RESUMO
Background: Metabolism of glutamine by glutaminase 1 (GLS1) plays a key role in tumor cell proliferation via the generation of ATP and intermediates required for macromolecular synthesis. We hypothesized that glutamine metabolism also plays a role in proliferation of autosomal-dominant polycystic kidney disease (ADPKD) cells and that inhibiting GLS1 could slow cyst growth in animal models of ADPKD. Methods: Primary normal human kidney and ADPKD human cyst-lining epithelial cells were cultured in the presence or absence of two pharmacologic inhibitors of GLS1, bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) and CB-839, and the effect on proliferation, cyst growth in collagen and activation of downstream signaling pathways were assessed. We then determined if inhibiting GLS1 in vivo with CB-839 in the Aqp2-Cre; Pkd1fl/fl and Pkhd1-Cre; Pkd1fl/fl mouse models of ADPKD slowed cyst growth. Results: We found that an isoform of GLS1 (GLS1-GAC) is upregulated in cyst-lining epithelia in human ADPKD kidneys and in mouse models of ADPKD. Both BPTES and CB-839 blocked forskolin-induced cyst formation in vitro. Inhibiting GLS1 in vivo with CB-839 led to variable outcomes in two mouse models of ADPKD. CB-839 slowed cyst growth in Aqp2-Cre; Pkd1fl/fl mice, but not in Pkhd1-Cre; Pkd1fl/fl mice. While CB-839 inhibited mammalian target of rapamycin (mTOR) and MEK activation in Aqp2-Cre; Pkd1fl/fl, it did not in Pkhd1-Cre; Pkd1fl/fl mice. Conclusion: These findings provide support that alteration in glutamine metabolism may play a role in cyst growth. However, testing in other models of PKD and identification of the compensatory metabolic changes that bypass GLS1 inhibition will be critical to validate GLS1 as a drug target either alone or when combined with inhibitors of other metabolic pathways.
Assuntos
Proliferação de Células/efeitos dos fármacos , Glutaminase/metabolismo , Glutamina/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Animais , Aquaporina 2/fisiologia , Benzenoacetamidas/farmacologia , Células Cultivadas , Feminino , Glutaminase/antagonistas & inibidores , Humanos , Masculino , Camundongos , Camundongos Knockout , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Tiadiazóis/farmacologiaRESUMO
Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that plays a critical role in cell motility. Movement and retraction of podocyte foot processes, which accompany podocyte injury, suggest focal adhesion disassembly. To understand better the mechanisms by which podocyte foot process effacement leads to proteinuria and kidney failure, we studied the function of FAK in podocytes. In murine models, glomerular injury led to activation of podocyte FAK, followed by proteinuria and foot process effacement. Both podocyte-specific deletion of FAK and pharmacologic inactivation of FAK abrogated the proteinuria and foot process effacement induced by glomerular injury. In vitro, podocytes isolated from conditional FAK knockout mice demonstrated reduced spreading and migration; pharmacologic inactivation of FAK had similar effects on wild-type podocytes. In conclusion, FAK activation regulates podocyte foot process effacement, suggesting that pharmacologic inhibition of this signaling cascade may have therapeutic potential in the setting of glomerular injury.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Glomerulonefrite/prevenção & controle , Podócitos/enzimologia , Proteinúria/prevenção & controle , Actinas/metabolismo , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Proteína-Tirosina Quinases de Adesão Focal/genética , Deleção de Genes , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Podócitos/efeitos dos fármacos , Podócitos/patologia , Proteinúria/metabolismo , Proteinúria/patologia , Pirimidinas/farmacologiaRESUMO
Naive T cell circulation is restricted to secondary lymphoid organs. Effector and memory T cells, in contrast, acquire the ability to migrate to nonlymphoid tissues. In this study we examined whether nonlymphoid tissues contribute to the differentiation of effector T cells to memory cells and the long-term maintenance of memory T cells. We found that CD4, but not CD8, effector T cell differentiation to memory cells is impaired in adoptive hosts that lack secondary lymphoid organs. In contrast, established CD4 and CD8 memory T cells underwent basal homeostatic proliferation in the liver, lungs, and bone marrow, were maintained long-term, and functioned in the absence of secondary lymphoid organs. CD8 memory T cells found in nonlymphoid tissues expressed both central and effector memory phenotypes, whereas CD4 memory T cells displayed predominantly an effector memory phenotype. These findings indicate that secondary lymphoid organs are not necessary for the maintenance and function of memory T cell populations, whereas the optimal differentiation of CD4 effectors to memory T cells is dependent on these organs. The ability of memory T cells to persist and respond to foreign Ag independently of secondary lymphoid tissues supports the existence of nonlymphoid memory T cell pools that provide essential immune surveillance in the periphery.
Assuntos
Diferenciação Celular , Memória Imunológica/imunologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Células Cultivadas , Homeostase , CamundongosRESUMO
Immune responses are suppressed in immunologically privileged sites, which may provide a unique opportunity to prolong allograft survival. However, it is unknown whether testicular immune privilege promotes transplantation tolerance. Mechanisms underlying immune privilege are also not well understood. Here we found that islet transplantation in the testis, an immunologically privileged site, generates much less memory CD8(+) T cells but induces more Ag-specific CD4(+)CD25(+) regulatory T cells than in a conventional site. These CD4(+)CD25(+) cells exhibited the suppression of alloimmune responses in vivo and in vitro. Despite the immune regulation, intratesticular islet allografts all were rejected within 42 days after transplantation although they survived longer than renal subcapsular islet allografts. However, blocking CD40/CD40L costimulation induced the tolerance of intratesticular, but not renal subcapsular, islet allografts. Tolerance to intratesticular islet allografts spread to skin allografts in the non-privileged sites. Either transfer of memory CD8(+) T cells or deletion of CD25(+) T cells in vivo broke islet allograft tolerance. Thus, transplantation tolerance requires both costimulatory blockade, which suppresses acute allograft rejection, and a favorable balance between memory and regulatory T cells that could favorably prevent late allograft failure. These findings reveal novel mechanisms of immune privilege and provide direct evidence that testicular immune privilege fosters the induction of transplantation tolerance to allografts in both immunologically privileged and non-privileged sites.
Assuntos
Memória Imunológica , Transplante das Ilhotas Pancreáticas/imunologia , Linfócitos T Reguladores/imunologia , Testículo/imunologia , Tolerância ao Transplante/imunologia , Animais , Anticorpos Bloqueadores/farmacologia , Apoptose/imunologia , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Inibidores do Crescimento/farmacologia , Transplante das Ilhotas Pancreáticas/patologia , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Interleucina-2/biossíntese , Linfócitos T Reguladores/metabolismo , Transplante Heterotópico/imunologia , Transplante Heterotópico/patologiaRESUMO
Foreign Ags that enter immunologically privileged sites such as the eye, brain, and testis persist for an extended period of time, whereas the same Ags are rapidly eliminated at conventional sites. Immune privilege, therefore, provides unwanted refuge for pathogens and tumor cells but is beneficial for the survival of allogeneic grafts. In this study, we asked whether memory T cells can eliminate foreign Ags deposited at an immunologically privileged site by studying CD8 memory T cell-mediated rejection of pancreatic islet allografts placed either in the testis (a privileged organ) or under the kidney capsule (a nonprivileged site) of diabetic mice. We found that CD8 memory T cells reject intratesticular grafts at a significantly slower rate than the rejection of intrarenal grafts. Delayed graft rejection in the testis was not due to reduced homing or proliferation of memory T cells but due to their increased apoptosis at that site. Apoptosis was mediated by the combined actions of two TNFR family members that are up-regulated on activated memory T cells, Fas, and CD30. Therefore, memory T cells survey immunologically privileged tissues but are subject to the immunosuppressive mechanisms present at these sites.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Testículo/citologia , Testículo/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/transplante , Movimento Celular/genética , Movimento Celular/imunologia , Epitopos de Linfócito T/imunologia , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Transplante das Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/métodos , Transplante das Ilhotas Pancreáticas/patologia , Antígeno Ki-1/fisiologia , Rim/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Testículo/patologia , Transplante Heterotópico/imunologia , Transplante Heterotópico/métodos , Transplante Heterotópico/patologia , Receptor fas/fisiologiaRESUMO
The location of immune activation is controversial during acute allograft rejection and unknown in xenotransplantation. To determine where immune activation to a xenograft occurs, we examined whether splenectomized alymphoplastic mice that possess no secondary lymphoid organs can reject porcine skin xenografts. Our results show that these mice rejected their xenografts, in a T cell-dependent fashion, at the same tempo as wild-type recipients, demonstrating that xenograft rejection is not critically dependent on secondary lymphoid organs. Furthermore, we provide evidence that immune activation in the bone marrow did not take place during xenograft rejection. Importantly, immunity to xenoantigens was only induced after xenotransplantation and not by immunization with porcine spleen cells, as xenografted mutant mice developed an effector response, whereas mutant mice immunized by porcine spleen cells via i.p. injection failed to do so. Moreover, we provide evidence that antixenograft immunity occurred via direct and indirect Ag presentation, as recipient T cells could be stimulated by either donor spleen cells or recipient APCs. Thus, our data provide evidence that direct and indirect Ag presentation by a xenograft induces immunity in the absence of secondary lymphoid organs. These results have important implications for developing relevant xenotransplantation protocols.
Assuntos
Apresentação de Antígeno/imunologia , Tecido Linfoide/imunologia , Transplante Heterólogo/imunologia , Animais , Formação de Anticorpos/genética , Apresentação de Antígeno/genética , Antígenos Heterófilos/administração & dosagem , Antígenos Heterófilos/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Divisão Celular/genética , Divisão Celular/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Rejeição de Enxerto/prevenção & controle , Imunidade Inata/genética , Memória Imunológica/genética , Injeções Intraperitoneais , Interfase/genética , Interfase/imunologia , Células Matadoras Naturais/imunologia , Tecido Linfoide/anormalidades , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos SCID , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transplante de Pele/imunologia , Transplante de Pele/patologia , Baço/citologia , Baço/imunologia , Baço/transplante , Esplenectomia , Suínos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/transplante , Transplante Heterólogo/métodos , Transplante Heterólogo/patologiaRESUMO
Transplanted organs fail due to either acute or chronic rejection. The prevailing view is that the nature or magnitude of the recipient's immune response to donor Ags determines the type of rejection. In variance with this view, we show in this study that the status of the graft itself plays a dominant role in defining the type of rejection even in the face of an established alloimmune response. Using adoptive transfer mouse models in which the graft is exposed to a constant number of effector lymphocytes, we found that newly transplanted heart allografts are rejected acutely, while healed-in allografts undergo chronic rejection. Acute rejection of healed-in allografts was largely recapitulated by subjecting the grafts to ischemia-reperfusion injury similar to that present in newly transplanted organs. Ischemia-Reperfusion injury altered the outcome of rejection by enhancing the accumulation of effector T cells within the graft. The accumulation of effector T cells in the graft was dependent on the presence of both ischemia-reperfusion injury (inflammation) and alloantigens. These findings demonstrate that the graft plays a dominant role in shaping the outcome of rejection by controlling the trafficking of effector T cells.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Imunidade Celular , Linfócitos T , Doença Aguda , Transferência Adotiva , Animais , Moléculas de Adesão Celular/biossíntese , Movimento Celular , Quimiocinas/biossíntese , Doença Crônica , Rejeição de Enxerto/diagnóstico , Camundongos , Camundongos Endogâmicos , Traumatismo por Reperfusão , Fatores de Tempo , Transplante HomólogoRESUMO
CD4(+)CD25(+) regulatory T (Treg) cells suppress naive T cell responses, prevent autoimmunity, and delay allograft rejection. It is not known, however, whether Treg cells suppress allograft rejection mediated by memory T cells, as the latter mount faster and stronger immune responses than their naive counterparts. Here we show that antigen-induced, but not naive, Treg cells suppress allograft rejection mediated by memory CD8(+) T cells. Suppression was allospecific, as Treg cells induced by third-party antigens did not delay allograft rejection. In vivo and in vitro analyses revealed that the apoptosis of allospecific memory CD8(+) T cells is significantly increased in the presence of antigen-induced Treg cells, while their proliferation remains unaffected. Importantly, neither suppression of allograft rejection nor enhanced apoptosis of memory CD8(+) T cells was observed when Treg cells lacked CD30 or when CD30 ligand-CD30 interaction was blocked with anti-CD30 ligand Ab. This study therefore provides direct evidence that pathogenic memory T cells are amenable to suppression in an antigen-specific manner and identifies CD30 as a molecule that is critical for the regulation of memory T cell responses.