Il 27

All experiments involving mice were approved by the Animal Care and Use Committee at the University of Miami and were carried out in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision research. The retinal degeneration 10 (rd10) mouse model was purchased from Jackson Laboratory and housed with a 12-h light–dark cycle, free access to food and water, and all cages were maintained at similar distances from the overhead lights. Experimental and control treatments were from the same litters.
Mice were divided randomly into experimental groups and were injected intravitreously with sterile saline (control) or IL-27 (10, 20, 40 ng/µl) (BioLegend, San Diego, CA) on post-natal day (P) 18, as follows. The mice were anesthetized using isoflurane, the corneas were anesthetized with a drop of 0.5% proparacaine hydrochloride and the mice were placed under a surgical microscope on a heated pad to maintain body temperature at 37 °C. The eyes were intravitreally injected with 1 µl of solution using a 33-gauge Hamilton needle (Hamilton Company, Reno, NV) passed through the sclera into the vitreous cavity and angled to avoid the lens. The fellow eye was uninjected. Mice with bleeding or swelling were excluded from further study. Investigators were masked to the treatment for all subsequent analyses.

Normal human epidermal keratinocytes (NHEKs) were purchased from Thermo Fisher Scientific and maintained for up to 6 passages in T-75 flasks or used earlier. Cells were grown in serum-free EpiLife cell culture medium with EpiLife Defined Growth Supplement containing 0.06 mM Ca²⁺ (Gibco, Waltham, MA) or Keratinocyte serum-free-media (KSFM) with supplements provided by manufacturer (Gibco) and additional 0.06mM Ca²⁺. NHEKs were grown to approximately 75-80% confluence. For experiments, cells between passage 3-6 were plated at approximately 200,000 cells/well in 6-well plates and 75,000 cells/chamber in 2-chamber slides, respectively (LabTek, Bloomington, IN). For some experiments, IL-27 was used at a concentration of 100 ng/mL; IFN-α was used at a concentration of 50 U/mL (BioLegend). The cells were collected for quantitative RT-PCR or immunofluorescence at various time points.