Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Nov 28, 2017; 23(44): 7830-7839
Published online Nov 28, 2017. doi: 10.3748/wjg.v23.i44.7830
Composition and immuno-stimulatory properties of extracellular DNA from mouse gut flora
Ce Qi, Ya Li, Ren-Qiang Yu, Sheng-Li Zhou, Xing-Guo Wang, Guo-Wei Le, Qing-Zhe Jin, Hang Xiao, Jin Sun
Ce Qi, Jin Sun, The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu Province, China
Ce Qi, Ya Li, Xing-Guo Wang, Guo-wei Le, Jin Sun, School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu Province, China
Ren-Qiang Yu, Wuxi Maternal and Child Health Hospital, Wuxi 212422, Jiangsu Province, China
Sheng-Li Zhou, Quality of Research and Development Department, COFCO Fortune Food Sales & Distribution Co., Ltd. Tianjin 300452, China
Hang Xiao, Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States
Author contributions: Sun J and Li Y designed the research; Qi C, Li Y, Wang XG, Le GW, and Zhou SL performed the research; Sun J, Li Y, Yu RQ, and Xiao H analyzed the data and wrote the article; Xiao H and Yu RQ revised the paper; all authors have read and approved the final version to be published.
Supported by China Postdoctoral Science Foundation, No. 172774; Fund of Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, No. KLCCB-KF201603; and National Natural Science Foundation of China, No. 31201805.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Jiangnan University (IACUC protocol number: No. 8/2014/JU).
Conflict-of-interest statement: No potential conflicts of interest.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Jin Sun, PhD, Associate Professor, School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu Province, China. sunj@jiangnan.edu.cn
Telephone: +86-510-85917780
Received: September 22, 2017
Peer-review started: September 23, 2017
First decision: October 11, 2017
Revised: October 14, 2017
Accepted: October 27, 2017
Article in press: October 27, 2017
Published online: November 28, 2017
Processing time: 65 Days and 12.8 Hours
Abstract
AIM

To demonstrate that specific bacteria might release bacterial extracellular DNA (eDNA) 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 eDNA in the mucus layers of the small intestine and colon in healthy Male C57BL/6 mice. Composition difference of eDNA and intracellular DNA (iDNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism (T-RFLP). Stimulation of cytokine production by eDNA was studied in RAW264.7 cells in vitro.

RESULTS

TOTO-1 iodide staining confirmed existence of eDNA 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 eDNA in the small intestinal mucus was significantly different from that of the iDNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the eDNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Gram-positive bacteria. Both eDNA and iDNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The eDNA induced significantly lower tumor necrosis factor-α/interleukin-10 (IL-10) and IL-6/IL-10 ratios than iDNA, 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.

Keywords: Bacterial extracellular DNA; Flora; Immune-stimulatory property; Gut microbiota; Mouse; Small intestine

Core tip: Our results revealed 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 genomic DNA of total gut flora. Our study highlights that bacteria derived DNA plays an important role in modulating local immune response in mouse gut.