Abstract
Introduction/Aim
Increasing evidence highlights that cellular senescence contributes to idiopathic pulmonary fibrosis (IPF). The DNA damage response (DDR) in senescence also leads to escalated mitochondrial superoxide production to reinforce senescence. This study characterises senescence in lung fibroblasts from IPF patients (IPF-LFs) and age-matched controls (Ctrl-LFs), delineating the role of mitochondria in senescence stabilization.
Methods
Fibroblast senescence was assessed using a composite set of markers, including p21 expression and senescence-associated-β-galactosidase activity, which were measured by PCR and cytochemical staining, respectively. The DDR was detected by formation of phosphorylated-p53 nuclear foci using immunofluorescence. A senescent-associated secretory phenotype (SASP) was characterised by measuring increases in cytokine production and inflammatory gene expression with ELISA and Nanostring technology, respectively. Mitochondrial dysfunction was monitored by increases in mitochondrial stress, mass and superoxide using the fluorogenic dyes, n-nonyl-acridine orange, Mitotracker Green and MitoSOX, respectively.
Results
Even at early passage, IPF-LFs were more senescent-like than Ctrl-LFs, exhibiting an intensified DDR, a SASP and mitochondrial dysfunction. The DNA damaging agent etoposide augmented senescence in Ctrl-LFs accompanied by heightened mitochondrial stress, mass and superoxide production. However, it had no effect on IPF-LFs. Mitochondrial perturbation by rotenone also evoked a DDR and senescence in Ctrl-LFs. Inhibition of mTORC1, a regulator of mitochondrial function or incubation with the antioxidant, N-acetyl cysteine attenuated pharmacological-induced senescence.
Conclusion
IPF-LFs exhibit senescent-like features and mitochondrial dysfunction reinforces the senescent phenotype. Understanding the mechanisms by which mitochondria contribute to fibroblast senescence in IPF has potentially important therapeutic implications.