Interactions between thymic endothelial cells and thymocytes are influenced by growth hormone.

Growth hormone (GH), in addition to its classic actions on growth and metabolism in the body, exerts pleiotropic effects on the immune system, particularly on the thymus. The aim of this study was to evaluate the influence of GH on the interactions between mature thymocytes and the thymic endothelium involved in the migratory process. To this end, fresh thymocytes (C57BL/6 mice) and the thymic endothelial cell line (tEnd.1) were used. In the cell adhesion assay, the GH-treated thymocytes adhered more to tEnd.1 cells. Additionally, there was an improvement in the deposition of fibronectin by tEnd.1 cells when co-cultured with GH-pre-treated thymocytes. Furthermore, GH induced thymocyte F-actin polymerization. In the transendothelial migration assay, a large number of GH-treated thymocytes, mainly the CD4-CD8+ subset, migrated towards the endothelium under the stimulus of insulin-like growth factor 1. In conclusion, we demonstrated the positive actions of GH in thymocyte/thymic endothelium interactions, including transendothelial migration.

Decline of FOXN1 gene expression in human thymus correlates with age: possible epigenetic regulation.

BACKGROUND: Thymic involution is thought to be an important factor of age related immunodeficiency. Understanding the molecular mechanisms of human thymic senescence may lead to the discovery of novel therapeutic approaches aimed at the reestablishment of central and peripheral T cell repertoire. RESULTS: As an initial approach, here we report that the decline of human thymic FOXN1 transcription correlates with age, while other genes, DLL1, DLL4 and WNT4, essential for thymopoiesis, are constitutively transcribed. Using a human thymic epithelial cell line (hTEC), we show that FOXN1 expression is refractory to signals that induce FOXN1 transcription in primary 3D culture conditions and by stimulation of the canonical WNT signaling pathway. Blockage of FOXN1 induceability in the hTEC line may be mediated by an epigenetic mechanism, the CpG methylation of the FOXN1 gene. CONCLUSION: We showed a suppression of FOXN1 transcription both in cultured human thymic epithelial cells and in the aging thymus. We hypothesize that the underlying mechanism may be associated with changes of the DNA methylation state of the FOXN1 gene.

Intrathymic somatotropic circuitry: consequences upon thymus involution.

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.

Metallic nanoparticles reduce the migration of human fibroblasts in vitro.

Nanoparticles have extremely wide applications in the medical and biological fields. They are being used in biosensors, local drug delivery, diagnostics, and medical therapy. However, the potential effects of nanoparticles on target cell and tissue function, apart from cytotoxicity, are not completely understood. Thus, the aim of this study was to investigate the in vitro effects of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) on human fibroblasts with respect to their interaction with the extracellular matrix and in cell migration. Immunofluorescence analysis revealed that treatment with AgNPs or AuNPs decreased collagen and laminin production at all the concentrations tested (0.1, 1, and 10 µg/mL). Furthermore, cytofluorometric analysis showed that treatment with AgNPs reduced the percentage of cells expressing the collagen receptor very late antigen 2, α2ß1 integrin (VLA-2) and the laminin receptor very late antigen 6, α6ß1 integrin (VLA-6). In contrast, AuNP treatment increased and decreased the percentages of VLA-2-positive and VLA-6-positive cells, respectively, as compared to the findings for the controls. Analysis of cytoskeletal reorganization showed that treatment with both types of nanoparticles increased the formation of stress fibres and number of cell protrusions and impaired cell polarity. Fibroblasts exposed to different concentrations of AuNPs and AgNPs showed reduced migration through transwell chambers in the functional chemotaxis assay. These results demonstrated that metal nanoparticles may influence fibroblast function by negatively modulating the deposition of extracellular matrix molecules (ECM) and altering the expression of ECM receptors, cytoskeletal reorganization, and cell migration.

Regulação epigenética de FOXN1 no epitélio tímico humano durante a senescência / Epigenetic regulation of foxn1 in human thymic epithelium during senescence

A involução do timo ao longo do envelhecimento se caracteriza por alterações nas células que compõem o seu microambiente, contudo as suas causas ainda não estão completamente esclarecidas. No intuito de contribuir para conhecimento acerca desse fenômeno, esta tese teve por objetivos avaliar a expressão de genes importantes para o desenvolvimento e função linfopoiética do timo como também investigar o mecanismo de regulação da expressão gênica de FOXN1 através de metilação de DNA. Para isso, foram utilizadas amostras de timo humano de diferentes idades provenientes de pacientes submetidos à cirurgia cardíaca. Quanto aos aspectos morfológicos, os timos de doadores entre cinco dias e um ano deidade (grupo pós-natal) apresentaram uma arquitetura microscópica característica do órgão, sem sinais de involução. Nessas amostras, também foi verificada uma deposição normal de citoqueratina, distribuída pelo órgão como uma fina rede de filamentos intracelulares. Já nos timos de doadores acima de 49 anos (grupo adulto idoso),observou-se a perda da arquitetura tímica, presença de uma grande quantidade de tecido adiposo e uma deposição de citoqueratina irregular, quandopresente, com regiões semelhantes a áreas livres de células epiteliais tímicas (TEC).Nos ensaios de PCRq, foi verificado um aumento na expressão dos genes DLL1 eDLL4, e uma diminuição na expressão de FOXN1 em amostras de timo adulto-idosoem comparação com amostras de timo pós-natal. Além disso, não foi possíveldetectar a presença da proteína FOXN1 em cortes congelados de timo adulto-idosoem relação às amostras de timo pós-natal...

Thymic involution during aging is accompanied by alterations on itsmicroenvironment, however little is known about the mechanisms responsible forthese modifications. In this study we aimed to evaluate the expression of genescritical for thymopoiesis and the FOXN1 gene epigenetic regulation by DNAmethylation. We obtained human thymic samples from patients ongoing cardiacsurgery with different ages. In the morphological evaluation, thymus from donors withfive days-old to one year-old (posnatal group) showed a typical thymic architecture,without involution signs, and a normal cytokeratin labelling pattern distributed as athin filament network on the organ. When thymuses from adult-old donors (49 to 78years-old) were analyzed, it was seen a disruption on thymus microenvironment, withlarge adipose tissue infiltration and an irregular cytokeratin pattern revealing someepitelial-free areas. The qPCR assays showed that DLL1, DLL4, FOXN1 e WNT4genes were expressed during aging on thymus, but DLL1 and DLL4 presented highexpression in thymuses from adult-old samples regarding the postnatal group, whilethe FOXN1 expression was lower in adult-old group. By immunofluorescence, wewere not able to detect FOXN1 staining in thymus sections from adult-old sampleswhen compared with samples from postnatal thymus. In human thymic epithelial cellline (THPN), we show that FOXN1 expression is refractory to signals that induceFOXN1 transcription in primary 3D culture conditions and by stimulation of thecanonical WNT signaling pathway. Next, we analyzed the DNA methylation patternon FOXN1 gene by bisulfite sequencing using converted DNA from human thymusand THPN cell line. It was observed differential methylation on intron 1 and exon 2selected regions on the gene sequence. These results demonstrate for the first timethat FOXN1 gene expression in thymus may be regulated through an epigeneticmechanism during aging...