Cooperation of Gastric Mononuclear Phagocytes with Helicobacter pylori during Colonization

Abstract

Helicobacter pylori, the dominant member of the human gastric microbiota, elicits immunoregulatory responses implicated in protective versus pathological outcomes. To evaluate the role of macrophages during infection, we employed a system with a shifted proinflammatory macrophage phenotype by deleting PPARγ in myeloid cells and found a 5- to 10-fold decrease in gastric bacterial loads. Higher levels of colonization in wild-type mice were associated with increased presence of mononuclear phagocytes and in particular with the accumulation of CD11b⁺F4/80^hiCD64⁺CX₃CR1⁺ macrophages in the gastric lamina propria. Depletion of phagocytic cells by clodronate liposomes in wild-type mice resulted in a reduction of gastric H. pylori colonization compared with nontreated mice. PPARγ-deficient and macrophage-depleted mice presented decreased IL-10-mediated myeloid and T cell regulatory responses soon after infection. IL-10 neutralization during H. pylori infection led to increased IL-17-mediated responses and increased neutrophil accumulation at the gastric mucosa. In conclusion, we report the induction of IL-10-driven regulatory responses mediated by CD11b⁺F4/80^hiCD64⁺CX₃CR1⁺ mononuclear phagocytes that contribute to maintaining high levels of H. pylori loads in the stomach by modulating effector T cell responses at the gastric mucosa.

Figure 1. Loss of peroxisome proliferator-activated receptor γ (PPARγ) in myeloid cells results in lower colonization with Helicobacter pylori and altered myeloid compartment

(A) WT, PPARγ-myeloid cell deficient (LysMcre) or PPARγ-T cell deficient (CD4cre) mice were infected with H. pylori SS1. Bacterial burden measured weekly up to 6 months post-infection show a significant reduction in colonization due to the loss of PPARγ in myeloid cells. Data represents mean ± SEM (n=10). (B) Loss of PPARγ in myeloid cells results in significantly lower percentage of CD64⁺CD11b⁺F4/80^hiCX₃CR1⁺ macrophages. Data represents mean ± SEM (n=5). (C) Phenotypic analysis of gastric myeloid cells in WT and LysMcre shows a significant depletion in myeloid cells in LysMcre mice including CD11b⁺F4/80^hiCD64⁺, neutrophils (CD11b^hi) and DC. (D) Phenotypic analysis of myeloid cells in naïve CX₃CR1-GFP^+/+ reporter mice. Macrophages, defined by CD64 expression, were found to be CX₃CR1⁺, DC were analyzed in the CD64⁻ fraction, after negative gating of CD3 and CD19 expression, and defined based on MHCII, CD11c and CD103 expression. Top panel represents CX₃CR1⁺ cells and bottom panel the CX₃CR1⁻ cells. Results from B, C and D are representative of 3 independent replicate experiments with same results. Points with an asterisk are significantly different when compared to the control (WT) group (P<0.05).

Figure 2. Helicobacter pylori infection causes accumulation of CD11b⁺F4/80^hiCD64⁺CX₃CR1+ macrophages in the gastric mucosa

Time-course FACS analysis of gastric myeloid cells after H. pylori infection of WT and LysMcre mice. The analysis was performed at the indicated times in cells isolated from mouse stomachs. Plots represent percentages (top row) and absolute numbers (middle row) of CD11b⁺F4/80^hiCD64⁺CX₃CR1⁺ macrophages (A, E) neutrophils, (B, D) CD11c⁺CD103⁻ (C, G) and CD11c⁺CD103⁺ DC (D, H) subsets. DC gating was done on MHCII⁺CD64⁻ cells. Results are averages of 5 mice per time-point and are presented as mean ± SEM (n=5/time point). (I, J) BrdU incorporation was measured by flow cytometry in CD11b⁺F4/80^hiCD64⁺CX₃CR1⁺ and F4/80^loCD11b⁺ cells isolated from the stomach of H. pylori infected mice on days 15, 18 and 21 post-infection. Points with an asterisk are significantly different when compared to the control group (P<0.05). Results are representative of 5 independent replicate experiments with same results. Points with an asterisk are significantly different when compared to the control (WT) group (P<0.05).

Figure 3. Macrophage depletion reduces Helicobacter pylori loads and suppresses IL-10-mediated regulatory responses in the stomach of WT mice

(A) WT mice were infected with SS1 and received three doses of either clodronate liposomes or PBS liposomes on days 11, 14, 17, and 20 post-infection. Analyses were performed prior to the first injection, day 11 (before treatment started), or one day after each injection. SS1-infected, non-treated LysMcre mice were used for reference. (B) Macrophage depletion suppressed bacterial loads to levels of untreated LysMcre mice. (C) Clodronate treatment depleted F4/80^hiCD11b⁺CX₃CR1⁺ and (D) F4/80^hiCD11b⁺CD64⁺ cells from the stomach of WT mice. (E) Neutrophil levels ware significantly increased in clodronate-treated mice. (F, G) Differences were observed in CD11c⁺CD103⁺, but not on CD11c⁺CD103⁺ DC (gated on CD64⁻MHCII⁺ cells). IL-10 production was measured in CD4⁺ T cells (H), CD4⁻ cells (I) during the time course study, and on (J) CD11b⁺ cell on day 18 post-infection. Data represents mean ± SEM (n=8). Points with an asterisk are significantly different when compared to the control group (P<0.05). Results are representative of 2 independent replicate experiments with same results. Points with an asterisk are significantly different when compared to the control (WT) group (P<0.05).

Figure 4. Phenotypic characterization of IL-10-producing cells during Helicobacter pylori infection

WT and LysMcre mice were infected with H. pylori SS1 strain. Stomachs were collected at days 0 (prior to infection), 18 and 24 post-infection. (A) Representative dot plots of IL-10 production by F8/40⁺ cells in WT and LysMcre mice. (B) Average IL-10 producing cells within CD11b⁺F4/80^lo and CD11b⁺F4/80^hiCD64⁺CX₃CR1⁺ macrophages. (C) Representative plots of IL-10 production by CD4⁺ T cells and B cells from H. pylori SS1-infected WT and LysMcre mice. Average IL-10 production by (D) CD4 T cells, (E) B cells, (F) Foxp3⁺ regulatory CD4 T cells and (G) PD-1⁺ Treg cells. Average result data represent mean ± SEM (n=8). Points with an asterisk are significantly different when compared to the control group (P<0.05). Results are representative of 4 independent replicate experiments with same results. Points with an asterisk are significantly different when compared to the control (WT) group (P<0.05).

Figure 5. IL-10 neutralization during Helicobacter pylori infection

WT mice were infected with H. pylori and on days 17, 19 and 21 post-infection they were treated with 100 μg of either neutralizing anti-mouse IL-10 or Rat IgG1 isotype control antibodies. Mice (n=6), were euthanized on day 22 post-infection to measure (A) bacterial burden in the stomach, and percentages of (B) CD11b⁺F4/80^hiCD64⁺CX₃CR1⁺, (C) neutrophils, and (D) DC. Cells from the gastric lymph nodes of 3–4 mice of the same gender and treatment were stimulated ex-vivo with RPMI (unstimulated) or 5 μg/ml of formalin-inactivated H. pylori SS1. Production of (E) IFNγ, (F) IL-10, (G) IL-17 and (H) IL-6 were measured in 72-hour cell culture supernatant using a cytometric bead array. Results are expressed as average of 3–5 samples of pooled cells and data represent mean ± SEM. Points with an asterisk are significantly different when compared to the control group (P<0.05). Results are representative of 2 independent replicate experiments with same results. Points with an asterisk are significantly different when compared to the control (WT) group (P<0.05).