Identification of mTEC precursor cells

Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire+ mTECs) is unclear. Here, we describe novel embryonic precursors of Aire+ mTECs. We found the candidate precursors of Aire+ mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire+ mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire+ mTECs and efficiently suppressed the onset of autoimmunity induced by Aire+ mTEC deficiency. Mechanistically, pMECs differentiated into Aire+ mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire+ mTECs

Hypergravity Provokes a Temporary Reduction in CD4+CD8+ Thymocyte Number and a Persistent Decrease in Medullary Thymic Epithelial Cell Frequency in Mice.

Gravity change affects many immunological systems. We investigated the effects of hypergravity (2G) on murine thymic cells. Exposure of mice to 2G for three days reduced the frequency of CD4+CD8+ thymocytes (DP) and mature medullary thymic epithelial cells (mTECs), accompanied by an increment of keratin-5 and keratin-8 double-positive (K5+K8+) TECs that reportedly contain TEC progenitors. Whereas the reduction of DP was recovered by a 14-day exposure to 2G, the reduction of mature mTECs and the increment of K5+K8+ TEC persisted. Interestingly, a surgical lesion of the inner ear's vestibular apparatus inhibited these hypergravity effects. Quantitative PCR analysis revealed that the gene expression of Aire and RANK that are critical for mTEC function and development were up-regulated by the 3-day exposure and subsequently down-regulated by the 14-day exposure to 2G. Unexpectedly, this dynamic change in mTEC gene expression was independent of the vestibular apparatus. Overall, data suggest that 2G causes a temporary reduction of DP and a persistent reduction of mature mTECs in a vestibular system-dependent manner, and also dysregulates mTEC gene expression without involving the vestibular system. These data might provide insight on the impact of gravity change on thymic functions during spaceflight and living

Expressions of mTEC-related molecules in the thymus and plasma of mice exposed to 2G.

<p>(A) Expressions of RANK, Aire, and RANKL in the whole thymus of mice exposed to 2G (2G) for 3 days. “C” indicates 1G control. Vestibular apparatus are surgically disrupted in some groups of mice (labeled as VL). Expression of RANK (<i>Rank</i>), Aire (<i>Aire</i>), and RANKL (<i>Rankl</i>) mRNAs was evaluated by qPCR analysis. N = 5 each C, 2G, C with VL, and 2G with VL groups. The asterisks indicate statistical significance at **P < 0.01 and *P < 0.05 (Student’s <i>t</i>-test). (B) Expressions of RANK and Aire in the whole thymus of mice exposed to 2G for 14 days. Expression of RANK and Aire mRNAs was evaluated by qPCR analysis. N = 6 each C, C with VL, and 2G with VL groups. N = 5 for 2G. The asterisks indicate statistical significance at *P < 0.05, *P < 0.01, and ***P < 0.001 (Student’s <i>t</i>-test). NS indicates that the difference is not significant (Student’s <i>t</i>-test). (C) RANKL and OPG protein levels in plasma of mice exposed to 2G for 3 days. Plasma concentration of RANKL (left) and OPG (middle) protein was determined by ELISA. Ratio of RANKL to OPG (RANKL/OPG) was exhibited in the right figure. N = 5 each C, C with VL, and 2G with VL groups. N = 4 for 2G. The asterisks indicate statistical significance at **P < 0.01 (Student’s <i>t</i>-test). (D) Corticosterone level in plasma of mice exposed to 2G for 3 days. Concentration of corticosterone was determined by ELISA. N = 6 each C, G, C with VL, and 2G with VL groups. NS indicates that the difference is not significant (Student’s <i>t</i>-test).</p

Increment of keratin-5 keratin-8-double positive cells induced by hypergravity is persistent.

<p>Immunostaining of thymic section from mice exposed to 2 <i>g</i> gravity (2G) for 14 days. Thymic frozen sections were immunostained with a combination of anti-keratin-8 antibodies (upper panels) and anti-keratin-5 (middle panels). Merged images are shown in lower panels. Mice were exposed to 2 <i>g</i> gravity (2G) for 3 days or left under 1G (Control). Vestibular apparatus are surgically disrupted in some groups of mice (VL). Data are representatives of 6 independent mice samples (N = 6). Scale bars indicate 100 μm. Data are representatives of 3 independent mice samples (N = 3).</p

The short-term hypergravity exposure causes an increment of keratin-5 keratin-8-double positive cells in a vestibular apparatus-dependent manner.

<p>(A) Immunostaining of thymic section from mice exposed to 2G for 3 days. Thymic frozen sections were immunostained with a combination of anti-keratin-8 antibodies (upper panels) and anti-keratin-5 (upper middle panels). Merged images are shown in lower middle panels. Magnified images (indicated as x40) of white dot rectangles are shown in lower panels. Mice were exposed to 2 <i>g</i> gravity (2G) for 3 days or left under 1G (Control). Vestibular apparatus are surgically disrupted in some groups of mice (VL). Data are representatives of 6 independent mice samples (N = 6). Scale bars indicate 100 μm. (B) Expression of keratin-5 and keratin-8 mRNA in the total thymus from mice exposed to 2G for 3 days. Expression of keratin-5 (<i>Krt5</i>) and keratin-8 (<i>Krt8</i>) mRNAs was evaluated by qPCR analysis. N = 5 each C, 2G, C with VL, and 2G with VL groups. The asterisks indicate statistical significance at *P < 0.05 (Student’s <i>t</i>-test).</p

The short-term hypergravity exposure causes a reduction of CD4⁺CD8⁺ double positive thymocytes in a vestibular apparatus-dependent manner.

<p>(A) Ratio of thymic weight to body weight (left) and total thymic cell number (right) of mice exposed to 2 <i>g</i> gravity (labeled as 2G) for 3 day or normal gravity control (labeled as C). Vestibular apparatus were surgically disrupted in some groups of mice (labeled as VL). N = 10 for C and 2G, N = 8 for C with VL, and N = 9 for 2G with VL. The asterisks indicate statistical significance at ***P < 0.001 and **P < 0.01 (Student’s <i>t</i>-test). NS indicates that the difference is not significant (Student’s <i>t</i>-test). (B) Flow cytometric analysis of thymocytes in mice exposed to 2G for 3 days. Thymocytes were analyzed by staining with CD4 and CD8α antibodies (left figures). Numbers in panels indicates percentage of each fraction. Percentages of CD4- and CD8-double positive thymocytes (CD4⁺CD8⁺DP) in total thymocytes are summarized in right figures. Mice were exposed to 2 <i>g</i> gravity (2G) for 3 days. “C” indicates 1G control. Vestibular apparatus are surgically disrupted in some groups of mice (VL). N = 10 for C and 2G, N = 8 for C with VL, and N = 9 for 2G with VL. The asterisks indicate statistical significance at ***P < 0.001 and *P < 0.05 (Student’s <i>t</i>-test). NS indicates that the difference is not significant (Student’s <i>t</i>-test). (C) Cell numbers of CD4⁺CD8⁺ (CD4⁺CD8⁺DP), CD4⁺CD8^- (CD4SP), CD4^-CD8⁺ (CD8SP), and CD4^-CD8^- (DN) fractions in mice exposed to 2G for 3days. N = 10 for C and 2G, N = 8 for C with VL, and N = 9 for 2G with VL. The asterisks indicate statistical significance at ***P < 0.001 and *P < 0.05 (Student’s <i>t</i>-test). NS indicates that the difference is not significant (Student’s <i>t</i>-test).</p

Thymic weight and thymocytes were normal in mice exposed to 2G for 14 days.

<p>(A) Ratio of thymic weight to body weight (left) and total thymic cell number (right) of mice exposed to 2 <i>g</i> gravity (2G) for 14 day or normal gravity control (C). Vestibular apparatus are surgically disrupted in some groups of mice (labeled as VL). N = 6 each C, C with VL, and 2G with VL groups. N = 5 for 2G. NS indicates that the difference is not significant (Student’s <i>t</i>-test). (B) Flow cytometric analysis data of thymocytes in mice exposed to 2G for 14 days. Mice were exposed to 2 <i>g</i> gravity (labeled as 2G) for 14 days or left under 1G (control). Vestibular apparatus are surgically disrupted in some groups of mice (VL). Thymocytes were analyzed by staining with CD4 and CD8α antibodies. Numbers in panels indicate percentage of each fraction. Percentage and numbers of CD4- and CD8-double positive thymocytes (DP) are summarized in right figures. N = 6 each C, C with VL, and 2G with VL groups. N = 5 for 2G. The asterisks indicate statistical significance at *P < 0.05 (Student’s <i>t</i>-test). NS indicates that the difference is not significant (Student’s <i>t</i>-test).</p

The short-term hypergravity exposure causes a reduction in frequency of mature medullary thymic epithelial cells in a vestibular apparatus-dependent manner.

<p>(A) Flow cytometric analysis of thymic epithelial cells (TECs) in mice exposed to 2G for 3 days. Mice were exposed to 2 <i>g</i> gravity (2G) for 3 days or left under 1G (Control). Vestibular apparatus are surgically disrupted in some mice (labeled as VL). TECs (CD45^–TER119^– EpCAM⁺) in total thymic cells were analyzed by staining with UEA-1-lectin, an mTEC marker, and CD80 antibody. Numbers in panels indicates percentage of each fraction. The percentages of UEA-1^– CD80^lo (containing cTECs), UEA-1⁺CD80^high (mTEC^hi), and UEA-1⁺CD80^low (mTEC^lo) cells among thymic stroma cells in the thymus are summarized in right figures. “C” in graphs indicates 1G control. N = 5 each C, 2G, C with VL, and 2G with VL groups. The asterisks indicate statistical significance at **P < 0.01 (Student’s <i>t</i>-test). (B) Cell numbers of total TECs (CD45^–TER119^– EpCAM⁺), UEA-1^– CD80^lo TECs (containing cTECs), mTECs (UEA-1⁺), mTEC^hi (UEA-1⁺CD80^high), and mTEC^lo (UEA-1⁺CD80^low) in the thymus are summarized in figures. N = 5 each C, 2G, C with VL, and 2G with VL groups.</p

Long-term 2G exposure causes a reduction in cell number of mTECs.

<p>(A) Flow cytometric analysis of thymic epithelial cells (TECs) in mice exposed to 2G for 14 days. Mice were exposed to 2 <i>g</i> gravity (2G) for 14 days or left under 1G (control). Vestibular apparatus are surgically disrupted in some mice (VL). TECs (CD45^–TER119^– EpCAM⁺) in total thymic cells were analyzed by staining with UEA-1-lectin, an mTEC marker, and CD80 antibody. Numbers in panels indicates percentage of each fraction. The percentages of UEA-1^– CD80^lo (containing cTECs), UEA-1⁺CD80^high (mTEC^hi), and UEA-1⁺CD80^low (mTEC^lo) cells among thymic stroma cells are summarized in right figures. “C” in graphs indicates 1G control. N = 6 each C, C with VL, and 2G with VL groups. N = 5 for 2G. NS indicates that the difference is not significant (Student’s <i>t</i>-test). NS indicates that the difference is not significant (Student’s <i>t</i>-test). (B) Cell numbers of total TECs (CD45^–TER119^– EpCAM⁺), UEA-1^– CD80^lo TECs (containing cTECs), mTECs (UEA-1⁺), mTEC^hi (UEA-1⁺CD80^high), and mTEC^lo (UEA-1⁺CD80^low) in the thymus are summarized in figures. N = 6 each C, C with VL, and 2G with VL groups. N = 5 for 2G. The asterisks indicate statistical significance at *P < 0.05 (Student’s <i>t</i>-test). NS indicates that the difference is not significant (Student’s <i>t</i>-test).</p