Helicobacter pylori DNA's anti-inflammatory effect on experimental colitis

Abstract

Our laboratory has demonstrated a clinical inverse association between H. pylori infection and inflammatory bowel disease (IBD). In our most recent work we described a possible mechanism by which H. pylori can reduce the risk of developing IBD. Specifically, we were able to demonstrate the immuno-regulatory properties of the H. pylori genome and its ability to downregulate inflammatory responses through interaction with mucosal dendritic cells both in an in vitro and in vivo model. Furthermore, we were able to demonstrate the ability of H. pylori DNA to downregulate dendritic cell production of IL-12 and type I interferon, two pro-inflammatory cytokines. In the present work, we conducted further studies to examine the unique properties of the H. pylori genome and the exact mechanism through which it interacts with dendritic cells. Our data highlight a specific immuno-regulatory sequence (IRS), TTTAGGG, which occurs significantly more frequently as compared with other IRS sequences and is unique to the H. pylori genome. Additionally, we illustrate that H. pylori DNA has no effect on modulating the TLR-4 dependent LPS-induction of dendritic cell IL-12 production. This indicates that the inhibitory effect of H. pylori genomic DNA is restricted to the TLR-9 signaling pathway that senses bacterial DNA. In conclusion, the findings of this addendum strengthen the evidence for unique immunoregulatory properties of the H. pylori genome and revealed the importance of TLR-9 mediated mechanism in the pathogenesis of IBD.

Figure 1. A proposed mechanism of H. pylori-induced protection against intestinal inflammation. H. pylori colonizes the stomach and releases H. pylori DNA into the intestinal lumen. Intestinal lamina propria dendritic cells that sample luminal antigen may be influenced by the H. pylori immune-regulatory DNA sequences (IRS) by decreasing their type I IFN production. This results in a decreased severity of gut inflammation.

Figure 2. The occurrence of known G-Rich octomers within various H. pylori genome. H. pylori genome sequences were obtained from NCBI gene bank. The number of times each sequence appeared in each genome was counted using MacVector. This number was then divided by the total size of the genome (in base pairs) to obtain the actual frequency. The actual frequency was then divided by the expected frequency (1/4⁸) to normalize the genome size, resulting in the occurrence, which is shown in the figure. The expected frequency is the frequency that a particular octomer sequence will occur randomly by chance.

Figure 3. H. pylori does not inhibit TLR-4 ligand stimulated dendritic cell IL-12 production. Bone marrow-derived dendritic cells (BMDCs, 1x10⁶ cells/mL) from C57BL/6 mice were stimulated for 18 h with (A) bacterial DNA (E. coli K27, H. pylori 26695, or J99), or (B) TLR-4 ligand LPS with or without increasing doses of bacterial DNA (E. coli K27, H. pylori 26695, or J99). IL-12 production by dendritic cells was measured by ELISA. Bacterial genomic DNA does not modulate TLR-4 mediated inflammatory signaling pathways. Data shown are obtained from three independent experiments. Isolation of genomic bacterial DNA was performed using the Wizard genomic DNA purification kit (Promega, Madison, Wisconsin, USA). Endotoxin contamination was removed from the DNA preparation using the MiraCLEAN endotoxin removal kit (Mirus, Madison, Wisconsin, USA). All DNA was suspended in endotoxin-free TE buffer (Qiagen, Valencia, CA, USA).