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Infection and Immunity, February 2007, p. 810-819, Vol. 75, No. 2
0019-9567/07/$08.00+0     doi:10.1128/IAI.00228-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Chronic Exposure to Helicobacter pylori Impairs Dendritic Cell Function and Inhibits Th1 Development

Peter Mitchell,^1,5 Conrad Germain,¹ Pier Luigi Fiori,² Wafa Khamri,⁵ Graham R. Foster,³ Subrata Ghosh,⁴ Robert I. Lechler,⁵ Kathleen B. Bamford,^6, and Giovanna Lombardi^5*,

Department of Immunology, Division of Medicine, Faculty of Medicine, Imperial College at Hammersmith Hospital, London, United Kingdom,¹ Department of Biomedical Sciences, University of Sassari, Sassari, Italy,² Hepatobiliary Group, Centre for Adult and Paediatric Gastroenterology, Bart's and The London, Queen Mary's School of Medicine and Dentistry, London, United Kingdom,³ Department of Gastroenterology, Division of Medicine, Faculty of Medicine, Imperial College at Hammersmith Hospital, London, United Kingdom,⁴ Department of Nephrology and Transplantation, Kings College London School of Medicine, Guy's Hospital Campus, London, United Kingdom,⁵ Department of Infectious Diseases and Immunity, Division of Investigative Sciences, Faculty of Medicine, Imperial College at Hammersmith Hospital, London, United Kingdom⁶

Received 10 February 2006/ Returned for modification 2 April 2006/ Accepted 13 October 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Helicobacter pylori causes chronic gastric infection that affects the majority of the world's population. Despite generating an inflammatory response, the immune system usually fails to clear the infection. Since dendritic cells (DCs) play a pivotal role in shaping the immune response, we investigated the effects of H. pylori on DC function. We have demonstrated that H. pylori increased the expression of activation markers on DCs while upregulating the inhibitory B7 family molecule, PD-L1. Functionally, H. pylori-treated DCs resulted in the production of interleukin-10 (IL-10) and IL-23 but not of alpha interferon (IFN-). While very little or no IL-12 was produced to H. pylori alone, simultaneous ligation of CD40 on DCs induced IL-12 release. We also demonstrated that DCs treated with H. pylori-induced IFN- production by allogeneic naive T cells. However, stimulation of DCs with H. pylori for an extended period of time impaired their ability to produce cytokines after CD40 ligation and limited their ability to promote IFN- release, suggesting that the DCs had become exhausted by the prolonged stimulation. The effect of chronic infection with H. pylori on DC function was further investigated by focusing on DC development. Demonstrating that monocytes differentiated into DCs in the presence of H. pylori exhibited an exhausted phenotype with an impaired ability to produce IL-12 and a downregulation of CD1a. Our results raise the possibility that in chronic H. pylori infection DCs become exhausted after prolonged antigen exposure leading to suboptimal Th1 development. This effect may contribute to persistence of H. pylori infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Helicobacter pylori is known to cause chronic gastric infection in at least 50% of the world population. In addition to the inflammation caused by H. pylori, this pathogen is a risk factor for a number of other diseases, including gastric cancer, peptic ulcers, and gastritis (14, 23). Infection with strains containing the Cag pathogenicity island (CagPAI) has been identified as a particular risk factor for the development of these diseases in many populations (4, 24). This pathogenicity island contains the genes for a type IV secretory system that delivers CagA to the target cell (5). CagPAI-positive H. pylori have been shown to elevate interleukin-8 (IL-8) secretion in mucosal cells, exacerbating the local inflammatory response (42). Despite eliciting an inflammatory response with resulting tissue damage, the immune response fails to eradicate the bacteria, leaving the host with a prolonged infection. Some recent studies have suggested that the type of T-cell responses to H. pylori may be responsible for the host's inability to clear the infection and that development of regulatory T cells modulates the immune response, thus preventing eradication of the pathogen (34, 36).

Dendritic cells (DCs) play an important role in shaping the immune response to pathogens. Responding to danger signals, these cells mature and migrate to the lymph nodes presenting antigens harvested from the site of infection (8, 27, 47). In addition to presenting pathogen-specific antigen to T cells, DCs can shape the T-cell response according to their state of activation. Different pathogens can interact with DCs via their Toll-like receptors (TLRs), influencing the DCs' surface proteins and cytokine production (26, 41). Some of these cytokines determine the outcome of subsequent T-cell activation after encounter with the primed DC; IL-12 drives a Th1 response, while IL-10 may promote a Th2/Treg response (7). Maturation of DCs is further influenced through CD40 ligation by CD40L expressed predominantly on surrounding T cells. This interaction leads to an amplification of cytokine production and, in mice, has been shown to be necessary for significant production of IL-10 and IL-12 by DCs when responding to microbial triggers (13, 48, 49). CD40L can be found in the periphery on T cells in the local tissue or on activated platelets (21) and in lymph nodes primarily on T cells. Recent studies have shown that IL-12 is produced by DCs in response to H. pylori, although in one of these reports the levels released were reduced compared to IL-12 production in response to Salmonella enterica serovar Typhimurium (18, 30). However, the influence of CD40 ligation on the ability of H. pylori to stimulate IL-10 and IL-12 production by DCs has not been investigated. Importantly, and relevant to a naturally chronic infection, the effect of persistent exposure to H. pylori on DC cytokine production is not understood.

There are other factors potentially involved in the DC response to H. pylori that could influence the development of a Th1 response. One is the production of IL-23, a new member of the IL-12 family of cytokines with a novel role in regulating T helper balance (25). IL-23 has been shown to increase IL-17 secretion, although it is not involved in the differentiation of IL-17-producing T cells (1, 53). Type I interferons (IFNs) have also been shown to have a role in modulating the immune system and in particular can regulate IL-12 production by DCs (15, 22). Whether these mediators contribute to the development of T-cell responses in H. pylori infection is not known.

H. pylori causes chronic infection that would result in prolonged antigen exposure. It has been shown that prolonged exposure to other stimuli cause DCs to become "exhausted" (29, 32), leading to a reduction in cytokine production and alteration in the stimulation of naive T cells so that they predominantly generate Th2 and nonpolarized T cells. This leads to the possibility that the time taken by the infected DCs to reach the lymph nodes may play an important role in chronic infection. While the predominant T-helper response in natural human infections with H. pylori is of a Th1 type, there is evidence that the strength of the Th1 response may be suboptimal. This might be a response to impaired activation of T cells by DCs (39, 44).

The initial interaction of residential DCs with pathogens is important in DCs' role in the immune response. However, there is also evidence that pathogens can affect how monocytes entering the site of infection differentiate into DCs (46). Work with Mycobacterium tuberculosis has shown that DCs generated from M. tuberculosis-infected monocytes have an altered phenotype (16, 37, 38).

Growing evidence indicates the involvement of DCs in response to H. pylori infection. Recently, Kao et al. demonstrated the recruitment of DCs to the gastric mucosa after H. pylori infection in mice (28).

The aim of the present study was to investigate whether an impaired DC function may be responsible for the chronicity observed in H. pylori infection. We have analyzed here the effects in vitro of short-term and extended exposure to live and paraformaldehyde-fixed H. pylori on differentiating and already differentiated DCs and the functional consequences for naive T cells. The results suggest that prolonged exposure of DCs to H. pylori leading to DC exhaustion may contribute to the chronicity of infection in vivo.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial preparations. The CagPAI-positive strain NCTC12455 was grown on Columbia base blood agar plate (Oxoid, Hampshire, United Kingdom) for 3 days at 37°C in microaerophilic (5% H₂, 10% CO₂, 5% O₂, 80% N₂) conditions in a Macs-VA 500 workstation (Don Whitley, West Yorkshire, United Kingdom). Confluent plates of bacteria were harvested in phosphate-buffered saline (PBS) and washed twice by centrifugation at 1,000 x g for 5 min. The number of bacteria was determined by calculating the A₅₅₀, with a optical density of 0.8 corresponding to 10⁸ CFU/ml. The collected bacteria were either used directly as live bacteria or fixed for 1 h in 4% paraformaldehyde, washed four times in PBS, and adjusted to the desired final concentration. Escherichia coli (enteropathogenic E. coli strain, characterized by the presence of eaeA and bfpA genes by PCR) was cultured in Luria-Bertani broth at 37°C, and bacteria were harvested, washed, and fixed for 4 h in 4% paraformaldehyde. The cells were then washed and stored in PBS (Oxoid).

Generation of DCs. Monocytes were separated from Buffy coats provided by the National Blood Transfusion Centre (South Thames, United Kingdom) using CD14-Beads (Miltenyi Biotec Surrey, United Kingdom). These cells were then cultured in RPMI 1640 (Invitrogen, Paisley United Kingdom) with 10% fetal calf serum (FCS; FCS SeraQ; Sussex, United Kingdom), penicillin, streptomycin, and L-glutamine (PSG; PAA Laboratories GmbH, Pasching, Austria). To develop DCs, IL-4 (First Link, West Midlands, United Kingdom) at 10 ng/ml and granulocyte-macrophage colony-stimulating factor (GM-CSF; kindly donated by S. Brett, GlaxoSmithKline, Stevenage, United Kingdom) at 20 ng/ml were added every 2 days before the cells were harvested at day 5.

DCs were also generated in the presence of pathogens or lipopolysaccharide (LPS) by adding the optimal dose of E. coli LPS (100 ng/ml; Sigma, St. Louis, MO), E. coli (10⁸ CFU/ml), or H. pylori (10⁶ CFU/ml) in addition to the IL-4 and GM-CSF on the first day (day 0) of monocyte culture and then every 2 days thereafter before the DCs were harvested on day 5.

DC activation. DCs (5 x 10⁵ cells) were incubated in the presence of 10⁸ to 10⁴ CFU of H. pylori antigen or 100 ng of E. coli LPS/ml for 48 h in 500 µl of RPMI 1640 supplemented with 10% FCS and PSG. The cells were then stained for CCR7 (R&D Systems, Minneapolis, MN), CD1a (Autogen-Bioclear, Wiltshire, United Kingdom), CD11c (Caltag, Burlingame, CA), CD14 (Caltag), CD40 (Caltag), CD83 (Caltag), CD86 (Caltag), HLA-DR (Sigma), ILT-3 (Immunotech, Marseille, France), and PD-L1 (eBioscience, San Diego, CA) using fluorescein isothiocyanate (FITC), R-Phycoerythrin and R-Phycoerythrin-cyanine 5-conjugated mouse anti-human monoclonal antibodies. The cells were then analyzed by using a FACScalibur flow cytometer and CellQuest software (Becton Dickinson, San Jose, CA).

CD40 ligation of DCs. CD40L-transfected L cells (CD40L-Tx) (49) were grown in RPMI 1640 with 10% FCS and PSG. Once growth was confluent, cells were harvested by incubating them with EDTA (Cambrex, Verviers, Belgium) for 15 min. They were then removed from the flask, washed in PBS, and resuspended in RPMI 1640 with 10% FCS and PSG. DCs (6 x 10⁴ cells) were incubated in 500 µl of RPMI 1640 with 10% FCS and PSG, alone or in the presence of CD40L-transfected L cells (1.5 x 10⁵ cells). These DCs were then incubated with H. pylori, E. coli, LPS, or medium alone. Supernatants were collected at the desired time points and stored at –20°C until analysis.

Naive T-cell purification. Naive T cells were separated from Buffy coats by negative selection. Using a cocktail of antibodies against CD8, CD45RO, CD33 (Caltag), CD14, CD16, CD19, CD56 (Diaclone Research, Besaucon, France), and -TCR (Becton Dickinson), unwanted cells were removed by using BioMag goat anti-mouse immunoglobulin G Fc (QIAGEN, Germantown, MD). Naive T-cell purity was analyzed by flow cytometry.

Mixed lymphocyte reaction. DCs (10⁶ cells) were incubated in the presence of H. pylori CagPAI-positive strains, E. coli, or 100 ng of E. coli LPS/ml for 8 or 48 h, and the DCs were then harvested and washed. Different numbers of DCs ranging between 10⁵ and 10³ cells were cocultured in triplicate with naive T cells (2 x 10⁴ cells) in a final volume of 250 µl of 10% FCS in RPMI for 5 days in 96-well plates. On day 5, 75 µl of supernatant was removed for IFN- and IL-5 enzyme-linked immunosorbent assay (ELISA) and 1 µCi of [³H]thymidine (GE Healthcare UK, Ltd., Buckinghamshire, United Kingdom)/well was added overnight before harvesting and counting with a ß-counter (Perkin-Elmer, Wellesley, MA) the following day.

ELISA. IL-5, IL-10, IL-12, and IFN- in supernatants were analyzed by an indirect sandwich ELISA, using purified and biotinylated anti-IL-5, anti-IL-10, anti-IL-12, and anti-IFN- (BD Pharmingen, San Diego, CA), and visualized with TMB (Zymed, San Francisco, CA) measuring the optical density at 450 nm on an automatic plate reader (Titertek Multiscan PLUS; Titertek, Helsinki, Finland). The amount of each cytokine was calculated from a standard curve. IL-23 was measured by using an IL-23 ELISA commercial assay (Bender Medsystems, Vienna, Austria) according to the manufacturer's instructions.

Luciferase assay for type I IFN detection. IFN- production was measured by using a luciferase bioassay. Briefly, human fibrocarcinoma HT1080 cells containing a luciferase cDNA under the control of the IFN- receptor. These cells were grown in 96-well plates until confluent. Samples, or standards from 100 U of IFN-2a (Hoffman-LaRoche, Welwyn Garden City, United Kingdom)/ml were added to the plate in duplicate and incubated for 6 h at 37°C. Supernatants were then discarded, and the cells were lysed with lysis buffer (Promega, Southampton, United Kingdom). The plates were frozen at –80°C and then thawed; 50 µl of Luciferin (Promega) was then dispensed, and the luminescence was measured by using a Lumuniskan Ascent luminometer (Thermo Labsystem, Franklin, MA).

Statistical analysis. Statistical analyses were performed by using StatView version 5.0.1 for Windows (SAS Institute, Inc., Cary, NC). Parametric data were analyzed by using the Student t test, and nonparametric data were analyzed by using the Wilcoxon signed-rank tests. Statistical significance was assumed for P values of <0.05.

Top Abstract Introduction Materials and Methods Results Discussion References

 
H. pylori induces phenotypic maturation of DCs. The effect of H. pylori on monocyte-derived DCs was determined by exposing in vitro differentiated DCs to different doses (10⁴ to 10⁸ CFU/ml) of paraformaldehyde-fixed H. pylori. In addition, E. coli LPS (100 ng/ml) or intact paraformaldehyde-fixed E. coli (10⁸ CFU/ml) were used as positive controls (49). The expression of activation markers was evaluated. DCs exposure to the optimal dose of paraformaldehyde-fixed H. pylori (10⁶ CFU/ml) for 48 h led to the upregulation of HLA-DR, CD86, and CD40 (Fig. 1 and Table 1). In addition, CD83 and CCR7 were also upregulated, whereas there was a slight reduction in the expression of CD1a (data not shown). Similar results were obtained with LPS and E. coli (Table 1). All of the DC markers analyzed were significantly upregulated by H. pylori, E. coli, and LPS treatment compared to the use of medium alone (P < 0.05; Table 1).

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FIG. 1. DCs upregulate stimulatory and inhibitory molecules in response to H. pylori.DCs were treated at day 6 for 48 h with whole paraformaldehyde-fixed H. pylori (106 CFU/ml). Cells were stained for HLA-DR, CD86, CD40, and PD-L1 and compared to their isotype controls. The mean fluorescence intensity above the isotype control is given in parentheses. This analysis is representative of five experiments with similar results.

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TABLE 1. Upregulation of cell surface markers on DCs treated with paraformaldehyde-fixed H. pylori

Expression of the recently described member of the B7 family of molecules, PD-L1, has been implicated in the inhibition of T-cell function (9). Expression of ILT3 has also been associated with tolerogenic properties in human DCs (10, 11). PD-L1 (Fig. 1 and Table 1) and, marginally, ILT-3 (data not shown) were upregulated compared to medium alone at 48 h in response to paraformaldehyde-fixed H. pylori. Upregulation of both PD-L1 and ILT-3 were also observed in response to E. coli and LPS.

Although paraformaldehyde-fixed pathogens were selected to evaluate the function of intact H. pylori in the absence of bacterial soluble factors, the effect of live H. pylori was compared. With the exception of PD-L1 expression at 10⁵ and 10⁶ CFU/ml (P < 0.05), similar upregulation of markers was observed with no significant differences between the live and paraformaldehyde-fixed H. pylori-treated DCs (Table 2).

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TABLE 2. Upregulation of cell surface markers on DCs treated with live and paraformaldehyde-fixed (dead) H. pylori

H. pylori induces the production of IL-10 by DCs, whereas CD40 ligation is required for IL-12 release. To investigate the functional consequences of DCs exposure to H. pylori, the production of IL-10 and IL-12 by DCs was measured after 24 h of culture, as previously described (49). DCs produced IL-10 in response to a range of doses of paraformaldehyde-fixed H. pylori. The peak of IL-10 production was seen in response to doses between 10⁶ and 10⁷ CFU/ml (data not shown). In contrast, no IL-12 release was observed in response to any dose of paraformaldehyde-fixed H. pylori (data not shown). In contrast, DCs stimulated with E. coli produced both IL-10 and IL-12 with response peaks at doses of 10⁵ and 10⁷ CFU/ml, respectively, although previous results have suggested that a dose of 10⁸ CFU/ml was necessary for efficient DC activation (49).

CD40 ligation has been shown to be necessary for significant production of IL-10 and IL-12 by DCs when responding to microbial triggers (48, 49). To model the second signal provided by CD40L-CD40 interaction, DCs were cocultured with CD40L-transfected cells (CD40L-Tx) with or without paraformaldehyde-fixed H. pylori antigen. In the presence of CD40L-Tx, DCs produced IL-12 in response to H. pylori, which peaked between 10⁵ and 10⁶ CFU/ml, although the amount produced was lower compared to E. coli (data not shown). Ligation of CD40 on DCs also leads to the upregulation of IL-10 in response to H. pylori and E. coli (data not shown). Similar to the observation with H. pylori, LPS was also found to induce production of IL-12 in the presence of CD40L-Tx (data not shown) Based on the optimal cytokine secretion for IL-10 and IL-12, a concentration of 10⁶ CFU of paraformaldehyde-fixed H. pylori/ml was selected for the subsequent experiments.

The production of IL-12 and IL-10 in DCs treated with paraformaldehyde-fixed H. pylori (10⁶ CFU/ml), E. coli (10⁸ CFU/ml), and E. coli LPS (100 ng/ml) in the presence of CD40L-Tx from at least 10 experiments is depicted in Fig. 2A and B. As previously shown, H. pylori stimulated production of IL-10 and while little or no IL-12 was observed with DCs alone, in the presence of CD40L-Tx cells IL-12 production by DCs was observed in response to H. pylori and LPS (Fig. 2A and B). The production of IL-10 was observed in the presence of CD40L-Tx alone, whereas IL-12 production in response to CD40L-Tx was minimal compared to the use of medium alone. These differences were significant as determined by analysis with a Wilcoxon signed-rank test (P < 0.05).

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FIG. 2. H. pylori-treated DCs can produce IL-12 in response to H. pylori when CD40 is ligated. (A and B) DCs were incubated with medium (diamonds), H. pylori (106 CFU/ml; squares), E. coli (108 CFU/ml; circles), and LPS (100 ng/ml; triangles) for 24 h in the presence of CD40L-Tx (filled symbols) or in the absence of CD40L stimulation (open symbols). IL-10 (A) and IL-12 (B) production was measured by ELISA. Each dot represents one experiment for a total of 10. *, P < 0.05 (Wilcoxon signed-rank test compared to medium alone); #, P < 0.05 (Wilcoxon signed-rank test compared to medium plus CD40L-Tx). (C and D) DCs were incubated alone (medium) or with H. pylori live or dead (105 to 107 CFU/ml) in the absence or in the presence of CD40L-Tx cells for 24 h before the supernatants were removed, and IL-10 and IL-12 levels were analyzed by ELISA.

When paraformaldehyde-fixed H. pylori was compared to live H. pylori, similar amounts of IL-10 and IL-12 were observed with an increased release of IL-12 when CD40 was ligated on DCs simultaneous with H. pylori infection (Fig. 2C and D).

H. pylori-treated DCs produce IL-23 but no type I IFNs. In addition to IL-12, we also studied IL-23, a new member of the IL-12 family of cytokines that shares the p40 subunit with IL-12 and is important in the survival and expansion of IL-17-producing T cells (1, 25, 53). Paraformaldehyde-fixed H. pylori stimulated DCs produced IL-23 at the highest doses of H. pylori in a manner that was comparable to the production seen in response to E. coli. Simultaneous ligation of CD40 on DCs amplified the production of IL-23 with paraformaldehyde-fixed H. pylori and was comparable to E. coli (Fig. 3A). The amount of IL-23 produced by DCs treated with H. pylori and E. coli was significant (at least from five experiments) as determined by the Wilcoxon signed-rank test (P < 0.05).

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FIG. 3. H. pylori stimulates the production of IL-23 but no type I IFNs. DCs treated with H. pylori (104 to 108 CFU/ml; squares) and E. coli (104 to 108 CFU/ml; circles) in the absence (open symbols) or presence (filled symbols) of CD40L-Tx. IL-23 (A) and IFN- (B) production in the supernatants were measured by using ELISA and luciferase assays, respectively.

Type I IFNs are another family of cytokines that have been implicated in affecting T helper development (33, 45). While type I IFNs were detectable in response to both E. coli and LPS stimulation of DCs, paraformaldehyde-fixed H. pylori did not lead to IFN-/ß production directly or in the presence of CD40L-Tx stimulation (Fig. 3B). Only E. coli treatment of DCs induced production of significant amounts of IFN-/ß compared to medium alone (at least from six experiments) as determined by the Wilcoxon signed-rank test (P < 0.05).

IFN- production by allogeneic T cells stimulated by H. pylori-treated DCs decreases with the time of H. pylori exposure. Having established that the exposure of differentiated DCs to H. pylori predominantly gives rise to IL-10 production, we investigated the effect of paraformaldehyde-fixed and live H. pylori-treated DCs on naive T cells. DCs were preincubated with H. pylori, E. coli, and LPS for either 8 or 48 h before being washed and cocultured with allogeneic naive T cells. These time points were chosen to represent DC migration away from the source of antigen exposure to the local lymph node. It has been shown that after stimulation by LPS, DCs reached the lymph nodes between 6 and 24 h later (35). In addition, data from mouse models have demonstrated that T-cell interaction with DCs is maximal between 6 and 48 h (40). In line with previous ex vivo observations with gut biopsies (6), DCs exposed to H. pylori for a short period of time, in this case 8 h, induced a Th1 response with the production of IFN- (Fig. 4C) and little IL-5 (data not shown), along with T-cell proliferation (Fig. 4A). However, there was impaired IFN- production by naive T cells responding to DCs treated with H. pylori for a prolonged period of time (48 h) (Fig. 4D), together with a small increase in IL-5 production (data not shown). This reduction in IFN- production by T cells was observed in response to DCs treated with both live and paraformaldehyde-fixed H. pylori and was found to be significant as determined by the Student t test (P < 0.05). In contrast, proliferation of naive T cells in response to the DC stimulation was still observed after prolonged exposure, suggesting that DCs incubated with pathogens for 48 h are still alive and capable of stimulating T cells (Fig. 4A and B). The IFN- production in response to H. pylori was also compared to the response to E. coli and LPS. Both E. coli- and LPS-treated DCs stimulated IFN- production by T cells, and this response was impaired when DCs were treated for 48 h (P < 0.05 [Student t test]).

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FIG. 4. IFN- production by allogeneic naive T cells stimulated by DCs decreases with increased time of exposure to H. pylori. DCs were incubated with 106 CFU of live () or paraformaldehyde-fixed () H. pylori/ml, LPS (100 ng/ml; ), and E. coli (108 CFU/ml; •) for 8 h (A and C) and 48 h (B and D) or left untreated DCs (). The DCs were then washed and cocultured with 2 x 104 allogeneic naive T cells for 5 days; supernatants were then obtained, and the IFN- (C and D) production was measured by ELISA. The cells were pulsed on day 5 with [3H]thymidine, and the cells were harvested the following day to measure proliferation by determining the thymidine incorporation (A and B). These same DC preparations were also cultured in the presence of CD40L-Tx for 24 h. The supernatant was then removed, and IL-10 and IL-12 (E, F, G, and H) production was measured by ELISA. *, P < 0.05 (Student t test comparing cytokine production at 8 and 48 h).

DCs stimulated with H. pylori become exhausted. In light of the different T-cell responses observed after incubation with H. pylori for 8 and 48 h, we looked at IL-10 and IL-12 production by DCs incubated with H. pylori for 8 and 48 h, followed by CD40 ligation (incubation with CD40L-Tx for further 24 h). After 8 h of exposure to H. pylori, followed by subsequent CD40 cross-linking, DCs produced IL-12 and IL-10 in amounts comparable to the cytokine levels released by DCs incubated for 8 h in medium alone (Fig. 4E and G). However, after 48 h exposure to H. pylori the DCs appeared to be exhausted in that cells treated with H. pylori produced little IL-10 or IL-12 (Fig. 4F and H). Identical cytokine profiles were observed with DCs treated with live and paraformaldehyde-fixed H. pylori. The exhausted phenotype and function of DCs was not restricted to H. pylori only, but similar results were obtained with DCs incubated with E. coli and LPS (Fig. 4).

The possibility that DCs were dead rather than exhausted was investigated by annexin staining using DCs incubated with paraformaldehyde-fixed H. pylori (Table 3). The results obtained suggest that DC viability was only partially affected, and no differences were observed between the incubations of DCs with H. pylori for 8 and 48 h.

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TABLE 3. Apoptosis of paraformaldehyde-fixed H. pylori treated DCs

DC differentiation from monocytes is affected by exposure to H. pylori. During an inflammatory response, pathogens at the site of infection can influence not only DCs that are already differentiated but also newly recruited monocytes. We investigated the effect of H. pylori on monocytes differentiation. Freshly separated monocytes were cultured with GM-CSF and IL-4 in the presence or absence of paraformaldehyde-fixed H. pylori and E. coli as well as LPS. DCs had upregulated HLA-DR and marginally upregulated CD86 or PD-L1 when derived from paraformaldehyde-fixed H. pylori-treated monocytes. However, it is important to note that the CD1a expression was downregulated and the CD14 expression was maintained (Fig. 5).

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FIG. 5. DCs differentiated from monocytes in the presence of H. pylori have an altered phenotype and impaired cytokine production. DCs were differentiated from monocytes in the presence of H. pylori, E. coli, and LPS. After 5 days these cells were stained for CD14, CD1a, HLA-DR, CD86, and PD-L1 and then compared to their isotype controls (shaded histograms). The mean fluorescence intensities are given in parentheses.

The DCs generated in the presence of paraformaldehyde-fixed H. pylori also exhibited impaired production of both IL-10 and IL-12 when stimulated for 24 h with CD40L-Tx compared to DCs generated in the absence of pathogens or DCs stimulated simultaneously with paraformaldehyde-fixed H. pylori or LPS and CD40L-Tx at day 5 (Fig. 6A and B).

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FIG. 6. The DCs differentiated with H. pylori (Hp-moDCs), E. coli (Ec-MoDCs), or LPS (LPS-moDCs) were also incubated in the absence or in the presence of CD40L-Tx for 24 h, and supernatants were harvested. In addition, day 6 immature DCs were simultaneously treated with H. pylori (106 CFU/ml), E. coli (108 CFU/ml), LPS (100 ng/ml), or medium alone in the presence or absence of CD40L-Tx cells; supernatants were harvested at 24 h. IL-10 (A) and IL-12 (B) production was assessed by ELISA. *, P < 0.05 (Student t test comparing cytokine production by DCs differentiated with pathogens and immature already-differentiated DCs treated with pathogens). DCs differentiated from monocytes in the presence of H. pylori (), E. coli (•), and LPS () at day 5 were cocultured with naive T cells (C and D). The proliferation in counts per minute (C and E) and the IFN- levels (D and F) were compared to day 6 immature DCs () and immature DCs treated with E. coli (•), H. pylori () and LPS () for 8 h (E and F).

Monocytes differentiated in the presence of paraformaldehyde-fixed H. pylori and E. coli as well as LPS were used as stimulators in mixed lymphocyte reactions and compared to DCs incubated with paraformaldehyde-fixed H. pylori, E. coli, and LPS for 8 h. While T cells cultured with already differentiated DCs pretreated for 8 h with paraformaldehyde-fixed H. pylori produced IFN-, T cells cultured with DCs generated in the presence of paraformaldehyde-fixed H. pylori produced low amounts of IFN- and only with the highest number of DCs (Fig. 6D and F). The difference observed was significant (P < 0.05 [Student t test]). A slight increase in IL-5 production was observed (data not shown). In contrast, T-cell proliferation in response to DCs treated with paraformaldehyde-fixed H. pylori for 8 h only was comparable to DCs generated in the presence of paraformaldehyde-fixed H. pylori (Fig. 6C and E).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results presented in this study indicate that H. pylori is capable of activating DCs and that these DCs can produce cytokines that lead to a Th1 response. However, the sustained exposure to H. pylori impairs DC function and inhibits the Th1 response. Furthermore, newly recruited monocytes that differentiate into DCs in the presence of H. pylori show an altered phenotype and have a further inhibitory effect on Th1 development. Altogether these data suggest that during H. pylori infection DC function can be impaired and Th1 development inhibited.

These data confirm a recent report that H. pylori exposure leads to upregulation of DCs costimulatory molecules (31). To extend this, we have also examined the influence of H. pylori on the expression of PD-L1 and ILT-3, molecules that are implicated in regulatory effects through the inhibition of T-cell responses (9-11), and shown that these are upregulated to different degrees. We have also demonstrated that E. coli and LPS can also lead to the upregulation of PD-L1 and ILT-3, suggesting that this is a general response to pathogens rather than specific to H. pylori. Thus, since DC maturation can lead to the upregulation of both costimulatory and inhibitory molecules, the balance between the two types of molecules may influence the resulting T-cell responses.

DCs produce predominantly IL-10 in response to H. pylori, whereas E. coli induces IL-12, IFN-, and IL-10 release in vitro (49). Our experiments show that cross-linking DCs with CD40L is required for IL-12 production in response to H. pylori. This is only partially consistent with previous results, where production of IL-12 by human monocyte-derived DCs treated with H. pylori was observed (18, 30, 31). Furthermore, no significant IL-10 production by DCs in response to H. pylori was observed in one of these studies (18). The differences in cytokine profiles observed between this and other studies may be due to the use of different bacterial preparations. In the present study we used paraformaldehyde-killed H. pylori, which contrasts with the sonicated gentamicin-killed or live bacteria used in a previous report (19). To address this, we also carried out experiments to compare the effect of live versus killed bacteria in this setting which showed that the DC responses to paraformaldehyde-killed and live H. pylori had an identical profile. Our data are confirmed in a more recent publication wherein the two types of preparations of H. pylori gave rise to the same cytokine pattern in contrast to a sonicated preparation that led to a reduction in IL-12 production by DCs (31). Our results are also consistent with previous findings in which DCs treated with H. pylori produced lower amounts of IL-12 than did DCs treated with S. enterica serovar Typhimurium (18). This suggests that active (energy-requiring) suppression of DCs is not the only mechanism H. pylori uses to modulate DCs function and that bacterial surface molecules are likely to play a role since paraformaldehyde-fixed H. pylori preparations have similar consequences on DC phenotype and function. It has been suggested that H. pylori can evade an effective immune response by poorly stimulating TLRs. LPS and flagellin from H. pylori have been shown to be less potent than the same components in E. coli or other pathogens (12, 17), which may in part explain the poor cytokine response to H. pylori. Interestingly, we did show that H. pylori was capable of stimulating IL-23. Although its influence on IL-12 and IFN- production remains unclear (25), IL-23 has been shown to expand T cells producing IL-17 (1, 53).

In our study, H. pylori-treated DCs activated allogeneic naive T cells and initially favored a Th1 response. This confirms previous in vitro observations (20) and is consistent with in vivo studies (6, 51). IL-12 and the development of a Th1 response have been shown to be essential for an effective response against H. pylori (2, 43). Although the very low production of IL-12 may reduce the effectiveness of the host response, we have now shown that in a human system the additional interaction between CD40 and CD40L can overcome this limitation. This is supported by previous studies in the murine system (10, 48).

Recent reports have shown that the duration of antigen exposure can influence the outcome of T-cell activation. In these experiments longer exposure led to a predominantly Th2/Th0 response (32). Additional work has shown that DCs treated with low levels of LPS for 24 h subsequently have an impaired response to further stimulation with a higher dose of LPS (29). This is relevant to our work because H. pylori causes a chronic infection in situations where there is continuous exposure to the infecting agent. We therefore investigated the hypothesis that continuous exposure to H. pylori could exhaust DCs and lead to impaired function. Here we show that certain functions of DCs exposed to H. pylori for 48 h are reduced in a dose-dependent manner, producing less IL-12 in response to CD40L-Tx cells. This effect was also observed in the T-cell response to pretreated DCs with these cells showing reduced IFN- release with a slight upregulation of IL-5 production from the background level. Interestingly, naive T cells were still able to proliferate in response to these DCs, suggesting a dissociation of cytokine from proliferative responses. The effect of DC exhaustion may play an important role in H. pylori infection. It is clear from this and previous work on the effect on exhaustion that an increase in the time it takes for DCs to reach the lymph node can lead to an impaired Th1 response (32). While in vivo analysis indicates that there is IFN- production in the site of infection, this production is not limited to T cells, since natural killer cells have been shown to produce IFN- in response to H. pylori (20). In addition, in vivo evidence suggests that, while a Th1 response has been detected (50, 52) in H. pylori-infected individuals, this response is probably suboptimal (39, 44).

Monocytes entering the site of infection can have their development into DCs impaired by the presence of LPS or whole bacteria (46). Other work with Leishmania has shown that after 28 days of infection DCs have defective CCR7 expression, reducing their capacity to migrate in response to the infection (3). Having shown that chronic exposure to H. pylori has a detrimental effect on the ability of DCs to induce a Th1 response, we went on to examine the effect of prolonged exposure on DC development. We have shown that generating DCs in the presence of H. pylori leads to an inhibition of CD1a expression, a lack of CCR7 upregulation, maintenance of CD14 expression, and limited IL-12 and IL-10 production, indicating that DCs produced in an H. pylori-rich environment may develop an "exhausted" DC phenotype. Similar results have been observed in vitro with DCs generated from monocytes in the presence of M. tuberculosis (16, 37, 38).

These results indicated that the persistent presence of pathogens can influence DC differentiation and subsequent function. DCs generated in the continued presence of bacteria in the gut may have an altered phenotype due to the presence of the infection. This in turn could lead to exhausted DCs presenting to naive T cells. The role that DCs play late in infection may be as important as their role in initiating the immune response. Although this effect of "exhaustion" was observed with both H. pylori and E. coli, indicating a general response to pathogen, it is important to consider the nature of each infection. While the stomach is normally a relatively sterile site, H. pylori is uniquely adapted to this niche. Is may also be unique among noninvasive mucosal pathogens in requiring a Th1 IL-12-dependent response for effective eradication and thus be more dependent on DC presentation to T cells. In the case of H. pylori the low immunogenicity of a number of bacterial components, coupled with repeated exposure, may enhance the bacterium's ability to persist, since any DCs generated or matured during ongoing exposure to H. pylori would have impaired function, thus limiting any sustained response. Future immunotherapy or vaccine strategies should consider interventions that might overcome DC exhaustion.

 
This study was in part financed by the Wexham Gastrointestinal Trust. Peter Mitchell's was supported by an MRC studentship.

We thank M. Thursz (Imperial College Faculty of Medicine, London) for providing stocks of H. pylori.

 
* Corresponding author. Mailing address: Immunoregulation Laboratory, Department of Nephrology and Transplantation, 5th Fl., Thomas Guy House, King's College London School of Medicine at Guy's, King's College and St. Thomas' Hospitals, London SE1 9RT, United Kingdom. Phone: 44 (0) 2071887674. Fax: 44 (0) 2071887675. E-mail: giovanna.lombardi{at}kcl.ac.uk.

Published ahead of print on 13 November 2006.

Editor: J. L. Flynn

K.B.B. and G.L. contributed equally to this study.

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Infection and Immunity, February 2007, p. 810-819, Vol. 75, No. 2
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