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1.
Nat Commun ; 10(1): 4698, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619674

RESUMO

T helper 17 (Th17) cells have crucial functions in mucosal immunity and the pathogenesis of several chronic inflammatory diseases. The lineage-specific transcription factor, RORγt, encoded by the RORC gene modulates Th17 polarization and function, as well as thymocyte development. Here we define several regulatory elements at the human RORC locus in thymocytes and peripheral CD4+ T lymphocytes, with CRISPR/Cas9-guided deletion of these genomic segments supporting their role in RORγt expression. Mechanistically, T cell receptor stimulation induces cyclosporine A-sensitive histone modifications and P300/CBP acetylase recruitment at these elements in activated CD4+ T cells. Meanwhile, NFAT proteins bind to these regulatory elements and activate RORγt transcription in cooperation with NF-kB. Our data thus demonstrate that NFAT specifically regulate RORγt expression by binding to the RORC locus and promoting its permissive conformation.


Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição NFATC/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Elementos Reguladores de Transcrição/genética , Células Th17/metabolismo , Timócitos/metabolismo , Ativação Transcricional , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas , Linhagem da Célula , Citometria de Fluxo , Células HEK293 , Código das Histonas , Humanos , Células Jurkat , Células Th17/citologia , Timócitos/citologia , Fatores de Transcrição de p300-CBP/metabolismo
2.
Food Chem Toxicol ; 133: 110748, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31377140

RESUMO

Hexavalent chromium raises high concern because of its wide industrial applications and reported toxicity. Long-term (135 days) oral exposure of Wistar rats to chromium in the form of K2Cr2O7 (exposed group~20 mg/kg/day) led to a decrease in thymus mass and thymocytes' number and caused structural and functional changes in the lymph nodes and spleen, namely lymphoreticular hyperplasia and plasmocytic macrophage transformation. Programmed cell death was increased in both thymocytes and splenocytes and decreased in lymphocytes in the T-zones of spleen and lymph nodes. Moreover, Cr (VI) administration decreased myeloid cells' and neutrophils' number, while it increased lymphoid and erythroid cells' number in bone marrow. Cr (VI) immune system effects seem to be related to oxidative stress induction, as depicted by the increased levels of diene conjugates and malondialdehyde in the spleen and liver and by the decreased activity of catalase and superoxide dismutase in rats' erythrocytes. In addition, exposure to Cr (VI) decreased copper, nickel and iron concentrations in blood and liver, while Cr levels in blood, spleen and liver were increased, as expected. The observed changes in the series of immunological parameters studied contribute to the development of new approaches for the prevention of low level Cr exposure toxicity.


Assuntos
Cromo/toxicidade , Linfonodos/efeitos dos fármacos , Baço/efeitos dos fármacos , Timo/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Imuno-Histoquímica , Linfonodos/metabolismo , Contagem de Linfócitos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos Wistar , Baço/metabolismo , Linfócitos T/metabolismo , Timócitos/metabolismo , Timo/metabolismo , Timo/patologia
3.
Aquat Toxicol ; 214: 105253, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31352076

RESUMO

Diclofop-methyl (DM) is widely used in agriculture and may lead to serious toxicity. However, a limited number of studies have been performed to evaluate the toxicity of DM in the immune and nervous systems of animals. Here, we utilized a good vertebrate model, zebrafish, to evaluate the toxicity of DM during the developmental process. Exposure of zebrafish embryos to 0.1, 0.3 and 0.5 mg/l DM from 6 h post fertilization (hpf) to 72 hpf induced developmental abnormalities, such as shorter body lengths and yolk sac edemas. The number of immune cells in zebrafish larvae was significantly reduced, but the inflammatory response was not influenced by DM treatment. The expression of immune-related genes were downregulated and the levels of oxidative stress were upregulated by DM exposure. Moreover, locomotor behaviors were inhibited by DM exposure. Therefore, our results suggest that DM has the potential to induce immunotoxicity and cause behavioral changes in zebrafish larvae. This study provides new evidence of the influence of DM exposure on aquatic ecosystems.


Assuntos
Comportamento Animal/efeitos dos fármacos , Embrião não Mamífero/imunologia , Exposição Ambiental , Éteres Difenil Halogenados/toxicidade , Peixe-Zebra/embriologia , Animais , Encéfalo/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Timócitos/efeitos dos fármacos , Timócitos/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genética
4.
Biomed Res Int ; 2019: 6261951, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183371

RESUMO

Background/Aims: Voltage-dependent potassium channels (Kv1.3) are predominantly expressed in lymphocyte plasma membranes. These channels are critical for the activation and proliferation of lymphocytes. Since second-generation antihistamines are lipophilic and exert immunomodulatory effects, they are thought to affect the lymphocyte Kv1.3-channel currents. Methods: Using the patch-clamp whole-cell recording technique in murine thymocytes, we tested the effects of second-generation antihistamines, such as cetirizine, fexofenadine, azelastine, and terfenadine, on the channel currents and the membrane capacitance. Results: These drugs suppressed the peak and the pulse-end currents of the channels, although the effects of azelastine and terfenadine on the peak currents were more marked than those of cetirizine and fexofenadine. Both azelastine and terfenadine significantly lowered the membrane capacitance. Since these drugs did not affect the process of endocytosis in lymphocytes, they were thought to have interacted directly with the plasma membranes. Conclusions: Our study revealed for the first time that second-generation antihistamines, including cetirizine, fexofenadine, azelastine, and terfenadine, exert suppressive effects on lymphocyte Kv1.3-channels. The efficacy of these drugs may be related to their immunomodulatory mechanisms that reduce the synthesis of inflammatory cytokine.


Assuntos
Antagonistas dos Receptores Histamínicos H1/farmacologia , Imunomodulação/efeitos dos fármacos , Canal de Potássio Kv1.3/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Timócitos/metabolismo , Animais , Masculino , Camundongos , Técnicas de Patch-Clamp
5.
Genes Cells ; 24(7): 485-495, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31125466

RESUMO

Mammalian STE20-like protein kinase 1/2 (MST1/2) and nuclear Dbf2-related kinase 1/2 (NDR1/2) are core components of Hippo signaling that are also known to be important regulators of lymphocyte trafficking. However, little is understood about the roles of other Hippo pathway molecules in these cells. Here, we present the first analysis of the function of Mps one binder kinase activator-1 (MOB1) in T lymphocytes in vivo. T-cell-specific double knockout (DKO) of MOB1A/B in mice [tMob1 DKO mice] reduces the number of naïve T cells in both the circulation and secondary lymphoid organs, but leads to an accumulation of CD4+ CD8- and CD4- CD8+ single-positive (SP) cells in the thymus. In vitro, naïve MOB1A/B-deficient T cells show increased apoptosis and display impaired trafficking capacity in response to the chemokine CCL19. These defects are linked to suppression of the activation of MST and NDR kinases, but are independent of the downstream transcriptional co-activator Yes-associated protein 1 (YAP1). Thus, MOB1 proteins play an important role in thymic egress and T-cell survival that is mediated by a pathway other than conventional Hippo-YAP1 signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Proteínas Quinases/fisiologia , Linfócitos T/imunologia , Timócitos/imunologia , Animais , Apoptose , Proteínas de Ciclo Celular , Células Cultivadas , Quimiotaxia , Feminino , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/metabolismo , Linfócitos T/patologia , Timócitos/metabolismo , Timócitos/patologia
6.
Nat Commun ; 10(1): 2220, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101805

RESUMO

Both medullary thymic epithelial cells (mTEC) and dendritic cells (DC) present tissue-restricted antigens (TRA) to thymocytes to induce central tolerance, but the relative contributions of these antigen-presenting cell (APC) subsets remain unresolved. Here we developed a two-photon microscopy approach to observe thymocytes interacting with intact APCs presenting TRAs. We find that mTECs and DCs cooperate extensively to induce tolerance, with their relative contributions regulated by the cellular form of the TRA and the class of major histocompatibility complex (MHC) on which antigen is presented. Even when TRA expression is restricted to mTECs, DCs still present self-antigens at least as frequently as mTECs. Notably, the DC subset cDC2 efficiently acquires secreted mTEC-derived TRAs for cross-presentation on MHC-I. By directly imaging interactions between thymocytes and APCs, while monitoring intracellular signaling, this study reveals that distinct DC subsets and AIRE+ mTECs contribute substantially to presentation of diverse self-antigens for establishing central tolerance.


Assuntos
Tolerância Central/imunologia , Células Dendríticas/imunologia , Timócitos/imunologia , Timo/imunologia , Animais , Apresentação do Antígeno/imunologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Transplante de Medula Óssea , Separação Celular/métodos , Células Dendríticas/metabolismo , Células Epiteliais/imunologia , Feminino , Citometria de Fluxo/métodos , Microscopia Intravital/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Linfócitos T Reguladores/imunologia , Timócitos/metabolismo , Timo/citologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Quimeras de Transplante/imunologia
7.
Toxicol Lett ; 311: 49-57, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31014974

RESUMO

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxins, is a persistent and ubiquitous environmental contaminant. Although the immunotoxic effects of TCDD have been reported, the mechanisms underlying these effects are still unclear. In this study, we have determined the toxic effects of TCDD on thymocytes and splenic T cells with in vitro cell culture systems. Magnetically isolated mouse splenic Th cells, Treg cells and the mixed spleen lymphocytes (SLC) were cultured and treated with TCDD and the differentiation of CD4 Th cells was determined by flow cytometery. Our results showed that different concentrations of TCDD caused immunotoxic effects through different toxicological mechanisms in both the purified mouse splenic Th cells and the mixed SLC. The low dose exposure to TCDD triggered regulatory effects in the immune system, while the high dose TCDD exposure resulted in severe immune toxicity. Notably, a decline of Treg subset was observed, suggesting an imbalanced immune regulation by TCDD treatment, as well as a possible decrease of TCDD's indirect effects on bystander immune cells. Our CD4 Th subset co-culture experiments showed that TCDD-induced pathobiology depended on immune cell balance, suggesting that cytokine-induced microenvironments further modulated toxic effects associated with TCDD exposure.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Dibenzodioxinas Policloradas/toxicidade , Baço/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Timócitos/efeitos dos fármacos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Microambiente Celular , Técnicas de Cocultura , Citocinas/imunologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Baço/imunologia , Baço/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Timócitos/imunologia , Timócitos/metabolismo
8.
Biochem Biophys Res Commun ; 511(4): 935-940, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30853180

RESUMO

BACKGROUND/AIMS: The expression of transcription factor Zbtb1 is essential for the maintenance and development of various blood cells in the hematopoietic system. In the current study, we found that the total number of thymocytes in PLZF deficient mice was reduced compared with thymocytes in wild-type mice, and the number of early T-cell progenitors decreased. However, the decrease of thymocytes in PLZF deficient mice was not cell intrinsic. This study adds new information regarding the regulation of the PLZF gene in the development and self-renewal of T cells. METHODS: The thymus was isolated from newborn mice, and the two lobes of each thymus were physically separated. Each host received a thymus, two lobes, each placed at one end of the kidney, as described in the literature. Tail vein blood was periodically collected from some of the recipients and analyzed for the presence of peripheral blood T cells. RESULTS: In PLZF-EGFP reporter mice and neonatal thymus transplantation to the kidney, we found that PLZF was highly expressed in DN1 (Lineage-CD44+CD25-) cells of thymic grafts of Rag2/γc-/- recipient mice. We found that the proportion of PLZF wild-type and mutant-derived cells in the thymocytes of recipient mice after bone marrow transplantation is approximately equal to the competitive bone marrow chimeric mouse model, and all mice contain a normal thymus. CONCLUSION: The development of T cells suggests that the effect of the PLZF gene on T cell differentiation and development is not cell intrinsic. However, in the neonatal mouse thymic transplant model in the Rag2/γc-/- recipient mouse, deletion of the PLZF gene results in a significant decrease in the proportion of DN1 cells from the donor in the thymic graft.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Linfócitos T/citologia , Animais , Contagem de Células , Autorrenovação Celular , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Timo/citologia , Timo/crescimento & desenvolvimento , Timo/metabolismo
9.
PLoS Biol ; 17(3): e2006716, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30856173

RESUMO

The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.


Assuntos
MicroRNAs/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Citometria de Fluxo , Camundongos , Camundongos Knockout , MicroRNAs/genética , Microscopia Confocal , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Timócitos/metabolismo
10.
Immunity ; 50(2): 348-361.e4, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30737145

RESUMO

NF-κB (nuclear factor κB) signaling is considered critical for single positive (SP) thymocyte development because loss of upstream activators of NF-κB, such as the IKK complex, arrests their development. We found that the compound ablation of RelA, cRel, and p50, required for canonical NF-κB transcription, had no impact upon thymocyte development. While IKK-deficient thymocytes were acutely sensitive to tumor necrosis factor (TNF)-induced cell death, Rel-deficient cells remained resistant, calling into question the importance of NF-κB as the IKK target required for thymocyte survival. Instead, we found that IKK controlled thymocyte survival by repressing cell-death-inducing activity of the serine/threonine kinase RIPK1. We observed that RIPK1 expression was induced during development of SP thymocytes and that IKK was required to prevent RIPK1-kinase-dependent death of SPs in vivo. Finally, we showed that IKK was required to protect Rel-deficient thymocytes from RIPK1-dependent cell death, underscoring the NF-κB-independent function of IKK during thymic development.


Assuntos
Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Timócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase I-kappa B/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Timócitos/citologia , Timócitos/efeitos dos fármacos , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
11.
Chem Biol Interact ; 302: 143-148, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30779908

RESUMO

N-(3-oxododecanoyl)-l-homoserine-lactone (ODHL), a quorum sensing molecule, affects intracellular Zn2+ concentration ([Zn2+]i) and cellular levels of nonprotein thiols ([NPT]i) of rat thymic lymphocytes, both of which are assumed to affect cell vulnerability to oxidative stress. Therefore, it is interesting to examine the effects of ODHL on the cells under oxidative stress. ODHL augmented the cytotoxicity of H2O2, but not calcium ionophore A23187. ODHL potentiated the H2O2-induced elevation of [Zn2+]i, wherein, it greatly attenuated the H2O2-induced increase in intracellular Ca2+ concentration. ODHL did not affect [NPT]i in the presence of H2O2. Therefore, we conclude that the elevation of [Zn2+]i is involved in the ODHL-induced potentiation of H2O2 cytotoxicity. Our findings suggest that ODHL modifies cell vulnerability to oxidative stress in host cells.


Assuntos
4-Butirolactona/análogos & derivados , Estresse Oxidativo/efeitos dos fármacos , Timócitos/efeitos dos fármacos , 4-Butirolactona/farmacologia , Animais , Calcimicina/farmacologia , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Masculino , Percepção de Quorum/efeitos dos fármacos , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Zinco/metabolismo
12.
Biochem Biophys Res Commun ; 510(2): 272-277, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30711250

RESUMO

Activation of the receptor activator of nuclear factor-κB (RANK) by its ligand (RANKL) is involved in both solid and hematological malignancies, including multiple myeloma, acute myeloid leukemia and B-cell leukemia. Although RANKL expression has been described in normal T cells, a potential role in T-cell leukemia remains undefined. Here, we used a model of immature T-cell leukemia/lymphoma, the TEL-JAK2 transgenic mice, to assess RANKL expression in leukemic cells and its regulatory mechanisms. We found that Rankl mRNA was significantly overexpressed in leukemic T cells when compared to wild-type thymocytes, their nonmalignant counterparts. Moreover, Rankl mRNA and RANKL surface expression in leukemic cells was induced by T-cell receptor (TCR) signaling activation, dependently on the NF-κB signaling pathway. These results indicate that TCR-activated leukemic T cells express high levels of RANKL and are potential inducers of RANK signaling in microenvironmental cells.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia de Células T/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Ligante RANK/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Subunidade p50 de NF-kappa B/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Timócitos/metabolismo , Microambiente Tumoral
13.
Toxicol Lett ; 304: 30-38, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30605750

RESUMO

Clinical study showed that smoking during pregnancy deceased the thymus size in newborns. However, the long-term effect remains unclear. This study was aimed to observe the effects of prenatal nicotine exposure (PNE) on the development of thymus and the T-lymphocyte subpopulation in mice offspring from the neonatal to adulthood. Both the thymus weight and cytometry data indicated that PNE caused persistent thymic hypoplasia in male offspring from neonatal to adult period and transient changes in female offspring from neonatal to prepuberal period. Flow cytometry analysis disclosed a permanent decreased proportion and number of mature CD4 single-positive (SP) T cells in thymus of both sex. In addition, the PNE male offspring showed a more serious thymus atrophy in the ovalbumin (OVA)-sensitized model. Moreover, increased autophagic vacuole and elevated mRNA expression of Beclin 1 were noted in PNE fetal thymus. In conclusion, PNE offspring showed thymus atrophy and CD 4 SP T cell reduction at different life stages. Mechanically, PNE induced excessive autophagy in fetal thymocytes might be involved in these changes. All the results provided evidence for elucidating the PNE-induced programmed immune diseases.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Doenças do Sistema Imunitário/induzido quimicamente , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Timócitos/efeitos dos fármacos , Timo/efeitos dos fármacos , Fatores Etários , Animais , Animais Recém-Nascidos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Feminino , Doenças do Sistema Imunitário/imunologia , Doenças do Sistema Imunitário/metabolismo , Doenças do Sistema Imunitário/patologia , Masculino , Exposição Materna , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Fenótipo , Gravidez , Timócitos/imunologia , Timócitos/metabolismo , Timócitos/patologia , Timo/imunologia , Timo/metabolismo , Timo/patologia
14.
Chem Biol Interact ; 300: 35-39, 2019 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-30629953

RESUMO

Azoxystrobin, a broad-spectrum fungicide, has been increasingly used in the agricultural industry. In Japan in 2018, azoxystrobin at five times the normal limit was detected in a shipment of Australian barley that had been used in food products. Therefore, the effects of azoxystrobin need to be carefully examined to predict potential adverse reactions in humans. In this study, the effects of azoxystrobin on the membrane potential and intracellular Ca2+ levels of thymocytes have been photochemically examined using flow cytometry. Azoxystrobin hyperpolarized plasma membrane potential. This hyperpolarization appeared to be due to the activation of Ca2+-dependent K+ channels, as both the removal of extracellular Ca2+ and addition of charybdotoxin attenuated the observed hyperpolarization. In the presence of quinine, an anti-malarial drug that blocks Ca2+-dependent K+ channels, azoxystrobin depolarized the membranes instead. Azoxystrobin increased intracellular Ca2+ levels in a concentration-dependent manner through the influx of extracellular Ca2+ and intracellular release of Ca2+, as confirmed by reduction in azoxystrobin-induced response in the absence of extracellular Ca2+. It appears likely that azoxystrobin at micromolar concentrations modifies membrane ion permeability in thymocytes. Since changes in membrane potential and intracellular Ca2+ levels occur during typical physiological lymphocyte responses, azoxystrobin may disturb lymphocyte function.


Assuntos
Fungicidas Industriais/farmacologia , Pirimidinas/farmacologia , Estrobilurinas/farmacologia , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Masculino , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Canais de Potássio Cálcio-Ativados/metabolismo , Quinina/farmacologia , Ratos , Ratos Wistar , Timócitos/citologia , Timócitos/efeitos dos fármacos , Timócitos/metabolismo
15.
PLoS Pathog ; 15(1): e1007456, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30608984

RESUMO

Innate CD8+ T cells express a memory-like phenotype and demonstrate a strong cytotoxic capacity that is critical during the early phase of the host response to certain bacterial and viral infections. These cells arise in the thymus and depend on IL-4 and IL-15 for their development. Even though innate CD8+ T cells exist in the thymus of WT mice in low numbers, they are highly enriched in KO mice that lack certain kinases, leading to an increase in IL-4 production by thymic NKT cells. Our work describes that in C57BL/6 WT mice undergoing a Th1 biased infectious disease, the thymus experiences an enrichment of single positive CD8 (SP8) thymocytes that share all the established phenotypical and functional characteristics of innate CD8+ T cells. Moreover, through in vivo experiments, we demonstrate a significant increase in survival and a lower parasitemia in mice adoptively transferred with SP8 thymocytes from OT I-T. cruzi-infected mice, demonstrating that innate CD8+ thymocytes are able to protect against a lethal T. cruzi infection in an Ag-independent manner. Interestingly, we obtained similar results when using thymocytes from systemic IL-12 + IL-18-treated mice. This data indicates that cytokines triggered during the acute stage of a Th1 infectious process induce thymic production of IL-4 along with IL-15 expression resulting in an adequate niche for development of innate CD8+ T cells as early as the double positive (DP) stage. Our data demonstrate that the thymus can sense systemic inflammatory situations and alter its conventional CD8 developmental pathway when a rapid innate immune response is required to control different types of pathogens.


Assuntos
Interleucina-15/metabolismo , Interleucina-4/metabolismo , Timo/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/imunologia , Citocinas/metabolismo , Feminino , Imunidade Inata/genética , Interleucina-12/metabolismo , Interleucina-15/genética , Interleucina-18/metabolismo , Interleucina-4/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia , Transdução de Sinais , Células Th1/imunologia , Timócitos/metabolismo , Timo/metabolismo , Timo/patologia
16.
J Immunol ; 202(4): 1033-1038, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626694

RESUMO

Intricate life-versus-death decisions are programmed during T cell development, and the regulatory mechanisms that coordinate their activation and repression are still under investigation. In this study, HDAC3-deficient double-positive (DP) thymocytes exhibit a severe decrease in numbers. The thymic cortex is rich in ATP, which is released by macrophages that clear apoptotic DP thymocytes that fail to undergo positive selection. We demonstrate that HDAC3 is required to repress expression of the purinergic receptor P2X7 to prevent DP cell death. HDAC3-deficient DP thymocytes upregulate the P2X7 receptor, increasing sensitivity to ATP-induced cell death. P2rx7/HDAC3-double knockout mice show a partial rescue in DP cell number. HDAC3 directly binds to the P2rx7 enhancer, which is hyperacetylated in the absence of HDAC3. In addition, RORγt binds to the P2rx7 enhancer and promotes P2X7 receptor expression in the absence of HDAC3. Therefore, HDAC3 is a critical regulator of DP thymocyte survival and is required to suppress P2X7 receptor expression.


Assuntos
Morte Celular , Histona Desacetilases/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Timócitos/citologia , Timócitos/enzimologia , Animais , Histona Desacetilases/deficiência , Camundongos , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores Purinérgicos P2X7/genética , Timócitos/metabolismo
17.
J Exp Med ; 216(1): 231-243, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30545902

RESUMO

Expression of Rag1 and Rag2 is tightly regulated in developing T cells to mediate TCR gene assembly. Here we have investigated the molecular mechanisms governing the assembly and disassembly of a transcriptionally active RAG locus chromatin hub in CD4+CD8+ thymocytes. Rag1 and Rag2 gene expression in CD4+CD8+ thymocytes depends on Rag1 and Rag2 promoter activation by a distant antisilencer element (ASE). We identify GATA3 and E2A as critical regulators of the ASE, and Runx1 and E2A as critical regulators of the Rag1 promoter. We reveal hierarchical assembly of a transcriptionally active chromatin hub containing the ASE and RAG promoters, with Rag2 recruitment and expression dependent on assembly of a functional ASE-Rag1 framework. Finally, we show that signal-dependent down-regulation of RAG gene expression in CD4+CD8+ thymocytes depends on Ikaros and occurs with disassembly of the RAG locus chromatin hub. Our results provide important new insights into the molecular mechanisms that orchestrate RAG gene expression in developing T cells.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica/fisiologia , Loci Gênicos/fisiologia , Proteínas de Homeodomínio/biossíntese , Timócitos/metabolismo , Transcrição Genética/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Antígenos CD4/genética , Antígenos CD4/metabolismo , Antígenos CD8/genética , Antígenos CD8/metabolismo , Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Proteínas de Homeodomínio/genética , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Camundongos , Elementos de Resposta/fisiologia , Timócitos/citologia
18.
Immunol Lett ; 206: 41-48, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30503823

RESUMO

Tolerance induction is a goal of clinical transplantation to prevent graft rejection without the lifelong use of immunosuppressive drugs. In a series of mouse studies, we previously reported that the establishment of mixed chimerism by treatment with a ligand for invariant natural killer T (iNKT) cells with CD40 signal blockade makes it possible to prevent allograft rejection without immunosuppressants, and this approach fails in thymectomized recipient mice. In this study, we showed that iNKT cells in murine thymocyte cultures are indispensable for the expansion of CD4+CD25+Foxp3+ regulatory T (Treg) cells as well as CD4+CD25+Foxp3- cells, which contained precursor Tregs (preTregs). After the culture of BALB/c mouse-derived thymocytes in the presence of α-galactosylceramide (α-GalCer), a representative ligand for iNKT cells, the ratio of CD4+CD25+Foxp3- preTregs to total CD4+CD8- T cells was much higher than that of CD4+CD25+Foxp3+ Treg cells, regardless of anti-CD40 L mAb treatment. The proliferation of CD4+CD25+Foxp3- cells, but not Treg cells, was significantly augmented, and the stability of Treg cells was not affected by α-GalCer. The expansion of thymocyte-derived Tregs was not inhibited by cytokine neutralization. However, in vitro thymus-derived CD4+CD25+Foxp3- cells expressed Foxp3 after IL-2 stimulation in a dose-dependent manner. These results collectively suggest that in vitro thymus-derived Treg cell expansion by α-GalCer treatment was caused by the proliferation of CD4+CD25+Foxp3- preTregs but not existing Treg cells.


Assuntos
Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Linfócitos T Reguladores/imunologia , Timócitos/imunologia , Animais , Biomarcadores , Comunicação Celular , Células Cultivadas , Citocinas/biossíntese , Imunofenotipagem , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/metabolismo , Linfócitos T Reguladores/metabolismo , Timócitos/metabolismo
19.
Am J Physiol Cell Physiol ; 316(1): C70-C80, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30404560

RESUMO

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder, affecting the quality of life of millions of people worldwide. The present study aims to determine the relationship between micro-RNA-143 (miR-143) and C-X-C motif chemokine 13 (CXCL13) and whether it influences the pathogenesis of myasthenia gravis (MG). Thymus specimens were resected from patients with thymic hyperplasia combined with MG and then infused into normal mouse cavities to establish MG mouse models. Immunohistochemistry, reverse transcription-quantitative PCR, in situ hybridization detection, and Western blot analysis were employed to identify the expression of miR-143 and CXCL13 in MG and normal mice. The obtained thymocytes were cultured in vitro and transfected with a series of miR-143 mimic, miR-143 inhibitor, overexpression of CXCL13, or siRNA against CXCL13. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and flow cytometry assays were employed to assess cell viability, cycle entry, and apoptosis of the thymocytes. Dual-luciferase reporter assay provided verification, confirming that CXCL13 was the target gene of miR-143. Low miR-143 expression in the thymus tissues of the MG mice was detected, which presented with a reciprocal relationship with the expression rate of CLCX13. Observations in relation to the interactions between miR-143 mimic or siRNA-CXCL13 exposure showed reduced cell viability, with a greater number of cells arrested at the G0/G1 phase and a greater rate of induced apoptosis. Furthermore, overexpression of CXCL13 rescued miR-143 mimic-induced apoptosis. The findings have identified the potential role of miR-143 as a MG development mediator by targeting CXCL13. The key results obtained provide a promising experimental basis for targeted intervention treatment with miR-143.


Assuntos
Proliferação de Células/fisiologia , Quimiocina CXCL13/biossíntese , Modelos Animais de Doenças , MicroRNAs/biossíntese , Miastenia Gravis/metabolismo , Timócitos/metabolismo , Adolescente , Adulto , Animais , Apoptose/fisiologia , Células Cultivadas , Quimiocina CXCL13/antagonistas & inibidores , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Miastenia Gravis/patologia , Timócitos/patologia , Adulto Jovem
20.
Chem Biol Interact ; 298: 52-56, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30391638

RESUMO

Benzophenone-3 (BP-3) is often used as an ultraviolet (UV) light filter in sunscreen products. Although BP-3 protects the human skin and hair from damage caused by excessive UV radiation, it is reported to exhibit toxic effects in human. However, the cytotoxicity of BP-3 on various cells is still not well understood. In the present study, the cytotoxicity of BP-3 against rat thymocytes was evaluated using a flow cytometric technique with fluorescent probes. Cell mortality increased significantly after 3 h of exposure to 300 µM BP-3, whereas the mean intensity of 5-chloromethylfluorescein diacetate (5-CMF) fluorescence and cellular content of non-protein thiols decreased significantly. However, the membrane potential of thymocytes was not change by BP-3 treatment. Moreover, intracellular Zn2+ levels increased significantly in a concentration-dependent manner in response to 30 µM BP-3 or higher. The BP-3-induced changes in intracellular Zn2+ levels and non-protein thiol content increased the vulnerability of thymocytes to oxidative stress. We concluded that BP-3-induced cytotoxicity may be caused by oxidative stress associated with an increase in intracellular Zn2+ levels.


Assuntos
Benzofenonas/toxicidade , Protetores Solares/toxicidade , Timócitos/efeitos dos fármacos , Timócitos/metabolismo , Zinco/metabolismo , Animais , Sobrevivência Celular , Sinergismo Farmacológico , Peróxido de Hidrogênio/toxicidade , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos Wistar , Compostos de Sulfidrila/metabolismo
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