Periodic Reporting for period 1 - Th17 tissue metab (Metabolic imprinting of Th17 cells in disease pathogenesis)
Período documentado: 2022-09-01 hasta 2024-12-31
Autoimmune diseases are estimated to affect 3-9% of the population1 and cause major personal and socio-economic consequences. Aberrant activation of the immune system occurs and results in immune cell recruitment and inflammation in tissues2. One cell type that is involved in pathogenesis of multiple autoimmune diseases are IL-17-producing CD4+ T cells (Th17), which also produce additional pathogenic cytokines3. The overall aim of this project is to gain insight into Th17 cell functions, their involvement in autoimmune disease pathogenesis and identification of novel targets for drug development. Targets will be validated in the context of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS) and leading cause of non-traumatic disability in young adults. Currently, no cure exists but a variety of treatments are available that improve life quality and reduce relapse rates4. Multiple therapies were based on findings from the mouse model for MS, experimental autoimmune encephalomyelitis (EAE)5. Th17 cells accumulate in CNS lesions of MS patients6 and drive disease pathogenesis in mice with EAE7,8. In general, Th17 cells are found in lymphoid tissues and inflamed tissue sites (e.g. CNS). However, Th17 cells were more recently also described in non-lymphoid tissues (termed “tissue Th17 cells”), where they exert homeostatic functions in the healthy state9-12. Thus, Th17 cells might be distinct in different tissues. How tissue Th17 cells acquire their specialized characteristics in diverse tissues remains elusive. We hypothesize that Th17 cells require metabolic cues from their tissue environment that instruct their tissue-specific phenotype. Thus, metabolic manipulation would be a promising approach to turn pathogenic tissue Th17 cells into homeostatic ones, reducing their pathogenicity. Several studies suggest that Th17 cells reside in or are primed at different tissue locations from where they can be recruited to sites of autoimmune inflammation, such as the CNS13-16. Thus, the metabolic environment in the tissue of origin might imprint pathogenic Th17 potential. Th17 cells are indeed implicated in disease pathogenesis in a variety of tissues, including skin, lung and gut17-19. Identifying tissue-specific metabolic switches for Th17 pathogenicity might thus pave the way for the development of novel treatment options for MS and other autoimmune diseases.
Metabolism was recently shown to play a role in Th17 cell function20-24, among others by the Kuchroo lab25,26. However, only isolated metabolic pathways have been examined, often restricted to single tissues or in vitro studies. Thus, a comprehensive, unbiased study on Th17 cells across all tissues is required, which provides a systematic understanding of tissue specific Th17 metabolism during homeostasis and an autoimmune reaction. Building on sound preliminary data from the gut, I will expand to a cross-tissue explorative single cell RNA-seq (scRNAseq) study with metabolic flux analysis and will validate targets in mouse models, as defined in the following objectives:
O1) What defines Th17 cells in different tissues and what are their metabolic requirements in health and disease?
O2) Which metabolic pathways are regulators of tissue specific Th17 cell function?
O3) Can specific metabolic regulators prevent Th17 mediated diseases and if so how?
Metabolism was recently shown to play a role in Th17 cell function20-24, among others by the Kuchroo lab25,26. However, only isolated metabolic pathways have been examined, often restricted to single tissues or in vitro studies. Thus, a comprehensive, unbiased study on Th17 cells across all tissues is required, which provides a systematic understanding of tissue specific Th17 metabolism during homeostasis and an autoimmune reaction. Building on sound preliminary data from the gut, I will expand to a cross-tissue explorative single cell RNA-seq (scRNAseq) study with metabolic flux analysis and will validate targets in mouse models, as defined in the following objectives:
O1) What defines Th17 cells in different tissues and what are their metabolic requirements in health and disease?
O2) Which metabolic pathways are regulators of tissue specific Th17 cell function?
O3) Can specific metabolic regulators prevent Th17 mediated diseases and if so how?
Based on the lab’s previous finding that Th17 cells from different tissues are transcriptionally distinct, we hypothesized that Th17 cells could have tissue-specific metabolic signatures and require different metabolic cues. To explore this hypothesis, we sorted Th17 cells by flow cytometry for large-scale scRNAseq. Building on sound preliminary data from the gut, we thus expanded to a cross-tissue explorative scRNAseq study with metabolic flux analysis (bioinformatics tool Compass) in steady state and EAE. In addition to the transcriptional data that give good indications of expression of individual genes per se, the Compass analysis provided a good measure of the metabolic state or directional fluxes within metabolic pathways. Supporting our original hypothesis, we identified tissue specific metabolic molecules for each tissue studied. We thus provide a comprehensive, unbiased study of Th17 cells across all tissues, which provides a systematic understanding of tissue specific Th17 metabolism during homeostasis and in autoimmunity.
We additionally provide metabolic characterization of current Th17 cells compared to ex-Th17 cells. IL-17cre-gfp x Rosa26tdTomato mice were used to distinguish current (IL-17cre-gfp+) from ex-Th17 cells (Rosa26tdT+gfp-, any cell that ever expressed IL-17 is Rosa26tdT+). In addition to studying Th17 cells in these tissues, we generated a more broad dataset by including all IL-17 producers (as opposed to restricting it to CD4+ IL-17 producers only) in the skin and lung tissues. This approach results in additional characterization of all IL-17 producing cell types (eg. ILC3s, gd T cells) within these tissues and for our Th17 study allows conclusions on cell type specificity of findings.
Metabolic hits shortlisted from the Compass analysis (O1) have a high potential to affect tissue Th17 cell function. We identified a metabolic enzyme that is particularly enriched in Th17 cells of the intestinal compartment, suggesting a gut tissue specific role. We thus wanted to study its role in Th17 cells from the steady state intestine using genetically modified mice. We found that this metabolic molecule is particularly enriched in CXCR6+ Th17 cells, a population our lab has recently characterized to be present in steady state with properties that support their inflammatory role in autoimmune diseases. This finding suggested a role of this enzyme in autoimmune disease context, which we studied in O3.
To study our identified target in autoimmunity, the well-established EAE mouse model was induced via active immunization with myelin oligodendrocyte glycoprotein (MOG) peptide in global and cell-specific KO mice. To study potential spontaneous disease development and T cell priming independent effects, both mouse strains were further crossed with T cell receptor transgenic mice (originally created in the Kuchroo lab) to induce EAE by transfer of transgenic “2D2” T cells specific for MOG. In both EAE models, disease severity was determined by an established clinical scoring system and CNS tissue damage quantified via histology. To confirm functional changes in Th17 cells or if Th17 cell numbers and residency are changed, high dimensional flow cytometry analyses was run. We discovered deletion of each of these molecules has significant effects on the disease, suggesting to further investigate the surrounding pathways to lay groundwork for potential therapeutic use. To explore mechanistically, how the metabolic regulators function, Th17 cells isolated from the CNS of EAE mice were subjected to scRNAseq and Compass analysis. In addition to effects observed in EAE and due to the fact that the metabolic molecules are highly expressed in the gut, we also focused on another Th17 cell involving disease state, intestinal inflammation. The transfer colitis model has recently intensely been used in the lab and was applied in this context with significant phenotypic effects.
We additionally provide metabolic characterization of current Th17 cells compared to ex-Th17 cells. IL-17cre-gfp x Rosa26tdTomato mice were used to distinguish current (IL-17cre-gfp+) from ex-Th17 cells (Rosa26tdT+gfp-, any cell that ever expressed IL-17 is Rosa26tdT+). In addition to studying Th17 cells in these tissues, we generated a more broad dataset by including all IL-17 producers (as opposed to restricting it to CD4+ IL-17 producers only) in the skin and lung tissues. This approach results in additional characterization of all IL-17 producing cell types (eg. ILC3s, gd T cells) within these tissues and for our Th17 study allows conclusions on cell type specificity of findings.
Metabolic hits shortlisted from the Compass analysis (O1) have a high potential to affect tissue Th17 cell function. We identified a metabolic enzyme that is particularly enriched in Th17 cells of the intestinal compartment, suggesting a gut tissue specific role. We thus wanted to study its role in Th17 cells from the steady state intestine using genetically modified mice. We found that this metabolic molecule is particularly enriched in CXCR6+ Th17 cells, a population our lab has recently characterized to be present in steady state with properties that support their inflammatory role in autoimmune diseases. This finding suggested a role of this enzyme in autoimmune disease context, which we studied in O3.
To study our identified target in autoimmunity, the well-established EAE mouse model was induced via active immunization with myelin oligodendrocyte glycoprotein (MOG) peptide in global and cell-specific KO mice. To study potential spontaneous disease development and T cell priming independent effects, both mouse strains were further crossed with T cell receptor transgenic mice (originally created in the Kuchroo lab) to induce EAE by transfer of transgenic “2D2” T cells specific for MOG. In both EAE models, disease severity was determined by an established clinical scoring system and CNS tissue damage quantified via histology. To confirm functional changes in Th17 cells or if Th17 cell numbers and residency are changed, high dimensional flow cytometry analyses was run. We discovered deletion of each of these molecules has significant effects on the disease, suggesting to further investigate the surrounding pathways to lay groundwork for potential therapeutic use. To explore mechanistically, how the metabolic regulators function, Th17 cells isolated from the CNS of EAE mice were subjected to scRNAseq and Compass analysis. In addition to effects observed in EAE and due to the fact that the metabolic molecules are highly expressed in the gut, we also focused on another Th17 cell involving disease state, intestinal inflammation. The transfer colitis model has recently intensely been used in the lab and was applied in this context with significant phenotypic effects.
We provide a comprehensive, unbiased study of Th17 cells across all tissues, which provides a systematic understanding of tissue specific Th17 metabolism during homeostasis and in autoimmunity. We further characterize 2 metabolic molecules and show that they affect disease severity in EAE and colitis.