In the eye, inflammation is tightly regulated to provide the eye with protection against pathogens, while at the same time, protecting the visual axis from sight-destroying inflammation. These mechanisms are collectively called immune privilege.
Dr. Gregory-Ksander’s early work focused on elucidating the mechanisms involved in the maintenance of ocular immune privilege and limiting inflammation in the eye. Her work on Fas ligand (FasL) provided a significant advance in our understanding of (i) how the membrane-bound and soluble forms of FasL maintain immune privilege and control innate immunity in the eye and (ii) how dysregulation of the FasL signaling pathway can contribute to tissue damage and ocular disease. Dr. Gregory-Ksander’s current research focuses on understanding how age-related changes in immune privilege and subsequent inflammation contribute to the development of glaucoma and age-related macular degeneration (AMD). While the importance of inflammation in glaucoma and AMD has been recognized, how it is triggered in and how it is controlled is largely unknown. The Gregory-Ksander laboratory is focused on identifying key signaling pathways that contribute to the disruption of immune privilege and development of inflammation associated with glaucoma and AMD. She hopes her work ultimately will help researchers identify new targets to protect the retina and preserve vision in patients with glaucoma and/or AMD.
Glaucoma is one of the most common causes of blindness worldwide and, while there are many different forms of glaucoma that differ significantly in clinical presentation and disease progression, they all share a common endpoint, which is the loss of retinal ganglion cells (RGCs). New treatments are needed, since current therapies can delay, but not stop disease progression. One of the major barriers to the development of novel treatments is the incomplete understanding of the disease pathogenesis.
Working with several mouse models of glaucoma, including the pigmentary dispersion model in DBA/2J mice, TNFα inducible model, open angle glaucoma model in SGC α1-deficient mice, and the microbead occlusion model, Dr. Gregory-Ksander’s laboratory has studied how disruption of immune privilege and subsequent glial activation and inflammation contributes to neurodegeneration and the development of glaucoma. This work has lead to the identification of the Fas ligand and NLRP3 signaling pathways as critical mediators in the pathogenesis of glaucoma. The current work in Dr. Gregory-Ksander’s lab focuses on (i) understanding how and why the Fas ligand and NLRP3 pathways become dysregulated in the development of glaucoma, and (ii) developing and testing novel inhibitors of these pathways as potential neuroprotective therapies in glaucoma.
The ultimate goal of this research is to develop an IOP-independent treatment approach for glaucoma aimed at neuroprotection and preservation of vision.
Age-Related Macular Degeneration (AMD)
AMD is a leading cause of blindness worldwide. Loss of vision is due to the degeneration of photoreceptors which can occur in two forms of AMD: Wet AMD develops with choroidal neovascularization (CNV), vascular leakage, hemorrhaging, and retinal detachment, while Dry AMD develops with a geographic loss of RPE cells and loss of the overlaying photoreceptors
Only patients with wet AMD develop CNV, which is associated with more severe vision loss. In wet AMD, the loss of vision is caused primarily by the leakage of fluid and blood from the choroidal neovascularization. Anti-VEGF therapy has emerged as the treatment of choice for patients with wet AMD and has been shown to preserve vision in many patients by preventing leakage from the existing choroidal neovascularization and inhibiting the growth of new vessels. However, some patients never respond to anti-VEGF therapy, while still others stop responding after initial success, highlighting the need for new therapies.
Working with a murine model of laser induced CNV, Dr. Gregory-Ksander’s laboratory has generated data that suggest a novel mechanism by which a protein called Fas ligand (FasL) prevents vascular leakage via normalization of the leaky vessels. During development and wound healing, macrophages produce soluble factors that drive the formation and maturation of blood vessels. In disease such as AMD, macrophages produce factors that drive formation, but not maturation, resulting in the development of immature and fragile vessels that leak blood and fluid. The vascular leakage causes damage to the retinal tissue and loss of vision. However, recent data from Dr. Gregory-Ksander’s laboratory suggest that FasL can “polarize” these macrophages by binding to the Fas receptor and triggering the production of vessel “maturation” factors, resulting in the normalization of the new vessels and the inhibition of vascular leakage. Current work in Dr. Gregory-Ksander’s laboratory focuses on determining (i) how Fas ligand polarizes these macrophages and (ii) whether these polarized macrophages can be used to prevent vascular leakage in an animal model of AMD. The ultimate goal of this work is to elucidate the mechanism by which FasL inhibits vascular leakage and determine if FasL can be used as a novel therapeutic to prevent vascular leakage in AMD.
- Mechanisms of immune privilege in the retina
- How age-related changes in immune-privilege and inflammation contribute to glaucoma and AMD
Fas Ligand as a target for neuroprotection in Glaucoma
In 2003 Dr. Gregory-Ksander made the first connection between the form of FasL (membrane (mFasL) or soluble (sFasL)) and the pathogenesis of glaucoma in DBA/2J mice (spontaneous mouse model of glaucoma). Working in collaboration with Dr. Simon John at The Jackson Laboratory in Bar Harbor, ME. Dr. Gregory-Ksander demonstrated that ocular immune privilege was terminated in DBA/2J mice prior to the development of glaucoma. Dr. Gregory-Ksander went on to demonstrate that the development of glaucoma also coincided with increased expression of mFasL in the eye. To further examine the role of mFasL and sFasL in ocular homeostasis and glaucoma, Dr. Gregory-Ksander, in collaboration with Dr. Ann Marshak-Rothstein at University Massachusetts School of Medicine in Worcester, constructed a membrane-only FasL gene-targeted mouse in which the FasL metalloproteinase cleavage sites were mutated. These mice only express mFasL and no sFasL. Using these mice in an inducible and spontaneous model of glaucoma Dr. gregroy-Ksander was able to specifically address how membrane and soluble forms of FasL contribute to the development of glaucoma. The results of these studies were significant because they: (i) elucidated the mechanism of retinal ganglion cell and nerve fiber destruction in glaucoma; (ii) identified cleavage of the FasL molecule as a major mechanism controlling FasL activity in the eye; and (ii) demonstrated the protective function of the soluble form of FasL, thereby identifying a potentially useful therapeutic strategy. Using a gene therapy approach, Dr. Gregory-Ksander demonstrated that a single intravitreal injection of AAV2.sFasL provided complete and sustained neuroprotection in both the chronic DBA/2J and acute microbead-induced models of elevated IOP. Dr. Gregory-Ksander’s lab is currently testing the effectiveness of the AAV2.sFasL gene therapy in protecting retinal ganglion cells in larger animal models of glaucoma. They are also using a combination of bone-marrow chimeras and tissue-specific Fas and FasL knock-out mice to (i) determine the function of Fas/FasL signaling in the amplification of inflammation that occurs in the ONH and neural retina following elevated IOP and (ii) elucidate the mechanism by which sFasL provides such complete and sustained protection of the retinal ganglion cell body and axons of the optic nerve, even in the presence of sustained elevated IOP.
NLRP3 as a target for neuroprotection in Glaucoma
Dr. Gregory-Ksander recently made the discovery that NLRP3 was constitutively expressed in the optic nerve head of normal human and mouse eyes. Using the microbead-induced mouse model of elevated IOP, Dr. Gregory-Ksander demonstrated the requirement of inflammasome activation in axonal damage and death of retinal ganglion cells. The use of the microbead mouse model of elevated IOP in conjunction with inflammasome deficient mice and NLRP3 inhibitors, Dr. Gregory-Ksander has demonstrated the essential role that NLRP3 inflammasome activation plays in the development of inflammation in the optic nerve head (ONH), resulting in axonal damage and death of RGCs. The current goal of this project is to (i) understand how and why the NLRP3 inflammasome pathway becomes dysregulated in the development of glaucoma, and (ii) develop and test novel inhibitors of the NLRP3 inflammasome pathway as a potential neuroprotective therapy in glaucoma.
Fas Ligand and Vascular Leakage in Age-Related Macular Degeneration
Age-related macular degeneration (AMD) is the primary cause of blindness in patients 60 years of age and older. The growth of abnormal blood vessels into the retina is the main cause of severe vision loss, in part due to vessel leakage of blood and fluid that causes damage to the surrounding retinal tissue.
In collaboration with Dr. Ann Marshak-Rothstein at University Massachusetts School of Medicine in Worcester, Dr. Gregory-Ksander has identified a novel function for membrane Fas ligand in preventing vascular leakage. The goal of this study is to:
- Determine whether the administration of this protein can be used to prevent vascular leakage in a mouse model of laser-induced choroidal neovascularization
- Elucidate the mechanism by which this protein functions