Granzyme B degrades extracellular matrix and promotes inflammation and choroidal neovascularization.

Age-related macular degeneration (AMD) is a common retinal neurodegenerative disease among the elderly. Neovascular AMD (nAMD), a leading cause of AMD-related blindness, involves choroidal neovascularization (CNV), which can be suppressed by anti-angiogenic treatments. However, current CNV treatments do not work in all nAMD patients. Here we investigate a novel target for AMD. Granzyme B (GzmB) is a serine protease that promotes aging, chronic inflammation and vascular permeability through the degradation of the extracellular matrix (ECM) and tight junctions. Extracellular GzmB is increased in retina pigment epithelium (RPE) and mast cells in the choroid of the healthy aging outer retina. It is further increased in donor eyes exhibiting features of nAMD and CNV. Here, we show in RPE-choroidal explant cultures that exogenous GzmB degrades the RPE-choroid ECM, promotes retinal/choroidal inflammation and angiogenesis while diminishing anti-angiogenic factor, thrombospondin-1 (TSP-1). The pharmacological inhibition of either GzmB or mast-cell degranulation significantly reduces choroidal angiogenesis. In line with our in vitro data, GzmB-deficiency reduces the extent of laser-induced CNV lesions and the age-related deterioration of electroretinogram (ERG) responses in mice. These findings suggest that targeting GzmB, a serine protease with no known endogenous inhibitors, may be a potential novel therapeutic approach to suppress CNV in nAMD.

Granzyme B promotes matrix metalloproteinase-1 (MMP-1) release from gingival fibroblasts in a PAR1- and Erk1/2-dependent manner: A novel role in periodontal inflammation.

OBJECTIVE: To gain insights into how proteases signal to connective tissues cells in the periodontium. BACKGROUND: The connective tissue degradation observed in periodontitis is largely due to matrix metalloproteinase (MMP) release by gingival fibroblasts. Granzyme B (GzmB) is a serine protease whose role in periodontitis is undefined. METHODS: Human gingival crevicular fluid (GCF) samples were obtained from sites with periodontal disease and healthy control sites. GzmB was quantified in the GCF ([GzmB]GCF ) by ELISA. Gingival fibroblasts (GF) were cultured in the presence or absence of recombinant GzmB. Culture supernatants were analyzed by ELISA to quantify GzmB-induced release of interstitial collagenase (MMP-1). In some experiments, cells were pre-treated with the inhibitor PD98059 to block MEK/ERK signaling. The protease-activated receptor-1 (PAR-1) was blocked with ATAP-2 neutralizing antibody prior to GzmB stimulation. Systemic MMP-1 levels were measured in plasma from wild-type (WT) and granzyme-B-knockout (GzmB-/- ) mice. RESULTS: The [GzmB]GCF in human samples was ~4-5 fold higher at sites of periodontal disease (gingivitis/periodontitis) compared to healthy control sites, suggesting an association between GzmB and localized matrix degradation. GzmB induced a ~4-5-fold increase in MMP-1 secretion by cultured fibroblasts. GzmB induced phosphorylation of Erk1/2, which was abrogated by PD98059. GzmB-induced upregulation of MMP-1 secretion was also reduced by PD98059. Blockade of PAR-1 function by ATAP-2 abrogated the increase in MMP-1 secretion by GF. Circulating MMP-1 was similar in WT and GzmB-/- mice, suggesting that GzmB's effects on MMP-1 release are not reflected systemically. CONCLUSION: These data point to a novel GzmB-driven signaling pathway in fibroblasts in which MMP-1 secretion is upregulated in a PAR1- and Erk1/2-dependent manner.

Noncytotoxic Roles of Granzymes in Health and Disease.

Granzymes are serine proteases previously believed to play exclusive and somewhat redundant roles in lymphocyte-mediated target cell death. However, recent studies have challenged this paradigm. Distinct substrate profiles and functions have since emerged for each granzyme while their dysregulated proteolytic activities have been linked to diverse pathologies.

Granzyme B Contributes to Choroidal Neovascularization and Age-Related Macular Degeneration Through Proteolysis of Thrombospondin-1.

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly. The pathology of neovascular age-related macular degeneration (nAMD), also known as wet AMD, is associated with an abnormal blood vessel growth in the eye and involves an imbalance of proangiogenic and antiangiogenic factors. Thrombospondin (TSP)-1 and TSP-2 are endogenous matricellular proteins that inhibit angiogenesis. TSP-1 is significantly diminished in eyes with AMD, although the mechanisms involved in its reduction are unknown. Granzyme B (GzmB) is a serine protease with an increased extracellular activity in the outer retina and choroid of human eyes with nAMD-related choroidal neovascularization (CNV). This study investigated whether TSP-1 and TSP-2 are GzmB substrates using in silico and cell-free cleavage assays and explored the relationship between GzmB and TSP-1 in human eyes with nAMD-related CNV and the effect of GzmB on TSP-1 in retinal pigment epithelial culture and an explant choroid sprouting assay (CSA). In this study, TSP-1 and TSP-2 were identified as GzmB substrates. Cell-free cleavage assays substantiated the GzmB proteolysis of TSP-1 and TSP-2 by showing dose-dependent and time-dependent cleavage products. TSP-1 and TSP-2 proteolysis were hindered by the inhibition of GzmB. In the retinal pigment epithelium and choroid of human eyes with CNV, we observed a significant inverse correlation between TSP-1 and GzmB, as indicated by lower TSP-1 and higher GzmB immunoreactivity. In CSA, the vascular sprouting area increased significantly with GzmB treatment and reduced significantly with TSP-1 treatment. Western blot showed significantly reduced expression of TSP-1 in GzmB-treated retinal pigment epithelial cell culture and CSA supernatant compared with that in controls. Together, our findings suggest that the proteolysis of antiangiogenic factors such as TSP-1 by extracellular GzmB might represent a mechanism through which GzmB may contribute to nAMD-related CNV. Future studies are needed to investigate whether pharmacologic inhibition of extracellular GzmB can mitigate nAMD-related CNV by preserving intact TSP-1.

γδ Intraepithelial Lymphocytes Facilitate Pathological Epithelial Cell Shedding Via CD103-Mediated Granzyme Release.

BACKGROUND & AIMS: Excessive shedding of apoptotic enterocytes into the intestinal lumen is observed in inflammatory bowel disease and is correlated with disease relapse. Based on their cytolytic capacity and surveillance behavior, we investigated whether intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IELs) are actively involved in the shedding of enterocytes into the lumen. METHODS: Intravital microscopy was performed on GFP γδ T cell reporter mice treated with intraperitoneal lipopolysaccharide (10 mg/kg) for 90 minutes to induce tumor necrosis factor-mediated apoptosis. Cell shedding in various knockout or transgenic mice in the presence or absence of blocking antibody was quantified by immunostaining for ZO-1 funnels and cleaved caspase-3 (CC3). Granzyme A and granzyme B release from ex vivo-stimulated γδ IELs was quantified by enzyme-linked immunosorbent assay. Immunostaining for γδ T cell receptor and CC3 was performed on duodenal and ileal biopsies from controls and patients with Crohn's disease. RESULTS: Intravital microscopy of lipopolysaccharide-treated mice revealed that γδ IELs make extended contact with shedding enterocytes. These prolonged interactions require CD103 engagement by E-cadherin, and CD103 knockout or blockade significantly reduced lipopolysaccharide-induced shedding. Furthermore, we found that granzymes A and B, but not perforin, are required for cell shedding. These extracellular granzymes are released by γδ IELs both constitutively and after CD103/E-cadherin ligation. Moreover, we found that the frequency of γδ IEL localization to CC3-positive enterocytes is increased in Crohn's disease biopsies compared with healthy controls. CONCLUSIONS: Our results uncover a previously unrecognized role for γδ IELs in facilitating tumor necrosis factor-mediated shedding of apoptotic enterocytes via CD103-mediated extracellular granzyme release.

Granzyme K contributes to endothelial microvascular damage and leakage during skin inflammation.

BACKGROUND: Granzyme K (GzmK) is a serine protease with minimal presence in healthy tissues while abundant in inflamed tissues. Initially thought to play an exclusive role in immune-mediated cell death, extracellular GzmK can also promote inflammation. OBJECTIVES: To evaluate the role of GzmK in the pathogenesis of atopic dermatitis (AD), the most common inflammatory skin disease. METHODS: A panel of human AD and control samples was analysed to determine if GzmK is elevated. Next, to determine a pathological role for GzmK in AD-like skin inflammation, oxazolone-induced dermatitis was induced in GzmK-/- and wild-type (WT) mice. RESULTS: In human lesional AD samples, there was an increase in the number of GzmK+ cells compared with healthy controls. GzmK-/- mice exhibited reduced overall disease severity characterized by reductions in scaling, erosions and erythema. Surprisingly, the presence of GzmK did not notably increase the overall pro-inflammatory response or epidermal barrier permeability in WT mice; rather, GzmK impaired angiogenesis, increased microvascular damage and microhaemorrhage. Mechanistically, GzmK contributed to vessel damage through cleavage of syndecan-1, a key structural component of the glycocalyx, which coats the luminal surface of vascular endothelia. CONCLUSIONS: GzmK may provide a potential therapeutic target for skin conditions associated with persistent inflammation, vasculitis and pathological angiogenesis.

Granzyme B Induces IRF-3 Phosphorylation through a Perforin-Independent Proteolysis-Dependent Signaling Cascade without Inducing Cell Death.

Granzyme B (GrB) is an immune protease implicated in the pathogenesis of several human diseases. In the current model of GrB activity, perforin determines whether the downstream actions of GrB occur intracellularly or extracellularly, producing apoptotic cytotoxicity or nonapoptotic effects, respectively. In the current study, we demonstrate the existence of a broad range of GrB-dependent signaling activities that 1) do not require perforin, 2) occur intracellularly, and 3) for which cell death is not the dominant outcome. In the absence of perforin, we show that GrB enzymatic activity still induces substoichiometric activation of caspases, which through nonlethal DNA damage response signals then leads to activity-associated phosphorylation of IFN regulatory factor-3. These findings illustrate an unexpected potential interface between GrB and innate immunity separate from the traditional role of GrB in perforin-dependent GrB-mediated apoptosis that could have mechanistic implications for human disease.

Granzyme B in epithelial barrier dysfunction and related skin diseases.

The predominant function of the skin is to serve as a barrier-to protect against external insults and to prevent water loss. Junctional and structural proteins in the stratum corneum, the outermost layer of the epidermis, are critical to the integrity of the epidermal barrier as it balances ongoing outward migration, differentiation, and desquamation of keratinocytes in the epidermis. As such, epidermal barrier function is highly susceptible to upsurges of proteolytic activity in the stratum corneum and epidermis. Granzyme B is a serine protease scarce in healthy tissues but present at high levels in tissues encumbered by chronic inflammation. Discovered in the 1980s, granzyme B is currently recognized for its intracellular roles in immune cell-mediated apoptosis as well as extracellular roles in inflammation, chronic injuries, tissue remodeling, as well as processing of cytokines, matrix proteins, and autoantigens. Increasing evidence has emerged in recent years supporting a role for granzyme B in promoting barrier dysfunction in the epidermis by direct cleavage of barrier proteins and eliciting immunoreactivity. Likewise, granzyme B contributes to impaired epithelial function of the airways, retina, gut, and vessels. In the present review, the role of granzyme B in cutaneous epithelial dysfunction is discussed in the context of specific conditions with an overview of underlying mechanisms as well as utility of current experimental and therapeutic inhibitors.

Implications of Sm22α-Cre expression in keratinocytes and unanticipated inflammatory skin lesion in a model of atherosclerosis.

Genetically modified mice are widely used to recapitulate human diseases. Atherosclerosis can be induced in mice with low-density lipoprotein receptor (Ldlr)-deficiency and a high-fat diet (HFD). Disintegrin and metalloproteinase-17 (ADAM17) in the smooth muscle cell (SMC) contribute to vascular pathologies, and hence its role in atherosclerosis was investigated. Adam17 deletion in SMCs by Sm22α-Cre driver (Ldlr-/-/Adam17Sm22Cre) and HFD resulted in severe skin lesions in >70% of mice, associated with skin inflammation, which was not observed in Ldlr-/--HFD, nor in mice with SMC deficiency of Adam17 by a different Cre driver (Ldlr-/-/Adam17Myh11Cre). We found that Sm22α is highly expressed in keratinocytes (compared with SMCs), which could underlie the observed skin lesion in Ldlr-/-/Adam17Sm22Cre-HFD. Although expression of Sm22α in non-SMCs has been reported, this is the first study demonstrating a severe side effect resulting from the off-target expression of Sm22α-Cre, resulting in ADAM17 loss in keratinocytes that led to a moribund state.NEW & NOTEWORTHY Although Sm22α-Cre is commonly used to target gene deletion in smooth muscle cells, Sm22α-derived Adam17 deletion resulted in unexpected severe skin lesions following high-fat diet feeding in a model of atherosclerosis. Adam17 deletion by a different SMC driver, Myh11-Cre, did not result in skin lesions in the same atherosclerosis model. Sm22α is highly expressed in keratinocytes, causing ectopic loss of ADAM17 in keratinocytes that caused significant epidermal lesions when combined with a high-fat diet.

Podocalyxin is required for maintaining blood-brain barrier function during acute inflammation.

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of Podxl in vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood-brain barrier (BBB) in PodxlΔTie2Cre mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primed PodxlΔTie2Cre mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

Experimental high thoracic spinal cord injury impairs the cardiac and cerebrovascular response to orthostatic challenge in rats.

Spinal cord injury (SCI) impairs the cardiovascular responses to postural challenge, leading to the development of orthostatic hypotension (OH). Here, we apply lower body negative pressure (LBNP) to rodents with high-level SCI to demonstrate the usefulness of LBNP as a model for experimental OH studies, and to explore the effect of simulated OH on cardiovascular and cerebrovascular function following SCI. Male Wistar rats (n = 34) were subjected to a sham or T3-SCI surgery and survived into the chronic period postinjury (i.e., 8 wk). Cardiac function was tracked via ultrasound pre- to post-SCI to demonstrate the clinical utility of our model. At study termination, we conducted left-ventricular (LV) catheterization and insonated the middle cerebral artery to investigate the hemodynamic, cardiac, and cerebrovascular response to a mild dose of LBNP that is sufficient to mimic clinically defined OH in rats with T3-SCI but not sham animals. In response to mimicked OH, there was a greater decline in stroke volume, cardiac output, maximal LV pressure, and blood pressure in SCI compared with sham (P < 0.034), whereas heart rate was increased in sham but decreased in SCI (P < 0.029). SCI animals also had an exaggerated reduction in peak, minimum and mean middle cerebral artery flow, for a given change in blood pressure, in response to LBNP (P < 0.033), implying impaired dynamic cerebral autoregulation. Using a preclinical SCI model of OH, we demonstrate that complete high thoracic SCI impairs the cardiac response to OH and disrupts dynamic cerebral autoregulation.NEW & NOTEWORTHY This is the first use of LBNP to interrogate the cardiac and cerebrovascular responses to simulated OH in a preclinical study of SCI. Here, we demonstrate the utility of our simulated OH model and use it to demonstrate that SCI impairs the cardiac response to simulated OH and disrupts dynamic cerebrovascular autoregulation.

Pathogenic mechanisms and the potential of drug therapies for aortic aneurysm.

Aortic aneurysm is a permanent focal dilation of the aorta. It is usually an asymptomatic disease but can lead to sudden death due to aortic rupture. Aortic aneurysm-related mortalities are estimated at ∼200,000 deaths per year worldwide. Because no pharmacological treatment has been found to be effective so far, surgical repair remains the only treatment for aortic aneurysm. Aortic aneurysm results from changes in the aortic wall structure due to loss of smooth muscle cells and degradation of the extracellular matrix and can form in different regions of the aorta. Research over the past decade has identified novel contributors to aneurysm formation and progression. The present review provides an overview of cellular and noncellular factors as well as enzymes that process extracellular matrix and regulate cellular functions (e.g., matrix metalloproteinases, granzymes, and cathepsins) in the context of aneurysm pathogenesis. An update of clinical trials focusing on therapeutic strategies to slow abdominal aortic aneurysm growth and efforts underway to develop effective pharmacological treatments is also provided.

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.

Lipids, adiposity and tendinopathy: is there a mechanistic link? Critical review.

Being overweight or obese is associated with an elevated risk of tendon pathology. However, for sportspeople the epidemiological data linking weight or adiposity on one hand, and risk of tendon pathology on the other, are less consistent. Indeed, the mechanistic links between diet, adiposity and tendon pathology remain largely unexamined. Recent studies have begun to examine the effects of dietary interventions on outcomes such as tendon biomechanics or pain. Oxidised low-density lipoprotein has been shown to (A) accumulate in the tendon tissues of mice that eat a fatty diet and (B) induce a pathological phenotype in human tendon cells. This paper addresses the current debate: is excessive body mass index (causing increased load and strain on tendon tissue) per se the underlying mechanism? Or do local or systemic influences of fat on tendons predispose to tendon pathology? This narrative review argues that excessive blood lipids may be an important avenue for clinical investigations.

Granzyme B releases vascular endothelial growth factor from extracellular matrix and induces vascular permeability.

The formation of unstable, leaky neovessels underlies the pathogenesis of many chronic inflammatory diseases. Granzyme B (GZMB) is an immune-derived serine protease that accumulates in the extracellular matrix (ECM) during chronic inflammation and is capable of cleaving fibronectin (FN). Vascular endothelial growth factor (VEGF) is a potent vascular permeabilizing agent that is sequestered in the ECM through its interaction with FN. As GZMB levels are elevated in chronic inflammatory diseases that are associated with increased vascular permeability, the role of GZMB in the regulation of VEGF bioavailability and vascular permeability were assessed. GZMB was added to either VEGF bound to FN or VEGF bound to endothelial cell (EC)-derived ECM. Supernatants containing released VEGF were assessed to determine VEGF activity by treating EC and evaluating VEGF receptor-2 (VEGFR2) phosphorylation. GZMB released VEGF from both FN and from EC-derived matrix, whereas GZMB inhibition prevented FN cleavage and VEGF release. GZMB-mediated VEGF release resulted in significant phosphorylation of VEGFR2. The role of GZMB-mediated VEGF release in altering vascular permeability was also assessed in vivo using Miles/Evans blue permeability assay. GZMB induced a significant VEGF-dependent increase in vascular permeability in vivo that was reduced in the presence of an anti-VEGF-neutralizing antibody. Inflammatory-mediated vascular leakage was also assessed in GZMB-KO mice using a delayed-type hypersensitivity model. GZMB-KO mice exhibited reduced microvascular leakage compared with C57\B6 controls. GZMB increases vascular permeability in part through the proteolytic release of ECM-sequestered VEGF, leading to VEGFR2 activation and increased vascular permeability in vivo. These findings present a novel role for GZMB as a modulator of vascular response during chronic inflammation.

Inflammation in myocardial diseases.

Inflammatory processes underlie a broad spectrum of conditions that injure the heart muscle and cause both structural and functional deficits. In this article, we address current knowledge regarding 4 common forms of myocardial inflammation: myocardial ischemia and reperfusion, sepsis, viral myocarditis, and immune rejection. Each of these pathological states has its own unique features in pathogenesis and disease evolution, but all reflect inflammatory mechanisms that are partially shared. From the point of injury to the mobilization of innate and adaptive immune responses and inflammatory amplification, the cellular and soluble mediators and mechanisms examined in this review will be discussed with a view that both beneficial and adverse consequences arise in these human conditions.

Granzyme serine proteases in inflammation and rheumatic diseases.

Granzymes (granule-secreted enzymes) are a family of serine proteases that have been viewed as redundant cytotoxic enzymes since their discovery more than 30 years ago. Predominantly produced by cytotoxic lymphocytes and natural killer cells, granzymes are delivered into the cytoplasm of target cells through immunological synapses in cooperation with the pore-forming protein perforin. After internalization, granzymes can initiate cell death through the cleavage of intracellular substrates. However, evidence now also demonstrates the existence of non-cytotoxic, pro-inflammatory, intracellular and extracellular functions that are granzyme specific. Under pathological conditions, granzymes can be produced and secreted extracellularly by immune cells as well as by non-immune cells. Depending on the granzyme, accumulation in the extracellular milieu might contribute to inflammation, tissue injury, impaired wound healing, barrier dysfunction, osteoclastogenesis and/or autoantigen generation.

Granzyme K mediates IL-23-dependent inflammation and keratinocyte proliferation in psoriasis.

Psoriasis is an inflammatory disease with systemic manifestations that most commonly presents as itchy, erythematous, scaly plaques on extensor surfaces. Activation of the IL-23/IL-17 pro-inflammatory signaling pathway is a hallmark of psoriasis and its inhibition is key to clinical management. Granzyme K (GzmK) is an immune cell-secreted serine protease elevated in inflammatory and proliferative skin conditions. In the present study, human psoriasis lesions exhibited elevated GzmK levels compared to non-lesional psoriasis and healthy control skin. In an established murine model of imiquimod (IMQ)-induced psoriasis, genetic loss of GzmK significantly reduced disease severity, as determined by delayed plaque formation, decreased erythema and desquamation, reduced epidermal thickness, and inflammatory infiltrate. Molecular characterization in vitro revealed that GzmK contributed to macrophage secretion of IL-23 as well as PAR-1-dependent keratinocyte proliferation. These findings demonstrate that GzmK enhances IL-23-driven inflammation as well as keratinocyte proliferation to exacerbate psoriasis severity.