David Granville

Radiation dermatitis (RD) is observed in up to 90% of patients receiving radiation therapy. Symptoms of RD include redness, scaling, and crusted wounds. Severe symptoms can prevent future radiation treatments. Current treatments are not effective and may cause severe side effects. Granzyme B (GzmB) was originally characterized as a serine protease secreted by cytotoxic lymphocytes to promote target cell apoptosis. It is now recognized that GzmB can be secreted by other immune cell and non-immune cell populations. GzmB is elevated in a number of inflammatory skin disorders, such as atopic dermatitis, autoimmune blistering, impaired wound healing and scarring. Previous studies suggest that extracellular GzmB cleaves E-cadherin, filaggrin, and decorin leading to reduced epithelial barrier function and scarring. Based on these findings, I hypothesized that GzmB contributes to increased RD severity and delayed healing through the cleavage of epithelial barrier proteins and/or matrix proteins.GzmB expression and cell source, E-cadherin, filaggrin, and decorin levels were assessed using immunohistochemical analysis of biopsies from RD patients. The role of GzmB was investigated in an established RD murine model, comparing GzmB knockout (GzmB-/-) to wild type (WT) mice. The dorsal skin was exposed to a single 40 gray dose of radiation. RD severity was blindly scored. Tissues were harvested at days 4, 14, and 46 post-radiation exposure. Tissues were examined for GzmB, immune cell infiltrate, scarring, and decorin. Macrophages and mast cells expressed GzmB while E-cadherin, filaggrin, and decorin were reduced in human RD skin compared to healthy skin. GzmB-/- mice exhibited a decrease in RD severity compared to WT mice from day 4 to day 12. CD3+ T-cell levels were reduced and neutrophil infiltration was observed at day 14. GzmB was expressed by resident mast cells in WT mice. GzmB-/- mice exhibited less scar area and more decorin than WT mice at day 46. In summary, GzmB may contribute to RD severity through E-cadherin, filaggrin, and decorin cleavage. The RD murine model suggests that depletion of GzmB attenuates severity of RD and could mitigate scarring. These studies provide a rationale for pursuing GzmB as a novel therapeutic target for the treatment of RD.

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The epidermis, consisting of keratinocytes, is the first defense against the exterior environmentof the skin. Integral to maintaining the homeostasis of skin function by aidingin thermoregulation, fluid retention, and pathogen defense, the extracellular matrix compositionaids in regulating keratinocyte migration after injury. Impaired healing processes can progressinto chronic non-healing wounds, leading to infection and mortality. Granzyme B (GzmB) is aserine protease, significantly elevated in chronic non-healing wounds. GzmB impairment ofwound healing is attributed to its ability to sustain its proteolytic activity and downstreaminflammation, inhibiting wound repair and tissue remodeling following injury. GzmB isclassically known for its intracellular role in cytotoxic lymphocyte-mediated apoptosis. Recentevidence demonstrates that extracellular GzmB has the ability to cleave a variety of importantextracellular proteins. Thrombospondin-2 (TSP-2) is an extracellular glycoprotein secreted by avariety of cells known to be involved in cell-cell and cell-matrix interactions. TSP-2 has beenreported to impair viability, adhesion, and migration in endothelial cells and squamouscarcinoma cells. Although extensive studies have been done with TSP-2, literature investigatingthe role of TSP-2 in the skin has largely been neglected. I hypothesized that GzmB cleaves TSP2, and GzmB cleavage of TSP-2 mediates extracellular matrix disorganization, alteredkeratinocyte viability, and reduced adhesion. To investigate this hypothesis purposed two aims.In the first aim, cell-free protease assays were utilized to determine whether TSP-2 is aproteolytic substrate of GzmB. In the second aim, the impact of TSP-2-mediated cleavage wasassessed using cultured human keratinocytes. TSP-2 was cleaved by GzmB in a time- and dose-dependent manner. Further, while GzmB-mediated TSP-2 cleavage did not affect cell viability, it did reduce cellular adhesion in a dose-dependent manner. In summary, TSP-2 is a proteolyticsubstrate of GzmB and such cleavage interferes with keratinocyte adhesion.

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Background - A major factor limiting survival for patients that have undergone cardiac transplantation is cardiac allograft vasculopathy (CAV). CAV is a fibroproliferative inflammatory form of vascular rejection mediated by immune cells and which is initiated upon damage to the graft endothelium and medial smooth muscle cells (SMC). Granzymes are a family of five serine proteases in humans. Granzymes have been shown to exert roles in cell death, endothelial dysfunction, inflammation and matrix remodeling. Granzyme K (GzmK) specifically can promote endothelial dysfunction and the production of inflammatory mediators IL-1β, IL-6, and monocyte chemotactic protein-1. As such, we hypothesized that GzmK contributes to CAV.Methods - An infrarenal aortic interposition graft was completed across a complete major histocompatibility complex (MHC)-mismatch with recipients being either wildtype or GzmK-KO mice. Allografts were then assessed for CAV severity and compared to human CAV samples. The effects of GzmK on human SMC were also assessed in vitro. Given that atherosclerosis shares similar underlying mechanisms to CAV, GzmK deposition was also characterized in atherosclerosis.Results - Human CAV samples demonstrated increased neointimal GzmK deposition as compared to unaffected native coronaries (p= 0.017). GzmK primarily localized to the medial and neointimal layers. Similar deposition patterns were observed in murine transplants. When the role of GzmK was examined, GzmK deficiency resulted in reduced CAV with less neointima formation (p=0.019) and less luminal obstruction (p
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The skin is comprised of multiple layers of keratinocytes which together form a barrier to the external environment, regulating temperature, water loss, and pathogen exposure. As such the skin barrier is vital for health as well as disease prevention. Disruption of the epithelial barrier can result in infection, allergen exposure, and inflammation, culminating into severe conditions. Many autoimmune conditions, such as pemphigus, involve a dysregulation and accumulation of immune cells, this results in a disruption in skin barrier causing a loss of function. Granzyme B (GzmB) is a serine protease that is expressed and secreted by a variety of immune and non-immune cells. It can accumulate in the extracellular milieu and retain its proteolytic functions resulting in chronic inflammation and impaired tissue repair due to extracellular matrix (ECM) remodeling. As such, I hypothesized that GzmB disrupts epithelial barrier function through the proteolytic cleavage of cell junction proteins. The present study investigated the impact of GzmB on epithelial barrier dysfunction using Electric Cell-substrate Impedance Sensing (ECIS) and western blot analyses of intercellular junction cleavage fragments. Human formalin fixed, paraffin embedded blistered skin tissue was assessed for the presence of GzmB. GzmB treatment resulted in a loss of E-cadherin staining on the cell membrane which was supported by western blot analysis of the cell supernatants. Additionally, we observed a dose-dependent increase in E-cadherin fragmentation in GzmB-treated cells compared to controls. HaCaT cells exhibited a significant decrease in barrier function when treated with GzmB while cells treated with GzmB in the presence of a specific GzmB inhibitor remained unaffected. While absent in normal skin, GzmB was observed in abundance within the intra-epidermal blister in addition to the surrounding epithelium. In summary, GzmB contributes to a decline in epithelial barrier function in part through the proteolytic cleavage of cell-cell junctions.

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The inflammatory cascade following spinal cord injury (SCI) involves multiple cellular and molecular responses that can both aid and impede recovery. A large component of the wound response is the infiltration of immune cells that secrete pro-inflammatory cytokines and proteases. Granzyme B (GzmB) is a serine protease released by immune cells that negatively affects wound healing through its intracellular and extracellular protease activity. GzmB is abundant in neuroinflammatory conditions and contributes to neuron and oligodendrocyte cell death. In this study we investigate the role of GzmB in tissue injury, inflammation and functional recovery following SCI. An SCI was induced in wild-type (WT) and GzmB knockout (GzmB-KO) mice at thoracic level 9. Mouse locomotion was observed over the course of 6 weeks using three behaviour tests (Basso mouse scale, rotarod and horizontal ladder). Lesions were harvested for histological analysis and sections stained with markers for neurons (NeuN) and dyed for myelin (Eriochrome Cyanine). A second cohort of mice were maintained for 1 week after SCI and probed for GzmB expression and cellular localization. GzmB expression was probed using markers for macrophages or microglia (CD68). GzmB-KO mice exhibited significantly improved motor scores, increased myelin and neural survival compared to WT controls. GzmB expression was observed in macrophages at 7 days post injury. In summary, GzmB is elevated and contributes to neurotoxicity, demyelination and impaired functional recovery following SCI.

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Granzymes are a family of serine proteases that were once thought to function exclusively as mediators of cytotoxic lymphocyte-induced target cell death. However, non-lethal roles for granzymes, including Granzyme K (GzK), have been recently proposed. As recent studies have observed elevated levels of GzK in plasma of patients diagnosed with sepsis, we hypothesized that extracellular GzK induces a pro-inflammatory response in endothelial cells. In the present study, extracellular GzK proteolytically activated Protease Activated Receptor-1 (PAR-1) leading to increased IL-6 and MCP-1 production in Human Umbilical Venous Endothelial Cells (HUVEC). Enhanced expression of ICAM-1 along with an increased capacity for adherence of THP-1 cells was also observed. Characterization of downstream pathways implicated the MAPK p38 pathway for ICAM-1 expression, and both the p38 and the ERK1/2 pathways in cytokine production. GzK also increased TNFα–induced inflammatory adhesion molecule expression. Furthermore, the physiological inhibitor of GzK, IαIp, significantly inhibited GzK activity in vitro. In summary, extracellular GzK is not cytotoxic but promotes a pro-inflammatory response in endothelial cells.

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Chronic skin ulceration is a common complication and cause of morbidity in the elderly, diabetic, obese and/or immobile populations. Effective therapies that adequately promote efficient closure and remodelling of chronic wounds are lacking. Inflammation and excessive protease accumulation and activity are thought to play key roles in the impairment of normal wound healing. Granzyme B (GzmB) is a serine protease that was, until recently, believed to function exclusively in cytotoxic lymphocyte-mediated apoptosis. However, during dysregulated and/or chronic inflammation, GzmB can accumulate in the extracellular milieu, retain its activity, and cleave a number of important extracellular proteins. Epidermal growth factor receptor (EGFR) is a transmembrane receptor involved in cellular processes such as proliferation and migration. EGFR signaling is integral to the wound healing process. I hypothesized that GzmB impairs EGF-induced keratinocyte migration by impairing EGFR signaling. The present study investigated the effects of GzmB on keratinocyte cell migration. Using Electric Cell Substrate Impedance Sensing (ECIS) and other in vitro wound healing assays, the present study demonstrates that GzmB inhibits keratinocyte migration by interfering with the EGFR pathway. GzmB limited cell transition into a migratory morphology and was found to reduce ligand-induced EGFR phosphorylation. Inhibition of GzmB reversed the aforementioned effects in HaCaT cells. In summary, data from the present study suggests a key role for GzmB in the pathogenesis of impaired wound healing through the reduction of EGFR signaling and cell migration.

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Chronic, non-healing wounds are a major complication of diabetes and are characterized by chronic inflammation and excessive protease activity. While once thought to function primarily as a pro-apoptotic serine protease, granzyme B (GzmB) can also accumulate in the extracellular matrix during chronic inflammation and cleave extracellular matrix (ECM) proteins that are essential for proper wound healing, including fibronectin. We hypothesized that GzmB contributes to the pathogenesis of impaired diabetic wound healing through excessive degradation of the ECM. In the first part of the thesis, we demonstrated that the majority of GzmB was secreted by mast cells and localized in the wound edges and granulation tissues of completely reepithelialized diabetic mouse wounds at higher levels. Subsequently, we observed that GzmB induced detachment of mouse embryonic fibroblasts and also showed that co-incubation with a mouse serine protease inhibitor, serpina3n (SA3N), abrogated this effect. Finally, we administered SA3N to diabetic mouse wounds and found that wound closure including both reepithelialization and contraction were significantly increased in wounds treated with SA3N. Histological and immunohistochemical analyses of the SA3N-treated wounds revealed a more mature, proliferative granulation tissue phenotype as indicated by increased cells with proliferative activity, vascularization, contractile myofibroblasts, as well as collagen deposition in remodeling tissues. Skin homogenates from SA3N-treated wounds also exhibited greater levels of full-length intact fibronectin when compared to control wounds. In summary, our findings suggested that GzmB contributes to the pathogenesis of diabetic wound healing through the proteolytic cleavage of fibronectin that are essential for normal wound closure, and that inhibition of GzmB can promote granulation tissue maturation and collagen deposition. These results offer preliminary evidence that a GzmB inhibitor may be a relevant therapeutic target in wound management therapy.

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