Targeting mitochondrial drivers of AT2 cell reprogramming and cellular crosstalk in lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal, age-related lung disease with a median survival of 2-3 years. IPF is characterized by pathological reprogramming of alveolar epithelial type 2 (AT2) cells, progenitors of AT1 cells, into transitional cell states that activate fibroblasts and drive pathological tissue remodeling. The molecular mechanisms underlying this reprogramming, and mechanisms that could be therapeutically targeted to reverse it, remain incompletely understood. This project investigates the mitochondrial transcription factor A (TFAM), a key regulator of mitochondrial DNA (mtDNA) maintenance that has not been explored in IPF, as a driver of AT2 cell reprogramming and lung fibrosis. Preliminary data show that TFAM expression is significantly reduced in AT2 cells in human IPF and experimental lung fibrosis, and that genetic TFAM deficiency in AT2 cells increases transitional AT2 cell number and causes spontaneous lung fibrosis. TFAM-deficient AT2 cells exhibit mitochondrial dysfunction, mtDNA release, and cGAS-STING-mediated inflammation, and express a distinct secretome sufficient to activate fibroblasts in vitro. Based on these findings, we hypothesize that TFAM reduction drives AT2 cell reprogramming and the emergence of metabolically altered transitional AT2 cells, thereby promoting lung fibrosis, and we will test this hypothesis in three aims. Aim 1 will define the intracellular mechanisms, testing whether TFAM loss triggers mtDNA instability and cGAS-STING/interferon activation in a dose-dependent manner, using heterozygous and homozygous TFAM-deficient mice in young and aged cohorts crossed with STING and interferon-stimulated response element reporter lines. Aim 2 will characterize the TFAM-dependent AT2 cell mitochondria-induced stress secretome ("MISS") and its impact on fibroblast activation and pro-fibrotic subtype differentiation, focusing on IGFBP2 as a candidate paracrine mediator. Aim 3 will test whether restoring TFAM function, genetically or pharmacologically, or neutralizing IGFBP2, can prevent, attenuate, or reverse established lung fibrosis, using a novel conditional TFAM-overexpression mouse model and human-derived precision-cut lung slices, including from IPF lungs. Together, these aims will establish TFAM as a novel regulator of AT2 cell fate and a druggable node in IPF, defining the mechanistic link between mitochondrial dysfunction, epithelial-fibroblast crosstalk, and fibrosis, and providing genetic and pharmacologic proof-of-concept for reversing this process therapeutically.