Elevated levels of plasma mitochondrial DNA DAMPs are linked to clinical outcome in severely injured human subjects

Abstract

Objective: Our objective was to execute a prospective cohort study to determine relationships between plasma mtDNA DAMP levels and the occurrence of systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), and mortality.

Background: Mitochondrial DNA damage-associated molecular patterns (DAMPs) accumulate in the circulation after severe injury. Observations in animal models demonstrate that mtDNA DAMPs contribute to organ dysfunction; however, the link between plasma mtDNA DAMPs and outcome in severely injured human subjects has not been established.

Methods: DNA was isolated from plasma samples taken from severely injured patients at hospital days 0, 1, and 2. Real-time PCR was used to quantify selected ≈200 base pair sequences of mtDNA within the COX1, ND1, and ND6 genes, as well as from the D-Loop transcriptional regulatory region. MODS was defined as a Denver Multiple Organ Failure score of 4 or greater.

Results: MtDNA DAMPs were quantified as PCR threshold cycle number. Lower threshold cycles indicate increased mtDNA DAMP content. Patients with SIRS had significantly increased mtDNA DAMP levels in all 4 sequences examined (32.14 ± 0.90 vs 29.00 ± 1.15 for COX1, 31.90 ± 0.47 vs 30.16 ± 1.42 for ND1, 32.40 ± 0.61 vs 28.94 ± 1.13 for ND6, and 33.12 ± 0.83 vs 28.30 ± 1.14 for D-Loop). Patients who developed MODS also had elevated mtDNA DAMP levels compared with those who did not (32.57 ± 0.74 vs 27.12 ± 0.66 for COX1, 32.45 ± 0.65 vs 28.20 ± 0.73 for ND1, 32.52 ± 0.56 vs 27.60 ± 0.79 for ND6, and 32.85 ± 0.75 vs 27.86 ± 1.27 for D-Loop). Patients with above-median mtDNA DAMP levels had a significantly elevated relative risk for mortality. Four patients died secondary to severe MODS.

Conclusions: These findings comprise the first observational evidence that plasma mtDNA DAMPs is associated with the evolution of SIRS, MODS, and mortality in severely injured human subjects.

Trial registration: ClinicalTrials.gov NCT01812941.

FIGURE 1

Associations of Plasma mtDNA DAMP Levels with MODS/Mortality. There were only 4 patients in our cohort who developed MODS, and these 4 patients were the only nonsurvivors. DAMP levels were significantly elevated in patients who developed MODS/Death when compared to survivors. All four sequences exhibited statistically significant differences at P < 0.05 by nonpara-metric analyses. Day 6 data were excluded to minimize survivor bias. The relative abundances of plasma mtDNA DAMPs were expressed as threshold cycles (Tc). Of note, higher Tc represents lower levels of mtDNA DAMPs.

FIGURE 2

Serum mtDNA DAMP levels (quantified as the PCR Tc) expressed as a function of the Denver Organ Failure Score. For all 4 mtDNA sequences examined, in patients with greater Denver scores, Tc tended to be lower indicating greater amounts of circulating mtDNA DAMPs.

FIGURE 3

Serum mtDNA DAMP levels displayed as a function of time after hospital admission. The number of PCR Tc, and thus mtDNA DAMP levels, did not significantly change during patients’ hospital stays for both survivors and nonsurvivors. Tc greater than 35 are considered equivalent and indicative of negligible mtDNA content for sequences of interest. All mortality occurred before day 6 in the present cohort.

FIGURE 4

Tc and clinical markers for 2 survivors with initially elevated serum mtDNA DAMPs. Two patients who presented with relatively greater plasma mtDNA DAMP levels among survivors improved clinically with concomitant decreases in mtDNA DAMPs; one patient (top) noted a modest normalization of serum creatinine and a dramatic improvement in pulmonary function, whereas a second patient (bottom) revealed a modest improvement in creatinine and maintenance of a normal P/F with subsequent uneventful removal from mechanical ventilation.