What question did this study set out to answer?

This research investigates how the loss of TFAM in alveolar type 2 cells affects lung fibrosis and epithelial cell communication.

May 20, 2026

D95-04 Loss of TFAM Drives Impaired Epithelial Mesenchymal Crosstalk in Pulmonary Fibrosis

Key Points

This research investigates how the loss of TFAM in alveolar type 2 cells affects lung fibrosis and epithelial cell communication.
Used AT2-specific Tfam conditional knockout mice and wild type littermates under varying conditions.
Performed survival analysis, lung mechanics assessment, histological evaluation, and RNA sequencing.
Conducted experiments to measure mitochondrial function and secreted factors impacting fibroblast behavior.
Adult Tfam cKO mice showed high mortality (92.9%) after bleomycin and developed spontaneous lung fibrosis by 18 weeks.
Significant mitochondrial dysfunction was noted in AT2 cells after Tfam deletion, with a reduced NAD+/NADH ratio and increased mitochondrial superoxide.
IGFBP2 levels were elevated in both conditioned media from cKO cells and IPF patient samples, enhancing fibroblast activation.

Abstract

Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is characterized by alveolar type 2 (AT2) cells obtaining transitional and senescent states. Mitochondrial integrity is central to epithelial fate, and its disruption contributes to IPF pathogenesis. Transcription Factor A, Mitochondrial (TFAM) maintenance mtDNA and we previously reported reduced TFAM in human and experimental fibrosis. We therefore hypothesize that TFAM loss in AT2 cells reprograms epithelial fate and amplifies profibrotic intercellular crosstalk. Methods We used AT2-specific Tfam conditional knockout (cKO) mice and WT littermates in adult and aging cohorts, at baseline (no injury) and after bleomycin. We assessed survival, lung mechanics, histology, and transitional epithelial markers by immunostaining. Whole-lung bulk and single-nucleus (sn) RNA-seq were performed. Primary mouse AT2 (pmAT2) cells underwent adenoviral Cre-mediated Tfam deletion. We assessed mitochondrial phenotypes by Seahorse, NAD+/NADH assays, MitoTracker/MitoSOX staining, Tomm20 immunofluorescence, and transmission EM. Senescence and AT2 states were measured by SA-β-gal and p21/Krt8 immunostaining. Cytokine arrays profiled conditioned medium from KO cells and bronchoalveolar lavage from IPF patients. Conditioned media were applied to fibroblasts to evaluate activation and migration. Results Adult AT2Tfam cKO mice exhibited high mortality after bleomycin challenge (92.9%). Deletion of Tfam in aged mice induced similar high mortality without challenge (83.3%). Adult cKO mice without bleomycin developed spontaneous distal lung fibrosis by 18 weeks, with impaired lung mechanics and histopathology. This was mediated by an expanded transitional AT2 population (p21+, Krt8+) as early as 3 weeks after Tfam deletion, as identified by bulk RNAseq and immunofluorescence. snRNA-seq further revealed transitional AT2 cells in cKO and identified amplified intercellular communication consistent with profibrotic signaling networks. In primary AT2 cells, Tfam deletion caused significant reduction of mitochondrial respiration (basal and maximal OCR), a reduced NAD+/NADH ratio, increased mitochondrial superoxide, and cristae disruption, while preserving mitochondrial mass as shown via MitoTracker and Tomm20. In vitro, AT2 losing Tfam showed large changes in secretome and conditioned media accelerated fibroblast activation and migration in indirect co-culture model. IGFBP2 was abundant in supernatants from Tfamdeficient AT2, elevated in IPF bronchoalveolar lavage by cytokine array, and in IPF AT2 by immunofluorescence. Recombinant IGFBP2 enhanced fibroblast activation, supporting a TFAM-IGFBP2-fibroblast signaling axis. Conclusion Loss of Tfam in AT2 cells causes spontaneous, lethal lung fibrosis. TFAM deficiency induces profound mitochondrial dysfunction while preserving mitochondrial mass. Functionally impaired AT2 cells contribute to fibrosis by secreting SASP factors, which promotes fibroblast activation and mediates impaired intercellular crosstalk in pulmonary fibrosis. This abstract is funded by: None

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