Background and Aims

Mucosal dendritic cells (DCs) play a key role in initiating the Th1 response toH. pylori. To further elucidate the mucosal response toH. pylori, we examined whether gastric stromal factors condition DCs to support tolerance toH. 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 (S-CM). We then analyzed the capacity of S-CM-treated monocyte-derived DCs and primary human gastric and intestinal DCs pulsedin vitro withH. pylori to induce T cell proliferation and IFN-γ secretion.

Results

Stromal factors in gastric mucosa suppressedH. pylori-stimulated DC activation and the ability of DCs to drive a Th1 proliferative and cytokine response toH. 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. S-CM-induced reduction in DC-stimulated Th1 responses was associated with reduced DC release of IL-12.

Conclusion

Gastric stromal factors down-regulate DC responsiveness toH. 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.

Keywords: Dendritic cells, mucosa,H. pylori, stomach, gastritis

Human subjects

Gastric and small intestinal tissue specimens were obtained with institutional review board approval from healthy human subjects undergoing elective gastric bypass surgery for obesity or from non-obese transplant donors. Heparinized blood samples were obtained from the same subjects or other healthy adult volunteers. All subjects were free ofH. pylori infection as determined by CLO-Test and/or serology.

Cell isolation and enrichment

Gastric and intestinal lamina propria cells were isolated from surgical specimens using our previously described protocol³. Briefly, mucosa was dissected from the underlying muscularis propria and submucosa, washed twice for 20 min in HBSS (Mediatech Inc., Manassas, VA) supplemented with 0.2 mg/mL dithiothreitol (DTT, Sigma, St. Louis, MO), and epithelial cells were removed by incubation in Hank’s buffered salt solution containing 1.25 mM EDTA plus 0.2 mg/mL DTT (3×30min). The tissue then was minced and transferred to a solution containing 0.5 FALGPA U/mL collagenase L (Sigma), 0.2 mg/mL DNAse (Sigma), 20 mM HEPES (Mediatech Inc.) plus antibiotics in RPMI (Mediatech). After each of three 45 min collagenase treatments, lamina propria mononuclear cells (lymphocytes, macrophages and DCs) were collected by centrifugation. To enrich human leukocyte antigen (HLA) DR^high DCs, positive MACS® selection was used with a low concentration of anti-HLA-DR beads (0.2 µL per 10⁶ cells; Miltenyi Biotec, Auburn, CA). For T cell stimulation assays, mucosal DCs were further purified to >95% by FACS sorting (FACSVantage Diva, Becton Dickinson, San Jose, CA) using fluorescent-labeled anti-HLA-DR antibodies with gates set to include HLA-DR^high cells and to exclude lymphocytes, debris and propidium iodide-positive dead cells.

Stroma-conditioned media and tissue-conditioned media

Stroma- and tissue-conditioned media were prepared as described previously^14,16. After tissue digestion (see previous paragraph), the remaining stroma consisted of cell-depleted extracellular matrix (collagen and proteoglycans), which contained only few residual epithelial cells, fibroblasts, endothelial cells and leukocytes, as determined by H&E staining. The stroma was cultured in serum-free RPMI (containing 20 mM HEPES and antibiotics) at 37°C for 24 h (1 g tissue wet weight/mL). The resultant supernatants were harvested, sterile-filtered (0.2 µm syringe filter, Corning Inc., NY), and frozen at −70°C. T-CM was derived from fresh intact gastric or intestinal mucosa that had been minced into 0.25 cm² pieces and cultured in RPMI at 37°C for 24 h at 0.5 g tissue/mL. Endotoxin and protein concentrations were determined by the Limulus Amebocyte Lysate assay (Cambrex, Walkersville, MD) and the BCA™ Protein Assay Kit (Thermo Scientific, Rockford, IL), respectively. Since some S-CM samples contained LPS (up to 15 endotoxin units/mL), polymyxin B (5 µg/mL, Sigma) was added to all cell cultures to block residual LPS effects on DCs.

Monocyte-derived DCs (MoDCs)

MoDCs were differentiated from MACS-isolated CD14⁺ blood monocytes by culturing 2×10⁶ monocytes per well in 24-well plates in complete medium (RPMI1640, 10% heat-inactivated human AB serum and antibiotics) supplemented with recombinant human granulocyte-monocyte colony stimulating factor (GM-CSF, 25 ng/mL) and interleukin 4 (IL-4, 17 ng/mL), both from R&D Systems, Minneapolis, MN. To recapitulate DC differentiation in the gastric or intestinal mucosal microenvironment, MoDC cultures were maintained in the presence or absence of gastric or intestinal S-CM or gastric T-CM at the previously determined optimal concentration of 500 µg protein/mL. Non-adherent cells were harvested as MoDCs by vigorous pipetting after four days of culture.

DC treatment withH. pylori

To elucidate the DC response toH. pylori, 3 – 5×10⁵ MoDCs were transferred into fresh medium (RPMI with 10% human Ab serum and IL-4/GM-CSF) with or without S-CM for 1 h, after whichH. pylori 60190 (CagA⁺, VacAs1m1) was added at a multiplicity of infection (MOI) of 10 for 48 h, as previously described³. Surface marker expression was determined by flow cytometry.

TGF-β neutralization and replacement

For antibody neutralization experiments, S-CM (500 µg protein/mL) was incubated with anti-human TGF-β1/2/3 antibody (R&D Systems, 100 µg/mL) for 30 min at 37°C before adding the S-CM to the MoDC cultures. To recapitulate the potential effects of TGF-β on MoDCs, active rhTGF-β1 (R&D Systems) was added to MoDC cultures at 6.25 – 100 pg/mL, a range that includes the TGF-β1 concentrations routinely measured in gastric and intestinal S-CM at 500 µg protein/mL.

PGE₂ neutralization and replacement

To determine the effect of exogenous PGE₂ on DC differentiation, PGE₂ (Sigma) was added to MoDC cultures at 0.06 – 1,000 ng/mL, a range that includes the PGE₂ concentrations in gastric and intestinal S-CM at 500 µg protein/mL. For antibody neutralization, S-CM was incubated with anti-PGE₂ (45 µg/mL, clone 2B5¹⁹, Cayman Chemicals, Ann Arbor, MI) or an irrelevant isotype-matched control (BD Pharmingen, San Diego, CA) for 60 min at 37°C before adding the S-CM to the MoDC cultures.

T cell stimulation assays

For DC-T cell stimulation assays, primary, FACS-purified gastric and intestinal HLA-DR^high DCs or day 4 MoDCs were pulsed withH. pylori (MOI=10) for 2 h at 37°C, washed twice and plated at 2×10⁴/well on 96-well plates. Autologous T cells were isolated by MACS using anti-CD4 microbeads or the Naive CD4⁺ T Cell Isolation Kit II (Milteny Biotec) and were added to the DCs at a DC:T cell ratio of 1:10, unless indicated otherwise. In some experiments, recombinant human IL-12p70 (1.25 – 20 ng/mL, R&D Systems) was added to the co-cultures. After 4 days, cell proliferation was determined by bromodeoxyuridine (BrdU) ELISA (Roche Diagnostics, Mannheim, Germany), and cell-free supernatants were collected for cytokine analysis.

Cell staining and flow cytometry

The following mAbs labeled with fluorescein isothiocyanate, phycoerythrin, peridinin-chlorophyll-protein, allophycocyanin or allophycocyanin-Cy™7, were used: CD11c (B-ly6), CD45 (2D1), CD80 (BB1), CD83 (HB15e), CD86 (FUN-1), HLA-DR (L243), DC-SIGN (DCN46), and appropriate isotype control antibodies, all from Becton Dickinson (San Jose, CA). For flow cytometric analysis, 0.3 – 1×10⁶ cells were blocked with 10% human AB serum for 20 min, then treated with the mAbs for 20 min at 4°C, washed, fixed in Cytofix, and analyzed on an LSRII flow cytometer (Becton Dickinson). Data from 10,000 cells per sample were analyzed with FlowJo 7.5.5 software (TreeStar, Ashland, OR). To compare results from different experiments, relative geometric mean was calculated asf(x) = (geomean fluorescence [stained sample]/geomean fluorescence [isotype control of sample]) / (geomean fluorescence [stained control cells]/geomean fluorescence [isotype control of control cells]).

Cytokine levels

The amounts of IL-10, IL-12p70, interferon-γ (IFN-γ), PGE₂, TGF-β1 and thymic stromal lymphopoietin (TSLP) in the S-CMs or culture supernatants were determined by ELISA (R&D Systems); the TGF-β1 was assayed after acid activation of the S-CM.

Statistical analysis

Data were analysed using Microsoft® Excel 2003 and Analyse-it® for Excel, version 1.73. Results are presented as mean ± SEM. Differences between values were analysed for statistical significance by 1-way analysis of variance (ANOVA) with pairwise comparisons and Tukey’s post hoc test, the two-tailed Studentt test, or the Mann-Whitney U test, as indicated. Differences were considered significant atP<0.05.

Gastric and intestinal stromal factors inhibit DC activation

DCs differentiate from monocytes or DC precursors under the influence of tissue-specific stromal factors^20,21. Using our establishedin vitro model of the mucosal microenvironment^14–16, MoDCs were generated in GM-CSF plus IL-4 in the presence or absence of conditioned media prepared from cell-depleted mucosal stroma/extracellular matrix (S-CM). DCs then were exposed toH. pylori bacteria in the continuous presence of S-CM. As shown inFigure 1A,H. pylori stimulation caused the up-regulation of CD80, CD83 and CD86 expression on MoDCs cultured in medium, consistent with previous findings^3,22–24. Importantly, both gastric and intestinal S-CM blocked inducible CD80, CD83 and CD86, as well as differentiation (GM-CSF plus IL-4)-induced CD86 expression. Gastric and intestinal S-CM were nearly equivalent in their capacity to suppress MoDC differentiation andH. pylori-induced activation. Both gastric and intestinal S-CM also suppressed DC activation bySalmonella enterica Typhimurium (MOI = 50, data not shown), indicating that inducible activation was not specific forH. pylori.

To dissect whether S-CM inhibition of DCs occurred during differentiation or activation, MoDCs were generated in the presence or absence of S-CM (days 1–4) and then stimulated withH. pylori, also in the presence or absence of S-CM (days 4–6) (Figure 1B,C). The presence of either gastric or intestinal S-CM exclusively during MoDC differentiation (days 1–4) suppressed CD80, CD83 and CD86 expression (Figure 1B) to the same level as the continuous presence of S-CM during both MoDC generation andH. pylori stimulation (days 1–6) (Figure 1A). In contrast, the presence of S-CM exclusively during stimulation withH. pylori (days 4–6) reduced DC activation marker expression by only a small amount (Figure 1C). Supernatants generated from intact gastric mucosa (T-CM) had the same suppressive effects as gastric S-CM, indicating that inhibition of DC activation by S-CM was not caused by collagen fragments or other products resulting from the collagenase treatment (Figure 2). These findings suggest that stromal conditioning of DCs occurs primarily during DC differentiation, rather than during activation, and that such conditioning has lasting inhibitory effects on the differentiation of mucosal DCs, consistent with the notion that mucosal DCs retain their phenotype after they leave the mucosa and migrate to draining lymph nodes.

Stromal inhibition of DCs is independent of TGF-β, IL-10,TSLP and PGE₂

To explore the mechanism by which gastric and intestinal stromal factors suppress DCs, we determined whether TGF-β, IL-10, TSLP or PGE₂, soluble mediators that regulate immune cell functions at mucosal sites^{11,14,15,25–27}, contribute to stromal regulation of DCs. Both gastric S-CM (3,043 ± 327 µg protein/mL; n=12) and intestinal S-CM (3,115 ± 390 µg protein/mL; n=12) contained the anti-inflammatory cytokine TGF-β1, although the concentration of TGF-β1 was significantly lower in gastric S-CM (39 ±15 pg/mL) than in intestinal S-CM (146 ± 42 pg/mL;P<0.01) (Figure 3A). In contrast, gastric S-CM contained significantly higher levels of PGE₂ (17,020 ± 3,528 pg/mL) than intestinal S-CM (4,030 ± 1,422 pg/mL;P<0.01). IL-10 and TSLP were undetectable in both gastric and intestinal S-CM. We first examined whether the inhibitory effect of S-CM on DC activation marker expression was due to stromal TGF-β. Neutralization of TGF-β with anti-TGF-β1/2/3 antibody (100 µg/mL), shown previously to inhibit TGF-β activity in intestinal S-CM^14,15, did not prevent gastric or intestinal S-CM inhibition ofH. pylori-induced MoDC expression of CD83 (Figure 3B) or CD80 and CD86 (data not shown). Furthermore, the addition of 6.25 – 100 pg/mL rhTGF-β1 to MoDC cultures, which was equivalent to the amount of TGF-β1 present in gastric and intestinal S-CM at a concentration of 500 µg protein/mL, did not inhibitH. pylori-induced up-regulation of CD80, CD83 and CD86 (data not shown).

To determine whether PGE₂ contributes to S-CM suppression of DC activation, we next showed that exogenous PGE₂ added during DC differentiation blockedH. pylori-induced DC expression of CD83 (Figure 3C), CD80 and CD86 (not shown) in a dose-dependent manner, with maximum suppression seen at ≥10 ng/mL PGE₂. However, gastric and intestinal S-CM diluted to 500 µg protein/mL routinely contained less PGE₂ than the amount of exogenous PGE₂ needed to achieve the same level of suppression. Consequently, antibody neutralization of PGE₂ in gastric and intestinal S-CM failed to restore the ability of the MoDCs to upregulate CD83 (Figure 3D), CD80 and CD86 (not shown), although antibody neutralization of 2.7 ng/mL of exogenous PGE₂, a dose calculated to match the PGE₂ concentration in the gastric S-CM used in the experiment, abolished the suppressive effects of the PGE₂. Exogenous PGE₂ at 0.06 ng/mL, the concentration present in the intestinal S-CM used in the experiment, did not suppressH. pylori-induced DC activation. These findings indicate that stromal factors other than TGF-β1, IL-10, TSLP and PGE₂ suppress DCs in gastric and small intestinal mucosa.

Intestinal and gastric stromal factors reduce the ability of DCs to drive a Th1 response to H. pylori

We next determined whether S-CM inhibition ofH. pylori-induced DC activation was associated with inhibition of inducible responses by CD4⁺ T cells, the predominant cell type in inflammatory infiltrates inH. pylori gastritis⁴. MoDCs differentiated in the presence or absence of gastric or intestinal S-CM were loaded withH. pylori, co-cultured with autologous CD4⁺ T cells, and the proliferative and cytokine responses of the T cells were determined 4 days later. MoDCs loaded withH. pylori drove a strong proliferative response, whereas MoDCs generated in the presence of either gastric or intestinal S-CM induced significantly lessH. pylori-specific T cell proliferation (Figure 4A).

The Th1 response driven byH. pylori is characterized by T cell secretion of high levels of IFN-γ^3–5. Therefore, we examined whether S-CM modulates the ability ofH. pylori-pulsed MoDCs to induce T cell IFN-γ secretion. Control MoDCs loaded withH. pylori elicited a strong T cell IFN-γ response, but MoDCs generated in the presence of either gastric or intestinal S-CM were significantly impaired in their ability to induce T cell IFN-γ secretion in response toH. pylori (Figure 4B). In contrast, neither gastric nor intestinal S-CM altered the ability ofH. pylori-pulsed MoDCs to stimulate IL-10 secretion by T cells (Figure 4C). T cell proliferation and cytokine responses did not differ between total CD4⁺ T cells and naïve CD4⁺ T cells (data not shown).

Because DCs induce Th1 responses through IL-12²⁸, and becauseH. pylori stimulates DC IL-12 production^22,23,29, we next evaluated the effect of S-CM onH. pylori-stimulated MoDC secretion of IL-12p70. Importantly, both gastric and intestinal S-CM potently down-regulatedH. pylori-induced MoDC secretion of IL-12 (Figure 4D). Addition of rhIL-12p70 to co-cultures of S-CM-treated,H. pylori-pulsed DCs and CD4⁺ T cells partially restored T cell IFN-γ secretion (Figure 4E). Thus, both gastric and intestinal S-CM significantly reduced the ability ofH. pylori-pulsed MoDCs to induce an IFN-γ-mediated Th1 response, likely through impaired DC IL-12 release.

Reduced capacity of primary gastric and intestinal DCs to induce T cell expansion in response to H. pylori

Dendritic cells that reside in normal gastric and intestinal mucosa are exposed to mucosal stromal factors in their tissue environment. Therefore, we next determined whether primary human mucosal DCs are impaired in their response toH. pylori, as suggested by the precedingin vitro findings. Primary gastric and intestinal HLA-DR^high/CD13^low DCs isolated by our previously described protocols^3,30 constituted 5.3 ± 1.2% and 5.3 ± 1.8% of the total gastric and intestinal lamina propria cells, respectively (n=4). Gastric and intestinal HLA-DR^high DCs also expressed similar levels of CD11c, DC-SIGN and CD206 (Figure 5). Both DC populations displayed equally low baseline expression of CD80, CD83 and CD86, consistent with an immature phenotype and low level activation. Having previously shown thatH. pylori infection enhances primary mucosal DC maturation and activation³, we assessed the ability of primary gastric and intestinal DCs isolated from the same tissue donor and pulsed withH. pylori to induce T cell proliferation and cytokine secretion.H. pylori-pulsed primary gastric and intestinal HLA-DR^high DCs triggered only marginal T cell expansion (Figure 6A), consistent with the ability of S-CM to down-regulate MoDC-induced proliferative responses (Figure 4A). However, both mucosal DC populations induced T cell secretion of IFN-γ, indicative of a Th1 response (Figure 6B and C). In all experiments,H. pylori-pulsed gastric DCs and intestinal DCs induced nearly identical T cell responses. Thus, recapitulating ourin vitro results with S-CM-treated MoDCs, primary DCs isolated from gastric and intestinal mucosa are phenotypically similar and induce similar levels of T cell IFN-γ secretion in the absence of strong T cell proliferation in response toH. pylori.

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