RESUMO
Aims: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis. Methods: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times. Results: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα. Conclusions: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology.
Assuntos
Granzimas/antagonistas & inibidores , Peritonite/tratamento farmacológico , Peritonite/enzimologia , Sepse/tratamento farmacológico , Sepse/enzimologia , Idoso , Idoso de 80 Anos ou mais , Animais , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Granzimas/sangue , Granzimas/deficiência , Granzimas/genética , Humanos , Mediadores da Inflamação/sangue , Interleucina-6/biossíntese , Células Matadoras Naturais/enzimologia , Macrófagos/enzimologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Peritonite/etiologia , Medicina de Precisão , Sepse/etiologia , Serpinas/farmacologia , Receptor 4 Toll-Like/metabolismoRESUMO
Mothers living near high-traffic roads before or during pregnancy are more likely to have children with asthma. Mechanisms are unknown. Using a mouse model, here we showed that maternal exposure to diesel exhaust particles (DEP) predisposed offspring to allergic airway disease (AAD, murine counterpart of human asthma) through programming of their NK cells; predisposition to AAD did not develop in DEP pups that lacked NK cells and was induced in normal pups receiving NK cells from WT DEP pups. DEP NK cells expressed GATA3 and cosecreted IL-13 and the killer protease granzyme B in response to allergen challenge. Extracellular granzyme B did not kill, but instead stimulated protease-activated receptor 2 (PAR2) to cooperate with IL-13 in the induction of IL-25 in airway epithelial cells. Through loss-of-function and reconstitution experiments in pups, we showed that NK cells and granzyme B were required for IL-25 induction and activation of the type 2 immune response and that IL-25 mediated NK cell effects on type 2 response and AAD. Finally, experiments using human cord blood and airway epithelial cells suggested that DEP might induce an identical pathway in humans. Collectively, we describe an NK cell-dependent endotype of AAD that emerged in early life as a result of maternal exposure to DEP.
Assuntos
Asma/imunologia , Granzimas/imunologia , Células Matadoras Naturais/imunologia , Exposição Materna/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/imunologia , Emissões de Veículos/toxicidade , Animais , Asma/genética , Asma/patologia , Modelos Animais de Doenças , Feminino , Granzimas/deficiência , Humanos , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Células Matadoras Naturais/patologia , Camundongos , Camundongos Knockout , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/patologia , Células Th2/imunologia , Células Th2/patologiaRESUMO
Cytotoxic CD8+ T (Tc) cells are the main executors of transformed and cancer cells during cancer immunotherapy. The latest clinical results evidence a high efficacy of novel immunotherapy agents that modulate Tc cell activity against bad prognosis cancers. However, it has not been determined yet whether the efficacy of these treatments can be affected by selection of tumoural cells with mutations in the cell death machinery, known to promote drug resistance and cancer recurrence. Here, using a model of prophylactic tumour vaccination based on the LCMV-gp33 antigen and the mouse EL4 T lymphoma, we analysed the molecular mechanism employed by Tc cells to eliminate cancer cells in vivo and the impact of mutations in the apoptotic machinery on tumour development. First of all, we found that Tc cells, and perf and gzmB are required to efficiently eliminate EL4.gp33 cells after LCMV immunisation during short-term assays (1-4 h), and to prevent tumour development in the long term. Furthermore, we show that antigen-pulsed chemoresistant EL4 cells overexpressing Bcl-XL or a dominant negative form of caspase-3 are specifically eliminated from the peritoneum of infected animals, as fast as parental EL4 cells. Notably, antigen-specific Tc cells control the tumour growth of the mutated cells, as efficiently as in the case of parental cells. Altogether, expression of the anti-apoptotic mutations does not confer any advantage for tumour cells neither in the short-term survival nor in long-term tumour formation. Although the mechanism involved in the elimination of the apoptosis-resistant tumour cells is not completely elucidated, neither necroptosis nor pyroptosis seem to be involved. Our results provide the first experimental proof that chemoresistant cancer cells with mutations in the main cell death pathways are efficiently eliminated by Ag-specific Tc cells in vivo during immunotherapy and, thus, provide the molecular basis to treat chemoresistant cancer cells with CD8 Tc-based immunotherapy.
Assuntos
Antígenos Virais/imunologia , Resistencia a Medicamentos Antineoplásicos/genética , Glicoproteínas/imunologia , Neoplasias/terapia , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/transplante , Proteínas Virais/imunologia , Animais , Apoptose , Caspase 3/metabolismo , Linhagem Celular Tumoral , Granzimas/deficiência , Granzimas/genética , Granzimas/metabolismo , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/imunologia , Neoplasias/mortalidade , Perforina/deficiência , Perforina/genética , Perforina/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Taxa de Sobrevida , Linfócitos T Citotóxicos/imunologia , Transplante Heterólogo , Proteína bcl-X/metabolismoRESUMO
Calcification is a major risk factor for vascular integrity. This pathological symptom and the underlying mechanisms in hypoxic pulmonary artery hypertension remain elusive. Here we report that pulmonary vascular medial calcification is elevated in pulmonary artery hypertension models as a result of an osteoblastic phenotype change of pulmonary arterial smooth muscle cells induced by hypoxia. Notably, inhibiting store-operated calcium channels significantly decreased osteoblastic differentiation and calcification of pulmonary arterial smooth muscle cells under hypoxia. We identified granzyme B, a major constituent of cytotoxic T lymphocytes/natural killer cell granules involved in apoptosis, as the main regulator of pulmonary arterial calcification. Overexpression of granzyme B blocked the mineralization through its effect on store-operated calcium channels in cultured pulmonary arterial smooth muscle cells under hypoxic conditions. Mice with overexpression of granzyme B exposed to hypoxia for 3 weeks showed attenuated vascular calcification and pathological progression of hypoxic pulmonary arterial hypertension. Our findings emphasize the central function of granzyme B in coordinating vascular calcification in hypoxic pulmonary arterial hypertension.
Assuntos
Calcinose/enzimologia , Granzimas/genética , Hipertensão Pulmonar/enzimologia , Hipóxia/enzimologia , Miócitos de Músculo Liso/enzimologia , Osteoblastos/enzimologia , Animais , Apoptose/genética , Calcinose/induzido quimicamente , Calcinose/genética , Calcinose/patologia , Canais de Cálcio , Diferenciação Celular , Regulação da Expressão Gênica , Granzimas/deficiência , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipóxia/induzido quimicamente , Hipóxia/genética , Hipóxia/patologia , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monocrotalina/administração & dosagem , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Osteoblastos/patologia , Cultura Primária de Células , Artéria Pulmonar/enzimologia , Artéria Pulmonar/patologia , Ratos Sprague-Dawley , Ratos Wistar , Transdução de Sinais , Linfócitos T Citotóxicos/enzimologia , Linfócitos T Citotóxicos/patologiaRESUMO
Granzyme A is a protease implicated in the degradation of intracellular DNA. Nucleotide complexes are known triggers of systemic autoimmunity, but a role in organ-specific autoimmune disease has not been demonstrated. To investigate whether such a mechanism could be an endogenous trigger for autoimmunity, we examined the impact of granzyme A deficiency in the NOD mouse model of autoimmune diabetes. Granzyme A deficiency resulted in an increased incidence in diabetes associated with accumulation of ssDNA in immune cells and induction of an interferon response in pancreatic islets. Central tolerance to proinsulin in transgenic NOD mice was broken on a granzyme A-deficient background. We have identified a novel endogenous trigger for autoimmune diabetes and an in vivo role for granzyme A in maintaining immune tolerance.
Assuntos
Diabetes Mellitus Tipo 1/etiologia , Granzimas/fisiologia , Tolerância Imunológica , Interferon Tipo I/fisiologia , Animais , DNA de Cadeia Simples/metabolismo , Feminino , Granzimas/deficiência , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
Inflammatory bowel disease (IBD) is an immunoregulatory disorder, associated with a chronic and inappropriate mucosal immune response to commensal bacteria, underlying disease states such as ulcerative colitis (UC) and Crohn's disease (CD) in humans. Granzyme M (GrzM) is a serine protease expressed by cytotoxic lymphocytes, in particular natural killer (NK) cells. Granzymes are thought to be involved in triggering cell death in eukaryotic target cells; however, some evidence supports their role in inflammation. The role of GrzM in the innate immune response to mucosal inflammation has never been examined. Here, we discover that patients with UC, unlike patients with CD, display high levels of GrzM mRNA expression in the inflamed colon. By taking advantage of well-established models of experimental UC, we revealed that GrzM-deficient mice have greater levels of inflammatory indicators during dextran sulfate sodium (DSS)-induced IBD, including increased weight loss, greater colon length reduction and more severe intestinal histopathology. The absence of GrzM expression also had effects on gut permeability, tissue cytokine/chemokine dynamics, and neutrophil infiltration during disease. These findings demonstrate, for the first time, that GrzM has a critical role during early stages of inflammation in UC, and that in its absence colonic inflammation is enhanced.
Assuntos
Colite Ulcerativa/imunologia , Colite/imunologia , Doença de Crohn/imunologia , Granzimas/imunologia , Imunidade Inata , Animais , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Colo/imunologia , Colo/patologia , Doença de Crohn/genética , Doença de Crohn/patologia , Sulfato de Dextrana , Feminino , Expressão Gênica , Granzimas/deficiência , Granzimas/genética , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Permeabilidade , RNA Mensageiro/genética , RNA Mensageiro/imunologiaRESUMO
Foxp3(+) regulatory T cells (Treg) playing a crucial role in the maintenance of immune tolerance and prevention of autoimmune diseases consist of thymus-derived naturally occurring CD4(+)Foxp3(+) Treg cells (nTreg) and those that can be induced ex vivo with TGF-ß (iTreg). Although both Treg subsets share similar phenotypes and functional characteristics, they also have potential biologic differences on their biology. The role of iTreg in regulating B cells remains unclear so far. The suppression assays of Treg subsets on activation, proliferation, and Abs production of B cells were measured using a Treg and B cell coculture system in vitro. Transwell and Ab blockade experiments were performed to assess the roles of cell contact and soluble cytokines. Treg were adoptively transferred to lupus mice to assess in vivo effects on B cells. Like nTreg, iTreg subset also directly suppressed activation and proliferation of B cells. nTreg subset suppressed B cell responses through cytotoxic manner related to expression of granzyme A, granzyme B, and perforin, whereas the role of iTreg subset on B cells did not involve in cytotoxic action but depending on TGF-ß signaling. Furthermore, iTreg subset can significantly suppress Ab produced by lupus B cells in vitro. Comparison experiments using autoantibodies microarrays demonstrated that adoptive transfer of iTreg had a superior effect than nTreg subset on suppressing lupus B cell responses in vivo. Our data implicate a role and advantage of iTreg subset in treating B cell-mediated autoimmune diseases, boosting the translational potential of these findings.
Assuntos
Linfócitos B/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/imunologia , Transferência Adotiva , Animais , Autoanticorpos/biossíntese , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Granzimas/deficiência , Granzimas/genética , Granzimas/metabolismo , Tolerância Imunológica , Interleucina-2/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/fisiologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Granzyme B (GzmB) has previously been shown to be critical for CD8(+) T cell-mediated graft-versus-host disease (GVHD) but dispensable for GVHD mediated by CD4(+) T cells. However, previous studies used high doses of CD4(+) T cells in MHC-mismatched models that caused rapid and lethal GVHD. Because of the hyperacute lethality, it is possible that the role of GzmB was concealed by the system. Therefore, in this study, we have titrated down the T cell dose to precisely determine the contribution of GzmB in GVHD mediated by CD4(+)CD25(-) T cells. Surprisingly, we have found that GzmB(-/-)CD4(+)CD25(-) T cells cause more severe GVHD compared with wild-type CD4(+)CD25(-) T cells in both MHC-matched and mismatched models. Mechanistic analyses reveal that although GzmB does not affect donor T cell engraftment, proliferation or tissue-specific migration, GzmB(-/-) CD4(+)CD25(-) T cells exhibit significantly enhanced expansion because of GzmB-mediated activation-induced cell death of wild-type CD4(+)CD25(-) T cells. As a result of enhanced expansion, GzmB(-/-) T cells produced higher amounts of proinflammatory cytokines (e.g., TNF-α and IFN-γ) that may contribute to the exacerbated GVHD. These results reveal that GzmB diminishes the ability of CD4(+) T cells to cause acute GVHD, which contradicts its established role in CD8(+) T cells. The differential roles suggest that targeting GzmB in selected T cell subsets may provide a strategy to control GVHD.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Granzimas/imunologia , Ativação Linfocitária/imunologia , Doença Aguda , Animais , Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/métodos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Morte Celular/genética , Morte Celular/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Proliferação de Células/genética , Citocinas/imunologia , Citocinas/metabolismo , Citometria de Fluxo , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/patologia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/genética , Granzimas/deficiência , Granzimas/genética , Histocompatibilidade/genética , Histocompatibilidade/imunologia , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/citologia , Baço/imunologia , Baço/metabolismoRESUMO
RATIONALE: CD4(+) natural killer T (NKT) cells augment atherosclerosis in apolipoprotein E-deficient (ApoE)(-/-) mice but their mechanisms of action are unknown. OBJECTIVES: We investigated the roles of bystander T, B, and NK cells; NKT cell-derived interferon-γ, interleukin (IL)-4, and IL-21 cytokines; and NKT cell-derived perforin and granzyme B cytotoxins in promoting CD4(+) NKT cell atherogenicity. METHODS AND RESULTS: Transfer of CD4(+) NKT cells into T- and B-cell-deficient ApoE(-/-)Rag2(-/-) mice augmented aortic root atherosclerosis by ≈75% that was ≈30% of lesions in ApoE(-/-) mice; macrophage accumulation similarly increased. Transferred NKT cells were identified in the liver and atherosclerotic lesions of recipient mice. Transfer of CD4(+) NKT cells into T-, B-cell-deficient, and NK cell-deficient ApoE(-/-)Rag2(-/-)γC(-/-) mice also augmented atherosclerosis. These data indicate that CD4(+) NKT cells can exert proatherogenic effects independent of other lymphocytes. To investigate the role of NKT cell-derived interferon-γ, IL-4, and IL-21 cytokines and perforin and granzyme B cytotoxins, CD4(+) NKT cells from mice deficient in these molecules were transferred into NKT cell-deficient ApoE(-/-)Jα18(-/-) mice. CD4(+) NKT cells deficient in IL-4, interferon-γ, or IL-21 augmented atherosclerosis in ApoE(-/-)Jα18(-/-) mice by ≈95%, ≈80%, and ≈70%, respectively. Transfer of CD4(+) NKT cells deficient in perforin or granzyme B failed to augment atherosclerosis. Apoptotic cells, necrotic cores, and proinflammatory VCAM-1 (vascular cell adhesion molecule) and MCP-1 (monocyte chemotactic protein) were reduced in mice receiving perforin-deficient NKT cells. CD4(+) NKT cells are twice as potent as CD4(+) T cells in promoting atherosclerosis. CONCLUSIONS: CD4(+) NKT cells potently promote atherosclerosis by perforin and granzyme B-dependent apoptosis that increases postapoptotic necrosis and inflammation.
Assuntos
Aterosclerose/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Granzimas/deficiência , Células T Matadoras Naturais/metabolismo , Proteínas Citotóxicas Formadoras de Poros/deficiência , Seio Aórtico/metabolismo , Transferência Adotiva/métodos , Animais , Aterosclerose/imunologia , Aterosclerose/patologia , Linfócitos T CD4-Positivos/imunologia , Masculino , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Seio Aórtico/imunologia , Seio Aórtico/patologiaRESUMO
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.
Assuntos
Matriz Extracelular/metabolismo , Granzimas/metabolismo , Pele/metabolismo , Pele/efeitos da radiação , Raios Ultravioleta , Animais , Contagem de Células , Colágeno/metabolismo , Decorina/metabolismo , Derme/patologia , Derme/efeitos da radiação , Relação Dose-Resposta à Radiação , Feminino , Fibroblastos/enzimologia , Fibroblastos/efeitos da radiação , Fibronectinas/metabolismo , Granzimas/deficiência , Mastócitos/enzimologia , Mastócitos/efeitos da radiação , Metaloproteinase 1 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Transporte Proteico/efeitos da radiação , Proteólise/efeitos da radiação , Envelhecimento da Pele/efeitos da radiaçãoRESUMO
Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmBW) common in wild mouse. While retaining 'Asp-ase' activity, GzmBW has substrate preferences that differ considerably from GzmBP, which is common to all inbred strains. In vitro, GzmBW preferentially cleaves recombinant Bid, whereas GzmBP activates pro-caspases directly. Recombinant GzmBW and GzmBP induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmBW were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmBW-expressing CD8 T cells isolated from infected mice were unable to kill MCMV-infected targets in vitro. Our results suggest that known virally-encoded inhibitors of the intrinsic (mitochondrial) apoptotic pathway account for the increased susceptibility of GzmBW mice to MCMV. We conclude that different natural variants of GzmB have a profound impact on the immune response to a common and authentic viral pathogen.
Assuntos
Variação Genética/genética , Granzimas/genética , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/mortalidade , Muromegalovirus/imunologia , Viroses/imunologia , Viroses/mortalidade , Alelos , Sequência de Aminoácidos , Animais , Apoptose , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Caspases/metabolismo , Modelos Animais de Doenças , Granzimas/análise , Granzimas/deficiência , Infecções por Herpesviridae/patologia , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Viroses/patologiaRESUMO
The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4-TRIF-p65 NF-κB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress.
Assuntos
Regulação da Expressão Gênica/imunologia , Granzimas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Transdução de Sinais/imunologia , Estresse Fisiológico/fisiologia , Animais , Apoptose/efeitos dos fármacos , Medula Óssea/fisiologia , Proliferação de Células/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Primers do DNA/genética , Tratamento Farmacológico , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fluoruracila/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Granzimas/deficiência , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Imuno-Histoquímica , Lipopolissacarídeos/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise em Microsséries , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Granzymes are generally recognized for their capacity to induce various pathways of perforin-dependent target cell death. Within this serine protease family, Granzyme M (GrzM) is unique owing to its preferential expression in innate effectors such as natural killer (NK) cells. During Listeria monocytogenes infection, we observed markedly reduced secretion of macrophage inflammatory protein-1 alpha (MIP-1α) in livers of GrzM-deficient mice, which resulted in significantly impaired NK cell recruitment. Direct stimulation with IL-12 and IL-15 demonstrated that GrzM was required for maximal secretion of active MIP-1α. This effect was not due to reduced protein induction but resulted from heightened intracellular accumulation of MIP-1α, with reduced release. These results demonstrate that GrzM is a critical mediator of innate immunity that can regulate chemotactic networks and has an important role in the initiation of immune responses and pathogen control.
Assuntos
Quimiocina CCL3/metabolismo , Granzimas/metabolismo , Imunidade Inata , Células Matadoras Naturais/enzimologia , Listeriose/enzimologia , Animais , Células Cultivadas , Quimiotaxia de Leucócito , Técnicas de Cocultura , Modelos Animais de Doenças , Granzimas/deficiência , Granzimas/genética , Humanos , Interleucinas/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/microbiologia , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Listeriose/genética , Listeriose/imunologia , Listeriose/microbiologia , Camundongos , Camundongos Knockout , Fatores de TempoRESUMO
Human and mouse granzyme (Gzm)B both induce target cell apoptosis in concert with pore-forming perforin (Pfp); however the mechanisms by which other Gzms induce non-apoptotic death remain controversial and poorly characterised. We used timelapse microscopy to document, quantitatively and in real time, the death of target cells exposed to primary natural killer (NK) cells from mice deficient in key Gzms. We found that in the vast majority of cases, NK cells from wild-type mice induced classic apoptosis. However, NK cells from syngeneic Gzm B-deficient mice induced a novel form of cell death characterised by slower kinetics and a pronounced, writhing, 'worm-like' morphology. Dying cells initially contracted but did not undergo membrane blebbing, and annexin-V staining was delayed until the onset of secondary necrosis. As it is different from any cell death process previously reported, we tentatively termed this cell death 'athetosis'. Two independent lines of evidence showed this alternate form of death was due to Gzm A: first, cell death was revealed in the absence of Gzm B, but was completely lost when the NK cells were deficient in both Gzm A and B; second, the athetotic morphology was precisely reproduced when recombinant mouse Gzm A was delivered by an otherwise innocuous dose of recombinant Pfp. Gzm A-mediated athetosis did not require caspase activation, early mitochondrial disruption or generation of reactive oxygen species, but did require an intact actin cytoskeleton and was abolished by latrunculin B and mycalolide B. This work defines an authentic role for mouse Gzm A in granule-induced cell death by cytotoxic lymphocytes.
Assuntos
Apoptose/efeitos dos fármacos , Granzimas/metabolismo , Células Matadoras Naturais/imunologia , Perforina/metabolismo , Citoesqueleto de Actina , Animais , Apoptose/imunologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Granzimas/deficiência , Granzimas/genética , Células HeLa , Humanos , Células Matadoras Naturais/citologia , Toxinas Marinhas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Oxazóis/farmacologia , Tiazolidinas/farmacologia , Imagem com Lapso de TempoRESUMO
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.
Assuntos
Lesão Pulmonar Aguda/imunologia , Granzimas/imunologia , Células Matadoras Naturais/enzimologia , Pulmão/enzimologia , Pneumonia/imunologia , Síndrome do Desconforto Respiratório/imunologia , Linfócitos T Reguladores/enzimologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Lesão Pulmonar Aguda/mortalidade , Administração Intranasal , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Bleomicina , Estudos de Casos e Controles , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Expressão Gênica , Granzimas/deficiência , Granzimas/genética , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Pneumonia/induzido quimicamente , Pneumonia/enzimologia , Pneumonia/mortalidade , Síndrome do Desconforto Respiratório/enzimologia , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/patologia , Análise de Sobrevida , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
MS (multiple sclerosis) is the most prevalent autoimmune disease of the CNS (central nervous system) historically characterized as an inflammatory and demyelinating disease. More recently, extensive neuronal pathology has lead to its classification as a neurodegenerative disease as well. While the immune system initiates the autoimmune response it remains unclear how it orchestrates neuronal damage. In our previous studies, using in vitro cultured embryonic neurons, we demonstrated that MBP (myelin basic protein)-specific encephalitogenic CD4 T-cells induce early neuronal damage. In an extension of those studies, here we show that polarized CD4 Th1 and Th17 cells as wells as CD8 T-cells and NK (natural killer) cells induce microtubule destabilization within neurites in a contact-independent manner. Owing to the cytotoxic potential of these immune cells, we isolated the luminal components of lytic granules and determined that they were sufficient to drive microtubule destabilization. Since lytic granules contain cytolytic proteins, we determined that the induction of microtubule destabilization occurred prior to signs of apoptosis. Furthermore, we determined that microtubule destabilization was largely restricted to axons, sparing dendrites. This study demonstrated that lymphocytes with cytolytic activity have the capacity to directly drive MAD (microtubule axonal destabilization) in a bystander manner that is independent of neuronal death.
Assuntos
Axônios/fisiologia , Encefalomielite Autoimune Experimental/patologia , Linfócitos/metabolismo , Microtúbulos/metabolismo , Neurônios/fisiologia , Animais , Proteínas de Bactérias/genética , Linfócitos T CD4-Positivos , Morte Celular/imunologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Citocinas/farmacologia , Embrião de Mamíferos , Encefalomielite Autoimune Experimental/imunologia , Granzimas/deficiência , Marcação In Situ das Extremidades Cortadas , Células Matadoras Naturais , Proteínas Luminescentes/genética , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Modelos Biológicos , Proteína Básica da Mielina/genética , Neurônios/citologia , Perforina/deficiência , Células Th1 , Células Th17/metabolismoRESUMO
Allogeneic hematopoietic cell transplantation is an established treatment for hematologic and other malignancies. Donor-derived immune cells can identify and attack host tumor cells, producing a graft-versus-tumor (GVT) effect that is crucial to the treatment. Using multiple tumor models and diverse donor-host combinations, we have studied the role of granzyme B (GzmB) in GVT effect. We first confirmed previous findings that GzmB deficiency diminished the ability of a high dose of CD8(+) T cells to cause lethal graft-versus-host disease. However, when GVT studies were performed using a moderate cell dose that the hosts could tolerate, GzmB(-/-) CD8(+) T cells demonstrated a significantly enhanced GVT effect. GzmB-mediated, activation-induced cell death in wild-type CD8(+) T cells was found responsible for their reduced GVT activity. Conversely, GzmB(-/-) CD8(+) T cells exhibited enhanced expansion, skewed toward an effector or effector memory phenotype, and produced higher amounts of IFN-γ and Fas ligand that might contribute to GzmB-independent tumor control. These findings demonstrate for the first time, to our knowledge, that GzmB-mediated damage of CD8(+) T cells impairs the desired GVT effect. This study suggests that inhibiting donor-derived GzmB function may represent a promising strategy to improve GVT effect without exacerbating graft-versus-host disease.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Efeito Enxerto vs Tumor/fisiologia , Granzimas/fisiologia , Animais , Apoptose/imunologia , Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/imunologia , Divisão Celular , Linhagem Celular Tumoral/transplante , Proteína Ligante Fas/biossíntese , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Granzimas/deficiência , Granzimas/genética , Memória Imunológica , Interferon gama/biossíntese , Interferon gama/genética , Interferon gama/imunologia , Ativação Linfocitária , Linfoma/imunologia , Linfoma/patologia , Linfoma/cirurgia , Camundongos , Camundongos Endogâmicos , Quimera por Radiação , Carga TumoralRESUMO
Protective immunity against Salmonella infection is known to require CD4 Th1 cells and B cells, but the role of MHC class-I-restricted CD8 T cells is less clear. Previous studies have suggested that CD8 T cells participate in secondary, but not primary, bacterial clearance. However, these studies have used experimental models that are difficult to interpret and do not clearly isolate the role of MHC class-I-restricted CD8 T cells from other cell populations. Here, we examined the role of class-I-restricted T cells in protection against Salmonella infection using mice lacking all classical MHC class-Ia molecules, perforin, or granzyme B. Immunized K(b)D(b)-, perforin-, granzyme B-, or perforin/granzyme B-deficient mice were able to resolve secondary infection with virulent Salmonella, demonstrating that class-I-restricted CTLs are not required for acquired immunity. However, during primary infection with attenuated bacteria, bacterial clearance was delayed in each of these mouse strains when compared to wild-type mice. Taken together, these data demonstrate that CD8 T cells are not required for acquired immunity to Salmonella, but can play a protective role in resolving primary infection with attenuated bacteria.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Salmonelose Animal/imunologia , Salmonella typhi/imunologia , Animais , Carga Bacteriana , Citotoxicidade Imunológica/imunologia , Granzimas/deficiência , Granzimas/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Perforina/deficiência , Perforina/genética , Salmonella typhi/patogenicidadeRESUMO
CTLs and NK cells use the perforin/granzyme cytotoxic pathway to kill virally infected cells and tumors. Human regulatory T cells also express functional granzymes and perforin and can induce autologous target cell death in vitro. Perforin-deficient mice die of excessive immune responses after viral challenges, implicating a potential role for this pathway in immune regulation. To further investigate the role of granzyme B in immune regulation in response to viral infections, we characterized the immune response in wild-type, granzyme B-deficient, and perforin-deficient mice infected with Sendai virus. Interestingly, granzyme B-deficient mice, and to a lesser extent perforin-deficient mice, exhibited a significant increase in the number of Ag-specific CD8(+) T cells in the lungs and draining lymph nodes of virally infected animals. This increase was not the result of failure in viral clearance because viral titers in granzyme B-deficient mice were similar to wild-type mice and significantly less than perforin-deficient mice. Regulatory T cells from WT mice expressed high levels of granzyme B in response to infection, and depletion of regulatory T cells from these mice resulted in an increase in the number of Ag-specific CD8(+) T cells, similar to that observed in granzyme B-deficient mice. Furthermore, granzyme B-deficient regulatory T cells displayed defective suppression of CD8(+) T cell proliferation in vitro. Taken together, these results suggest a role for granzyme B in the regulatory T cell compartment in immune regulation to viral infections.