DC-LAMP+ dendritic cells are recruited to gastric lymphoid follicles in Helicobacter pylori-infected individuals

Abstract

Infection with Helicobacter pylori is associated with development of ulcer disease and gastrointestinal adenocarcinoma. The infection leads to a large infiltration of immune cells and the formation of organized lymphoid follicles in the human gastric mucosa. Still, the immune system fails to eradicate the bacteria, and the substantial regulatory T cell (Treg) response elicited is probably a major factor permitting bacterial persistence. Dendritic cells (DCs) are professional antigen-presenting cells that can activate naive T cells, and maturation of DCs is crucial for the initiation of primary immune responses. The aim of this study was to investigate the presence and localization of mature human DCs in H. pylori-infected gastric mucosa. Gastric antral biopsy specimens were collected from patients with H. pylori-associated gastritis and healthy volunteers, and antrum tissue was collected from patients undergoing gastric resection. Immunohistochemistry and flow cytometry showed that DCs expressing the maturation marker dendritic cell lysosome-associated membrane glycoprotein (DC-LAMP; CD208) are enriched in the H. pylori-infected gastric mucosa and that these DCs are specifically localized within or close to lymphoid follicles. Gastric DC-LAMP-positive (DC-LAMP(+)) DCs express CD11c and high levels of HLA-DR but little CD80, CD83, and CD86. Furthermore, immunofluorescence analyses demonstrated that DC-LAMP(+) DCs are in the same location as FoxP3-positive putative Tregs in the follicles. In conclusion, we show that DC-LAMP(+) DCs with low costimulatory capacity accumulate in the lymphoid follicles in human H. pylori-infected gastric tissue, and our results suggest that Treg-DC interactions may promote chronic infection by rendering gastric DCs tolerogenic.

Fig 1

DC-LAMP⁺ DCs in the human antrum mucosa. Biopsy specimens were collected from 8 H. pylori-infected (Hp+) and 10 uninfected (Hp−) individuals, and expression of DC-LAMP was determined by immunoperoxidase staining. (A) Frequencies of DC-LAMP⁺ DCs, expressed as a percentage of the stained area in the gastric mucosa. Symbols represent individual values, and lines indicate the median. (B to E) Representative staining of DC-LAMP from uninfected (B) and H. pylori-infected (C to E) individuals. (F) Isotype control for staining in panel E. **, P < 0.01. Magnifications, ×20.

Fig 2

Phenotype of gastric DC-LAMP⁺ DCs from H. pylori-infected subjects. Gastric DCs from uninfected (Hp−) and H. pylori-infected (Hp+) individuals were analyzed by flow cytometry for expression of HLA-DR and DC-LAMP (A) or CD11c and DC-LAMP (B). The lower dot plots show staining with an isotype control. Numbers by the gates indicate cell frequencies among all live (7AAD⁻) cells. (C) Percentage of DC-LAMP⁺ DCs, gated as described for panels A and B, in the gastric lamina propria of uninfected and H. pylori-infected subjects. Symbols represent individual values, which were calculated from several stainings in each individual, and horizontal lines indicate the median. All staining with the isotype control was subtracted from the DC-LAMP staining. (D) Flow cytometric analysis of the phenotype of gastric DC-LAMP⁺ HLA-DR^high cells identified as described for panels A and B. Thick lines, staining with the indicated antibody; dotted lines, staining with isotype controls. The data shown are from two H. pylori-infected individuals.

Fig 3

Immunofluorescence staining of DCs in the antrum mucosa. Biopsy specimens were collected from H. pylori-infected individuals, and expression of DC-LAMP, FoxP3, and CD68 was determined by immunofluorescence. (A) Representative double staining of DC-LAMP (red) and FoxP3 (green) in two lymphoid follicles from H. pylori-infected individuals. Magnification, ×20. Nuclei are stained blue with DAPI, and follicles are encircled. (B) Representative double staining with DC-LAMP (red) and CD68 (green) close to a lymphoid follicle in an H. pylori-infected individual.

Fig 4

Expression of CCL19 and CCL21 in human antrum mucosa. Biopsy specimens were collected from 9 uninfected (Hp−) and 10 H. pylori-infected (Hp+) individuals and used for mRNA and protein purification. Relative expression of CCL19 (A) and CCL21 (B) mRNA was determined by real-time PCR. (C) The CCL19 protein concentration in relation to the total amount of protein was determined by ELISA. Symbols represent individual values, and lines indicate the median. **, P < 0.01; ***, P < 0.001.