Expression of TCTP antisense in CD25high regulatory T cells aggravates cuff-injured vascular inflammation

doi:10.1016/j.atherosclerosis.2008.07.041

Atherosclerosis

Volume 203, Issue 2, April 2009, Pages 401-408

https://doi.org/10.1016/j.atherosclerosis.2008.07.041 Get rights and content

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.

Introduction

CD4+CD25^highFoxp3+ regulatory T cells (Tregs), comprising 5–10% of CD4+ T cells [1], exhibit potent immunosuppressive functions [2] in the regulation of autoimmunity and inflammatory atherosclerosis [3], [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 responses [5] 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 cells [6], especially to interleukin-2 (IL-2) withdrawal-induced apoptosis. Tregs are poor IL-2 producers [7], 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 apoptosis [8]. 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 cells [9], and that higher expression of Bax is responsible for higher susceptibility of Tregs to apoptosis [10]. 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 apoptosis [11]. A recent report showed that TCTP inhibits apoptosis by antagonizing pro-apoptotic function of Bax [12], 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 atherosclerosis [3], [4]. In addition, IL-2 knock-out mice have a deficiency in Tregs, and spontaneously develop autoimmune diseases including vasculitis [13], 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 cells [14], 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 therapeutics [15]. 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 promoter [16] 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 Tregs [14].

Section snippets

Construction of CD25+ T cell-targeting mouse IL-2Rα promoter-TCTP-AS transgenic mice

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. Nabholz [16]. The construction of the CD25+ T cell targeting vector pCD25-Tg was described previously [9]. The transgenic vector pCD25-TCTP-AS-Tg was verified by DNA sequencing by SeqWright Company (Houston, TX). The transgenic DNA fragment “NruI-CD25 promoter-TCTP-AS-Sex AI” was prepared by digesting the pCD25-TCTP-AS-Tg vector

Tregs have lower expression of TCTP, which is associated with Tregs’ higher apoptotic rates, than CD4+CD25− T cells after IL-2 withdrawal

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 (Fig. 1A1 and A2). In contrast, the expression of pro-apoptotic protein Bax in activated T cells was not changed, suggesting that

Discussion

Previous reports have shown that Tregs are highly susceptible to apoptosis [6], [14], [30]. Since Tregs generate endogenous IL-2 poorly [7], 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

Funding

This work was partially supported by funds from National Institutes of Health Grant AI054514 and Temple University Fund.

Conflict of interest

None declared.

Acknowledgements

We are grateful to Drs. P. Reichenbach and M. Nabholz at the Swiss Cancer Center for providing mouse CD25 promoter.

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¹: These authors contribute equally to this work.

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