Periodic Reporting for period 1 - REPROGRAMIT (Control of T cell differentiation and plasticity through mitochondrial reprogramming)
Okres sprawozdawczy: 2019-08-01 do 2021-07-31
This project aimed at understanding whether mitochondrial morphology and function regulate the transcriptional and epigenetic landscape of the distinct T cell subsets. The combination of pharmacologic and genetic approaches in in vitro and in vivo models of T cell differentiation and inflammation, revealed a role for mitochondrial membrane organization and metabolism in sustaining the effector function of Th17 cells. Genetic targeting of the proteins involved in the fusion or fission of mitochondrial membranes (mitochondrial dynamics) using in vitro and in in vivo models of disease, together with immune and metabolic assays, let me to identify and characterize the role of the mitochondrial profusion protein OPA1 in Th17 effector function. During the course of this project, I found that deletion of OPA1 in T cells promotes profound metabolic, transcriptional and proteomic alterations which restrain Th17 pathogenicity, while keeping intact the effector function across other T cell subsets (i.e. Th1, Th2 or regulatory T cells, Tregs). These findings highlight the requirement of mitochondrial function in Th17 cells and reveal novel metabolic vulnerabilities of Th17 cells that can be exploited therapeutically in Th17-cell related pathologies.
1.2.1 Work Package 1. Define the role of mitochondrial dynamics in T cell differentiation
To characterize whether distinct T cells subsets show specific configurations of their mitochondrial membranes, we first analysed the mitochondrial morphology in naive CD4 T cells differentiated in vitro towards distinct T cell subsets (Th1, Th2, Th17 and Treg). As opposed to other CD4 T cell subsets, Th17 cells show fused mitochondrial network, tight cristae organization, and reduced metabolic activity, a similar configuration as found in quiescent, low-metabolically memory T cells. Genetic manipulation of mitochondrial inner membrane by deletion of OPA1 reduced mitochondrial OXPHOS and increased glycolysis across T cell subsets. However, this metabolic rewiring induced by OPA1-deficiency, only restrained Th17 function. Using in vivo models of Th17 function (experimental autoimmune encephalomyelitis, EAE, or CD3 antibody (Ab) monoclonal injection), we found that OPA1 and mitochondrial inner membrane fusion are required for establishing Th17 autoimmune responses.
1.2.2 Work package 2
Elucidate how mitochondria shapes the transcriptional and epigenetic networks of T cell lineages
To investigate whether manipulation of mitochondrial membrane controls chromatin remodeling and gene expression in T cells, I first interrogated the transcriptome of the distinct T cell subsets upon specific deletion of OPA1 during in vitro polarization experiments. OPA1 deletion up- and downregulated a number of different genes involved in T cell differentiation, cellular metabolism and inflammatory disease.
1.2.3 Work Package 3
Identify mitochondria-to-nucleus signaling pathways controlling T cell differentiation
The third objective aimed to identify metabolites regulating the epigenome and T cell transcriptional programs. To this end, I performed extensive characterization of the metabolome of in vitro differentiated T cells with specific deletion of Opa1. Global metabolite analysis revealed an accumulation of TCA metabolites, concomitant with an impairment in NAD/NADH levels, reduced aspartate biosynthesis, and increased lactate and serine biosynthesis. Notably, we detected the accumulation of a byproduct of the TCA metabolism, the oncometabolite 2-hydroxyglutarate (2-HG) upon OPA-1 deletion, whose accumulation in cancer cells inhibits DNA and histone modifying enzymes and triggers epigenetic reprogramming. Proteomic analysis identified upstream regulators of this metabolic rewiring, being LKB1 a major candidate. OPA-1 deletion activates LKB1 signaling and CRISPR-Cas9 knockdown of LKB1 in OPA1-deficient cells, restores IL-17A production in Th17 cells. In all, these results pointed to a crucial role of LKB1 in controlling OPA1 phenotype. In this line, silencing of LKB1 by CRISPR-Cas9 on OPA1-deficient cells restored the metabolic deficiencies quantified in OPA1 cells (i.e. accumulation of TCA metabolites and 2-HG).
1.2.3 Work Package 4
Identify and validate metabolites changing T cell transcriptional networks in vitro and in vivo
Intrigued by the high specificity of the effect of OPA1 deficiency in Th17 cells and the lack of effect on the effector function in other subsets, we speculated that the different metabolism of these cells would determine their response to OPA1 deficiency (mitochondrial dysfunction). We found that reducing metabolic activity of Th1 cells by culturing them with decreasing amounts of glucose, made Th1 cells susceptible to OPA1 deficiency. These results support the notion that cellular metabolic activity and distinct substrate availability are responsible for the vulnerability of T cells to mitochondrial function/dysfunction. To investigate whether mitochondrial membrane remodeling sustain T cell responses in vivo, we focused our analysis in immune models mediated by Th17 cells and we did not perform any of the cancer models originally proposed. To this end, we generated mice in which OPA1 is deleted specifically in IL17-expressing cells by crossing OPA1 flox mice with a Th17 fate reporter mouse (IL17Cre x R26eYFP, Hirota et al. 2009). In these mice, cells that express IL-17A become fluorescently labelled and show targeted OPA1 expression. OPA1 deletion in Th17 cells fully abrogated disease incidence in a mouse model of multiple sclerosis (EAE), a Th17-related pathology. In addition, mice in which OPA1 and LKB1 are deleted specifically in Th17 cells, recover signs of disease upon immunization, supporting a role for LKB1 as a sensor of OPA1-deficiency in vivo.
To characterize whether distinct T cells subsets show specific configurations of their mitochondrial membranes, we first analysed the mitochondrial morphology in naive CD4 T cells differentiated in vitro towards distinct T cell subsets (Th1, Th2, Th17 and Treg). As opposed to other CD4 T cell subsets, Th17 cells show fused mitochondrial network, tight cristae organization, and reduced metabolic activity, a similar configuration as found in quiescent, low-metabolically memory T cells. Genetic manipulation of mitochondrial inner membrane by deletion of OPA1 reduced mitochondrial OXPHOS and increased glycolysis across T cell subsets. However, this metabolic rewiring induced by OPA1-deficiency, only restrained Th17 function. Using in vivo models of Th17 function (experimental autoimmune encephalomyelitis, EAE, or CD3 antibody (Ab) monoclonal injection), we found that OPA1 and mitochondrial inner membrane fusion are required for establishing Th17 autoimmune responses.
1.2.2 Work package 2
Elucidate how mitochondria shapes the transcriptional and epigenetic networks of T cell lineages
To investigate whether manipulation of mitochondrial membrane controls chromatin remodeling and gene expression in T cells, I first interrogated the transcriptome of the distinct T cell subsets upon specific deletion of OPA1 during in vitro polarization experiments. OPA1 deletion up- and downregulated a number of different genes involved in T cell differentiation, cellular metabolism and inflammatory disease.
1.2.3 Work Package 3
Identify mitochondria-to-nucleus signaling pathways controlling T cell differentiation
The third objective aimed to identify metabolites regulating the epigenome and T cell transcriptional programs. To this end, I performed extensive characterization of the metabolome of in vitro differentiated T cells with specific deletion of Opa1. Global metabolite analysis revealed an accumulation of TCA metabolites, concomitant with an impairment in NAD/NADH levels, reduced aspartate biosynthesis, and increased lactate and serine biosynthesis. Notably, we detected the accumulation of a byproduct of the TCA metabolism, the oncometabolite 2-hydroxyglutarate (2-HG) upon OPA-1 deletion, whose accumulation in cancer cells inhibits DNA and histone modifying enzymes and triggers epigenetic reprogramming. Proteomic analysis identified upstream regulators of this metabolic rewiring, being LKB1 a major candidate. OPA-1 deletion activates LKB1 signaling and CRISPR-Cas9 knockdown of LKB1 in OPA1-deficient cells, restores IL-17A production in Th17 cells. In all, these results pointed to a crucial role of LKB1 in controlling OPA1 phenotype. In this line, silencing of LKB1 by CRISPR-Cas9 on OPA1-deficient cells restored the metabolic deficiencies quantified in OPA1 cells (i.e. accumulation of TCA metabolites and 2-HG).
1.2.3 Work Package 4
Identify and validate metabolites changing T cell transcriptional networks in vitro and in vivo
Intrigued by the high specificity of the effect of OPA1 deficiency in Th17 cells and the lack of effect on the effector function in other subsets, we speculated that the different metabolism of these cells would determine their response to OPA1 deficiency (mitochondrial dysfunction). We found that reducing metabolic activity of Th1 cells by culturing them with decreasing amounts of glucose, made Th1 cells susceptible to OPA1 deficiency. These results support the notion that cellular metabolic activity and distinct substrate availability are responsible for the vulnerability of T cells to mitochondrial function/dysfunction. To investigate whether mitochondrial membrane remodeling sustain T cell responses in vivo, we focused our analysis in immune models mediated by Th17 cells and we did not perform any of the cancer models originally proposed. To this end, we generated mice in which OPA1 is deleted specifically in IL17-expressing cells by crossing OPA1 flox mice with a Th17 fate reporter mouse (IL17Cre x R26eYFP, Hirota et al. 2009). In these mice, cells that express IL-17A become fluorescently labelled and show targeted OPA1 expression. OPA1 deletion in Th17 cells fully abrogated disease incidence in a mouse model of multiple sclerosis (EAE), a Th17-related pathology. In addition, mice in which OPA1 and LKB1 are deleted specifically in Th17 cells, recover signs of disease upon immunization, supporting a role for LKB1 as a sensor of OPA1-deficiency in vivo.
With this project we have evidenced a critical role for mitochondrial function and morphology in the biology of Th17 cells. Our results provide mechanistic understanding on how mitochondrial shaping proteins are critical for sustaining mitochondrial metabolism and the transcriptional landscape of Th17 cells during in vitro experiments and in in vivo models relevant for human pathology. Specifically, we found that the inner mitochondrial fusion protein OPA1 is a critical mediator of Th17 pathogenicity that sustains metabolic quiescence and effector function of Th17 cells. Moreover, our unbiased -omic approaches identified LKB1 signaling as a rheostat of mitochondrial integrity, linking mitochondrial metabolism to cytokine expression in T cells. In all, our results propose a model where distinct T cell subsets rely on different mitochondrial configurations for their effector function, and reveal novel metabolic vulnerabilities with relevance for therapeutic purposes in T cell-related pathologies.