Periodic Reporting for period 1 - FRC BioEnergetics (Functional dissection of metabolic checkpoints in lymph node fibroblastic reticular cells)
Periodo di rendicontazione: 2020-06-01 al 2022-05-31
Efficient interactions between immune cells and antigens, necessary for the induction of immune responses, are initiated in secondary lymphoid organs (SLOs). Due to their unique position and structure, lymph nodes (LNs) collect extracellular fluids from peripheral tissues and display tissue and foreign antigens to circulating lymphocytes facilitating their activation. LNs structure and function are defined by fibroblastic reticular cells (FRCs) that build highly structured cellular scaffolds that, at the same time, provide migration clues (e.g. the chemokines CCL19, CCL21, CXCL13) and survival factors (e.g. the cytokines IL-7, IL-15) for incoming lymphocytes to position and to nurture them in defined microenvironmental niches. During the initiation of an immune response, the increased influx and extensive proliferation of activated lymphocytes require substantial changes in the form and function of the FRC infrastructure leading to a 10-fold increase in LN size. This process relies on the physical elasticity, cell stretching mechanisms, and proliferation of LN stromal cells. While the molecular signals inducing FRC activation and functional adaptation are being uncovered, bioenergetic demands of these cellular processes remain elusive.
Cellular metabolism has emerged as one of the main processes underlying immune regulation. The possibility of manipulating the metabolic decisions of hematopoietic cells and those cells that control their activity offers new ways to optimize immune cell performance and treat immunological diseases. LN FRCs act as the significant coordinators of immune processes and need to change form and function to support LN growth and hematopoietic cell activation. The ability of cells to swiftly respond to such demands is energetically very expensive. FRC BioEnergetics has addressed the energetic needs of FRCs as coordinators of immune responsiveness and explored how metabolism regulates FRC function during the initiation of the immune response. Combining state-of-the-art genetic models for in vivo FRC targeting with high-throughput metabolic and transcriptional data profiling, we revealed swift rewiring of FRC metabolism upon lymph node swelling. To support immune response initiation, LN FRCs must increase mitochondria mass and membrane polarization and reshape the mitochondrial network using OPA1 protein. Genetic targeting of the proteins involved in mitochondrial fusion using in vivo models of bacterial infection and immune and metabolic assays revealed the critical role of the mitochondrial profusion protein OPA1 in regulating FRCs biology during infection. The inability of FRCs to rewire mitochondrial networks due to genetic deficiency in OPA1 abolished LN swelling reaction, thereby reducing overall immune responsiveness and pathogen control. This project has unveiled hitherto unknown regulatory circuits in cell-specific energetics that critically impinge on immune responsiveness and pathogen control, thereby opening new avenues for treating immunological diseases by targeting stromal cell metabolism.
Cellular metabolism has emerged as one of the main processes underlying immune regulation. The possibility of manipulating the metabolic decisions of hematopoietic cells and those cells that control their activity offers new ways to optimize immune cell performance and treat immunological diseases. LN FRCs act as the significant coordinators of immune processes and need to change form and function to support LN growth and hematopoietic cell activation. The ability of cells to swiftly respond to such demands is energetically very expensive. FRC BioEnergetics has addressed the energetic needs of FRCs as coordinators of immune responsiveness and explored how metabolism regulates FRC function during the initiation of the immune response. Combining state-of-the-art genetic models for in vivo FRC targeting with high-throughput metabolic and transcriptional data profiling, we revealed swift rewiring of FRC metabolism upon lymph node swelling. To support immune response initiation, LN FRCs must increase mitochondria mass and membrane polarization and reshape the mitochondrial network using OPA1 protein. Genetic targeting of the proteins involved in mitochondrial fusion using in vivo models of bacterial infection and immune and metabolic assays revealed the critical role of the mitochondrial profusion protein OPA1 in regulating FRCs biology during infection. The inability of FRCs to rewire mitochondrial networks due to genetic deficiency in OPA1 abolished LN swelling reaction, thereby reducing overall immune responsiveness and pathogen control. This project has unveiled hitherto unknown regulatory circuits in cell-specific energetics that critically impinge on immune responsiveness and pathogen control, thereby opening new avenues for treating immunological diseases by targeting stromal cell metabolism.
The project's overarching goal is to elucidate the metabolic regulation of FRC function and determine to which extent these processes impact global immune responsiveness. In particular, we aimed to define the metabolic adaptations that support FRCs immune activation and elucidate the immune signals that trigger metabolic switching supporting FRCs activation. We have performed global metabolic profiling of FRCs isolated from homeostatic and activated LNs using a battery of metabolic tests, including targeted metabolomics analysis, which revealed substantial changes between homeostatic and immune-activated FRCs, with increased accumulation of amino acid and TCA cycle metabolites upon activation. Next, we inspected the characteristics of the mitochondria as the main hubs of cellular metabolism. Using confocal and electron microscopy to determine mitochondrial morphology and cristae organization, we revealed a change in mitochondrial shape upon FRCs activation. Investigating proteins determining the mitochondria shape, we uncovered differential regulation of profusion protein OPA1 upon FRCs activation. Concomitantly, activated FRCs have increased mitochondrial mass and membrane polarization. Broad metabolic profiling revealed a substantial shift of metabolic needs from homeostatic to immune-activated FRCs. Next, we have addressed the requirement of mitochondrial OPA1 for FRCs and lymph node function. While mesenteric lymph nodes of naïve animals with FRC specific OPA1-deficiency showed unaltered immune cell composition, upon intestinal bacterial infection, FRC-specific OPA1 deficiency resulted in impaired control of bacterial infection. Importantly, OPA1-deficient FRCs failed to support lymph node swelling, leading to strongly reduced lymph node mass and immune cell numbers. Transcriptional analysis revealed that OPA1 deletion up- and downregulated several genes involved in FRC cellular metabolism and inflammatory disease. In addition, analysis of the differentially expressed genes exposed a core genetic signature induced by OPA1 deletion related to metabolic pathways, which included the downregulation of OXPHOS and the tricarboxylic acid cycle (TCA) and the upregulation of one-carbon metabolism. Results of our analysis evidence a critical role for mitochondrial processes and morphology in supporting FRC immune function with implications for global immune responsiveness
To elucidate whether and how immunological signals impinge on metabolic regulation of FRC function, we have first performed in vitro screen for possible immune mediators of metabolic rewiring in FRCs. A comprehensive library of Toll-like receptor (TLR)-ligands and cytokines has been assessed for the ability to induce metabolic reprogramming in cultured FRCs measured by the increase in mitochondrial mass, membrane polarization, and OPA1-driven change of mitochondrial shape. This analysis revealed the IL1b-OPA1 axis as the core of immune-mediated metabolic reprogramming in FRCs.
To elucidate whether and how immunological signals impinge on metabolic regulation of FRC function, we have first performed in vitro screen for possible immune mediators of metabolic rewiring in FRCs. A comprehensive library of Toll-like receptor (TLR)-ligands and cytokines has been assessed for the ability to induce metabolic reprogramming in cultured FRCs measured by the increase in mitochondrial mass, membrane polarization, and OPA1-driven change of mitochondrial shape. This analysis revealed the IL1b-OPA1 axis as the core of immune-mediated metabolic reprogramming in FRCs.
A growing body of evidence demonstrates the critical role FRCs have in regulating immune responses in SLOs and peripheral tissues, including the brain, gut, lung, and tumors, hence making the biology of FRCs one of the leading interests in immunology. However, how FRCs manage their energetic needs remains a blind spot. Results of the action evidence a critical role for mitochondrial processes and morphology in determining FRC function with implications for global immune responsiveness. Our results provide a mechanistic understanding of how mitochondrial shaping proteins sustain mitochondrial metabolism and influence the transcriptional landscape and function of FRCs, thereby affecting the function of secondary lymphoid organs. Moreover, our results identify metabolic vulnerabilities of chemokine CCL19/CCL21-producing cells, opening new avenues for treating immune-mediated diseases by targeting FRC metabolism. The translational potential of the action goes beyond options to control immune responses at the site of immune activation. It unlocks the possibility of regulating late processes such as fibrosis or directing the action of cancer-associated and immune-stimulating fibroblasts within the tumor microenvironment by targeting the metabolic weakness of fibroblastic stromal cells.