Efeito pleiotrópico da semaforina-3A sobre timócitos e células estromais tímicas

Abstract

Thymus, a primary lymphoid organ, is responsible for the differentiation of thymocytes into immunocompetent T lymphocytes. For this purpose, their microenvironment and the several types of cell, collectively referred as thymic stromal cells (such as thymic epithelial cells, macrophages, dendritic cells, and fibroblasts), actively cooperate for thymocyte differentiation. This microenvironment can be influenced by extrinsic factors such as hormones and neuropeptides. Among these molecules, semaphorin- 3A (Sema3A) is described as a multifunctional protein involved in neurite migration however, there are few data about its effects on thymic microenvironment cells. In order to contribute to this knowledge, we aimed to evaluate the effects of Sema3A on thymocytes and thymic stromal cells. Therefore, stromal cells were obtained by enzymatic digestion of murine thymus. Firstly, cell types obtained from the thymic stroma were characterized mainly as thymic epithelial cells (67%), but others cell types were also present, as indicate the expression of cell markers to macrophages, fibroblasts, and dendritic cells. In addition, the expression of the receptor for Sema3A, neuropilin-1, was observed on the surface of these cells. Besides that, the treatment with Sema3A modulated the morphology of thymic cells. Also, Sema3A promoted the projection of filopodia by thymocytes and altered F-actin polymerization in stroma cells. Secondly, through the analysis of immunocytochemistry images, it was verified that the fibronectin fibers produced by the stromal cells were reorganized by Sema3A and the laminin production was increased by this treatment. Using flow cytometry, it was observed that the expression of VLA-5 was reduced in both total and double- positive thymocytes, however, there was no change in VLA-6 levels after the treatment with Sema3A. In the cell adhesion scenario, an inverse effect was observed on cells pretreated with Sema3A, whose thymocyte adhesion was increased when stroma was treated and decreased when only thymocytes were treated. Finally, when Sema3A was applied during the migratory process, the number of migrant thymocytes was reduced. Together these data indicate the pleiotropy of Sema3A effects on thymic microenvironment (thymocytes and stromal cells), classifying this protein as an important molecule to modulate thymic functions.