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Aging and disease    2019, Vol. 10 Issue (3) : 497-509     DOI: 10.14336/AD.2018.0608
Orginal Article |
Th1 Biased Progressive Autoimmunity in Aged Aire-Deficient Mice Accelerated Thymic Epithelial Cell Senescence
Jie Zhang, Yuqing Wang, Abudureyimujiang Aili, Xiuyuan Sun, Xuewen Pang, Qing Ge, Yu Zhang*, Rong Jin*
Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Medical Immunology, Ministry of Health (Peking University), Beijing 100191, China
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Abstract  

Although autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, are frequently associated with premature aging of the thymus, a direct link is missing between autoimmunity and thymic atrophy. Here we monitored the progression of thymic involution in Aire-deficient mice, in which defective negative selection causes spontaneous and progressive development of autoimmunity. In young and middle-aged mice, Aire deficiency appeared to be protective as supported by the reduced β-gal+ epithelial cells and the enhanced thymic output. However, once the autoimmune phenotype was fully developed in aged Aire-deficient mice, their thymuses underwent accelerated involution. In comparison to the age-matched wildtype littermates, old Aire-deficient mice showed lower numbers of total thymocytes and recent thymic emigrants but more β-gal+ thymic epithelial cells. This phenomenon may partly be attributable to the increased number of activated Th1 cells homing to the thymus. This speculation was further supported by the enhanced thymic aging following repeated challenges with complete Freund’s adjuvant immunization. Taken together, the present study highlights a unique mechanism by which autoimmunity facilitates the senescence of thymic epithelial cells through returning Th1 cells.

Keywords Aire      thymic senescence      thymic epithelial cells      autoimmune disease      Th1     
Corresponding Authors: Zhang Yu,Jin Rong   
Issue Date: 23 March 2018
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Jie Zhang
Yuqing Wang
Abudureyimujiang Aili
Xiuyuan Sun
Xuewen Pang
Qing Ge
Yu Zhang
Rong Jin
Cite this article:   
Jie Zhang,Yuqing Wang,Abudureyimujiang Aili, et al. Th1 Biased Progressive Autoimmunity in Aged Aire-Deficient Mice Accelerated Thymic Epithelial Cell Senescence[J]. Aging and disease, 2019, 10(3): 497-509.
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http://www.aginganddisease.org/EN/10.14336/AD.2018.0608     OR     http://www.aginganddisease.org/EN/Y2019/V10/I3/497
Figure 1.  Decreased senescent cells in young <i>Aire</i>-deficient mice

(A) Senescence-associated β-galactosidase (β-gal) was stained in thymic frozen slices from 6-week-old mice. Scale bars: 50μm. Representative images from three independent experiments are shown. (B) The number of β-gal+ senescent cells (blue) in 10 randomly chosen sections were counted using Image J. The statistical data are shown as average β-gal+ spots/mm2±SD (n=3 pairs). Statistical differences between groups were determined by the Student’s t test. **p < 0.01.

Figure 2.  The autoimmune disorders in early middle-aged <i>Aire</i>-deficient mice

(A) Lymphocyte infiltration in the salivary gland, retina, pancreas, stomach and ovary in 12-month-old WT (n=4) and Aire-/- (n=4) mice. The circles represent individual mice and summarize the degree and pattern of immune infiltration in the indicated organs. Mouse body weight (B), thymus weight (C), the cell number of thymocytes (D), lymph node cells (E) and splenocytes (F) of 6-week-old, 6- and 12-month old Aire-/- mice and WT littermates are shown. Each dot represents an individual mouse. (G, H) The percentage of double negative (DN), double positive (DP), CD4 and CD8 single positive (SP) thymocytes in 6-(G) and 12-month-old (H) mice were gated on CD45+ thymocytes. The statistical data are shown as Mean ± SD (n=8 pairs). No significant difference was detected in the above figures. (I, J) β-gal was stained in thymic frozen slices from 12-month-old mice (I). Scale bars: 50μm. (J) The number of β-gal+ senescent cells (blue) in 10 randomly chosen sections were counted using Image J. The statistical data are shown as average β-gal+ spots/mm2 ± SD (n=3 pairs). The experiments were repeated 3 times. Statistical differences between groups were determined by the Student’s t test. **p < 0.01.

Figure 3.  The alteration of recent thymic emigrants (RTEs) in early middle-aged <i>Aire</i>-deficient mice

(A, B) The proportion of GFP+ cells in peripheral CD4+ and CD8+ cells in 6- and 12-month-old Aire-/- mice and WT littermates. Representative dot plots (A) and the statistical data (B) are shown. (C, D) Splenocytes from 6-month-old Aire-/- and WT littermates were stimulated with anti-CD3 and anti-CD28 for 6h. IL-2 and IFN-γ producing cells in the GFP+CD4+ and GFP-CD4+ T cells was determined by intracellular staining. Representative plots (C) and the percentage of IL-2+ and IFN-γ+ cells (D) are shown. Statistical data are presented as Mean ± SD (n=8 pairs). The experiments were repeated four times. Statistical differences between groups were determined by the Student’s t test. *p< 0.05, **p < 0.01, and NS, no significance.

Figure 4.  The activation of peripheral CD4<sup>+</sup> T cells and the accumulation of Tregs in early middle-aged <i>Aire<sup>-/-</sup></i> mice

(A) CD62L and CD44 staining for splenic CD4+ T cells in 6- and 12- month-old mice. The number indicates the percentages of CD4+CD62LhighCD44low (naïve), CD4+CD62LlowCD44high (effector/memory) among CD4+ T cells (left). The statistical data are shown on the right as Mean ± SD (n=8 pairs). (B) CD69 staining for splenic CD4+ T cells in 6- and 12-month-old mice (left). The percentage of CD69+ T cells in CD4+ T cell are presented as Mean ± SD (right). Each dot represents an individual mouse. (C) Flow cytometric analysis of Tregs in the splenocytes of 6- and 12-month-old mice. Dot plots show representative profiles for CD25/Foxp3 staining (gated on CD4+ cells). (D) The percentage of Foxp3+CD25- and Foxp3+CD25+ Tregs are shown as Mean ± SD (n=8 pairs). The experiments were repeated 3 times. Statistical differences between groups were determined by the Student’s t test. **p< 0.01, ***p < 0.001, and NS, no significance.

Figure 5.  Deteriorated autoimmune disorder in aged <i>Aire</i>-deficient mice

(A) Lymphocyte infiltration in the salivary gland, retina, pancreas, stomach and ovary in 18-month-old WT (n=4) and Aire-/- (n=4) mice. The circles represent individual mice and summarize the degree and pattern of immune infiltration in the indicated organs. (B) Mouse body weight (left), thymus weight (middle) and the cell number of thymocytes, lymph node cells and splenocytes (right) of 18-month-old Aire-/- mice and WT littermates. Data are presented as Mean ± SD. Each dot represents an individual mouse. (C) Flow cytometric analysis of T cell activated markers in the spleen of 18-month-old mice. CD62L and CD44 profile (left) and the percentage of CD25+, CD69+ and CD44+ T cells (right) of CD4+ splenocytes are shown. Data are presented as Mean ± SD (n=3 pairs). (D) BrdU incorporation (gated on CD4+ cells) in the spleen of 18-month-old mice was detected by flow cytometry. The number indicates the percentage of cells in each quadrant. (E) The percentage of GFP+ RTEs in splenic CD4+ and CD8+ T cells in 18-month-old mice. (F) β-gal was stained in thymic frozen slices from 18-month-old mice (left). Scale bars: 50μm. The number of β-gal+ senescent cells (blue) in 10 randomly chosen sections were counted using Image J. The statistical data are shown as average β-gal+ spots/mm2±SD (right, n=5 pairs). The experiments were repeated at least three times. Statistical differences between groups were determined by the Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.  Elevated returning of IFN-γ-producing cells in aged <i>Aire</i>-deficient mice

(A, B) Flow cytometric analysis of GFP- returning T cells in the thymus of 6-, 12- and 18-month-old Aire-/- Rag2p-GFP mice and WT littermates. Representative dot plots (A) and the proportion of GFP- T cells in CD4+ and CD8+ T cells in the thymus (B) are shown. (C) The expression levels of CD44 and PD-1 measured by flow cytometry in GFP+ CD4 SP thymocytes and GFP- returning CD4+ T cells from18-month-old Aire-/- mice and WT littermates. Representative histograms are shown. (D, E) Isolated CD4+GFP- returning T cells were stimulated with anti-CD3 and anti-CD28 for 12 hours. IFN-γ-, IL-4- and IL-17A-producing cells in CD4+GFP- returning T cells were analyzed by flow cytometry. Representative density plots (D) and the proportion of IFN-γ-, IL-4- and IL-17A-producing cells in CD4+GFP- returning T cells (E) are shown. The experiments were repeated three times. Data are presented as Mean ± SD (n=8 pairs). Statistical differences between groups were determined by the Student’s t test. **p < 0.01.

Figure 7.  Th1 biased condition accelerates senescence of thymic epithelial cells

(A) Mice were immunized subcutaneously with 5μg of OVA in saline or CFA for a total 6 times at 6-, 8-, 10-, 17-, 19- and 21-week-old and analyzed at 16-, 27- and 39-week-old. (B) Mouse body weight of immunized mice at indicated time points. (C) IFN-γ- and IL-4-producing CD4+ cells in the spleen of immunized mice at indicated time points were detected by intracellular staining. Data are showed as the ratio of IFN-γ+%:IL-4+%. (D-H) The numbers of total thymocytes (D), DN (E), DP (F), CD4SP (G) and CD8SP (H) thymocytes in the thymus are shown. (I, J) The percentage of GFP+hi and GFP-lo cells in the lymph nodes (I) and spleen (J) after OVA immunization. Each dot represents an individual mouse. (K) EpCam+Ly51lo mTEC and EpCam+Ly51hi cTEC in the thymus of two immunized groups were detected by EpCam and Ly51 staining in CD45- gate. Representative density plots (left) and ratio of mTECs versus cTECs (right) are shown. (L) The expression of MHCII in mTEC from two immunized groups. Representative density plots (left) and the proportion of MHCII+ mTECs (right) are shown. (M) K5 (upper left) and β-gal (upper right) was stained in thymic frozen slices from CFA group. Representative images (up) and the percentage of β-gal+ cells (down) are shown. The white arrows pointed cells coexpressed K5 and β-gal. Scale bars: 20μm. The experiments were conducted twice with 5 mice for each group. Data are presented as Mean ± SD. Statistical differences between groups were determined by the Student’s t test. *p<0.05, **p<0.01, ***p < 0.001.

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