Periodic Reporting for period 1 - SPArKLE (New players in immunometabolism: investigating the role of succinate and pyruvate kinase in T cell immunity)
Période du rapport: 2018-04-01 au 2019-07-31
During its 16-month duration, the SPArKLE training programme provided an experienced researcher (ER) with expertise in immunology with a high-quality training in the field of immunometabolism. This field represents an emerging frontier in immunological research, and over the past 15 years it revolutionised our view on how the immune system works. It is indeed now clear that immune cells regulate the generation and strength of immune responses by fine-tuning their intracellular metabolic profile, and that alteration of metabolic pathways in specific immune cell subsets can impair their activation, polarisation and fate. In particular, a large body of evidence indicate that intracellular metabolism controls the activation and inflammatory potential of CD4+ T cells. This immune cell population is essential for pathogen clearance and long-term immunity, for example after vaccination. On the other hand, over-activation of CD4+ T cells may be harmful for the body, leading to uncontrolled systemic and local inflammation, and causing the development of immune-related pathologies such as autoimmune and allergic diseases, which have a huge socio-economic impact on national and international health agencies. Of note, the inflammatory activity of CD4+ T cells represents a major target of many disease-modifying drugs used for the treatment of such pathologies. It is therefore evident that a better understanding of the biological processes regulating CD4+ T cell functions would lead to the development of novel therapeutics specifically affecting T cell activation and detrimental activity. Given the increasing incidence of immune-related disorders worldwide, these new therapeutic approaches would have a major impact on the global community.
During the SPArKLE project, the ER investigated the role of the metabolite succinate and the enzyme pyruvate kinase (PK) in CD4+ T cells. Previous reports showed that succinate and PK play an important role in the functionality of certain immune cell subsets and in the development of inflammatory pathologies, but no studies have specifically evaluated if such molecules regulate CD4+ T cell activation and pathogenicity. Therefore, the overall aim of the SPArKLE research programme was to evaluate whether succinate and PK play a role in the activation, polarisation and functionality of murine and human CD4+ T cells, with the final goal to identify novel potential targets with therapeutic utility in inflammatory and autoimmune pathologies. The results obtained during the development of the SPArKLE project indicate that while succinate plays a minor role in the functionality of CD4+ T lymphocytes, modulation of the activity of the enzyme PK, in particular of its isoform PKM2, could limit CD4+ T cell pro-inflammatory activity and the development of autoimmunity. The project thus identified PKM2 as a potential therapeutic target for the treatment of T cell-mediated inflammatory diseases.
During the SPArKLE project, the ER investigated the role of the metabolite succinate and the enzyme pyruvate kinase (PK) in CD4+ T cells. Previous reports showed that succinate and PK play an important role in the functionality of certain immune cell subsets and in the development of inflammatory pathologies, but no studies have specifically evaluated if such molecules regulate CD4+ T cell activation and pathogenicity. Therefore, the overall aim of the SPArKLE research programme was to evaluate whether succinate and PK play a role in the activation, polarisation and functionality of murine and human CD4+ T cells, with the final goal to identify novel potential targets with therapeutic utility in inflammatory and autoimmune pathologies. The results obtained during the development of the SPArKLE project indicate that while succinate plays a minor role in the functionality of CD4+ T lymphocytes, modulation of the activity of the enzyme PK, in particular of its isoform PKM2, could limit CD4+ T cell pro-inflammatory activity and the development of autoimmunity. The project thus identified PKM2 as a potential therapeutic target for the treatment of T cell-mediated inflammatory diseases.
The project started with an initial evaluation on the role of succinate in CD4+ T cells, in particular the generation of different CD4+ T cell populations. The research activity focused on the pro-inflammatory CD4+ subsets named T helper 1 (Th1), Th17 and Th2 cells, which are important players in autoimmune and atopic disease. In parallel, the importance of succinate on the generation of the CD4+ anti-inflammatory subset named regulatory T cells (Tregs) was evaluated. To this purpose, two different approaches were used: CD4+ T cells were treated with extracellular succinate or with cell-permeable inhibitors of succinate dehydrogenase (SDH), the enzyme that degrades succinate into the cell. The latter approach was used to induce intracellular succinate accumulation in T cells. It was first observed that, despite the fact that CD4+ T cell express on their surface the cellular receptor for succinate, extracellular succinate did not affect the activation or functionality of T cells. In particular, succinate did not significantly modulate the in vitro generation of pro-inflammatory Th1, Th17 and Th2 cells or anti-inflammatory Tregs. Pharmacological inhibition of SDH moderately increased the pro-inflammatory potential of Th1 cells, even though this effect was mild. Overall, the results indicate a minor role for extracellular or intracellular succinate in the modulation of CD4+ T cell activity.
In parallel with the above experiments, the expression of different isoforms of the enzyme PK was evaluated in CD4+ T cells. The results indicate that, upon in vitro activation, two different isoforms of PK (PKM1 and PKM2) are upregulated in CD4+ T cells. In particular, the PKM2 isoform undergoes a complex regulation upon activation, which involves its phosphorylation at multiple sites and accumulation in the nucleus of activated CD4+ T cells. Of note, at all phases of activation, this enzyme is present in equilibrium between a monomeric/dimeric form (i.e. one/two enzyme subunits joined together) and a tetrameric form (i.e. four enzyme subunits joined together). Treatment of CD4+ T cells with PKM2 pharmacological activators, which induce the tetramerisation of the PKM2 enzyme, limited PKM2 nuclear translocation and strongly inhibited T cell activation, proliferation and cytokine production. These activators prevented the engagement of glycolysis, a metabolic pathway that was previously shown to be essential for T cell activation. Importantly, this is associated with a block in the generation of both pro-inflammatory Th1 and Th17 cells and parallel induction of anti-inflammatory Tregs in vitro. Finally, treatment with PKM2 activators ameliorated experimental autoimmune encephalomyelitis, the mouse model of human multiple sclerosis, by inhibiting Th1- and Th17-mediated inflammation in the central nervous system. Overall, these results suggest that pharmacological targeting of PKM2 may represent a valuable therapeutic approach in T cell-mediated inflammation and autoimmunity.
In parallel with the above experiments, the expression of different isoforms of the enzyme PK was evaluated in CD4+ T cells. The results indicate that, upon in vitro activation, two different isoforms of PK (PKM1 and PKM2) are upregulated in CD4+ T cells. In particular, the PKM2 isoform undergoes a complex regulation upon activation, which involves its phosphorylation at multiple sites and accumulation in the nucleus of activated CD4+ T cells. Of note, at all phases of activation, this enzyme is present in equilibrium between a monomeric/dimeric form (i.e. one/two enzyme subunits joined together) and a tetrameric form (i.e. four enzyme subunits joined together). Treatment of CD4+ T cells with PKM2 pharmacological activators, which induce the tetramerisation of the PKM2 enzyme, limited PKM2 nuclear translocation and strongly inhibited T cell activation, proliferation and cytokine production. These activators prevented the engagement of glycolysis, a metabolic pathway that was previously shown to be essential for T cell activation. Importantly, this is associated with a block in the generation of both pro-inflammatory Th1 and Th17 cells and parallel induction of anti-inflammatory Tregs in vitro. Finally, treatment with PKM2 activators ameliorated experimental autoimmune encephalomyelitis, the mouse model of human multiple sclerosis, by inhibiting Th1- and Th17-mediated inflammation in the central nervous system. Overall, these results suggest that pharmacological targeting of PKM2 may represent a valuable therapeutic approach in T cell-mediated inflammation and autoimmunity.
The project identified PKM2 as a potential target to limit T cell activation, Th1/Th17-mediated inflammation and the development of autoimmunity. The results obtained are novel and represent an important step forward to understand the molecular basis of T cell activation and pathogenicity. Previous work indeed suggested a role for PKM2 in the pro-inflammatory potential of immune cells, but no studies investigated in detail the role of this enzyme in T cells. Interestingly, succinate appears to have a minor role in T cell biology. This was an unexpected result, as CD4+ T cells express the succinate receptor, and this metabolite was previously shown to significantly modulate the functionality of innate immune cells, such as pro-inflammatory macrophages. However, these results confirm that, despite the fact that all immune cell subsets undergo similar metabolic reprograming upon activation and express a partially overlapping profile of surface receptors, the ability of different metabolites and metabolic enzymes to regulate the functionality of different immune cells varies. This notion is of importance for the developing of drugs able to modulate the activity of specific subsets of pro- and anti-inflammatory immune cells. Of note, these results have a potential high clinical and industrial impact, as they may lead to the development of drugs targeting PKM2 for the treatment of T cell-mediate inflammation and autoimmunity.