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This study examines our hypothesis that translationally controlled tumor protein (TCTP) expression in CD4+CD25high 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 CD25high 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 (total words: 207).
CD4+CD25highFoxp3+ regulatory T cells (Tregs), comprising 5–10% of CD4+ T cells1, exhibit potent immunosuppressive functions2 in the regulation of autoimmunity and inflammatory atherosclerosis3, 4. Naturally occurring Treg cells (thymus-generated, nTreg cells), as an independent subset, are engaged in the maintenance of immunological self-tolerance and down-regulation of various immune responses5 and inflammation. nTregs (Tregs in the rest of paper) appear to have specific apoptosis pathways since Treg cells have higher susceptibility to apoptosis than CD4+CD25− T cells6, especially to interleukin-2 (IL-2) withdrawal-induced apoptosis. Tregs are poor IL-2 producers7, implying that insufficient paracrine IL-2 supply to Tregs in inflammatory conditions could be responsible for the higher susceptibility of Tregs to apoptosis. However, intracellular regulation of IL-2 insufficiency-triggered Treg apoptosis remains poorly defined. The Bcl-2/Bcl-xL protein family members play a central role in the regulation of apoptosis8. Our recent reports showed that expression of a prototypic pro-apoptotic protein Bax in Bcl-2 family is higher in Tregs than in CD4+CD25− T cells9, and that higher expression of Bax is responsible for higher susceptibility of Tregs to apoptosis10. We previously identified a novel Bcl-xL-interacting anti-apoptotic protein, translationally controlled tumor protein (TCTP)11. Although TCTP is not a Bcl-2/Bcl-xL family member, down-regulation of endogenous TCTP protein expression by TCTP antisense cDNA results in the increase of T cell apoptosis11. A recent report showed that TCTP inhibits apoptosis by antagonizing pro-apoptotic function of Bax12, implying that Tregs with higher expression of Bax may express lower levels of TCTP. However, the pathological significance of TCTP downregulation in Tregs remains unknown.
Tregs play an important role in controlling pathogenesis of inflammatory atherosclerosis3, 4. In addition, IL-2 knock-out mice have a deficiency in Tregs, and spontaneously develop autoimmune diseases including vasculitis13, suggesting the possibility that a deficiency or decrease of Tregs promotes the development of vascular inflammation. However, it remains to be determined whether a deficiency in Treg generation in IL-2 knock-out mice, or higher susceptibility of mature Tregs to apoptosis induced by IL-2 deficiency, contributes to accelerated acute vasculitis. Since Tregs with high affinity IL-2 receptor have higher susceptibility to apoptosis in comparison to CD4+CD25− T cells14, characterization of the IL-2 withdrawal-induced apoptosis pathway in Tregs would be important for developing a strategy in generating a substantial quantity of well-survived Tregs for Treg-based immune therapeutics15. Therefore, three important questions remain to be answered toward this goal. First, whether the expression of anti-apoptotic protein TCTP in Tregs and CD4+CD25− T cells is associated with the susceptibility of these two groups of T cells to apoptosis induced by IL-2 withdrawal; second, whether the expression levels of TCTP are critical for Treg survival; and third, what the pathological significance of TCTP expression in Tregs is in the development of acute vascular inflammation. To examine these questions, we established a mouse CD25 promoter16-directed TCTP antisense transgenic mouse model (TCTP-AS Tg). We found that TCTP-AS Tg resulted in enhanced apoptosis of Tregs. Interestingly, we also found that Treg apoptosis led to accelerated development of cuff-induced vasculitis, presumably due to promotion of Treg apoptosis and failure of decreased Tregs in inhibiting inflammatory cells. These results have demonstrated the proof of principle that novel therapeutics can be developed for treating acute vasculitis, atherosclerosis and other inflammatory diseases via enhancing TCTP expression in Tregs and promoting survival of Tregs14.
Mouse IL-2 receptor α chain (CD25) promoter −2539 to +93 (GenBank Accession Number: M16398) vector pmIL2Rα-CAT1 (6.9 kb) was generously provided by P. Reichenbach and M. Nabholz16. The construction of the CD25+ T cell targeting vector pCD25-Tg was described previously9. The transgenic vector pCD25-TCTP-AS-Tg was verified by DNA sequencing by SeqWright Company (Houston, TX). The transgenic DNA fragment “Nru I-CD25 promoter-TCTP-AS-Sex AI” was prepared by digesting the pCD25-TCTP-AS-Tg vector with three restriction enzymes, Nru I, Sex AI, and Pvu I (New England Biolabs, Ipswich, MA), gel-purified, and microinjected into zygotes from C57BL/6 mice in the Baylor College of Medicine Genetically Engineered Mouse Core. ApoE-KO mice on a C57BL/6 background and their wild-type littermates were purchased from Jackson Laboratories (Bar Harbor, ME). Advanced atherosclerotic lesions were induced by feeding ApoE−/− mice with high fat diet for three months as reported17. All animal experiments in this study were performed according to the US National Institutes of Health guideline and the protocols were approved by Baylor College of Medicine and Temple University Institutional Animal Care and Use Committees.
Transgenic mice were identified by polymerase chain reaction (PCR) with mouse tail DNA as the templates using the special primers (Fig. 3A). The amplified PCR products were subcloned into TOPO TA plasmid (Invitrogen) and confirmed by DNA sequencing. Enforced expression of TCTP-AS in CD25+ T cell-specific manner in these transgenic mice was confirmed by reverse transcription (RT)-PCR using the primers (3xFLAG primer and TCTP anti-sense primer) that distinguish transgenic and endogenous RNA transcripts18. All mice used in the experiments were age- and sex-matched. Total RNA isolation and RT-PCR were performed as previously reported19.
Single cell suspensions of samples were treated with ammonium chloride to remove erythrocytes, washed, and then stained for various cell markers. Cells (1 × 106) were suspended in 100 μl of phosphorate buffered saline (PBS) containing 2% bovine serum albumin (BSA) and stained with 1 μg of various fluorescent antibodies (Abs) for 30 minutes (min) at 4°C, and analyzed on a FACSCalibar flow cytometer (BD Biosciences, San Jose, CA). Forward and side scatter gating were used to exclude debris from the analysis. Cells were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or PE-Cy7-conjugated mAbs against: CD4, CD8a, CD3ε, and CD25 (BD Pharmingen, San Diego, CA). PE-, PE-Cy7- and FITC-conjugated rat and hamster IgG were used as controls. Intracellular staining for Foxp3 with FITC-anti-Foxp3 antibody (eBioscience, San Diego, CA) was performed as we previously reported10
Splenocytes were normally cultured in the RPMI 1640 supplemented with 10% heat-inactivated FCS (Hyclone) in the presence or absence of mouse IL-2 at 5 ng/ml (Sigma-Aldrich, St. Louis, MO). IL-2 was withdrawn for 12 hours (hrs) to induce apoptosis. After 12 hrs, cell viabilities were determined by flow cytometry on the FACSCalibur flow cytometer (BD) with Annexin V staining (BD PharMingen) 20.
The femoral artery was looped with a ligature. A non-flow obstructing polyethylene cuff (0.3 mm inner diameter, 0.70 mm outer diameter) was placed on the femur artery to induce stenosis, and closed with a ligature. Finally, the skin incision was closed with a running suture21.
Images of lymphoid tissues were taken with a Zeiss Axioskopz Plus microscope (Oberkochen, Germany). The tissue areas in the images were analyzed by the software Image-Pro Plus 6.0 and used as indices of the size of tissues as reported for the indices of tumor sizes22. Femoral arteries with cuff-induced stenosis were removed, snap frozen in liquid nitrogen, and stored at −80°C. Serial cryostatic sections (5-μm thick) were cut consecutively: hematoxylin-eosin (H&E)-stained slides were used to examine the vessel affected by induced vasculitis with a microscope (Zeiss LSM 310). Smooth muscle cells were quantited by determining the numbers of anti-smooth muscle α-actin-antibody staining positive cells (1:200) (Abcam, Cambridge, MA)21. Cells with activated nuclear factor-κB (NF-κB) pathway were determined by staining cells with anti phosphorylated IκBα antibody (1:200) (Santa Cruz Biotech, Santa Cruz, CA) 23.
We first determined whether upregulation of anti-apoptotic protein TCTP is associated with increased production of IL-2 in the T cell proliferation activated by T cell antigen receptor (TCR) ligation and CD28 co-stimulation. TCTP protein expression was significantly upregulated by TCR ligation and CD28 co-stimulation with plate-bound anti-CD3 and anti-CD28 antibodies (Figs.1A1–A2). In contrast, the expression of pro-apoptotic protein Bax in activated T cells was not changed, suggesting that upregulated TCTP antagonizes the pro-apoptotic function of Bax and supports T cell proliferation (Fig. 1C). Interestingly, TCTP upregulation by TCR ligation and CD28 co-stimulation, at least in the first 12 hours, was associated with increased secretion of IL-2 (Fig. 1B), suggesting that these two molecules may share some regulatory mechanism for their expression in T cells. Our bioinformatic analyses showed that both TCTP promoter and IL-2 promoter contain a previously characterized NF-κB-like cis-acting transcription regulatory sequence termed CD28-resposive element (CD28RE) (not shown). To elucidate the function of upregulated TCTP, we transfected TCTP antisense cDNA directed by a T cell-specific lck promoter in Jurkat T cells. As shown in Fig. 1D, overexpression of TCTP antisense cDNA increased apoptotic rates from 3.16% (vector-transfected) to 45.7%, which is correlated with our previous report that TCTP inhibits T cell apoptosis, and that overexpression of TCTP antisense decreases TCTP protein expression and promotes T cell apoptosis 11.
Tregs have higher susceptibility to apoptosis than CD4+CD25− T cells 14, which is correlated with our report that Tregs have higher expression of pro-apoptotic protein Bax than CD4+CD25− T cells 9. To examine whether Tregs have higher susceptibility to apoptosis induced by IL-2 withdrawal, we cultured wild-type mouse splenic T cells in the absence of IL-2 for 12 hrs. As shown in Fig. 2A, IL-2 withdrawal induced higher rates of apoptosis in CD4+CD25+ T cells, especially in CD4+CD25high Tregs (nearly 20%), than in CD4+CD25− T cells (about 5%). Since TCTP antagonizes the pro-apoptotic function of Bax 12, we hypothesized that TCTP may be expressed at lower levels in CD4+CD25+ Tregs than in CD4+CD25− T cells. As expected, we found that TCTP was expressed at lower levels (nearly 50%) in purified CD4+CD25+ Tregs than in CD4+CD25− T cells (Fig. 2B). Lower expression of TCTP in CD4+CD25+ Tregs may be correlated with the lower IL-2 production in Tregs 7 potentially due to their shared CD28RE transcription mechanism. Based on our results, we propose the following working model (Fig. 2C): lower expression of TCTP in Tregs is associated with higher susceptibility of Tregs to apoptosis induced by insufficient supply of IL-2.
We reported that overexpression of TCTP antisense in T cells decreases TCTP protein expression and increases T cell apoptosis, which demonstrated that the TCTP antisense approach can efficiently downregulate TCTP expression in T cells11. We hypothesized that the expression levels of TCTP is critical for Treg survival. To test this hypothesis, we constructed CD25 promoter-driven TCTP antisense transgenic mice (Fig. 3A) by using the same TCTP antisense structure we reported previously11, since CD25high is a reliable marker for Tregs. The detailed justification for use of CD25 promoter to direct the expression of TCTP-AS transgene (Tg), rather than another promoter, is presented in the Discussion. The results of genomic DNA PCR analysis with mouse tail DNAs showed that two founders of TCTP-AS Tg were generated (Fig. 3B). The RT-PCR analysis with RNAs prepared from three lymphoid tissues (thymus, lymph nodes (LN), spleen), and two non-lymphoid control tissues (liver and kidney), of TCTP-AS Tg showed that transgenic TCTP-AS transcripts are expressed in lymphoid tissues but not in non-lymphoid tissues of TCTP-AS Tg mice, nor in all the tissues from wild-type littermate controls (Fig. 3C), suggesting that TCTP-AS transgene is expressed as the CD25 promoter directs.
Since our previous report showed that TCTP is important for T cell survival and proliferation11 (also see Figs. 1C and D), we hypothesized that a decrease in TCTP expression by TCTP antisense inhibits positive selection of thymocytes at double-positive stages (DP). The results show that TCTP antisense inhibits the positive selection of DP thymocytes (Fig. 3D) from 83.13% to 74.15%. In addition, TCTP antisense decreases CD4+CD25high thymocyte population. Moreover, CD4+CD25high Treg population in spleens and lymph nodes were also decreased. In correlation with the decrease of CD4+CD25high Tregs, glucocorticoid-induced tumour necrosis factor receptor (GITR)+ Tregs, another Treg marker24, were also decreased. Of note, phenotypic analysis of lymphoid cells of the two founders of TCTP-AS transgenic mice (Fig. 3B) indicated that both founders shared the phenotypic changes, suggesting that these changes result from overexpression of TCTP but not the insertion of TCTP-AS transgenic construct. Since 96% of CD4+CD25high Tregs express Treg specific transcription factor Foxp325, use of CD25high as a Treg marker is justified. These results suggest that TCTP expression levels are critical for Treg survival.
In Figs. 2A and B, we showed that higher susceptibility of Tregs to apoptosis induced by IL-2 withdrawal is associated with lower expression of TCTP in Tregs. We hypothesized that knock-down TCTP expression in Tregs may further increase the susceptibility of Tregs to apoptosis induced by IL-2 withdrawal. As shown in Fig. 3E, TCTP antisense overexpression in Tregs significantly increases the susceptibility of Tregs to apoptosis induced by IL-2 withdrawal from 19% to 30%, which is suppressed by exogenous IL-2 supply. These results suggest that the expression levels of TCTP in Tregs are critical for Treg survival. Treg apoptosis induced by IL-2 withdrawal in both wild-type Tregs and TCTP antisense Tregs is inhibited by IL-2 supply, suggesting that exogenous IL-2 supply may inhibit Treg apoptosis at least partially via increasing TCTP expression in Tregs. A previous report showed that Foxp3 per se promotes Treg apoptosis26. Thus, we exmained a possibility whether IL-2 withdrawal for 12 hours induces a change in Foxp3 expression in Tregs from TCTP-AS Tg mice in comparison to that from wild-type controls. The results in Fig. 3F showed that IL-2 withdrawal for 12 hours did not result in significant change in Foxp3 expression in Tregs from TCTP-AS Tg mice and from wild-type controls. These results are correlated with a recent report that Foxp3 is required for Treg dependence on IL-227.
Tregs play a critical role in suppressing development of chronic atherosclerotic plaques in ApoE−/− mice presumably via inhibiting autoimmune atherogenic inflammatory responses3. The IL-2 expression, a key Treg survival factor, was absent in 50% of human atherosclerotic plaques28, suggesting that insufficient supply of IL-2 can induce Treg apoptosis. As reported4, we also found that Tregs were decreased in atherosclerotic ApoE−/− mice (lesions not shown) in comparison to wild-type control mice (Fig. 3G)17. However, the question remains as to whether insufficient paracrine IL-2 supply triggered-TCTP downregulation affects the development of acute vascular inflammation in ApoE+/+ background. To examine this issue we adopted a well-characterized perivascular cuff-injury induced vasculitis 21 (Figs. 4A and B). The results show that a decrease of TCTP expression in Tregs significantly promotes inflammatory responses to loose placement of a non-flow obstructing cuff around the femoral artery, which leads to partial closure of the vasculitis-affected artery (Fig. 4B). In contrast, loose placement of a non-flow obstructing cuff does not induce severe vasculitis in wild-type control mice (Fig. 4B). The results suggest that the inflamed vessel wall in TCTP-AS Tg, after placing the cuff, is significantly thicker than that in wild-type mice. Correlating with the thickened vessel wall, the infiltrated inflammatory cells in the vessel from TCTP-AS Tg, after cuff placement, were significantly higher than that in wild-type mice (p < 0.05) (Fig. 4C). Associating with increased infiltration of inflammatory cells, cell numbers with cytosolic staining of p-IκBα were significantly higher in cuff-injured vessel from TCTP-AS Tg than in that from wild-type control (p < 0.05) (Figs. 4E and F), which suggests that cuff-injured vascular inflammation is mediated by NF-κ B activation pathway23. In addition, smooth muscle cell (SMC)-specific α-actin+ SMCs were increased but not very significantly (p > 0.05) (Fig. 4D), which may be due to relatively short period of inflammation21. In correlation with increased inflammatory cells and immune cells in the vessel from TCTP-AS, CD8+ T cells in the spleens and lymph nodes in TCTP-AS mice were significantly increased in comparison to that in wild-type mice (Fig. 3D). Moreover, since CD4+CD25+ Tregs can also exert direct suppressive effects on monocytes/macrophages in addition to suppressing effector T cells29, our results therefore suggest that the decrease of TCTP expression in Tregs reduces the striking threshold of inflammatory pathogenesis of vasculitis due to higher rates of Treg apoptosis and failure of decreased Tregs in suppressing inflammatory cells (Fig. 4G). Of note, our previous report suggested that a decrease of Treg suppression mainly resulted from reduced numbers of Treg due to overexpression of apoptotic protein rather than decreased Treg suppressive function.
Previous reports have shown that Tregs are highly susceptible to apoptosis6, 14, 30. Since Tregs generate endogenous IL-2 poorly7, Tregs require an exogenous IL-2 supply for survival by expressing higher levels of IL-2 receptor. However, IL-2-regulated Treg apoptotic pathways that underlie homeostatic mechanisms of Tregs remain poorly defined. Since homeostasis of Tregs is critical in maintaining immune tolerance and regulation of immune responses, elucidation of Treg-specific apoptotic pathways is important14. Our recent report showed that expression of pro-apoptotic protein Bax in Tregs is higher than that in CD4+CD25− T cells in the absence of any stimuli, which may be responsible for higher susceptibility to apoptosis of Tregs than that of CD4+CD25− T cells9. Since TCTP inhibits apoptosis by antagonizing the pro-apoptotic function of Bax12, our report implied that Bax antagonist protein TCTP is expressed at lower levels in Tregs than in CD4+CD25− T cells. Our current study has demonstrated, for the first time, the following findings: (a) TCTP upregulation in T cells activated by TCR ligation and CD28 co-stimulation is associated with increased IL-2 secretion and T cell proliferation, presumably via a shared CD28RE-mediated mechanism. This result suggests that (a) in Tregs with poor IL-2 production, TCTP expression is low; (b) a decrease of TCTP expression by overexpression of TCTP antisense leads to increased apoptosis, which reduces the positive selection of thymocytes and CD4+CD25high mature thymocytes. Since our previous report showed that overexpression of the same TCTP antisense construct significantly decreases the expression of TCTP protein11, our new result suggests that TCTP expression levels are critical for the same positive selection pathway shared by Tregs and other thymocytes; and (c) TCTP-AS transgene also promotes Treg apoptotic rates triggered by IL-2 withdrawal, which is inhibited by exogenous IL-2 supply, at least partially, via increasing TCTP expression in Tregs. Finally, our results show that decreased TCTP expression in Tregs lowers the striking threshold of inflammatory vasculitis due to promotion of Treg apoptosis and failure of decreased Tregs in suppressing anti-vascular T cells and other inflammatory cells. Taken together, our data suggest that modulation of Tregs apoptosis/survival could be used as a therapeutic approach for inflammatory cardiovascular diseases14.
nTregs are a special lineage of CD4+ T cells developed in thymus rather than a functional status, which makes a transgenic approach feasible. In addition to a marker for Tregs, low levels (but not high levels) of CD25 can also be a T cell activation marker31. However, up to 96% of CD4+CD25high Treg cells express Foxp325, suggesting that CD25high is an essential marker for Foxp3+ Tregs32, 33. Our TCTP-AS transgenic data show that transgenic TCTP antisense promotes more apoptosis of CD25highFoxp3+ Tregs than that of CD4+CD25low, suggesting that TCTP-AS transgene expresses higher in Tregs than in CD4+CD25− T cells. Of note, although Foxp3 plays a critical role in Treg development and function, Foxp3 per se promotes Treg apoptosis26, and Foxp3 expression in activated T cells does not necessarily lead to acquisition of suppression function34. These results suggest that use of the CD25 promoter to generate TCTP-AS transgenic mice in this study was well justified.
Phosphorylation of IκBα (p-IκBα) is a prerequisite for nuclear factor-κB (NF-κB) activation, and therefore, identification of cytosolic p-IκBα is an excellent surrogate marker of NF-κB activation23. Our results showed that cuff-injured vascular inflammation is mediated by NF-κB activation pathway. Previous reports showed that antisense and siRNA can function as agonists of toll-like receptors and activate NF-κB pathway35. However, increased p-IκBα+ inflammatory cells in cuff-injured vessel from TCTP-AS Tg did not result from potential activation of antisense-mediated toll-like receptor pathway. This conclusion is supported by following two findings: (1) TCTP antisense transgene was only expressed in Tregs but not in inflammatory cells; and (2) TCTP AS expression induced apoptosis whereas NF-κB activation inhibits apoptosis36.
Inflammatory atherosclerosis and vasculitis share the inflammatory responses against vessel wall components. Tregs play a critical role in suppressing the inflammatory atherogenic process since depletion of Tregs accelerates atherosclerosis3. Similarly, IL-2 knock-out mice have a deficiency in Tregs and spontaneously develop autoimmune diseases, including vasculitis13. These reports suggest that the IL-2 pathway is essential in maintaining survival of apoptosis-prone Tregs and suppressing inflammatory responses against vascular cell components. However, several important questions remain poorly defined including what mechanism underlies higher apoptosis of Tregs in the absence of a sufficient supply of IL-2; and whether the Treg apoptosis pathway is therapeutically significant in modulating the pathogenesis of inflammatory vasculitis. Our results demonstrate that Bax plays an important role in the IL-2 withdrawal-induced apoptosis pathway of Tregs. Proliferation and migration of vascular smooth muscle cells (VSMCs) during neointima formation induced by arterial injury represent a critical component of restenosis after angioplasty of human coronary arteries and an important feature of atherosclerotic lesions37. Peri-vascular cuff placement induces neointima formation, and this system represents the early features of atherosclerosis, such as proliferation of VSMCs but not foam cell formation. In this system, upregulated secretion of proinflammatory cytokines and increased inflammatory cell infiltration38 may further lead to the exhausted availability of Tregs. As outlined in Fig. 4G, our working model shows that lower expression of TCTP in transgenic Tregs “amplifies” cuff-induced inflammation. Future definition of the mechanisms of attenuating regulation of TCTP in Tregs may lead to development of new therapeutics for inflammatory vasculitis and atherosclerosis.
This work was partially supported by funds from National Institutes of Health grant AI054514 and Temple University Fund.
We are grateful to Drs. P. Reichenbach and M. Nabholz at the Swiss Cancer Center for providing mouse CD25 promoter.
Conflict of Interest
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