RNA interference-mediated knockdown of CD49e (α5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Abstract Background The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin α5β1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

We thank Dr. Laurent Beck (Université Paris Descartes) for providing some primers for real-time quantitative PCR and for technical support, and Dr. Ana Tereza R. Vasconcelos (Bioinformatics Group, Brazilian National Laboratory on Computational Sciences) for critically reading the manuscript. The cDNA clones used to prepare the cDNA microarrays were ceded by Dr. Catherine Nguyen from INSERM U928, Marseille, France. This work was developed in the context of the CNRS-Fiocruz International Associated Laboratory of Immunology and Immunopathology. It was further supported by grants from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), Capes (Coordenação de aperfeiçoamento de pessoal de nível superior), Faperj (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro), Fapesp (Fundação de Amparo a Pesquisa do Estado de São Paulo) and Oswaldo Cruz Foundation (Brazil).

We thank Dr. Laurent Beck (Université Paris Descartes) for providing some primers for realtime quantitative PCR and for technical support, and Dr. Ana Tereza R. Vasconcelos (Bioinformatics Group, Brazilian National Laboratory on Computational Sciences) for critically reading the manuscript. The cDNA clones used to prepare the cDNA microarrays were ceded by Dr. Catherine Nguyen from INSERM U928, Marseille, France. This work was developed in the context of the CNRSFiocruz International Associated Laboratory of Immunology and Immunopathology. It was further supported by grants from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), Capes (Coordenação de aperfeiçoamento de pessoal de nível superior), Faperj (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro), Fapesp (Fundação de Amparo a Pesquisa do Estado de São Paulo) and Oswaldo Cruz Foundation (Brazil).

This article has been published as part of BMC Genomics Volume 11 Supplement 5, 2010: Proceedings of the 5th International Conference of the Brazilian Association for Bioinformatics and Computational Biology. The full contents of the supplement are available online at http://www.biomedcentral.com/14712164/11?issue=S5.

This article has been published as part of BMC Genomics Volume 11 Supplement 5, 2010: Proceedings of the 5th International Conference of the Brazilian Association for Bioinformatics and Computational Biology. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2164/11?issue=S5.