The role of RIP3 in innate immune responses and death of bystander cells during MCMV retinitis

Abstract

Purpose: Recent studies suggest that RIP kinases, which are key decision makers in cell death and innate immunity against invading pathogens, are activated during MCMV ocular MCMV infection. The purpose of this study is to determine if RIP3 plays a significant role in innate immune responses and death of bystander cells by comparing MCMV retinal infection, innate immune response and cell death in Rip3^-/- mice and control mice.

Methods: RIP3^-/- and RIP3^+/+ mice were immunosuppressed (IS) with methylprednisolone acetate (2mg per mouse, i.m. every 4 days) and inoculated with 5 Χ10³ PFU of MCMV K181 strain, via the supraciliary route. Virus injected and control eyes were removed at days 4, 7 and 10 post-infection and analyzed by plaque assay, electron microscopy (EM), H & E staining, TUNEL assay, western blot (for caspase 3, caspase 1, caspase 12, NFκB), as well as immunohistochemical staining for MCMV early antigen, cleaved caspase 3 and AIF.

Results: Compared to RIP3^+/+ mice, significantly more MCMV was recovered and more MCMV infected RPE cells were observed in injected eyes of RIP3^-/- mice at day 4 and day 7 p.i. In contrast, fewer TUNEL-stained photoreceptors were observed in injected RIP3^-/- eyes than in injected RIP3^+/+ eyes at these times. EM results also confirm that significantly more apoptotic photoreceptor cells were observed in the outer nuclear layer of RIP3^+/+ wild type mice than in the outer nuclear layers of RIP3^−/− mice. Furthermore, the results of immunohistochemistry showed that the majority of TUNEL-stained photoreceptors died via AIF-mediated, caspase 3-independent apoptosis, although significantly lower levels of cleaved caspase 3 were also detected in injected eyes of RIP3^-/- mice than in injected eyes of RIP3^+/+ mice by western blot. Expression of cleaved caspase 1 was increased in RIP3^+/+ eyes after MCMV infection as early as day 4 p.i. In contrast, increased cleaved caspase 1 expression was observed in RIP3^-/- eyes only at day 10 p.i. In addition, significantly higher levels of activated NFκB were observed in RIP3^+/+ eyes compared to RIP3^-/- eyes at days 4, 7 and 10 p.i.

Conclusions: Our results suggest that RIP3 enhances innate immune responses against ocular MCMV infection via activation of the inflammasome and NF-κB, which, also leads to inflammation and death of bystander cells by multiple pathways including apoptosis and necroptosis.