Paul R. Hiebert

Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma.

The cytotoxic granzyme B (GrB)/perforin pathway has been traditionally viewed as a primary mechanism that is used by cytotoxic lymphocytes to eliminate allogeneic, virally infected and/or transformed cells. Although originally proposed to have intracellular and extracellular functions, upon the discovery that perforin, in combination with GrB, could induce apoptosis, other potential functions for this protease were, for the most part, disregarded. As there are 5 granzymes in humans and 11 granzymes in mice, many studies used perforin knockout mice as an initial screen to evaluate the role of granzymes in disease. However, in recent years, emerging clinical and biochemical evidence has shown that the latter approach may have overlooked a critical perforin-independent, pathogenic role for these proteases in disease. This review focuses on GrB, the most characterized of the granzyme family, in disease. Long known to be a pro-apoptotic protease expressed by cytotoxic lymphocytes and natural killer cells, it is now accepted that GrB can be expressed in other cell types of immune and nonimmune origin. To the latter, an emerging immune-independent role for GrB has been forwarded due to recent discoveries that GrB may be expressed in nonimmune cells such as smooth muscle cells, keratinocytes, and chondrocytes in certain disease states. Given that GrB retains its activity in the blood, can cleave extracellular matrix, and its levels are often elevated in chronic inflammatory diseases, this protease may be an important contributor to certain pathologies. The implications of sustained elevations of intracellular and extracellular GrB in chronic vascular, dermatological, and neurological diseases, among others, are developing. This review examines, for the first time, the multiple roles of GrB in disease pathogenesis.

Granzyme B in injury, inflammation, and repair

Fragile skin and susceptibility to skin tearing are major problems among the elderly and can be complicated further by impaired wound healing. Non-healing wounds fail to progress through the normal stages of healing and enter a state of chronic inflammation featuring increased proteolytic activity. Increased expression of the serine protease granzyme B is observed during prolonged inflammation and is implicated in the pathogenesis of several chronic inflammatory diseases. Although its role in cytotoxic lymphocyte-mediated apoptosis is well established, granzyme B can also degrade extracellular matrix proteins and alter inflammation if present in the extracellular milieu. The present review focuses on the emerging evidence for the involvement of granzyme B in chronic inflammation, impaired wound healing, and age-related skin fragility.

Granzyme B contributes to extracellular matrix remodeling and skin aging in apolipoprotein E knockout mice.

Apolipoprotein E knockout (apoE-KO) mice have been utilized for decades as a model of atherosclerosis. However, in addition to atherosclerosis, apoE-KO mice develop extensive cutaneous xanthomatosis, accelerated skin aging and frailty when fed a high fat diet. Granzyme B (GrB) is a pro-apoptotic serine protease that has recently been shown to exhibit extracellular proteolytic activity in certain pathologies. In the present study, the role of GrB in skin aging and pathology was assessed using the apoE-KO model of skin aging. Male C57BL/6 wild type and apoE-KO mice were grown for 0, 5, 15 or 30 weeks on either a high fat (21.2% fat, 0.2% cholesterol) or regular chow diet (7% fat). ApoE/GrB double knockout (DKO) mice were also generated and assessed after being fed either diet for 30 weeks. Skin was removed from the mid to lower back and examined for age-related changes such as hair loss, skin thinning and collagen remodeling and disorganization. ApoE-KO mice exhibited signs of frailty, hair graying, hair loss, skin thinning, loss of collagen density and increased skin pathologies featuring collagen remodeling and reduced decorin compared to wild type controls. These phenotypes occurred earlier and were more severe when fed a high fat diet. In addition, we also observed increased GrB expression in proximity to areas of decorin degradation and reduced collagen density in the skin of apoE-KO mice. DKO mice exhibited protection against skin thinning, ECM degradation and loss of dermal collagen density. In summary, our results provide novel insights into the effects of a high fat diet and apoE deficiency on skin aging and pathology and suggest a role for GrB in age-related skin thinning and frailty.

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-1β, 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.

Granzyme B degrades extracellular matrix and contributes to delayed wound closure in apolipoprotein E knockout mice

Chronic inflammation and excessive protease activity have a major role in the persistence of non-healing wounds. Granzyme B (GzmB) is a serine protease expressed during chronic inflammation that, in conjunction with perforin, has a well-established role in initiating apoptotic cell death. GzmB is also capable of acting extracellularly, independent of perforin and can degrade several extracellular matrix (ECM) proteins that are critical during wound healing. We used apolipoprotein E (ApoE) knockout (AKO) mice as a novel model of chronic inflammation and impaired wound healing to investigate the role of GzmB in chronic wounds. Wild-type and AKO mice were grown to 7 weeks (young) or 37 weeks (old) of age on a regular chow or high-fat diet (HFD), given a 1-cm diameter full thickness wound on their mid dorsum and allowed to heal for 16 days. Old AKO mice fed a HFD exhibited reduced wound closure, delayed contraction, chronic inflammation and altered ECM remodeling. Conversely, GzmB/ApoE double knockout mice displayed improved wound closure and contraction rates. In addition, murine GzmB was found to degrade both fibronectin and vitronectin derived from healthy mouse granulation tissue. In addition, GzmB-mediated degradation of fibronectin generated a fragment similar in size to that observed in non-healing mouse wounds. These results provide the first direct evidence that GzmB contributes to chronic wound healing in part through degradation of ECM.

Mucosal production of uric acid by airway epithelial cells contributes to particulate matter-induced allergic sensitization

Exposure to particulate matter (PM), a major component of air pollution, contributes to increased morbidity and mortality worldwide. PM induces innate immune responses and contributes to allergic sensitization, although the mechanisms governing this process remain unclear. Lung mucosal uric acid has also been linked to allergic sensitization. The links among PM exposure, uric acid, and allergic sensitization remain unexplored. We therefore investigated the mechanisms behind PM-induced allergic sensitization in the context of lung mucosal uric acid. PM10 and house dust mite exposure selectively induced lung mucosal uric acid production and secretion in vivo, which did not occur with other challenges (lipopolysaccharide, virus, bacteria, or inflammatory/fibrotic stimuli). PM10-induced uric acid mediates allergic sensitization and augments antigen-specific T-cell proliferation, which is inhibited by uricase. We then demonstrate that human airway epithelial cells secrete uric acid basally and after stimulation through a previously unidentified mucosal secretion system. Our work discovers a previously unknown mechanism of air pollution-induced, uric acid-mediated, allergic sensitization that may be important in the pathogenesis of asthma.

Perforin and granzyme B have separate and distinct roles during atherosclerotic plaque development in apolipoprotein E knockout mice.

The granzyme B/perforincytotoxic pathway is a well established mechanism of initiating target cell apoptosis. Previous studies have suggested a role for the granzyme B/perforin cytotoxic pathway in vulnerable atherosclerotic plaque formation. In the present study, granzyme B deficiency resulted in reduced atherosclerotic plaque development in the descending aortas of apolipoprotein E knockout mice fed a high fat diet for 30 weeks while perforindeficiency resulted in greater reduction in plaque development with significantly less plaque area than granzyme Bdeficient mice. In contrast to the descending aorta, no significant change in plaque size was observed in aortic roots from either granzyme Bdeficient or perforindeficient apolipoprotein E knockout mice. However, atherosclerotic plaques in the aortic roots did exhibit significantly more collagen in granzyme B, but not perforin deficient mice. Together these results suggest significant, yet separate roles for granzyme B and perforin in the pathogenesis of atherosclerosis that go beyond the traditional apoptotic pathway with additional implications in plaque development, stability and remodelling of extracellular matrix.

Granzyme B Deficiency Exacerbates Lung Inflammation in Mice after Acute Lung Injury

Granzyme B (GzmB) is a serine protease with intracellular and extracellular activities capable of regulating inflammation through cytokine processing and the apoptosis of effector cells. We tested the hypothesis that GzmB expression in T regulatory cells (Tregs) is required for the control of inflammatory responses and pathology during acute lung injury. To substantiate the clinical relevance of GzmB during lung injury, we performed GzmB immunohistochemistry on lung tissue from patients with acute respiratory distress syndrome (ARDS) and healthy control subjects. We also performed in vivo experiments with wild-type (WT) C57BL/6 and GzmB(-/-) mice exposed to a single intranasal instillation of bleomycin to model lung injury. Our results demonstrate that the expression of GzmB was elevated in ARDS lung sections, relative to healthy control samples. Bleomycin-exposed GzmB(-/-) mice exhibited greater morbidity and mortality, which was associated with increased numbers of lung lymphocytes. Bleomycin induced an equal increase in CD4(+)/CD25(+)/FoxP3(+) Treg populations in WT and GzmB(-/-) mice. GzmB expression was not significant in Tregs, with the majority of the expression localized to natural killer (NK)-1.1(+) cells. The expression of GzmB in NK cells of bleomycin-exposed WT mice was associated with greater lymphocyte apoptosis, reduced total lymphocyte numbers, and reduced pathology relative to GzmB(-/-) mice. Our data demonstrate that GzmB deficiency results in the exacerbation of lymphocytic inflammation during bleomycin-induced acute lung injury, which is associated with pathology, morbidity, and mortality.