Plasma Mitochondrial DNA and Necroptosis as Prognostic Indicators in Critically Ill Patients with Sepsis

Mitochondrial DNA (mtDNA) has been identified as a biomarker for predicting sepsis mortality. Although preclinical studies suggested that necroptosis could explain the mechanistic link of mtDNA in sepsis, this is not yet evident in patients with sepsis. This study evaluated the association between mtDNA and essential necroptosis mediators in prospectively enrolled patients with sepsis. Plasma mtDNA copy number was measured using quantitative PCR assay and necroptosis mediators, including receptor-interacting protein kinase-3 (RIPK3), mixed lineage domain-like pseudokinase (MLKL), and high-mobility group box 1 (HMGB1), were measured by ELISA. Receiver operating characteristic (ROC) analysis was conducted to evaluate the predictive ability of mtDNA copy number as a predictor of hospital mortality. Among the 142 patients with sepsis, the mtDNA copy number was significantly higher in non-survivors than in survivors (median, 4040 copies/µL vs. 2585 copies/µL; p < 0.001), and the area under the ROC curve was 0.73 (95% CI, 0.64–0.82) for the relationship between mtDNA and hospital mortality. Furthermore, the correlation between mtDNA copy number and each necroptosis mediator was excellent (p < 0.001 for all): RIPK3 (r = 0.803), MLKL (r = 0.897), and HMGB1 (r = 0.603). The plasma mtDNA copy number was highly correlated with essential necroptosis mediators, suggesting that mtDNA propagates necroptosis and increases sepsis mortality.


Introduction
Sepsis has been identified as a life-threatening organ dysfunction caused by a dysregulated host response to infection [1]. Sepsis and septic shock are major healthcare problems, affecting millions of people worldwide each year and killing approximately 17-33% of affected patients [2,3]. Considering the high mortality in sepsis, accurately identifying high-risk patients with sepsis and focusing treatment on this population is deemed crucial. In this regard, there have been many biomarkers identified for predicting morbidity and mortality in sepsis in the critical care setting [4,5].
Plasma mitochondrial DNA (mtDNA) was assessed as a biomarker for predicting sepsis mortality [6]. Previous studies revealed that plasma mtDNA copy number is significantly higher in patients with sepsis or septic shock than in healthy controls [7][8][9]. Furthermore, mtDNA copy number has also been associated with mortality in patients with sepsis [9,10]. However, increasing evidence has shown that mtDNA is not only a marker for sepsis mortality but also a key player in organ dysfunction and mortality in patients with sepsis. This is because circulating mtDNA can trigger innate immunity through multiple mechanisms, such as activating the Toll-like receptor 9/NF-κB pathway or the NALP3 inflammasome [11,12]. Consequently, mtDNA may contribute to the persistent and dysregulated inflammatory response and amplify organ dysfunction in sepsis [10]. mtDNA serves as damage-associated molecular patterns (DAMPs) that drive an immune or inflammatory response in sepsis [6]. The DAMPs are often released during extensive, infection-induced immune cell death, which is linked with multiple organ dysfunction [13].
Necroptosis is a type of cell death that combines apoptosis and necrosis. It is morphologically similar to necrosis, but it can also be regulated by underlying genetic programs [14]. Previous studies suggested that the necroptotic pathway and its regulatory proteins are crucial mediators of sepsis-induced organ injury [15][16][17]. Although a mouse study showed that mtDNA was translocated to the extracellular space by necroptosis [18], clinical studies exploring the association between mtDNA and necroptosis remain to be limited. Figure 1 schematically depicts the correlation between necroptosis and extracellular mtDNA. marker for sepsis mortality but also a key player in organ dysfunction and mortality in patients with sepsis. This is because circulating mtDNA can trigger innate immunity through multiple mechanisms, such as activating the Toll-like receptor 9/NF-κB pathway or the NALP3 inflammasome [11,12]. Consequently, mtDNA may contribute to the persistent and dysregulated inflammatory response and amplify organ dysfunction in sepsis [10]. mtDNA serves as damage-associated molecular patterns (DAMPs) that drive an immune or inflammatory response in sepsis [6]. The DAMPs are often released during extensive, infection-induced immune cell death, which is linked with multiple organ dysfunction [13].
Necroptosis is a type of cell death that combines apoptosis and necrosis. It is morphologically similar to necrosis, but it can also be regulated by underlying genetic programs [14]. Previous studies suggested that the necroptotic pathway and its regulatory proteins are crucial mediators of sepsis-induced organ injury [15][16][17]. Although a mouse study showed that mtDNA was translocated to the extracellular space by necroptosis [18], clinical studies exploring the association between mtDNA and necroptosis remain to be limited. Figure 1 schematically depicts the correlation between necroptosis and extracellular mtDNA.

Study Design and Population
The Samsung Medical Center Registry of Critical Illness (SMC-RoCI) is a prospective observational study conducted at the Samsung Medical Center (1989-bed, university-affiliated, tertiary referral hospital in Seoul, South Korea) [16]. This ongoing prospective study was initiated in April 2014 and aims to establish a human sample repository and develop new biological markers for critical illness [16]. The cohort profile was described This study aimed to evaluate the usefulness of mtDNA as a biomarker to predict mortality in critically ill patients with sepsis and to assess the association between plasma mtDNA levels and necroptosis mediators.

Study Design and Population
The Samsung Medical Center Registry of Critical Illness (SMC-RoCI) is a prospective observational study conducted at the Samsung Medical Center (1989-bed, universityaffiliated, tertiary referral hospital in Seoul, South Korea) [16]. This ongoing prospective study was initiated in April 2014 and aims to establish a human sample repository and develop new biological markers for critical illness [16]. The cohort profile was described previously [15][16][17]19]. This study was approved by the Institutional Review Board of Samsung Medical Center, and written informed consent was obtained from all patients or their legally authorized representatives prior to enrollment.
All consecutive adult patients (aged ≥ 19 years) admitted to the medical intensive care unit (ICU) were eligible for inclusion in this registry. The exclusion criteria from eligibility were as follows: (1) cognitive impairment, (2) inability to provide informed consent, (3) ICU admission for a simple procedure or postsurgical care, (4) transfer from other hospitals, (5) end-of-life decision or admission to facilitate palliative care, (6) hemoglobin < 8 g/dL upon admission or persistent bleeding, and (7) discharge within 24 h of ICU admission [16].
In total, 1367 patients were admitted to the medical ICU from April 2014 to August 2016. After excluding 1172 patients who met the exclusion criteria and 1 who missed screening (transferred to the general ward before the screening process), 194 were initially identified. After further excluding 46 patients who were not diagnosed with sepsis, 4 who withdrew consent, and 2 whose samples expired, 142 were ultimately included in the analysis ( Figure 2).
previously [15][16][17]19]. This study was approved by the Institutional Review Board of Samsung Medical Center, and written informed consent was obtained from all patients or their legally authorized representatives prior to enrollment.
All consecutive adult patients (aged ≥ 19 years) admitted to the medical intensive care unit (ICU) were eligible for inclusion in this registry. The exclusion criteria from eligibility were as follows: (1) cognitive impairment, (2) inability to provide informed consent, (3) ICU admission for a simple procedure or postsurgical care, (4) transfer from other hospitals, (5) end-of-life decision or admission to facilitate palliative care, (6) hemoglobin < 8 g/dL upon admission or persistent bleeding, and (7) discharge within 24 h of ICU admission [16].
In total, 1367 patients were admitted to the medical ICU from April 2014 to August 2016. After excluding 1172 patients who met the exclusion criteria and 1 who missed screening (transferred to the general ward before the screening process), 194 were initially identified. After further excluding 46 patients who were not diagnosed with sepsis, 4 who withdrew consent, and 2 whose samples expired, 142 were ultimately included in the analysis (Figure 2).

Data Collection
Clinical data on patient demographics, the reason for ICU admission, the severity of illness score, and laboratory data were obtained at the time of enrollment. Illness severity was assessed using the Acute Physiology and Chronic Health Evaluation II (APACHE II) [20], Simplified Acute Physiology Score 3 (SAPS 3) [21], and Sequential Organ Failure Assessment (SOFA) scores [22]. Sepsis was defined according to the third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) [1]. Since the SMC-RoCI was initiated in April 2014, patients enrolled before the release of the new definition were reclassified using Sepsis-3.

Measurement of Plasma Mitochondrial DNA and Necroptosis-Related Markers
Blood samples were collected as 19 mL of whole blood within 48 h of study enrollment and centrifuged within 4 h of collection. Plasma was separated and stored at −80 °C for further analysis. In order to measure the mtDNA copy number, the plasma samples

Data Collection
Clinical data on patient demographics, the reason for ICU admission, the severity of illness score, and laboratory data were obtained at the time of enrollment. Illness severity was assessed using the Acute Physiology and Chronic Health Evaluation II (APACHE II) [20], Simplified Acute Physiology Score 3 (SAPS 3) [21], and Sequential Organ Failure Assessment (SOFA) scores [22]. Sepsis was defined according to the third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) [1]. Since the SMC-RoCI was initiated in April 2014, patients enrolled before the release of the new definition were reclassified using Sepsis-3.

Measurement of Plasma Mitochondrial DNA and Necroptosis-Related Markers
Blood samples were collected as 19 mL of whole blood within 48 h of study enrollment and centrifuged within 4 h of collection. Plasma was separated and stored at −80 • C for further analysis. In order to measure the mtDNA copy number, the plasma samples were pre-filtered using 0.8 µm syringe filters, and additional centrifugation was performed to eliminate residual cellular material. DNA was isolated from 1 mL of plasma using the QI-Aamp Circulating Nucleic Acids kit (QIAGEN, Germany) and eluted in 50 µL of kit elution buffer. The mtDNA copy numbers were then assessed by measuring the NADH dehydrogenase 1 gene using quantitative real-time PCR with a PRISM 7300 sequence detection system (Applied Biosystems, Austin, TX, USA) with the following primers: human NADH dehydrogenase 1 gene (mtDNA): forward 5 -ATACCCATGGCCAACCTCCT-3 , reverse 5 -GGGCCTTTGCGTAGTTGTAT-3 ; and human β-globin (nuclear DNA): forward 5 -GTGCACCTGACTCCTGAGGAGA-3 , reverse 5 -CCTTGATACCAACCTGCCCAG-3 [23]. The thermal profile for detecting mtDNA was as follows: denaturation at 95 • C for 10 min,

Statistical Analysis
Categorical variables were compared using the Chi-square test or Fisher's exact test, whereas continuous variables were compared using the Mann-Whitney U test. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic accuracy of mtDNA in predicting survival among critically ill patients with sepsis. Pearson's correlation analysis was used to assess the association between plasma mtDNA copy numbers and necroptosisrelated markers. All tests were two-sided, and p < 0.05 was considered significant. Data were analyzed using STATA version 16 (Stata Corp., College Station, TX, USA).

Discussion
In this observational study, plasma mtDNA satisfactorily predicted in-hospital mortality in critically ill patients with sepsis. Furthermore, the mtDNA copy number showed excellent correlations with those of necroptosis mediators, including RIPK3, MLKL, and HMGB1, which implies a link between plasma mtDNA and necroptosis in sepsis.
This study revealed that the mtDNA copy number was significantly higher in non-

Discussion
In this observational study, plasma mtDNA satisfactorily predicted in-hospital mortality in critically ill patients with sepsis. Furthermore, the mtDNA copy number showed excellent correlations with those of necroptosis mediators, including RIPK3, MLKL, and HMGB1, which implies a link between plasma mtDNA and necroptosis in sepsis.
This study revealed that the mtDNA copy number was significantly higher in nonsurvivors than in survivors among critically ill patients with sepsis, although no intergroup differences were noted in CRP and lactate levels. In agreement with our results, previous studies found that the mtDNA copy number was elevated in patients with sepsis [7][8][9] and was associated with mortality [9,10]. In this regard, plasma mtDNA may be a suitable biomarker for diagnosing sepsis and predicting mortality in patients with sepsis. However, mtDNA copy number was not significantly associated with mortality in some sepsis studies [8,24]. This discrepancy might be explained by differences in terms of the protocols used to measure mtDNA copy numbers across previous studies, thus warranting the standardization of mtDNA protocols to firmly establish the clinical usefulness of mtDNA in sepsis [25].
In order to serve as a DAMP driving the inflammatory response in sepsis, mtDNA needs to be released from the mitochondria into the cytosol and extracellular compartments [6]. The underlying mechanisms of the phenomenon include the generation of mitochondrial reactive oxygen species [26,27] and destabilized mtDNA packaging [28]. In addition to these two potential mechanisms, necroptosis can translocate mtDNA to the extracellular space, which was then suggested in a mouse study [18]. Excellent correlations between mtDNA level and necroptosis mediators (RIPK3, MLKL, and HMGB1) in this study show that a large proportion of plasma mtDNA may be released by necroptosis in sepsis.
Necroptosis is often induced by specific ligand binding, including the binding of tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) to several death receptors, including TNF receptor 1 and TRAIL receptors [29]. Subsequently, the necroptosis pathway is regulated by distinct proteins, such as RIPK1 and RIPK3, and downstream substrate MLKL [14,30]. The ruptured, dying cell induces the release of several necrosis-associated DAMPs, including HMGB1 [31,32]. Previous animal and clinical studies demonstrated the crucial role of necroptosis in driving mortality during sepsis [15,17,33,34]. In the same vein, this study also showed that plasma RIPK3, MLKL, and HMGB1 levels were significantly higher in non-survivors than in survivors among critically ill patients with sepsis. Furthermore, considering the correlation between plasma mtDNA and necroptosis mediators, mtDNA as a DAMP may propagate a proinflammatory response and consequent necroptosis pathway activation, which may be followed by mortality in patients with sepsis. Overall, mtDNA and necroptosis affect each other, and these interactions could increase mortality in patients with sepsis.
This study has several limitations that should be acknowledged. First, although excellent correlations were observed between plasma mtDNA copy number and necroptosis mediators, this study could not evaluate the causal relationships among them. Future studies are thus warranted to elucidate the mechanistic link between mtDNA and necroptosis. Second, no protocol for measuring mtDNA copy numbers was established. Hence, measuring the mtDNA copy number in serum, not in plasma, or using a different protocol might lead to different results. Third, this study was conducted at a single center in Korea, which might limit the generalization of our findings to other institutions or ethnic groups.

Conclusions
Plasma mtDNA copy number was determined to be a predictive factor of mortality in critically ill patients with sepsis and was strongly correlated with essential necroptosis mediators, suggesting that mtDNA propagates necroptosis and increases sepsis mortality. Informed Consent Statement: Written informed consent was obtained from patients or their legally authorized representatives prior to enrollment.

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Conflicts of Interest:
The authors declare that they have no competing interests.