Final Report Summary - MENTORINGTREGS (Unravelling paradoxes in regulatory T cell biology: the molecular basis for an mTOR-dependent oscillatory metabolic switch controlling immune tolerance and autoimmunity)
We also determined the molecular basis of metabolic regulation of Tconv and Treg cell homestasis. Indeed we performed high throughput proteomic analysis (MudPIT), on human freshly isolated and in vitro cultured Treg and Tconv cells and we found that ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate. All these data were also confirmed at functional level. In parallel, we also observed that this scenario was altered in MS subjects and that glycolysis and oxidative phosphorylation (measured by Seahorse assays) were impaired during T cell activation in MS patients when compared with healthy controls. In this context, the treatment with interferon beta-1a (IFN beta-1a) was able to restore both pathways in MS patients. Also at transcriptional level, we found that metabolic signals of "starvation" are able to elicit transcriptional modulation of crucial cellular processes including proliferation, epigenetic control of transcription and metabolic asset, specifically in Treg cells, thus impinging on transcriptional fitness and plasticity of human pTreg cells linking micro-environmental cues with Treg cell homestasis.
Mechanistically, we found that engagement of glycolysis during Tconv cell activation was necessary for inducible Treg (iTreg) cell generation and we found that Tconv cells from MS subjects had reduced glycolysis, which together with reduced mTOR-pathway led to altered expression of specific splicing variant of Foxp3 containing Exon2 (FoxP3E2) and impaired suppressive function of iTreg cells. These data indicated that specific metabolic programs are required for the generation and function of Treg cells. Moreover these events associated with specific epigenetic modifications, altered engagement of autophagy and clock genes expression, strongly impacting again Treg cell homeostasis and function.
We confirmed the relevance of mTOR pathway in the control of Treg cell responsiveness also in vivo, in experimental autoimmune encephalomyielitis (EAE)-affected mice, in which we showed that acute rapamycin treatment strongly expanded Treg cells and improved EAE, while chronic rapamycin treatment reduced both Treg cells and Tconv cells. In line with these evidence, we also showed that high fat diet (HFD) worsened EAE score and brain inflammation (via mTOR overactivation), by decreasing the number and function of Treg cells, while caloric restriction (CR) improved EAE by increasing mTOR oscillations, boosting Treg cell expansion and repressing inflammatory cytokines production. Moreover impaired mTOR activation (secondary to LepR mutation), altered the metabolic profile of both Treg cells, which displayed increased proliferative potential at systemic and pancreatic level, by engaging glycolysis at higher level than the WT counterpart. Finally, we also demonstrated the role of autophagy and oscillation of clock genes in mTOR-mediated control of Treg cell function and biology in physiological and pthological conditions.
Taken together our data demonstrate that metabolism can modulate autoimmune diseases susceptibility, by regulating Treg cell function. Therefore, our findings might pave the way to identify factors or compounds that could selectively affect, through modulation of mTOR pathway, the crosstalk between immune system and metabolic status.