Systemic Inflammation Induces Release of Cell-Free DNA from Hematopoietic and Parenchymal Cells in Mice and Human
Authors: Kroeze Anna , van der Meer Anne Jan , Hoogendijk Arie J , Ait Soussan Aicha , van der Schoot C. Ellen , Wuillemin Walter A , Voermans Carlijn , van der Poll Tom , Zeerleder Sacha S 
Published: October 2019

Abstract
Cell-free DNA (cfDNA) and DNA binding proteins are released in the circulation upon systemic inflammation, the extent of which is proportional to the severity of the systemic inflammation. There is an ongoing debate on the cellular source of cfDNA during systemic inflammatory conditions. During infection, circulating histones and DNA in the form of nucleosomes have been argued to be released by activated neutrophils in the process of NETosis. However, cfDNA has also been attributed to the cell death of other cell types, such as lymphocytes and endothelial cells. The objective of the current study is to investigate the compartmental source of extracellular DNA released into the circulation during a systemic inflammatory response.
We determined the plasma levels of nucleosomes as a measure of cfDNA and DNA binding proteins, and of elastase-α1-antitrypsin (EA) complexes as a measure of neutrophil activation in different systemic inflammatory responses. First, we induced a low-grade endotoxemia in eight healthy human subjects by injection of lipopolysaccharide (LPS). In these subjects, nucleosome levels increased to a maximum at 3 hours after LPS injection (mean 529 ± SE 112 AU/ml), after which they returned towards baseline levels. Interestingly, the release of EA showed similar kinetics as that of nucleosomes, reaching a maximal level of 499 ± 58 ng/ml at 3 hours. Furthermore, plasma EA levels positively correlated with nucleosome levels during LPS challenge (r=0,79; p<0.0001), suggesting that during a mild, transient inflammatory response, neutrophils are the predominant source of nucleosomes within the plasma. We next measured nucleosomes and neutrophil activation in twenty patients with severe systemic inflammation caused by sepsis. Surprisingly, sequential measurements in sepsis patients did not show a correlation between plasma EA levels and nucleosome levels (r=0,155; p=0,13), suggestive of a non-neutrophil source of cfDNA. Thus, there might be other potential cellular sources of cfDNA than neutrophils in a severe systemic inflammation.
To further investigate the difference of cfDNA release between a mild and a severe systemic inflammation, we used chimeric mice transplanted with bone marrow of the opposite sex. After hematopoietic recovery, mice were challenged with LPS. We were able to follow up on the release of cfDNA from different compartmental sources, that is either the hematopoietic or the non-hematopoietic compartment, by using a specific mouse SRY qPCR as a measure for male DNA, and IL3 qPCR as a measure for total DNA. CfDNA from the hematopoietic compartment was present within the plasma of the mice at 6 and 20 hours after LPS challenge, whereas cfDNA from a parenchymal source could be detected at 20 hours but less at 6 hours after LPS challenge. This suggests that the hematopoietic compartment acts as a source of cfDNA both early and late after LPS challenge, whereas the parenchymal compartment only releases DNA at later stages of severe systemic inflammation.
Collectively, our results show that in severe systemic inflammation, circulating cfDNA originates from both hematopoietic cells (e.g. neutrophils and lymphocytes) and parenchymal cells (e.g. endothelial cells). Understanding the kinetics of extracellular DNA may improve knowledge on duration and severity of sepsis and may provide a point of interference during systemic inflammation.

Disclosures
No relevant conflicts of interest to declare.