Composition and immuno-stimulatory properties of extracellular DNA from mouse gut flora

AIM To demonstrate that specific bacteria might release bacterial extracellular DNA(e DNA) to exert immunomodulatory functions in the mouse small intestine.METHODS Extracellular DNA was extracted using phosphate buffered saline with 0.5 mmol/L dithiothreitol combined with two phenol extractions. TOTO-1 iodide, a cell-impermeant and high-affinity nucleic acid stain, was used to confirm the existence of e DNA in the mucus layers of the small intestineand colon in healthy Male C57 BL/6 mice. Composition difference of e DNA and intracellular DNA(i DNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism(T-RFLP). Stimulation of cytokine production by e DNA was studied in RAW264.7 cells in vitro.RESULTS TOTO-1 iodide staining confirmed existence of e DNA in loose mucus layer of the mouse colon and thin surface mucus layer of the small intestine. Illumina sequencing analysis and T-RFLP revealed that the composition of the e DNA in the small intestinal mucus was significantly different from that of the i DNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the e DNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Grampositive bacteria. Both e DNA and i DNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The e DNA induced significantly lower tumor necrosis factor-α/interleukin-10(IL-10) and IL-6/IL-10 ratios than i DNA, suggesting the predominance for maintaining immune homeostasis of the gut.CONCLUSION Our results indicated that degraded bacterial genomic DNA was mainly released by Gram-negative bacteria, especially Bacteroidales-S24-7 and Stenotrophomonas genus in gut mucus of mice. They decreased pro-inflammatory activity compared to total gut flora genomic DNA.