Mitochondrial DNA damage-associated molecular patterns mediate a feed-forward cycle of bacteria-induced vascular injury in perfused rat lungs

Abstract

Fragments of the mitochondrial genome released into the systemic circulation after mechanical trauma, termed mitochondrial DNA damage-associated molecular patterns (mtDNA DAMPs), are thought to mediate the systemic inflammatory response syndrome. The close association between circulating mtDNA DAMP levels and outcome in sepsis suggests that bacteria also might be a stimulus for mtDNA DAMP release. To test this hypothesis, we measured mtDNA DAMP abundance in medium perfusing isolated rat lungs challenged with an intratracheal instillation of 5 × 10(7) colony-forming units of Pseudomonas aeruginosa (strain 103; PA103). Intratracheal PA103 caused rapid accumulation of selected 200-bp sequences of the mitochondrial genome in rat lung perfusate accompanied by marked increases in both lung tissue oxidative mtDNA damage and in the vascular filtration coefficient (Kf). Increases in lung tissue mtDNA damage, perfusate mtDNA DAMP abundance, and Kf were blocked by addition to the perfusion medium of a fusion protein targeting the DNA repair enzyme Ogg1 to mitochondria. Intra-arterial injection of mtDNA DAMPs prepared from rat liver mimicked the effect of PA103 on both Kf and lung mtDNA integrity. Effects of mtDNA and PA103 on Kf were also attenuated by an oligodeoxynucleotide inhibitor of Toll-like receptor 9 (TLR-9) by mitochondria-targeted Ogg1 and by addition of DNase1 to the perfusion medium. Collectively, these findings are consistent with a model wherein PA103 causes oxidative mtDNA damage leading to a feed-forward cycle of mtDNA DAMP formation and TLR-9-dependent mtDNA damage that culminates in acute lung injury.

Keywords: Ogg1; damage-associated molecular patterns; lung injury; mitochondrial DNA; oxidant stress.

Copyright © 2015 the American Physiological Society.

Figures

Fig. 1.

Pseudomonas aeruginosa (P. aeruginosa) causes time-dependent increases in vascular permeability accompanied by mitochondrial DNA (mtDNA) damage-associated molecular pattern (DAMP) release into the perfusion medium. Rat lungs were isolated, mechanically ventilated, and perfused at a constant flow rate with physiological salt solution containing albumin as a colloid. P. aeruginosa, strain 103 [PA103; 5 × 107 colony-forming units (CFU) in 100 μl physiological salt solution] was instilled as a bolus into the trachea. As shown in A, relative to control preparations not challenged with bacteria (n = 12), instillation of PA103 (n = 9) caused time-dependent increases in the vascular filtration coefficient (Kf). *Different from control at P < 0.05; **different from both control and 15 min post-PA103 at P < 0.05. In results of companion experiments shown in B, perfusate contents of 200-bp sequences of the indicated mtDNA regions measured by qRT-PCR were increased at 15 min after PA103 instillation relative to controls although not all sequences were increased to the same extent (n = 4 for both groups). *Different from control at P < 0.05. Finally, in results of separate experiments shown in C, formamidopyrimidine DNA glycosylase (Fpg)-detectable oxidative base damage in the mtDNA genome, determined by quantitative Southern blot analysis, was increased in lung tissue challenged 15 min previously with PA103 relative to damage levels in control perfused lung tissue (n = 4 for both groups). *Different from control at P < 0.05. Cox2, cytochrome C oxidase subunit II; ND4, NADH dehydrogenase subunit 4; D-loop, displacement loop.

Fig. 2.

Mitochondria-targeted 8-oxoguanine DNA glycosylase (Ogg1) reduces PA103-induced increases in Kf and perfusate mtDNA sequences and decreases oxidative mtDNA damage. Rat lungs were isolated, mechanically ventilated, and perfused at a constant flow rate with physiological salt solution containing albumin as a colloid. In some preparations, a fusion protein targeting the DNA repair enzyme, Ogg1, was added to the perfusate to achieve a final concentration of 10 μg/ml and allowed to recirculate for 30 min before intratracheal instillation of P. aeruginosa (PA103; 5 × 107 CFU in 100 μl physiological salt solution). As shown in A, relative to control and PA103-challenged preparations (n = 12 and 9), treatment with mt-targeted Ogg1 alone (n = 4) failed to alter baseline Kf but prevented the increase normally evoked by PA103 (n = 4). B demonstrates that, whereas Ogg1 alone (n = 4) failed to alter baseline perfusate mtDNA DAMP levels, the fusion protein inhibited the increase normally evoked by PA103. In addition, neither PA103 nor Ogg1, given alone or in concert, altered accumulation of a ≈200-bp nuclear DNA sequence encoding 28S rRNA (n = 4). C shows that, although Ogg1 alone (n = 4) failed to alter the baseline level of Fpg-detectable oxidative mtDNA damage, the fusion protein inhibited the increase normally evoked by PA103 (n = 4). Note that, in all three panels, bars depicting control and PA103 responses in the absence of mt-targeted Ogg1 are identical to those displayed in Fig. 1. *Significantly different from all other groups at P < 0.05.

Fig. 3.

Evidence that mtDNA DAMPs mediate PA103 effects on lung endothelial permeability. Results shown in A–F were generated from experiments in isolated, mechanically ventilated rat lungs perfused at a constant flow rate with physiological salt solution containing albumin as a colloid. A shows that 16 μg exogenous mtDNA DAMPs (n = 4) increased Kf to a level similar to that evoked by intratracheal administration of 5 × 107 CFU PA103. B and C show that enhanced DNA degradation with DNase 1 (0.5 U/ml) added to the perfusion medium 15 before mtDNA or PA103 challenge, respectively, inhibited the increase in Kf evoked by both stimuli. Similarly, D and E show that the effects of mtDNA DAMPs and PA103 on Kf are both suppressed by a Toll-like receptor 9 inhibitory oligodeoxynucleotide (ODN; added to the perfusate reservoir to achieve a final concentration of 5.3 μg/ml; n = 4 for all groups, except control PA103, for which n = 12 and 9, respectively). F shows that the antioxidant N-acetylcysteine (NAC) added to the perfusate reservoir to attain a final concentration of 1 mM also inhibited increases in Kf evoked by PA103 and mtDNA. *Significantly different from all other groups at P < 0.05.

Fig. 4.

Exogenous mtDNA DAMPs cause oxidative mtDNA damage-dependent increases in Kf. Results shown in A indicate that mtDNA isolated from lungs perfused for 15 min after challenge with 16 μg intra-arterial bolus injections of exogenous mtDNA DAMPs displayed more oxidative damage relative to mtDNA isolated from control lungs. Recirculating perfusion with 10 μg/ml mitochondrially targeted Ogg1 for 15 min failed to alter baseline oxidative mtDNA damage but attenuated the increase normally evoked by mtDNA DAMPs, n = 4/group. *Significantly increased from control at P < 0.05; **significantly different from control and mtDNA DAMPs alone at P < 0.05. B displays results of similarly designed studies, except Kf was measured instead of oxidative mtDNA damage. Here, too, treatment with mitochondrially targeted Ogg1, while not impacting baseline Kf, inhibited the increase evoked by exogenous mtDNA DAMPs; n = 4/group. *Significantly different from all other groups at P < 0.05.

Fig. 5.

Proposed feed-forward cycle linking oxidative mtDNA damage and DAMP formation to injury propagation. Proposed model linking oxidant stress induced by bacteria, trauma, or ischemia-reperfusion injury to oxidative mtDNA damage, mtDNA DAMP formation, and regenerative mtDNA damage in a feed-forward cycle culminating in inflammation and tissue damage. The model, if valid, explains why treatment of the initial insult, regardless of its specific etiology, often fails to prevent propagation of the insult to distant organs and the occurrence of delayed organ dysfunction. The model also points to two new isolated targets for pharmacological intervention. Both inhibition (A) of oxidative mtDNA damage and enhanced degradation (B) of mtDNA DAMPs would be expected to forestall injury progression. Note that the model fails to consider the potentially complex interactions between alveolar macrophages, resident neutrophils, and vascular endothelial cells in driving the postulated feed-forward pathway. See text for additional details.