Cell-free nuclear, but not mitochondrial, DNA concentrations correlate with the early host inflammatory response after severe trauma

Severe blunt trauma is associated with an early ‘genomic storm’ which causes simultaneous up- and down-regulation of host protective immunity. Excessive inflammation can lead to organ injury. In the absence of infection, the inflammatory response is presumably driven by release of endogenous alarmins called danger-associated molecular patterns (DAMPs), which initiate immune responses through pattern-recognition receptors (PRR). Here we examined the relationship between concentrations of cell-free (cf) nuclear DNA (ncDNA) and mitochondrial DNA (mtDNA) within 24 hours post trauma with circulating leukocyte transcriptomics and plasma IL-6 concentrations, as well as the patients’ clinical trajectories. In 104 patients enrolled from two level-1 trauma centers, ncDNA and mtDNA concentrations were increased within 24 hours of severe trauma, but only ncDNA concentrations correlated with leukocyte gene expression and outcomes. Surprisingly, ncDNA, not mtDNA concentrations, were significantly elevated in trauma patients who developed chronic critical illness versus rapid clinical recovery. Plasma IL-6 and leukocyte transcriptomics were better predictors of outcomes than cfDNA levels. Although mtDNA and ncDNA are significantly increased in the immediate post-trauma period, the dramatic inflammatory and gene expression changes seen after severe trauma are only weakly correlated with ncDNA concentrations, and more importantly, mtDNA concentrations are not associated with adverse clinical trajectories.

Overall, the total length of hospital and ICU stays for all patients were 18 [11,29] and 8 [4,15] days, respectively ( Table 2). As a whole, average 'time to recovery' (TTR) was 8.5 [4.5, 29] days. Over one third of subjects (36%) had noninfectious complications, while 42% had nosocomial infections. The primary infectious complication was pneumonia (21%) followed by surgical site infection (13%). Forty percent were discharged home, either with or without home healthcare.
As expected, patients who developed CCI had a significantly higher incidence of both infectious (86%) and noninfectious (73%) complications compared to patients who rapidly recovered (32% and 24%, respectively; p < 0.001). Finally, while nearly 70% of RAP patients were discharged to home or to rehabilitation facilities, more than half of patients who developed CCI died or were discharged to facilities associated with poor long-term outcomes (i.e. skilled nursing facilities, long-term acute care) ( Table 2).
Plasma cell-free DNA (cfDNA) and transcriptomic responses. Mitochondrial cytochrome C oxidase subunit III (MT-CO3) and human rhodopsin (RHO) copy number were used to represent mtDNA and ncDNA plasma cfDNA concentrations, respectively. Compared to age, gender and race-ethnicity-matched healthy subjects, both mtDNA and ncDNA copy number were significantly elevated in trauma patients at ≤12 and 24 hours ( Fig. 1 panels A,B). Similarly, plasma IL-6 concentration, a well-accepted biomarker of the magnitude of the systemic inflammatory response, was also significantly increased as compared to controls at both ≤12 and 24 hours ( Fig. 1 panel C) 21 . Although statistical analysis of the transcriptomic response cannot be performed since the metric is derived from the difference between healthy control and the trauma subjects, and therefore is not independent, there was a five-fold increase in the transcriptional metric at both ≤12 and 24 hours ( Fig. 1

panel D).
Differences in early cfDNA and inflammatory responses in trauma patients with alternate clinical trajectories. Since 28-day mortality was low in this cohort (6 of 104, 6%), we used the development of CCI as our primary clinical outcome variable. Unexpectedly, when patients were dichotomized based on their clinical trajectories, differences in mtDNA concentrations were not significantly different at either ≤12 or 24 hours in patients with CCI versus rapid recovery ( Fig. 1 panel A). In contrast, ncDNA concentrations were significantly higher at ≤12 and 24 hours in CCI versus rapid recovery patients ( Fig. 1 panel B). Similarly, both plasma IL-6 concentrations and the leukocyte transcriptomic metric (S63) were significantly higher in CCI patients at both ≤12 and 24 hours than in those who rapidly recovered ( Fig. 1 panel C,D).
Correlations and ability of cfDNA, IL-6 and transcriptomics to predict clinical trajectory. Since mtDNA has been proposed to serve as an endogenous alarmin responsible for the immediate and early inflammatory responses, including cytokine and genomic 'storms', we initially looked at univariate correlations between cfDNA concentrations and inflammatory and genomic markers (Table 3). We focused on 24 hour measurements, since the correlations obtained within 12 hours post trauma were consistently less than seen at 24 hours. This is likely due to the hemodynamic instability of these patients in the immediate post-trauma period. As anticipated, the strongest correlations among alarmins and inflammatory markers were between the concentrations of ncDNA and mtDNA (rho = 0.646, p < 0.001). Surprisingly, mtDNA concentrations were not correlated with either ISS or APACHE II scores, while ncDNA was correlated with injury burden (ISS; rho = 0.203, p = 0.040). Both ncDNA and mtDNA correlated with an abnormal leukocyte transcriptomic response (ncDNA; rho = 0.219, p = 0.031 and mtDNA; rho = 0.214, p = 0.036) but neither were correlated with plasma IL-6 nor maximal lactate concentrations (Table 3). In contrast, the S63 leukocyte transcriptomic metric was most strongly correlated with shock severity, physiologic derangement, and elevated IL-6 levels.
Subsequently, the ability of cfDNA concentrations, plasma IL-6 and transcriptomic changes measured at 24 hours after blunt trauma to predict clinical trajectory over 14 days was assessed using area under the receiver-operating curves (Fig. 2). The leukocyte transcriptomic response (S63), followed by plasma IL-6 concentrations, and then ncDNA were nearly identical in their predictive ability. In contrast, mtDNA concentrations could not distinguish RAP from CCI at 24 hours (OR 1.00 [95% CI 1.000-1.001]) (Fig. 2).

Discussion
In this report, we demonstrate that in the immediate 24-hour period following severe blunt trauma, there is a significant increase in the amount of circulating cell-free ncDNA and mtDNA, as measured with a highly quantitative droplet digital PCR (ddPCR) technology. Importantly, ncDNA, but not mtDNA, copy number was significantly greater in patients who subsequently developed CCI versus RAP, and were as predictive of clinical trajectory as IL-6 concentrations and an abnormal leukocyte transcriptomic response.
Previously, authors have suggested that plasma mtDNA concentrations in particular could serve as a diagnostic biomarker for the severity of the injury response [9][10][11][12][13][14] . In fact, some investigators have recently speculated that removal of circulating mtDNA could be a therapeutic option in this patient population 22 . All of these proposals are based on the well-known properties of mtDNA as endogenous alarmins 3 . Recognized by PRRs, primarily intracellular, mtDNA in particular can activate innate immunity and inflammation through multiple intracellular signaling pathways 23,24 .
Our failure to detect any association between mtDNA copy number and injury severity or clinical outcomes is in contrast to several earlier publications [9][10][11][12][13][14] . There are two potential explanations. First, we excluded subjects www.nature.com/scientificreports www.nature.com/scientificreports/ with severe traumatic brain injury and those who died within 48 hours of injury. This eliminated the neurologically impaired or mortally-injured patient secondary to uncontrollable hemorrhage or refractory shock, since our focus was on the association between cfDNA concentrations, the early inflammatory response and long-term clinical trajectories. In this manner, our patients were less lethally injured and more homogenous since we only included immediate survivors with the potential for recovery, but at high risk of a complicated clinical course. Secondly, we used a highly precise ddPCR technology that does not rely on exponential quantitation based on the number of PCR cycles. We purposefully removed any whole cells, whole mitochondrion and microaggregates by repeated high speed centrifugation prior to assay to assure that the mtDNA and ncDNA measured were indeed cell-free, which was not always the case in earlier studies.  www.nature.com/scientificreports www.nature.com/scientificreports/ We would also conclude that extracellular cfDNA probably does not directly contribute to the systemic inflammatory response; rather, it more likely serves as a biomarker of cellular stress or cell death. Furthermore, the release of cfDNA is more likely to be representative of injury burden secondary to direct tissue damage as opposed to shock severity or early physiologic derangement. Interestingly, ncDNA and mtDNA concentrations were detectable in the plasma of healthy subjects, presumably in the absence of any systemic inflammatory response. The presence of mtDNA and ncDNA in the cell-free plasma of healthy controls has been previously reported, but why it does not elicit inflammation is unclear [9][10][11] . Unlike ncDNA, mtDNA does not circulate bound to nucleosomes, and is highly charged, binding to a variety of plasma proteins. Cellular receptors recognizing single and double-stranded DNA are primarily located intracellularly on the endoplasmic reticulum, and are therefore physically separated from their plasma ligand 25,26 . Poli et al. have argued that plasma cfDNA must bind to IL-26, a member of the IL-10 superfamily, for intracellular transport and binding to intracellular PRRs 27 . Others have speculated that circulating and intracellular mtDNA may preferentially bind to multiple extra-or intracellular proteins including LL37, HMGB1, TFAM and other cationic proteins to signal through either cytosolic RAGE, TLR, STING or cGAS signaling pathways 23,24 . This would imply that the ability of plasma or extracellular cfDNA to serve as an injured tissue alarmin requires the trauma-induced increased production of transporter proteins across the cell membrane or intracellularly.
Plasma ncDNA may better predict clinical trajectories because, in contrast to mtDNA, the presence of ncDNA has been directly attributed to cell death and release of cellular contents [28][29][30] . The exact cellular source of this ncDNA remains undetermined in trauma patients, although necroptosis and NETosis of blood neutrophils clearly contributes to extracellular DNA 31 . Snyder et al. and Ulz et al. deep-sequenced plasma cfDNA from healthy control subjects, and based on the nucleosome patterns, concluded that plasma cfDNA likely results from the death of lymphoid and myeloid cells 32,33 . If this is the case, then circulating cell-free ncDNA should be common in both healthy and trauma patients, and is the result of the normal death and/or release from hematopoietic cells. Whether the increase in ncDNA concentrations after trauma in patients with CCI is from increased lymphocyte, macrophage and dendritic cell death or from injured parenchymal tissues is presently unknown. Interestingly, our data shows that ncDNA is most strongly associated with tissue injury burden, specifically ISS, rather than other metabolic stressors such as shock severity or acute physiologic derangement. This would support the hypothesis that CCI is due to increased immune cell death leading to immunosuppression, placing these patients at increased risk for secondary infections 34 . Regardless of its source, this ncDNA is likely not inherently proinflammatory or immunogenic because of its tight binding to nucleosomes 35 .
Unlike ncDNA, which is almost exclusively released upon cell death, mtDNA can be released by both cell death and active secretory processes [36][37][38] . Mast cells are known to secrete cell-free mtDNA 39 . In addition, whole www.nature.com/scientificreports www.nature.com/scientificreports/ mitochondria and mitochondrial fragments are often released during necroptosis and NETosis 40,41 . These organelles or cell fragments are then phagocytosed by myeloid cell populations and mtDNA is released intracellularly from endo-lysosomes for binding to intracellular receptors 41 . Therefore, the reduced ability of cell-free mtDNA concentrations to predict the host inflammatory response and clinical trajectories when compared to ncDNA may reflect differences in the origins and release of these cfDNA.
Our study has multiple limitations that require discussion. The first sample for trauma patients was collected within the first 12 hours after injury due to variability in patient presentation at the emergency department following injury. This may introduce significant variation in plasma cytokine and cfDNA concentrations given the variable number of hours between injury and initial sample collection. Additionally, we did not quantify cfDNA levels beyond 24 hours post-injury, limiting our ability to predict how cfDNA concentration changes following the first day. Further work is necessary to determine how cfDNA concentrations change beyond one day after injury, and whether these levels are additionally related to volume resuscitation or major surgical interventions. In addition, our isolation procedure removes cells, mitochondria and microaggregates, it does not remove microparticles, exosomes or ectosomes, and therefore, the source of the cfDNA remains unknown, and how it is affected by blunt trauma.
The findings here both contribute to our understanding of what drives the host inflammatory response, and the utility of various biomarkers of clinical outcome after severe trauma. We found that cfDNA concentrations have only limited association with either the magnitude of the inflammatory response or clinical trajectory, although ncDNA does appear to outperform mtDNA in this regard. The complex host response to severe blunt trauma is likely driven by the release of multiple alarmins simultaneously, and the concentration of any individual alarmin is unlikely to be an effective predictor of clinical outcomes. Therefore, further refinement using a multi-factor biomarker approach is likely necessary to enhance prediction modeling and to improve patient selection for targeted immunotherapies to treat severely-injured trauma patients.  Table 3. Correlations Among Biomarkers And Clinical Determinants at 24 Hours a . Abbreviations: ncDNA = nuclear DNA; mtDNA = mitochondrial DNA; WBC = white blood cell; ISS = injury severity score; APACHE II = acute physiology and chronic health evaluation II; DFR = distance from reference a Spearman correlation coefficients (rho) were used to determine the relationships between variables. Corresponding p-values along with the number of patients upon which these correlations were derived are also listed. b Maximum lactate was the greatest lactate (mmol/L) within 24 hours of injury. (2019) 9:13648 | https://doi.org/10.1038/s41598-019-50044-z www.nature.com/scientificreports www.nature.com/scientificreports/

Materials and Methods
Study sites and design. This prospective, observational cohort study was conducted over a 3-year period (October 2013 to August 2016) at two United States Level 1 trauma and tertiary care centers: UF Health Hospital, Gainesville, Florida, and Harborview Medical Center, Seattle, Washington. The institutional review board (IRB) of each institution granted approval prior to study initiation. All research was performed in accordance with relevant guidelines/regulations. The study was prospectively registered with clinicaltrials.gov (NCT01810328), and clinical outcomes previously reported 42 . enrollment and informed consent. Subjects were initially enrolled under a 96-hour waiver of informed consent protocol, which was previously approved and implemented by both institutions. The IRB considered critically injured patients to be a 'vulnerable' population, and therefore the purpose of the delayed consent was to permit study patients and their next of kin sufficient time to understand the nature of the study, its risks and benefits. All patients were consented after regaining capacity, or informed consent was provided by the patient proxy in any cases in which patients remained incapacitated.
Inclusion/exclusion criteria. Inclusion criteria included patients ≥18 years with severe blunt traumatic injury and hemorrhagic shock (systolic blood pressure <90 mm Hg or base deficit of ≥6 meq/L within 60 min of arrival of trauma center). Patients expected to survive ≤48 hours and those with severe traumatic brain injury (Glasgow Coma Scale <8 and abnormal head CT) were excluded. All consecutive patients meeting these criteria in which informed consent was obtained within 96 hours were enrolled.
Definition of outcomes. Development of CCI was the primary clinical outcome variable. CCI was defined as prolonged ICU admission (≥14 days during index hospitalization) with evidence of persistent organ www.nature.com/scientificreports www.nature.com/scientificreports/ dysfunction. This definition is based upon prior data that patients meeting these criteria demonstrate a prolonged, dysregulated genomic response to injury, persistent organ dysfunction, and adverse outcomes 2,19,20 . Persistent organ dysfunction was defined using the Modified Marshal Score criteria requiring either greater than or equal to 2 in the renal (serum creatinine ≥1.9 mg/dl [without dialysis]) or pulmonary (PaO 2 /FIO 2 ≤ 300) categories, or greater than or equal to 1 in the cardiac category (systolic blood pressure <90 mm Hg, or use of vasopressors). MOF was defined as a maximum Denver MOF score ≥3. Patients with an ICU length of stay <14 days without persistent organ dysfunction were classified as RAP. TTR in these trauma patients was defined as the first day meeting organ failure recovery criteria in all organ systems without subsequent days of organ failure (Table 4 outlines organ failure recovery criteria). Finally, early death was defined as death within 7 days of injury in patients surviving greater than 48 hours.
Sample collection and processing. EDTA-anticoagulated blood samples were collected from trauma patients within 12 hours following trauma, and again at 24 hours. A single blood collection was performed on thirty-seven age, gender and race/ethnicity-matched healthy control subjects who provided written informed consent. In order to measure IL-6 and the levels of freely circulating DNA in plasma, samples were centrifuged first at 200 × g and stored at −80 °C. For cfDNA, the plasma was then thrice centrifuged at 5000 × g to remove whole mitochondria, residual leukocytes, and apoptotic vesicles. At this speed, exosomes, microparticles and ectosomes are likely not removed. For transcriptomic measurements, total blood leukocytes were processed and extracted as previously described 43,44 . An aliquot of whole blood was initially centrifuged at 500 × g and the buffy coat layer was removed; residual erythrocytes were then lysed with 15 ml of erythrocyte lysis buffer (Qiagen, Valencia, CA) and the leukocyte-rich fraction was collected by centrifugation 45 . Cells were washed twice with cold lysis buffer, and after the final centrifugation, the dry cell pellet was lysed with RLT buffer (Qiagen, Valnecia, CA), and stored at −80 °C until analysis.
Analytical methods. The number of copies of a representative mtDNA and ncDNA sequence was quantified using the Bio-Rad QX 200 Droplet Digital PCR (ddPCR) System with EvaGreen ™ fluorescent dye (Hercules, California, USA) as previously described 46 . Briefly, primers and probes targeting representative mtDNA and ncDNA were selected for quantification of absolute copies/µL. Enzyme activation followed by denaturation and annealing cycles and enzyme deactivation were performed per the manufacturer's instructions. At least 50,000 droplets were read and quantified with the number of positive droplets determining the absolute copy number and concentration. Human mitochondrial cytochrome C oxidase subunit III (MT-CO3), and rhodopsin (RHO) DNA sequences were used to represent mtDNA and ncDNA, respectively. Primer sequences are provided in Supplementary Table 1.
To quantify the transcriptomic response from blood leukocytes, we used a metric comprised of the expression of 63 genes, using the NanoString FLEX ™ (Seattle, Washington, USA) platform that has been prospectively validated to predict clinical trajectories in a similar population of severe trauma patients Supplementary Table 2 43 .
Briefly, leukocyte RNA captured from EDTA-anticoagulated blood samples, was extracted, the NanoString ™ platform was performed per the manufacturer's guidelines, and fold-changes of the 63 genes of interest were calculated and reported as the difference-from-related (DFR) metric. Very briefly, the Nanostring platform uses specific capture probes for the 63 genes (and 7 housekeeping genes) and unique fluorescently labeled probes to quantitate the number of captured transcripts simultaneously. Unlike, qPCR, Nanostring requires no amplification, is linear and has analytical reproducibility less than 5-10% depending upon the quantity of mRNA. Plasma IL-6 concentrations were determined using the Luminex Magpix ™ (Austin, Texas, USA) platform according to the manufacturer's recommendations. Clinical outcomes were prospectively adjudicated by the investigators, and the clinical dataset locked before predictive modeling commenced.
Statistics. The genomic metric, S63, was calculated as a difference from reference, as previously described 43 .
Very briefly, the single metric is derived from the sum of the square root differences in gene expression from age, gender matched healthy controls, adjusted for the individual gene variance, and squared to eliminate positive and negative differences from control.
Definition of Time to Recovery (TTR) a First day meeting organ failure recovery criteria in all systems listed below, without any subsequent days with further organ system failure.  Table 4. Parameters for Time to Recovery. a A surviving patient who did not recover by day 28 or died prior to day 28 was assigned a TTR value of 30 days. Parameters used to define TTR were established by Cuschieri et al. 19 .