Abstract
Purpose :
Immune homeostasis and unregulated inflammation drive the development of age-related macular degeneration (AMD), in which the role of one of the innate immune system ‘master regulators’, the mast cell (MC), remains ill-defined. Our aim is through mechanisms of MC biology understand their role in governing retinal vascular integrity and the implications for development of AMD.
Methods :
RPE(B6-RPE07) and bone marrow derived mast cells(BMMC) were assayed by RT-PCR and Western Blot. Co-cultures of BMMC and RPE or mouse retinal microvascular endothelial cells(MRMEC) were established. Choroidal sprouting assay and laser-induced CNV were used as models of ocular angiogenesis. WT, ST2-/- and mast cell deficient c-kit-/- mice were employed in the laser-induced CNV model.
Results :
We first determined whether interaction with RPE triggers immediate changes in MC immunophenotype. Stimulation of BMMC with toll-like receptor stimulated-RPE supernatant led to a robust inflammatory cascade, increasing the expression of IL-6, IL-1b, TNF and MCP-1. Treatment of BMMC with calcimycin, a secretagogue for mast cells, led to degranulation and increase in glycolysis. By contract, stimulation of MC with IL-33, a novel signaling protein secreted by RPE, did not result in their degranulation or increase in glycolysis or oxidative phosphorylation, sustaining their metabolism and granules. Furthermore, when we stimulated MC with IL-33, we found a marked upregulation of IL-4 and IL-13, and release of PGE2 and PGD2, demonstrating that mast cells can act as a source of anti-angiogenic factors. To support this finding, we utilised the well-established laser-induced CNV model. Choroidal MC infiltrated the sites of laser injury. Intravitreal administration of IL-33 significantly increased MC infiltration, accompanied by attenuated CNV formation. Inhibition of MC reversed the anti-angiogenic effect of IL-33.
To support MC role in maintaining vascular beds we generated co-culture of MRMEC with IL-33 treated MC that resulted in a significant reduction in VEGF secretion and VEGFR2 expression. The co-culture also increased the expression of occludin and ZO-1 in MRMEC, providing evidence of a protective effect and maintaining vascular integrity.
Conclusions :
Immunomodulation of mast cells with IL-33 governs the choroidal microenvironment by restricting excessive responses to inflammation, vascular permeability and development of angiogenesis.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.