DNA-Fragments Are Transcytosed across CaCo-2 Cells by Adsorptive Endocytosis and Vesicular Mediated Transport

<div><p>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 <em>in vivo</em> conditions it could be hypothesized that this transport mechanism has a function in the immune system.</p> </div

Effect of inhibition of endocytosis on transcytosis of DNA-fragment (5 nM).

<p>Transcytosis of DNA fragments and Lucifer yellow (LY) in the apical to basolateral direction were quantified as described in Materials and Methods and tested statistically with a linear mixed-effects model. Inhibition factor is calculated as control divided by treated. The number <i>n</i> is the number of independent experiments and the number <i>m</i> is the total number of wells analysed (observations). Control for ice-treatment is 37 °C and control for bafilomycin A1 (BafA1)-treatment is with vehicle (DMSO). TEER = trans-epithelial electric resistance.</p

Trans-epithelial electric resistance (TEER) in the presence of compounds increasing Lucifer yellow (LY)-transport.

<p>TEER was measured at the end of the experiment. Deviation (dev) is calculated as the absolute value of the difference between the two experiments divided by 2. The number <i>n</i> is the number of independent experiments and number <i>m</i> is the total number of wells analysed (observations). CpG ODN = short oligonucleotide rich in CG dinucleotide motifs. Control for Cytochalasin D (CytD)-treatment is with vehicle (DMSO).</p

Competition for transcytosis of DNA-fragment (5 nM) by nucleic acids and anionic compounds.

<p>Transcytosis of DNA fragments in the apical to basolateral direction with (treated) and without (control) competitor were quantified after 90 minutes of incubation as described in Materials and Methods and tested statistically with a linear mixed-effects model. Factor is calculated as control divided by treated. The number <i>n</i> is the number of independent experiments and the number <i>m</i> is the total number of wells analysed (observations). pUC19 lin = linearized pUC19 plasmid. CpG and GpC ODN = short oligonucleotides rich in CpG and GpC dinucleotide motifs, respectively.</p

Transcytosis of Lucifer yellow (LY) in the presence of compounds affecting transcytosis of DNA-fragment.

<p>Transcytosis of LY in the apical to basolateral direction with (treated) and without (control) competitor was quantified after 90 minutes of incubation as described in Materials and Methods and tested statistically with a linear mixed-effects model. Factor is calculated as control divided by treated. The number <i>n</i> is the number of independent experiments and the number <i>m</i> is the total number of wells analysed (observations). pUC19 lin = linearized pUC19 plasmid. CpG ODN = short oligonucleotide rich in CG dinucleotide motifs. Control for Cytochalasin D (CytD)-treatment is with vehicle (DMSO).</p

Differentiation of CaCo-2 cells.

<p>A: Trans-epithelial electric resistance (TEER) was measured on CaCo-2 cells on filters during their differentiation. Measurements were performed before change of medium. TEER (Ω x cm²) was plotted against time. One representative experiment is shown with mean +/−SD from nine wells. B: Intestinal alkaline phosphatase (IAP) expression at mRNA level detected by reverse transcription followed by PCR in CaCo-2 cells, CaCo-2/HT29-MTX Mix (3∶1) and HT29-MTX cells. Control is HeLa total mRNA from the Superscript III cellsdirect cDNA synthesis kit (Invitrogen). One representative experiment out of two is shown.</p

Expression and subcellular localization of the S1-encoded σ3 and p13 proteins in mammalian VERO and salmonid CHSE cells.

<p>Immunofluorescent staining of σ3 or p13 (green colour), and staining with the trans-Golgi marker WGA (red colour). Transfected VERO cells (A) and CHSE cells (B) expressing both σ3 and p13 from the large S1 ORF (upper panels), and p13 expression from the S1 internal ORF (lower panels). Nuclei are stained with DAPI (blue colour). Yellow colour indicates colocalization of p13 and WGA. Non-transfected cells stained with WGA and anti-p13 serum was used as controls (CTRL).</p

The PRV genome.

<p>Gene segments are assigned according to mammalian reoviruses. Open reading frames (ORFs) and putative encoded proteins are indicated by regions in grey, with start and end positions indicated. Non-translated regions (UTR’s) at gene segment ends are shown in black. Gene segments L2, S1 and S2 are possibly polycistronic.</p

Multiple sequence alignment of PRV σ1 with MRV T3D σ1.

<p>Black lines represent putative nuclear export signals (NEP) in MRV and PRV, respectively, as predicted by NetNes 1.1. ▪ = L₁₄₉ in the MRV protein involved in a second predicted NES. ▴ = residues in the MRV protein involved in binding to sialic acid residues. The alignment has been manually adjusted.</p

Percentage amino acid identity among all ungapped positions between pairs; predicted PRV proteins and the homologues proteins from three reovirus prototype strains.

a,b,c<p>Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070075#pone-0070075-t001" target="_blank">Table 1</a> for gene segment annotations and names of homologues proteins in MRV, ARV and GCRV. Identity values are from separate pairwise alignments of the protein sequences.</p>d<p>Value from a manually adjusted pairwise alignment of the two proteins.</p>f<p>GCRV does not appear to have a cell attachment protein homologue to σ1/σC.</p