Mucosal vaccination increases local chemokine production attracting immune cells to the stomach mucosa of Helicobacter pylori infected mice

doi:10.1016/j.vaccine.2011.12.111

Vaccine

Volume 30, Issue 9, 21 February 2012, Pages 1636-1643

https://doi.org/10.1016/j.vaccine.2011.12.111 Get rights and content

Abstract

Background

Vaccination is an attractive approach for the prevention of Helicobacter pylori infection and disease. In a mouse model, infection induces an accumulation of dendritic cells, macrophages, granulocytes, and B- and T cells to the stomach mucosa, which is further heightened when the infection is preceded by a mucosal immunization. We have studied the chemokines and chemokine receptors guiding infection- and vaccination-induced immune cells to the stomach and their relation to protection against H. pylori infection in mice.

Materials and methods

C57BL/6 mice were immunized sublingually with H. pylori lysate antigens and cholera toxin adjuvant or left unimmunized, and then challenged with live H. pylori bacteria. Stomach tissue was taken at 3, 7, 14 and 21 days after challenge and bacterial colonization, chemokine and chemokine receptor gene expression, and influx of cells into the stomach mucosa were evaluated.

Results

RT-PCR array screening revealed differential expression of a broad range of chemokine and chemokine receptor genes between immunized and unimmunized mice. A significant upregulation of chemokines known to attract, among other cells, eosinophils (CCL8), T cells (CXCL10, CXCL11) and neutrophils (CXCL2, CXCL5) and of their cognate receptors CCR3, CXCR3 and CXCR2, preceded or coincided with vaccine-induced protection, which was first evident 7 days after infection and was then sustained at the later time-points. Consistent with the increase in chemokines and chemokine receptors flow cytometric analysis indicated a sequential accumulation of CD4⁺ T cells, eosinophils, neutrophils and CD103⁺ dendritic cells in the gastric lamina propria of immunized mice.

Conclusions

This study provides insights into vaccination-induced chemokines that guide the influx of protective immune cells into the stomach of H. pylori infected mice.

Highlights

► Sublingual vaccination protects against H. pylori infection in the stomach. ► The relationship between vaccine-induced gastric chemokine responses and protection against H. pylori infection is not known. ► Vaccination strongly induced chemokines in the stomach known to attract eosinophils, T cells and neutrophils. ► Protection coincided with accumulation of CD4⁺ T cells, eosinophils, neutrophils and CD103⁺ dendritic cells in the stomach. ► Our results will guide in the testing of vaccines against H. pylori infection in humans.

Introduction

Infection of the stomach with Helicobacter pylori elicits a specific local immune response, which suppresses but rarely eradicates the infection [1], and in a significant proportion of the infected individuals diseases such as gastritis, peptic ulcers and gastric cancer develop. The current treatment for eradication of H. pylori infection in symptomatic individuals is a combination of a proton pump inhibitor and antibiotics. Although effective in individual cases, the treatment is associated with increased development of antibiotic resistance and provides no protection against reinfection. A vaccine against H. pylori infection would therefore be a desirable alternative or complement to the current treatment options available. Indeed, vaccination of experimental animals has been shown to reinforce the infection-induced immune responses and further reduce H. pylori colonization in the stomach ultimately leading to eradication [2].

Immune-mediated protection against H. pylori infection and its reinforcement by vaccination appears to largely, if not exclusively, depend on immune cells migrating to the stomach mucosa. Of the various cell types in the stomach mucosa, CD4⁺ T cells have been found to be essential for the generation of these protective immune responses whereas CD8⁺ T cells and B cells seem to be dispensable [3]. Both Th1 and Th17 types of CD4⁺ T cells have been suggested to contribute to the protective immune responses in mice [4], [5], [6], [7], [8]. The mechanisms through which activated Th1 and Th17 effector cells can promote eradication of the H. pylori bacteria in the stomach are not well understood although neutrophils were recently suggested to be mediators of the protective Th17 responses [5]. However, vaccine-induced protective immune mechanisms involving other recruited cell types may also occur in parallel.

The recruitment of cells from the blood to a target tissue, such as the stomach mucosa, is a complex process in which chemokines play a central role. Transient interactions between immune cells in the blood and the activated endothelium cause the cells in the blood stream to slow down and roll along the vessel wall. The rolling allows the immune cells to bind to immobilized chemokines via their chemokine receptors. This interaction facilitates the firm adhesion of the immune cells via integrins on the immune cells and cell adhesion molecules (CAM) on endothelial cells, leading to the transendothelial migration into the stomach. Once there in the stomach, soluble chemokines further guide the immune cells by establishing chemotactic gradients. Thus an appropriate chemokine response in the stomach tissue is a prerequisite for an efficacious vaccine-induced immune response.

Earlier we have shown that the mucosal addressin cell adhesion molecule-1 (MadCAM-1) and the T and B cell attracting chemokines CXCL10 and CCL28 are upregulated in the stomach of immunized and H. pylori challenged mice. The upregulation of CCL28 was accompanied by an accumulation of B cells and increased IgA production in the stomach mucosa [9]. However, information about a broader range of chemokines produced that may be involved in the recruitment of immune cells to the stomach of immunized-protected mice is scarce. In this study we have used real-time PCR screening arrays to measure the expression of chemokine and chemokine receptor genes in the stomach mucosa and more specifically differences in expression between immunized and unimmunized mice at various time points after challenge with an infectious dose of H. pylori. Our studies reveal an up-regulation of a range of lymphocyte-, granulocyte- and dendritic cell (DC)-attracting chemokines in the stomach of immunized mice, which was associated with an increased expression of cognate chemokine receptor genes, supporting the influx of CD4⁺ T cells, neutrophils, eosinophils and CD103⁺ DCs into the stomach mucosa observed by flow cytometry.

Section snippets

Animals

Female 6- to 8-week-old C57BL/6 mice were purchased from Taconic Farms, Denmark and housed in microisolators at the Laboratory for Experimental Biomedicine, University of Gothenburg, during the study. All experiments were approved by the ethical committee for animal experiments (Gothenburg, Sweden).

Cultivation of H. pylori used for challenge

The mouse-adapted H. pylori Sydney strain 1 (SS1) (CagA⁺ VacA⁺ Le^y) was cultured on Colombia iso-agar plates (BD Biosciences, San Jose, CA) under microaerophilic conditions (10% CO₂, 6%O₂, 84% N₂)

A broad vaccine-induced up-regulation of chemokines and chemokine receptors in the stomach after H. pylori challenge

The stomach of both immunized and unimmunized mice was taken to prepare RNA for cDNA synthesis and subsequent real-time PCR analyses of chemokines and chemokine receptors as well as for quantification of the number of colonizing H. pylori bacteria.

As we have reported earlier [6], the effect of vaccination on bacterial colonization was first seen in mice 7 days after H. pylori challenge (70% reduction, P < 0.05) and was maintained at day 14 (85% reduction, P < 0.05) and day 21 (96% reduction, P <

Discussion

H. pylori infection-induced inflammation is characterized by dense infiltration of immune cells into the stomach [21]. The migration of cells from the blood to an inflamed tissue is mediated through an orchestrated sequence of events involving chemokines and chemokine receptors [22]. The chemokine–chemokine receptor network is very complex due to the fact that chemokines show redundancy in function and are pluripotent, having the ability to attract different cell types. Still very little is

Acknowledgements

This work was supported by grants from the Swedish Research Council (Medicine), Marianne and Marcus Wallenberg Foundation, Åke Wiberg Foundation, Adlerbert Foundations and the Swedish Foundation for Strategic Research (to the Mucosal Immunobiology and Vaccine Center). We would like to acknowledge the excellent technical assistance with mice provided by Margareta Blomquist and Annelie Ekman.

Conflict of interest statement: The authors declare no conflict of interest.

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Mucosal vaccination increases local chemokine production attracting immune cells to the stomach mucosa of Helicobacter pylori infected mice

Abstract

Background

Materials and methods

Results

Conclusions

Highlights

Introduction

Section snippets

Animals

Cultivation of H. pylori used for challenge

A broad vaccine-induced up-regulation of chemokines and chemokine receptors in the stomach after H. pylori challenge

Discussion

Acknowledgements

Gastroenterology

Mucosal Immunol

Gastroenterology

Immunity

Gastroenterology

Microbes Infect

Helicobacter pylori persistence: biology and disease

J Clin Invest

Clearance of Helicobacter pylori Infection and resolution of postimmunization gastritis in a kinetic study of prophylactically immunized mice

Infect Immun

Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class II-restricted responses

J Exp Med

Protection against Helicobacter pylori infection following immunization is IL-12-dependent and mediated by Th1 cells

J Immunol

Proinflammatory cytokine gene expression in the stomach correlates with vaccine-induced protection against Helicobacter pylori infection in mice: an important role for interleukin-17 during the effector phase

Infect Immun

The CD4+ T cell-mediated IFN-gamma response to Helicobacter infection is essential for clearance and determines gastric cancer risk

J Immunol

Local recall responses in the stomach involving reduced regulation and expanded help mediate vaccine-induced protection against Helicobacter pylori in mice

Eur J Immunol

Sublingual immunization protects against Helicobacter pylori infection and induces T and B cell responses in the stomach

Infect Immun

Effects of oral vaccination and immunomodulation by cholera toxin on experimental Helicobacter pylori infection, reinfection, and gastritis

Infect Immun

Enhanced M1 macrophage polarization in human Helicobacter pylori-associated atrophic gastritis and in vaccinated mice

PLoS One