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1.
Scand J Immunol ; 94(4): e13094, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34780092

RESUMO

The thymus produces self-limiting and self-tolerant T cells through the interaction between thymocytes and thymus epithelial cells (TECs), thereby generating central immune tolerance. The TECs are composed of cortical and medullary thymic epithelial cells, which regulate the positive and negative selection of T cells, respectively. During the process of negative selection, thymocytes with self-reactive ability are deleted or differentiated into regulatory T cells (Tregs). Tregs are a subset of suppressor T cells that are important for maintaining immune homeostasis. The differentiation and development of Tregs depend on the development of TECs and other underlying molecular mechanisms. Tregs regulated by thymic epithelial cells are closely related to human health and are significant in autoimmune diseases, thymoma and pregnancy. In this review, we summarize the current molecular and transcriptional regulatory mechanisms by which TECs affect the development and function of thymic Tregs. We also review the pathophysiological models of thymic epithelial cells regulating thymic Tregs in human diseases and specific physiological conditions.


Assuntos
Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Imunidade Adaptativa , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Epiteliais/classificação , Células Epiteliais/citologia , Células Epiteliais/imunologia , Feminino , Homeostase , Humanos , Masculino , Modelos Imunológicos , Poliendocrinopatias Autoimunes/genética , Poliendocrinopatias Autoimunes/imunologia , Gravidez , Transdução de Sinais/imunologia , Linfócitos T Reguladores/classificação , Timócitos/classificação , Timócitos/citologia , Timócitos/imunologia , Timoma/imunologia , Timo/citologia , Timo/imunologia , Neoplasias do Timo/imunologia
2.
J Vis Exp ; (175)2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34633363

RESUMO

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt signaling operates via a dosage-driven mechanism, motivating the need to study and measure Wnt signaling in various cell types. Although several reporter models have been proposed to represent physiological Wnt expression, either the genetic context or the reporter protein highly influenced the validity, accuracy, and flexibility of these tools. This paper describes methods for acquiring and analyzing data obtained with the Axin2-mTurquoise2 mouse Wnt reporter model, which contains a mutated Axin2em1Fstl allele. This model facilitates the study of endogenous canonical Wnt signaling in individual cells over a wide range of Wnt activity. This protocol describes how to fully appreciate Axin2-mTurquoise2 reporter activity using cell population analysis of the hematopoietic system, combined with cell surface markers or ß-catenin intracellular staining. These procedures serve as a base for implementation and reproduction in other tissues or cells of interest. By combining fluorescence-activated cell sorting and confocal imaging, distinct canonical Wnt expression levels can be visualized. The recommended measurement and analysis strategies provide quantitative data on the fluorescent expression levels for precise assessment of canonical Wnt signaling. These methods will be useful for researchers who want to use the Axin2-mTurquise2 model for canonical Wnt expression patterns.


Assuntos
Timócitos , Via de Sinalização Wnt , Animais , Proteína Axina/genética , Citometria de Fluxo , Indicadores e Reagentes , Camundongos
3.
Int J Mol Sci ; 22(18)2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34576301

RESUMO

Organochlorine pesticides, such as DDT, methoxychlor, and their metabolites, have been characterized as endocrine disrupting chemicals (EDCs); suggesting that their modes of action involve interaction with or abrogation of endogenous endocrine function. This study examined whether embryonic thymocyte death and alteration of differentiation induced by the primary metabolite of methoxychlor, HPTE, rely upon estrogen receptor binding and concurrent T cell receptor signaling. Estrogen receptor inhibition of ERα or GPER did not rescue embryonic thymocyte death induced by HPTE or the model estrogen diethylstilbestrol (DES). Moreover, adverse effects induced by HPTE or DES were worsened by concurrent TCR and CD2 differentiation signaling, compared with EDC exposure post-signaling. Together, these data suggest that HPTE- and DES-induced adverse effects on embryonic thymocytes do not rely solely on ER alpha or GPER but may require both. These results also provide evidence of a potential collaborative signaling mechanism between TCR and estrogen receptors to mediate adverse effects on embryonic thymocytes, as well as highlight a window of sensitivity that modulates EDC exposure severity.


Assuntos
Diferenciação Celular , Disruptores Endócrinos/toxicidade , Receptor alfa de Estrogênio/metabolismo , Fenóis/toxicidade , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Timócitos/efeitos dos fármacos , Animais , Antígenos CD2/metabolismo , Morte Celular , Células Cultivadas , Receptor alfa de Estrogênio/antagonistas & inibidores , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Estrogênio/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais , Timócitos/citologia , Timócitos/metabolismo
4.
Int J Mol Sci ; 22(18)2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34575873

RESUMO

Sjögren's syndrome (SjS) is a chronic autoimmune disease primarily involving the exocrine glands in which the involvement of the innate immune system is largely uncharacterized. Mer signaling has been found to be protective in several autoimmune diseases but remains unstudied in SjS. Here, we investigated the role of Mer signaling in SjS. Mer knockout (MerKO) mice were examined for SjS disease criteria. SjS-susceptible (SjSS) C57BL/6.NOD-Aec1Aec2 mice were assessed for defective Mer signaling outcomes, soluble Mer (sMer) levels, A disintegrin and metalloprotease 17 (ADAM17) activity, and Rac1 activation. In addition, SjS patient plasma samples were evaluated for sMer levels via ELISA, and sMer levels were correlated to disease manifestations. MerKO mice developed submandibular gland (SMG) lymphocytic infiltrates, SMG apoptotic cells, anti-nuclear autoantibodies (ANA), and reduced saliva flow. Mer signaling outcomes were observed to be diminished in SjSS mice, as evidenced by reduced Rac1 activation in SjSS mice macrophages in response to apoptotic cells and impaired efferocytosis. Increased sMer was also detected in SjSS mouse sera, coinciding with higher ADAM17 activity, the enzyme responsible for cleavage and inactivation of Mer. sMer levels were elevated in patient plasma and positively correlated with focus scores, ocular staining scores, rheumatoid factors, and anti-Ro60 levels. Our data indicate that Mer plays a protective role in SjS, similar to other autoimmune diseases. Furthermore, we suggest a series of events where enhanced ADAM17 activity increases Mer inactivation and depresses Mer signaling, thus removing protection against the loss of self-tolerance and the onset of autoimmune disease in SjSS mice.


Assuntos
Regulação Enzimológica da Expressão Gênica , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , c-Mer Tirosina Quinase/genética , Proteína ADAM17/metabolismo , Animais , Anticorpos Antinucleares/química , Apoptose , Autoanticorpos/metabolismo , Autoimunidade , Modelos Animais de Doenças , Feminino , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Fenótipo , Saliva/metabolismo , Transdução de Sinais , Timócitos/metabolismo
5.
J Immunol ; 207(4): 1055-1064, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34312259

RESUMO

Central tolerance aims to limit the production of T lymphocytes bearing TCR with high affinity for self-peptide presented by MHC molecules. The accumulation of thymocytes with such receptors is limited by negative selection or by diversion into alternative differentiation, including T regulatory cell commitment. A role for the orphan nuclear receptor NR4A3 in negative selection has been suggested, but its function in this process has never been investigated. We find that Nr4a3 transcription is upregulated in postselection double-positive thymocytes, particularly those that have received a strong selecting signal and are destined for negative selection. Indeed, we found an accumulation of cells bearing a negative selection phenotype in NR4A3-deficient mice as compared with wild-type controls, suggesting that Nr4a3 transcriptional induction is necessary to limit accumulation of self-reactive thymocytes. This is consistent with a decrease of cleaved caspase-3+-signaled thymocytes and more T regulatory and CD4+Foxp3-HELIOS+ cells in the NR4A3-deficient thymus. We further tested the role for NR4A3 in negative selection by reconstituting transgenic mice expressing the OVA Ag under the control of the insulin promoter with bone marrow cells from OT-I Nr4a3 +/+ or OT-I Nr4a3 -/- mice. Accumulation of autoreactive CD8 thymocytes and autoimmune diabetes developed only in the absence of NR4A3. Overall, our results demonstrate an important role for NR4A3 in T cell development.


Assuntos
Diabetes Mellitus Tipo 1 , Receptores de Esteroides , Animais , Proteínas de Ligação a DNA , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso , Receptores dos Hormônios Tireóideos , Timócitos , Fatores de Transcrição
6.
Development ; 148(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34323272

RESUMO

During positive selection at the transition from CD4+CD8+ double-positive (DP) to single-positive (SP) thymocyte, TCR signalling results in appropriate MHC restriction and signals for survival and progression. We show that the pioneer transcription factors Foxa1 and Foxa2 are required to regulate RNA splicing during positive selection of mouse T cells and that Foxa1 and Foxa2 have overlapping/compensatory roles. Conditional deletion of both Foxa1 and Foxa2 from DP thymocytes reduced positive selection and development of CD4SP, CD8SP and peripheral naïve CD4+ T cells. Foxa1 and Foxa2 regulated the expression of many genes encoding splicing factors and regulators, including Mbnl1, H1f0, Sf3b1, Hnrnpa1, Rnpc3, Prpf4b, Prpf40b and Snrpd3. Within the positively selecting CD69+DP cells, alternative RNA splicing was dysregulated in the double Foxa1/Foxa2 conditional knockout, leading to >850 differentially used exons. Many genes important for this stage of T-cell development (Ikzf1-3, Ptprc, Stat5a, Stat5b, Cd28, Tcf7) and splicing factors (Hnrnpab, Hnrnpa2b1, Hnrnpu, Hnrnpul1, Prpf8) showed multiple differentially used exons. Thus, Foxa1 and Foxa2 are required during positive selection to regulate alternative splicing of genes essential for T-cell development, and, by also regulating splicing of splicing factors, they exert widespread control of alternative splicing.


Assuntos
Processamento Alternativo/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/genética , Splicing de RNA/genética , Timócitos/fisiologia , Animais , Éxons/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Processamento de RNA/genética , Linfócitos T/fisiologia , Timo/fisiologia
7.
Front Immunol ; 12: 669943, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34211466

RESUMO

Major Histocompatibility Complex (MHC) class II (MHCII) deficiency (MHCII-D), also known as Bare Lymphocyte Syndrome (BLS), is a rare combined immunodeficiency due to mutations in genes regulating expression of MHCII molecules. MHCII deficiency results in impaired cellular and humoral immune responses, leading to severe infections and autoimmunity. Abnormal cross-talk with developing T cells due to the absence of MHCII expression likely leads to defects in thymic epithelial cells (TEC). However, the contribution of TEC alterations to the pathogenesis of this primary immunodeficiency has not been well characterized to date, in particular in regard to immune dysregulation. To this aim, we have performed an in-depth cellular and molecular characterization of TEC in this disease. We observed an overall perturbation of thymic structure and function in both MHCII-/- mice and patients. Transcriptomic and proteomic profiling of murine TEC revealed several alterations. In particular, we demonstrated that impairment of lymphostromal cross-talk in the thymus of MHCII-/- mice affects mTEC maturation and promiscuous gene expression and causes defects of central tolerance. Furthermore, we observed peripheral tolerance impairment, likely due to defective Treg cell generation and/or function and B cell tolerance breakdown. Overall, our findings reveal disease-specific TEC defects resulting in perturbation of central tolerance and limiting the potential benefits of hematopoietic stem cell transplantation in MHCII deficiency.


Assuntos
Células Epiteliais/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Tolerância Imunológica , Imunodeficiência Combinada Severa/imunologia , Timo/imunologia , Adolescente , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Europa (Continente) , Feminino , Transplante de Células-Tronco Hematopoéticas , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Lactente , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , América do Norte , Proteoma , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/metabolismo , Imunodeficiência Combinada Severa/cirurgia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Timócitos , Timo/metabolismo , Transcriptoma , Adulto Jovem
8.
Front Immunol ; 12: 668528, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34220815

RESUMO

The microenvironments formed by cortical (c) and medullary (m) thymic epithelial cells (TECs) play a non-redundant role in the generation of functionally diverse and self-tolerant T cells. The role of TECs during the first weeks of the murine postnatal life is particularly challenging due to the significant augment in T cell production. Here, we critically review recent studies centered on the timely coordination between the expansion and maturation of TECs during this period and their specialized role in T cell development and selection. We further discuss how aging impacts on the pool of TEC progenitors and maintenance of functionally thymic epithelial microenvironments, and the implications of these chances in the capacity of the thymus to sustain regular thymopoiesis throughout life.


Assuntos
Diferenciação Celular , Proliferação de Células , Células Epiteliais/fisiologia , Células-Tronco/fisiologia , Timo/fisiologia , Fatores Etários , Animais , Microambiente Celular , Células Epiteliais/imunologia , Humanos , Tolerância a Antígenos Próprios , Células-Tronco/imunologia , Linfócitos T/imunologia , Timócitos/imunologia , Timo/imunologia
9.
Front Immunol ; 12: 682220, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34305914

RESUMO

Regnase-1 is an RNA-binding protein with ribonuclease activities, and once induced it controls diverse immune responses by degrading mRNAs that encode inflammatory cytokines and costimulatory molecules, thus exerting potent anti-inflammatory functions. However, Regnase-1 is extremely sensitive to degradation by proteases and therefore short-lived. Here, we constructed a mutant Regnase-1 that is resistant to degradation and expressed this mutant in vivo as a transgene specifically in T cells. We found that the mutant Regnase-1 transgenic mice exhibited profound lymphopenia in the periphery despite grossly normal spleen and lymph nodes, and spontaneously accepted skin allografts without any treatment. Mechanistic studies showed that in the transgenic mice thymic T cell development was disrupted, such that most of the developing thymocytes were arrested at the double positive stage, with few mature CD4+ and CD8+ T cells in the thymus and periphery. Our findings suggest that interfering with the dynamic Regnase-1 expression in T cells disrupts T cell development and functions and further studies are warranted to uncover the mechanisms involved.


Assuntos
Expressão Gênica , Linfopoese , Mutação , Ribonucleases/genética , Linfócitos T/metabolismo , Diferenciação Celular , Ativação Enzimática , Pele , Baço , Linfócitos T/citologia , Timócitos
10.
Elife ; 102021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34240701

RESUMO

Signals from the pre-T cell receptor and Notch coordinately instruct ß-selection of CD4-CD8-double negative (DN) thymocytes to generate αß T cells in the thymus. However, how these signals ensure a high-fidelity proteome and safeguard the clonal diversification of the pre-selection TCR repertoire given the considerable translational activity imposed by ß-selection is largely unknown. Here, we identify the endoplasmic reticulum (ER)-associated degradation (ERAD) machinery as a critical proteostasis checkpoint during ß-selection. Expression of the SEL1L-HRD1 complex, the most conserved branch of ERAD, is directly regulated by the transcriptional activity of the Notch intracellular domain. Deletion of Sel1l impaired DN3 to DN4 thymocyte transition and severely impaired mouse αß T cell development. Mechanistically, Sel1l deficiency induced unresolved ER stress that triggered thymocyte apoptosis through the PERK pathway. Accordingly, genetically inactivating PERK rescued T cell development from Sel1l-deficient thymocytes. In contrast, IRE1α/XBP1 pathway was induced as a compensatory adaptation to alleviate Sel1l-deficiency-induced ER stress. Dual loss of Sel1l and Xbp1 markedly exacerbated the thymic defect. Our study reveals a critical developmental signal controlled proteostasis mechanism that enforces T cell development to ensure a healthy adaptive immunity.


Assuntos
Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Receptores Notch/metabolismo , Timócitos/metabolismo , Animais , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Feminino , Inflamação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/metabolismo , Proteostase , Ubiquitina-Proteína Ligases/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo
11.
Commun Biol ; 4(1): 681, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083746

RESUMO

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. In the absence of CCT-controlled protein folding, Th2 polarization diverges from normal differentiation with paradoxical continued IFN-γ expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity.


Assuntos
Chaperonina com TCP-1/imunologia , Proteostase/imunologia , Linfócitos T/imunologia , Timócitos/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Chaperonina com TCP-1/genética , Chaperonina com TCP-1/metabolismo , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteoma/imunologia , Proteoma/metabolismo , Proteostase/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Transcriptoma/genética , Transcriptoma/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
12.
Chin Med J (Engl) ; 134(15): 1855-1865, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34133355

RESUMO

BACKGROUND: Endotoxin tolerance (ET) is a protective phenomenon in which pre-treatment with a tolerance dose of lipopolysaccharide (LPS) leads to dramatically elevated survival. Accumulating evidence has shown that peripheral T cells contribute to the induction of ET. However, what happens to T cell development in the thymus under ET conditions remains unclear. The purpose of this study was to analyze the alterations in thymocyte populations (double-positive [DP] and single-positive [SP] cells) under ET conditions. METHODS: Mice were intraperitoneally injected with LPS at a concentration of 5 mg/kg to establish an LPS tolerance model and were divided into two groups: a group examined 72 h after LPS injection (72-h group) and a group examined 8 days after LPS injection (8-day group). Injection of phosphate-buffered saline was used as a control (control group). Changes in thymus weight, cell counts, and morphology were detected in the three groups. Moreover, surface molecules such as CD4, CD8, CD44, CD69, and CD62L were analyzed using flow cytometry. Furthermore, proliferation, apoptosis, cytokine production, and extracellular signal-regulated kinase (ERK) pathway signaling were analyzed in thymocyte populations. The polymorphism and length of the T-cell receptor (TCR) ß chain complementarity-determining region 3 (CDR3) were analyzed using capillary electrophoresis DNA laser scanning analysis (ABI 3730). RESULTS: Thymus weight and cell counts were decreased in the early stage but recovered by the late stage in a murine model of LPS-induced ET. Moreover, the proportions of DP cells (control: 72.130 ±â€Š4.074, 72-h: 10.600 ±â€Š3.517, 8-day: 84.770 ±â€Š2.228), CD4+ SP cells (control: 15.770 ±â€Š4.419, 72-h: 44.670 ±â€Š3.089, 8-day: 6.367 ±â€Š0.513), and CD8+ SP cells (control: 7.000 ±â€Š1.916, 72-h: 34.030 ±â€Š3.850, 8-day: 5.133 ±â€Š0.647) were obviously different at different stages of ET. The polymorphism and length of TCR ß chain CDR3 also changed obviously, indicating the occurrence of TCR rearrangement and thymocyte diversification. Further analysis showed that the expression of surface molecules, including CD44, CD69, and CD62L, on thymocyte populations (DP and SP cells) were changed to different degrees. Finally, the proliferation, apoptosis, cytokine production, and ERK pathway signaling of thymocyte populations were changed significantly. CONCLUSION: These data reveal that alterations in thymocyte populations might contribute to the establishment of ET.


Assuntos
Endotoxinas , Timócitos , Animais , Linfócitos T CD4-Positivos , Diferenciação Celular , Endotoxinas/toxicidade , Citometria de Fluxo , Camundongos , Transdução de Sinais , Timo
13.
Nat Commun ; 12(1): 3933, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168132

RESUMO

Thymic T cell development and T cell receptor repertoire selection are dependent on essential molecular cues provided by thymic epithelial cells (TEC). TEC development and function are regulated by their epigenetic landscape, in which the repressive H3K27me3 epigenetic marks are catalyzed by polycomb repressive complex 2 (PRC2). Here we show that a TEC-targeted deficiency of PRC2 function results in a hypoplastic thymus with reduced ability to express antigens and select a normal repertoire of T cells. The absence of PRC2 activity reveals a transcriptomically distinct medullary TEC lineage that incompletely off-sets the shortage of canonically-derived medullary TEC whereas cortical TEC numbers remain unchanged. This alternative TEC development is associated with the generation of reduced TCR diversity. Hence, normal PRC2 activity and placement of H3K27me3 marks are required for TEC lineage differentiation and function and, in their absence, the thymus is unable to compensate for the loss of a normal TEC scaffold.


Assuntos
Epigênese Genética , Células Epiteliais/citologia , Complexo Repressor Polycomb 2/genética , Timo/citologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Epiteliais/fisiologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Repressor Polycomb 2/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologia , Linfócitos T/fisiologia , Timócitos/citologia , Timócitos/fisiologia , Timo/fisiologia
14.
Front Immunol ; 12: 669893, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34140950

RESUMO

Down syndrome (DS) patients prematurely show clinical manifestations usually associated with aging. Their immune system declines earlier than healthy individuals, leading to increased susceptibility to infections and higher incidence of autoimmune phenomena. Clinical features of accelerated aging indicate that trisomy 21 increases the biological age of tissues. Based on previous studies suggesting immune senescence in DS, we hypothesized that induction of cellular senescence may contribute to early thymic involution and immune dysregulation. Immunohistochemical analysis of thymic tissue showed signs of accelerated thymic aging in DS patients, normally seen in older healthy subjects. Moreover, our whole transcriptomic analysis on human Epcam-enriched thymic epithelial cells (hTEC), isolated from three DS children, which revealed disease-specific transcriptomic alterations. Gene set enrichment analysis (GSEA) of DS TEC revealed an enrichment in genes involved in cellular response to stress, epigenetic histone DNA modifications and senescence. Analysis of senescent markers and oxidative stress in hTEC and thymocytes confirmed these findings. We detected senescence features in DS TEC, thymocytes and peripheral T cells, such as increased ß-galactosidase activity, increased levels of the cell cycle inhibitor p16, telomere length and integrity markers and increased levels of reactive oxygen species (ROS), all factors contributing to cellular damage. In conclusion, our findings support the key role of cellular senescence in the pathogenesis of immune defect in DS while adding new players, such as epigenetic regulation and increased oxidative stress, to the pathogenesis of immune dysregulation.


Assuntos
Proliferação de Células , Senescência Celular , Síndrome de Down/metabolismo , Células Epiteliais/metabolismo , Imunossenescência , Estresse Oxidativo , Timócitos/metabolismo , Timo/metabolismo , Fatores Etários , Estudos de Casos e Controles , Proliferação de Células/genética , Senescência Celular/genética , Criança , Pré-Escolar , Síndrome de Down/genética , Síndrome de Down/imunologia , Síndrome de Down/patologia , Epigênese Genética , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Imunossenescência/genética , Lactente , Masculino , Estresse Oxidativo/genética , Timócitos/imunologia , Timócitos/patologia , Timo/imunologia , Timo/patologia , Transcriptoma
15.
Cell Death Dis ; 12(6): 526, 2021 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-34023853

RESUMO

Thymic atrophy in sepsis is a critical disadvantage because it induces immunosuppression and increases the mortality rate as the disease progresses. However, the exact mechanism of thymic atrophy has not been fully elucidated. In this study, we discovered a novel role for VSIG4-positive peritoneal macrophages (V4(+) cells) as the principal cells that induce thymic atrophy and thymocyte apoptosis. In CLP-induced mice, V4(+) cells were activated after ingestion of invading microbes, and the majority of these cells migrated into the thymus. Furthermore, these cells underwent a phenotypic shift from V4(+) to V4(-) and from MHC II(low) to MHC II(+). In coculture with thymocytes, V4(+) cells mainly induced apoptosis in DP thymocytes via the secretion of TNF-α. However, there was little effect on CD4 or CD8 SP and DN thymocytes. V4(-) cells showed low levels of activity compared to V4(+) cells. Thymic atrophy in CLP-induced V4(KO) mice was much less severe than that in CLP-induced wild-type mice. In addition, V4(KO) peritoneal macrophages also showed similar activity to V4(-) cells. Taken together, the current study demonstrates that V4(+) cells play important roles in inducing immunosuppression via thymic atrophy in the context of severe infection. These data also suggest that controlling the function of V4(+) cells may play a crucial role in the development of new therapies to prevent thymocyte apoptosis in sepsis.


Assuntos
Macrófagos Peritoneais/fisiologia , Receptores de Complemento/metabolismo , Sepse/patologia , Timócitos/fisiologia , Animais , Apoptose/genética , Ceco/patologia , Ceco/cirurgia , Modelos Animais de Doenças , Feminino , Ligadura , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Punções , Receptores de Complemento/genética , Sepse/genética , Sepse/metabolismo , Timócitos/metabolismo , Timócitos/patologia , Fator de Necrose Tumoral alfa/metabolismo
16.
Exp Cell Res ; 405(1): 112662, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34022203

RESUMO

The cytoophidium, a filamentous structure formed by metabolic enzymes, has emerged as a novel regulatory machinery for certain proteins. The rate-limiting enzymes of de novo CTP and GTP synthesis, cytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH), are the most characterized cytoophidium-forming enzymes in mammalian models. Although the assembly of CTPS cytoophidia has been demonstrated in various organisms including multiple human cancers, a systemic survey for the presence of CTPS cytoophidia in mammalian tissues in normal physiological conditions has not yet been reported. Herein, we examine major organs of adult mouse and observe that CTPS cytoophidia are displayed by a specific thymocyte population ranging between DN3 to early DP stages. Most of these cytoophidium-presenting cells have both CTPS and IMPDH cytoophidia and undergo rapid cell proliferation. In addition, we show that cytoophidium formation is associated with active glycolytic metabolism as the cytoophidium-presenting cells exhibit higher levels of c-Myc, phospho-Akt and PFK. Inhibition of glycolysis with 2DG, however, disrupts most of cytoophidium structures and impairs cell proliferation. Our findings not only indicate that the regulation of CTPS and IMPDH cytoophidia are correlated with the metabolic switch triggered by pre-TCR signaling, but also suggest physiological roles of the cytoophidium in thymocyte development.


Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Citidina Trifosfato/metabolismo , Citoesqueleto/fisiologia , IMP Desidrogenase/metabolismo , Timócitos/citologia , Animais , Proliferação de Células , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Transdução de Sinais , Timócitos/metabolismo
17.
Nucleic Acids Res ; 49(10): 5760-5778, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34037780

RESUMO

Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.


Assuntos
Processamento Alternativo/genética , Carcinogênese/metabolismo , Desenvolvimento Embrionário/genética , Hematopoese/genética , Melanoma/metabolismo , Timócitos/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Apoptose/genética , Carcinogênese/genética , Proliferação de Células/genética , Genômica , Células HEK293 , Humanos , Lectinas Tipo C/metabolismo , Linfopenia/genética , Linfopenia/metabolismo , Melanoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Timo/embriologia , Timo/metabolismo
18.
Eur J Immunol ; 51(7): 1698-1714, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33949677

RESUMO

CD4+ CD8+ double-positive thymocytes give rise to both conventional TCRαß+ T cells and invariant natural killer T cells (iNKT cells), but these two kinds of cells display different characteristics. The molecular mechanism underlying iNKT cell lineage development and function acquisition remain to be elucidated. We show that the loss of chromatin assembly factor 1B (CHAF1b) maintains the normal development of conventional TCRαß+ T cells but severely impairs early development of iNKT cells. This dysregulation is accompanied by the impairment in chromatin activation and gene transcription at Vα14-Jα18 locus. Notably, ectopic expression of a Vα14-Jα18 TCR rescues Chaf1b-deficient iNKT cell developmental defects. Moreover, cytokine secretion and antitumor activity are substantially maintained in Vα14-Jα18 TCR transgene-rescued Chaf1b-deficient iNKT cells. Our study identifies CHAF1b as a critical factor that controls the early development but not function acquisition of iNKT cells via lineage- and stage-specific regulation.


Assuntos
Fator 1 de Modelagem da Cromatina/imunologia , Células T Matadoras Naturais/imunologia , Animais , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Linhagem da Célula/imunologia , Montagem e Desmontagem da Cromatina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Timócitos/imunologia
19.
Histochem Cell Biol ; 156(2): 133-146, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33993340

RESUMO

Immunosuppressive drugs such as cyclosporine A (CSA) can disrupt thymic structure and functions, ultimately inducing syngeneic/autologous graft-versus-host disease together with involuted medullas. To elucidate the effects of CSA on the thymus more precisely, we analyzed the effects of CSA on the thymus and T cell system using rats. In addition to confirming the phenomena already reported, we newly found that the proportion of recent thymic emigrants also greatly decreased, suggesting impaired supply. Immunohistologically, the medullary thymic epithelial cells (mTECs) presented with a relative decrease in the subset with a competent phenotype and downregulation of class II major histocompatibility complex molecules. In control rats, thymic dendritic cells (DCs) comprised two subsets, XCR1+SIRP1α-CD4- and XCR1-SIRP1α+CD4+. The former had a tendency to selectively localize in the previously-reported epithelium-containing areas of the rat medullas, and the number was significantly reduced by CSA treatment. The epithelium-free areas, another unique domains in the rat medullas, contained significantly more Foxp3+ thymic Tregs. With CSA treatment, the epithelium-free areas presented strong involution, and the number and distribution of Tregs in the medulla were greatly reduced. These results suggest that CSA inhibits the production of single-positive thymocytes, including Tregs, and disturbs the microenvironment of the thymic medulla, with a decrease of the competent mTECs and disorganization of epithelium-free areas and DC subsets, leading to a generation of autoreactive T cells with selective medullary involution.


Assuntos
Ciclosporina/farmacologia , Células Epiteliais/efeitos dos fármacos , Fatores de Transcrição Forkhead/análise , Imunossupressores/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Timo/efeitos dos fármacos , Animais , Ciclosporina/administração & dosagem , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Células Epiteliais/patologia , Fatores de Transcrição Forkhead/metabolismo , Imuno-Histoquímica , Imunossupressores/administração & dosagem , Injeções Subcutâneas , Masculino , Imagem Óptica , Ratos , Ratos Endogâmicos Lew , Receptores de Quimiocinas/análise , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/metabolismo , Linfócitos T Reguladores/patologia , Timócitos/efeitos dos fármacos , Timócitos/patologia , Timo/patologia
20.
Sci Rep ; 11(1): 10439, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001954

RESUMO

The thymus facilitates mature T cell production by providing a suitable stromal microenvironment. This microenvironment is impaired by radiation and aging which lead to immune system disturbances known as thymic involution. Young adult thymus shows thymic recovery after such involution. Although various genes have been reported for thymocytes and thymic epithelial cells in such processes, the roles of stromal transcription factors in these remain incompletely understood. MafB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is a transcription factor expressed in thymic stroma and its expression was induced a day after radiation exposure. Hence, the roles of mesenchymal MafB in the process of thymic regeneration offers an intriguing research topic also for radiation biology. The current study investigated whether MafB plays roles in the adult thymus. MafB/green fluorescent protein knock-in mutant (MafB+/GFP) mice showed impaired thymic regeneration after the sublethal irradiation, judged by reduced thymus size, total thymocyte number and medullary complexity. Furthermore, IL4 was induced after irradiation and such induction was reduced in mutant mice. The mutants also displayed signs of accelerated age-related thymic involution. Altogether, these results suggest possible functions of MafB in the processes of thymic recovery after irradiation, and maintenance during aging.


Assuntos
Fator de Transcrição MafB/metabolismo , Regeneração/efeitos da radiação , Timócitos/fisiologia , Timo/fisiologia , Envelhecimento/genética , Animais , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Técnicas de Introdução de Genes , Fator de Transcrição MafB/genética , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Regeneração/genética , Timócitos/efeitos da radiação , Timo/citologia , Timo/efeitos da radiação , Irradiação Corporal Total
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