Recombinant Decorin Fusion Protein Attenuates Murine Abdominal Aortic Aneurysm Formation and Rupture.

Decorin (DCN) is a small-leucine rich proteoglycan that mediates collagen fibrillogenesis, organization, and tensile strength. Adventitial DCN is reduced in abdominal aortic aneurysm (AAA) resulting in vessel wall instability thereby predisposing the vessel to rupture. Recombinant DCN fusion protein CAR-DCN was engineered with an extended C-terminus comprised of CAR homing peptide that recognizes inflamed blood vessels and penetrates deep into the vessel wall. In the present study, the role of systemically-administered CAR-DCN in AAA progression and rupture was assessed in a murine model. Apolipoprotein E knockout (ApoE-KO) mice were infused with angiotensin II (AngII) for 28 days to induce AAA formation. CAR-DCN or vehicle was administrated systemically until day 15. Mortality due to AAA rupture was significantly reduced in CAR-DCN-treated mice compared to controls. Although the prevalence of AAA was similar between vehicle and CAR-DCN groups, the severity of AAA in the CAR-DCN group was significantly reduced. Histological analysis revealed that CAR-DCN treatment significantly increased DCN and collagen levels within the aortic wall as compared to vehicle controls. Taken together, these results suggest that CAR-DCN treatment attenuates the formation and rupture of Ang II-induced AAA in mice by reinforcing the aortic wall.

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-inhibitor serpina3n induces neuroprotection in vitro and in vivo.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune inflammatory and neurodegenerative disease of the central nervous system (CNS). It is widely accepted that inflammatory cells play major roles in the pathogenesis of MS, possibly through the use of serine protease granzyme B (GrB) secreted from the granules of cytotoxic T cells. We have previously identified GrB as a mediator of axonal injury and neuronal death. In this study, our goal was to evaluate the effect of GrB inhibition in the human system in vitro, and in vivo in EAE using the newly isolated GrB-inhibitor serpina3n. METHODS: We used a well-established in vitro model of neuroinflammation characterized by a co-culture system between human fetal neurons and lymphocytes. In vivo, we induced EAE in 10- to 12-week-old female C57/BL6 mice and treated them intravenously with serpina3n. RESULTS: In the in vitro co-culture system, pre-treatment of lymphocytes with serpina3n prevented neuronal killing and cleavage of the cytoskeletal protein alpha-tubulin, a known substrate for GrB. Moreover, in EAE, 50 µg serpina3n substantially reduced the severity of the disease. This dose was administered intravenously twice at days 7 and 20 post EAE induction. serpina3n treatment reduced axonal and neuronal injury compared to the vehicle-treated control group and maintained the integrity of myelin. Interestingly, serpina3n treatment did not seem to reduce the infiltration of immune cells (CD4(+) and CD8(+) T cells) into the CNS. CONCLUSION: Our data suggest further studies on serpina3n as a potentially novel therapeutic strategy for the treatment of inflammatory-mediated neurodegenerative diseases such as MS.

Design and characterization of a novel human Granzyme B inhibitor.

The intracellular roles of Granzyme B (GrB) in immune-mediated cell killing have been extensively studied. Recent data also implicate GrB in extracellular pathways of inflammation, cytokine activation and autoimmunity. Targeting (GrB) provides a new pharmaceutical agent for various inflammatory disorders. Serpina3n is a mouse extracellular inhibitor of GrB. There is no apparent equivalent in humans. In this study, we used a novel applied genetics approach to engineer a new extracellular GrB serpin. A chimeric protein was generated in which the reactive center loop (RCL) of human extracellular antichymotrypsin (ACT) was replaced with that of serpina3n. This serpin contained 27 amino acid residues from the serpina3n RCL and the remaining 395 residues from human ACT. The insertion converted human ACT into a GrB-inhibitory serpin. Several critical residues were identified by scanning mutagenesis on the chimera and serpina3n. Targeted mutagenesis was conducted on wild-type human ACT by specifically substituting those critical residues, creating a novel inhibitor that contains 99.3% human ACT sequence with only three point mutations. Wild-type human ACT had a kass for GrB of 2.26 × 10(4) M(-1) s(-1), whereas the novel inhibitor binds GrB with a kass of 7.65 × 10(5) M(-1) s(-1). This new drug candidate can be developed in animal models and further tested in clinical trials to help us understand the role of GrB in numerous disorders.

Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia.

Recent studies demonstrated that autophagy is an important regulator of innate immune response. However, the mechanism by which autophagy regulates natural killer (NK) cell-mediated antitumor immune responses remains elusive. Here, we demonstrate that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitro via the activation of autophagy. This impairment was not related to a defect in target cell recognition by NK cells but to the degradation of NK-derived granzyme B in autophagosomes of hypoxic cells. Inhibition of autophagy by targeting beclin1 (BECN1) restored granzyme B levels in hypoxic cells in vitro and induced tumor regression in vivo by facilitating NK-mediated tumor cell killing. Together, our data highlight autophagy as a mechanism underlying the resistance of hypoxic tumor cells to NK-mediated lysis. The work presented here provides a cutting-edge advance in our understanding of the mechanism by which hypoxia-induced autophagy impairs NK-mediated lysis in vitro and paves the way for the formulation of more effective NK cell-based antitumor therapies.

Granzyme H induces cell death primarily via a Bcl-2-sensitive mitochondrial cell death pathway that does not require direct Bid activation.

Natural killer and T cell-mediated cytotoxicity is important for the elimination of viruses and transformed cells. The granule lytic pathway utilizes perforin and granzymes to induce cell death, while receptor-mediated lytic pathways rely on molecules such as FasL. Pro-apoptotic activities of Granzyme B (GrB) and Fas are well-established, and many of their cellular targets have been identified. However, humans express additional related granzymes - GrA, GrM, GrK, and GrH. Neither the cytotoxic potential of GrH, nor the mechanism by which GrH may induce target cell death is currently understood. We proposed that GrH would have pro-apoptotic activity that would be distinct from that of GrB and FasL, which could be relevant when Fas/FasL or GrB activity or death pathways were impaired. Our results, using a purified recombinant form of GrH, revealed that GrH induced cell death via a Bcl-2-sensitive mitochondrial pathway without direct processing of Bid. Additionally, neither the apoptosome nor caspase-3 was essential to the induction of GrH-mediated cell death. However, GrH did directly process DFF45, potentially leading to DNA damage. Our findings support the idea that multiple, non-redundant death pathways may be initiated by cytotoxic cells to counteract various immune evasion strategies.

The unmet need in the elderly: how immunosenescence, CMV infection, co-morbidities and frailty are a challenge for the development of more effective influenza vaccines.

Influenza remains the single most important cause of excess disability and mortality during the winter months. In spite of widespread influenza vaccination programs leading to demonstrated cost-savings in the over 65 population, hospitalization and death rates for acute respiratory illnesses continue to rise. As a person ages, increased serum levels of inflammatory cytokines are commonly recorded (TNF-α, IL-1, IL-6). Termed "inflammaging", this has been linked to persistent cytomegalovirus (CMV) infection and immune senescence, while increased anti-inflammatory cytokines (IL-10, TGF-ß) are possibly associated with more healthy aging. Paradoxically, a shift with aging toward an anti-inflammatory (IL-10) response and decline in the IFN-γ:IL-10 ratio in influenza-challenged peripheral blood mononuclear cells is associated with a decline in the cytolytic capacity of CD8+ T cells responsible for clearing influenza virus from infected lung tissue. Thus, it is seemingly counter intuitive that the immune phenotype of healthy aging predicts a poor cell-mediated immune response and more serious outcomes of influenza. Herein we postulate a mechanistic link between the accumulation of late-stage, potentially terminally differentiated T cells, many or most of which result from CMV infection, and the immunopathogenesis of influenza infection, mediated by granzyme B in older adults. Further, adjuvanted influenza vaccines that stimulate inflammatory cytokines and suppress the IL-10 response to influenza challenge, would be expected to enhance protection in the 65+ population.

Seasonal H1N1 influenza virus infection induces cross-protective pandemic H1N1 virus immunity through a CD8-independent, B cell-dependent mechanism.

During the 2009 H1N1 influenza virus pandemic (pdmH1N1) outbreak, it was found that most individuals lacked antibodies against the new pdmH1N1 virus, and only the elderly showed anti-hemagglutinin (anti-HA) antibodies that were cross-reactive with the new strains. Different studies have demonstrated that prior contact with the virus can confer protection against strains with some degree of dissimilarity; however, this has not been sufficiently explored within the context of a pdmH1N1 virus infection. In this study, we have found that a first infection with the A/Brisbane/59/2007 virus strain confers heterologous protection in ferrets and mice against a subsequent pdmH1N1 (A/Mexico/4108/2009) virus infection through a cross-reactive but non-neutralizing antibody mechanism. Heterologous immunity is abrogated in B cell-deficient mice but maintained in CD8(-/-) and perforin-1(-/-) mice. We identified cross-reactive antibodies from A/Brisbane/59/2007 sera that recognize non-HA epitopes in pdmH1N1 virus. Passive serum transfer showed that cross-reactive sH1N1-induced antibodies conferred protection in naive recipient mice during pdmH1N1 virus challenge. The presence or absence of anti-HA antibodies, therefore, is not the sole indicator of the effectiveness of protective cross-reactive antibody immunity. Measurement of additional antibody repertoires targeting the non-HA antigens of influenza virus should be taken into consideration in assessing protection and immunization strategies. We propose that preexisting cross-protective non-HA antibody immunity may have had an overall protective effect during the 2009 pdmH1N1 outbreak, thereby reducing disease severity in human infections.

Granzyme B inhibits vaccinia virus production through proteolytic cleavage of eukaryotic initiation factor 4 gamma 3.

Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD(1408)S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery.

Protection of porcine islet xenografts in mice using sertoli cells and monoclonal antibodies.

BACKGROUND: To remedy the shortage of human donor islets, xenotransplantation of neonatal porcine islets (NPI) provides an attractive alternative source of donor tissue so long as graft rejection can be circumvented. Thus, in this study, we sought to determine whether cotransplantation of NPI with Sertoli cells (SC) combined with a short-course treatment of monoclonal antibody (mAb) could provide long-term islet xenograft survival. METHODS: NPI alone or NPI cotransplanted with neonatal porcine SC were transplanted into diabetic C57BL/6 mice. These mice were left untreated or were treated with a short course of antileukocyte function associated antigen-1 (LFA-1), anti-CD154, or anti-CD45RB mAb. Blood glucose levels were monitored twice a week to assess graft function. At more than 100 days posttransplantation or on the day of rejection, graft-bearing kidneys were collected for characterization using immunohistochemistry. RESULTS: None of the untreated control mice transplanted with NPI alone (0/5) or NPI cotransplanted with SC (0/8) achieved normoglycemia. However, of the mice receiving NPI alone, 3 of 7 treated with anti-LFA-1 mAb, 2 of 7 treated with anti-CD154 mAb, and 1 of 7 treated with anti-CD45RB mAb achieved long-term graft survival (>100 days). These proportions improved considerably when NPI were cotransplanted with SC, as 15 of 15 mice treated with anti-LFA-1 mAb, 7 of 8 mice treated with anti-CD154 mAb, and 4 of 9 mice treated with anti-CD45RB mAb achieved long-term graft survival. CONCLUSIONS: These results show that transient administration of anti-LFA-1 mAb or anti-CD154 mAb is efficacious in prolonging NPI xenograft survival when islets are cotransplanted with SC. Interleukin-4 and Serpina3n may be important mediators of protection observed in this model.

Granule-derived granzyme B mediates the vulnerability of human neurons to T cell-induced neurotoxicity.

Multiple sclerosis (MS) is considered an autoimmune disease of the CNS and is characterized by inflammatory cells infiltrating the CNS and inducing demyelination, axonal loss, and neuronal death. Recent evidence strongly suggests that axonal and neuronal degeneration underlie the progression of permanent disability in MS. In this study, we report that human neurons are selectively susceptible to the serine-protease granzyme B (GrB) isolated from cytotoxic T cell granules. In vitro, purified human GrB induced neuronal death to the same extent as the whole activated T cell population. On the contrary, activated T cells isolated from GrB knockout mice failed to induce neuronal injury. We found that following internalization through various parts of neurons, GrB accumulated in the neuronal soma. Within the cell body, GrB diffused out of endosomes possibly through a perforin-independent mechanism and induced subsequent activation of caspases and cleavage of α-tubulin. Inhibition of caspase-3, a well-known substrate for GrB, significantly reduced GrB-mediated neurotoxicity. We demonstrated that treatment of neurons with mannose-6-phosphate prevented GrB entry and inhibited GrB-mediated neuronal death, suggesting mannose-6-phosphate receptor-dependent endocytosis. Together, our data unveil a novel mechanism by which GrB induces selective neuronal injury and suggest potential new targets for the treatment of inflammatory-mediated neurodegeneration in diseases such as MS.

Pyruvate dehydrogenase inhibition by the inflammatory cytokine TNFα contributes to the pathogenesis of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMC). This apoptosis resistance is characterized by PASMC mitochondrial hyperpolarization [in part, due to decreased pyruvate dehydrogenase (PDH) activity], decreased mitochondrial reactive oxygen species (mROS), downregulation of Kv1.5, increased [Ca(++)](i), and activation of the transcription factor nuclear factor of activated T cells (NFAT). Inflammatory cells are present within and around the remodeled arteries and patients with PAH have elevated levels of inflammatory cytokines, including tumor necrosis factor-α (TNFα). We hypothesized that the inflammatory cytokine TNFα inhibits PASMC PDH activity, inducing a PAH phenotype in normal PASMC. We exposed normal human PASMC to recombinant human TNFα and measured PDH activity. In TNFα-treated cells, PDH activity was significantly decreased. Similar to exogenous TNFα, endogenous TNFα secreted from activated human CD8(+) T cells, but not quiescent T cells, caused mitochondrial hyperpolarization, decreased mROS, decreased K(+) current, increased [Ca(++)](i), and activated NFAT in normal human PASMC. A TNFα antibody completely prevented, while recombinant TNFα mimicked the T cell-induced effects. In vivo, the TNFα antagonist etanercept prevented and reversed monocrotaline (MCT)-induced PAH. In a separate model, T cell deficient rats developed less severe MCT-induced PAH compared to their controls. We show that TNFα can inhibit PASMC PDH activity and induce a PAH phenotype. Our work supports the use of anti-inflammatory therapies for PAH.

Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.

How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

CD4+CD25+CD127dimFoxp3+ T cells are cytotoxic for human neurons.

MS lesions are characterized by destruction of myelin and significant neuronal and axonal loss. Preliminary studies with the use of T(regs) in the mouse model of MS have been extremely encouraging. However, recent studies with human cells have shown the presence of different subpopulations of T cells within the CD4(+)CD25(+)Foxp3(+) T cell phenotype, some of which do not have regulatory functions. These findings suggest a potential difference between mouse and human in the regulatory phenotype. Here, we show that human activated CD4(+)CD25(+)Foxp3(+) T cells are neurotoxic in vitro. These cells expressed high levels of the cytotoxic molecule GrB and had no suppressive effect. On the contrary, they produced IFN-γ and low IL-17, suggesting a shift toward a T(H)1 phenotype. Thus, our data confirm the presence of a nonregulatory cytotoxic subpopulation within the human CD4(+)CD25(+)Foxp3(+) T cells and suggest further studies on the human regulatory phenotype prior to any potential therapeutic application.

The human cathelicidin, LL-37, induces granzyme-mediated apoptosis in cytotoxic T lymphocytes.

LL-37 is a human cationic host defense peptide (antimicrobial peptide) belonging to the cathelicidin family of peptides. In this study, LL-37 was shown to kill stimulated CD8(+) T cells (Cytotoxic T lymphocytes; CTLs) via apoptosis, while having no cytotoxic effect on non-stimulated CD8(+) or CD4(+) T cells or stimulated CD4(+) T cells. Of interest, the CD8(+) cells were much more sensitive to LL-37 than many other cell types. LL-37 exposure resulted in DNA fragmentation, chromatin condensation, and the release of both granzyme A and granzyme B from intracellular granules. The importance of granzyme family members in the apoptosis of CTLs following LL-37 treatment was analyzed by using C57BL/6 lymphocytes obtained from mice that were homozygous for null mutations in the granzyme B gene, the granzyme A gene, or both granzymes A and B. Granzymes A and B were both shown to play an important role in LL-37-induced apoptosis of CTLs. Further analysis revealed that apoptosis occurred primarily through granzyme A-mediated caspase-independent apoptosis. However, caspase-dependent cell death was also observed. This suggests that LL-37 induces apoptosis in CTLs via multiple different mechanisms, initiated by the LL-37-induced leakage of granzymes from cytolytic granules. Our results imply the existence of a novel mechanism of crosstalk between the inflammatory and adaptive immune systems. Cells such as neutrophils, at the site of a tumor for example, could influence the effector, activity of CTL through the secretion of LL-37.

The human host defense peptide LL-37 induces apoptosis in a calpain- and apoptosis-inducing factor-dependent manner involving Bax activity.

LL-37 is a human cationic host defense peptide (antimicrobial peptide) belonging to the cathelicidin family of peptides. In this study, LL-37 was shown to kill Jurkat T leukemia cells via apoptosis. A loss of mitochondrial membrane potential, DNA fragmentation, and phosphatidylserine externalization were detected following LL-37 exposure, whereas apoptosis was independent of caspase family members. The specific apoptotic pathway induced by LL-37 was defined through the utilization of Jurkat cells modified to express antiapoptotic proteins, as well as cells deficient in various proteins associated with apoptosis. Of interest, both Bcl-2-overexpressing cells and cells deficient in Bax and Bak proteins displayed a significant reduction in LL-37-induced apoptosis. In addition, Jurkat cells modified in the Fas receptor-associated pathway showed no reduction in apoptosis when exposed to LL-37. Analysis of the involvement of apoptosis-inducing factor (AIF) in LL-37-mediated apoptosis revealed that AIF transferred from the mitochondria to the nucleus of cells exposed to LL-37, where it may lead to large-scale DNA fragmentation and chromatin condensation. AIF knockdown analysis resulted in LL-37-resistant cells. This suggests that AIF is mandatory in LL-37-mediated killing. Lastly, chelation or inhibition of Ca(2+) or calpains inhibited LL-37-mediated killing. Further analysis revealed that calpains were required for LL-37-mediated Bax translocation to mitochondria. Together, these data show that LL-37-induced apoptosis is mediated via the mitochondria-associated pathway in a caspase-independent and calpain- and AIF-dependent manner that involves Bax activation and translocation to mitochondria.

Granzyme B: Correlates with protection and enhanced CTL response to influenza vaccination in older adults.

This study compared serum antibody titers and granzyme B (GrzB) levels in virus-stimulated peripheral blood mononuclear cells following influenza vaccination. Twelve of 239 older adults who subsequently developed laboratory-diagnosed influenza illness (LDI) had significantly lower GrzB levels compared to subjects without LDI (p=0.004). Eight subjects with LDI in the previous year showed an enhanced GrzB response to vaccination (p=0.02). Serum antibody titers following vaccination did not distinguish those older adults who developed LDI from those who did not. These results suggest that GrzB levels could be combined with antibody titers to more effectively predict vaccine efficacy in older adults.

PPP1R9B (Neurabin 2): involvement and dynamics in the NK immunological synapse.

The NK immunological synapse (NKIS) is a dynamic structure dependent on the assembly of membrane, cytoskeletal and signaling components. These serve to focus and generate stimuli for adhesion and orientation of the cytoskeleton for targeted cytolytic granule release. Previous studies have demonstrated the importance of the cytoskeleton in these processes. We previously identified PPP1R9B (neurabin 2, spinophilin) as a cytoskeletal component of the NK-like cell line YTS. We demonstrate that (i) PPP1R9B gradually accumulates at the NKIS in a maturation stage-dependent manner; (ii) it mimics the early kinetics of actin recruitment to the NKIS but it precedes actin departure from the site; (iii) it is recruited by CD18 stimulation but not by CD28 ligation; (iv) it is required for the maintenance of the cortical F-actin organization in the YTS cells and knocking down PPP1R9B reduces the frequency of YTS-target cell conjugation, possibly due to the collapsed F-actin cytoskeleton in these cells. These results indicate that PPP1R9B is required for synapse formation in the NK cells and suggest that it may be involved in the maintenance of cellular architecture by regulation of actin assembly, possibly acting to stabilize the NKIS until granule release is eminent.