Necroptosis pathway activation in the retina after ocular alkali burn

Abstract

Purpose : Previous studies have shown that ocular alkali injury leads to peripheral monocyte infiltration into the retina within 24 hours and subsequent release of TNF-α and IL1b. This was shown to cause Caspase3 and Endonuclease G -mediated apoptosis in retinal neurons and subsequent neurodegeneration. Here we investigate the differential role of engrafted monocytes and microglia in mediating retinal cell death through the necroptosis pathway by analyzing receptor interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) expression.

Methods : Retinal expression of RIPK1, RIPK3 and MLKL was evaluated using western blot, qPCR, and immunofluorescence staining (IF) 24 hours after alkali injury to the eye. Bone marrow chimeras were employed to differentiate microglia from infiltrating periphery monocytes for subsequent flow shorting and analysis of the regulation of necroptosis pathway using assay for transposase-accessible chromatin with sequencing (ATAC-seq).

Results : Ocular alkali burn caused significant upregulation of IL-1b, IL-6, Mcp-1, and Nlrp3 in the retina at 24 hours and increase in TUNEL+ cells. mRNA and protein levels of RIPK1, RIPK3, and MLKL were dramatically increased, while immunostaining of flat mount retinal tissues showed increase in RIPK3 expression in engrafted monocytes, but not in microglia. Upon engraftment into the retina, monocytes exhibited increase in chromatin accessibility for TNF receptor superfamily gene (tnfrsf8, tnfrsf11a and tnfrsf17), which is upstream of RIPK1-RIPK3-MLKL axis, and TNF receptor-associated factor 2(TRAF2) gene, which mediates TNF signaling transduction. Chromatin accessibility for RIPK1 and RIPK3 gene was similar between circulating and engrafted monocytes, but only engrafted monocytes had open chromatin for MLKL gene.

Conclusions : Necroptosis is another mediator of retinal damage after ocular alkali injury. To this end, engrafted monocytes play a key role in activating this pathway by changing chromatin accessibility for genes encoding for necroptosis pathway. Further work is required to understand the implications of these results in retinal damage.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.