Stromal regulation of human gastric dendritic cells restricts the Th1 response to Helicobacter pylori

Abstract

Background & aims: Mucosal dendritic cells (DCs) play a key role in initiating the T-helper (Th)1 response to Helicobacter pylori. To further elucidate the mucosal response to H pylori, we examined whether gastric stromal factors condition DCs to support tolerance to H pylori, analogous to intestinal stromal factor-driven macrophage tolerance to commensal bacteria.

Methods: To model mucosal DC development, we isolated and cultured cell-depleted human stroma/extracellular matrix from fresh gastric and intestinal mucosa to generate stroma-conditioned media. We then analyzed the capacity of stroma-conditioned media-treated monocyte-derived DCs and primary human gastric and intestinal DCs pulsed in vitro with H pylori to induce T-cell proliferation and interferon gamma secretion.

Results: Stromal factors in gastric mucosa suppressed H pylori-stimulated DC activation and the ability of DCs to drive a Th1 proliferative and cytokine response to H pylori. The ability of gastric stromal factors to down-regulate DC function was similar to that of intestinal stromal factors and was independent of transforming growth factor β, prostaglandin E₂, interleukin (IL)-10, and thymic stromal lymphopoietin. Stroma-conditioned media-induced reduction in DC-stimulated Th1 responses was associated with reduced DC release of IL-12.

Conclusions: Gastric stromal factors down-regulate DC responsiveness to H pylori, resulting in a dampened gastric Th1 response. We speculate that stroma-induced down-regulation of DC function contributes to the permissiveness of both gastric and intestinal mucosa to colonization by persistent residential microbes.

Figure 1

Conditioned media from gastric and intestinal stroma (S-CM) down-modulate H. pylori-induced MoDC activation. MoDCs were differentiated from blood monocytes, stimulated with H. pylori and then analyzed for activation marker expression by FACS. DCs were exposed to gastric or intestinal S-CM (500 µg protein/mL) during (A) both DC differentiation (days 1–4) and H. pylori-induced activation (days 4–6) (n=8), (B) DC differentiation only (n=4) or (C) activation only (n=3). Bars represent geometric mean fluorescence normalized to medium control samples (where control=1) and corrected for isotype control antibody. Values are shown as mean ± SEM; statistical significance *P≤.05 was determined by 1-way ANOVA with Tukey’s post hoc test.

Figure 2

T-CM derived from gastric mucosal tissue down-modulate H. pylori-induced MoDC activation. MoDCs were exposed to medium alone, gastric S-CM or gastric T-CM (500 µg protein/mL) during DC differentiation, stimulated with H. pylori and then analyzed for activation marker expression by FACS (n=3). Bars represent geometric mean fluorescence normalized to medium control samples (where control=1) and corrected for isotype control antibody. Values are represented as mean ± SEM; statistical significance *P≤.05 was determined by 1-way ANOVA with Tukey’s post hoc test.

Figure 3

(A) Gastric and intestinal S-CM were analyzed for the presence of anti-inflammatory mediators TGF-β1 (n=13), IL-10 (n=5), TSLP (n=5) and PGE₂ (n=8) by ELISA. Values represent mean ± SEM; Mann-Whitney U test: **P≤.01. Detection limits for IL-10 and TSLP were 3.9 pg/mL and 3.5 pg/mL, respectively. (B) MoDCs were differentiated in the presence of medium, gastric or intestinal S-CM (500 µg protein/mL), or gastric or intestinal S-CM (500 µg protein/mL) pre-incubated with anti-TGF-β1/2/3 (100 µg/mL), stimulated with H. pylori and then analyzed for CD83 expression. Black histogram and legend correspond to cells treated with H. pylori, grey histogram and legend to cells treated with medium, and filled grey histogram to cells plus isotype control antibody; n=2. (C) MoDCs were differentiated from blood monocytes in the presence of medium, gastric or intestinal S-CM (500 µg/mL) or PGE₂ (0.1 – 1,000 ng/mL), stimulated with H. pylori and then analyzed for CD83 expression. Mean ± SEM of two independent experiments. (D) MoDCs were generated in the presence of medium, PGE₂ (2.7 or 0.06 ng/mL), gastric S-CM (2.7 ng/mL PGE₂, 500 µg protein/mL) or intestinal S-CM (0.06 ng/mL PGE₂, 500 µg protein/mL), all either pre-incubated with anti-PGE₂ (45 µg/mL) or an irrelevant isotype control for 1 h at 37°C. DCs then were stimulated with H. pylori and analyzed for CD83 expression. Results from a representative experiment (n=2).

Figure 4

Gastric and intestinal stromal factors inhibit the ability of DCs to prime a Th1 response through suppression of DC IL-12p70 secretion. (A) MoDCs were differentiated in medium alone or in the presence of gastric or intestinal S-CM (500 µg protein/mL), harvested, and pulsed with H. pylori bacteria for 2 h. Proliferation of autologous CD4⁺ T cells in response to untreated or H. pylori-pulsed DCs was determined after 4 days by BrdU ELISA. Proliferation is expressed as T cell proliferation index, which was calculated as absorption of DC-T cell co-culture relative to absorption of CD4⁺ T cells alone. (B) IFN-γ and (C) IL-10 secretion by autologous CD4⁺ T cells in response to untreated DCs or H. pylori-pulsed DCs was determined for the 4-day culture supernatants by ELISA. (D) Gastric and intestinal S-CM block H. pylori-induced IL-12p70 secretion by MoDCs. MoDCs differentiated in the presence or absence of S-CM were stimulated with H. pylori for 48 h, and culture supernatants were analyzed for IL-12p70 by ELISA (detection limit 5 pg/mL). (A – D) Diamonds represent cumulative data from three experiments with one or two S-CM each; lines represent mean values. *P ≤ .05, 1-way ANOVA with Tukey’s post hoc test. (E) Co-cultures of naïve CD4⁺ T cells and S-CM-treated or untreated DCs pulsed with H. pylori were established as described above, with rhIL-12p40 added at 5 ng/mL. T cell IFN-γ secretion was determined by ELISA analysis of 4-day culture supernatants. Results from a representative experiment (n=4).

Figure 5

Phenotype profile of primary human gastric and intestinal DCs. DCs were isolated from healthy human stomach and jejunum by collagenase digestion followed by HLA-DR MACS purification as described in the Materials and Methods. Surface marker expression was determined on DCs gated as HLA-DR^high cells. (A) Representative dotplots and (B) mean ± SEM proportion of cells that expressed the indicated surface markers (n=3–5).

Figure 6

Primary gastric and intestinal DCs are weak inducers of T cell proliferation but trigger T cell IFN-γ secretion. Freshly isolated gastric and intestinal HLA-DR^high DCs derived from healthy subjects were pulsed with H. pylori bacteria for 2 h and then co-cultured with autologous T cells. (A) Proliferation of autologous blood T cells in response to DCs treated with medium alone or with H. pylori was determined after 4 days by BrdU ELISA. Proliferation is expressed as T cell proliferation index and was calculated as absorption of DC-T cell co-cultures relative to absorption of T cells alone. Data are representative of 3 experiments with cells from separate subjects. (B,C) IFN-γ secretion by autologous T cells in response to H. pylori-pulsed or medium-treated DCs for 4-day culture supernatants was determined by ELISA. (B) Cumulative data from 5 experiments at a DC-T cell ratio of 1:10 (dots) with mean values shown as bars, and (C) the DC dilution curve for values from the subject shown in (A). Values correspond to mean ± SEM.