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1.
Diabetologia ; 63(8): 1576-1587, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32500289

RESUMO

AIMS/HYPOTHESIS: Self-antigen-specific T cell responses drive type 1 diabetes pathogenesis, but alterations in innate immune responses are also critical and not as well understood. Innate immunity in human type 1 diabetes has primarily been assessed via gene-expression analysis of unstimulated peripheral blood mononuclear cells, without the immune activation that could amplify disease-associated signals. Increased responsiveness in each of the two main innate immune pathways, driven by either type 1 IFN (IFN-1) or IL-1, have been detected in type 1 diabetes, but the dominant innate pathway is still unclear. This study aimed to determine the key innate pathway in type 1 diabetes and assess the whole blood immune stimulation assay as a tool to investigate this. METHODS: The TruCulture whole blood ex vivo stimulation assay, paired with gene expression and cytokine measurements, was used to characterise changes in the stimulated innate immune response in type 1 diabetes. We applied specific cytokine-induced signatures to our data, pre-defined from the same assays measured in a separate cohort of healthy individuals. In addition, NOD mice were stimulated with CpG and monocyte gene expression was measured. RESULTS: Monocytes from NOD mice showed lower baseline vs diabetes-resistant B6.g7 mice, but higher induced IFN-1-associated gene expression. In human participants, ex vivo whole blood stimulation revealed higher induced IFN-1 responses in type 1 diabetes, as compared with healthy control participants. In contrast, neither the IL-1-induced gene signature nor response to the adaptive immune stimulant Staphylococcal enterotoxin B were significantly altered in type 1 diabetes samples vs healthy control participants. Targeted gene-expression analysis showed that this enhanced IFN response was specific to IFN-1, as IFN-γ-driven responses were not significantly different. CONCLUSIONS/INTERPRETATION: Our study identifies increased responsiveness to IFN-1 as a feature of both the NOD mouse model of autoimmune diabetes and human established type 1 diabetes. A stimulated IFN-1 gene signature may be a potential biomarker for type 1 diabetes and used to evaluate the effects of therapies targeting this pathway. DATA AVAILABILITY: Mouse gene expression data are found in the gene expression omnibus (GEO) repository, accession GSE146452 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146452 ). Nanostring count data from the human experiments were deposited in the GEO repository, accession GSE146338 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146338 ). Data files and R code for all analyses are available at https://github.com/rodriguesk/T1D_truculture_diabetologia . Graphical abstract.


Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Imunidade Inata/fisiologia , Leucócitos Mononucleares/metabolismo , Monócitos/metabolismo , Animais , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Enterotoxinas/farmacologia , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Interleucina-1/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NOD , Monócitos/efeitos dos fármacos
2.
F1000Res ; 7: 318, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29707204

RESUMO

Background: We previously identified the transcriptional regulator Zbtb32 as a factor that can promote T cell tolerance in the Non-Obese Diabetic (NOD) mouse, a model of Type 1 diabetes. Antigen targeted to DCIR2 + dendritic cells (DCs) in vivo inhibited both diabetes and effector T cell expansion in NOD mice. Furthermore, Zbtb32 was preferentially induced in autoreactive CD4 T cells stimulated by these tolerogenic DCIR2 + DCs, and overexpression of Zbtb32 in islet-specific T cells inhibited the diabetes development by limiting T cell proliferation and cytokine production. Methods: To further understand the role of Zbtb32 in T cell tolerance induction, we have now used CRISPR to target the Zbtb32 gene for deletion directly in NOD mice and characterized the mutant mice. We hypothesized that the systemic loss of Zbtb32 in NOD mice would lead to increased T cell activation and increased diabetes pathogenesis. Results: Although NOD.Zbtb32 -/- male NOD mice showed a trend towards increased diabetes incidence compared to littermate controls, the difference was not significant. Furthermore, no significant alteration in lymphocyte number or function was observed. Importantly, in vitro stimulation of lymphocytes from NOD.Zbtb32 -/- mice did not produce the expected hypersensitive phenotype observed in other genetic strains, potentially due to compensation by homologous genes. Conclusions: The loss of Zbtb32 in the NOD background does not result in the expected T cell activation phenotype.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Diabetes Mellitus Experimental/epidemiologia , Tolerância Imunológica/imunologia , Ativação Linfocitária/imunologia , Proteínas Repressoras/fisiologia , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Citocinas/metabolismo , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Feminino , Incidência , Masculino , Camundongos , Camundongos Endogâmicos NOD , Proteínas Repressoras/antagonistas & inibidores
3.
JCI Insight ; 3(3)2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29415894

RESUMO

Type I IFN (IFN-I) dysregulation contributes to type 1 diabetes (T1D) development, and although increased IFN-I signals are pathogenic at the initiation of autoimmune diabetes, IFN-I dysregulation at later pathogenic stages more relevant for therapeutic intervention is not well understood. We discovered that 5 key antigen-presenting cell subsets from adult prediabetic NOD mice have reduced responsiveness to IFN-I that is dominated by a decrease in the tonic-sensitive subset of IFN-I response genes. Blockade of IFNAR1 in prediabetic NOD mice accelerated diabetes and increased Th1 responses. Therefore, IFN-I responses shift from pathogenic to protective as autoimmunity progresses, consistent with chronic IFN-I exposure. In contrast, IL-1-associated inflammatory pathways were elevated in prediabetic mice. These changes correlated with human T1D onset-associated gene expression. Prostaglandin E2 (PGE2) and prostaglandin receptor 4 (PTGER4), a receptor for PGE2 that mediates both inflammatory and regulatory eicosanoid signaling, were higher in NOD mice and drive innate immune dysregulation. Treating prediabetic NOD mice with a PTGER4 antagonist restored IFNAR signaling, decreased IL-1 signaling, and decreased infiltration of leukocytes into the islets. Therefore, innate cytokine alterations contribute to both T1D-associated inflammation and autoimmune pathogenesis. Modulating innate immune balance via signals such as PTGER4 may contribute to treatments for autoimmunity.


Assuntos
Autoimunidade/efeitos dos fármacos , Diabetes Mellitus Tipo 1/imunologia , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Compostos de Sulfonilureia/administração & dosagem , Células Th1/imunologia , Administração Oral , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Diabetes Mellitus Tipo 1/tratamento farmacológico , Dinoprostona/imunologia , Dinoprostona/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Interleucina-1/imunologia , Interleucina-1/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/imunologia , Receptor de Interferon alfa e beta/metabolismo , Receptores de Prostaglandina E Subtipo EP4/imunologia , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/metabolismo
4.
PLoS Biol ; 16(1): e2004111, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29357353

RESUMO

Nuclear factor of activated T cells (NFAT) transcription factors are required for induction of T-cell cytokine production and effector function. Although it is known that activation via the T-cell antigen receptor (TCR) results in 2 critical steps, calcineurin-mediated NFAT1 dephosphorylation and NFAT2 up-regulation, the molecular mechanisms underlying each are poorly understood. Here we find that T cell p38, which is activated by an alternative pathway independent of the mitogen-activated protein (MAP) kinase cascade and with different substrate specificities, directly controls these events. First, alternatively (but not classically) activated p38 was required to induce the expression of the AP-1 component c-Fos, which was necessary for NFAT2 expression and cytokine production. Second, alternatively (but not classically) activated p38 phosphorylated NFAT1 on a heretofore unidentified site, S79, and in its absence NFAT1 was unable to interact with calcineurin or migrate to the nucleus. These results demonstrate that the acquisition of unique specificities by TCR-activated p38 orchestrates NFAT-dependent T-cell functions.


Assuntos
Fatores de Transcrição NFATC/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Calcineurina , Comunicação Celular , Humanos , Imunidade Celular/genética , Imunidade Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Fosforilação , Proteólise , Proteínas Proto-Oncogênicas c-fos , Receptores de Antígenos de Linfócitos T/fisiologia , Especificidade por Substrato , Linfócitos T , Fatores de Transcrição
5.
J Immunol ; 198(6): 2223-2231, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28264998

RESUMO

Immune tolerance is necessary to prevent the immune system from reacting against self, and thus to avoid the development of autoimmune diseases. In this review, we discuss key findings that position dendritic cells (DCs) as critical modulators of both thymic and peripheral immune tolerance. Although DCs are important for inducing both immunity and tolerance, increased autoimmunity associated with decreased DCs suggests their nonredundant role in tolerance induction. DC-mediated T cell immune tolerance is an active process that is influenced by genetic variants, environmental signals, as well as the nature of the specific DC subset presenting Ag to T cells. Answering the many open questions with regard to the role of DCs in immune tolerance could lead to the development of novel therapies for the prevention of autoimmune diseases.


Assuntos
Doenças Autoimunes/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica , Imunoterapia/métodos , Linfócitos T/imunologia , Animais , Apresentação de Antígeno , Autoimunidade , Humanos , Imunoterapia/tendências , Ativação Linfocitária
6.
Eur J Immunol ; 47(3): 575-584, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28083937

RESUMO

Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and T cells, causing Type 1 Diabetes in non-obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well-characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus-like particles produced by co-expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes-resistant mice developed anti-Env autoantibodies that increase in titer as disease progresses. A lentiviral-based RNA interference knockdown of Gag revealed that Gag contributes to the MV-induced T-cell response, whose diabetogenic function can be demonstrated via cell-transfer into immune-deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet-derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes-resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti-retroviral responses to trigger autoimmunity.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Retrovirus Endógenos/imunologia , Produtos do Gene env/metabolismo , Produtos do Gene gag/metabolismo , Ilhotas Pancreáticas/imunologia , Células-Tronco Mesenquimais/metabolismo , Linfócitos T/imunologia , Transferência Adotiva , Animais , Autoanticorpos/sangue , Autoimunidade , Micropartículas Derivadas de Células/imunologia , Células Cultivadas , Feminino , Produtos do Gene env/genética , Produtos do Gene gag/genética , Humanos , Ilhotas Pancreáticas/metabolismo , Ativação Linfocitária , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , RNA Interferente Pequeno/genética , Linfócitos T/transplante
7.
J Immunol ; 196(5): 2031-40, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26826238

RESUMO

Innate immune signals help break self-tolerance to initiate autoimmune diseases such as type 1 diabetes, but innate contributions to subsequent regulation of disease progression are less clear. Most studies have measured in vitro innate responses of GM-CSF dendritic cells (DCs) that are functionally distinct from conventional DCs (cDCs) and do not reflect in vivo DC subsets. To determine whether autoimmune NOD mice have alterations in type 1 IFN innate responsiveness, we compared cDCs from prediabetic NOD and control C57BL/6 (B6) mice stimulated in vivo with the TLR9 ligand CpG, a strong type 1 IFN inducer. In response to CpG, NOD mice produce more type 1 IFN and express higher levels of CD40, and NOD monocyte DCs make more TNF. However, the overall CpG-induced transcriptional response is muted in NOD cDCs. Of relevance the costimulatory proteins CD80/CD86, signals needed for regulatory T cell homeostasis, are upregulated less on NOD cDCs. Interestingly, NOD Rag1(-/-) mice also display a defect in CpG-induced CD86 upregulation compared with B6 Rag1(-/-), indicating this particular innate alteration precedes adaptive autoimmunity. The impaired response in NOD DCs is likely downstream of the IFN-α/ß receptor because DCs from NOD and B6 mice show similar CpG-induced CD86 levels when anti-IFN-α/ß receptor Ab is added. IFN-α-induced nuclear localization of activated STAT1 is markedly reduced in NOD CD11c(+) cells, consistent with lower type 1 IFN responsiveness. In conclusion, NOD DCs display altered innate responses characterized by enhanced type 1 IFN and activation of monocyte-derived DCs but diminished cDC type 1 IFN response.


Assuntos
Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Fator de Transcrição STAT1/imunologia , Tolerância a Antígenos Próprios/imunologia , Transporte Ativo do Núcleo Celular , Animais , Western Blotting , Linhagem da Célula , Núcleo Celular/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Microscopia Confocal , Monócitos/citologia , Monócitos/imunologia , Oligodesoxirribonucleotídeos/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT1/metabolismo , Receptor Toll-Like 9/agonistas
8.
J Immunol Methods ; 432: 4-12, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26344574

RESUMO

The role of antigen presenting cells (APCs) in the pathogenesis of autoimmune and other inflammatory diseases is now better understood due to advances in multicolor flow cytometry, gene expression analysis of APC populations, and functional correlation of mouse to human APC populations. A simple but informative nomenclature of conventional and plasmacytoid dendritic cell subsets (cDC1, cDC2, pDC) and monocyte-derived populations incorporates these advances, but accurate subset identification is critical. Ambiguous gating schemes and alterations of cell surface markers in inflammatory condition can make comparing results between studies difficult. Both acute inflammation, such as TLR-ligand stimulation, and chronic inflammation as found in mouse models of autoimmunity can alter DC subset gating. Here, we address these issues using in vivo CpG stimulation as an example of acute inflammation and the non-obese diabetic (NOD) mouse as a model of chronic inflammation.We provide a flow cytometric antibody panel and gating scheme that differentiate 2 monocytic and 3DC subsets in the spleen both at steady state and after CpG stimulation. Using this method, we observed differences in the composition of NOD DCs that have been previously reported, and newly identified increases in the number of NOD monocyte-derived DCs. Finally, we established a protocol for DC phosphoflow to measure the phosphorylation state of intracellular proteins, and use it to confirm functional differences in the identified subsets. Therefore, we present optimized methods for distinguishing monocytic and DC populations with and without inflammation and/or autoimmunity associated with NOD mice.


Assuntos
Doenças Autoimunes/imunologia , Autoimunidade , Separação Celular/métodos , Células Dendríticas/imunologia , Citometria de Fluxo , Inflamação/imunologia , Monócitos/imunologia , Baço/imunologia , Doença Aguda , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Biomarcadores/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Imunofenotipagem , Inflamação/induzido quimicamente , Inflamação/metabolismo , Interferon gama/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Oligodesoxirribonucleotídeos , Fenótipo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas/imunologia , Proteínas/metabolismo , Baço/efeitos dos fármacos , Baço/metabolismo , Receptor Toll-Like 9/agonistas
9.
Eur J Immunol ; 43(10): 2588-97, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23817982

RESUMO

Exosomes (EXOs) are nano-sized secreted microvesicles that can function as potent endogenous carriers of adjuvant and antigens. To examine a possible role in autoimmunity for EXOs, we studied EXO-induced immune responses in nonobese diabetic (NOD) mice, an autoimmune-prone strain with tissue-specific targeting at insulin-secreting beta cells. EXOs released by insulinoma cells can activate various antigen-presenting cells to secrete several proinflammatory cytokines and chemokines. A subset of B cells responded to EXO stimulation in culture by proliferation, and expressed surface markers representing marginal zone B cells, which was independent of T helper cells. Importantly, splenic B cells from prediabetic NOD mice, but not diabetic-resistant mice, exhibited increased reactivity to EXOs, which was correlated with a high level of serum EXOs. We found that MyD88-mediated innate TLR signals were essential for the B-cell response; transgenic B cells expressing surface immunoglobulin specific for insulin reacted to EXO stimulation, and addition of a calcineurin inhibitor FK506 abrogated the EXO-induced B-cell response, suggesting that both innate and antigen-specific signals may be involved. Thus, EXOs may contribute to the development of autoimmunity and type 1 diabetes in NOD mice, partially via activating autoreactive marginal zone-like B cells.


Assuntos
Linfócitos B/imunologia , Diabetes Mellitus Experimental/imunologia , Exossomos/imunologia , Células Precursoras de Linfócitos B/imunologia , Animais , Autoantígenos/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Suscetibilidade a Doenças , Exossomos/metabolismo , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Mediadores da Inflamação/metabolismo , Insulina/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos NOD , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tacrolimo/farmacologia , Receptores Toll-Like/metabolismo
10.
Clin Diagn Lab Immunol ; 11(6): 1022-7, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15539500

RESUMO

We have previously demonstrated that Mycobacterium bovis BCG-specific immunoglobulin G antibodies in lymphocyte secretions (ALS) can be employed as a marker for active tuberculosis (TB). We aimed to determine whether the ALS method allows detection of subclinical TB infection in asymptomatic individuals. A prospective study of family contacts (FCs) of patients with active TB and healthy controls was performed. Thirteen of 42 FCs had high ALS responses, including 6 FCs who subsequently developed active TB. No correlation was observed between the tuberculin skin test and the ALS responses in the FCs (r = 0.1, P = 0.23). Among patients with active TB, BCG-specific ALS responses steadily declined from the time of diagnosis through 6 months following antimycobacterial chemotherapy (P = 0.001). The ALS assay enabled detection of infection in exposed symptom-free contacts, who are at greater risk for developing active TB. The method may also allow discrimination between effective treatment of active infection and suboptimal response to therapy.


Assuntos
Anticorpos Antibacterianos/análise , Antígenos de Bactérias/imunologia , Imunoglobulina G/análise , Linfócitos/imunologia , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Escarro/imunologia , Tuberculose Pulmonar/diagnóstico , Anticorpos Antibacterianos/imunologia , Células Cultivadas , Imunoglobulina G/imunologia , Valor Preditivo dos Testes , Escarro/citologia , Tuberculose Pulmonar/patologia
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