RESUMO
ABSTRACT: Neointimal hyperplasia causes the failure of coronary artery bypass grafting. Our previous studies have found that endothelial dysfunction is 1 candidate for triggering neointimal hyperplasia, but which factors are involved in this process is unclear. Glutathione S-transferase α4 (GSTA4) plays an important role in metabolizing 4-hydroxynonenal (4-HNE), a highly reactive lipid peroxidation product, which causes endothelial dysfunction or death. Here, we investigated the role of GSTA4 in neointima formation after arteriovenous grafts (AVGs) with or without high-fat diet (HFD). Compared with normal diet, HFD caused endothelial dysfunction and increased neointima formation, concomitantly accompanied by downregulated expression of GSTA4 at the mRNA and protein levels. In vitro, overexpression of GSTA4 attenuated 4-HNE-induced endothelial dysfunction and knockdown of GSTA4 aggravated endothelial dysfunction. Furthermore, silencing GSTA4 expression facilitated the activation of 4-HNE-induced endoplasmic reticulum stress and inhibition of endoplasmic reticulum stress pathway alleviated 4-HNE-induced endothelial dysfunction. In addition, compared with wild-type mice, mice with knockout of endothelial-specific GSTA4 (GSTA4 endothelial cell KO) exhibited exacerbated vascular endothelial dysfunction and increased neointima formation caused by HFD. Together, these results demonstrate the critical role of GSTA4 in protecting the function of endothelial cells and in alleviating hyperlipidemia-induced vascular neointimal hyperplasia in arteriovenous grafts.
Assuntos
Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Glutationa Transferase , Hiperlipidemias , Hiperplasia , Camundongos Endogâmicos C57BL , Neointima , Animais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glutationa Transferase/metabolismo , Glutationa Transferase/genética , Masculino , Hiperlipidemias/enzimologia , Derivação Arteriovenosa Cirúrgica/efeitos adversos , Camundongos Knockout , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Transdução de Sinais , Humanos , Aldeídos/metabolismo , Aldeídos/farmacologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Camundongos , Células Cultivadas , Dieta HiperlipídicaRESUMO
Neointima formation is the leading cause of arteriovenous fistula (AVF) failure. We have shown that CKD accelerates this process by transforming the vascular smooth muscle cells (SMCs) lining the AVF from a contractile to the synthetic phenotype. However, the underlying mechanisms affecting this transformation are not clear. Previous studies have shown that the α-class glutathione transferase isozymes have an important role in regulating 4-hydroxynonenal (4-HNE)-mediated proliferative signaling of cells. Here, using both the loss- and gain-of-function approaches, we investigated the role of glutathione S-transferase α4 (GSTA4) in modulating cellular 4-HNE levels for the transformation and proliferation of SMCs. Compared with non-CKD controls, mice with CKD had downregulated expression of GSTA4 at the mRNA and protein levels, with concomitant increase in 4-HNE in arteries and veins. This effect was associated with upregulated phosphorylation of MAPK signaling pathway proteins in proliferating SMCs. Overexpressing GSTA4 blocked 4-HNE-induced SMC proliferation. Additionally, inhibitors of MAPK signaling inhibited the 4-HNE-induced responses. Compared with wild-type mice, mice lacking GSTA4 exhibited increased CKD-induced neointima formation in AVF. Transient expression of an activated form of GSTA4, achieved using a combined Tet-On/Cre induction system in mice, lowered levels of 4-HNE and reduced the proliferation of SMCs. Together, these results demonstrate the critical role of GSTA4 in blocking CKD-induced neointima formation and AVF failure.
Assuntos
Aldeídos/metabolismo , Glutationa Transferase/genética , Miócitos de Músculo Liso/metabolismo , Neointima/genética , Neointima/patologia , Insuficiência Renal Crônica/fisiopatologia , Túnica Íntima/patologia , Animais , Artérias/metabolismo , Derivação Arteriovenosa Cirúrgica , Proliferação de Células , Regulação para Baixo , Expressão Gênica , Glutationa Transferase/metabolismo , Hiperplasia/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Fenótipo , Fosforilação/genética , RNA Mensageiro/metabolismo , Insuficiência Renal Crônica/terapia , Veias/metabolismoRESUMO
Regulatory T (Treg) cells play an important role in the maintenance of immune self-tolerance and homeostasis. We previously reported that neonatal CD4(+) T cells have an intrinsic 'default' mechanism to become Treg (neoTreg) cells in response to T-cell receptor (TCR) stimulation. However, the underlying mechanisms are unclear and the effects of neoTreg cells on regulating immune responses remain unknown. Due to their involvement in Foxp3 regulation, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b during the induction of neoTreg cells in the Foxp3(gfp) mice. The function of neoTreg cells was assessed in an acute allograft rejection model established in RAG2(-/-) mice with allograft cardiac transplantation and transferred with syngeneic CD4(+) effector T cells. Following ex vivo TCR stimulation, the DNMT activity was increased threefold in adult CD4(+) T cells, but not significantly increased in neonatal cells. However, adoptively transferred neoTreg cells significantly prolonged cardiac allograft survival (mean survival time 47 days, P < 0.001) and maintained Foxp3 expression similar to natural Treg cells. The neoTreg cells were hypomethylated at the conserved non-coding DNA sequence 2 locus of Foxp3 compared with adult Treg cells. The DNMT antagonist 5-aza-2'-deoxycytidine (5-Aza) induced increased Foxp3 expression in mature CD4(+) T cells. 5-Aza-inducible Treg cells combined with continuous 5-Aza treatment prolonged graft survival. These results indicate that the 'default' pathway of neoTreg cell differentiation is associated with reduced DNMT1 and DNMT3b response to TCR stimulus. The neoTreg cells may be a strategy to alleviate acute allograft rejection.
Assuntos
Metilação de DNA , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Transplante de Coração , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Regiões não Traduzidas , Transferência Adotiva , Animais , Animais Recém-Nascidos , Azacitidina/análogos & derivados , Azacitidina/farmacologia , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Decitabina , Fatores de Transcrição Forkhead/genética , Sobrevivência de Enxerto/efeitos dos fármacos , Imunofenotipagem , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Transplante Homólogo , DNA Metiltransferase 3BRESUMO
OBJECTIVE: Endothelial dysfunction is one candidate for triggering neointima formation after arteriovenous grafts (AVGs), but the factors mediating this process are unclear. The purpose of this study was to investigate the role of endoplasmic reticulum stress (ERS)-induced endothelial dysfunction in neointima formation following AVGs in high-fat diet (HFD) mice. METHODS: CCAAT-enhancer-binding protein-homologous protein (CHOP) knockout (KO) mice were created. Mice were fed with HFD to produce HFD model. AVGs model were applied in the groups of WT ND, WT HFD, and CHOP KO HFD. Human umbilical vein endothelial cells (HUVECs) were cultured with oxidized low density lipoprotein (ox-LDL) (40 mg/L) for the indicated time lengths (0, 6, 12, 24 h). ERS inhibitor tauroursodeoxycholic acid (TUDCA) was used to block ERS. Immunohistochemical staining was used to observe the changes of ICAM1. Changes of ERS were detected by real-time RT-PCR. Protein expression levels and ERS activation were detected by Western blotting. Endothellial cell function was determined by endothelial permeability assay and transendothelial migration assay. RESULTS: HFD increased neointima formation in AVGs associated with endothelial dysfunction. At the same time, ERS was increased in endothelial cells (ECs) after AVGs in mice consuming the HFD. In vitro, ox-LDL was found to stimulate ERS, increase the permeability of the EC monolayer, and cause endothelial dysfunction. Blocking ERS with TUDCA or CHOP siRNA reversed the EC dysfunction caused by ox-LDL. In vivo, knockout of CHOP (CHOP KO) protected the function of ECs and decreased neointima formation after AVGs in HFD mice. CONCLUSION: Inhibiting ERS in ECs could improve the function of AVGs.
Assuntos
Dieta Hiperlipídica , Neointima , Humanos , Animais , Camundongos , Neointima/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Estresse do Retículo EndoplasmáticoRESUMO
Eicosapentaenoic acid (EPA) is one of n-3 polyunsaturated fatty acids that possesses a wide array of anti-inflammatory effects but its effects, on transplantation in general and on Tregs and IL-17(+) T cells in particular, are not well studied. We treated recipient mice of heart transplantation with EPA and examined the effect of EPA on the ratio of Tregs/IL-17(+) T cells in an allogeneic heart transplant model. The hearts from BALB/c (H-2d) mice were transplanted into C57BL/6 (H-2b) mice, and the recipients were administered EPA (500 mg/kg/d, 250 mg/kg/d, or 100 mg/kg/d) from d 1 to 3 post-transplant. The survival of cardiac allografts in mice treated with EPA was significantly protracted. Further examination of donor hearts in EPA-treated group demonstrated that infiltrating Foxp3(+) T cells were increased, IL-17(+) T cells were decreased, and expression of PPARγ was up-regulated. In mixed lymphocytes reaction (MLR), incubation with EPA significantly inhibited the proliferation of IL-17(+) T cells and promoted the proliferation of Tregs, while PPARγ antagonists GW9662 could reverse the results. Our study demonstrated that EPA can effectively protect cardiac allografts and disrupt the balance between Tregs and IL-17(+) T cells in a murine model. This effect is partially mediated by PPARγ nuclear receptor activation.
Assuntos
Ácido Eicosapentaenoico/farmacologia , Ácido Eicosapentaenoico/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Transplante de Coração/fisiologia , Interleucina-17/metabolismo , PPAR gama/efeitos dos fármacos , Linfócitos T Reguladores/patologia , Linfócitos T/patologia , Anilidas/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Rejeição de Enxerto/fisiopatologia , Sobrevivência de Enxerto/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais , PPAR gama/antagonistas & inibidores , PPAR gama/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Transplante HomólogoRESUMO
Previously, studies suggest that CD4(+) effector T-cell subsets participate in allograft rejection. However, the dynamic changes and relative roles of these CD4(+) effector T-cell subsets, especially Th17 cells, have not been systemically examined in patients with acute rejection after cardiac transplantation. In this study, we have studied and compared these CD4(+) T-cell subsets in peripheral blood and endomyocardial biopsies (EMB) in patients with stable-graft and acute cellular rejection. We observed that the gene expressions including T-bet, IFN-γ, RORγt, IL-17, IL-23, and FoxP3, the functional marker of Th1, Th17, and FoxP3(+) CD4(+) T cells, were elevated in EMB samples from patients with acute graft rejection. Accordingly, the percentages of circulating Th1, Th17, and FoxP3(+) CD4(+) T cells were also significantly increased. The data suggest that Th1, Th17, and FoxP3(+) CD4(+) T cells are associated with acute graft rejection in patients with cardiac transplantation.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Interleucina-17/imunologia , Miocárdio/patologia , Células Th1/imunologia , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Técnicas Imunoenzimáticas , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-23/imunologia , Interleucina-23/metabolismo , Miocárdio/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th1/metabolismoRESUMO
BACKGROUND: The mammalian target of rapamycin (mTOR) inhibitors are valuable immunosuppressants in clinical transplantation; however, the mTOR regulation of allogeneic T-cell responses is not fully understood yet. Therefore, the objective of this study is to investigate the effects of T-cell-specific mTOR deletion on the allogeneic T-cell responses and heart transplant survival. METHODS: BALB/c heart allografts, with or without BALB/c skin sensitization, were transplanted in the wild-type C57BL/6, Mtorfl/flCd4-Cre, Stat3fl/flCd4-Cre, and Mtorfl/flStat3fl/flCd4-Cre mice. Graft survival and histology, as well as T-cell frequencies and phenotypes, were evaluated after transplantation. RESULTS: In the absence of donor skin sensitization, long-term heart allograft survival was achieved in the Mtorfl/flCd4-Cre recipients, which was associated with significantly decreased frequencies of CD62L-CD44+ effector T cells and BCL-6+CXCR5+ T follicular helper (Tfh) cells in the periphery. Long-term heart allograft survival was also achieved in the donor skin-sensitized Mtorfl/flStat3fl/flCd4-Cre mice, whereas the heart allograft survival was prolonged in the donor skin-sensitized Mtorfl/flCd4-Cre and Stat3fl/flCd4-Cre mice. CONCLUSIONS: mTOR is required for Tfh cell response in murine heart transplantation. T-cell-specific deletion of both mTOR and Stat3 abrogates the memory response to heart transplants. These findings help us to better understand the molecular mechanisms underlying the T cell immunity to transplanted organs.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Coração , Imunidade Celular , Linfócitos T Auxiliares-Indutores/imunologia , Serina-Treonina Quinases TOR/metabolismo , Aloenxertos , Animais , Modelos Animais de Doenças , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The regulation of hypoxic response elements on the expression of vascular endothelial growth factor (VEGF) gene transfected to primary cultured rat skeletal myoblasts under hypoxic environment was investigated. pEGFP-C3-9HRE-CMV-VEGF vector was constructed with molecular biology technique and transfected to primary cultured rat skeletal myoblasts by lipofectamine in vitro. Gene expression of transfected myoblasts was detected by RT-PCR, Western blot and fluorescence microscope under different oxygen concentrations and different hypoxia time. The results showed that in hypoxia group, the VEGF gene bands were seen and with the decrease of oxygen concentrations and prolongation of hypoxia time, the expression of VEGF mRNA was obviously increased. Under hypoxic environment, the expression of VEGF protein in the transfected myoblasts was significantly increased. EGFP was expressed only under hypoxic environment but not under normoxic environment. It was concluded that hypoxia promoter could be constructed with HRE and regulate the expression of VEGF gene under hypoxic and normoxic environment, which could enhance the reliability of gene therapy.
Assuntos
Hipóxia/genética , Mioblastos Esqueléticos/metabolismo , Elementos de Resposta/genética , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Células Cultivadas , Mioblastos Esqueléticos/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Pancreatic cancer is among the most aggressive human cancers, and is resistant to regular chemotherapy and radiotherapy. The AT-rich interactive domain containing protein 1A (ARID1A) gene, a crucial chromatin remodeling gene, mutates frequently in a broad spectrum of cancers, including pancreatic cancer. Recent evidence suggests that ARID1A acts as tumor suppressor and plays an important role in DNA damage repair (DDR). However, the effect of ARID1A on the radiosensitivity of pancreatic cancer remains unclear. Herein, we investigated the involvement of ARID1A depletion in the radioresistance of pancreatic cancer cells, and explored the underlying mechanisms. The results reveal that knockdown of ARID1A enhances the radioresistance of pancreatic cancer cells through suppressing apoptosis, impairing G2-M checkpoint arrest, strengthening DDR, and accompanying activation of PI3K/AKT signaling pathway. Moreover, upon inhibition of PI3K/AKT pathway by PI3K-inhibitor LY294002 or AKT-inhibitor mk2206, the radiosensitivity of ARID1A-deficient pancreatic cancer cells is improved in vitro via increased apoptosis and weakened DDR. Taken together, these data suggest that loss of ARID1A expression enhances radioresistance of pancreatic cancer through activation of PI3K/AKT pathway, which maybe a promising target for radiosensitization of ARID1A-deficient pancreatic cancer.
RESUMO
Insulin-producing ß cells in patients with type 1 diabetes (T1D) are destroyed by T lymphocytes. We investigated whether targeting the T-cell receptor (TCR) with a monoclonal antibody (mAb) abrogates T-cell response against residual and newly formed islets in overtly diabetic nonobese diabetic (NOD) mice. NOD mice with blood glucose levels of 250 to 350 mg/dL or 350 to 450 mg/dL were considered as new-onset or established overt diabetes, respectively. These diabetic NOD mice were transiently treated with an anti-TCR ß chain (TCRß) mAb, H57-597, for 5 days. Two weeks later, some NOD mice with established overt diabetes further received hepatic gene therapy using the islet-lineage determining gene Neurogenin3 (Ngn3), in combination with the islet growth factor gene betacellulin (Btc). We found that anti-TCRß mAb (50 µg/d) reversed >80% new-onset diabetes in NOD mice for >14 weeks by reducing the number of effector T cells in the pancreas. However, anti-TCRß mAb therapy alone reversed only â¼20% established overt diabetes in these mice. Among those overtly diabetic NOD mice whose diabetes was resistant to anti-TCRß mAb treatment, â¼60% no longer had diabetes when they also received Ngn3-Btc hepatic gene transfer 2 weeks after initial anti-TCRß mAb treatment. This combination of Ngn3-Btc gene therapy and anti-TCRß mAb treatment induced the sustained formation of periportal insulin-producing cells in the liver of overtly diabetic mice. Therefore, directly targeting TCRß with a mAb potently reverses new-onset T1D in NOD mice and protects residual and newly formed gene therapy-induced hepatic neo-islets from T-cellâmediated destruction in mice with established overt diabetes.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diabetes Mellitus Tipo 1/terapia , Terapia Genética/métodos , Proteínas do Tecido Nervoso/genética , Receptores de Antígenos de Linfócitos T alfa-beta/antagonistas & inibidores , Animais , Betacelulina/genética , Terapia Combinada , Diabetes Mellitus Tipo 1/imunologia , Feminino , Imunoterapia/métodos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/imunologia , Fígado/citologia , Camundongos , Camundongos Endogâmicos NOD , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologiaRESUMO
BACKGROUND: Myocardial infarction results in tissue necrosis, leading to cell loss and ultimately to cardiac failure. Implantation of skeletal muscle satellite cells into the scar area may compensate for the cell loss and provides a new strategy for infarct therapy. Vascular endothelial growth factor (VEGF) is a promising reagent for inducing myocardial angiogenesis. Skeletal myoblast transplantation has been shown to improve cardiac function in chronic heart failure models by regenerating muscle. We hypothesized that VEGF expression and vascular regeneration increased in infarcted myocardium by skeletal muscle satellite cells, which can promote vascular producing and improve survival environment in infarcted myocardium. METHODS: The skeletal muscle satellite cells were implanted into the infarcted myocardium in a model through ligated left anterior artery in Louis Inbrad Strain rat. Specimens were got for identifying the expression of VEGF and the density of vascular by immunochemical method at two weeks after implantation. RESULTS: The proliferation and differentiation of the skeletal muscle satellite cell was very well. The expression of VEGF was higher in the implanted group (146.83 +/- 2.49) than that in the control group (134.26 +/- 6.84) (P < 0.05). The vascular density in the implanted group (13.00 +/- 1.51) was also higher than that in the control (10.68 +/- 1.79) (P < 0.05). CONCLUSION: The implanted satellite cell could excrete growth factor that would induce angiogenesis and improve cell survival environment in infarcted myocardium.
Assuntos
Infarto do Miocárdio/terapia , Células Satélites de Músculo Esquelético/transplante , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Proliferação de Células , Células Cultivadas , Infarto do Miocárdio/fisiopatologia , Neovascularização Fisiológica , RNA Mensageiro/análise , Ratos , Regeneração , Células Satélites de Músculo Esquelético/citologiaRESUMO
Memory T cells resist co-stimulatory blockade and present a unique therapeutic challenge in transplantation and autoimmune diseases. Herein, we determined whether memory T cells express less "tolerogenic" genes than naïve T cells to reinforce a proliferative response under the deprivation of co-stimulatory signals. The expression of â¼40 tolerogenic genes in memory and naïve CD4(+) T cells was thus assessed during an in vitro TCR stimulation without co-stimulation. Briefly, upon TCR stimulation with an anti-CD3 mAb alone, memory CD4(+) T cells exhibited more proliferation than naïve CD4(+) T cells. To our surprise, at 24h upon anti-CD3 mAb stimulation, memory CD4(+) T cells expressed more than a 5-fold higher level of the transcription factor Egr2 and a 20-fold higher level of the transmembrane E3 ubiquitin ligase GRAIL than those in naïve T cells. Hence, the high-level expression of tolerogenic genes, Egr2 and GRAIL, in memory CD4(+) T cells does not prevent cell proliferation. Importantly, anti-CD3 mAb-stimulated memory CD4(+) T cells expressed high protein/gene levels of phosphorylated STAT5, Nedd4, Bcl-2, and Bcl-XL. Therefore, co-stimulation-independent proliferation of memory CD4(+) T cells may be due to elevated expression of molecules that support cell proliferation and survival, but not lack of tolerogenic molecules.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Tolerância Imunológica/genética , Memória Imunológica/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais/farmacologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
A breakdown in self-tolerance underlies autoimmune destruction of ß-cells and type 1 diabetes. A cure by restoring ß-cell mass is limited by the availability of transplantable ß-cells and the need for chronic immunosuppression. Evidence indicates that inhibiting costimulation through the PD-1/PD-L1 pathway is central to immune tolerance. We therefore tested whether induction of islet neogenesis in the liver, protected by PD-L1-driven tolerance, reverses diabetes in NOD mice. We demonstrated a robust induction of neo-islets in the liver of diabetic NOD mice by gene transfer of Neurogenin3, the islet-defining factor, along with betacellulin, an islet growth factor. These neo-islets expressed all the major pancreatic hormones and transcription factors. However, an enduring restoration of glucose-stimulated insulin secretion and euglycemia occurs only when tolerance is also induced by the targeted overexpression of PD-L1 in the neo-islets, which results in inhibition of proliferation and increased apoptosis of infiltrating CD4(+) T cells. Further analysis revealed an inhibition of cytokine production from lymphocytes isolated from the liver but not from the spleen of treated mice, indicating that treatment did not result in generalized immunosuppression. This treatment strategy leads to persistence of functional neo-islets that resist autoimmune destruction and consequently an enduring reversal of diabetes in NOD mice.
Assuntos
Antígeno B7-H1/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Ilhotas Pancreáticas/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Apoptose , Antígeno B7-H1/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linfócitos T CD4-Positivos/fisiologia , Proliferação de Células , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Terapia de Imunossupressão , Ilhotas Pancreáticas/citologia , Camundongos , Camundongos Endogâmicos NOD , Proteínas do Tecido Nervoso/genética , Baço/citologiaRESUMO
The immune system provides defenses against invading pathogens while maintaining immune tolerance to self-antigens. This immune homeostasis is harmonized by the direct interactions between immune cells and the cytokine environment in which immune cells develop and function. Herein, we discuss three non-redundant paradigms by which cytokines maintain or break immune tolerance. We firstly describe how anti-inflammatory cytokines exert direct inhibitory effects on immune cells to enforce immune tolerance, followed by discussing other cytokines that maintain immune tolerance through inducing CD4(+)Foxp3(+) regulatory T cells (Tregs), which negatively control immune cells. Interleukin (IL)-2 is the most potent cytokine in promoting the development and survival of Tregs, thereby mediating immune tolerance. IL-35 is mainly produced by Tregs, but its biology function remains to be defined. Finally, we discuss the actions of proinflammatory cytokines that breach immune tolerance and induce autoimmunity, which include IL-7, IL-12, IL-21, and IL-23. Recent genetic studies have revealed the role of these cytokines (or their cognate receptors) in susceptibility to autoimmune diseases. Taken together, we highlight in this review the cytokine regulation of immune tolerance, which will help in further understanding of human diseases that are caused by dysregulated immune system.
RESUMO
Optimal T cell activation and expansion require binding of the common gamma-chain (γc) cytokine Interleukin-2 (IL-2) to its cognate receptor that in turn engages a γc/Janus tyrosine kinase (Jak)3 signaling pathway. Because of its restricted expression by antigen-activated T cells and its obligatory role in promoting their survival and proliferation, IL-2 has been considered as a selective therapeutic target for preventing T cell mediated diseases. However, in order to further explore IL-2 targeted therapy, it is critical to precisely understand its role during early events of T cell activation. In this study, we delineate the role of IL-2 and other γc cytokines in promoting the survival of CD4 and CD8 T cells during early phases of priming. Under IL-2 inhibitory conditions (by neutralizing anti-IL-2 mAbs), the survival of activated CD8⺠T cells was reduced, whereas CD4⺠T cells remained much more resistant. These results correlated with reduced Bcl-2 expression, and mitochondrial membrane potential in CD8⺠T cells in comparison to CD4⺠T cells. However, using transwell co-culture assays we have found that CD4⺠T cells could rescue the survival of CD8⺠T cells even under IL-2 deprived conditions via secretion of soluble factors. A cytokine screen performed on CD8⺠T cells cultured alone revealed that IL-21, another γc cytokine, was capable of rescuing their survival under IL-2 deprivation. Indeed, blocking the IL-21 signaling pathway along with IL-2 neutralization resulted in significantly reduced survival of both CD4⺠and CD8⺠T cells. Taken together, we have shown that under IL-2 deprivation conditions, IL-21 may act as the major survival factor promoting T cell immune responses. Thus, investigation of IL-2 targeted therapies may need to be revisited to consider blockade of the IL-21 signaling pathways as an adjunct to provide more effective control of T cell immune responses.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Apresentação Cruzada/efeitos dos fármacos , Interleucina-2/farmacologia , Interleucinas/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
BACKGROUND: Regulated on activation normal T-cell expressed and secreted (RANTES)-chemokine ligand 5 plays a key role in mediating heart transplant rejection. Suppression of RANTES-mediated signals can reduce leukocyte recruitment and mitigate transplant rejection severity. The present study describes the construction of an adenovirus overexpression vector encoding a natural S24F RANTES variant as a means of reducing leukocyte recruitment, resulting in the prevention of allograft rejection. METHODS: The in vitro transendothelial chemotaxis assay was used to compare RANTES-induced transmigration of peripheral blood mononuclear cells across human umbilical vein endothelial cells cultured on the upper Transwell chamber. Intracoronary delivery of Ad-S24F, Ad-Null, or phosphate-buffered saline was performed in BALB/c donor hearts that were transplanted into the abdominal cavity of C57BL/6 recipients as a measure of allograft survival. Intragraft inflammatory cell infiltrates and associated proinflammatory cytokine expression profiles were detected by immunohistochemistry and quantitative real-time polymerase chain reaction on day 6 after transplantation, respectively. RESULTS: Regulated on activation normal T-cell expressed and secreted-induced peripheral blood mononuclear cell transendothelial chemotaxis is inhibited by S24F (Ad-S24F, 9.2%±0.02%; Ad-Null, 17.7%±0.02%; medium control, 15.1%±0.01%; P<0.05). Cardiac allograft survival was prolonged after delivery of 1×10 plaque-forming units of Ad-S24F (13.00±0.33 days compared with 9.38±0.60 and 9.00±0.38 days after Ad-Null or phosphate-buffered saline treatment, respectively, P<0.05). S24F gene transfer reduced the number of intragraft CD8 T lymphocytes, monocyte-macrophages, and T-cell receptor αß cell infiltrates (P<0.05) and decreased transcripts for RANTES and interferon-γ (P<0.05). CONCLUSION: S24F is an important component of the chemokine network involved in regulating the biologic activity of RANTES, and its expression can be used in the prevention and treatment of cardiac allograft rejection.
Assuntos
Quimiocina CCL5/metabolismo , Terapia Genética/métodos , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Transplante de Coração/efeitos adversos , Linfócitos T/metabolismo , Adenoviridae/genética , Animais , Células Cultivadas , Quimiocina CCL5/genética , Quimiotaxia , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Fatores de Tempo , Migração Transendotelial e Transepitelial , TransfecçãoRESUMO
BACKGROUND: Clinical application of islet transplantation to treat type 1 diabetes has been limited by islet allograft destruction by both allogeneic and autoimmune diabetogenic T-cell responses. The current study aims at determining whether an anti-T-cell receptor (TCR) monoclonal antibody (mAb) has potential as a novel and potent induction immunotherapy for islet transplantation. METHODS: We have investigated the therapeutic efficacy and mechanisms of action of anti-TCR therapy in four different murine models, which comprise either allo- or autoimmune responses alone or both together. RESULTS: T-cell response to islet allografts was potently abrogated by a brief treatment with an anti-TCRß mAb (clone H57-597), resulting in long-term survival of BALB/c islet allografts in streptozotocin-induced diabetic B6 mice. Moreover, transient anti-TCR treatment permanently prevented BALB/c skin allograft rejection on Rag1 B6 recipients that were reconstituted with Foxp3 cell-depleted B6 splenocytes, but did not impair the reconstituted cells' ability to reject the later transplanted C3H skin allografts (transplanted at 120 days after BALB/c skin grafting). Transient anti-TCR treatment was also able to completely prevent diabetes onset in NOD.SCID.γc mice that were transferred with lymphocytes from diabetic NOD mice. Next, transient anti-TCR treatment significantly prolonged the survival of transplanted BALB/c islets in overtly diabetic NOD mice, which comprise both allogeneic and autoimmune diabetogenic T-cell responses to the transplanted islets. CONCLUSIONS: Overall, anti-TCR mAb induced peripheral tolerance to specific alloantigens even in the absence of Foxp3-expressing natural regulatory T cells. These findings reveal the potential for using TCR-targeting mAbs as induction immunotherapy for islet transplantation.
Assuntos
Anticorpos Monoclonais/farmacologia , Diabetes Mellitus Experimental/cirurgia , Diabetes Mellitus Tipo 1/cirurgia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante das Ilhotas Pancreáticas , Isoantígenos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Tolerância ao Transplante/efeitos dos fármacos , Transferência Adotiva , Aloenxertos , Animais , Células Cultivadas , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Fatores de Transcrição Forkhead/imunologia , Rejeição de Enxerto/sangue , Rejeição de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/efeitos adversos , Isoantígenos/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Transplante de Pele/efeitos adversos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Fatores de Tempo , Transplante HomólogoRESUMO
BACKGROUND: In a previous study, activation of the peroxisome proliferator-activated receptor γ (PPARγ) inhibited chronic cardiac rejection. However, because of the complexity of chronic rejection and the fact that PPARγ is widely expressed in immune cells, the mechanism of the PPARγ-induced protective effect was unclear. MATERIALS AND METHODS: A chronic rejection model was established using B6.C-H-2bm12KhEg (H-2bm12) mice as donors, and MHC II-mismatched T-cell-specific PPARγ knockout mice or wild type (WT) littermates as recipients. The allograft lesion was assessed by histology and immunohistochemistry. T cells infiltrates in the allograft were isolated, and cytokines and subpopulations were detected using cytokine arrays and flow cytometry. Transcription levels in the allograft were measured by RT-PCR. In vitro, the T cell subset differentiation was investigated after culture in various polarizing conditions. PPARγ-deficient regulatory T cells (Treg) were cocultured with monocytes to test their ability to induce alternatively activated macrophages (AAM). RESULTS: T cell-specific PPARγ knockout recipients displayed reduced cardiac allograft survival and an increased degree of pathology compared with WT littermates. T cell-specific PPARγ knockout resulted in more CD4+ T cells infiltrating into the allograft and altered the Th1/Th2 and Th17/Treg ratios. The polarization of AAM was also reduced by PPARγ deficiency in T cells through the action of Th2 and Treg. PPARγ-deficient T cells eliminated the pioglitazone-induced polarization of AAM and reduced allograft survival. CONCLUSIONS: PPARγ-deficient T cells influenced the T cell subset and AAM polarization in chronic allograft rejection. The mechanism of PPARγ activation in transplantation tolerance could yield a novel treatment without side effects.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , PPAR gama/genética , PPAR gama/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Transplante de Coração , Ativação de Macrófagos , Camundongos , Monócitos/citologiaRESUMO
Type 1 diabetes (T1D) is an autoimmune disease for which there is no cure. The pancreatic beta cells are the source of insulin that keeps blood glucose normal. When susceptible individuals develop T1D, their beta cells are destroyed by autoimmune T lymphocytes and no longer produce insulin. T1D patients therefore depend on daily insulin injections for survival. Gene therapy in T1D aims at the induction of new islets to replace those that have been destroyed by autoimmunity. A major goal of T1D research is to restore functional beta cell mass while eliminating diabetogenic T cells in the hope of achieving insulin independence. Multiple therapeutic strategies for the generation of new beta cells have been under intense investigations. However, newly formed beta cells would be immediately destroyed by diabetogenic T cells. Therefore, successful islet induction therapy must be supported by potent immunotherapy that will protect the newly formed beta cells. Herein, we will summarize the current information on immunotherapies that aim at modifying T cell response to beta cells. We will first outline the immune mechanisms that underlie T1D development and progression and review the scientific background and rationale for specific modes of immunotherapy. Numerous clinical trials using antigen-specific strategies and immune-modifying drugs have been published, though most have proved too toxic or have failed to provide long-term beta cell protection. To develop an effective immunotherapy, there must be a continued effort on defining the molecular basis that underlies T cell response to pancreatic islet antigens in T1D.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Imunoterapia , Terapia Genética , Humanos , Terapia de Imunossupressão , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Linfócitos T/imunologiaRESUMO
T cells play a major role in allograft rejection, which occurs after T cell activation by the engagement of several functional molecules to form an immune synapse with alloantigen presenting cells. In this study, the immune synapse was targeted using mAbs directed to the TCR beta-chain (TCRß) and lymphocyte function-associated antigen-1 (LFA1) to induce long-term allograft survival. Evaluation of antigen-specific T cell responses was performed by adoptively transferring CFSE labeled transgenic OT-II cells into wild-type mice and providing OVA peptide by intravenous injection. Graft survival studies were performed in mice by transplanting BALB/c ear skins onto the flanks of C57BL/6 recipients. The anti-TCRß plus anti-LFA1 mAb combination (but not either mAb alone) abrogated antigen-specific T cell responses invitro and invivo. Transient combination therapy with these agents resulted in significantly prolonged skin allograft survival in mice (51±10 days; p<0.01) when compared to treatment with either anti-TCRß mAb (24±5 days) or anti-LFA1 mAb (19±3 days) alone or no treatment (10±1 days). When lymphoid tissues from these mice were analyzed at different times post-transplant, only those receiving the combination of anti-TCRß and anti-LFA1 mAbs demonstrated long-lasting reductions in total T cell numbers, cellular and humoral anti-donor responses, and expression of CD3 on the surface of T cells. These results demonstrate that transient anti-TCRß and anti-LFA1 mAb combination therapy abrogates antigen-reactive T cell responses with long-lasting effects that significantly prolong allograft survival.