Atypical cytomegalovirus retinal disease in pyroptosis-deficient mice with murine acquired immunodeficiency syndrome

Abstract

Pyroptosis is a caspase-dependent programmed cell death pathway that initiates and sustains inflammation through release of pro-inflammatory cytokines interleukin (IL)-1β and IL-18 following formation of gasdermin D (GSDMD)-mediated membrane pores. To determine the possible pathogenic contributions of pyroptosis toward development of full-thickness retinal necrosis during AIDS-related human cytomegalovirus retinitis, we performed a series of studies using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS). Initial investigations demonstrated significant transcription and translation of key pyroptosis-associated genes within the ocular compartments of MCMV-infected eyes of mice with MAIDS. Subsequent investigations compared MCMV-infected eyes of groups of wildtype MAIDS mice with MCMV-infected eyes of groups of caspase-1^-/- MAIDS mice, GSDMD^-/- MAIDS mice, or IL-18^-/- MAIDS mice to explore a possible contribution of pyroptosis towards the pathogenesis of MAIDS-related MCMV retinitis. Histopathologic analysis revealed typical full-thickness retinal necrosis in 100% of MCMV-infected eyes of wildtype MAIDS mice. In sharp contrast, none (0%) of MCMV-infected eyes of MAIDS mice that were deficient in either caspase-1, GSDMD, or IL-18 developed full-thickness retinal necrosis but instead exhibited an atypical pattern of retinal disease characterized by thickening and proliferation of the retinal pigmented epithelium layer with relative sparing of the neurosensory retina. Surprisingly, MCMV-infected eyes of all groups of deficient MAIDS mice harbored equivalent intraocular amounts of infectious virus as seen in MCMV-infected eyes of groups of wildtype MAIDS mice despite failure to develop full-thickness retinal necrosis. We conclude that pyroptosis plays a significant role in the development of full-thickness retinal necrosis during the pathogenesis of MAIDS-related MCMV retinitis. This observation may extend to the pathogenesis of AIDS-related HCMV retinitis and other AIDS-related opportunistic virus infections.

Keywords: AIDS; Cell death pathway; Human cytomegalovirus; MAIDS; Murine cytomegalovirus; Pyroptosis; Retinitis.

Fig. 1.

GSDMD mRNA and caspase-11 mRNA are stimulated intraocularly within MCMV-infected eyes of mice with MAIDS. Whole MCMV-infected eyes and mock-infected eyes were collected at 3, 6, and 10 days postinfection from groups (n = 3 – 5) of wildtype mice with MAIDS and assessed for (A) GSDMD mRNA expression and (B) caspase 11 mRNA expression by real-time RT-PCR assay using the comparative 2Λ-ΔΔCT method. *p < 0.05, MCMV-infected eyes were compared with mock-infected eyes at the same time point.

Fig. 2.

Proteins associated with the canonical pyroptosis pathway (pro-caspase-1, cleaved caspase-1, GSDMD, cleaved GSDMD, IL-1β, and IL-18) and the noncanonical pyroptosis pathway (pro-caspase-11 and caspase-11) are stimulated intraocularly within MCMV-infected eyes of mice with MAIDS. Whole MCMV-infected eyes and mock-infected eyes were collected at 3, 6, and 10 days postinfection from groups (n = 4 – 6) of wildtype mice with MAIDS, pooled, and subjected to western blot assay. The figure represents a composite of individual western blot assays. GAPDH served as a control.

Fig. 3.

MCMV-infected eyes from groups of MAIDS mice deficient in the production of a pyroptosis-associated protein show prominent RPE proliferation but relative preservation of the neurosensory retina. MCMV-infected eyes were collected at 10 days postinfection from groups of wildtype (WT) mice with MAIDS (B, D, and F), caspase-1^−/− mice with MAIDS (C), GSDMD^−/− mice with MAIDS (E), and IL-18^−/− mice with MAIDS (G) and subjected to H&E staining for histopathologic analysis. Full-thickness retinal necrosis within sections of MCMV-infected eyes of WT mice with MAIDS is indicated using brackets. Thickening and proliferation of the RPE layer within sections of MCMV-infected eyes of caspase-1^−/− mice with MAIDS, GSDMD^−/− mice with MAIDS, and IL-18^−/− mice with MAIDS is indicated using black arrows. Representative RPE showing virus-induced inclusions within sections of MCMV-infected eyes of WT MAIDS and MCMV-infected eyes of GSDMD^−/− mice with MAIDS are indicated using white arrows. A section of retina from an uninfected and unmanipulated eye of a WT mouse with MAIDS showing normal retinal architecture is shown for comparison. Original magnification = 200X

Fig. 4.

MCMV-infected eyes of caspase-1^−/− mice with MAIDS, GSDMD^−/− mice with MAIDS, and IL-18^−/− mice with MAIDS harbor equivalent amounts of intraocular infectious MCMV when compared with MCMV-infected wildtype mice with MAIDS. Whole MCMV-infected eyes were collected at 10 days after subretinal MCMV inoculation from groups (n = 4 – 6) of wildtype (WT) mice with MAIDS, caspase-1^−/− mice with MAIDS, GSDMD^−/− mice with MAIDS, and IL-18^−/− mice with MAIDS, individually homogenized, and individually subjected to standard plaque assay for quantification of infectious virus.