal structure and function suggesting that it has therapeutic potential for the treatment of autoimmune uveitis.

Fig 1. IFNα-C suppressed the release of IL-1β from ARPE-19 cells induced with TNFα.

ARPE-19 cells were grown overnight in 12-well plates. They were placed in low serum medium and pre-treated with IFNα-C (3 μM) for 2 hr followed by treatment with TNFα (50 ng/ml) for 24 hr. Supernatants were collected and assayed for IL-1β using ELISA. N = 6. The error bars indicate standard deviation. *, P = 0.01.

Fig 2. Suppression of NF-κB signaling by IFNα-C.

ARPE-19 cells were seeded on 8 well microscopic slides and grown overnight. They were taken in low serum medium and treated with IFNα-C (3 μM) for 2 hr followed by treatment with TNFα (50 ng/ml) for 30 min. Cells were fixed, permeabilized with 1% Triton X-100 (in PBS) and stained with an antibody to p65, followed by staining with Cy3-conjugated secondary antibody and DAPI and imaged using fluorescence microscopy at 40x.

Fig 3. IFNα-C prevented signaling from IL-17A.

ARPE-19 cells were seeded on 8 well microscopic slides and grown overnight. They were placed in low serum medium and treated with IFNα-C (3 μM) for 2 hr followed by treatment with IL-17A (50 ng/ml) for 30 min. Cells were fixed, permeabilized with 1% Triton X-100 (in PBS) and stained with an antibody to pSTAT3, followed by staining with Alexa 488-conjugated secondary antibody and DAPI and imaged in a fluorescence microscope at 40x magnification.

Fig 4. IFNα-C prevented the loss of tight junctions caused by TNFα or IL-17A.

ARPE-19 cells grown in 8 well slides in low serum for 4 weeks were pre-treated with IFNα-C (3 μM) for 4 hr followed by TNFα or IL-17A (both at 50 ng/ml) for 48 hr. These were compared to untreated cells and cell treated with TNFα alone. Cells were fixed, permeabilized and stained with an antibody to ZO-1, followed by staining with Cy3-conjugated secondary antibody, and imaged by fluorescence microscopy at 40x magnification.

Fig 5. IFNα-C prevents the reduction of transepithelial resistance caused by TNFα.

ARPE-19 cells grown in 24 well transwell plates in low serum medium for 4 weeks were treated with IFNα-C (3 μM) for 4 hr followed by treatment with TNFα (50 ng/ml) for 48 hr. Untreated cells or those treated with TNFα alone were included alongside. TEER, in individual cells in triplicate was measured using EVOM2 voltohmmeter. The error bars indicate standard deviation. *, p = 0.001; **, p = 0.009.

Fig 6. Protection against EAU by oral gavage with IFNα-C.

Eight-week old female B10.RIII mice (n = 8) were gavaged with IFNα-C (200 μg/mouse) or solvent (PBS) in 200 μl on days -2, -1 and 0. On day 0, mice were immunized with IRBP peptide emulsified in complete Freund’s adjuvant. Treatment with IFNα-C or solvent was continued for 3 weeks. A. Optical coherence tomography on day 14. Mice treated with solvent showed influx of inflammatory cells and swelling of retina, which were not seen in mice treated with IFNα-C. B. Fundoscopic images on day 14 show engorged blood vessels and hemorrhage in solvent treated mice. C. Hematoxylin and eosin staining of eyes harvested on day 14 after immunization. Infiltrating cells and retinal folding seen in solvent were not observed in mice treated with IFNα-C. Images were taken at 40x magnification. D. A clinical score, as described in Materials and Methods was used. Mice treated with IFNα-C were protected against the symptoms of EAU. The error bars represent standard deviation. The symptoms did not develop in the following two weeks after termination of treatment. **, p<0.01.

Fig 7. IFNα-C prevented the loss of ERG a- and b- wave amplitudes in EAU mice.

Dark adapted and light adapted electroretinograms were recorded in B10.RIII mice before immunization (pre) and 3 weeks after immunization with IRBP and treatment with solvent (top row) or IFNα-C (bottom row). Average a-wave and b-wave amplitudes are shown. The error bars represent standard deviation. The same cohort of mice were used for all data points. P values were determined by 2-way ANOVA using the Sidak method for multiple comparisons. N = 8.

Fig 8. IFNα-C suppressed the IRBP-induced proliferation of splenocytes.

Splenocytes were harvested from IRBP-immunized B10.RIII mice 3 weeks after immunization. Splenocytes (5x10⁵ cells/well) in 96 well plates were grown in RPMI with 10% FBS. IFNα-C (3 μM) was added for 4 hr followed by addition of IRBP (50 μg/ml) and cells were grown for 72 hrs. Cell proliferation was measured as described in Material and Methods. The error bars indicate standard deviation. P = 0.02.