Mitochondrial DNA damage associated molecular patterns in ventilator-associated pneumonia: Prevention and reversal by intratracheal DNase I

Abstract

Background: Previous studies in isolated perfused rat lungs have revealed that endothelial barrier disruption after intratracheal administration of Pseudomonas aeruginosa (strain 103; PA103) only occurs after accumulation of extracellular mitochondrial DNA (mtDNA) damage-associated molecular patterns (DAMPs) in the perfusate and is suppressed by addition of DNase to the perfusion medium. Herein, we tested the hypothesis that intratracheal DNase-a route of administration readily translatable to patient with ventilator-associated pneumonia (VAP)-also enhances degradation of mtDNA and prevents bacteria-induced lung injury.

Methods: Intratracheal DNase was administered to isolated rat lungs either before or after intratracheal challenge with PA103 to determine if bacteria-induced mtDNA DAMP-dependent lung injury could be prevented or reversed by enhanced mtDNA degradation. To explore whether this concept is translatable to patients with VAP, consecutive patients suspected of VAP were prospectively enrolled. All patients suspected of VAP received a bronchoalveolar lavage (BAL) with quantitative culture for the diagnosis of VAP. Mitochondrial and nuclear DNAs were measured from the BAL. MtDNA DAMPs (i.e., ND6) were measured from serum at time of suspected diagnosis and at 24 to 48 hours afterward.

Results: Intratracheal PA103 caused significantly increased the vascular filtration coefficient (Kf) and perfusate mtDNA DAMPs. In contrast, lungs pretreated or posttreated with intratracheal DNase were protected from increases in Kf and mtDNA DAMPs. Patients with the diagnosis of VAP had significantly higher mtDNA DAMPs in the BAL (248.70 ± 109.7 vs. 43.91 ± 16.61, p < 0.05, respectively) and in the serum at 24 hours (159.60 ± 77.37 vs. 10.43 ± 4.36, p < 0.05; respectively) when compared with patients that did not have VAP.

Conclusion: These findings in isolated perfused rat lungs and a cohort of severely injured patients reveal an association between bacterial pneumonia and accumulation of mtDNA DAMPs in the lung and serum. Furthermore, administration of intratracheal DNase I prevented and reversed pulmonary endothelial dysfunction evoked by PA103.

Conflict of interest statement

DISCLOSURE

The authors deny all other conflicts of interest regarding the research presented within this manuscript. Meeting: 2015 AAST, Las Vegas, NV.

Figures

Figure 1

DNase prevents and reverses pulmonary vascular endothelial barrier injury in a rat model of bacterial pneumonia. The Kfs labeled in this figure correlate with Figure 1. Negative controls: NS (Kf1): NS was given at time zero and Kf was checked after 30 minutes (A). NS + NS (Kf2): NS was given at time zero and again at 30 minutes (A). Prevention Model: NS (Kf1): NS was given at time zero and Kf was checked after 30 minutes (A). NS + PA103 =NS was given at time zero and PA103 at 30minutes (A). DNase (Kf1) = DNase was given at time zero and Kf was checked at 30minutes (A). DNase + PA103 = DNase was given at time zero and PA103 at 30 minutes (A). Reversal Model: PA103 (Kf1): PA103 was given after 15 minutes and Kf was checked 15 minutes later (B). PA103 + DNase (Kf2): PA103 was given at 15minutes and DNase was given at 30minutes (B). PA103 (Kf1): PA103 was given after 15 minutes and Kf was checked 15 minutes later (B). PA103 + NS (Kf2): PA103 was given at 15 minutes and NS was given at 30 minutes (B). NS: normal saline, PA103: Pseudomonas aeruginosa strain 103, Kf: vascular filtration coefficient (i.e., lung injury).

Figure 2

Pseudomonas aeruginosa causes increases in vascular permeability accompanied by mtDNA DAMP release into the perfusionmedium. As shown in A, administration of intratracheal saline (i.e., positive control, n = 4) 30minutes before instillation of PA103 caused increases in the vascular filtration coefficient (Kf), which was prevented when DNase (i.e., experimental group, n = 4) was administered. As shown in B, intratracheal DNase administered 15 minutes after PA103 causes reversal of mtDNA DAMP-dependent lung injury compared to the positive control. *Different from control at p < 0.05.

Figure 3

VAP is associated with a time-dependent increase in serum mtDNA DAMPs in a cohort of severely injured patients. *Different from patients without VAP at 24 hours, p < 0.05.

Figure 4

VAP is accompanied by mtDNA DAMPs in the bronchoalveolar lavage. *Different from No VAP at p < 0.05.