Date Published: June 27, 2018
Publisher: Public Library of Science
Author(s): Heather E. Stefanski, Yan Xing, Patricia A. Taylor, Stefano Maio, Jorge Henao-Meija, Adam Williams, Richard A. Flavell, Georg A. Hollander, Bruce R. Blazar, Geraldo A Passos.
MicroRNAs (miRNAs) have been shown to be key modulators of post-transcriptional gene silencing in many cellular processes. In previous studies designed to understand the role of miRNAs in thymic development, we globally deleted miRNA exclusively in thymic epithelial cells (TECs), which are critical in thymic selection. This resulted in the loss of stromal cells that instruct T cell lineage commitment and affect thymocyte positive selection, required for mature T cell development. Since murine miR-181 is expressed in the thymus and miR-181 deficiency disrupts thymocyte development, we first quantified and thereby demonstrated that miR181a1 and miR181b1 are expressed in purified TECs. By generating mice with TEC targeted loss of miR-181a1 and miR-181b1 expression, we observed that neither TEC cellularity nor thymocyte number nor differentiation was adversely affected. Thus, disrupted thymopoiesis in miR-181 deficient mice was not due to miR-181 loss of expression in TECs. Importantly, in mice with restricted TEC deficiency of miR-181a1 and miR-181b1, there were similar numbers of mature T cells in the periphery in regards to frequencies, differentiation, and function as compared to controls. Moreover miR-181a1 and miR-181b1 were not required for maintenance of thymus integrity over time, as thymic involution was not accelerated in gene-targeted mice. Taken together our data indicate that miR-181a1 and miR-181b1 are dispensable for TEC differentiation, their control of thymocyte development and mature T cell export to and homeostasis within the periphery.
A normal thymic stromal composition and arrangement are essential for growth, differentiation and T cell receptor repertoire selection of thymocytes. The thymus is composed of thymic epithelial cells (TECs), fibroblasts, B cells and macrophage/dendritic cells with the predominant population being thymocytes. TECs form a three-dimensional matrix that reaches from the subcapsular cortical area to the core of the medulla with cortical TEC (cTEC) and medullary TEC (mTEC) subsets defining these compartments according to their functional, structural and antigenic features [2, 3]. Bidirectional interactions between developing thymocytes and the stroma are critical to maintain a normally structured and regularly functioning microenvironment able to support thymocyte development[2, 4, 5]. Developing thymocytes can be distinguished by their CD4 and CD8 cell surface expression. CD4−CD8− (double-negative, DN) thymocytes mature to become CD4+CD8+ (double-positive, DP) thymocytes and undergo positive selection on cTECs. DP thymocytes continue their maturation into CD4+ or CD8+(single-positive, SP) thymocytes which then are subjected to negative selection first by cTECs and, then, upon migration to the medulla, by mTECs and medullary dendritic cells (DCs). mTECs can be further characterized based on their MHC II cell surface expression into largely immature and terminally differentiated mTEClo and mature mTEChi populations. The mTEClo population are heterogeneous with some cells giving rise to mTEChi or to cells that express involucrin and contribute to Hassal’s corpuscles while others express CCL21 that attracts positively selected thymocytes to the medulla[6–8]. The mTEChi population is also heterogeneous, not least with regards to expression of the autoimmune regulator (AIRE), critical for establishing self-tolerance of medullary thymocytes. AIRE is a transcription factor that controls the ectopic expression of a large set of peripheral tissue antigen genes in mTECs. SP thymocytes complete their maturation in the medulla after which they are exported into the periphery as mature T cells.
Statistical analysis was performed using Student t test (unpaired, two-tailed) using the Graph Pad Prism software (version 6). Probability values were classified into four categories: p > 0.05 (NS), *0.05 ≥ p > 0.01, **0.01 ≥ p > 0.001, and ***p ≤ 0.0001.
MiR-181a has been shown to be expressed at high levels in the thymus[19, 21]. Our studies addressed the role of miR-181a1 and miR-181b1 in TECs and the effects on TEC development. In all subsets of TECs, expression of miR-181a1 and miR-181b1 was present at levels equal to thymocytes. Interestingly, there was a decrease in the percentage of total TECs in thymii from Foxn1-Cre::Mir181a1/b1fl/fl mice, which was due to a decrease in the cTEC compartment. Although this did not result in a decrease an absolute number of total TECS, but did result in a lower number of cTECS. Despite this difference, positive selection was not affected in these mice.