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DNA as a molecule seems relatively familiar, in fact we still have a lot to learn about it. Also thanks to scientists from the Institute of Molecular Biomedicine (IMBM), Faculty of Medicine of the Comenius University, we are learning more and more about this molecule. In this article, we will introduce the concept of extracellular DNA and what role it plays in inflammatory diseases - Crohn's disease and ulcerative colitis, also known as IBD (Inflammatory Bowel Diseases).  

 

 

DNA outside the cell? But we didn't learn this...

Extracellular DNA (ecDNA) is a component of DNA that is found outside the cell and therefore in plasma, saliva, breast milk, urine and semen. These are fragments of genomic (chromosomal) and mitochondrial DNA that normally occur in the organism. 

 

When does the concentration of ecDNA increase?

An increase in the concentration of ecDNA mainly occurs when organs are damaged, such as myocardial infarction, burns or stroke, or damage caused by an accident. 

 

White blood cells and their traps

Neutrophils make up the majority of white blood cells, yet we are only now discovering their previously hidden abilities. Under specific conditions, inflammatory activation of neutrophils results in a specific type of cell death - NETosis (Neutrophil Extracellular Trap). NETs contain DNA and are therefore a form of ecDNA.

 

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Since released NETs are formed primarily by histones and chromatin, their elimination is possible through the use of the enzyme DNase I, which is capable of cleaving dinucleotide chains. 

 

Inflammatory bowel diseases and ecDNA

Crohn's disease and ulcerative colitis, both diseases are characterized by the presence of an inflammatory process in the area of the gastrointestinal tract. But these are diseases that arise on the basis of the interaction between the intestinal microbiome and the immune system, genetic predisposition, lifestyle and environmental factors. So far, only limited evidence is available for the role of ecDNA in IBD. However, there are data suggesting reduced DNase activity in patients suffering from this type of disease. Therefore, Dr. Bábíčková and the team decided to investigate the relationship between ecDNA and IBD. 

 

Experimental model and methodology

For the purpose of this research, the research team used an animal model, namely mice. Before starting the experiment, it was necessary to obtain permission from the ethics committee (EK), which also evaluates the benefit and cost of such an experiment. In addition, the entire experiment had to be carried out in accordance withthe Declaration of Helsinkidescribing the ethical principles of medical research. After EC approval, the researchers randomly allocated the mice into four groups, with some receiving only saline (placebo); others received dextran sodium sulfate (DSS), which induced acute colitis. The two remaining groups received either a combination of DSS and DNase I or saline and DNase I. The dose of DNase was 7 mg/kg and the animals received it twice (third and seventh day) during the duration of the experiment. 

 

Evaluation of the experiment

In mice, parameters such as body weight, stool consistency, rectal bleeding and fluid intake were monitored on a daily basis. On the sixth and final day (killing), mice were bled for biochemical analysis. After decapitation, other parameters such as the weight and size of some organs were also determined. The data obtained in the research were later subjected to statistical evaluation. 

 

What were the conclusions? 

 Dextra sodium sulfate used to induce colitis caused a statistically significant decrease in weight and intestinal shortening  in mice compared to saline. Administration of DNase I, however, did not cause statistically significant differences between the results of the treated and control groups. 

ecDNA concentrations across experimental groups were not statistically significantly different.

 Biochemical analysis demonstrated that the DSS group had a statistically significantly higher concentration of TNF-α when compared to the control group that received saline. TNF-α is a cytokine, a signaling molecule used by the immune system, which is increased by inflammation. The group of mice treated with DNase I had a slightly reduced level of this marker when compared to the control DSS group (they did not receive DNase). However, this difference between the groups did not reach statistical significance, so it is not clear to what extent this decrease was really caused by DNase.

 

The results are clear, but what do they mean?

The results did not support the hypothesis of a beneficial benefit of DNase I in the treatment of IBD. Similarly, they did not prove that its concentration would increase in colitis induced by sodium dextra sulfate. Every result - whether positive or negative - is a result that is accompanied by some kind of discovery and a topic for further continuation of scientific work. In this case, it is worth mentioning the aforementioned TNF-α and its reduced level after DNase administration. This particular observation could be worth verifying. 

 

Every research has limits

Like other research, the research of Dr. Bábíčková and her team has certain limits. These limits indicate the extent to which the results can be generalized. Limiting factors in this case can be, for example, the dose of DNase, the time intervals between the doses themselves and the tests, but also the method of DNase administration. Since intravenous administration has a quick effect, the effect may not be long-lasting. Another factor may be the rapid degradation of DNase after administration, so it is sometimes advisable to try other ways of administering the substance. 

Being aware of the limits of your own study can help identify additional areas of research and consider previously unforeseeable factors. 

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Study authors: J. Bábíčková, J. Čonka , L. Janovičová, M. Boris, B. Final, R. Gardlík

Link to full research from IMBM

Source of the illustration

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