51 - PINK1 deficiency in AECII triggers release of mitochondrial DAMPS that initiate pro-inflammatory and pro-fibrotic responses in the lung

Abstract

Rationale Mitochondrial abnormalities are often observed with aging, and mitochondrial dysfunction has been identified as key pathogenic mechanism in several age-related diseases. Our previous studies have shown that alterations in mitochondria homeostasis by low expression of PTEN inducible kinase 1 (PINK1) in the aging lung type II epithelial cell (AECII) can influence the susceptibility to Idiopathic Pulmonary Fibrosis. Deficiency of PINK1 leads to accumulation of swollen dysfunctional mitochondria and increased apoptosis. Mitochondrial components released during cellular injury and necrosis or apoptosis have been identified as damage associated molecular patterns (DAMPs) including mtDNA. We hypothesize that PINK1 deficiency increase the release of mtDNA that can function as a DAMP and autonomously activate TLR9 to initiate downstream inflammatory and pro-fibrotic signaling. Methods mtDNA release was determined by quantitative PCR in supernatant of A549 cells exposed to the ER stressor tunicamycin and in cells transfected with shPINK1 or scramble vector. Similarly, mtDNA was analyzed in brochoalveolar lavage (BAL) of wild type and PINK1 deficient mice and from donor control and IPF patients. Expression of pro-inflammatory and pro-fibrotic mediators by mtDNA stimulation was analyzed by ELISA in cell culture supernatants and by determination of transcript levels by qRT-PCR, with and without TLR9 antagonist and pre-treatment with DNAse. Results Knockdown of PINK1 expression in A549 cells caused significant increase in mtDNA copies in the culture media and increase in TGF-b and IL-6 expression. Also, significant increases in mtDNA copies were found in the supernatant of cells exposed to tunicamycin associated with reduced PINK1 expression. A549 cells and primary human lung epithelial cells exposed to mtDNA significantly increased secretion of IL-6 and TGFb, but not with nuclear genomic DNA. Pre-treatment with DNAse or the presence of a TLR9 antagonist oligonucleotide reduced the expression of pro-fibrotic and pro-inflammatory mediators induced by mtDNA. Furthermore, mtDNA stimulation of primary human lung fibroblasts induced expression of a-SMA. In vivo, copy numbers of mtDNA were increased in BAL of PINK1 deficient mice in comparison with wild type mice. More importantly, mtDNA copy numbers in BAL of IPF patients was significantly elevated compared to donor controls and inversely correlated with total lung capacity. Conclusion Collectively, our data show PINK1 depletion mediates release of mitochondrial DAMP triggering pro-inflammatory and pro-fibrotic responses via TLR9. These data suggest that the release of mtDNA fragments might have a role in the progression of pulmonary fibrosis and identify PINK1 and mtDNA DAMPs as potential new therapeutic targets.