SCCM Resource Library-Nucleosomes and Nuclear DNA, but Not Mitochondrial DNA, Are Associated With Pediatric ARDS Mortality

Video Transcription

Hi everybody, today we're going to be talking about nucleosomes and nuclear DNA but not mitochondrial DNA being associated with pediatric ARDS mortality.  My name is Dr. Yeya, these are my disclosures. So cell-free DNA is a known damp in critical illness.  Damage-associated molecular patterns or DAMPs are endogenous moieties which are released from dead and dying cells.  They're recognized by pattern recognition receptors like toll-like receptors or TLRs and they aggravate inflammation.  Cell-free DNA has been recognized primarily as mitochondrial DNA, which is recognized by TLR9 due to its hypomethylated CPG which mimics bacterial DNA.  Nuclear DNA, which is also a form of cell-free DNA, that is to say DNA which came from the  nucleus, is not really recognized as a DAMP primarily because it doesn't have an identified receptor.  So some differences between mitochondrial and nuclear DNA as they're circulating in the body. Mitochondrial DNA is prognostic in sepsis and ARDS.  It's a known contributor to innate immune activation as well as to shock and organ failure. Nuclear DNA is inconsistently prognostic in critical illness syndromes.  And when it is prognostic, it's not clear whether it's prognostic because it correlates with tissue damage and immune cell turnover or whether it itself is pathogenic.  There's no actual known mechanism for direct damage by nuclear DNA. Nuclear DNA can be released as either free nuclear DNA or complex with nucleosomes.  And nucleosomes are DNA and histone complexes. Histones are in fact pathogenic and known to be DAMPs as well.  One final property of nuclear DNA, which is interesting and which we're going to exploit later on in this talk, is that it retains the methylation pattern of its tissue of origin,  suggesting that you could actually use that methylation pattern to potentially identify where that nuclear DNA that's circulating in the blood came from.  Cell-free DNA is not characterized in pediatric ARDS. We don't know if it's there. We don't know if it's relevant.  So the knowledge gap that we're trying to address with our study is the relevance of mitochondrial DNA and the relevance of nuclear DNA.  Therefore, we aim to determine the prognostic utility of cell-free DNA in pediatric ARDS. We measured mitochondrial DNA, nuclear DNA, and nucleosomes.  Finally, we want to assess the feasibility of deconvolution of the nuclear DNA to identify its tissue of origin by exploiting its retained methylation pattern.  This was a prospective cohort study carried out in the CHOP pediatric ICU between 2014 and 2019. We had children with intubated Berlin-defined ARDS.  On day zero, we had 279 subjects with plasma samples. By day seven, we had 207 subjects.  The drop-off in that time frame reflects approximately equal numbers of patients who improved rapidly before day seven, as well as those who died before day seven.  The outcomes we were looking at were ICU mortality and the correlation of cell-free DNA with Berlin ARDS categories, as well as with non-pulmonary organ failure.  For mitochondrial DNA, we performed PCRs of COX-1 and ND-1. For nuclear DNA, we performed PCR of COX-4. And for nucleosomes, we performed a commercial ELISA.  We performed univariate as well as multivariable analysis, and multivariable analysis required us to log transform as well as adjust for known confounders.  The deconvolution analysis for the nuclear DNA of origin was piloted in eight subjects. Both mitochondrial DNA moieties were highly correlated with each other, with COX-1 and  ND-1 having a correlation coefficient of 0.67. Nucleosomes and COX-4, the nuclear DNA, were also highly correlated with a correlation coefficient of 0.65.  Nucleosomes but neither cell-free DNA correlated with ARDS severity. With increasing levels of nucleosomes across increasing levels of ARDS severity.  Nucleosomes in nuclear DNA but not mitochondrial DNA correlated with non-pulmonary organ failures.  Finally, nucleosomes in nuclear DNA but not mitochondrial DNA were higher and remained higher over the first seven days of ARDS in non-survivors.  In our deconvolution analysis, and keeping in mind this was only eight patients, if you add up all of the immune cell contributions that we were able to assign, then approximately  half of the circulating nuclear DNA came from an immune cell origin. 16% came from endothelial cell, there was a minority 6% which came from lung epithelia,  and finally there were 19% of methylations that could not be assigned a cell type. These are the eight patients and their clinical characteristics, I don't expect you guys to  read all this, but there's a span of ages, there's a span of comorbidities, there's a fair amount of heterogeneity even in these eight subjects.  I do want to focus on the last subject in the bottom right hand corner, and this subject had ARDS as a result of CAR-T therapy, and what I want to do with this patient is that  44% of this patient's circulating nuclear DNA was identified mapped back to B cells, and this was nice because this patient's CAR-T therapy was actually directed against B cells,  and so this actually served as a nice unintentional positive control which gave us some confidence that what we're looking at wasn't completely made up.  Overall we conclude that nucleosomes in nuclear DNA but not mitochondrial DNA are associated  with ARDS severity, with organ failures, and with mortality, and the origin of the nuclear DNA, half of it appeared to be from immune cell turnover, and there was three-fold greater  endothelial rather than epithelial contribution. The implications of this are that nuclear DNA is primarily prognostic in pediatric ARDS  due to immune cell turnover and organ failures rather than being directly pathogenic. Mitochondrial DNA was high but was not associated with either organ failures or with outcomes.  Finally we conclude that endotheliopathy appears to be a significant contributor to nuclear DNA in pediatric ARDS.  The limitations of our study were that it was single center, it's a small sample size for deconvolution, this was really just a proof of concept at this stage, although we  think it's pretty cool proof of concept, and we haven't yet done any sort of longitudinal deconvolution.  So we have samples on day 0, 3, and 7, and we haven't done the day 3 and 7, or even more than the first 8 on day 0, frankly.  And so there's a lot of work left to be done, and it would be interesting to see whether or not we would see some sort of change in the source of the nuclear DNA over time, which  we're very interested in.

Video Summary

In this video, Dr. Yeya discusses the role of nucleosomes and nuclear DNA in pediatric ARDS (acute respiratory distress syndrome) mortality. He explains that cell-free DNA, specifically mitochondrial DNA, is known to contribute to inflammation in critical illness. However, the role of nuclear DNA as a DAMP (damage-associated molecular pattern) is unclear, as it lacks a recognized receptor. The study aims to determine the prognostic value of cell-free DNA in pediatric ARDS and explore the potential identification of the tissue of origin through its methylation pattern. The results show that nucleosomes and nuclear DNA, but not mitochondrial DNA, are associated with ARDS severity, organ failures, and mortality. Furthermore, the origin of nuclear DNA is primarily immune cell turnover, with endothelial cells playing a significant role. However, the study has limitations, including a small sample size and the need for longitudinal analysis.

Asset Subtitle

Pulmonary, Quality and Patient Safety, Pediatrics, 2022

Asset Caption

INTRODUCTION: Plasma cell-free DNA (cfDNA) has been proposed as a prognostic, and potentially causative, biomarker in acute respiratory distress syndrome (ARDS). However, this has not been investigated in children. We therefore determined the kinetics of plasma mitochondrial (mtDNA) and nuclear (nDNA) over the first 7 days of pediatric ARDS and assessed the association with pediatric intensive care unit (PICU) mortality. Given that nDNA can circulate alone or complexed with histones as nucleosomes, we determined the kinetics and prognostic utility of nucleosomes. METHODS: This was a prospective cohort study of intubated children with ARDS (Berlin), with blood collection on day 0, 3, and 7 after ARDS onset. We measured mtDNA, nDNA, and nucleosomes using RT-PCRs and ELISA. Day 0 biomarkers were correlated with ARDS severity and number of organ failures, and mortality discrimination assessed by computing areas under the receiver operating characteristic (AUROC) curve. Log-transformed biomarkers were tested for association with mortality using mixed models adjusting for age, ARDS etiology, immunocompromised status, and ARDS severity. We tested for overall differences in biomarker levels between survivors and non-survivors over the first 7 days, as well as differences in trajectories between survivors and non-survivors over the first 7 days. RESULTS: There were 279 ARDS subjects (64 [23%] non-survivors). Nucleosomes and nDNA levels were correlated (pearson r = 0.65). Day 0 nucleosomes, but not nDNA or mtDNA, were associated with ARDS severity (trend p = 0.022). Higher levels of day 0 nucleosomes and nDNA, but not mtDNA, were associated with more organ failures (both p < 0.001) and modestly discriminated mortality (both AUROC 0.76). Nucleosomes and nDNA, but not mtDNA, were persistently elevated in non-survivors over the first 7 days of ARDS (both p < 0.001), without differences in trajectory. Levels of all cfDNAs, but not nucleosomes, increased over the first 7 days. CONCLUSIONS: Nucleosomes and nDNA, but not mtDNA, were associated with mortality in pediatric ARDS. These findings differ from the established utility of mtDNA in adult ARDS, highlighting the importance of pediatric-specific investigations. Whether nucleosomes or nDNA themselves induce damage or are markers of organ failure requires further investigation.

Meta Tag

Content Type Presentation

Knowledge Area Pulmonary

Knowledge Area Quality and Patient Safety

Knowledge Area Pediatrics

Knowledge Level Advanced

Membership Level Select

Tag Acute Respiratory Distress Syndrome ARDS

Tag Mortality

Tag Pediatrics

Year 2022

Keywords

nucleosomes

nuclear DNA

pediatric ARDS

cell-free DNA

mitochondrial DNA

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