Expression of TCTP antisense in CD25(high) regulatory T cells aggravates cuff-injured vascular inflammation

Abstract

This study examines our hypothesis that translationally controlled tumor protein (TCTP) expression in CD4+ CD25(high) regulatory T cells (Tregs) is critical for the interleukin-2 (IL-2) withdrawal-triggered apoptosis pathway in Tregs, and modulation of Treg apoptosis pathway affects development of vascular inflammation. To test this hypothesis, we established a Tregs-specific TCTP antisense transgenic mouse model. Lower TCTP expression in Tregs than in CD4+ CD25- T cells is associated with the higher susceptibility of Tregs to apoptosis induced by IL-2 withdrawal. Overexpression of TCTP antisense in Tregs leads to decreased positive selection of CD25(high) thymic Tregs and reduced survival of peripheral Tregs, which is correlated to our previous report that TCTP antisense knocks-down TCTP protein expression and promotes apoptosis. In addition, TCTP antisense transgene confers higher susceptibility of Tregs to apoptosis induced by IL-2 withdrawal than wild-type Tregs, which can be suppressed by exogenous supply of IL-2, suggesting that IL-2 promotes Treg survival at least partially due to promoting TCTP expression. Finally, decreased expression of TCTP in Tregs aggravates experimental vascular inflammation, presumably due to increased Treg apoptosis and failure of decreased Tregs in suppressing inflammatory cells and immune cells. These results suggest that the modulation of Tregs apoptosis/survival may be used as a new therapeutic approach for inflammatory cardiovascular diseases.

Fig. 1. Association of TCTP upregulation with IL-2 secretion in T cells activated by TCR ligation and CD28 co-stimulation

A1. As detected by Western blot using anti-TCTP antibodies and anti-Bax antibodies, TCTP expression was upregulated by TCR ligation and CD28 co-stimulation with anti-CD3 and anti-CD28 antibodies, respectively, in a time course from 0, 12, 24, and 48 hours (hrs) where Bax expression in activated T cells was not changed. A2. Quantitation of TCTP upregulation and Bax expression in activated T cells was performed by normalizing the protein band intensities of TCTP and Bax with that of b-actin. The relative densitometric units were plotted against time course (hrs). B. IL-2 secretion was measured in a time course in wild-type mouse splenic T cells activated by anti-CD3 and anti-CD28 antibodies. The experiments were repeated three times and representative data presented. C. Mouse splenic T cell proliferation was measured in the presence and absence of T cell activation with anti-CD3 and anti-CD28 antibodies, as judged by [3H]-labeled thymidine incorporation. The experiments were repeated three times and representative data presented. D. TCTP antisense cDNA was constructed in a pW120 vector under the direction of T cell-specific lck distal promoter. The Jurkat T cells were transfected with pW120 vector control and pW120-TCTP antisense using the Amaxa Jurkat cell nucleofector transfection kit. The apoptotic rates of transfected Jurkat T cells were measured by FITC-conjugated annexin V apoptosis kit followed by flow cytometrical analysis. The experiments were repeated three times and representative data presented.

Fig. 2. Association of higher susceptibility of CD4+CD25+ T cells to apoptosis and lower expression of TCTP

A.The apoptotic rates of splenic CD4+CD25− T cells, CD4+CD25low T cells and CD4+CD25high T cells induced by IL-2 withdrawal at 0 hrs and 12 hrs were measured by FACS analysis of apoptotic cells with FITC-Annexin V. The experiments were repeated three times and the statistical data are presented. B. Semi-quantitative RT-PCR analysis of TCTP expression in purified CD4+CD25− T cells and CD4+CD25+ T cells. In the lower panel, the relative densitometric units of TCTP are presented as the expression levels of TCTP after normalizing the band density of TCTP with that of β-actin. C. A working model of association of decreased IL-2 secretion and reduced expression of TCTP. Increased susceptibility of Tregs to apoptosis induced by IL-2 withdrawal may result from decreased TCTP expression in Tregs.

Fig. 3. Decrease of TCTP expression in Tregs leads to increase of apoptotic rates of Tregs induced by IL-2 withdrawal

A. TCTP antisense transgenic construct. An antisense oriented 3X FLAG expression tag-fused TCTP cDNA (TCTP-AS) was placed under the direction of mouse CD25 promoter. The locations of primers used for PCR detection of transgenic TCTP-AS DNA, and RT-PCR detection of transgenic TCTP-AS transcripts and β-actin transcripts are depicted with arrows. In the lower panel, the primer sequences are presented. B. Detection of TCTP-AS transgenic mice by PCR using mouse tail genomic DNAs as templates. No DNA template control and non-transgenic mouse (wild-type) tail DNA control are included. C. Detection of transgenic TCTP transcripts by RT-PCR. Equal amounts of RNAs prepared from non-lymphoid tissues (kidney and liver) from TCTP transgenic mice and counterpart tissues from non-transgenic wild-type control mice were used in the experiment. D. FACS analysis of lymphoid cells from TCTP-AS transgenic mice and wild-type control mice (littermates). The data shown are representative of one of three independent experiments with groups of three to four mice at the age of 8 to 12 weeks. E. The apoptotic rates of splenic CD4+CD25− T cells, CD4+CD25^low T cells and CD4+CD25^high T cells induced by IL-2 withdrawal at 0 hrs and 12 hrs or in the presence of IL-2 supply were measured by FACS analysis of apoptotic cells with FITC-Annexin V in wild-type control mice and TCTP-AS transgenic mice. The experiments were repeated three times and the statistical data are presented. F. Foxp3 expression in CD4+CD25^high Tregs. The percentages of Foxp3+ CD4+CD25^high Tregs from wild-type control mice and TCTP-AS Tg mice, in the presence of IL-2 or absence of IL-2 supply for 0 hour and 12 hours, were measured by FACS analysis. The experiments were repeated three times and the representative results were presented. G. The ratio of CD4+CD25^high Tregs in wild-type control mice and mutant mice (ApoE−/− deficient mice and TCTP AS Tg mice) over CD4+CD25^high Tregs in wild-type (WT) mice. The experiments were repeated three times and the representative results were presented.

Fig. 4. Decreased striking threshold of cuff-injured vasculitis in TCTP-AS transgenic mice

A.A schematic representation of the experimental model of non-constrictive cuff placement-induced vasculitis in a single side of the mouse femoral artery. Non-constrictive cuff placement did not block the blood supply in the femoral artery before the tissue preparation (left panel). The left leg, without cuff placement, from the same mouse served as the control (right panel). B. Histological analysis of the vessel wall sections of TCTP-AS transgenic mice (TCTP-AS) and wild-type control mice (WT) without (upper panel) or with cuff placement (lower panel). C. The infiltrated inflammatory cell counts over the unit area were calculated for 10 mice in each group. The mean cell counts and standard deviation for each group are presented. D. The smooth muscle cells (SMCs), stained by immunofluorescence with anti-SMC-specific α-actin antibody, in the unit area were calculated for 10 mice in each group. E. Immuofluorescence staining was performed for analyzing phosphorylated IκBα+ (P- IκBα) cells in red fluorescence among the infiltrated inflammatory cells in cuff-injured vessel (upper panels). In order to confirm the cytosolic localization of phosphorylated IκBα DAPI co-staining of nucleus in blue fluorescence was performed (middle panels). Phosphorylated IκBα+ cells in red fluorescence were superimposed over DAPI-stained nucleus (lower panels). Cells with cytosolically localized phosphorylated IκBα were indicated by yellow arrows. F. The phosphorylated IκBα+ cell counts over the unit area were calculated for 10 mice in each group. The mean cell counts and standard deviation for each group are presented. G. A working model of how downregulated TCTP in Tregs lowers the striking threshold of experimental vasculitis. Our results show that downregulated TCTP expression leads to higher susceptibility of Tregs to apoptosis and decreased numbers of Tregs. Decreased Tregs presumably cannot maintain immune tolerance and suppression of inflammatory cells, which lowers the striking threshold and results in accelerated cuff placement-induced vasculitis.