Organ specific, plasma CPG DNA motifs are TLR9 dependent immune/regenerative response activators and stem cell mobilisers

Background: Circulating, plasma, free-DNA (cpfDNA) is increased in IBD and neoplastic colorectal alterations (CRC). Non-eukaryotic, not-methylated CpG DNA motifs are recognised by human Toll-like receptors on immune cells. Aims: Our aims were to prove the phyisiological role of cpfDNA as endogenous ligand of TLR9 and to analyze its activator and mobiliser effects on immune and regenerative cells. Materials and methods: Using 5% dextrane-sodium-sulfate (DSS), colitis was induced in mice. CpfDNA was isolated from 1ml PBL. The isolated DNA was injected into the tail vein of mices. Total RNA from PBL was isolated 1.4 and 24 hours after the DNA injection. Quantitative RT-PCRs for TLR9, TNF-alpha, CD133, Myd88, IRF3, TRAF6, NFKB, IL-6 were performed. 14 days after the injection of isolated cpfDNA, the colon, liver, skin, kidney of mice were removed and analysed. The regeneration of the colitis was evaluated by quantitative microsopic analysis and the number of infiltrating CD133+ cells was determined. CpfDNA was isolated from peripheral blood (PBL) of 20 healthy, adenoma, colorectal cancer (CRC) and active IBD patiens. Whole genomic sequencing was performed for determination of disease specific fragments. PCR amplified, SEPTIN 9 fragments were intravenously injected into mice. PBL qRT-PCRs were performed for the above gene set. Results: Heterologous cpfDNA induced and enhanced the healing. CpfDNA from DDS-colitis resulted in significant crypt density elevation (5.5±0.5/6.8±0.4 pieces/100µm in non-treated vs. treated healthy colon; 1.7±0.2/2.6±0.6 pieces/cm in non-treated vs. treated DSS-colitis, p<0.05). It mobilised CD133+ cells into the colon and not into other organs. In colonic tissue significantly (p<0.01) increased number of intraepithelial CD133+cells was found in the DNA treated healthy mice (9.86±0.84%) compared to the control (7.36±1.1%) animals. CpfDNA fragments were different in healthy, adenoma, CRC and IBD cases. Methylated S9 was found to be a CRC specific marker. Depending on the methylation status of the synthesized S9 fragments, regenerative or immune dominant PBL mobilisation and response could be observed.

Conclusions: CpfDNA is endogenous ligand of TLR9+ stem and immune cells and contains organ damage specific DNA motifs. Methylation status of the determined fragments influences immune/regenerative cell response.