Identification of MiR-205 As a MicroRNA That Is Highly Expressed in Medullary Thymic Epithelial Cells

<div><p>Thymic epithelial cells (TECs) support T cell development in the thymus. Cortical thymic epithelial cells (cTECs) facilitate positive selection of developing thymocytes whereas medullary thymic epithelial cells (mTECs) facilitate the deletion of self-reactive thymocytes in order to prevent autoimmunity. The mTEC compartment is highly dynamic with continuous maturation and turnover, but the genetic regulation of these processes remains poorly understood. MicroRNAs (miRNAs) are important regulators of TEC genetic programs since miRNA-deficient TECs are severely defective. However, the individual miRNAs important for TEC maintenance and function and their mechanisms of action remain unknown. Here, we demonstrate that miR-205 is highly and preferentially expressed in mTECs during both thymic ontogeny and in the postnatal thymus. This distinct expression is suggestive of functional importance for TEC biology. Genetic ablation of miR-205 in TECs, however, neither revealed a role for miR-205 in TEC function during homeostatic conditions nor during recovery from thymic stress conditions. Thus, despite its distinct expression, miR-205 on its own is largely dispensable for mTEC biology.</p></div

Validation of miR-205 ablation in TECs.

<p><b>(A)</b> mTECs from 4–6 week old <i>miR-205</i>^CTRL and <i>miR-205</i>^ΔTEC mice were sorted to analyze miR-205 expression by qPCR. Reactions were normalized to sno202 and normalized to CD45⁺ cells. Values depict mean ±SD. Data is representative of two independent experiments. <b>(B)</b><i>in situ</i> hybridizations were performed using a miR-205 probe on frozen thymic sections from 4–6 week old <i>miR-205</i>^CTRL and <i>miR-205</i>^ΔTEC mice to confirm the uniform deletion of miR-205 in mTECs. Scale bars = 200 μm (top), and 100 μm (bottom).</p

miR-205 deficient TECs show comparable sensitivity and recovery potential to poly(I:C) mediated thymic involution.

<p><b>(A)</b> 4-week old mice were treated with varying doses of poly(I:C) at day (-3) and day (0) before being harvested at day 4 of their recovery for enumeration of total thymic cellularity. <b>(B)</b> Mice were treated with 250μg of poly(I:C) as conducted in <b>(A)</b> and then harvested at 12 days of recovery to enumerate total thymic cellularity. <b>(C)</b> Enumeration of total mTEC and cTEC cellularity in 4-week old mice shown in <b>(B)</b>. cTECs were defined as CD45^-, EpCAM⁺, Ly51⁺, MHC II⁺ events. mTECs were defined as CD45^-, EpCAM⁺, Ly51^-, MHC II⁺ events. <b>(D)</b> Subset composition was assessed by flow cytometry of mTECs as defined in <b>(C)</b>. Quantification of total TEC cellularity and assessment of the proliferation marker Ki67 for the mTEC subsets shown on the left. Data in <b>(B-D)</b> are shown as mean ±SEM of 7 samples per group and are representative of at least two independent experiments. Gray bars in <b>(B-E)</b> indicate untreated <i>miR-205</i>^CTRL mice, gray bars indicate poly(I:C) treated <i>miR-205</i>^CTRL mice, and red bars indicate poly(I:C) treated<i>miR-205</i>^ΔTEC mice.</p

Radiation-induced thymic stress does not reveal a role for miR-205 in TECs.

<p><b>(A)</b> 6-week old mice were exposed to sub-lethal total body irradiation and then harvested after either 15 or 30 days of recovery to enumerate total thymic cellularity. <b>(B)</b> Enumeration of total mTEC and cTEC cellularity from mice shown in <b>(A)</b>. mTECs were defined as CD45^-, EpCAM⁺, Ly51^-, MHC II⁺ events. cTECs were defined as CD45^-, EpCAM⁺, Ly51⁺, MHC II⁺ events. <b>(C)</b> Subset composition of mTECs was assessed by flow cytometry of mTECs as defined in <b>(B)</b>. <b>(D)</b> Quantification of mTEC cellularity and assessment of the proliferation marker Ki67 for the mTEC subsets shown in <b>(C)</b>. Data in <b>(A-D)</b> are shown as mean ±SEM of 10 samples per group pooled from two independent experiments. Total cellularity plotted as percent of untreated <i>miR-205</i>^CTRL mice to allow for direct comparison between the two timepoints. Blue bars indicate untreated <i>miR-205</i>^CTRL mice, gray bars indicate treated <i>miR-205</i>^CTRL mice, and red bars indicate treated <i>miR-205</i>^ΔTEC mice. * denotes p≤0.05, Student’s <i>t</i>-test.</p

miR-205 is expressed during thymic ontogeny and maintained in the adult thymus.

<p><b>(A)</b> X-gal staining was performed on transverse sections of e14.5 <i>miR-205</i>^lacZ embryos to identify patterns of miR-205 transcription. <b>(B)</b> Whole-mount X-gal staining was performed on a dissected e18.5 <i>miR-205</i>^lacZ embryo (left) and positive lacZ reporter activity was observed in the thymus (arrow). A dissected and stained <i>miR-210</i>^lacZ embryo (right) is shown as a negative control for lacZ activity in the thymus. These images have been published previously at <a href="http://rna.keck.ucsf.edu/sites/rna.keck.ucsf.edu/files/205_E18.5_051510_24.jpg" target="_blank">http://rna.keck.ucsf.edu/sites/rna.keck.ucsf.edu/files/205_E18.5_051510_24.jpg</a>. <b>(C)</b> Thymic sections from an e18.5 <i>miR-205</i>^lacZ embryo were cut and then stained with X-gal. Arrows indicate positive lacZ reporter activity in a subset of cells in the thymus. <b>(D)</b><i>in situ</i> hybridization for miR-205 was performed on frozen thymic sections from 6–8 week old B6 wildtype mice. Serial sections were hybridized using either a miR-205 probe or a scramble probe. Image pairs from two samples are shown. Scale bars = 200 μm. <b>(E-F)</b> Sorted thymic subsets from either <i>Aire</i>^+/+<b>(E)</b> or <i>Aire</i>^-/-<b>(F)</b> mice were analyzed by qPCR for miR-205 expression. Both genotypes carried the Aire-GFP (<i>Adig</i>) allele to facilitate the sorting of Aire⁺/GFP⁺ and Aire^-/GFP^- mTEC subsets. Reactions were standardized to sno202 and then normalized to CD45⁺ cells with error bars depicting mean ±SD.</p

TEC miRNA profiling identifies miR-205 as highly expressed in mTECs.

<p><b>(A)</b> Thymic subsets were purified from 4–5 week old <i>NOD</i> wildtype mice for miRNA profiling by microarray analysis. The heatmap depicts the union of differentially expressed miRNAs from any comparison (FDR <0.05) with an absolute log2 fold change >1 relative to the signal intensity of CD45⁺ cells. <b>(B)</b> Plot depicts average log2 fold change (FC) between mTEC vs CD45⁺ cells on the <i>y</i>-axis and average log2 signal intensity across all samples on the <i>x</i>-axis. Red dots indicate genes that are differentially expressed in mTECs vs CD45⁺ cells with an FDR <0.05. <b>(C)</b> Thymic stromal subsets were FACS-purified from 4–6 week old <i>B6</i> wildtype mice to confirm the expression of miR-205 in mTECs by qPCR analysis. All reactions were standardized to sno202 and then normalized to CD45⁺ cells with error bars depicting mean ±SD.</p