Abstract Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive scarring of the alveolar tissue, resulting in shortness of breath, hypoxemia, and mortality. Ineffective lung repair and mitochondrial dysfunction have been implicated in IPF pathogenesis. Alveolar epithelial type 2 cells (AT2) play a crucial role in facilitating lung repair. IPF tissues have altered fatty acid beta oxidation (FAO), a key energy pathway that further affects alveolar repair. Lack of FAO induced mitochondrial stress and activated mitophagy, the removal of defective mitochondria. Adenine nucleotide translocase 2 (ANT2), a mitochondrial ATP/ADP transporter, plays an essential role in lung epithelial biology. Here, we determined the role of ANT2 in senescence and mitophagy in IPF. Human lung tissues were analyzed by qPCR for SLC25A5 (ANT2) gene expression and tissue staining. Wild-type (WT) and AT2-cell-specific ANT2-null mice (ANT2-Mut) were treated with via intratracheal bleomycin (2 U/kg) or vehicle and harvested on days 10 and 21. Bronchoalveolar lavage (BAL) fluid and lung tissues were processed for histology, fibrosis analysis, gene expression, and western blot analysis. BEAS-2b cells were treated with siRNA control or ANT2 siRNA for gene knockdown and CRISPR-Cas9 gene knockout for ANT2, then stimulated with or without TGF-beta (2 ng/mL) to determine alterations in markers of senescence, fibrosis, FAO, and mitophagy. Lentiviral-mt-Keima and MitoSox were used to measure mitophagy and reactive oxygen species (ROS), respectively, in control and ANT2KO cells using flow cytometry. SLC25A5 gene expression was reduced in AT2 cells from human IPF lung tissue. Mice with ANT2-Mut showed greater weight loss, immune cell infiltration, fibrosis score, and collagen deposition after bleomycin treatment at day 10. SLC25A5 gene expression was decreased after TGF-beta stimulation. Mitochondrial ROS levels were increased at baseline and further after TGF-beta stimulation at 24 hrs in ANT2 KO cells. Senescence marker p21 was increased in lung tissue from Mut-BL mice and in cells with ANT2 siRNA knockdown. Loss of ANT2 resulted in enhanced SMAD2/SMAD3 phosphorylation after TGF-beta stimulation. Expression of key mitophagy proteins were decreased with TGF-beta stimulation in ANT2KO cells compared to control cells, with changes in mitophagic flux by mt-Keima assessment. Our findings show that ANT2 deficiency in AT2 cells occurs in IPF and loss of ANT2 exacerbates lung fibrosis by promoting cell senescence, ROS production, enhancing TGF-beta signaling and SMAD activation, and altering mitophagy. ANT2 could be a potential therapeutic target for lung fibrosis by modulating mitochondrial function to improve lung repair. This abstract is funded by: None
Ting et al. (Fri,) studied this question.