Involvement of Toll-Like Receptors on Helicobacter pylori-Induced Immunity

Dendritic cells (DCs) play a major role in the innate immune response since they recognize a broad repertoire of PAMPs mainly via Toll-like receptors (TLRs). During Helicobacter pylori (H. pylori) infection, TLRs have been shown to be important to control cytokine response particularly in murine DCs. In the present study we analyzed the effect of blocking TLRs on human DCs. Co-incubation of human DCs with H. pylori resulted in the release of the pro-inflammatory cytokines IL-12p70, IL-6 and IL-10. Release of IL-12p70 and IL-10 was predominantly influenced when TLR4 signaling was blocked by adding specific antibodies, suggesting a strong influence on subsequent T cell responses through TLR4 activation on DCs. Co-incubation of H. pylori-primed DC with allogeneic CD4+ T cells resulted in the production of IFN-γ and IL-17A as well as the expression of Foxp3, validating a mixed Th1/Th17 and Treg response in vitro. Neutralization of TLR4 during H. pylori infection resulted in significantly decreased amounts of IL-17A and IFN-γ and reduced levels of Foxp3-expressing and IL-10-secreting T cells. Our findings suggest that DC cytokine secretion induced upon TLR4-mediated recognition of H. pylori influences inflammatory and regulatory T cell responses, which might facilitate the chronic bacterial persistence.


Introduction
Helicobacter pylori (H. pylori) is a gram-negative bacillus that selectively colonizes the human gastric mucosa. Since its discovery in 1984, it has been recognized as the principal cause of peptic ulcer disease and as the main risk factor for the development of gastric cancer [1]. In order to successfully colonize the human stomach, H. pylori must initially overcome multiple innate and adaptive host defenses. Indeed, during acute infection, a strong gastric mucosal inflammation caused by acute neutrophilic infiltration is accompanied by H. pylori-specific IgM and IgA immune response as well as an infiltration of CD4 + and CD8 + to the stomach [2]. However, despite the development of an acquired immune response, H. pylori can persistently colonize the stomach for decades.
Moreover, in the later course of the infection increased amounts of CD4 + /CD25 + regulatory T cells (T regs ) expressing Foxp3 are observed [3]. This cell type is known to further suppress effector T cell responses, thereby enabling H. pylori persistence and pathology. Interestingly, the pathogenic persistence is presumably facilitated through the induction of tolerance. In this context, DCs have been show to play a crucial role. Beside their ability to enter the gastric epithelium in order to take up bacteria, DCs play an exceptional role in the regulation of T cell-mediated immune responses which are influenced by the strength of antigen presentation, intensity of costimulation [4] and by the milieu of secreted cytokines [5].
An involvement of TLR signaling on the cytokine response of murine DCs has been shown already a few years ago [15]. However, there is no clear evidence how TLR activation influences intracellular DC signaling during H. pylori infection. In the present study we demonstrate the involvement of TLRs in inflammatory cytokine response and maturation of human DCs during H. pylori infection, thereby influencing adaptive immune responses.

Ethics Statement
DCs were generated from fresh blood samples taken from healthy volunteers for the purpose of this study as well as for serological analysis. The study was approved by the ethics committee of the Technische Universität München. All volunteers gave written informed consent.

Flow Cytometry
Cells were stained with Ethidium Monoazide bromide (EMA) (Anaspec, USA) for 30 min on ice for live/dead discrimination, and then resuspended in cold FACS buffer (PBS-1 % BSA). Fluorescence-labeled antibodies recognizing CD14, CD11c, HLA-DR, CD80, CD83, CD86, CD4, CD25 and Foxp3 (eBioscience, Germany) were added following manufacturer's instructions for 30 minutes at 4uC in the dark. After incubation, cells were washed and resuspended in FACS buffer before either analysis or staining of intracellular Foxp3 expression. Intracellular staining was performed by incubating the cells with Foxp3 antibody diluted in permeabilization buffer (eBioscience) according to manufacturer's protocol. After 30 minute incubation at room temperature cells were washed, resuspended in FACS buffer and analyzed. Analysis was performed with the FACS CyAn (Beckman Coulter) and the FlowJo software.

ELISA
For determination of cytokine secretions, culture supernatants were harvested and cytokine concentrations were assessed by sandwich ELISA according to the manufacturer's instructions (eBioscience, Germany).

CD4 + T cell isolation and co-cultivation with DCs
CD4 + T cells were separated from PBMCs by using the CD4 + T cell Isolation Kit II (Miltenyi Biotec, Germany). Their purity was determined by flow cytometry (anti-CD45 and anti-CD4). Immature DCs were infected with H pylori at MOI 5. After 24 h, H. pylori was killed by supplementation of Gentamycin (0,04 mg/ml) for 2-3 h, afterwards freshly isolated CD4 + T cells were added to the infected DCs at 2:1 ratio, as previously described [16]. DCs and CD4 + T cells were co-cultured for 3 days at 37uC in a humidified incubator containing 5% CO 2 . Foxp3 expression was analyzed by FACS and the released cytokines were measured by ELISA.

Statistical Analysis
For statistical analysis, ANOVA test with Dunnett's correction was performed.

H. pylori-mediated activation of TLR signaling influences DC maturation
The involvement of TLRs in the recognition of H. pylori by DCs was shown in mice [17], while little is known about their role in human cells. To investigate the effect of TLR signaling on human DC maturation upon H. pylori infection, we blocked the extracellular receptors TLR2, TLR4 and TLR5 with neutralizing antibodies prior H. pylori challenge. Maturation of DCs was characterized by analyzing the surface expression levels of the costimulatory molecule CD86 and the maturation marker CD83.
H. pylori infection induced maturation of human DCs, leading to increased expression of CD86 and CD83 compared to uninfected control cells. Interestingly, blocking of TLR signaling during H. pylori infection by using neutralizing antibodies affected the maturation status of DCs: the expression of CD86 and CD83 was significantly increased when TLR2 and TLR4 were blocked, whereas no effect was detected after inhibition of TLR5 ( Figure 1). These data indicate that TLR4 and to a lesser extent TLR2 signaling is involved in DC maturation during H. pylori infection.

Blocking of TLR2 and TLR4 influences DC cytokine response upon H. pylori infection
Maturation and antigen presentation of DCs is usually accompanied by cytokine secretion that can further influence T cell polarization, thereby shaping the adaptive immune response. Therefore, we next sought to investigate the influence of TLR activation on DC cytokine secretion in response to H. pylori infection.
Co-incubation of human DCs with H. pylori resulted in the release of the pro-inflammatory cytokines IL-12p70 and IL-6 that promote T helper cell type 1 (Th1) and Th17 polarization, respectively. Similarly, IL-10, an anti-inflammatory cytokine driving T reg differentiation, was also released upon H. pylori infection ( Figure 2).
Next, H. pylori-mediated cytokine secretion of DCs in the presence of neutralizing antibodies was determined. Secretion of IL-6 in response to H. pylori was mainly blocked upon neutralization of TLR2 and TLR4 (Figure 2), but was not affected by anti-TLR5 antibodies, suggesting that TLR2 and TLR4 prime Th17 responses. The release of IL-12p70, however, was predominantly influenced when TLR4 signaling was blocked, indicating that TLR4 is the main receptor inducing Th1 responses upon H. pylori infection. Furthermore, the secretion of anti-inflammatory IL-10 was mainly reduced upon TLR4 neutralization, suggesting that TLR4 does not only influence inflammatory T cell responses, but also regulatory immune responses. In summary these data indicate an important role of TLR4 for the cytokine release of human DCs in response to H. pylori that might influence the subsequent T cell response.

Blocking of TLRs on DCs during H. pylori infection influences T cell polarization
Since TLRs and specifically TLR4 deeply influence DC cytokine release, we next examined the effect of TLR inhibition on DCs on the subsequent T cell response towards H. pylori infection. Therefore, DCs were co-incubated with TLR-neutralizing antibodies and H. pylori for 24 h, afterwards CD4 + T cells were added and analyzed after 3 days of incubation. As a mixed Th1/Th17 response as well as a regulatory T cell response has been described in H. pylori-infected patients [18] [19] [20], the release of IFN-c and IL-17A as well as the expression of Foxp3 was determined after co-culture of DCs.
Co-incubation of H. pylori-primed DC with allogeneic CD4 + T cells resulted in the production of IFN-c and IL-17A as well as the expression of Foxp3 ( Figure 3A and B), validating a mixed Th1/ Th17 and T reg response also in vitro.
Blocking of TLR2 on DCs during H. pylori infection induced decreased secretion of IL-17A by co-incubated CD4 + T cells, however the release of IFN-c was less affected ( Figure 3A). Similarly, no significant differences were observed in the expression of Foxp3 by T cells (Figure 3B). These findings confirm an involvement of H. pylori-mediated TLR2-activation of human DCs on subsequent Th17 activation.
In contrast, neutralization of TLR4 during H. pylori infection, which led to reduced secretion of IL-6, IL-12p70 as well as IL-10 by human DCs, resulted in significantly decreased amounts of IL-17A and IFN-c ( Figure 3A). Furthermore, also the levels of Foxp3expressing and IL-10-secreting T cells were reduced ( Figure 3B), validating that DC cytokine secretion induced upon TLR4mediated recognition of H. pylori deeply influences inflammatory and regulatory T cell responses that might facilitate the pathogenic persistence of H. pylori.

Discussion
The adaptive immune response against bacteria is activated and regulated by several innate immune cells. However, especially during H. pylori infection, DCs seem to play a special role in the initiation of adaptive immune responses, since increased numbers of activated DCs have been detected in the lamina propria of H. pylori-infected individuals [21], while in murine models DCs have been shown to induce H. pylori-specific adaptive immune responses [22].
Several studies have pointed out a crucial role of TLR in the context of H. pylori infection and gastric disease [23] [24] [25]. Moreover, we showed that TLR-mediated recognition of H. pylori influences murine DC activation and cytokine secretion [26], however little is known in human DCs. We analyzed the involvement of TLR signaling in human DCs upon H. pylori infection by blocking the receptors with neutralizing antibodies and we found that distinct TLRs induce different cytokine secretion. While the release of IL-6 by DCs upon H. pylori stimulation was affected by the blockage of TLR2, TLR4 and TLR5, the release of IL-12p70 was mainly influenced by TLR4 neutralization, indicating that TLR4 but no other TLRs is essential for IL-12p70 secretion upon H. pylori infection. The release of IL-12p70 is synergistically enhanced by the activation of MyD88 and TRIF [26]. Importantly, TLR4 is the only receptor that can activate both, leading to the generation of inflammatory cytokines and type I interferons. The latter were shown to stimulate IL-12p70 production through an autocrine-paracrine loop [27]. In contrast, TLR2 activation can only induce the expression of messenger RNA (mRNA) encoding the p40 and p19 subunits of IL-23, but not the mRNA expression of the crucial p35 subunit of IL-12 [20].
Inhibition of TLR4 signaling does not only influence IL-12p70 secretion, but also the production of IL-10. TLR neutralization on DCs showed that TLR4 and in part TLR2 induced IL-10 secretion, suggesting that their activation might be responsible for the acquisition of a tolerogenic phenotype. Indeed, neutralization of TLR2 and TLR4 led to the restoration of DC maturation after H. pylori infection, indicating that the secretion of IL-10 was responsible for the weak expression of costimulatory molecules and activation markers. In line with our data, H. pylori was shown to induce IL-10 expression via p38 MAPK and NFkB activation downstream of DC-SIGN, TLR2 and TLR4 signaling in human DCs [28]. Furthermore, it has been shown that IL-10 is involved in the down-regulation of MHC class II and CD86 expression on the surface of DCs [29]. Similarly, our study shows that inhibited TLR4 recognition of H. pylori in DCs leads to increased DC maturation and decreased IL-10 production.
In H. pylori-infected gastric lamina propria increased levels of CD4 + T cells have been observed [30]. Besides IL-17-producing cells [19], IFN-c-secreting Th1 cells represent the largest group of infiltrating CD4 + T cells [18]. Nevertheless, this T cell response cannot successfully clear the infection. This deficiency might be explained by immune evasion mechanisms developed by H. pylori triggering the expansion of T regs . Indeed, emerging evidence suggests that H. pylori also induces a regulatory T cell response against T helper cell immunity, since increased expression of Foxp3 was observed in the gastric tissue of H. pylori-infected individuals compared to uninfected controls [20]. H. pylorispecific T regs have been shown to suppress IFN-c production [31] and memory T cell responses to H. pylori in infected individuals (3). In mice, they were capable of inhibiting Th17 differentiation [32].
We observed that co-cultivation of H. pylori-infected DCs with CD4 + T cells caused a weak secretion of IFN-c and IL-17A, whereas increasing amounts of Foxp3-expressing T cells accompanied by enhanced IL-10 secretion were observed, confirming previous observations [33]. Blocking of TLR4 on DCs reverted the tolerogenic phenotype of the cells, suggesting that TLR4 activation on DCs might influence the subsequent T cell response during H. pylori infection. Indeed, decreased amounts of T regs were found after co-culture experiments in the presence of TLR4-neutralizing antibodies, as measured by changes in Foxp3 expression and reduced IL-10 secretion by co-cultured CD4 + T cells. In addition, T cells also secreted lower levels of IFN-c and IL-17A upon coculture with TLR-4 neutralized H. pylori-infected DCs, indicating that H. pylori-induced DC cytokine secretion strongly influences T cell polarization during H. pylori infection.
Previous studies of our own group revealed that a reduction of T regs by using the specific anti-CD25 antibody PC61 induced increasing levels of IFN-c mRNA in the gastric mucosa of treated mice that was accompanied by a strong gastritis and lower bacterial colonization [34]. However, IFN-c-deficient mice did not achieve bacterial elimination, but showed no inflammatory symptoms, suggesting an important role for IFN-c in gastric inflammation induced by H. pylori [35]. In light of our results, we speculate that dysfunctional TLR4 signaling might contribute to a reduced regulatory T cell response in H. pylori infected individuals, while enhanced effector T cell response mediated by the activation of other TLRs as TLR2 may lead to the damage of epithelial cells and ulcerogenesis as it has been previously described [36].