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1.
Nat Immunol ; 20(10): 1299-1310, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31534238

RESUMO

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

2.
Nat Commun ; 10(1): 3545, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391467

RESUMO

Tens of millions suffer from insulin deficiency (ID); a defect leading to severe metabolic imbalance and death. The only means for management of ID is insulin therapy; yet, this approach is sub-optimal and causes life-threatening hypoglycemia. Hence, ID represents a great medical and societal challenge. Here we report that S100A9, also known as Calgranulin B or Myeloid-Related Protein 14 (MRP14), is a leptin-induced circulating cue exerting beneficial anti-diabetic action. In murine models of ID, enhanced expression of S100A9 alone (i.e. without administered insulin and/or leptin) slightly improves hyperglycemia, and normalizes key metabolic defects (e.g. hyperketonemia, hypertriglyceridemia, and increased hepatic fatty acid oxidation; FAO), and extends lifespan by at least a factor of two. Mechanistically, we report that Toll-Like Receptor 4 (TLR4) is required, at least in part, for the metabolic-improving and pro-survival effects of S100A9. Thus, our data identify the S100A9/TLR4 axis as a putative target for ID care.


Assuntos
Calgranulina B/metabolismo , Diabetes Mellitus Experimental/metabolismo , Hiperglicemia/metabolismo , Longevidade/fisiologia , Receptor 4 Toll-Like/metabolismo , Animais , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/etiologia , Toxina Diftérica/toxicidade , Ácidos Graxos/metabolismo , Humanos , Hiperglicemia/sangue , Hiperglicemia/etiologia , Insulina/deficiência , Leptina/administração & dosagem , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Oxirredução , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estreptozocina/toxicidade , Receptor 4 Toll-Like/genética
3.
Diabetologia ; 60(4): 679-689, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28062921

RESUMO

AIMS/HYPOTHESIS: Administration of anti-CD40 ligand (CD40L) antibodies has been reported to allow long-term islet allograft survival in non-human primates without the need for exogenous immunosuppression. However, the use of anti-CD40L antibodies was associated with thromboembolic complications. Targeting downstream intracellular components shared between CD40 and other TNF family co-stimulatory molecules could bypass these complications. TNF receptor associated factor 2 (TRAF2) integrates multiple TNF receptor family signalling pathways that are critical for T cell activation and may be a central node of alloimmune responses. METHODS: T cell-specific Traf2-deficient mice (Traf2TKO) were generated to define the role of TRAF2 in CD4+ T cell effector responses that mediate islet allograft rejection in vivo. In vitro allograft responses were tested using mixed lymphocyte reactions and analysis of IFN-γ and granzyme B effector molecule expression. T cell function was assessed using anti-CD3/CD28-mediated proliferation and T cell polarisation studies. RESULTS: Traf2TKO mice exhibited permanent survival of full MHC-mismatched pancreatic islet allografts without exogenous immunosuppression. Traf2TKO CD4+ T cells exhibited reduced proliferation, activation and acquisition of effector function following T cell receptor stimulation; however, both Traf2TKO CD4+ and CD8+ T cells exhibited impaired alloantigen-mediated proliferation and acquisition of effector function. In polarisation studies, Traf2TKO CD4+ T cells preferentially converted to a T helper (Th)2 phenotype, but exhibited impaired Th17 differentiation. Without TRAF2, thymocytes exhibited dysregulated TNF-mediated induction of c-Jun N-terminal kinase (JNK) and canonical NFκB pathways. Critically, targeting TRAF2 in T cells did not impair the acute phase of CD8-dependent viral immunity. These data highlight a specific requirement for a TRAF2-NFκB and TRAF2-JNK signalling cascade in T cell activation and effector function in rejecting islet allografts. CONCLUSION/INTERPRETATION: Targeting TRAF2 may be useful as a therapeutic approach for immunosuppression-free islet allograft survival that avoids the thromboembolic complications associated with the use of anti-CD40L antibodies.


Assuntos
Imunossupressão , Transplante das Ilhotas Pancreáticas/imunologia , Fator 2 Associado a Receptor de TNF/metabolismo , Animais , Western Blotting , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/genética , Proliferação de Células/fisiologia , Feminino , Citometria de Fluxo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Associado a Receptor de TNF/genética , Transplante Homólogo
4.
J Exp Med ; 212(8): 1239-54, 2015 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-26122662

RESUMO

The nuclear factor κB (NF-κB) pathway is a master regulator of inflammatory processes and is implicated in insulin resistance and pancreatic ß cell dysfunction in the metabolic syndrome. Whereas canonical NF-κB signaling is well studied, there is little information on the divergent noncanonical NF-κB pathway in the context of pancreatic islet dysfunction. Here, we demonstrate that pharmacological activation of the noncanonical NF-κB-inducing kinase (NIK) disrupts glucose homeostasis in zebrafish in vivo. We identify NIK as a critical negative regulator of ß cell function, as pharmacological NIK activation results in impaired glucose-stimulated insulin secretion in mouse and human islets. NIK levels are elevated in pancreatic islets isolated from diet-induced obese (DIO) mice, which exhibit increased processing of noncanonical NF-κB components p100 to p52, and accumulation of RelB. TNF and receptor activator of NF-κB ligand (RANKL), two ligands associated with diabetes, induce NIK in islets. Mice with constitutive ß cell-intrinsic NIK activation present impaired insulin secretion with DIO. NIK activation triggers the noncanonical NF-κB transcriptional network to induce genes identified in human type 2 diabetes genome-wide association studies linked to ß cell failure. These studies reveal that NIK contributes a central mechanism for ß cell failure in diet-induced obesity.


Assuntos
Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , Insulina/metabolismo , Obesidade/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Western Blotting , Primers do DNA/genética , Humanos , Imuno-Histoquímica , Secreção de Insulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/patologia , Peixe-Zebra
5.
Eur J Immunol ; 45(6): 1820-31, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25931426

RESUMO

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8(+) T-cell and NKT-cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naïve CD8(+) T-cell subsets. IL-15-dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8(+) CD44(hi) CD122(+) T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8(+) CD44(hi) T cells exhibited impaired dose-dependent proliferation to exogenous IL-15. In contrast, TRAF2TKO CD8(+) T cells proliferated normally to anti-CD3 and TRAF2TKO CD8(+) CD44(hi) T cells exhibited normal proliferation to exogenous IL-2. TRAF2TKO CD8(+) T cells expressed normal levels of IL-15-associated receptors and possessed functional IL-15-mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8(+) CD44(hi) CD122(+) and NKT cells was mechanistically linked to an inability to respond to IL-15. The reduced CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell populations in TRAF2TKO mice were rescued in the presence of high dose IL-15 by IL-15/IL-15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell homeostasis by modulating sensitivity to T-cell intrinsic growth factors such as IL-15.


Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/fisiologia , Homeostase , Interleucina-15/farmacologia , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/fisiologia , Fator 2 Associado a Receptor de TNF/genética , Animais , Microambiente Celular , Citocinas/farmacologia , Feminino , Expressão Gênica , Memória Imunológica , Imunofenotipagem , Contagem de Linfócitos , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/metabolismo , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Fenótipo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina-15/genética , Receptores de Interleucina-15/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/fisiologia , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo
6.
Cell Transplant ; 22(12): 2355-66, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23127588

RESUMO

Islet grafts can contribute to their own destruction via the elaboration of proinflammatory genes, many of which are transcriptionally regulated by nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB). Thus, NF-κB constitutes an enticing gene therapy candidate to improve the success of islet transplantation. To test this hypothesis in vivo, we blocked NF-κB in BALB/c (H2(d)) to C57/BL6 (H2(b)) mouse islet allografts by genetically engineering islets to express the NF-κB superrepressor, IκBα. Here we show by microarray and RTqPCR that islets exhibit an intrinsic early immediate proinflammatory response, with the most highly upregulated proinflammatory genes comprising the chemokines Cxcl1, Cxcl2, Cxcl10, and Ccl2; the cytokines Tnf-α and Il-6; and the adhesion molecule Icam1. Overexpression of IκBα inhibited the expression of these genes by 50-95% in islets and MIN6 ß-cells in vitro, by inhibiting NF-κB-dependent gene transcription. Histological and RTqPCR analysis at postoperative day (POD) 10 revealed that IκBα-transduced islet allografts exhibited improved islet architecture and strong insulin-labeling with decreased Ccl2 and Il-6 mRNA levels compared to the GFP-transduced control grafts. Despite these protective effects, NF-κB-blocked islet allografts were promptly rejected in our MHC-mismatched mouse model. However, IκBα-expressing grafts did harbor localized "pockets" of Foxp3(+) mononuclear cells not evident in the control grafts. This result suggested that the effect of the NF-κB blockade might synergize with regulatory T-cell-sparing rapamycin. Indeed, combining intragraft IκBα expression with low-dose rapamycin increased the mean survival time of islet allografts from 20 to 81 days, with 20% of the grafts surviving for greater than 100 days. In conclusion, rapamycin unmasks the protective potential of intragraft NF-κB blockade, which can, in some cases, permit permanent allograft survival without continuous systemic immunosuppression.


Assuntos
Diabetes Mellitus Experimental/cirurgia , Sobrevivência de Enxerto/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/citologia , NF-kappa B/antagonistas & inibidores , Sirolimo/farmacologia , Animais , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Diabetes Mellitus Experimental/mortalidade , Diabetes Mellitus Experimental/patologia , Sinergismo Farmacológico , Quinase I-kappa B/genética , Imunossupressores/farmacologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Suínos , Transplante Homólogo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
7.
J Biol Chem ; 286(26): 23121-31, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21558271

RESUMO

The peptide transporter (PTR) family represents a group of proton-coupled secondary transporters responsible for bulk uptake of amino acids in the form of di- and tripeptides, an essential process employed across species ranging from bacteria to humans. To identify amino acids critical for peptide transport in a prokaryotic PTR member, we have screened a library of mutants of the Escherichia coli peptide transporter YdgR using a high-throughput substrate uptake assay. We have identified 35 single point mutations that result in a full or partial loss of transport activity. Additional analysis, including homology modeling based on the crystal structure of the Shewanella oneidensis peptide transporter PepT(so), identifies Glu(56) and Arg(305) as potential periplasmic gating residues. In addition to providing new insights into transport by members of the PTR family, these mutants provide valuable tools for further study of the mechanism of peptide transport.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Periplásmicas , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Shewanella/química , Shewanella/genética , Shewanella/metabolismo , Homologia Estrutural de Proteína
8.
J Biol Chem ; 285(50): 39201-10, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-20889976

RESUMO

The serotonin transporter (SERT) is a member of the SLC6 family of solute carriers. SERT plays a crucial role in synaptic neurotransmission by retrieving released serotonin. The intracellular carboxyl terminus of various neurotransmitter transporters has been shown to be important for the correct delivery of SLC6 family members to the cell surface. Here we studied the importance of the C terminus in trafficking and folding of human SERT. Serial truncations followed by mutagenesis identified sequence spots (PG(601,602), RII(607-609)) within the C terminus relevant for export of SERT from the endoplasmic reticulum (ER). RI(607,608) is homologous to the RL-motif that in other SLC6 family members provides a docking site for the COPII component Sec24D. The primary defect resulting from mutation at PG(601,602) and RI(607,608) was impaired folding, because mutated transporters failed to bind the inhibitor [(3)H]imipramine. In contrast, when retained in the ER (e.g. by dominant negative Sar1) the wild type transporter bound [(3)H]imipramine with an affinity comparable to that of the surface-expressed transporter. SERT-RI(607,608)AA and SERT-RII(607-609)AAA were partially rescued by treatment of cells with the nonspecific chemical chaperone DMSO or the specific pharmacochaperone ibogaine (which binds to the inward facing conformation of SERT) but not by other classes of ligands (inhibitors, substrates, amphetamines). These observations (i) demonstrate an hitherto unappreciated role of the C terminus in the folding of SERT, (ii) indicates that the folding trajectory proceeds via an inward facing intermediate, and (iii) suggest a model where the RI-motif plays a crucial role in preventing premature Sec24-recruitment and export of incorrectly folded transporters.


Assuntos
Mutação , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Imipramina/química , Ligantes , Proteínas de Membrana/química , Microscopia de Fluorescência/métodos , Chaperonas Moleculares/química , Neurotransmissores/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Serotonina/química
9.
Pediatr Res ; 60(3): 321-6, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16857757

RESUMO

We present the clinical and laboratory features of a boy with a new syndrome of mitochondrial depletion syndrome and T cell immunodeficiency. The child suffered from severe recurrent infectious diseases, anemia, and thrombocytopenia. Clinically, he presented with severe psychomotor retardation, axial hypotonia, and a disturbed pain perception leading to debilitating biting of the thumb, lower lip, and tongue. Brain imaging showed hypoplasia of corpus callosum and an impaired myelinization of the temporo-occipital region with consecutive supratentorial hydrocephalus. Histologic examination of a skeletal muscle biopsy was normal. Biochemical investigation showed combined deficiency of respiratory chain complexes II+III and IV. MtDNA depletion was found by real-time PCR. No pathogenic mutations were identified in the TK2, SUCLA2, DGUOK, and ECGF1 genes. A heterozygous missense mutation was found in POLG1. The pathogenic relevance of this mutation is unclear. Interestingly, a lack of CD8(+) T lymphocytes as well as NK cells was also observed. The percentage of CD45RO-expressing cells was decreased in activated CD8(+) T lymphocytes. Activation of T lymphocytes via IL-2 was diminished. The occurrence of the immunologic deficiency in our patient with mtDNA depletion is a rare finding, implying that cells of the immune system might also be affected by mitochondrial disease.


Assuntos
Imunodeficiência de Variável Comum/imunologia , Disgamaglobulinemia/imunologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/deficiência , Doenças Mitocondriais/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Imunodeficiência de Variável Comum/metabolismo , Imunodeficiência de Variável Comum/patologia , DNA Mitocondrial/genética , Disgamaglobulinemia/metabolismo , Disgamaglobulinemia/patologia , Evolução Fatal , Humanos , Imunoglobulinas/biossíntese , Imunoglobulinas/sangue , Lactente , Recém-Nascido , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/patologia , Masculino , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Músculo Esquelético/química , Músculo Esquelético/metabolismo
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