Cell-free Mitochondrial DNA Levels Research Articles

Prognostic potential of circulating cell free mitochondrial DNA levels in COVID-19 patients.

In viral infections, mitochondria act as one of the main hubs of the pathogenesis. Recent findings present new insights into the potential role of circulating cell-free mitochondrial DNA (ccf-mtDNA) in COVID-19 pathogenesis by the induction of immune response and aggressive cytokine storm in SARS-CoV-2 infection. The levels of ccf-mtDNA were investigated in 102 hospitalized patients with COVID-19 using the quantitative PCR (q-PCR) method. Statistical analysis confirmed a strong association between the levels of ccf-mtDNA and and mortality, ICU admission, and intubation. Also, our findings highlighted the pivotal role of comorbidities as a risk factor for COVID-19 mortality and severity. Higher levels of ccf-mtDNA can serve as a potential early indicator for progress and poor prognosis of COVID-19.

Comparison of plasma mitochondrial DNA copy number in asymptomatic and symptomatic COVID-19 patients.

Since the beginning of the COVID-19 pandemic, a wide clinical spectrum, from asymptomatic infection to mild or severe disease and death, have been reported in COVID-19 patients. Studies have suggested several possible factors, which may affect the clinical outcome of COVID-19. A pro-inflammatory state and impaired antiviral response have been suggested as major contributing factors in severe COVID-19. Considering that mitochondria have an important role in regulating the immune responses to pathogens, pro-inflammatory signaling, and cell death, it has received much attention in SARS-CoV-2 infection. Recent studies have demonstrated that high levels of cell-free mitochondrial DNA (cf-mtDNA) are associated with an increased risk of COVID-19 intensive care unit (ICU) admission and mortality. However, there have been few studies on cf-mtDNA in SARS-CoV-2 infection, mainly focusing on critically ill COVID-19 cases. In the present study, we investigated cf-mtDNA copy number in COVID-19 patients and compared between asymptomatic and symptomatic cases, and assessed the clinical values. We also determined the cf-nuclear DNA (cf-nDNA) copy number and mitochondrial transcription factor A (TFAM) mRNA level in the studied groups. Plasma and buffy coat samples were collected from 37 COVID-19 patients and 33 controls. Briefly, after total DNA extraction, plasma cf-mtDNA, and cf-nDNA copy numbers were measured by absolute qPCR using a standard curve method. Furthermore, after total RNA extraction from buffy coat and cDNA synthesis, TFAM mRNA levels were evaluated by qPCR. The results showed that cf-mtDNA levels in asymptomatic COVID-19 patients were statistically significantly higher than in symptomatic cases (p value = 0.01). However, cf-nDNA levels were higher in symptomatic patients than in asymptomatic cases (p value = 0.00). There was no significant difference between TFAM levels in the buffy coat of these two groups (p value > 0.05). Also, cf-mtDNA levels showed good diagnostic potential in COVID-19 subgroups. cf-mtDNA is probably important in the outcome of SARS-CoV-2 infection due to its role in inflammation and immune response. It can also be a promising candidate biomarker for the diagnosis of COVID-19 subgroups. Further investigation will help understanding the COVID-19 pathophysiology and effective diagnostic and therapeutic strategies.

N-acetylcysteine modulates rotenone-induced mitochondrial Complex I dysfunction in THP-1 cells

Mitochondrial Complex I dysfunction and oxidative stress have been part of the pathophysiology of several diseases ranging from mitochondrial disease to chronic diseases such as diabetes, mood disorders and Parkinson’s Disease. Nonetheless, to investigate the potential of mitochondria-targeted therapeutic strategies for these conditions, there is a need further our understanding on how cells respond and adapt in the presence of Complex I dysfunction. In this study, we used low doses of rotenone, a classical inhibitor of mitochondrial complex I, to mimic peripheral mitochondrial dysfunction in THP-1 cells, a human monocytic cell line, and explored the effects of N-acetylcysteine on preventing this rotenone-induced mitochondrial dysfunction. Our results show that in THP-1 cells, rotenone exposure led to increases in mitochondrial superoxide, levels of cell-free mitochondrial DNA, and protein levels of the NDUFS7 subunit. N-acetylcysteine (NAC) pre-treatment ameliorated the rotenone-induced increase of cell-free mitochondrial DNA and NDUFS7 protein levels, but not mitochondrial superoxide. Furthermore, rotenone exposure did not affect protein levels of the NDUFV1 subunit but induced NDUFV1 glutathionylation. In summary, NAC may help to mitigate the effects of rotenone on Complex I and preserve the normal function of mitochondria in THP-1 cells.

Chronic Lung Allograft Dysfunction Is Associated with Increased Levels of Cell-Free Mitochondrial DNA in Bronchoalveolar Lavage Fluid of Lung Transplant Recipients.

Chronic Lung Allograft Dysfunction (CLAD) is a life-threatening complication that limits the long-term survival of lung transplantation patients. Early diagnosis remains the basis of efficient management of CLAD, making the need for distinctive biomarkers critical. This explorative study aimed to investigate the predictive power of mitochondrial DNA (mtDNA) derived from bronchoalveolar lavages (BAL) to detect CLAD. The study included 106 lung transplant recipients and analyzed 286 BAL samples for cell count, cell differentiation, and inflammatory and mitochondrial biomarkers, including mtDNA. A receiver operating curve analysis of mtDNA levels was used to assess its ability to detect CLAD. The results revealed a discriminatory pro-inflammatory cytokine profile in the BAL fluid of CLAD patients. The concentration of mtDNA increased in step with each CLAD stage, reaching its highest concentration in stage 4, and correlated significantly with decreasing FEV1. The receiver operating curve analysis of mtDNA in BAL revealed a moderate prediction of CLAD when all stages were grouped together (AUROC 0.75, p-value < 0.0001). This study has found the concentration mtDNA in BAL to be a potential predictor for the early detection of CLAD and the differentiation of different CLAD stages, independent of the underlying pathology.

CD36 is Associated With the Development of Coronary Artery Lesions in Patients With Kawasaki Disease.

Kawasaki disease (KD) is an autoimmune-like vasculitis of childhood involving the coronary arteries. Macrophages require scavenger receptors such as CD36 to effectively clear cellular debris and induce self-tolerance. In this study, we hypothesized that CD36 plays an important role in the immunopathogenesis of KD, by aiding in the clearance of plasma mitochondrial DNA, and by amplifying the immune response by activating the inflammasome pathway via AIM2. Fifty-two healthy controls, 52 febrile controls, and 102 KD patients were recruited for RT-PCR of target mRNA expression and plasma mitochondrial DNA. Blood samples were obtained 24 hours prior and 21 days after the administration of intravenous immunoglobulin (IVIG) therapy. Patients with acute KD had higher plasma levels of cell-free mitochondrial DNA (ND1, ND4, and COX1), and higher mRNA expressions of CD36 and AIM2 when compared to both healthy and febrile controls. A greater decrease in both CD36 and AIM2 mRNA expression after IVIG therapy was associated with the development of coronary artery lesions. Coronary artery lesions were associated with a larger decrease of CD36 expression following IVIG therapy, which may indicate that prolonged expression of the scavenger receptor may have a protective effect against the development of coronary artery lesions in KD.

Growth differentiation factor 15 protects against the aging-mediated systemic inflammatory response in humans and mice.

Mitochondrial dysfunction is associated with aging‐mediated inflammatory responses, leading to metabolic deterioration, development of insulin resistance, and type 2 diabetes. Growth differentiation factor 15 (GDF15) is an important mitokine generated in response to mitochondrial stress and dysfunction; however, the implications of GDF15 to the aging process are poorly understood in mammals. In this study, we identified a link between mitochondrial stress‐induced GDF15 production and protection from tissue inflammation on aging in humans and mice. We observed an increase in serum levels and hepatic expression of GDF15 as well as pro‐inflammatory cytokines in elderly subjects. Circulating levels of cell‐free mitochondrial DNA were significantly higher in elderly subjects with elevated serum levels of GDF15. In the BXD mouse reference population, mice with metabolic impairments and shorter survival were found to exhibit higher hepatic Gdf15 expression. Mendelian randomization links reduced GDF15 expression in human blood to increased body weight and inflammation. GDF15 deficiency promotes tissue inflammation by increasing the activation of resident immune cells in metabolic organs, such as in the liver and adipose tissues of 20‐month‐old mice. Aging also results in more severe liver injury and hepatic fat deposition in Gdf15‐deficient mice. Although GDF15 is not required for Th17 cell differentiation and IL‐17 production in Th17 cells, GDF15 contributes to regulatory T‐cell‐mediated suppression of conventional T‐cell activation and inflammatory cytokines. Taken together, these data reveal that GDF15 is indispensable for attenuating aging‐mediated local and systemic inflammation, thereby maintaining glucose homeostasis and insulin sensitivity in humans and mice.

P 164 - Circulating mtDNA levels as an early marker for metabolic syndrome

Chronic diseases, infection and physical activity are conditions related with metabolic or oxidative stress and the releasing of mitochondria to circulation by cells. This might be due to apoptosis or transference of mitochondria from one cell to another, either as survival or as tissue repair process. Circulating levels of cell-free mitochondrial DNA (ccf-mtDNA), although controversial, have been used for cancer and sepsis diagnose. The objective of this study was to investigate the relationship between ccf-mtDNA and metabolic syndrome. In this sense, after IRB approval, healthy subjects (n=32) and adults with diabetes (n=6) were invited to participate. Body mass index (BMI) was calculated and fasting glucose measured as biomarker of metabolic syndrome. In addition, DNA was extracted from serum and real time PCR used to detect ccf-mtDNA and both parameters were compared. Our data showed that ccf-mtDNA values ranged from 10 to 100 ng/ml in subjects with normal BMI while there were no detectable levels of ccf-mtDNA in samples from overweight subjects and diabetic patients. Glucose levels did not show differences independently of their BMI, but there was an inverse relationship between ccf-mtDNA and BMI. In conclusion, our results suggest that ccf-mtDNA might represent an early biomarker to identify people with metabolic syndrome or at risk for future develpment of diabetes.

Increased levels of cell-free mitochondrial DNA in the cerebrospinal fluid of patients with multiple sclerosis

Mitochondrial DNA (mtDNA) can act as damage-associated molecular pattern molecule (DAMP) and initiate an inflammatory response. We hypothesized that the concentration of mtDNA might reflect inflammatory activity in multiple sclerosis and investigated therefore levels of cell-free mitochondrial DNA in cerebrospinal fluid of patients with relapsing-remitting multiple sclerosis. Significantly higher levels of mtDNA were found in patients compared to controls and there was an inverse correlation between disease duration and mtDNA concentration. Our study suggests that mitochondria can be involved early in multiple sclerosis, but whether this is as an initiator of the inflammatory response or part of its maintenance is unclear. Further, our study suggests that changes in mtDNA may provide a novel marker for early disease activity.

Cerebrospinal fluid mitochondrial DNA in the Alzheimer's disease continuum

Low levels of cell-free mitochondrial DNA (mtDNA) in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients have been identified and proposed as a novel biomarker for the disease. The lack of validation studies of previous results prompted us to replicate this finding in a comprehensive series of patients and controls. We applied droplet digital polymerase chain reaction in CSF specimens from 124 patients representing the AD spectrum and 140 neurologically healthy controls. The following preanalytical and analytical parameters were evaluated: the effect of freeze-thaw cycles on mtDNA, the linearity of mtDNA load across serial dilutions, and the mtDNA levels in the diagnostic groups. We found a wide range of mtDNA copies, which resulted in a high degree of overlap between groups. Although the AD group presented significantly higher mtDNA counts, the receiver-operating characteristic analysis disclosed an area under the curve of 0.715 to distinguish AD patients from controls. MtDNA was highly stable with low analytical variability. In conclusion, mtDNA levels in CSF show a high interindividual variability, with great overlap within phenotypes and presents low sensitivity for AD.

The Cell-Free Mitochondrial DNA: A Novel Biomarker of Cardiovascular Risk?

Circulating cell-free mitochondrial DNA could find in healthy subjects and patients with neoplasia, trauma, infections, stroke, autoimmune, metabolic and rheumatic diseases. The triggers of cell-free mitochondrial DNA secretion are various impacts, i.e. microbial antigen stimulation, inflammatory cytokine, active molecules. It is speculated that the cell-free mitochondrial DNA might be a critical activator of inflammation, coagulation and the innate immune system linking mitochondrial dysfunction, cell death and target organ injury. There is evidence regarding that the cell-free mitochondrial DNA levels may elevate in healthy individuals depending aging and in cancer and non-cancer subjects at risk of CV diseases, as well as in persons with established CH disease. The results of several studies have shown that elevated circulating cell-free mitochondrial DNA has associated with cardiovascular (CV) diseases, while diagnostic and predictive value of this biomarker in non-cancer individuals is not fully clear. The mini review is devoted the biological role, diagnostic and predictive value of cellfree mitochondrial DNA in patients at CV risk.

Decline in relative plasma cell-free mitochondrial DNA levels in response to prolonged moderate aerobic and eccentric exercise (HUM1P.306)

Abstract Elevated plasma levels of cell-free mitochondrial DNA (cf-mDNA), a cell damage-associated molecular pattern (DAMP), contribute to neutrophil activation and inflammation in trauma patients, and may occur in cancer and autoimmunity. To further understand relationships between cf-mDNA released by tissue injury, inflammation, and health benefits of exercise, we conducted trial by time experiments for plasma cf-mDNA response to prolonged moderate aerobic exercise, and to eccentric exercise. Seven healthy moderately-trained young men (n=7) completed treadmill exercise trials for 90 min at 60% VO2 max, and resting control trials, and blood was sampled immediately prior to (Pre), during (at +18, +54, and +90 min), and after (R40) trials. A significant difference in cf-mDNA response was observed between exercise and control trials. Specifically, cf-mDNA levels differed at +54 and +90 (with or without plasma volume (PV) shift correction). A significant time effect was observed, with relative cf-mDNA levels declining at +54 and +90 during exercise. These results suggest increased clearance or reduced release of plasma cf-mDNA with exercise. Declines in cf-mDNA were also seen after eccentric exercise. Our finding of cf-mDNA decline with prolonged moderate treadmill and eccentric exercise contrasts with studies by others involving exhaustive short-term treadmill exercise, where cf-mDNA levels were unchanged, and highlights differences between exercise- and trauma-induced inflammation.