RATIONALE: Idiopathic pulmonary fibrosis (IPF) is an age-related disease with remodeling of alveolar epithelial cells. Transitional alveolar epithelial type 2 (AT2) cells become senescent, contributing to tissue remodeling. Mitochondrial hemostasis is pivotal for epithelial cell fate, and its dysfunction drives fibrosis after lung injury. Transcription Factor A, Mitochondrial (TFAM), is essential for mitochondrial DNA integrity (mtDNA). We hypothesize that TFAM deficiency in AT2 leads to the accumulation of transitional AT2, promoting lung fibrosis. METHODS: Datasets from Lung Genomics Research Consortium (LGRC) microarray and IPF Atlas single-cell RNA sequencing (scRNA-seq) were analyzed to assess TFAM expression. TFAM in IPF and bleomycin-treated mouse lungs were quantified and localized via immunofluorescence. qPCR and Western blotting measured Tfam in primary mouse AT2 (pmAT2) cells isolated from bleomycin-treated mice. Tfam was deleted in pmAT2 via adenovirus-mediated Cre recombination. Scratch assays assessed fibroblast proliferation and migration in response to supernatants from Tfam-deficient cells, and cytokine arrays identified cytokine profile changes. Senescence was evaluated with immunofluorescence and SA-β-gal staining. Tfam conditional knockout (cKO) mice (Sftpc ERT2-Cre/WT; Tfam fl/fl) and controls (Sftpc ERT2-Cre/WT; Tfam WT/WT) were subjected to bleomycin injury. Lung function was measured by flexiVent, and tissues were collected for histology and senescence/fibrotic marker assessment. RESULTS: Compared to healthy donors (n=137) and COPD patients (n=220), IPF patients (n=255) showed significantly lower TFAM expression in lung tissue in the LGRC datasets. IPF Atlas scRNA-seq data demonstrated a significant TFAM reduction in AT2. The reduction of TFAM in AT2 of IPF lungs was further confirmed via immunofluorescence. Similarly, in the bleomycin mouse model, Tfam in AT2 was reduced at RNA and protein levels 14 days post-injury. Deleting Tfam from pmAT2 promoted mitochondrial dysfunction, mtDNA alterations, and emergence of transitional AT2 markers, including senescence markers like increased SA-β-gal staining, P21+ cells, and an expression of Krt8+, among others. These cells secreted distinct profibrotic and pro-senescence cytokines, including IGFBP2, GDF-15, VEGF and CXCL10. Conditioned medium from Tfam-deficient AT2 stimulated fibroblast activation and migration. In vivo, Tfam cKO mice exhibited high mortality (92.9%) post-bleomycin injury. Notably, Tfam cKO mice developed spontaneous distal lung fibrosis without injury as early as 18 weeks, assessed by lung function decline, lung histology, and fibrosis markers. Baseline assessments of Tfam cKO mice revealed increased transitional AT2 cells, marked by P21 and Krt8 expression. CONCLUSION: Our findings suggest that Tfam deficiency drives the emergence of transitional AT2 cells, which contribute to impaired epithelial-mesenchymal crosstalk, fibroblast activation, and lung fibrosis development.

The question addressed by the study Good biological indicators capable of predicting chronic obstructive pulmonary disease (COPD) phenotypes and clinical trajectories are lacking. Because nuclear and mitochondrial genomes are damaged and released by cigarette smoke exposure, plasma cell-free mitochondrial and nuclear DNA (cf-mtDNA and cf-nDNA) levels could potentially integrate disease physiology and clinical phenotypes in COPD. This study aimed to determine whether plasma cf-mtDNA and cf-nDNA levels are associated with COPD disease severity, exacerbations, and mortality risk. Materials and methods We quantified mtDNA and nDNA copy numbers in plasma from participants enrolled in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE, n = 2,702) study and determined associations with relevant clinical parameters. Results Of the 2,128 participants with COPD, 65% were male and the median age was 64 (interquartile range, 59–69) years. During the baseline visit, cf-mtDNA levels positively correlated with future exacerbation rates in subjects with mild/moderate and severe disease (Global Initiative for Obstructive Lung Disease [GOLD] I/II and III, respectively) or with high eosinophil count (≥ 300). cf-nDNA positively associated with an increased mortality risk (hazard ratio, 1.33 [95% confidence interval, 1.01–1.74] per each natural log of cf-nDNA copy number). Additional analysis revealed that individuals with low cf-mtDNA and high cf-nDNA abundance further increased the mortality risk (hazard ratio, 1.62 [95% confidence interval, 1.16–2.25] per each natural log of cf-nDNA copy number). Answer to the question Plasma cf-mtDNA and cf-nDNA, when integrated into quantitative clinical measurements, may aid in improving COPD severity and progression assessment.

Mitochondria play essential roles in metabolic support and signaling within all cells. Congenital and acquired defects in mitochondria are responsible for several pathologies, including premature entrance to cellar senescence. Conversely, we examined the consequences of dysfunctional telomere-driven cellular senescence on mitochondrial biogenesis and function. We drove senescence in vitro and in vivo by deleting the telomere-binding protein TRF2 in fibroblasts and hepatocytes, respectively. Deletion of TRF2 led to a robust DNA damage response, global changes in transcription, and induction of cellular senescence. In vitro, senescent cells had significant increases in mitochondrial respiratory capacity driven by increased cellular and mitochondrial volume. Hepatocytes with dysfunctional telomeres maintained their mitochondrial respiratory capacity in vivo, whether measured in intact cells or purified mitochondria. Induction of senescence led to the upregulation of overlapping and distinct genes in fibroblasts and hepatocytes, but transcripts related to mitochondria were preserved. Our results support that mitochondrial function and activity are preserved in telomere dysfunction-induced senescence, which may facilitate continued cellular functions.