Age-associated changes in miRNA expression profiles in thymopoiesis

Abstract

During adult life, the thymus involutes and thymic output of mature T cells drastically declines. The molecular events underlying this process are not well understood. Here, we present evidence of the importance of miRNAs in regulating T cell differentiation in the aged. miRNAs are a wide-ranging regulatory element influencing gene expression throughout the lifetime of the organism. To establish whether they play a role in the age-specific thymic decline, the miRNA expression pattern was examined in TN subsets of young and aged mice. Fifty-two percent of the miRNAs exhibited elevated expression levels in the aged TN1 cells. This expression profile leads us to hypothesize that the large number of highly expressed miRNAs, indicative of rigidly controlled protein expression, limits the developmental potential of this population and results in the age-induced decline in thymopoiesis.

Figure 1. Fold difference in miRNA expression in aged relative to young total TN thymocytes

Two thymuses from 3 month old mice were dissected and the thymocytes pooled for the isolation of young total TN cells. Thymocytes from six animals (24 months old) were pooled for the isolation of aged total TN cells. Panel A. Each vertical bar corresponds to the fold difference in expression of a single miRNA. The columns extending above the horizontal line represent the miRNAs whose expression level is greater than 3 fold in the aged TN thymocytes. Panel B. The pie chart shows the distribution of miRNA’s whose fold expression is up regulated, down regulated or not significantly changed in aged compared to young TN thymocytes. The percentage comprising each group is indicated.

Figure 2. Fold difference in miRNA expression in aged relative to young TN1 thymocytes

For the isolation of the young TN1 subpopulation, thymocytes were pooled from five animals (2 months in age). Ten animals, 22 to 24 months old, were used for the purification of the aged thymic subsets. The Lin⁻ TN1 cells were selected by cell sorting as CD8⁻, CD4⁻, CD3⁻, CD11b⁻, B220⁻, Gr-1⁻ (Ly6G), and Ter-119⁻ (Ly-76), CD44⁺, and CD25⁻. Total RNA was isolated and the miRNA profiles generated as described for the TN population. Panel A. The setup of the graph is as detailed in Figure 1. Panel B. The organization of the pie chart is described in Figure 1.

Figure 3. Fold difference in miRNA expression in aged relative to young TN2/3

The cells were isolated as in Figure 2. The TN2/3 subset was generated during sorting by pooling the TN2 cells, CD8⁻, CD4⁻, CD3⁻, CD11b⁻, B220⁻, Gr-1⁻ (Ly6G), Ter-119⁻ (Ly-76), CD44⁺, CD25⁺ and the TN3 cells, CD8⁻, CD4⁻, CD3⁻, CD11b⁻, B220⁻, Gr-1⁻ (Ly6G), Ter-119⁻ (Ly-76), CD44⁻, CD25⁺. RNA isolation and miRNA profiling is as detailed for the TN population. Panel A. The graph organization is described in Figure 1. Panel B. The pie chart is as detailed in Figure 1.

Figure 4. Fold difference in miRNA expression in aged relative TN4 thymocytes

The thymocytes were obtained as described in Figure 2. The TN4 cells were purified by choosing the CD8⁻, CD4⁻, CD3⁻, CD11b⁻, B220⁻, Gr-1⁻ (Ly6G), Ter-119⁻ (Ly-76), CD44⁻, CD25⁻ population during cell sorting. RNA isolation and miRNA profiling is as detailed for the TN population. Panel A. The graph organization is described in Figure 1. Panel B. The set up for the pie chart is explained in Figure 1.

Figure 5. Comparison of the total number of miRNAs up or down regulated in all cell types analyzed

Panel A. The columns represent number of miRNAs whose expression level is greater than 3 fold in the aged. Panel B. The columns indicate the number of miRNAs whose expression is down regulated 3 fold or more in aged cells.