Metabolic Reprogramming of the Alveolar Stem Cell Niche in Pulmonary Fibrosis - PROJECT SUMMARY Cell metabolism regulates epigenetic reprograming to determine cellular identity and fate. Intermediary metabolites serve as essential cofactors for epigenetic modifying enzymes. Epithelial- mesenchymal crosstalk is critical for the maintenance of adult tissues/organs and in regenerative responses to tissue injury. In the lung, alveolar maintenance and regeneration are orchestrated by the interaction of alveolar type 2 (AT2) cells, a facultative stem/progenitor cell population, with the adjacent mesenchyme that contributes to “niche” support of the regenerating epithelium. These homeostatic type 2 niche-supporting stromal cells (T2NSCs) may transition to pathological mesenchymal states/fates during lung injury-repair. We have identified a metabolic enzyme, nicotinamide N-methyltransferase (NNMT), that regulates the plasticity of tissue-resident fibroblasts (FBs) and the transition of lipofibroblasts (lipo-FBs) into myofibroblasts (myo-FBs). NNMT catalyzes the N-methylation of nicotinamide and other pyridine compounds; by utilizing the universal methyl donor, SAM in this reaction, NNMT functions as a “methyl sink” in many tissues, while also depleting cellular NAD+ levels. Our data indicate that NNMT is upregulated in lung mesenchymal cells of idiopathic pulmonary fibrosis (IPF), and is induced by the pro-fibrotic cytokine, transforming growth factor-β1, in human lung fibroblasts. NNMT functions as a critical switch from lipo-FB to myo-FB differentiation that acquire apoptosis resistance, thus impairing fibrosis resolution. In this project we will test the hypothesis that metabolic-epigenetic reprogramming of activated stromal T2NSCs by targeting NNMT potentiates lung regenerative capacity and facilitates fibrosis resolution by augmenting cellular levels of NAD+ and/or SAM. Our specific aims are to: (1) identify T2NSCs subpopulations and characterize their regulation by NNMT; (2) determine mechanisms by which NNMT regulates lipo-FB to myo-FB transition; and (3) determine whether targeting NNMT accelerates fibrosis resolution in an animal model of lung injury-induced fibrosis. A combination of bulk and single-cell RNA-seq, ATAC-seq, metabolomics, bioenergetics, and epigenetic profiling in 3D alveolospheres, IPF lung FBs, and an in-vivo lung injury model will be employed. The studies proposed in this grant application will advance the field by identifying a critical regulatory switch in lipo-FB to myo-FB differentiation, linking metabolism to epigenetics by an enzyme that controls both cellular bioenergetics and protein methylation, defining a therapeutic strategy that achieves fibrosis resolution/reversal in established lung fibrosis, and elucidating a functional role of alveolar stem cell niche-supporting fibroblasts in stem cell rejuvenation and tissue regeneration.
