Abstract
Dietary DNA is degraded into shorter DNA-fragments and single nucleosides in the gastrointestinal tract. Dietary DNA is mainly taken up as single nucleosides and bases, but even dietary DNA-fragments of up to a few hundred bp are able to cross the intestinal barrier and enter the blood stream. The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown. Here we investigate the transport of DNA-fragments across the intestinal barrier, focusing on transport mechanisms and rates. The human intestinal epithelial cell line CaCo-2 was used as a model. As DNA material a PCR-fragment of 633 bp was used and quantitative real time PCR was used as detection method. DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB). After 90 min the difference in directionality AB vs. BA was >103 fold. Even undegraded DNA-fragments of 633 bp could be detected in the basolateral receiver compartment at this time point. Transport of DNA-fragments was sensitive to low temperature and inhibition of endosomal acidification. DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2. Our findings therefore suggest that vesicular transport is mediating transcytosis of dietary DNA-fragments across intestinal cells and that DNA binding proteins are involved in this process. If we extrapolate our findings to in vivo conditions it could be hypothesized that this transport mechanism has a function in the immune system.
Highlights
Most of the dietary DNA that is consumed by an organism is degraded to nucleosides and bases before being absorbed in the gastrointestinal tract, we and others have shown that up to 1% of dietary DNAs are able to pass the intestinal barrier as longer fragments and enter the blood stream of humans [1], chicken [2], pig [3], mice [4] and fish [5,6] These fragments are thereafter transported to different organs before they eventually become degraded
We present evidence that dietary DNA-fragments are actively transported across the intestine by DNA binding proteins using vesicular mediated transport
We demonstrate that DNA binding proteins not capable of binding short single stranded ODNs with a phosphothioate backbone, fucoidan, heparin, heparan sulphate and dextran sulphate are involved in the observed transcytosis of DNA-fragments
Summary
Most of the dietary DNA that is consumed by an organism is degraded to nucleosides and bases before being absorbed in the gastrointestinal tract, we and others have shown that up to 1% of dietary DNAs are able to pass the intestinal barrier as longer fragments and enter the blood stream of humans [1], chicken [2], pig [3], mice [4] and fish [5,6] These fragments are thereafter transported to different organs before they eventually become degraded. The observed transport of DNA across the intestinal barrier has been suggested to have an immunomodulatory role [7]. In the human intestinal cell line CaCo-2 transcellular transport of single stranded short oligodeoxynucleotides (ODNs) has been suggested [8]. Toll like receptor 9 (TLR9) [10] is a pattern recognition receptor (PRR) recognizing different pathogens by identifying molecular patterns (PAMPs) found on bacteria
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