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

Joanne A Matsubara,Yuan Tian,Matthew R Zeglinski,Christopher T Turner,Sho Hiroyasu,Jing Z Cui,David J Granville

doi:10.3389/fimmu.2020.00574

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, 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.

Highlights

Age-related macular degeneration (AMD) is a complex disease with many risk factors contributing to its pathogenesis [2,3,4]
Our premise is that extracellular Granzyme B (GzmB) contributes to the onset and/or progression of AMD via the cleavage of key extracellular proteins in Bruch’s Membrane (BM) resulting in retinal pigment epithelial (RPE) dysregulation and barrier function abnormalities
GzmB immunoreactivity is evident in the extracellular matrix of BM and the intercellular spaces between RPE cells

Summary

Introduction

Age-related macular degeneration (AMD) is a complex disease with many risk factors contributing to its pathogenesis [2,3,4]. Despite the fact that clinical and genetic data support an association of a chronic, low-grade inflammatory response in the outer retina during the development of AMD, the exact underlying mechanisms and triggers of inflammation remain elusive [5]. Genetic studies point to a central role of the innate immune response and the complement cascade in the development and progression of AMD [for review see [6]]. We recently reported that the RPE and immune cells (primarily mast cells) in the choroid express Granzyme B (GzmB) [7], a serine protease that was once thought to function exclusively as intracellular initiators of immune-mediated cell death, capable of inducing apoptosis (a process requiring perforin, a pore-forming protein) in target cells. Experimental degranulation of choroidal mast cells in rodents resulted in RPE abnormalities and outer retinal barrier breakdown [13], it is not yet known which of the many proteases, or other choroidal mast cell mediators, are involved [14]

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Conclusion