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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 28, 2004 as doi:10.1096/fj.04-2321fje. |
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Max-Planck Institute for Infection Biology, Department of Molecular Biology, Berlin, Germany
2Correspondence: Max-Planck Institute for Infection Biology, Department of Molecular Biology, Schumanstrasse 21/22 Berlin, Germany. E-mail: meyer{at}mpiib-berlin.mpg.de
SPECIFIC AIMS
An effective prophylactic or therapeutic vaccine against Helicobacter pylori would be a cost effective way to control chronic gastritis and gastric cancer. Experimental vaccines against H. pylori are effective in the mouse model, but a clear link to an effector mechanism cannot yet be made. A better understanding of the immune response in the stomach would facilitate design of a human vaccine.
The aim of this study was to identify genes specifically expressed in immunized and infected mice and shed light on what happens in vivo when the immunized epithelium is challenged with H. pylori. We determined the transcription profile in the gastric mucosa of mice immunized with recombinant Salmonella expressing H. pylori UreA/B on days 1, 3, and 14 after infection with H. pylori.
PRINCIPAL FINDINGS
1. Infection with H. pylori causes an immediate reaction at the transcriptional level in vivo
Three hundred genes passed the cut-off criteria and were differentially expressed in mice infected with H. pylori. This infection signature was different at all time points investigated, illustrating at least two major shifts in gene expression in the inflammatory response (Fig. 1
). The infection signature was characterized by down-regulation of innate immune response (defensin-related cryptdins, Defcr), B cell-related, and stress response genes. By day 14, gastrin was up-regulated and again (different) inflammatory response genes were down-regulated (Irf7,Tnf), as were genes controlling cell migration. This signature implies that H. pylori is able to suppress the early inflammatory response, which probably allows the bacterium to become established in the gastric niche.
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2. Immunization induces a unique transcriptional signature
When protected immunized and infected mice were compared with infected mice, 333 genes were differentially expressed only in immunized mice. This immunization signature must reflect the mechanisms responsible for protection, and our further studies focused on this.
This transcriptional signature could not be explained simply by infiltrating lymphocytes, as immunohistochemical studies using a panel of immune cell markers revealed only minor differences in the population of infiltrating cells between immunized and nonimmunized mice at time points up to 14 days postchallenge.
3. Immunization induces expression of innate epithelial mediators
Analysis of the immunization signature led us to make two interesting observations: first, the anti-microbial peptide Defcr4 were specifically up-regulated in immunized mice on day 1. The anti-microbial activities of Defcr4 may help to suppress growth of H. pylori in the first few days until more effective and persistent (T cell mediated) effects can accrue. Second, on day 14 the epithelial mediators crp-ductin (product of the Dmbt1 gene) and surfactant protein D (Sfptd) were up-regulated. Crp-ductin and surfactant protein D have both been shown to bind H. pylori, and we propose they act as effector molecules to reduce bacterial motility.
4. Immunization induces an IFN-
-dependent inflammatory response and expression of nonclassical immune mediators in the gastric mucosa
The immunization signature was characterized by increased IFN- expression on day 1 compared with infected mice and several genes involved in proliferation. By day 14 a new set of IFN--dependent, inflammation-related genes were expressed, reflecting the developing infiltrate of CD4+ T cells, which has been shown to mediate protection. Nonclassical inflammatory genes such as the proinflammatory adipokine Acrp30 (adiponectin), fatty acid binding protein lipocalin 2 (Lcn2) were up-regulated. It would appear that the immunized epithelium can respond more quickly to H. pylori than the naive epithelium and the early establishment of an inflammatory response facilitates bacterial killing.
5. Immunization causes an increase in epithelial proliferation
A significant proportion of the genes up-regulated in immunized mice were involved in cell turnover or proliferation, which prompted us to ask whether immunization induces a general increase proliferation in the epithelium as part of the protective mechanism. BrdU incorporation studies showed that on day 14 the neck gland cells of immunized mice had indeed incorporated more BrdU than those of infected-only mice. This effect was more distinct in the antrum region, where approximately twice as many BRDU positive nuclei were detected. This increased proliferation probably underlies increased turnover and sloughing of the epithelium, making it more difficult for H. pylori to become established.
CONCLUSIONS AND SIGNIFICANCE
It has been demonstrated that mucosal antibody is not responsible for vaccine-induced protection against H. pylori but that T cells and MHCII are required. For protective CD4+ T cells to have an effect on the luminal bacteria, a signal must be transmitted to the epithelial cells, stimulating them to produce the factors that ultimately suppress bacterial growth. As a result of our transcriptome studies, we were able to identify genes that may be playing these roles and have gained insight into how the protective mechanism might work.
It appears that the immunized stomach can respond more rapidly to H. pylori challenge, probably by overcoming the initial suppressive effects exerted by the bacterium. This immune response likely involves additional players such as adipokines. Involvement of such nonclassical immune mediators in protection may explain in part why immunological studies focusing on Th1/2 responses resulted in conflicting results. Our observations on a possible role for adipokines in the gastric immune response are consistent with results from another micorarray study by Müller et al., where protection against Helicobacter felis after 22 months was correlated with the expression of adipsin and other genes expressed by mature adipocytes.
That immunization stimulates the expression of innate epithelial mediators was unexpected and interesting. The Dmbt1 gene produces the sulfated mucin-like glycoprotein crp-ductin, which is expressed in many tissues (including the intestine), agglutinates bacteria, and binds to the mucosal collectin Surfactant protein D (Sftpd). Sftpd in turn has been shown to bind to Helicobacter and reduce its motility; its expression is increased in patients with H. pylori gastritis. We propose that these gene products play an important effector role in protection.
H. pylori infection is known to induce alterations in cell cycle in vitro and in vivo. We were able show increased epithelial cell proliferation in the corpus and antrum of immunized mice relative to infected mice, confirming the gene expression pattern we observed. A more rapid turnover of the gland cells, and probably increased epithelial sloughing, could be expected to create less favorable local conditions for H. pylori.
We now suggest a working model for the protection mechanism (Fig. 2
). Within 24 h of challenge with H. pylori in immunized mice, secretion of innate factors such as Defcr4 leads to at least transient suppression of bacterial growth. Within 3–5 days, a small population of specific CD4+ T cells migrate to the gastric mucosa where they secrete mediators such as IFN-, and possibly adipokines. These mediators act on epithelial cells, causing secretion of epithelial defense factors, including crp-ductin and Sfptd, which bind bacteria, reducing their motility. This coupled with increased epithelial turnover and possibly altered mucin secretion together make the gastric niche unfavorable for H. pylori. The combination of these effects results in the long-term suppression of H. pylori colonization (but not sterile immunity) observed in immunized mice.
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FOOTNOTES
1 Current address: Laboratoire de Virologie, Centre Hospitalier Regional Universitaire, Lille, France. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2321fje;
This article has been cited by other articles:
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  R. L. Ferrero, P. Ave, D. Ndiaye, J.-C. Bambou, M. R. Huerre, D. J. Philpott, and S. Memet NF-{kappa}B Activation during Acute Helicobacter pylori Infection in Mice Infect. Immun., February 1, 2008; 76(2): 551 - 561. [Abstract] [Full Text] [PDF]
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H. Suto, M. Zhang, and D. E. Berg Age-Dependent Changes in Susceptibility of Suckling Mice to Individual Strains of Helicobacter pylori Infect. Immun., February 1, 2005; 73(2): 1232 - 1234. [Abstract] [Full Text] [PDF]
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