Retinal Distribution and Extracellular Activity of Granzyme B: A Serine Protease That Degrades Retinal Pigment Epithelial Tight Junctions and Extracellular Matrix Proteins

Abstract

Granzymes are a family of serine proteases first shown to be intracellular initiators of immune-mediated cell death in target pathogenic cells. In addition to its intracellular role, Granzyme B (GzmB) has important extracellular functions in immune regulation and extracellular matrix (ECM) degradation. Verified substrates of extracellular GzmB activity include tight junctional and ECM proteins. Interestingly, little is known about the activity of GzmB in the outer human retina, a tissue in which the degradation of the tight junctional contacts of retinal pigment epithelial (RPE) cells and within the external limiting membrane, as well as remodeling of the ECM in Bruch's membrane, cause the breakdown of the blood-retinal barrier and slowing of metabolite transport between neuroretina and choroidal blood supply. Such pathological changes in outer retina signal early events in the development of age-related macular degeneration (AMD), a multifactorial, chronic inflammatory eye disease. This study is the first to focus on the distribution of GzmB in the outer retina of the healthy and diseased post-mortem human eye. Our results revealed that GzmB is present in RPE and choroidal mast cells. More immunoreactive cells are present in older (>65 years) compared to younger (<55 years) donor eyes, and choroidal immunoreactive cells are more numerous in eyes with choroidal neovascularization (CNV), while RPE immunoreactive cells are more numerous in eyes with soft drusen, an early AMD event. In vitro studies demonstrated that RPE-derived tight junctional and ECM proteins are cleaved by exogenous GzmB stimulation. These results suggest that the increased presence of GzmB immunoreactive cells in outer retina of older (healthy) eyes as well as in diseased eyes with CNV (from AMD) and eyes with soft drusen exacerbate ECM remodeling in the Bruch's membrane and degradation of the blood-retinal barrier. Currently there are no treatments that prevent remodeling of the Bruch's membrane and/or the loss of function of the outer blood-retinal barrier, Matsubara et al. Granzyme B in Outer Retina known to promote early AMD changes, such as drusen deposition, RPE dysfunction and pro-inflammation. Specific inhibitors of GzmB, already in preclinical studies for non-ocular diseases, may provide new strategies to stop these early events associated with the development of AMD.

Key Determinants of Cell-Mediated Immune Responses: A Randomized Trial of High Dose Vs. Standard Dose Split-Virus Influenza Vaccine in Older Adults

“…(2012), GrB accumulates extracellularly and retains its activity. Thus, in the absence of perforin, GrB is released into the extracellular space and degrades proteins key to maintaining cell-cell junctions Buzza et al(2005), disrupts epithelial barrier function Matsubara et al (2020), and increases susceptibility to infection, inflammation, and injury.…”

A Re-Appraisal of Pathogenic Mechanisms Bridging Wet and Dry Age-Related Macular Degeneration Leads to Reconsider a Role for Phytochemicals

“…Autophagy is also involved in selective degradation of the serine protease granzyme B [ 139 ], which is known to cleave RPE tight junctions and extracellular matrix (ECM) proteins, eventually contributing to a breakdown of the BRB and remodeling of the Bruch’s membrane. This is expected to consistently affect RPE-dependent transport between the retina and choroidal blood supply [ 140 ]. In line with this, granzyme B is increased in the RPE and choroidal mast cells in old, compared with young, donor eyes, and also in CNV patients’ eyes [ 140 ].…”

“…This is expected to consistently affect RPE-dependent transport between the retina and choroidal blood supply [ 140 ]. In line with this, granzyme B is increased in the RPE and choroidal mast cells in old, compared with young, donor eyes, and also in CNV patients’ eyes [ 140 ]. Again, in RPE cells, downregulation of thioredoxin-interacting protein (TXNIP) expression occurs under oxidative stress, which is associated with a block of the autophagy flux, leading to a decrease of RPE cell proliferation, disruption of RPE cell tight junctions, and a loss of oBRB integrity [ 138 ].…”

Hyperreflective foci, OCT progression indicators in age-related macular degeneration, include transdifferentiated retinal pigment epithelium

“…This investigation like our previous studies used high resolution comprehensive microscopy to link cells with characteristic RPE organelles ( 18, 19 ) to a time course of activity visible through longitudinal clinical imaging. Our cellular phenotyping system ( 18 ) built on prior literature and is used by others ( 43, 44 ). We posited that these phenotypes represented discrete steps towards death, migration, or transdifferentiation of individual cells that could be followed over time in OCT due to high RPE reflectivity.…”