Pre-Analytical Factors Affecting Extracellular DNA in Saliva.

Salivary DNA is widely used for genetic analyses because of its easy collection. However, its extracellular fraction in particular, similar to the extracellular DNA (ecDNA) in plasma, could be a promising biomarker for oral or systemic diseases. In contrast to genetics, the quantity of salivary ecDNA is of importance and can be affected by the pre-analytical processing of samples, but the details are not known. The aim of our study was to analyze the effects of centrifugation and freezing of saliva on the concentration of ecDNA in saliva. Fifteen healthy volunteers, free of any known systemic or oral diseases, were asked to collect unstimulated saliva samples. Aliquots were centrifuged at 1600× g and frozen or directly processed. The fresh or thawed cell-free saliva samples underwent subsequent centrifugation at 16,000× g. The supernatants were used for DNA isolation and quantification using fluorometry and real-time PCR. While freezing had minimal effects on the salivary ecDNA concentration, another centrifugation step decreased ecDNA considerably in both fresh and frozen samples (by 97.8% and 98.4%, respectively). This was mirrored in the quantitative PCR targeting a nuclear (decrease by 93.5%) and mitochondrial (decrease by 97.7%) ecDNA sequence. In conclusion, in this first study focusing on the technical aspects of salivary ecDNA quantitation, we show that, regardless of its subcellular origin, the concentration of ecDNA in saliva is mainly affected by additional centrifugation and not by the freezing of centrifuged cell-free saliva samples. This suggests that most salivary ecDNA likely is associated with cell debris and apoptotic bodies. Which fraction is affected by a particular disease should be the focus of further targeted studies.

Mitochondria-induced formation of neutrophil extracellular traps is enhanced in the elderly via Toll-like receptor 9.

Neutrophil extracellular traps are potent antimicrobial weapons; however, their formation during sterile inflammation is detrimental, and the mechanism of induction is still unclear. Since advanced age is the primary clinical risk factor for poor outcomes in inflammatory diseases, we hypothesized that sterile stimuli, represented by mitochondria, would induce neutrophil extracellular trap formation in an age-dependent manner. Therefore, we analyzed induction of neutrophil extracellular traps in patients grouped according to age or immune status and observed that neutrophils from elderly patients responded to the presence of mitochondria with enhanced neutrophil extracellular trap formation. These neutrophil extracellular traps were also found to be more oxidized and exhibited higher resistance to DNase I degradation. Additionally, a higher concentration of residual neutrophil extracellular traps was detected in the plasma of the elderly. This plasma was capable of priming neutrophils through TLR9-mediated signaling, leading to further neutrophil extracellular trap formation, which was successfully inhibited with chloroquine. Finally, in a mouse model of mitochondria-induced acute lung injury, we observed that neutrophils from aged mice displayed impaired chemotactic activity but exhibited a trend of higher neutrophil extracellular trap formation. Thus, we propose that residual neutrophil extracellular traps circulating in the elderly preactivate neutrophils, making them more prone to enhanced neutrophil extracellular trap formation when exposed to mitochondria during sterile inflammation. Further investigation is needed to determine whether this vicious circle could be a suitable therapeutic target.

Free heme exacerbates colonic injury induced by anti-cancer therapy.

Gastrointestinal inflammation and bleeding are commonly induced by cancer radiotherapy and chemotherapy but mechanisms are unclear. We demonstrated an increased number of infiltrating heme oxygenase-1 positive (HO-1+) macrophages (Mø, CD68+) and the levels of hemopexin (Hx) in human colonic biopsies from patients treated with radiation or chemoradiation versus non-irradiated controls or in the ischemic intestine compared to matched normal tissues. The presence of rectal bleeding in these patients was also correlated with higher HO-1+ cell infiltration. To functionally assess the role of free heme released in the gut, we employed myeloid-specific HO-1 knockout (LysM-Cre : Hmox1flfl), hemopexin knockout (Hx-/-) and control mice. Using LysM-Cre : Hmox1flfl conditional knockout (KO) mice, we showed that a deficiency of HO-1 in myeloid cells led to high levels of DNA damage and proliferation in colonic epithelial cells in response to phenylhydrazine (PHZ)-induced hemolysis. We found higher levels of free heme in plasma, epithelial DNA damage, inflammation, and low epithelial cell proliferation in Hx-/- mice after PHZ treatment compared to wild-type mice. Colonic damage was partially attenuated by recombinant Hx administration. Deficiency in Hx or Hmox1 did not alter the response to doxorubicin. Interestingly, the lack of Hx augmented abdominal radiation-mediated hemolysis and DNA damage in the colon. Mechanistically, we found an altered growth of human colonic epithelial cells (HCoEpiC) treated with heme, corresponding to an increase in Hmox1 mRNA levels and heme:G-quadruplex complexes-regulated genes such as c-MYC, CCNF, and HDAC6. Heme-treated HCoEpiC cells exhibited growth advantage in the absence or presence of doxorubicin, in contrast to poor survival of heme-stimulated RAW247.6 Mø. In summary, our data indicate that accumulation of heme in the colon following hemolysis and/or exposure to genotoxic stress amplifies DNA damage, abnormal proliferation of epithelial cells, and inflammation as a potential etiology for gastrointestinal syndrome (GIS).

DNA in fresh urine supernatant is not affected by additional centrifugation and is protected against deoxyribonuclease.

Urinary DNA is widely studied as a non-invasive marker for monitoring of kidneys after transplantation or the progression of urinary tract tumors. The quantity of urinary DNA especially of mitochondrial origin has been reported to mirror kidney damage in various renal diseases and their models. Processing of samples might affect urinary DNA concentrations but the details are not clear. Samples of urine were collected from fifteen healthy volunteers. DNA was extracted from the whole urine, but also from the supernatant after centrifugation at 1600 g and 16000 g. In addition, we have analyzed the DNA in the microparticles in the pellet after the last spin. DNA was measured using fluorometry and real time PCR targeting nuclear and mitochondrial sequences. Addition of deoxyribonuclease to aliquots of samples enabled the characterization of DNA protection. Centrifugation at 1600 g decreased the concentration of extracted DNA by 66% at least in samples with higher DNA in whole urine. Interestingly, the additional spin at 16000 g did not result in a significant decrease in DNA concentration in the supernatant despite detectable microparticle-associated DNA. Deoxyribonuclease decreases total and nuclear DNA by 26% and 31% in whole urine. The majority of urinary mitochondrial DNA seems to be protected against deoxyribonuclease. Our results indicate high variability in urinary DNA even in healthy probands. Extracellular urinary DNA is partially bound to cell debris or microparticles, but a considerable part is still in the supernatant and is protected against cleavage. Further research should identify the nature of the protection, especially for mitochondrial DNA. Better understanding of the biology of urinary DNA should help its clinical interpretation.

Endogenous DNase Activity in an Animal Model of Acute Liver Failure.

Deoxyribonucleases (DNases) cleave extracellular DNA (ecDNA) and are under intense research as interventions for diseases associated with high ecDNA, such as acute live injury. DNase I treatment decreases morbidity and mortality in this animal model. Endogenous DNase activity has high interindividual variability. In this study, we tested the hypothesis that high endogenous DNase activity is beneficial in an animal model of acute liver failure. DNase activity was measured in the plasma of adult male mice taken before i.p. injection of thioacetamide to induce acute liver failure. The survival of mice was monitored for 48 h. Mice were retrospectively divided into two groups based on the median DNase activity assessed using the gel-based single-radial enzyme diffusion assay. In acute liver failure, mice with a higher baseline DNase activity had lower mortality after 48 h (by 25%). Different protection of ecDNA against nucleases by vesicles or DNA-binding proteins could play a role and should be further evaluated. Similarly, the role of endogenous DNase activity should be analyzed in other disease models associated with high ecDNA.

Extracellular DNA concentrations in various aetiologies of acute kidney injury.

Extracellular DNA (ecDNA) in plasma is a non-specific biomarker of tissue damage. Urinary ecDNA, especially of mitochondrial origin, is a potential non-invasive biomarker of kidney damage. Despite prominent tissue damage, ecDNA has not yet been comprehensively analysed in acute kidney injury (AKI). We analysed different fractions of ecDNA, i.e. total, nuclear and mitochondrial, in plasma and urine of children, and different animal models of AKI. We also analysed the activity of the deoxyribonuclease (DNase), which is contributes to the degradation of ecDNA. Patients with AKI had higher total and nuclear ecDNA in both, plasma and urine (sixfold and 12-fold in plasma, and 800-fold in urine, respectively), with no difference in mitochondrial ecDNA. This was mainly found for patients with AKI due to tubulointerstitial nephritis and atypical haemolytic uremic syndrome. Increased plasma ecDNA was also found in animal models of AKI, including adenine nephropathy (fivefold), haemolytic uremic syndrome (fourfold), and ischemia-reperfusion injury (1.5-fold). Total urinary ecDNA was higher in adenine nephropathy and ischemia-reperfusion injury (1300-fold and twofold, respectively). DNase activity in urine was significantly lower in all animal models of AKI in comparison to controls. In conclusion, plasma total and nuclear ecDNA and urinary total ecDNA is increased in patients and animals with particular entities of AKI, suggesting a mechanism-dependent release of ecDNA during AKI. Further studies should focus on the dynamics of ecDNA and its potential role in the pathogenesis of AKI.

Heme oxygenase-1 mitigates liver injury and fibrosis via modulation of LNX1/Notch1 pathway in myeloid cells.

Activation of resident macrophages (Mϕ) and hepatic stellate cells is a key event in chronic liver injury. Mice with heme oxygenase-1 (HO-1; Hmox1)-deficient Mϕ (LysM-Cre:Hmox1 flfl ) exhibit increased inflammation, periportal ductular reaction, and liver fibrosis following bile duct ligation (BDL)-induced liver injury and increased pericellular fibrosis in NASH model. RiboTag-based RNA-sequencing profiling of hepatic HO-1-deficient Mϕ revealed dysregulation of multiple genes involved in lipid and amino acid metabolism, regulation of oxidative stress, and extracellular matrix turnover. Among these genes, ligand of numb-protein X1 (LNX1) expression is strongly suppressed in HO-1-deficient Mϕ. Importantly, HO-1 and LNX1 were expressed by hepatic Mϕ in human biliary and nonbiliary end-stage cirrhosis. We found that Notch1 expression, a downstream target of LNX1, was increased in LysM-Cre:Hmox1 flfl mice. In HO-1-deficient Mϕ treated with heme, transient overexpression of LNX1 drives M2-like Mϕ polarization. In summary, we identified LNX1/Notch1 pathway as a downstream target of HO-1 in liver fibrosis.

Variability of endogenous deoxyribonuclease activity and its pathophysiological consequences.

Deoxyribonucleases (DNases) are enzymes that cleave DNA. Some DNases are secreted outside of cells where they can cleave extracellular DNA (ecDNA). High concentration of ecDNA is associated with diseases such as sepsis, preeclampsia, and systemic lupus. DNA can be released from dying cells and is immunogenic. DNases cleave ecDNA and might prevent activation of the immune system. Low DNase activity could be disadvantageous in diseases where high amounts of ecDNA are released from dying cells. The relationship between DNase activity and ecDNA remains unknown. The lack of standard values in DNase activity makes the studies difficult to compare. This review focuses on summarizing methods for DNase activity measurements, the possible implication of decreased DNase activity in diseases, and the impact on diseases associated with a high concentration of ecDNA.

Labile Heme and Heme Oxygenase-1 Maintain Tumor-Permissive Niche for Endometriosis-Associated Ovarian Cancer.

Endometriosis, a painful gynecological condition accompanied by inflammation in women of reproductive age, is associated with an increased risk of ovarian cancer. We evaluated the role of peritoneal heme accumulated during menstrual cycling, as well as peritoneal and lesional macrophage phenotype, in promoting an oncogenic microenvironment. We quantified the heme-degrading enzyme, heme oxygenase-1 (HO-1, encoded by Hmox1) in normal peritoneum, endometriotic lesions and endometriosis-associated ovarian cancer (EAOC) of clear cell type (OCCC). HO-1 was expressed primarily in macrophages and increased in endometrioma and OCCC tissues relative to endometriosis and controls. Further, we compared cytokine expression profiles in peritoneal macrophages (PM) and peripheral blood mononuclear cells (PBMC) in women with endometriosis versus controls as a measure of a tumor-promoting environment in the peritoneum. We found elevated levels of HO-1 along with IL-10 and the pro-inflammatory cytokines (IL-1ß, IL-16, IFNγ) in PM but not in PBMC from endometriosis patients. Using LysM-Cre:Hmox1flfl conditional knockout mice, we show that a deficiency of HO-1 in macrophages led to the suppression of growth of ID8 ovarian tumors implanted into the peritoneum. The restriction of ID8 ovarian tumor growth was associated with an increased number of Mac3+ macrophage and B cells in LysM-Cre:Hmox1flfl mice compared to controls. Functional experiments in ovarian cancer cell lines show that HO-1 is induced by heme. Low levels of exogenous heme promoted ovarian cancer colony growth in soft agar. Higher doses of heme led to slower cancer cell colony growth in soft agar and the induction of HO-1. These data suggest that perturbation of heme metabolism within the endometriotic niche and in cancer cells themselves may be an important factor that influences tumor initiation and growth.

Circulating extracellular DNA is in association with continuous metabolic syndrome score in healthy adolescents.

Obesity is associated with chronic low-grade inflammation that eventually leads to metabolic complications. Extracellular DNA (ecDNA) is a damage-associated molecular pattern. Extracellular mitochondrial DNA can activate innate immunity. We hypothesized that ecDNA, especially of mitochondrial origin, could be associated with components of the metabolic syndrome in young healthy probands. In a cross-sectional study, healthy adolescents (n = 1,249) provided blood samples. Anthropometric data, blood pressure, and blood counts were assessed. In addition, biochemical analysis of sera or plasma was conducted, including the quantification of advanced oxidation protein products (AOPPs) as a marker of oxidative stress induced by neutrophil or monocyte activation. Plasma ecDNA was isolated and measured by fluorometry. Nuclear and mitochondrial DNA were quantified by real-time PCR. Males had higher total plasma ecDNA [15 (11-21) vs. 11 (8-17) ng/mL; median (interquartile range)], nuclear [1,760 (956-3,273) vs. 1,153 (600-2,292) genome equivalents (GE)/mL], and mitochondrial [37,181 (14,836-90,896) vs. 30,089 (12,587-72,286) GE/mL] DNA. ecDNA correlated positively with the continuous metabolic syndrome score (r = 0.158 for males and r = 0.134 for females). Stronger correlations were found between ecDNA of mitochondrial origin and AOPP (r = 0.202 and 0.186 for males and females, respectively). Multivariate regression analysis revealed associations of nuclear DNA with leukocyte and erythrocyte counts. The results of this study of healthy adolescents show that circulating ecDNA is associated with the risk of metabolic syndrome, not with obesity per se. The association between mitochondrial ecDNA and AOPP requires further attention as it supports a potential role of mitochondria-induced sterile inflammation in the pathogenesis of the metabolic syndrome.

Measurement of labile and protein-bound heme in fixed prostate cancer cells and in cellular fractions.

Labile heme is present in the cells at very low concentrations, either unbound or loosely bound to molecules, and accessible for signaling as alarmin. Our recent work suggests that extracellular heme can be taken up and detected in the nuclei of cancer cells. Here, we describe the detailed protocol for detection of labile and total heme in prostate cancer cells and its measurement in subcellular compartments in vitro. The protocol can be adapted to be used for other cell types. For complete details on the use and execution of this protocol, please refer to Canesin et al. (2020).

Deoxyribonuclease activity negative correlates with extracellular DNA in uncomplicated singleton pregnancies in the third trimester.

OBJECTIVES: It is not clear, which factors affect extracellular DNA (ecDNA) concentrations in healthy women with singleton uncomplicated pregnancies, although deoxyribonucleases (DNases) are hypothesized to be responsible for the cleavage of plasma ecDNA. The aim of this study was to analyze potential determinants of total ecDNA including plasma DNase activity. METHODS: Plasma samples were collected from 48 healthy women with singleton uncomplicated pregnancies in the third trimester (gestation week 37). DNA was isolated and quantified using fluorometry and real time PCR. DNase activity was assessed using the single radial enzyme-diffusion method. RESULTS: Neither ecDNA, nor DNase activity were affected by maternal age or BMI. DNase activity negatively correlated with total plasma ecDNA (r=-0.40, p=0.007). Similar associations were found for ecDNA of nuclear and mitochondrial origin, but not with fetal DNA quantified using Y-targeted PCR in male fetus-bearing pregnancies. CONCLUSIONS: The role of plasma ecDNA of fetal and maternal origin is studied in the pathogenesis of pregnancy-complications. The results indicate that plasma DNase activity could negatively regulate ecDNA concentrations and should, thus, be analyzed in preeclampsia, preterm birth and other ecDNA-related pregnancy complications.

Deoxyribonucleases and Their Applications in Biomedicine.

Extracellular DNA, also called cell-free DNA, released from dying cells or activated immune cells can be recognized by the immune system as a danger signal causing or enhancing inflammation. The cleavage of extracellular DNA is crucial for limiting the inflammatory response and maintaining homeostasis. Deoxyribonucleases (DNases) as enzymes that degrade DNA are hypothesized to play a key role in this process as a determinant of the variable concentration of extracellular DNA. DNases are divided into two families-DNase I and DNase II, according to their biochemical and biological properties as well as the tissue-specific production. Studies have shown that low DNase activity is both, a biomarker and a pathogenic factor in systemic lupus erythematosus. Interventional experiments proved that administration of exogenous DNase has beneficial effects in inflammatory diseases. Recombinant human DNase reduces mucus viscosity in lungs and is used for the treatment of patients with cystic fibrosis. This review summarizes the currently available published data about DNases, their activity as a potential biomarker and methods used for their assessment. An overview of the experiments with systemic administration of DNase is also included. Whether low-plasma DNase activity is involved in the etiopathogenesis of diseases remains unknown and needs to be elucidated.

Plasma Concentrations of Extracellular DNA in Acute Kidney Injury.

Current diagnostic methods of acute kidney injury (AKI) have limited sensitivity and specificity. Tissue injury has been linked to an increase in the concentrations of extracellular DNA (ecDNA) in plasma. A rapid turnover of ecDNA in the circulation makes it a potential marker with high sensitivity. This study aimed to analyze the concentration of ecDNA in plasma in animal models of AKI. Three different fractions of ecDNA were measured-total ecDNA was assessed fluorometrically, while nuclear ecDNA (ncDNA) and mitochondrial DNA (mtDNA) were analyzed using quantitative real-time PCR. AKI was induced using four different murine models of AKI-bilateral ureteral obstruction (BUO), glycerol-induced AKI (GLY), ischemia-reperfusion injury (IRI) and bilateral nephrectomy (BNx). Total ecDNA was significantly higher in BUO (p < 0.05) and GLY (p < 0.05) compared to the respective control groups. ncDNA was significantly higher in BUO (p < 0.05) compared to SHAM. No significant differences in the concentrations of mtDNA were found between the groups. The plasma concentrations of different fractions of ecDNA are dependent on the mechanism of induction of AKI and warrant further investigation as potential surrogate markers of AKI.

Isolation and Quantification of Extracellular DNA from Biofluids.

Extracellular DNA is studied as a diagnostic biomarker, but also as a factor involved in the pathophysiology of several diseases due to its pro-inflammatory properties. Extracellular DNA can be extracted from plasma, urine, saliva or other biofluids using standard DNA isolation procedures and specialized commercial kits. Sample preparation for isolation is important, freezing and thawing may affect the amount of extracellular DNA extracted. Subsequent centrifugations remove cells and cell debris from the samples to obtain true extracellular DNA. Small volume of samples especially from animal experiments is often an issue and it affects the DNA yield. Very short fragments ( 100 bp) can be lost during isolation and are difficult to quantify using PCR. Fluorometric methods asses all stained DNA fragments. Selecting the method for quantification of extracellular DNA is crucial and combination of at least two methods is ideal. Standardization of procedures or at least their reporting in research papers is of utmost importance for comparison of results.

Sex, Age, and Bodyweight as Determinants of Extracellular DNA in the Plasma of Mice: A Cross-Sectional Study.

Extracellular DNA (ecDNA) is studied as a possible biomarker, but also as a trigger of the immune responses important for the pathogenesis of several diseases. Extracellular deoxyribonuclease (DNase) activity cleaves ecDNA. The aim of our study was to describe the interindividual variability of ecDNA and DNase activity in the plasma of healthy mice, and to analyze the potential determinants of the variability, including sex, age, and bodyweight. In this experiment, 58 adult CD1 mice (41 females and 31 males) of a variable age (3 to 16 months old) and bodyweight (females 25.7 to 52.1 g, males 24.6 to 49.6 g) were used. The plasma ecDNA was measured using a fluorometric method. The nuclear ecDNA and mitochondrial ecDNA were quantified using real-time PCR. The deoxyribonuclease activity was assessed using the single radial enzyme diffusion method. The coefficient of variance for plasma ecDNA was 139%, and for DNase 48%. Sex differences were not found in the plasma ecDNA (52.7 ± 73.0 ηg/mL), but in the DNase activity (74.5 ± 33.5 K.u./mL for males, and 47.0 ± 15.4 K.u./mL for females). There were no associations between plasma ecDNA and bodyweight or the age of mice. Our study shows that the variability of plasma ecDNA and DNase in adult healthy mice is very high. Sex, age, and bodyweight seem not to be major determinants of ecDNA variability in healthy mice. As ecDNA gains importance in the research of several diseases, it is of importance to understand its production and cleavage. Further studies should, thus, test other potential determinants, taking into account cleavage mechanisms other than DNase.