The Helicobacter pylori HopQ outermembrane protein inhibits immune cell activities

Abstract

We previously showed that the colorectal cancer colonizing bacterium Fusobacterium nucleatum protects tumors from immune cell attack via binding of the fusbacterial Fap2 outer-membrane protein to TIGIT, a checkpoint inhibitory receptor expressed on T cells and NK cells. Helicobacter pylori, the causative agent for peptic ulcer disease, is associated with the development of gastric adenocarcinoma and MALT lymphoma. The HopQ outer-membrane adhesin of H. pylori was recently shown to bind carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) including CEACAM1, an inhibitory receptor expressed mainly by activated T and NK cells. Here we investigated the possibility that similar to Fap2, HopQ can also inhibit immune cell activities by interacting with CEACAM1. We used several approaches to confirm that HopQ indeed interacts with CEACAM1, and show that CEACAM1-mediated activation by HopQ, may inhibit NK and T cell functions.

Keywords: CEACAM1; Helicobacter pylori; immune cells.

Figure 1.

H. pylori activates CEACAM1. (a) Flow cytometry of BW cells expressing human NK cell inhibitory receptors: CEACAM1, KIR2DL1, TIGIT or human activating receptors: NKp44, NKp30, NKp46 and CD16, all fused to mouse zeta chain. The various BW cells were stained with the antibodies against the receptors indicated above the histograms. The black empty histograms represent the specific staining, and the grey filled histograms represent the control staining with the secondary antibody only. (b-c) The various BWs were incubated with the H. pylori strains W0508, W1354 (1354) and HP-P12 (b) or with S. gallolyticus isolates SG1, SG2 and SG3 (c), at bacteria to BW ratio of 100:1. The presence of mouse IL-2 in the supernatants was determined by ELISA 48hr later (represented by pg/ml). The figure shows one representative experiment out of four performed. The error bars are derived from triplicates. **** p < 0.0001 repeated measures one way ANOVA (compared with other groups).

Figure 2.

H. pylori binds directly to CEACAM1. (a) Flow cytometry of BW and 721.221 cells and of the same cells transfected to express CEACAM1 as indicated. Cells were stained with anti-human CEACAM1 mAb (black empty histograms) or with secondary antibodies only (control, grey filled histograms). (b) Flow cytometry of binding of FITC-labelled H. pylori W0508 to parental BW and BW cells expressing the human CEACAM1 receptor, or to parental 721.221 and 721.221 cells expressing the human CEACAM1 receptor. The bacteria to target (BW or 721.221) ratios are indicated above the dot plots. The left dot plots in each row show BW or 721.221 cells without bacteria (cells only). The Median Fluorescent Intensity (MFI) of the FITC-labelled H. pylori binding to the target cells are indicated in the dot plots. One representative experiment out of three performed is presented. (c) Summary of fold increase of binding of H. pylori W0508 (B) to target (T): BW/CEACAM1 versus BW, and to 721.221/CEACAM1 versus 721.221 (d) Flow cytometry of binding of FITC-labelled H. pylori P12 to parental BW and BW cells expressing the human CEACAM1 receptor. The bacteria to target (BW or BW/CEACAM1) ratios are indicated above the dot plots. The Median Fluorescent Intensity (MFI) of the FITC-labelled H. pylori binding to the target cells are indicated in the dot plots. One representative experiment out of three performed is presented.

Figure 3.

CEACAM1 interacts with the H. pylori protein HopQ. (a) WT (P12, upper) and HopQ deficient H. pylori (P12ΔhopQ, lower) were stained with 1µg of hCEACAM1-Ig (left) or Control-Ig (right). Empty black histograms represent the specific staining, and the filled grey histograms represent staining of secondary antibodies only. One representative staining out of three is shown. (b) Flow cytometry of parental BW (left), and BW cells expressing human CEACAM1 (right) stained with anti-human CEACAM1 (black empty histograms) or with secondary antibodies only (control, grey filled histograms). (c) The CEACAM1-expressing BW cells were incubated with the parental WT P12 H. pylori or its HopQ deficient mutant P12ΔhopQ at various bacteria to BW/CEACAM1 (B:T) ratios (as indicated at the X axis). Mouse IL-2 in the supernatants was determined by ELISA 48hr later (represented by pg/ml). Figure shows one representative experiment out of four performed. The error bars are derived from triplicates. **p = 0.007 10:1 ratio, **p = 0.001 50:1 ratio, ***p = 0.0009 100:1 ratio, ***p = 0.0005 300:1 ratio. Two tailed paired t test (differences between paired values). (d) Far western blotting analysis of P12 and P12ΔhopQ using CEACAM-Ig. D and N represents denaturative and native preparation (see materials and methods), respectively. M represents the protein marker. The marker molecular weights are indicated on the left. Protein bands are indicated with arrows on the right.

Figure 4.

Helicobacter Pylori inhibits activity of T and NK cells. (a) Flow cytometry of representative primary IL-2 activated human CD4 + T cells expressing CEACAM1, stained with anti-CD4 mAb, and anti-CEACAM1 mAb (empty histograms, left and right, respectively), or with secondary antibodies only (control, grey filled histograms). (b) Schematic representation of the redirected assay. Mastocytoma P815 cells expressing the FcγR receptor were pre-incubated with anti-CD3 mAb. The anti-CD3 mAb binds the P815 cells via its Fc portion and thus, when T cells are added it activates them in a re-directed manner. (c) The murine FcγR-positive mastocytoma P815 cells were pre-incubated with anti-CD3 mAb, and then incubated with or without WT P12 or with P12ΔhopQ H. pylori (as indicated in the x axis) at bacteria to P815 ratio of 10:1. IL-2 activated CD4 cells expressing CEACAM1 (indicated in a) were then added and incubated with the P815 cells in an E:T ratio of 1:1. The presence of the IFN-γ in the supernatants was determined by ELISA 48 hr later (represented by pg/ml). The error bars are derived from triplicates. **p < 0.005, Bonferroni corrected two tailed paired t test (differences between paired values). The figure represents data collected from three independent experiments. (d) Flow cytometry of representative primary IL-2 activated human CD8 + T cells expressing the human CEACAM1, stained with anti-CD8 mAb and anti-CEACAM1 mAb (empty histograms, left and right, respectively), or with secondary antibodies only (control, grey filled histograms). (e and g) CD8 and NK cell cytotoxicity was assayed by CD107a degranulation assay. (e) CD8 cells (25000/w) expressing the CEACAM1 receptor (indicated in d), were incubated with P815 cells pre-coated with anti CD3 mAb with or without P12 or P12ΔhopQ at target (p815) to bacteria ratio of 100:1. The effector (CD8) to target (P815) ratio was 1:1. The Y axis indicates the percentages of CD3-mediated redirected killing (represented as CD107a+) of the P815 cells by CEACAM1 positive CD8 + T cells. (f and g) IL-2 activated human NK cells (25000/w) expressing the human CEACAM1, stained with anti-CD56 mAb and anti-CEACAM1 mAb (empty histograms, left and right, respectively, f), were incubated with 721.221 cells that were pre-coated with P12 or P12ΔhopQ, at effector (NK) to bacteria ratio of 100:1. The effector to target (721.221) ratio was 1:1. The Y axis represents the percentages of killing of the 721.221 cells (represented as CD107a+) by CEACAM1 positive NK cells. Data are shown as means percentage and ±SD of three replicates. (e) *p < 0.05. (g) ***p = 0.0008 **p = 0.0024. Bonferroni corrected two tailed paired t test (differences between paired values). e and g represent data collected from two independent experiments.