Granzyme B Deficiency Protects against Angiotensin II-Induced Cardiac Fibrosis.

Cardiac fibrosis is observed across diverse etiologies of heart failure. Granzyme B (GzmB) is a serine protease involved in cell-mediated cytotoxicity in conjunction with the pore-forming protein, perforin. Recent evidence suggests that GzmB also contributes to matrix remodeling and fibrosis through an extracellular, perforin-independent process. However, the role of GzmB in the onset and progression of cardiac fibrosis remains elusive. The present study investigated the role of GzmB in the pathogenesis of cardiac fibrosis. GzmB was elevated in fibrotic human hearts and in angiotensin II-induced murine cardiac fibrosis. Genetic deficiency of GzmB reduced angiotensin II-induced cardiac hypertrophy and fibrosis, independently of perforin. GzmB deficiency also reduced microhemorrhage, inflammation, and fibroblast accumulation in vivo. In vitro, GzmB cleaved the endothelial junction protein, vascular endothelial (VE)-cadherin, resulting in the disruption of endothelial barrier function. Together, these results suggest a perforin-independent, extracellular role for GzmB in the pathogenesis of cardiac fibrosis.

Granzyme B inhibits keratinocyte migration by disrupting epidermal growth factor receptor (EGFR)-mediated signaling.

Chronic non-healing wounds including diabetic, venous, and decubitus skin ulcers are currently lacking effective therapies. Non-healing diabetic ulcers can lead to amputations as progress into a highly chronic state before detection and existing treatments for these wounds often fail. Granzyme B (GzmB) is a serine protease that was, until recently, believed to function exclusively in cytotoxic lymphocyte-mediated apoptosis. However, during excessive 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. The present study investigated the effects of GzmB on keratinocyte cell migration using HaCaT cell line. Using electric cell-substrate impedance sensing and scratch 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 summary, data from the present study suggest key role for GzmB in the pathogenesis of impaired wound healing through the impairment of EGFR signaling and cell migration.

The nucleotide-binding domain, leucine-rich repeat protein 3 inflammasome/IL-1 receptor I axis mediates innate, but not adaptive, immune responses after exposure to particulate matter under 10 µm.

Exposure to particulate matter (PM), a major component of air pollution, contributes to increased morbidity and mortality worldwide. Inhaled PM induces innate immune responses by airway epithelial cells that may lead to the exacerbation or de novo development of airway disease. We have previously shown that 10-µm PM (PM10) activates the nucleotide-binding domain, leucine-rich repeat protein (NLRP) 3 inflammasome in human airway epithelial cells. Our objective was to determine the innate and adaptive immune responses mediated by the airway epithelium NLRP3 inflammasome in response to PM10 exposure. Using in vitro cultures of human airway epithelial cells and in vivo studies with wild-type and Nlrp3(-/-) mice, we investigated the downstream consequences of PM10-induced NLPR3 inflammasome activation on cytokine production, cellular inflammation, dendritic cell activation, and PM10-facilitated allergic sensitization. PM10 activates an NLRP3 inflammasome/IL-1 receptor I (IL-1RI) axis in airway epithelial cells, resulting in IL-1ß, CC chemokine ligand-20, and granulocyte/macrophage colony-stimulating factor production, which is associated with dendritic cell activation and lung neutrophilia. Despite these profound innate immune responses in the airway epithelium, the NLRP3 inflammasome/IL-1RI axis is dispensable for PM10-facilitated allergic sensitization. We demonstrate the importance of the lung NLRP3 inflammasome in mediating PM10 exposure-associated innate, but not adaptive, immune responses. Our study highlights a mechanism by which PM10 exposure can contribute to the exacerbation of airway disease, but not PM10-facilitated allergic sensitization.

Extracellular granzyme K mediates endothelial activation through the cleavage of protease-activated receptor-1.

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-apoptotic roles for granzymes, including granzyme K (GzK), have been proposed. As recent studies have observed elevated levels of GzK in the plasma of patients diagnosed with clinical sepsis, we hypothesized that extracellular GzK induces a proinflammatory response in endothelial cells. In the present study, extracellular GzK proteolytically activated protease-activated receptor-1 leading to increased interleukin 6 and monocyte chemotactic protein 1 production in endothelial cells. Enhanced expression of intercellular adhesion molecule 1 along with an increased capacity for adherence of THP-1 cells was also observed. Characterization of downstream pathways implicated the mitogen-activated protein kinase p38 pathway for intercellular adhesion molecule 1 expression, and both the p38 and the extracellular signal-regulated protein kinases 1 and 2 pathways in cytokine production. GzK also increased tumour necrosis factor α-induced inflammatory adhesion molecule expression. Furthermore, the physiological inhibitor of GzK, inter-α-inhibitor protein, significantly inhibited GzK activity in vitro. In summary, extracellular GzK promotes a proinflammatory response in endothelial cells.

Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low-dose ultraviolet light irradiation.

Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases.

The effect of nano-scale topography on keratinocyte phenotype and wound healing following burn injury.

Topographic modulation of tissue response is an important consideration in the design and manufacture of a biomaterial. In developing new tissue therapies for skin, all levels of architecture, including the nanoscale need to be considered. Here we show that keratinocyte phenotype is affected by nanoscale changes in topography with cell morphology, proliferation, and migration influenced by the pore size in anodic aluminum oxide membranes. A membrane with a pore size of 300 nm, which enhanced cell phenotype in vitro, was used as a dressing to cover a partial thickness burn injury in the pig. Wounds dressed with the membrane showed evidence of advanced healing with significantly less organizing granulation tissue and more mature epidermal layers than control wounds dressed with a standard burns dressing. The results demonstrate the importance of nanoscale topography in modulating keratinocyte phenotype and skin wound healing.

The potential of nanoporous anodic aluminium oxide membranes to influence skin wound repair.

Cells respond to changes in the environment by altering their phenotype. The ability to influence cell behavior by modifying their environment provides an opportunity for therapeutic application, for example, to promote faster wound healing in response to skin injury. Here, we have modified the preparation of an aluminium oxide template to generate large uniform membranes with differing nano-pore sizes. Epidermal cells (keratinocytes) and dermal cells (fibroblasts) readily adhere to these nanoporous membranes. The pore size appears to influence the rate of cell proliferation and migration, important aspects of cell behavior during wound healing. The suitability of the membrane to act as a dressing after a burn injury was assessed in vivo; application of the membrane demonstrated adherence and conformability to the skin surface of a pig, with no observed degradation or detrimental effect on the repair. Our results suggest that keratinocytes are sensitive to changes in topography at the nanoscale level and that this property may be exploited to improve wound repair after tissue injury.