Stabilized beta-catenin in thymic epithelial cells blocks thymus development and function.

Thymic T cell development is dependent on a specialized epithelial microenvironment mainly composed of cortical and medullary thymic epithelial cells (TECs). The molecular programs governing the differentiation and maintenance of TECs remain largely unknown. Wnt signaling is central to the development and maintenance of several organ systems but a specific role of this pathway for thymus organogenesis has not yet been ascertained. In this report, we demonstrate that activation of the canonical Wnt signaling pathway by a stabilizing mutation of beta-catenin targeted exclusively to TECs changes the initial commitment of endodermal epithelia to a thymic cell fate. Consequently, the formation of a correctly composed and organized thymic microenvironment is prevented, thymic immigration of hematopoietic precursors is restricted, and intrathymic T cell differentiation is arrested at a very early developmental stage causing severe immunodeficiency. These results suggest that a precise regulation of canonical Wnt signaling in thymic epithelia is essential for normal thymus development and function.

Foxn1 is essential for vascularization of the murine thymus anlage.

We addressed whether vascularization of the thymus anlage depends on Foxn1 expression. In the thymus anlagen of wild-type mice, CD31(+) endothelial cells are initially observed between epithelial cells on embryonic day (Ed)12.5 and form luminal structure on Ed13. VEGF are produced in epithelial cells and mesenchymal cells which invaginate in the epithelial region of the anlagen on Ed13. However, in the nude thymus anlagen, neither CD31(+) cells nor VEGF producing mesenchymal cells is detected in the epithelial region. The present results indicate that Foxn1 dependent epithelial development is essential for vascularization of the thymus anlagen.

Moxibustion activates host defense against herpes simplex virus type I through augmentation of cytokine production.

Moxibustion is a technique used in traditional oriental medicine, the aim of which is to cure and/or prevent illness by activating a person's ability for self-healing. In this study, we assessed how moxibustion would affect the immune system and whether it would augment protective immunity. Mice were treated with moxibustion at Zusanli (ST36) acupoints; we analyzed mortality and cytokine activity in sera after infection with herpes simplex virus type 1 (HSV-1), and cytokine gene expression in the skin and the spleen without a virus challenge. Our study demonstrates that pretreatment of BALB/c mice with moxibustion resulted in a marked increase in the survival rate after infection with lethal doses of HSV-1, and elevated serum levels of IL-1ß and IFN-γ on days 1 and 6 post-infection with HSV-1. Semi-quantitative RT-PCR assay showed that moxibustion treatment augmented the expression of IL-1α, IL-1ß, IL-6, universal-IFN-α, MIP-1α, and TNF-α mRNA in the skin, and IL-1α, IL-1ß, IL-12p40, IL-15, u-IFN-α, MIP-1α, and TNF-α mRNA in the spleen. Moreover, moxibustion induces augmentation of natural killer cell activity. Collectively, our study demonstrates that moxibustion activates protective responses against HSV-1 infection through the activation of cytokine production including IFN, and of NK cells.

Expression of Dll4 and CCL25 in Foxn1-negative epithelial cells in the post-natal thymus.

Foxn1 transcription factor is known to be essential for development of the thymic organ. We analyzed whether Foxn1 expression in thymic epithelial cells is necessary for the expression of functional molecules such as Delta-like 4 (Dll4) and CCL25, and whether maintenance of these molecular expressions depends on the Foxn1 transcription factor. We show that almost all thymic epithelial cells in the early thymus anlagen express Foxn1, and Dll4 and CCL25 are limitedly expressed in Foxn1-positive epithelial cells. The results are consistent with previous reports suggesting the indispensability of Foxn1 for epithelial cell differentiation which enables these cells to induce the expressions of CCL25 (Bleul, C. C. and Boehm, T. 2000. Chemokines define distinct microenvironments in the developing thymus. Eur. J. Immunol. 30:3371), Dll1 and Dll4 (Tsukamoto, N., Itoi, M., Nishikawa, M. and Amagai, T. 2005. Lack of Delta like 1 and 4 expressions in nude thymus anlages. Cell. Immunol. 234:77). On the other hand, the expression of Foxn1 was not detectable in a large number of post-natal thymic epithelial cells. Both Foxn1-positive and -negative epithelial cells seem to express Dll4 and CCL25. Therefore, the expressions of Dll4 and CCL25 are independent of Foxn1 transcription factor in the post-natal thymus. These results indicate that in the post-natal thymus, epithelial cells may maintain the expressions of those functional molecules without the aid of Foxn1 transcription factor.

The perivascular space as a path of hematopoietic progenitor cells and mature T cells between the blood circulation and the thymic parenchyma.

It is known that selected populations of lymphoid cells migrate into and from the adult thymus through blood vessels at the cortico-medullary junction and in the medulla. Here, we show that in the perivascular spaces (PVS) of mice surrounding large blood vessels, CD117-positive hematopoietic progenitor cells, CD4 single-positive (SP) and CD8SP T cells are located. However, developing thymocytes, CD25-positive cells and CD4 and CD8 double-positive cells, are not detectable in the PVS. After intravenous (i.v.) injection of CD117-positive bone marrow (BM) cells from C57BL/6 mice into non-irradiated RAG2 mutant mice i.v., donor-derived cells first preferentially migrate into the PVS within 30 min, and then the number of donor-derived cells in the thymic parenchyma increases. Likewise, newly developed mature T cells in the thymic parenchyma of RAG2 mutant mice transferred with wild-type BM cells migrate to the PVS, before leaving the thymus to the circulation. Accumulation of mature T cells was observed after treatment with sphingosine-1 phosphate receptor agonist FTY720 not only in the medulla but also in the thymic PVS. These results suggest that the PVS is a transit pathway for progenitor cells to immigrate into the thymus and for mature T cells to emigrate from the thymus.

Mesenchymal cells are required for functional development of thymic epithelial cells.

Epithelial-mesenchymal interactions have essential roles in thymus organogenesis. Mesenchymal cells are known to be required for epithelial cell proliferation. However, the contribution of mesenchymal cells to thymic epithelial cell differentiation is still unclear. In the present study, we have investigated the roles of mesenchymal cells in functional development of epithelial cells in the thymus anlage in patch (ph) mutant mice, which have a primarily defect in mesenchymal cells caused by the absence of platelet-derived growth factor receptor alpha expression. In the ph/ph thymus anlage, T cell progenitors migrate normally among the epithelial cells, however, they are severely impaired to proliferate and differentiate to CD25-positive cells. Epithelial cells of the ph/ph thymus anlage show severely impaired proliferation and expression of functional molecules, such as SCF, Delta-like 4 and MHC class II, which have crucial roles in T cell development. Moreover, the cultured ph/ph thymus anlage fails to develop into a mature organ supporting full T cell development. Addition of intact thymic mesenchymal cells to organ culture induces development of the ph/ph thymus anlage. In the cultured lobes, added mesenchymal cells contribute to form not only the capsule but also the meshwork structure mingled with epithelial cells. Our present results strongly suggest the roles of mesenchymal cells in functional development of epithelial cells in thymus organogenesis. In addition, our data suggest that mesenchymal cells are required to create the thymic microenvironment and to maintain epithelial architecture and function.

Lack of Delta like 1 and 4 expressions in nude thymus anlages.

Notch signaling is required for the early steps in T cell development. However, distribution of Notch ligands in the thymus anlages is not clear. We investigated the expressions of Delta like (Dll) 1 and Dll4 in the mouse thymus anlages. In the normal thymus anlages on embryonic day 13, Dll4 is strongly expressed throughout the epithelial region, but Dll1 is expressed only in scattered cells. In contrast, epithelial cells of the nude thymus anlages express neither Dll1 nor Dll4. These results indicate that expressions of Dll1 and Dll4 in thymic epithelial cells are regulated by Foxn1 transcriptional factor.