The Role of Mitochondrial Uncoupling Protein 3 in T Cell Function

Abstract

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier superfamily that can mediate the transfer of protons into the mitochondrial matrix from the intermembrane space. Our laboratory has reported evidence of UCP1 and UCP3 expression in thymocytes and spleen mitochondria, while Krauss et al. (2002) reported expression of UCP2 in thymocytes. Thymocytes develop into mature T cells before exiting the thymus and travelling to the periphery where they wait to carry out an immune response. Expression of all three UCPs in thymocytes suggests their importance in thymic function, as well as a potential role in T cell selection, maturation, metabolism and/or function. Using quantitative RT-PCR, we have demonstrated that Ucp1, Ucp2 and Ucp3 are expressed in peripheral naive CD4 + T cells at the mRNA level before being markedly downregulated following cell activation under different polarizing conditions, the only exception being the expression of Ucp2 in regulatory T cells (T reg cells) which is not significantly altered following T cell activation until 120 h post-stimulation when it is significantly increased. Overall, the downregulation of Ucps in response to T cell activation is suggestive of a role for them in naive T cell maintenance. As our laboratory has previously provided evidence of an altered T cell profile in both thymus and spleen of Ucp3 -/- mice, we investigated the role of UCP3 in CD4 + T cell metabolism and function using the Seahorse XF Cell Mito Stress Test, flow cytometry and ELISAs. Following Ucp3 ablation, non-polarized, activated T cells (T H 0 cells) produced significantly more IL-2, which was reflected by an increase in CD25 and CD69 expression as well as an increase in cell proliferation. However, this was also followed by a decrease in cell viability and IFN-γ production 72 h post-stimulation. IL-2 promotes T cell activation, growth and survival but it is also known to prime T cells for activation-induced cell death (AICD). In this regard, it is thought that the increased IL-2 levels observed in Ucp3 -/- T H 0 cells are promoting early T cell activation but also subsequently inducing AICD. Interestingly, although Ucp3 -/- T H 0 cells underwent early activation, Ucp3 ablation did not have a major impact on T cell metabolism. IL-2 can influence the generation of T H 17 cells, a subset of T cells that are highly pro-inflammatory, and T reg cells; high levels of IL-2 promote T reg cell generation while inhibiting the generation of T H 17 cells. In line with this, the altered IL-2 expression observed between Ucp3 +/+ and Ucp3 -/- T H 0 cells appears to have a crucial impact on the generation of T H 17 and T reg cells in vitro. An increased frequency of FoxP3 + T reg cells was generated in vitro from Ucp3 -/- mice compared to Ucp3 +/+ mice, while Ucp3 -/- T H 17 cells generated in vitro displayed lower cell survival/viability and decreased IL-17A production compared to their Ucp3 +/+ counterparts. These results were also mirrored in vivo. Ucp3 -/mice immunized with keyhole limpet hemocyanin and cholera toxin displayed a higher frequency of FoxP3 + CD25 + T reg cells and significantly less production of IL-17A during antigen recall response compared to Ucp3 +/+ mice. We postulate that UCP3 is acting to restrict the activation of naive T cells. UCP3 may be acting as a rheostat to dampen signals following T cell receptor and CD28 co-receptor ligation, thereby preventing early activation and AICD. The fact that Ucp3 ablation alters the T H 17:T reg cell balance in vivo as well as in vitro implicates UCP3 as a potential target for the treatment of T H 17 cell-mediated forms of autoimmunity.