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.