Elsevier

Microbial Pathogenesis

Volume 159, October 2021, 105151
Microbial Pathogenesis

CD4+ CD8αα+ T cells in the gastric epithelium mediate chronic inflammation induced by Helicobacter felis

https://doi.org/10.1016/j.micpath.2021.105151Get rights and content

Highlights

  • The proportion of DP T cells is positively correlated with the degree of inflammation.

  • DP T cells have a regulatory phenotype.

  • DP T cells inhibit DC maturation and T lymphocyte proliferation.

  • Silk fibroin adjuvant vaccine reduces the proportion of DP T cells and Helicobacter felis colonization.

Abstract

CD4+ CD8αα+ double-positive intraepithelial T lymphocytes (DP T cells), a newly characterized subset of intraepithelial T cells, are reported to contribute to local immunosuppression. However, the presence of DP T cells in Helicobacter. pylori -induced gastritis and their relationship with disease prognosis has yet to be elucidated. In this study, a chronic gastritis model was established by infecting mice with Helicobacter felis. Gastric-infiltrating lymphocytes were isolated from these mice and analyzed by flow cytometry. The frequency of DP T cells in H. felis-induced gastritis mice was higher than that in uninfected mice. The gastric DP T cells were derived from lamina propria cells but were predominantly distributed in the gastric epithelial layer. These gastric DP T cells also exhibited anti-inflammatory functions, and they inhibited the maturation of dendritic cells and proliferation of CD4+ T lymphocytes in vitro. Elimination of DP T cells simultaneously resulted in severe gastritis and a reduction of H. felis load in vivo. Finally, vaccine mixed with different adjuvants was used to explore the relationship between vaccine efficacy and DP cells. Silk fibroin as the vaccine delivery system enhanced vaccine efficacy by reducing the number of DP T cells. This study demonstrated that DP T cells perform an immunosuppressive role in Helicobacter felis-induced gastritis, and consequently, DP T cells may affect disease prognosis and vaccine efficacy.

Introduction

Helicobacter pylori is a Gram-negative, spiral-shaped, microaerophilic bacterium that colonizes that gastric mucosa of more than half of the world's population [1]. This bacterium is involved in several diseases including chronic gastritis, gastric ulcer, and gastric cancer [2,3]. CD4+ T helper cells (Th cells) play an important role in diseases connected to H. pylori infection. For example, both Th1 and Th17 cells have been reported to have pro-inflammatory functions [4,5], and regulatory T cells (Treg) may have an anti-inflammatory role during H. pylori infection [6,7]. However, whether and how other CD4+ T cell subsets respond to H. pylori infection and affect antibacterial immunity remains unclear.

CD4+ CD8αα+ T cells (also known as double-positive T cells, DP T cells) are a distinctive subset of CD4+ T cells with critical functions in inflammatory diseases. Some studies report that CD4+ CD8αα+ intraepithelial lymphocytes (IELs) have cytotoxic potential [8,9]. Small intestinal epithelial cells (sIECs) with antigen presentation ability induced CD4+ CD8αα+ IELs to express large amounts of granzyme B and CD107a, also known as lysosomal associated membrane protein-1 (LAMP-1) [8]. In clinical studies [10], it was found that the numbers of DP T cells in patients with celiac disease were significantly reduced compared with those of healthy individuals, suggesting that DP T cells have potential regulatory functions. DP T cells can secrete cytokines similar to Treg cells. In addition, DP T cells inhibit Th1-induced intestinal inflammation in an IL-10-dependent manner [11]. Studies have also shown that DP IELs and Treg cells synergistically regulate intestinal inflammation. In mice with loss of FOXP3 function, the number of DP IELs was significantly increased, but inflammation and diarrhea induced by OVA infection were markedly reduced. However, after using anti-CD8a antibodies to deplete DP IELs, the inflammation and diarrhea were aggravated [12]. These studies demonstrated that DP T cells are an important subset of CD4+ T lymphocytes in the intestinal epithelium and have an irreplaceable role in intestinal inflammation. However, there are no reports on the effects of DP T cells on H. pylori persistence or vaccine efficacy.

The Helicobacter felis mouse infection model and the human H. pylori infection model are two systems used to study H. pylori-induced gastritis [13]. H. felis induces gastritis in mice and this gastritis is similar to that in humans infected with H. pylori. This is because they have similar colonization methods and disease characteristics [14]. Therefore, the current study utilized the H. felis infection model. Previous studies showed that the number of CD4+FOXP3+ Treg cells in H. pylori-infected patients and mice increased significantly [15,16], and there was a positive correlation between Treg and H. pylori colonization in the stomach [17]. In addition, it has been proposed that Treg cells in the stomach can inhibit the proliferation of T cells and the production of IFN-γ induced by H. pylori [18]. The current study identified a subset of mouse gastric T cells that co-express CD4 and CD8αα. These gastric DP T cells increased significantly in H. felis-induced gastritis mice. The location and phenotype of the gastric DP T cells were analyzed. In addition, the gastric DP T cells were found to inhibit maturation of dendritic cells (DCs) and proliferation of CD4+ T cells in vitro. The DP T cells also affected gastritis and H. felis load in vivo.

Liu et al. demonstrated that mice receiving the H. pylori vaccine in the gastric subserous layer (GSL) had considerable protection against H. pylori infection [19]. However, we found that vaccination in the GSL induced numerous DP T cells compared with vaccinations via other routes such as p.o. and subcutaneous administration. Recombinant attenuated Salmonella Typhimurium-based vaccination was reported to reduce the proportions of CD4+ Treg in the stomach of H. pylori-infected mice [20]. Studies in other models have shown that Treg are reported to affect vaccine efficacy by inhibiting the protective immune response induced by vaccination [21]. Hence, induction of DP T cells may affect vaccine efficacy. The current study compared two vaccine delivery systems used in the laboratory. Mice administered with silk fibroin (SF)-loaded vaccine had a lower proportion of DP cells in their gastric tissues. This suggests that the immunosuppression of H. felis-induced gastritis may be related to DP T cells, which may affect disease prognosis and vaccine efficacy.

Section snippets

Mice

Six-week-old female C57BL/6J mice were obtained from the Comparative Medicine Center of Yangzhou University and bred at the China Pharmaceutical University Animal Experimental Center. All animal experiments were approved by the Animal Ethical and Experimental Committee of China Pharmaceutical University. Mice were allowed to acclimatize in the Animal Facility for one week before being randomly assigned to experimental groups.

H. felis challenge

H. felis ATCC 49179 was cultured as previously described [22,23]. H.

DP T lymphocytes are present in the stomach after H. felis infection

To detect gastric DP T cells following H. felis infection, the co-expression of CD4 and either CD8α or CD8β by T cells isolated from H. felis induced-gastritis mice and uninfected mice was analyzed (Fig. S1). The mice harbored substantial numbers of DP T cells upon H. felis infection, whereas DP T cells were negligible or absent in uninfected (control) mice (Fig. 1a). After H. felis infection, DP T cells were observed on day 20 (Fig. 1b, Fig. S2) and stabilized after day 30 (data not shown),

Discussion

A reduction in DP T cells has been observed in patients with inflammatory bowel disease (IBD) [10,32]. Furthermore, DP T cells cooperate with Treg cells to regulate intestinal inflammation in mice [28]. However, there is currently limited evidence describing how DP T cells regulate gastritis. The present study demonstrated that DP T cells in the stomach of gastritis mice increased significantly, and the induction of DP T cells was coincident with the progression of chronic gastritis.

DP T cells

Conclusion

In summary, this study showed that gastric DP T cells were significantly increased in mice with gastritis induced by H. felis. DP T cells were located in the gastric epithelium and exhibited a Treg phenotype and anti-inflammatory function. Use of SF as an adjuvant enhanced vaccine efficacy by inhibiting the production of DP T cells. These findings suggested an immunosuppressive role for DP T cells in both the pathogenesis of H. felis-induced gastritis and vaccine efficacy. In the future,

Ethics declarations

The approval of all animal's treatments was obtained from the Animal Ethics Committee of China Pharmaceutical University

Submission declaration and verification

We declare that the work described has not been published previously

Authors' contributions

Yingying Xing conceived and designed the project. Guojing Ruan acquired and analyzed the data.Guojing Ruan drafted the article and revised it critically for important intellectual content. An Huang, Chupeng Hu, Ningyin Xu, Zhenxing Zhang, Menghui Fan and Yue Wang participated in some experiments. All authors contributed to discussion of the results, reviewed the manuscript, and approved the version to be submitted.

Funding information

This work was supported by National Key R&D Program of China (No.2017YFD0400303), National Natural Science Foundation of China (No. 81971562), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions and Six Talent Peaks Project in Jiangsu Province (No. 2018-WSW-003).

Author statement

We declare that the content of the article is true and valid. We declare that the article does not involve any conflict of interest.We declare that the work described has not been published previously.

Declaration of competing interest

The authors have no conflicts of interest to declare that are relevant to the content of this article.

References (35)

  • H. Grasberger et al.

    Dual oxidases control release of hydrogen peroxide by the gastric epithelium to prevent Helicobacter felis infection and inflammation in mice

    Gastroenterology

    (2013)
  • E. Godefroy et al.

    Expression of CCR6 and CXCR6 by gut-derived CD4(+)/CD8alpha(+) T-regulatory cells, which are decreased in blood samples from patients with inflammatory bowel diseases

    Gastroenterology

    (2018)
  • M. Michetti et al.

    Gastric mucosal alpha(4)beta(7)-integrin-positive CD4 T lymphocytes and immune protection against helicobacter infection in mice

    Gastroenterology

    (2000)
  • M. Plummer et al.

    Global burden of gastric cancer attributable to Helicobacter pylori

    Int. J. Canc.

    (2015)
  • F. Munari et al.

    Cytokine BAFF released by Helicobacter pylori-infected macrophages triggers the Th17 response in human chronic gastritis

    J. Immunol.

    (2014)
  • J. Carton et al.

    CD4+CD8+ human small intestinal T cells are decreased in coeliac patients, with CD8 expression downregulated on intra-epithelial T cells in the active disease

    Eur. J. Gastroenterol. Hepatol.

    (2004)
  • G. Das et al.

    An important regulatory role for CD4+CD8 alpha alpha T cells in the intestinal epithelial layer in the prevention of inflammatory bowel disease

    Proc. Natl. Acad. Sci. U. S. A.

    (2003)
  • Cited by (1)

    View full text