Circulating, plasma mucleic acids are endogenous activators of stem and immune cells through toll-like receptors

• Published in: Zeitschrift für Gastroenterologie
• Main Authors: Molnár, B, Spisák, S, Valcz, G, Sipos, F, Galamb, O, Tulassay, Z
• Format: Conference Proceeding
• Language: English; German
• Published: 15.05.2012
• ISSN: 0044-2771; 1439-7803
• DOI: 10.1055/s-0032-1312406

Background: The amount of circulating plasma free DNA (cpfDNA) is increased in IBD and neoplastic colorectal alterations (CRC). Their physiological role is not cleared, yet. Non-eukaryotic, non-methylated CpG DNA motifs are recognized by human Toll-like receptors on immune cells and stem cells. Aims: To prove the physiological role of cpfDNA as endogenous ligand of TLR9 and to analyze its activator and mobiliser effects on immune and regenerative cells. Materials and methods: 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. Using 5% dextrane sodium sulfate (DSS), colitis was induced in mice. CpfDNA was isolated. Isolated DNA was injected into tail vein of mice with DSS-induced colitis and controls. Total RNA from PBL was isolated 1, 4 and 24 hours after the DNA injection. PCR amplified, artificially methylated and non-methylated S9 fragments were also intravenously injected into mice. 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. Results: CpfDNA fragments were different in healthy, adenoma, CRC and IBD cases. Methylated S9 was found to be a CRC specific marker. 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. 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.