RESUMEN
To further understand the impact of deficiency of the autoimmune regulator (Aire) gene during the adhesion of medullary thymic epithelial cells (mTECs) to thymocytes, we sequenced single-cell libraries (scRNA-seq) obtained from Aire wild-type (WT) (Airewt/wt ) or Aire-deficient (Airewt/mut ) mTECs cocultured with WT single-positive (SP) CD4+ thymocytes. Although the libraries differed in their mRNA and long noncoding RNA (lncRNA) profiles, indicating that mTECs were heterogeneous in terms of their transcriptome, UMAP clustering revealed that both mTEC lines expressed their specific markers, i.e., Epcam, Itgb4, Itga6, and Casp3 in resting mTECs and Ccna2, Pbk, and Birc5 in proliferative mTECs. Both cocultured SP CD4+ thymocytes remained in a homogeneous cluster expressing the Il7r and Ccr7 markers. Comparisons of the two types of cocultures revealed the differential expression of mRNAs that encode transcription factors (Zfpm2, Satb1, and Lef1), cell adhesion genes (Itgb1) in mTECs, and Themis in thymocytes, which is associated with the regulation of positive and negative selection. At the single-cell sequencing resolution, we observed that Aire acts on both Aire WT and Aire-deficient mTECs as an upstream controller of mRNAs, which encode transcription factors or adhesion proteins that, in turn, are posttranscriptionally controlled by lncRNAs, for example, Neat1, Malat1, Pvt1, and Dancr among others. Under Aire deficiency, mTECs dysregulate the expression of MHC-II, CD80, and CD326 (EPCAM) protein markers as well as metabolism and cell cycle-related mRNAs, which delay the cell cycle progression. Moreover, when adhered to mTECs, WT SP CD4+ or CD8+ thymocytes modulate the expression of cell activation proteins, including CD28 and CD152/CTLA4, and the expression of cellular metabolism mRNAs. These findings indicate a complex mechanism through which an imbalance in Aire expression can affect mTECs and thymocytes during adhesion.
Asunto(s)
Proteína AIRE , Adhesión Celular , Células Epiteliales , ARN Largo no Codificante , Timocitos , Factores de Transcripción , Transcriptoma , ARN Largo no Codificante/genética , Animales , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ratones , Timocitos/metabolismo , Timocitos/inmunología , Timocitos/citología , Células Epiteliales/metabolismo , Células Epiteliales/inmunología , Timo/citología , Timo/inmunología , Timo/metabolismo , Análisis de la Célula Individual , Redes Reguladoras de Genes , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Técnicas de Cocultivo , Perfilación de la Expresión Génica , Ratones NoqueadosRESUMEN
Chagas disease, caused by the protozoan parasite T. cruzi, is a prevalent parasitic disease in Latin America. Presently, it is spreading around the world by human migration, thus representing a new global health issue. Chronically infected individuals reveal a dissimilar disease progression: while nearly 60% remain without apparent disease for life, 30% develop life-threatening pathologies, such as chronic chagasic cardiomyopathy (CCC) or megaviscerae. Inflammation driven by parasite persistence seems to be involved in the pathophysiology of the disease. However, there is also evidence of the occurrence of autoimmune events, mainly caused by molecular mimicry and bystander activation. In experimental models of disease, is well-established that T. cruzi infects the thymus and causes locally profound structural and functional alterations. The hallmark is a massive loss of CD4+CD8+ double positive (DP) thymocytes, mainly triggered by increased levels of glucocorticoids, although other mechanisms seem to act simultaneously. Thymic epithelial cells (TEC) exhibited an increase in extracellular matrix deposition, which are related to thymocyte migratory alterations. Moreover, medullary TEC showed a decreased expression of AIRE and altered expression of microRNAs, which might be linked to a disrupted negative selection of the T-cell repertoire. Also, almost all stages of thymocyte development are altered, including an abnormal output of CD4-CD8- double negative (DN) and DP immature and mature cells, many of them carrying prohibited TCR-Vß segments. Evidence has shown that DN and DP cells with an activated phenotype can be tracked in the blood of humans with chronic Chagas disease and also in the secondary lymphoid organs and heart of infected mice, raising new questions about the relevance of these populations in the pathogenesis of Chagas disease and their possible link with thymic alterations and an immunoendocrine imbalance. Here, we discuss diverse molecular mechanisms underlying thymic abnormalities occurring during T. cruzi infection and their link with CCC, which may contribute to the design of innovative strategies to control Chagas disease pathology.
Asunto(s)
Enfermedad de Chagas/inmunología , Timocitos/inmunología , Timo/inmunología , Animales , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Células Epiteliales/inmunología , Humanos , RatonesRESUMEN
To evaluate the levels of recent thymic emigrant (RTE) CD4+ T cells in HIV-infected children and to explore the associations among their frequency, antiretroviral treatment (ART) adherence, and CD4+ T cell restoration. The group evaluated comprised 85 HIV-infected patients classified as subjects with moderate or severe immunosuppression or as those with no evidence of immunosuppression. To evaluate the association between the frequency of RTE CD4+ T cells and ART adherence, 23 of the 85 patients were evaluated at two different time points during a one-year follow-up period. Children with severe immunosuppression had lower frequencies of RTE CD4+ T cells compared with children without evidence of immunosuppression (P < .001). The frequency of RTE CD4+ T cells in children with a high rate of adherence was significantly higher (P < .05) than that observed among those with suboptimal adherence. The latter group presented with infectious intercurrences on admission that decreased after initiation of treatment along with improved CD4+ and RTE naïve CD4+ T cells counts. The adequate ART adherence is essential for immune reconstitution, which might be reflected by the levels of RTE CD4+ T cells.
Asunto(s)
Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Timocitos/inmunología , Adolescente , Terapia Antirretroviral Altamente Activa , Biomarcadores , Linfocitos T CD4-Positivos/metabolismo , Movimiento Celular , Niño , Preescolar , Femenino , Infecciones por VIH/tratamiento farmacológico , Humanos , Inmunofenotipificación , Lactante , Masculino , Cumplimiento de la Medicación , Timocitos/metabolismo , Resultado del Tratamiento , Carga ViralRESUMEN
In this work, we demonstrate that adhesion between medullary thymic epithelial cells (mTECs) and thymocytes is controlled by miRNAs. Adhesion between mTECs and developing thymocytes is essential for triggering negative selection (NS) of autoreactive thymocytes that occurs in the thymus. Immune recognition is mediated by the MHC / TCR receptor, whereas adhesion molecules hold cell-cell interaction stability. Indeed, these processes must be finely controlled, if it is not, it may lead to aggressive autoimmunity. Conversely, the precise molecular genetic control of mTEC-thymocyte adhesion is largely unclear. Here, we asked whether miRNAs would be controlling this process through the posttranscriptional regulation of mRNAs that encode adhesion molecules. For this, we used small interfering RNA to knockdown (KD) Dicer mRNA in vitro in a murine mTEC line. A functional assay with fresh murine thymocytes co-cultured with mTECs showed that single-positive (SP) CD4 and CD8 thymocyte adhesion was increased after Dicer KD and most adherent subtype was CD8 SP cells. Analysis of broad mTEC transcriptional expression showed that Dicer KD led to the modulation of 114 miRNAs and 422 mRNAs, including those encoding cell adhesion or extracellular matrix proteins, such as Lgals9, Lgals3pb, Tnc and Cd47. Analysis of miRNA-mRNA networks followed by miRNA mimic transfection showed that these mRNAs are under the control of miR-181b-5p and miR-30b*, which may ultimately control mTEC-thymocyte adhesion. The expression of CD80 surface marker in mTECs was increased after Dicer KD following thymocyte adhesion. This indicates the existence of new mechanisms in mTECs that involve the synergistic action of thymocyte adhesion and regulatory miRNAs.
Asunto(s)
Adhesión Celular/inmunología , Células Epiteliales/inmunología , MicroARNs/inmunología , Timocitos/inmunología , Timo/inmunología , Animales , Antígeno B7-1/inmunología , Biomarcadores/sangre , Diferenciación Celular/inmunología , Femenino , Regulación de la Expresión Génica/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/inmunología , Autotolerancia/inmunología , Factores de Transcripción/inmunologíaRESUMEN
OBJECTIVE: To investigate the correlation between total lymphocyte and CD3+ T cell counts in peripheral blood in renal transplant patients treated with anti-thymocyte globulin, and discuss related outcomes. METHODS: A single-center, retrospective study involving 226 patients submitted to kidney transplant between 2008 and 2013, and treated with anti-thymocyte globulin for induction or treatment of cellular rejection. Doses were adjusted according to CD3+ T cell or total lymphocyte counts in peripheral blood. RESULTS: A total of 664 paired samples were analyzed. The Spearman's correlation coefficient was 0.416 (p<0.001) for all samples combined; the overall Kappa coefficient was 0.267 (p<0.001). Diagnostic parameters estimated based on total lymphocyte counts were also calculated using the number of CD3+ T cells (gold standard), with a cut off of >20 cells/mm3. CONCLUSION: Total lymphocyte and CD3+ T cell counts in peripheral blood are not equivalent monitoring strategies in anti-thymocyte globulin therapy.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Complejo CD3 , Rechazo de Injerto/terapia , Isoanticuerpos/uso terapéutico , Trasplante de Riñón , Timocitos/inmunología , Receptores de Trasplantes , Adulto , Femenino , Citometría de Flujo/métodos , Humanos , Inmunoterapia/métodos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Monitorización Inmunológica/instrumentación , Estudios Retrospectivos , Análisis de Supervivencia , Linfocitos T/inmunologíaRESUMEN
Aging is linked with a thymic oxidative damage and some infectious diseases such as Chagas' disease may aggravate this process. The aim of this study was to evaluate the production of distinct cytokines as well as the antioxidant/oxidant status of the thymus and thymocytes populations during Trypanosoma cruzi (T. cruzi) infection. Young (5â¯weeks old) and aged (18â¯weeks old) male Wistar rats were inoculated with blood trypomastigotes forms of the Y strain of T. cruzi. On the 16th day after T. cruzi infection, increased concentrations of transforming growth factor ß (TGF-ß), interleukin (IL)-12, IL-17 were detected in aged infected subjects as compared to young infected ones. Interestingly, a reduction in the production of tumor necrose factor (TNF)-α was observed in aged infected rats when compared to young infected subjects. Aged-infected rats presented increased O2- levels, compared to young counterparts. Significant raise in the generation of O2- in aged infected animals, as compared to uninfected counterparts was observed. Up-regulated expression of Nox2 in the thymus of young and aged infected animals was observed. An increased SOD2 expression was detected in the thymus of young animals infected with T. cruzi, when compared to uninfected young rats. Aged animals showed reduced thymus weight and the number of thymocytes. Decreased percentages of SPCD4+ and SPCD8+T cells were detected in aged and control groups when compared to young counterparts. In summary, this is the first data to directly examine the influence of aging on age-related dysfunctions during the acute phase of experimental Chagas disease. Concerning to oxidative stress, it is clear from our analysis that aged infected rats suffer a more intense oxidative damage when compared to young and infected ones. Age and infection triggered a dynamic interplay of cytokines, oxidative stress and thymic dysfunctions which led to impaired response from aged and infected rats. Such findings may have significant functional relevance in therapeutic strategies in order to reestablish the thymic immunological function which occurs in aged and T. cruzi infected subjects.
Asunto(s)
Envejecimiento/inmunología , Enfermedad de Chagas/inmunología , Citocinas/inmunología , Estrés Oxidativo/inmunología , Timo/inmunología , Trypanosoma cruzi/inmunología , Envejecimiento/patología , Animales , Enfermedad de Chagas/patología , Masculino , Ratas , Ratas Wistar , Timocitos/inmunología , Timocitos/patología , Timo/patologíaRESUMEN
The function of medullary thymic epithelial cells (mTECs) is associated with thymocyte adhesion, which is crucial for the negative selection of autoreactive thymocytes in the thymus. This process represents the root of central tolerance of self-components and prevents the onset of autoimmune diseases. Since thymic epithelia correspond to an important target of donor T cells during the onset of chronic graft-vs-host-disease, mTEC-thymocyte adhesion may have implications for alloimmunity. The Aire and Fezf2 genes function as transcriptome controllers in mTECs. The central question of this study is whether there is a mutual relationship between mTEC-thymocyte adhesion and the control of the mTEC transcriptome and whether Aire is involved in this process. Here, we show that in vitro mTEC-thymocyte adhesion causes transcriptome changes in mTECs and upregulates the transcriptional expression of Aire and Fezf2, as well as cell adhesion-related genes such as Cd80 or Tcf7, among others. Crispr-Cas9-mediated Aire gene disruption demonstrated that this gene plays a role in the process of mTEC-thymocyte adhesion. Consistent with the nuclear localization signal (NLS) encoded by Aire exon 3, which was targeted, we demonstrate that Aire KO-/- mTECs impair AIRE protein localization in the nucleus. Consequently, the loss of function of Aire reduced the ability of these cells to adhere to thymocytes. Their transcriptomes differed from their wild-type Aire+/+ counterparts, even during thymocyte adhesion. A set of mRNA isoforms that encode proteins involved in cell adhesion were also modulated during this process. This demonstrates that both thymocyte interactions and Aire influence transcriptome profiling of mTEC cells.
Asunto(s)
Células Epiteliales/metabolismo , Timocitos/metabolismo , Timo/citología , Factores de Transcripción/genética , Transcriptoma , Animales , Adhesión Celular , Diferenciación Celular/inmunología , Células Epiteliales/inmunología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Timocitos/inmunología , Timo/inmunología , Activación Transcripcional , Proteína AIRERESUMEN
ABSTRACT Objective: To investigate the correlation between total lymphocyte and CD3+ T cell counts in peripheral blood in renal transplant patients treated with anti-thymocyte globulin, and discuss related outcomes. Methods: A single-center, retrospective study involving 226 patients submitted to kidney transplant between 2008 and 2013, and treated with anti-thymocyte globulin for induction or treatment of cellular rejection. Doses were adjusted according to CD3+ T cell or total lymphocyte counts in peripheral blood. Results: A total of 664 paired samples were analyzed. The Spearman's correlation coefficient was 0.416 (p<0.001) for all samples combined; the overall Kappa coefficient was 0.267 (p<0.001). Diagnostic parameters estimated based on total lymphocyte counts were also calculated using the number of CD3+ T cells (gold standard), with a cut off of >20 cells/mm3. Conclusion: Total lymphocyte and CD3+ T cell counts in peripheral blood are not equivalent monitoring strategies in anti-thymocyte globulin therapy.
RESUMO Objetivo: Investigar a correlação entre a contagem de linfócitos totais e células T CD3+ no sangue periférico em receptores de transplante renal submetidos a tratamento com globulina antitimocitária, e discutir resultados relacionados. Métodos: Estudo retrospectivo de centro único envolvendo 226 pacientes submetidos a transplante renal entre 2008 e 2013 e tratados com globulina antitimocitária, para fins de indução ou tratamento de rejeição celular. As doses foram ajustadas de acordo com a contagem de células T CD3+ ou linfócitos totais no sangue periférico. Resultados: No total, 664 amostras pareadas foram analisadas. O coeficiente de correlação de Spearman para as amostras em geral foi de 0,416 (p<0,001) e o coeficiente Kappa, de 0,267 (p<0,001). Os parâmetros diagnósticos estimados com base na contagem de linfócitos totais foram recalculados, empregando-se o número de células T CD3+ (padrão-ouro) e adotando-se o ponto de corte >20 células/mm3. Conclusão: A contagem de linfócitos totais no sangue periférico não substitui a contagem de células T CD3+ enquanto estratégia de monitorização da terapia à base de globulina antitimocitária.
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Trasplante de Riñón , Complejo CD3 , Timocitos/inmunología , Receptores de Trasplantes , Rechazo de Injerto/terapia , Isoanticuerpos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Linfocitos T/inmunología , Monitorización Inmunológica/instrumentación , Análisis de Supervivencia , Estudios Retrospectivos , Recuento de Linfocitos , Citometría de Flujo/métodos , Inmunoterapia/métodos , Persona de Mediana EdadRESUMEN
The regulatory effect of allergic responses induced by IgG antibodies on human intra-thymic cells has not been reported in the literature. The aim of this study was to evaluate the possible differential effect of purified IgG from atopic and non-atopic individuals on human intra-thymic αßT cell cytokine production. Thymic tissues were obtained from 14 patients who were less than 7 d old. Additionally, blood samples were collected from atopic and non-atopic volunteers. Thymocytes and peripheral blood mononuclear cells were cultured with purified atopic or non-atopic IgG, and intracellular cytokine production was assessed. Purified IgG did not influence the frequency or viability of human intra-thymic αßT cells. Purified non-atopic IgG induced greater IFN-γ production by intra-thymic CD4+CD8+ T cells than did the mock treatment and atopic IgG. A similar effect of purified non-atopic IgG on TCD8 cells was observed compared with the mock treatment. Atopic IgG inhibited IFN-γ and TGF-ß production by intra-thymic TCD4 cells. Treatment with intravenous immunoglobulin resulted in intermediate levels of IFN-γ and TGF-ß in intra-thymic TCD4 cells compared with treatment with atopic and non-atopic IgG. Peripheral TCD4 cells from non-atopic individuals produced IFN-γ only in response to atopic IgG. This report describes novel evidence revealing that IgG from atopic individuals may influence intracellular IFN-γ production by intra-thymic αßT cells in a manner that may favor allergy development.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Hipersensibilidad Inmediata , Inmunoglobulinas Intravenosas/administración & dosificación , Factores Inmunológicos/administración & dosificación , Interferón gamma/metabolismo , Timocitos/inmunología , Técnicas de Cocultivo , Humanos , Recién Nacido , Timo/inmunologíaRESUMEN
We demonstrate that even a partial reduction of Aire mRNA levels by siRNA-induced Aire knockdown (Aire KD) has important consequences to medullary thymic epithelial cells (mTECs). Aire knockdown is sufficient to reduce Aire protein levels, impair its nuclear location, and cause an imbalance in large-scale gene expression, including genes that encode cell adhesion molecules. These genes drew our attention because adhesion molecules are implicated in the process of mTEC-thymocyte adhesion, which is critical for T cell development and the establishment of central self-tolerance. Accordingly, we consider the following: 1) mTECs contribute to the elimination of self-reactive thymocytes through adhesion; 2) Adhesion molecules play a crucial role during physical contact between these cells; and 3) Aire is an important transcriptional regulator in mTECs. However, its role in controlling mTEC-thymocyte adhesion remains unclear. Because Aire controls adhesion molecule genes, we hypothesized that the disruption of its expression could influence mTEC-thymocyte interaction. To test this hypothesis, we used a murine Aire(+) mTEC cell line as a model system to reproduce mTEC-thymocyte adhesion in vitro. Transcriptome analysis of the mTEC cell line revealed that Aire KD led to the down-modulation of more than 800 genes, including those encoding for proteins involved in cell adhesion, i.e., the extracellular matrix constituent Lama1, the CAM family adhesion molecules Vcam1 and Icam4, and those that encode peripheral tissue antigens. Thymocytes co-cultured with Aire KD mTECs had a significantly reduced capacity to adhere to these cells. This finding is the first direct evidence that Aire also plays a role in controlling mTEC-thymocyte adhesion.
Asunto(s)
Adhesión Celular/inmunología , Células Epiteliales/citología , Timocitos/citología , Timo/citología , Factores de Transcripción/metabolismo , Animales , Western Blotting , Diferenciación Celular/inmunología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Técnicas de Silenciamiento del Gen , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Autotolerancia/inmunología , Timocitos/inmunología , Timocitos/metabolismo , Timo/inmunología , Timo/metabolismo , Factores de Transcripción/inmunología , Transcriptoma/inmunología , Proteína AIRERESUMEN
Our understanding of how thymocytes differentiate into many subtypes has been increased progressively in its complexity. At early life, the thymus provides a suitable microenvironment with specific combination of stromal cells, growth factors, cytokines, and chemokines to induce the bone marrow lymphoid progenitor T-cell precursors into single-positive CD4(+) and CD8(+) T effectors and CD4(+)CD25(+) T-regulatory cells (Tregs). At postthymic compartments, the CD4(+) T-cells acquire distinct phenotypes which include the classical T-helper 1 (Th1), T-helper 2 (Th2), T-helper 9 (Th9), T-helper 17 (Th17), follicular helper T-cell (Tfh), and induced T-regulatory cells (iTregs), such as the regulatory type 1 cells (Tr1) and transforming growth factor-ß- (TGF-ß-) producing CD4(+) T-cells (Th3). Tregs represent only a small fraction, 5-10% in mice and 1-2% in humans, of the overall CD4(+) T-cells in lymphoid tissues but are essential for immunoregulatory circuits mediating the inhibition and expansion of all lineages of T-cells. In this paper, we first provide an overview of the major cell-intrinsic developmental programs that regulate T-cell lineage fates in thymus and periphery. Next, we introduce the SV40 immortomouse as a relevant mice model for implementation of new approaches to investigate thymus organogenesis, CD4 and CD8 development, and thymus cells tumorogenesis.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Tejido Linfoide/citología , Ratones , Células TH1/citología , Células TH1/inmunología , Células Th17/citología , Células Th17/inmunología , Células Th2/citología , Células Th2/inmunología , Timocitos/citología , Timocitos/inmunologíaRESUMEN
The physiology of the thymus, the primary lymphoid organ in which T cells are generated, is controlled by hormones. Data from animal models indicate that several peptide and nonpeptide hormones act pleiotropically within the thymus to modulate the proliferation, differentiation, migration and death by apoptosis of developing thymocytes. For example, growth hormone and prolactin can enhance thymocyte proliferation and migration, whereas glucocorticoids lead to the apoptosis of these developing cells. The thymus undergoes progressive age-dependent atrophy with a loss of cells being generated and exported, therefore, hormone-based therapies are being developed as an alternative strategy to rejuvenate the organ, as well as to augment thymocyte proliferation and the export of mature T cells to peripheral lymphoid organs. Some hormones (such as growth hormone and progonadoliberin-1) are also being used as therapeutic agents to treat immunodeficiency disorders associated with thymic atrophy, such as HIV infection. In this Review, we discuss the accumulating data that shows the thymus gland is under complex and multifaceted hormonal control that affects the process of T-cell development in health and disease.
Asunto(s)
Diferenciación Celular/inmunología , Hormona de Crecimiento Humana/inmunología , Prolactina/inmunología , Linfocitos T/inmunología , Timocitos/inmunología , Timo/inmunología , Animales , Movimiento Celular/inmunología , Proliferación Celular , Hormona Liberadora de Gonadotropina/uso terapéutico , Hormona del Crecimiento/inmunología , Infecciones por VIH/tratamiento farmacológico , Hormona de Crecimiento Humana/uso terapéutico , Humanos , Síndromes de Inmunodeficiencia/tratamiento farmacológico , Tejido Linfoide/inmunología , Precursores de Proteínas/uso terapéuticoRESUMEN
In autoimmune type 1 diabetes mellitus (T1D), auto-reactive clones of CD4+ and CD8+ T lymphocytes in the periphery evolve into pancreas-infiltrating T lymphocytes (PILs), which destroy insulin-producing beta-cells through inflammatory insulitis. Previously, we demonstrated that, during the development of T1D in non-obese diabetic (NOD) mice, a set of immune/inflammatory reactivity genes were differentially expressed in T lymphocytes. However, the posttranscriptional control involving miRNA interactions that occur during the evolution of thymocytes into PILs remains unknown. In this study, we postulated that miRNAs are differentially expressed during this period and that these miRNAs can interact with mRNAs involved in auto-reactivity during the progression of insulitis. To test this hypothesis, we used NOD mice to perform, for the first time, a comprehensive survey of miRNA and mRNA expression as thymocytes mature into peripheral CD3+ T lymphocytes and, subsequently, into PILs. Reconstruction of miRNA-mRNA interaction networks for target prediction revealed the participation of a large set of miRNAs that regulate mRNA targets related to apoptosis, cell adhesion, cellular regulation, cellular component organization, cellular processes, development and the immune system, among others. The interactions between miR-202-3p and the Ccr7 chemokine receptor mRNA or Cd247 (Cd3 zeta chain) mRNA found in PILs are highlighted because these interactions can contribute to a better understanding of how the lack of immune homeostasis and the emergence of autoimmunity (e.g., T1D) can be associated with the decreased activity of Ccr7 or Cd247, as previously observed in NOD mice. We demonstrate that these mRNAs are controlled at the posttranscriptional level in PILs.
Asunto(s)
Complejo CD3/genética , MicroARNs/genética , Páncreas/metabolismo , Interferencia de ARN , ARN Mensajero/genética , Receptores CCR7/genética , Linfocitos T/metabolismo , Regiones no Traducidas 3' , Animales , Sitios de Unión , Análisis por Conglomerados , Biología Computacional/métodos , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genes Reporteros , Ratones , Ratones Endogámicos NOD , Páncreas/inmunología , Procesamiento Postranscripcional del ARN , Reproducibilidad de los Resultados , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Timocitos/inmunología , Timocitos/metabolismo , TranscriptomaRESUMEN
A variety of mechanisms are involved in the regulation of offspring allergy development through maternal immunization with allergens. The passive transfer of antigens, antibodies, and cytokines, the induction of phenotypic alterations in offspring lymphocytes, and the induction of regulatory populations in offspring have been proposed, but these mechanisms remain incompletely understood. It is likely that maternal immunization could affect the intrathymic maturation of offspring TCD4+, TCD8+, γδT, nTreg, iNKT, and B lymphocytes, although there are currently no human maternal immunization protocols for the regulation of allergic responses in children. Some studies have suggested a direct interaction between the maternal immune status and the offspring intrathymic microenvironment; this interaction could influence the maturation of offspring regulatory cells and must be explored for the development of therapies to control allergy development in children.
Asunto(s)
Alérgenos/inmunología , Diferenciación Celular , Inmunidad Materno-Adquirida , Subgrupos Linfocitarios/citología , Subgrupos Linfocitarios/inmunología , Timocitos/citología , Timocitos/inmunología , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Niño , Femenino , Humanos , Hipersensibilidad/inmunología , Inmunización , Subgrupos Linfocitarios/metabolismo , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Timocitos/metabolismo , Timo/citología , Timo/inmunología , Timo/metabolismoRESUMEN
We investigated the consequences of mild maternal malnutrition in rat dams, in terms of thymocyte responses and the putative role of leptin. The young progeny of dams submitted to protein deprivation (PD) during lactation showed at 30 days of age lower body and thymus weights, significant alterations in CD4/CD8-defined T cell subsets without modifications in total thymocyte number as well as in proliferative response. Despite, the rats from PD group did not present alterations in leptin circulating levels, the expression of leptin receptor ObRb was enhanced in their thymocytes. This change was accompanied by an increase in leptin signaling response of thymocytes from PD rats, with an increase in JAK2 and STAT3 phosphorylation after leptin stimulation. Thymocytes from PD rats also presented a decreased rate of spontaneous apoptosis when compared to controls. Accordingly, higher expression of anti-apoptotic protein Bcl-2, and lower of pro-apoptotic protein Bax, with no change of pro-apoptotic Bad, and higher pro-caspase 3 content were detected in PD thymocytes. Moreover, thymocytes from PD group exhibited a constitutive higher nuclear content of p65 NF-kB associated to a lower IkB content in the cytoplasm. Finally, although there was no change in ob gene expression in PD thymocytes, a higher mRNA expression for the Ob gene was observed in the thymic microenvironment from PD animals. Taken together, the results show that mild maternal protein deprivation during lactation affects thymic homeostasis, enhancing leptin activity, which in turn protects thymocytes from apoptosis in the young progeny, with possible consequences upon the immune response of these animals in adult life.
Asunto(s)
Apoptosis , Lactancia , Leptina/metabolismo , Desnutrición Proteico-Calórica , Timocitos/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Peso Corporal , Microambiente Celular/genética , Microambiente Celular/inmunología , Dieta con Restricción de Proteínas , Femenino , Regulación de la Expresión Génica , Inmunofenotipificación , Janus Quinasa 2/metabolismo , Leptina/sangre , Leptina/genética , Masculino , FN-kappa B/metabolismo , Ratas , Receptores de Leptina/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Subgrupos de Linfocitos T/metabolismo , Timocitos/inmunología , Timo/citologíaRESUMEN
In the context of immunoneuroendocrine cross talk, growth hormone (GH) exerts pleiotropic effects in the immune system. For example, GH-transgenic mice, as well as animals and humans treated with GH, exhibit enhanced cellularity in the thymus. GH also stimulates the thymic microenvironment, augmenting chemokine and extracellular matrix (ECM) production, with consequent increase in ECM- and chemokine-driven thymocyte migratory responses. Peripheral T cell migration triggered by laminin or fibronectin was enhanced in cells from GH-transgenic versus wild-type control adult mice, as seen for CD4(+) and CD8(+) T cells from mesenteric lymph nodes. Migration of these T lymphocytes, triggered by the chemokine CXCL12, in conjunction with laminin or fibronectin, was also enhanced compared with control counterparts. Considering that GH can be used as an adjuvant therapy in immunodeficiencies, including AIDS, the concepts defined herein, that GH enhances developing and peripheral T cell migration, provide new clues for future GH-related immune interventions.
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Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Quimiotaxis de Leucocito/inmunología , Hormona del Crecimiento/inmunología , Timocitos/metabolismo , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/metabolismo , Animales , Quimiocina CXCL12/metabolismo , Fibronectinas/metabolismo , Hormona del Crecimiento/metabolismo , Humanos , Laminina/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Transgénicos , Timocitos/inmunologíaRESUMEN
A major concern in transplantation is the preservation of organ function. Ischemia time and microcirculatory disturbance of the organ cannot be avoided and may result in ischemia reperfusion injury (IRI), increasing the risk of delayed graft function (DGF) and acute and chronic rejection. Anti-thymocyte immunoglobulin (rATG) is a polyclonal antibody preparation with multiple effects when administered to recipients. Our objective has been to evaluate whether the administration of rATG to kidney donors instead of recipients, in an experimental model of syngeneic rat transplantation, ameliorates IRI and facilitates immediate graft function recovery. Urea and creatinine levels and necrosis severity scores were significantly lower in kidneys from donors that had received rATG (urea: control: 211±8mg/dl vs. treatment: 110±15mg/dl, p<0.001; creatinine: control: 4.6±0.24mg/dl vs. treatment: 2.6±0.22mg/dl, p<0.001; necrosis severity scores: control: 2.3 vs. treatment: 1.6, p<0.05). TUNEL staining showed 80±13 positive cells in control group and 9±3 (p<0.001) in treatment group. In situ expression of proinflammatory cytokines TNF-α, IL-6, IL-21 and TGF-ß1 was reduced in rATG group (p<0.01); the same was observed for KIM-1 and caspase 8 (p<0.001). Cytoprotective genes Bcl2 and HO-1 were upregulated in situ in treatment group (p<0.001). In situ expression of IL-17, caspase 9, IL-23a, CxCl3 and ICAM1 showed no difference between groups (p>0.05). Findings suggest ATG administered to donors may ameliorate the IRI process in kidney transplantation, expressed by lower necrosis and apoptosis scores and the improvement of renal function, which may be explained through the diminished in situ expression of inflammatory mediators.
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Suero Antilinfocítico/administración & dosificación , Trasplante de Riñón , Daño por Reperfusión/prevención & control , Timocitos/inmunología , Donantes de Tejidos , Animales , Apoptosis , Caspasa 8/metabolismo , Moléculas de Adhesión Celular/metabolismo , Creatinina/análisis , Perfilación de la Expresión Génica , Genes bcl-2 , Hemo-Oxigenasa 1/genética , Interleucina-6/metabolismo , Interleucinas/metabolismo , Riñón/inmunología , Masculino , Necrosis , Distribución Aleatoria , Ratas , Ratas Wistar , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Urea/análisisRESUMEN
Previous studies revealed a significant production of inflammatory cytokines together with severe thymic atrophy and thymocyte migratory disturbances during experimental Chagas disease. Migratory activity of thymocytes and mature T cells seem to be finely tuned by cytokines, chemokines and extracellular matrix (ECM) components. Systemic TNF-α is enhanced during infection and appears to be crucial in the response against the parasite. However, it also seems to be involved in disease pathology, since it is implicated in the arrival of T cells to effector sites, including the myocardium. Herein, we analyzed the role of TNF-α in the migratory activity of thymocytes in Trypanosoma cruzi (T. cruzi) acutely-infected mice. We found increased expression and deposition of TNF-α in the thymus of infected animals compared to controls, accompanied by increased co-localization of fibronectin, a cell migration-related ECM molecule, whose contents in the thymus of infected mice is also augmented. In-vivo studies showed an enhanced export of thymocytes in T. cruzi-infected mice, as ascertained by intrathymic injection of FITC alone or in combination with TNF-α. The increase of immature CD4(+)CD8(+) T cells in secondary lymphoid organs was even more clear-cut when TNF-α was co-injected with FITC. Ex-vivo transmigration assays also revealed higher number of migrating cells when TNF-α was added onto fibronectin lattices, with higher input of all thymocyte subsets, including immature CD4(+)CD8(+). Infected animals also exhibit enhanced levels of expression of both mRNA TNF-α receptors in the CD4(+)CD8(+) subpopulation. Our findings suggest that in T. cruzi acute infection, when TNF-α is complexed with fibronectin, it favours the altered migration of thymocytes, promoting the release of mature and immature T cells to different compartments of the immune system. Conceptually, this work reinforces the notion that thymocyte migration is a multivectorial biological event in health and disease, and that TNF-α is a further player in the process.
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Movimiento Celular/inmunología , Enfermedad de Chagas/inmunología , Timocitos/inmunología , Trypanosoma cruzi/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Atrofia , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Movimiento Celular/efectos de los fármacos , Enfermedad de Chagas/metabolismo , Enfermedad de Chagas/parasitología , Fibronectinas/inmunología , Fibronectinas/metabolismo , Citometría de Flujo , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timocitos/citología , Timocitos/metabolismo , Timo/inmunología , Timo/metabolismo , Timo/patología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Dendritic cells (DCs) are professional antigen-presenting cells able to induce immunity or tolerance. The interactions of immature DCs with naive T lymphocytes induce peripheral tolerance through mechanisms that include anergy or deletion of lymphocytes or the generation of regulatory T cells. Because of the central role of DCs in the immune response, they are potential targets for the induction of experimental tolerance. Thus, the generation of immature (tolerogenic) DCs able to capture and present alloantigens to T cells represents an important aim in our efforts to achieve better transplant acceptance. METHODS: In this work, we generated immature DCs by using vitamin D(3) (VD3) during the process of DC differentiation. RESULTS: The VD3-DCs showed an immature phenotype characterized by a low expression of major histocompatibility complex antigens of class II, CD86, and CD80 molecules and the secretion of a tolerogenic cytokine pattern. Furthermore, we showed that VD3-DCs phagocytose apoptotic allogeneic cells efficiently without inducing DC maturation or activation. Most important, our experiments demonstrated that mice treated with VD3 produce immature DCs in vivo, and that DCs from VD3-treated mice immunized with allogeneic apoptotic cells maintained their tolerogenic phenotype. CONCLUSION: Our results show that allogeneic apoptotic cells in combination with VD3 generate DCs with tolerogenic characteristics that could be used to induce tolerance towards alloantigens.
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Apoptosis , Células Dendríticas/inmunología , Tolerancia Inmunológica , Isoantígenos/inmunología , Fagocitosis , Timocitos/inmunología , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Antígeno B7-1/inmunología , Diferenciación Celular , Células Cultivadas , Colecalciferol/farmacología , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunofenotipificación , Masculino , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Necrosis , Fenotipo , Timocitos/patología , Timocitos/trasplante , Factores de Tiempo , TransfecciónRESUMEN
We have previously showed alterations in the thymus during experimental infection with Plasmodium berghei, the causative agent of Malaria. Such alterations comprised histological changes with loss of delimitation between cortical and medullar regions, a profound atrophy with depletion of CD4(+)CD8(+) double-positive (DP) thymocytes, and severe changes in the expression of cell migration-related molecules, belonging to the extracellular matrix and chemokine protein families. Taken together, these considerations prompted us to evaluate if the acute thymic atrophy observed during Plasmodium infection was correlated with increased apoptotic levels of thymocytes or with their premature emigration to the periphery. Our results confirmed that the marked reduction of the thymus weight in infected animals was accompanied by histological alterations, which included a very large number of cells showing nuclear condensation and karyorrhectic changes surrounded by histiocytes suggesting increased levels of apoptosis. This was confirmed by immunohistochemistry and flow cytometry techniques. In order to verify if an accelerated emigration of thymic cells to the peripheral lymphoid organs was also occurring we analyzed the spleen and mesenteric lymph nodes from control and infected mice. No significant differences were found in the spleen, but were seen after 14 days of infection between control and infected mice in the mesenteric lymph nodes. The main alteration was the presence of double negative (CD4(-)CD8(-)) and double positive (CD4(+)CD8(+)) cells. We concluded that both apoptosis of thymocytes and premature egress of immature cells take place during infection. Additional studies will be necessary to verify how such alterations might influence the systemic immune response to the parasite.