Helicobacter pylori modulates the T helper cell 1/T helper cell 2 balance through phase-variable interaction between lipopolysaccharide and DC-SIGN - PubMed (original) (raw)

Helicobacter pylori modulates the T helper cell 1/T helper cell 2 balance through phase-variable interaction between lipopolysaccharide and DC-SIGN

Mathijs P Bergman et al. J Exp Med. 2004.

Abstract

The human gastric pathogen Helicobacter pylori spontaneously switches lipopolysaccharide (LPS) Lewis (Le) antigens on and off (phase-variable expression), but the biological significance of this is unclear. Here, we report that Le+ H. pylori variants are able to bind to the C-type lectin DC-SIGN and present on gastric dendritic cells (DCs), and demonstrate that this interaction blocks T helper cell (Th)1 development. In contrast, Le- variants escape binding to DCs and induce a strong Th1 cell response. In addition, in gastric biopsies challenged ex vivo with Le+ variants that bind DC-SIGN, interleukin 6 production is decreased, indicative of increased immune suppression. Our data indicate a role for LPS phase variation and Le antigen expression by H. pylori in suppressing immune responses through DC-SIGN.

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Figures

Figure 1.

Figure 1.

Clinically isolated H. pylori and purified LPS bind to DC-SIGN. H. pylori strains (•) isolated from patients from different continents (reference 5) and purified LPS (○) were coated on ELISA plates, and binding of recombinant DC-SIGN-Fc was assessed with peroxidase-labeled goat anti–human Fc.

Figure 2.

Figure 2.

Le blood group and related antigens and some of their substructures bind to DC-SIGN. (A) Le antigens expressed by H. pylori and discussed in this paper. (B and C) Carbohydrates, representing blood group antigens or their substructures, conjugated to polyacrylamide (B) or ceramide (C), were coated and binding of recombinant DC-SIGN-Fc was measured after incubation with peroxidase-labeled goat anti–human Fc. The mean (+ SD) of three independent experiments is shown.

Figure 3.

Figure 3.

Binding of H. pylori is dependent on Le antigen expression. (A–C) H. pylori α3-fucosyltransferase mutants (A), strains (B), and phase variants (C) were coated, and binding of recombinant DC-SIGN-Fc was measured after incubation with peroxidase-labeled goat anti–human Fc. H. pylori were coated and incubated with mAbs specific for Le antigens indicated, and their serotype was determined after incubation with peroxidase-labeled anti–mouse Igs. The mean (+ SD) of three independent experiments is shown.

Figure 4.

Figure 4.

LPS phase variation in H. pylori occurs in vivo. (A) After a short time of culturing directly from the biopsy, followed by one single passage in fluid phase and distribution over solid media, Lex/y+ phase variants of J223 were detected by colony blotting with mAbs specific for the indicated Le antigens. C-tract sequencing was performed to determine the on and off status of genes futA (HP0379) and futB (HP0651). The J223.3 futB mutant was generated by natural transformation with construct containing a chloramphenicol resistance marker cassette inserted in gene HP0651, and serotyped as indicated for J223. (B) Consequences of the C-tract length for functional expression of futA and futB.

Figure 5.

Figure 5.

DC-SIGN is expressed on gastric DCs and is the major receptor for Le+ H. pylori. (A) A tissue section of stomach was fixed and stained with anti–DC-SIGN antibodies. A magnification of 20. Arrows indicate DC-SIGN+ DC-like cells in the lamina propria. (B–D) Monocyte-derived DCs and RAW 264 macrophages (B), monocyte-derived DCs (C), or K-562 cells transfected with DC-SIGN (D) were incubated with FITC-labeled H. pylori J223.3 or J223.8, and binding was analyzed using flow cytometry. In C and D, cells were preincubated with anti–DC-SIGN antibodies, mannan, EDTA, or anti-MR antibodies. One representative out of four experiments is shown.

Figure 6.

Figure 6.

Binding of H. pylori induces DC-SIGN–dependent increase of IL-10. (A) DCs were incubated with H. pylori J223.3 or J223.8 at an MOI of 20 in the presence or absence of anti–DC-SIGN antibodies for 1 h, washed, and cultured for 20 h. Supernatant was harvested and the amount of IL-10 was analyzed by ELISA. (B) Upon coculture of DCs with H. pylori J223.3 or J223.8, cells were incubated with CD40L-transfected J558 fibroblasts in the absence or presence of IFN-γ for 24 h. Supernatant was harvested and the amount of IL-12p70 was analyzed by ELISA. One representative experiment out of four is shown.

Figure 7.

Figure 7.

Binding of H. pylori to DC-SIGN blocks skewing of naive T cells to Th1 cells. After preincubation with anti–DC-SIGN antibodies, DCs were incubated with H. pylori J223.3 or J223.8 at an MOI of 10 for 48 h, washed, and then cocultured with highly purified CD45RA+ CD4+ T cells. Quiescent T cells were restimulated with PMA and ionomycin, and IL-4 and IFN-γ was analyzed on a single cell basis by intracellular flow cytometry. Data from two representative donors (A and B/C) are shown from nine independent experiments.

Figure 8.

Figure 8.

Ex vivo challenge with DC-SIGN–binding H. pylori induces lower expression of IL-6 in stomach biopsies. Gastric biopsies were cocultured with 10 × 106 H. pylori J223.3 or J223.8 for 48 h and supernatants were harvested for analysis of cytokines, as indicated in Materials and Methods, by ELISA.

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