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.

Introduction

Our laboratory has previously demonstrated an inverse association clinically between H. pylori infection and IBD. In a meta-analysis of 23 studies which examined the incidence of H. pylori infection in patients with and without IBD, we showed that 27.1% of IBD patients had evidence of H. pylori infection compared with 40.9% patients in the control group.¹ These data suggested that there are protective benefits of H. pylori infection against the development of IBD; however, a mechanism for this effect had yet to be described.

In our article recently published in Gut, we attempted to address a mechanism to explain the observed protective effect of H. pylori against IBD.² We turned our attention toward the HP DNA, given documentation of its presence in the stool and colon of infected patients.^3,4 Therefore, the DNA could offer a physical connection between a bacterium that strictly colonizes the stomach with an inflammatory condition that more commonly affects the small bowel and colon. Based on previous work which demonstrated the existence of certain nucleotide sequences that could either downregulate (immuno-regulatory sequences, IRS) or stimulate (immuno-stimulatory sequences, ISS) the immune system,⁵ we highlighted the uniquely high ratio of IRS:ISS within the genome of H. pylori as compared to other gram-negative bacteria.

Dendritic cells (DCs) are professional antigen-presenting cells that have the unique ability to traverse epithelial tight junctions in the intestine and interact with bacterial DNA through a TLR-9-dependent mechanism.^6,7 Further, it has been previously demonstrated that DCs play a critical role in the induction of colitis.⁸ Therefore, we next examined in vitro the interaction between H. pylori DNA and bone marrow-derived dendtritic cells (BMDCs) and plasmacyotid dendritic cells (pDCs). In these experiments we revealed the inability of H. pylori DNA to stimulatemouse BMDC or human plasmacytoid DC production of type I IFN or IL-12, both well-described inflammatory cytokines. Further, H. pylori DNA was also able to suppress E. coli DNA-induced production of type I IFN and IL-12, suggesting H. pylori DNA has the ability to inhibit pro-inflammatory responses.

We then conducted in vivo experiments in which the administration of H. pylori DNA before the induction of dextran sulfate sodium colitis significantly ameliorated the severity of colitis (both clinically and histologically) compared with E. coli DNA or vehicle control in both an acute and chronic model of colitis. These data supports our previous model in which administration of the whole bacterium can protect against Salmonella typhi-induced experimental colitis.⁹

Last, based on our in vitro data that suggested an alteration in type I interferon production, we examined the systemic levels of type I interferon in patients with and without H. pylori infection. We were able to demonstrate H. pylori-infected patients had significantly lower systemic levels of type I interferon as compared with patients without H. pylori infection. Based on our findings, we concluded H. pylori DNA is shed into the distal intestinal system in infected individuals and reduce inflammation through its interaction with dendritic cells in the intestinal mucosa. This depressed inflammatory response may lower one’s risk of developing chronic inflammation in the intestine (Fig. 1).

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.

The occurrence of known G-Rich octomers within various H. pylori genome

In our original work, we highlighted the elevated IRS to ISS ratios across various H. pylori strains, close to 30-fold in most instances.² In this addendum, we aimed to determine the presence of any dominant known IRS sequence⁵ in H. pylori and whether it is conserved across all H. pylori strains measured. We found that the specific IRS sequence TTTAGGGG occurs greater than 20 times in each H. pylori genome and this sequence dominates all other known IRS sequences (Fig. 2). The pattern of IRS sequence distribution was similar across all strains of H. pylori measured but differed from that found in other gram-negative bacteria measured indicating this observed distribution is unique to H. pylori.

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.

A lower IRS:ISS ratio correlated with lower DNA immunogenicity and H. pylori DNA inhibits TLR-9 ligand, but not TLR-4 ligand-stimulated dendritic cell activation

We showed that the IRS:ISS ratio for H. pylori J99 is 30% lower than that of H. pylori 26695 strain.² To investigate whether H. pylori J99 is more immuno-stimulatory than H. pylori 26695, mouse bone marrow-derived dendritic cells (BMDCs) were stimulated with genomic DNA of E. coli K27, H. pylori J99, or H. pylori 26695. We found that H. pylori J99 stimulated dendritic cell IL-12 production in a dose dependent fashion whereas H. pylori 26695 did not (Fig. 3A), indicating that a lower IRS:ISS ratio correlated with weaker immune stimulation. We also showed that H. pylori genomic DNA inhibited E. coli genomic DNA-stimulated dendritic cell IL-12 production in a dose dependent fashion.² We provide further evidence that H. pylori DNA does not inhibit LPS stimulation of DCs. BMDCs were stimulated with TLR-4 agonist, LPS, and then E. coli or H. pylori genomic DNA were added to determine their effect on LPS-stimulated BMDC IL-12 production. (Fig. 3A). We found both E. coli and H. pylori DNA to have no effect on modulating LPS- induced BMDC IL-12 production, suggesting the inhibitory effect of H. pylori genomic DNA may be restricted to a TLR-9 signaling pathway that senses bacterial DNA (Fig. 3B).

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).

Potential Applications

Further understanding the mechanism by which H. pylori alters the likelihood of developing IBD is critical for multiple reasons. First, these data provide further mechanistic insight into the development of IBD confirming the important role of dendritic cells in the pathogenesis. Specifically, IL-12 and type I IFN appear to be critical in the inflammatory cascade with contributes to ongoing inflammation as seen in IBD. Extrapolating our data to the development of future therapies would suggest targeting TLR-9-dependent activation of these inflammatory cytokines with the goal of suppressing immune activation.

Second, this work further confirms the immuno-regulatory properties of the H. pylori genome. It is not debatable that H. pylori can lead to peptic ulcer disease and gastric cancer; however, it is important to note that 85% of infected patients remain asymptomatic while only 15% develop symptomatic peptic ulcer disease and less than 1% go on to develop gastric cancer.¹⁰ Further, standard treatment fails in 25% of patients, so wide-spread use of antibiotics may result in future antibiotic resistant strains.¹¹ It is also important to note that up to 50% of patients may experience side effects associated with antibiotic treatment.¹² With emerging data suggesting possible benefits of H. pylori, the decision to eradicate infection in asymptomatic individuals and to develop vaccination against this bacterium should now be questioned. Further research aimed at identifying particular strains of H. pylori that are likely to provide more benefit than harm is ongoing in our laboratory. Until then, H. pylori DNA or specific IRS octomers could be a potential treatment for IBD.

Conclusion

In summary, our work indicates that H. pylori genomic DNA contributes to the beneficial anti-inflammatory effect of H. pylori colonization in patients with chronic inflammatory conditions. We have already shown the in vivo benefit of luminal delivery of H. pylori genomic DNA in ameliorating the severity of chronic experimental colitis. The findings of this addendum strengthen the evidence of the unique properties of the H. pylori genome and revealed the importance of TLR-9 mediated mechanism in the pathogenesis of IBD. Further research to determine the relative contributions of various immune signaling pathways is important to determine the benefit of targeting single vs. multiple TLR pathways in treating IBD.

Disclosure of Potential Conflicts of Interest

J.Y.K. is a Consultant for Otsuka Pharmaceutical America, Inc.

Luther J, Owyang SY, Takeuchi T, Cole TS, Zhang M, Liu M, et al. Helicobacter pylori DNA decreases pro-inflammatory cytokine production by dendritic cells and attenuates dextran sodium sulphate-induced colitis. Gut. 2011;60:1479–86. doi: 10.1136/gut.2010.220087.