ITGA6 gene silencing by RNA interference modulates the expression of a large number of cell migration-related genes in human thymic epithelial cells.

BACKGROUND: The thymic epithelium is the major microenvironmental component of the thymus, the primary lymphoid organ responsible for the generation of T lymphocytes. Thymic epithelial cells (TEC) control intrathymic T cell differentiation by means of distinct types of interactions. TEC constitutively produce chemokines and extracellular matrix ligands (such as laminin and fibronectin) and express corresponding receptors, which allow thymocytes to migrate in a very ordered fashion. We previously showed that laminin mediates TEC/thymocyte interactions in both mice and humans. More recently, we used RNAi technology to knock-down the ITGA5 gene (which encodes CD49e, the integrin α-chain subunit of the fibronectin receptor VLA-5) in cultured human TEC. Using a similar strategy, herein we knocked-down the ITGA6 gene, which encodes CD49f, the α-chain of two integrin-type laminin receptors, namely VLA-6 (α6ß1) and α6ß4. RESULTS: We first confirmed that RNAi-induced knock-down of the ITGA6 gene was successful, at both transcription and translational levels, with a significant decrease in the membrane expression of CD49f, apart from CD49b, CD49c and CD49d, ascertained by cytofluorometry on living TEC. We also demonstrated that such knock-down promotes a decrease in cell adhesion to laminin. Using quantitative PCR, we demonstrated that gene expression of other integrin α-chains were concomitantly down-regulated, particularly those which form other laminin receptors, including ITGA1, ITGA2 and ITGA7. Interestingly enough, LAMA1 gene expression (whose corresponding protein chain is part of laminin-111) was largely increased in ITGA6 knocked-down TEC cultures. Lastly, the network complexity of gene expression under ITGA6 influence is much broader, since we found that other cell migration-related genes, namely those coding for various chemokines, are also modulated when IGTA6 is knocked-down. CONCLUSION: The data presented herein clearly show that down regulation of ITGA6 gene in the human thymic epithelium triggers a complex cascade of effects upon the expression levels of several other cell migration-related genes, including extracellular matrix and chemokine ligands and receptors. Taken together, these data unravel the concept that the expression of genes involved in controlling of thymocyte migration by the thymic microenvironment should be regarded as complex networks, so that a defect in the expression of one single gene may reflect in an amplified cascade with functional consequences for TEC adhesion onto the natural ligand and potential consequences upon the normal patterns of TEC/thymocyte interactions.

Laminin database: a tool to retrieve high-throughput and curated data for studies on laminins.

The Laminin(LM)-database, hosted at http://www.lm.lncc.br, is the first database focusing a non-collagenous extracellular matrix protein family, the LMs. Part of the knowledge available in this website is automatically retrieved, whereas a significant amount of information is curated and annotated, thus placing LM-database beyond a simple repository of data. In its home page, an overview of the rationale for the database is seen and readers can access a tutorial to facilitate navigation in the website, which in turn is presented with tabs subdivided into LMs, receptors, extracellular binding and other related proteins. Each tab opens into a given LM or LM-related molecule, where the reader finds a series of further tabs for 'protein', 'gene structure', 'gene expression' and 'tissue distribution' and 'therapy'. Data are separated as a function of species, comprising Homo sapiens, Mus musculus and Rattus novergicus. Furthermore, there is specific tab displaying the LM nomenclatures. In another tab, a direct link to PubMed, which can be then consulted in a specific way, in terms of the biological functions of each molecule, knockout animals and genetic diseases, immune response and lymphomas/leukemias. LM-database will hopefully be a relevant tool for retrieving information concerning LMs in health and disease, particularly regarding the hemopoietic system.

NOD2 receptors in adenopituitary folliculostellate cells: expression and function.

Folliculostellate cells (FS cells) are non-endocrine cells from the pituitary gland that respond to bacterial endotoxins by producing cytokines. In immune cells, an important component of bacterial recognition are the toll-like receptors (TLRs). Previously, we showed that FS cells express TLR4. The TLR4 ligand lipopolysaccharide (LPS) stimulates interleukin-6 (IL6) production through nuclear factor kappaB (NFKB) induction. Binding of IL6 to gp130 receptor activates signal transducer and activator of transcription 3 (STAT3), an important mediator of inflammatory response. Another family involved in innate immune response following bacterial infection is the nucleotide-binding oligomerisation domain (NOD) intracellular receptor family. Herein, we describe for the first time the expression and function of NOD receptors in human pituitary and FS TtT/GF cell line. The NOD2 agonist muramyl dipeptide (MDP) increased Nf kappa b1-transcriptional activity, -protein expression and IL6 secretion in TtT/GF cells. Furthermore, these effects were potentiated by the combination of MDP and LPS. Silencing NOD2 abolished the action of LPS on NFKB transcriptional activity and IL6 production, indicating that, in TtT/GF cells, TLR4 transduces its signal through NOD2 receptor. We show here that in TtT/GF cells, Nod2 overexpression or stimulation by MDP increased STAT3 transcriptional activity. Furthermore, silencing STAT3 inhibited basal, LPS and MDP stimulated NFKB protein expression and overexpression of protein inhibitor of activated STAT3 (Pias3) markedly decreased basal NFKB activity. These data suggest that in TtT/GF cells, STAT3 acting upstream to NFKB mediates NOD2 receptor signalling pathway. In conclusion, the present study demonstrates that NOD molecules play a modulatory role in the pituitary by regulating the function and activation of FS cells in response to bacterial components.

Modulation of growth hormone and prolactin secretion in Trypanosoma cruzi-infected mammosomatotrophic cells.

OBJECTIVE: In a previous study, we reported an imbalance in the hypothalamus-pituitary-adrenal axis of mice acutely infected with the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. METHODS: Possible effects of this parasitic infection on the endocrine function of other pituitary cell types were studied, in particular regarding the production of prolactin (PRL) and growth hormone (GH). RESULTS: In the mammosomatotrophic cell line GH3, both GH and PRL secretion were decreased, reflecting the diminished PRL concentrations in the pituitary glands of infected mice. Additionally, expression of extracellular matrix proteins, e.g. laminin, was increased in T. cruzi-infected GH3 cells, which may be related to the diminished secretory function of these cells. Lastly, the expression of Pit-1, a major transcription factor for the PRL and GH genes, is also decreased in T. cruzi-infected cultures. CONCLUSION: T. cruzi infection downregulates PRL and GH production. Combined with our previous data showing increased glucocorticoid levels following T. cruzi infection, the immunosuppression induced by T. cruzi infection may be partially related to multiple endocrine changes involving the hypothalamus-pituitary axis and corresponding target endocrine glands.

Laminin-211 controls thymocyte--thymic epithelial cell interactions.

Thymocyte differentiation occurs within the thymic microenvironment, consisting of distinct cell types and extracellular matrix (ECM) elements. One of these ECM proteins is laminin. Previous experiments showed that laminin mediates interactions between thymocytes and thymic epithelial cells (TEC) in mice. Since, laminin comprises a family of related isoforms, we searched for laminin isoform expression in the human thymus. We found constitutive gene expression of various laminin chains in TEC preparations, comprising laminin-111 and laminin-211 isoforms. Immunocytochemistry revealed a selective laminin-211 distribution in the thymic lobules. In vitro functional assays revealed that laminin-211 enhances TEC/thymocyte adhesion and thymocyte release from thymic nurse cells, as well as the reconstitution of these complexes. Conversely, these interactions are blocked by monoclonal antibodies specific for laminin-211 and the laminin receptor VLA-6. Our results reinforce the notion that distinct laminin isoforms in the human thymus are relevant for lymphoepithelial interactions.

Immunoneuroendocrine interactions in Chagas disease.

We investigated immunoneuroendocrine interactions in vivo and in vitro following infection by Trypanosoma cruzi, the causative agent of Chagas disease. In a first set of experiments, we studied the hypothalamus-pituitary-adrenal axis. Nests of parasites were seen in the adrenal gland, whereas T. cruzi-specific PCR gene amplification product was found in both the adrenal and pituitary glands of infected mice. These endocrine glands also revealed alterations including vascular stasis, increase in the deposition of extracellular matrix (ECM), as well as T cell and macrophage infiltration. Functionally, we found a decrease in corticotrophin-releasing hormone and an increase in corticosterone contents, in hypothalamus and serum, respectively, whereas no significant changes were seen in serum adrenocortricotropic hormone of infected animals. Nevertheless, the serum levels of interleukin-6 (known to directly stimulate glucocorticoid secretion) were increased, as compared to controls. Considering the presence of T cells within the nervous tissue of chagasic animals, we performed a number of in vitro experiments co-culturing spleen-derived T cells from control or infected mice, with neuronal cells (being or not being directly infected in vitro). In particular, we looked for ECM-mediated interactions, known to affect T cell migration. We found an increase in ECM deposition in infected cultures, as compared to controls. Moreover, adhesion of T cells was enhanced when neuronal cells were infected in vitro, or when T cells were derived from T. cruzi-infected mice, events that could be abrogated with anti-ECM antibodies. Together, the data summarized above clearly reveal that neuroendocrine axes are altered in experimental Chagas disease.

Hypothalamus-pituitary-adrenal axis during Trypanosoma cruzi acute infection in mice.

Functional interactions between neuroendocrine and immune systems are mediated by similar ligands and receptors, which establish a bi-directional communication that is relevant for homeostasis. We investigated herein the hypothalamus-pituitary-adrenal (HPA) axis in mice acutely infected by Trypanosoma cruzi, the causative agent of Chagas' disease. Parasites were seen in the adrenal gland, whereas T. cruzi specific PCR gene amplification product was found in both adrenal and pituitary glands of infected mice. Histological and immunohistochemical analyses of pituitary and adrenal glands of infected animals revealed several alterations including vascular stasis, upregulation of the extracellular matrix proteins fibronectin and laminin, as well as T cell and macrophage infiltration. Functionally, we detected a decrease in CRH and an increase in corticosterone contents, in hypothalamus and serum respectively. In contrast, we did not find significant changes in the amounts of ACTH in sera of infected animals, whereas the serum levels of the glucocorticoid-stimulating cytokine, IL-6 (interleukin-6), were increased as compared to controls. When we analyzed the effects of T. cruzi in ACTH-producing AtT-20 cell line, infected cultures presented lower levels of ACTH and pro-opiomelanocortin production when compared to controls. In these cells we observed a strong phosphorylation of STAT-3, together with an increased synthesis of IL-6, suppressor of cytokine signaling 3 (SOCS-3) and inhibitor of activated STAT-3 (PIAS-3), which could explain the partial blockage of ACTH production. In conclusion, our data reveal that the HPA axis is altered during acute T. cruzi infection, suggesting direct and indirect influences of the parasite in the endocrine homeostasis.

Atrophy of mesenteric lymph nodes in experimental Chagas' disease: differential role of Fas/Fas-L and TNFRI/TNF pathways.

It is currently accepted that experimental acute infection by Trypanosoma cruzi promotes changes in secondary lymphoid organs, with general T and B lymphocyte polyclonal activation. Here we show that mesenteric lymph nodes (MLN) of acutely infected mice show severe atrophy due to extensive lymphocyte apoptosis. Accordingly, clusters of apoptotic cells are detected in the initial phase of infection in MLN but not in subcutaneous nodes. Moreover, such atrophy is independent of the infection route, parasite load or the mouse strain used. Studies in Fas-L deficient (BALB gld/gld+/+) and in TNF type 1 receptor (p55-/-) knockout mice indicate that both molecules are involved in MLN atrophy: Fas-L participates in cell death of CD4+ as well as B lymphocytes, whereas the TNF type 1 receptor is important for the apoptosis of CD4+ and CD8+ T lymphocytes. In contrast, perforin does not play a role, as lymph nodes from perforin-deficient mice do not behave differently from the corresponding wild types. Our data support the concept that, even in a systemic infection, differential (even opposing) responses can be found in different lymph node chains.