RESUMO
BACKGROUND: Thymic epithelial cells (TECs) are responsible for shaping the repertoires of T cells, where their postnatal regeneration depends on a subset of clonogenic TECs. Despite the implications for regenerative medicine, their cultivation and expansion remain challenging. Primary explant cell culture is a technique that allows the seeding and expansion of difficult-to-culture cells. Here, we report a reliable and simple culture system to obtain functional TECs and thymic interstitial cells (TICs). METHODS: To establish primary thymic explants, we harvested 1 mm cleaned fragments of thymus from 5-week-old C57/BL6 mice. Tissue fragments of a complete thymic lobe were placed in the center of a Petri dish with 1 mL of DMEM/F-12 medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillinâstreptomycin. To compare, thymic explants were also cultivated by using serum-free DMEM/F-12 medium supplemented with 10% KnockOut™. RESULTS: We obtained high numbers of functional clonogenic TECs and TICs from primary thymic explants cultivated with DMEM/F-12 with 20% FBS. These cells exhibited a highly proliferative and migration profile and were able to constitute thymospheres. Furthermore, all the subtypes of medullary TECs were identified in this system. They express functional markers to shape T-cell and type 2 innate lymphoid cells repertoires, such as Aire, IL25, CCL21 and CD80. Finally, we also found that ≥ 70% of lineage negative TICs expressed high amounts of Aire and IL25. CONCLUSION: Thymic explants are an efficient method to obtain functional clonogenic TECs, all mTEC subsets and different TICs Aire+IL25+ with high regenerative capacity.
Assuntos
Imunidade Inata , Linfócitos , Camundongos , Animais , Timo/metabolismo , Células Epiteliais/metabolismo , Linfócitos T , Diferenciação CelularRESUMO
Growth hormone (GH) is a classic pituitary-derived hormone crucial to body growth and metabolism. In the pituitary gland, GH production is stimulated by GH-releasing hormone and inhibited by somatostatin. GH secretion can also be induced by other peptides, such as ghrelin, which interacts with receptors present in somatotropic cells. It is well established that GH acts directly on target cells or indirectly by stimulating the production of insulin-like growth factors (IGFs), particularly IGF-1. Notably, such somatotropic circuitry is also involved in the development and function of immune cells and organs, including the thymus. Interestingly, GH, IGF-1, ghrelin, and somatostatin are expressed in the thymus in the lymphoid and microenvironmental compartments, where they stimulate the secretion of soluble factors and extracellular matrix molecules involved in the general process of intrathymic T-cell development. Clinical trials in which GH was used to treat immunocompromised patients successfully recovered thymic function. Additionally, there is evidence that the reduction in the function of the somatotropic axis is associated with age-related thymus atrophy. Treatment with GH, IGF-1 or ghrelin can restore thymopoiesis of old animals, thus in keeping with a clinical study showing that treatment with GH, associated with metformin and dehydroepiandrosterone, could induce thymus regeneration in healthy aged individuals. In conclusion, the molecules of the somatotrophic axis can be envisioned as potential therapeutic targets for thymus regeneration in age-related or pathological thymus involution.
Assuntos
Grelina , Fator de Crescimento Insulin-Like I , Animais , Fator de Crescimento Insulin-Like I/metabolismo , Hormônio do Crescimento , Timo , SomatostatinaRESUMO
BACKGROUND: Besides controlling the expression of peripheral tissue antigens, the autoimmune regulator (AIRE) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown that AIRE knockout (KO) mice have a small and disorganized thymic medulla; however, whether AIRE influences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed an in vitro model system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. RESULTS: The comparisons between AIRE wild type (AIREWT) and AIRE KO (AIRE-/-) 3D mTEC spheroid formation showed that the absence of AIRE: i) disorganizes the 3D structure of mTEC spheroids, ii) increases the proportion of cells at the G0/G1 phase of the cell cycle, iii) increases the rate of mTEC apoptosis, iv) decreases the strength of mTEC-mTEC adhesion, v) promotes a differential regulation of mTEC classical surface markers, and vi) modulates genes encoding adhesion and other molecules. CONCLUSIONS: Overall, the results show that AIRE influences the 3D structuring of mTECs when these cells begin the spheroid formation through controlling cell adhesion genes.
Assuntos
Células Epiteliais , Genes Reguladores , Animais , Adesão Celular , Diferenciação Celular/genética , Células Epiteliais/metabolismo , Camundongos , Camundongos KnockoutRESUMO
The existence of a crosstalk between the nervous and immune systems is well established. Neurotransmitters can be produced by immune cells, whereas cytokines can be secreted by cells of nervous tissues. Additionally, cells of both systems express the corresponding receptors. Herein, we discuss the thymus as a paradigm for studies on the neuroimmune network. The thymus is a primary lymphoid organ responsible for the maturation of T lymphocytes. Intrathymic T-cell development is mostly controlled by the thymic microenvironment, formed by thymic epithelial cells (TEC), dendritic cells, macrophages, and fibroblasts. Developing thymocytes and microenvironmental cells can be influenced by exogenous and endogenous stimuli; neurotransmitters are among the endogenous molecules. Norepinephrine is secreted at nerve endings in the thymus, but are also produced by thymic cells, being involved in controlling thymocyte death. Thymocytes and TEC express acetylcholine receptors, but the cognate neurotransmitter seems to be produced and released by lymphoid and microenvironmental cells, not by nerve endings. Evidence indicates that, among others, TECs also produce serotonin and dopamine, as well as somatostatin, substance P, vasoactive intestinal peptide (VIP) and the typical pituitary neurohormones, oxytocin and arg-vasopressin. Although functional data of these molecules in the thymus are scarce, they are likely involved in intrathymic T cell development, as exemplified by somatostatin, which inhibits thymocyte proliferation, differentiation, migration and cytokine production. Overall, intrathymic neuroimmune interactions include various neurotransmitters, most of them of non-neuronal origin, and that should be placed as further physiological players in the general process of T-cell development.
RESUMO
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.
Assuntos
Doença de Chagas/imunologia , Timócitos/imunologia , Timo/imunologia , Animais , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Células Epiteliais/imunologia , Humanos , CamundongosRESUMO
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.
Assuntos
Células Epiteliais/metabolismo , Timócitos/metabolismo , Timo/citologia , Fatores de Transcrição/genética , Transcriptoma , Animais , Adesão Celular , Diferenciação Celular/imunologia , Células Epiteliais/imunologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Timócitos/imunologia , Timo/imunologia , Ativação Transcricional , Proteína AIRERESUMO
The thymus supports differentiation of T cell precursors. This process requires relocation of developing thymocytes throughout multiple microenvironments of the organ, mainly with thymic epithelial cells (TEC), which control intrathymic T cell differentiation influencing the formation and maintenance of the immunological synapse. In addition to the proteins of the major histocompatibility complex (MHC), this structure is supported by several adhesion molecules. During the process of thymopoiesis, we previously showed that laminin-mediated interactions are involved in the entrance of T-cell precursors into the thymus, as well as migration of differentiating thymocytes within the organ. Using small interference RNA strategy, we knocked-down the ITGA6 gene (which encodes the CD49f integrin α-chain) in cultured human TEC, generating a decrease in the expression of the corresponding CD49f subunit, in addition to modulation in several other genes related to cell adhesion and migration. Thymocyte adhesion to TEC was significantly impaired, comprising both immature and mature thymocyte subsets. Moreover, we found a modulation of the MHC, with a decrease in membrane expression of HLA-ABC, in contrast with increase in the expression of HLA-DR. Interestingly, the knockdown of the B2M gene (encoding the ß-2 microglobulin of the HLA-ABC complex) increased CD49f expression levels, thus unraveling the existence of a cross-talk event in the reciprocal control of CD49f and HLA-ABC. Our data suggest that the expression levels of CD49f may be relevant in the general control of MHC expression by TEC and consequently the corresponding synapse with developing thymocytes mediated by the T-cell receptor.
Assuntos
Moléculas de Adesão Celular/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Sinapses Imunológicas/metabolismo , Integrina alfa6/genética , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Matriz Extracelular/metabolismo , Marcação de Genes/métodos , Humanos , Integrinas/metabolismoRESUMO
The human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4+T cells are the main target of HTLV-1, but other cell types are known to be infected, including immature lymphocytes. Developing T cells undergo differentiation in the thymus, through migration and interaction with the thymic microenvironment, in particular with thymic epithelial cells (TEC) the major component of this three dimensional meshwork of non-lymphoid cells. Herein, we show that TEC express the receptors for HTLV-1 and can be infected by this virus through cell-cell contact and by cell-free virus suspensions. The expression of anti-apoptosis, chemokine and adhesion molecules genes are altered in HTLV-1-infected TEC, although gene expression of antigen presentation molecules remained unchanged. Furthermore, HTLV-1-infected TEC transmitted the virus to a CD4+ T cell line and to CD4+ T cells from healthy donors, during in vitro cellular co-cultures. Altogether, our data point to the possibility that the human thymic epithelial cells play a role in the establishment and progression of HTLV-1 infection, functioning as a reservoir and transmitting the virus to maturing CD4+ T lymphocytes, which in turn will cause disease in the periphery.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Células Epiteliais/virologia , Infecções por HTLV-I/transmissão , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Timo/patologia , Apoptose/genética , Linfócitos T CD4-Positivos/virologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Técnicas de Cocultura , Progressão da Doença , Células Epiteliais/fisiologia , Regulação da Expressão Gênica , Infecções por HTLV-I/imunologia , Humanos , Leucemia-Linfoma de Células T do Adulto , Paraparesia Espástica , Paraparesia Espástica Tropical , Receptores Virais/metabolismo , Internalização do VírusRESUMO
Autoimmune regulator (Aire) is a transcriptional regulator of peripheral tissue antigens (PTAs) and microRNAs (miRNAs) in medullary thymic epithelial cells (mTECs). In this study, we tested the hypothesis that Aire also played a role as an upstream posttranscriptional controller in these cells and that variation in its expression might be associated with changes in the interactions between miRNAs and the mRNAs encoding PTAs. We demonstrated that downregulation of Aire in vivo in the thymuses of BALB/c mice imbalanced the large-scale expression of these two RNA species and consequently their interactions. The expression profiles of a large set of mTEC miRNAs and mRNAs isolated from the thymuses of mice subjected (or not) to small-interfering-induced Aire gene knockdown revealed that 87 miRNAs and 4,558 mRNAs were differentially expressed. The reconstruction of the miRNA-mRNA interaction networks demonstrated that interactions between these RNAs were under Aire influence and therefore changed when this gene was downregulated. Prior to Aire-knockdown, only members of the miR-let-7 family interacted with a set of PTA mRNAs. Under Aire-knockdown conditions, a larger set of miRNA families and their members established this type of interaction. Notably, no previously described Aire-dependent PTA interacted with the miRNAs, indicating that these PTAs were somehow refractory. The miRNA-mRNA interactions were validated by calculating the minimal free energy of the pairings between the miRNA seed regions and the mRNA 3' UTRs and within the cellular milieu using the luciferase reporter gene assay. These results suggest the existence of a link between transcriptional and posttranscriptional control because Aire downregulation alters the miRNA-mRNA network controlling PTAs in mTEC cells.
RESUMO
BACKGROUND: Several evidences indicate that hormones and neuropeptides function as immunomodulators. Among these, growth hormone (GH) is known to act on the thymic microenvironment, supporting its role in thymocyte differentiation. The aim of this study was to evaluate the effect of GH on human thymocytes and thymic epithelial cells (TEC) in the presence of laminin. RESULTS: GH increased thymocyte adhesion on BSA-coated and further on laminin-coated surfaces. The number of migrating cells in laminin-coated membrane was higher in GH-treated thymocyte group. In both results, VLA-6 expression on thymocytes was constant. Also, treatment with GH enhanced laminin production by TEC after 24 h in culture. However, VLA-6 integrin expression on TEC remained unchanged. Finally, TEC/thymocyte co-culture model demonstrated that GH elevated absolute number of double-negative (CD4(-)CD8(-)) and single-positive CD4(+) and CD8(+) thymocytes. A decrease in cell number was noted in double-positive (CD4(+)CD8(+)) thymocytes. CONCLUSIONS: The results of this study demonstrate that GH is capable of enhancing the migratory capacity of human thymocytes in the presence of laminin and promotes modulation of thymocyte subsets after co-culture with TEC.