Helicobacter pylori infection causes a chronic gastric mucosal inflammation, leading to peptic ulcer disease in 5–10% of the infected people, and to gastric adenocarcinoma and low-grade mucosa-associated lymphoid tissue (MALT) lymphoma in 1% of the cases. The pathogenesis of H. pylori infection has been linked to the strength of inflammation it promotes, which is correlated to the functionality of the cag pathogenicity island (PAI) in the bacteria (Censini et al., 1996). Gastric epithelial cells constitute the first line of defense against H. pylori; they produce interleukin (IL)-8, which promotes the recruitment of polynuclear cells. Immune cells including macrophages, dendritic cells (DC) and mucosa infiltrating lymphocytes take part in the innate and adaptative immune responses to the bacteria.

The pro-inflammatory properties of H. pylori strains have been mainly evaluated by measuring IL-8 production of the AGS gastric epithelial cell line, but they deserve to be investigated on immune cells due to their orchestrated pro- and anti-inflammatory cytokine production in response to pathogens. In vivo the professional antigen-presenting DC were found to be recruited into the gastric mucosa and, due to the emission of dendrites inserting themselves between epithelial cell tight junctions, they may be able to interact with H. pylori (Rescigno et al., 2008; Shiu and Blanchard, 2013). Their role in H. pylori-induced inflammatory response needs to be clarified.

Host defense against pathogens requires the induction of appropriate innate immune responses, as excessive or inappropriate activation of the immune system can be deleterious to the organism. Therefore, various immune regulators, including microRNAs (miRNA), also take part in the immune responses (Baltimore et al., 2008). MiRNA received considerable attention because of their implication in maintaining homeostasis in fundamental biological processes in non-pathological states, and their deregulation in pathological states (Sonkoly and Pivarcsi, 2009). Changes in miRNA expression in response to bacterial infection have been reported, including H. pylori infection in the gastric mucosa, in gastric epithelial cells and in immune cells (Zhang et al., 2008; Fassi Fehri et al., 2010; Belair et al., 2011; Matsushima et al., 2011).

In the present study, primary human DCs were used to explore the inflammatory effectors induced by H. pylori strains. Two fully sequenced H. pylori strains were included for this purpose, i.e., strains B38 (Thiberge et al., 2010) and B45 (Lehours et al., 2011) which are cagPAI negative and cagPAI positive, respectively. In parallel to cytokine production, miRNA changes induced by H. pylori were analyzed; they included measuring miR-146 and miR-155, to which specific immunomodulatory functions in H. pylori infection were assigned after performing loss of function experiments.

In this manuscript, we aimed at analyzing the initial steps of the immune response to H. pylori strains. We therefore developed an ex vivo co-culture cell system, in which we showed that H. pylori strains were able to activate primary human DCs and promote strong proinflammatory cytokine and miRNA responses. Although most of the observed effects were independent of the H. pylori cagPAI status, the cagPAI positive strain specifically upregulated IL-23, most likely to orientate DCs to promote a Th17 response.

Numerous articles have been published on the interaction between H. pylori and DCs, however, with a huge heterogeneity in the bacterial strains, MOI and experimental conditions (viable/sonicated/paraformaldehyde-treated bacteria) (Guiney et al., 2003; Hafsi et al., 2004; Kranzer et al., 2004, 2005; Kao et al., 2006; Mitchell et al., 2007; Rad et al., 2009; Andres et al., 2010; Kao et al., 2010; Fehlings et al., 2012). Our data were obtained using primary human DCs from several donors. All experiments were repeated with at least three different donors in order to ensure the reliability of the results. Significant productions of several cytokines (TNFα, IL-1β, IL-6, IL-8, IL-10, IL-12, and IL-23) were observed in H. pylori-activated DCs, TNFα and IL-6 being produced at the highest levels. In vivo, TNFα contributes to monocyte maturation after recruitment, whereas IL-6 supports the transition between the early stages of the infection and the sustained mononuclear influx into the infected gastric mucosa. Early production of TNFα and IL-6 upon LPS stimulation reflects the intrinsic property of E. coli LPS to trigger TLR4 signaling pathways, a property that is not shared by H. pylori LPS (Rad et al., 2009). Delayed secretion of TNFα and IL-6 by H. pylori-activated DCs as compared to LPS-activated ones may depend on bacterial DNA and RNA recognition by endosomal TLR9 and TLR8, respectively (Rad et al., 2009), a later process when compared to the recognition of pathogen associated-motifs by surface TLRs. At later time points (24 and 48 h), a switch in the inflammatory response occurred with an accumulation over time of IL-6, IL-10, IL-23, and IL-1β, without any significant induction of TGFβ. The late IL-10 and IL-12 production by H. pylori-activated DCs may coincide in vivo with their homing in lymph nodes (Kranzer et al., 2004). IL-10 was secreted at a higher level after DC activation by H. pylori than by E. coli LPS, in accordance with recent data, which showed that H. pylori interactions with both TLR2 and the lectin DC-SIGN contributed to an anti-inflammatory environment via the release of IL-10 (Rad et al., 2009; Fehlings et al., 2012). IL-12, which plays a role in the differentiation of naive T cells toward Th1 cells (Pellicano et al., 2007), was the least produced cytokine upon DC activation. In the present study, the high IL-10 and TNFα levels secreted by activated DCs could be responsible for the low IL-12 secretion due to their ability to inhibit its production (Langenkamp et al., 2000). In addition, IL-23 was the only cytokine dependent on the strain's cagPAI status. This result is still a matter of debate. Indeed several papers show that cagPAI could influence in vitro IL-23 release by DCs (Khamri et al., 2009; Tanaka et al., 2010). However, others argue that cagPAI does not regulate cytokine production in DCs cocultured with H. pylori (Kao et al., 2010; Horvath et al., 2012). Previous data from experiments on human gastric biopsies infected with H. pylori indicate that myeloid DCs co-localize with IL-23 and IL-17-producing, infiltrating lymphocytes (Khamri et al., 2010); one could therefore hypothesize that the H. pylori B45 strain may be able to induce a Th17 response, likely mediated by the cagPAI status. As IL-23 also plays an important role in sustaining Th17 responses in addition to acting on lymphoid cells to induce IL-17 (Shi et al., 2010; Hitzler et al., 2013), signaling pathways involved in this phenomenon deserve further investigation.

In parallel to the cytokine response profile, miR-155 and miR-146a were co-upregulated most intensely and invariably in activated DCs, independently of the cagPAI status of the bacterial strain. These two miRNA were previously expressed at high levels in H. pylori-infected gastric mucosa (Liu et al., 2010; Lario et al., 2012). Our results clearly show that the extent of their upregulation as well as their kinetics and MOI-dependence did not evolve in parallel with the time-course of H. pylori-activated DCs. MiR-146 upregulation takes part in the early immune program activated in DCs by H. pylori, as it reached 50% of its maximal 10 fold stimulation as early as 6 h post-infection, while miR-155 was induced at a striking rate 24 h post-infection, reaching 200 fold the stimulated levels at 48 h. The massive induction of miR-155 in DCs over the time-course of their activation by both LPS and H. pylori is consistent with previous findings (O'Connell et al., 2007; Tili et al., 2007; Martinez-Nunez et al., 2009). Indeed, miR-155 can be induced by pathogen-associated ligands via TLR-, NF-κ B-, and MyD 88-dependent pathways or by several cytokines such as IL-1β (Ceppi et al., 2009) and TNFα (Tili et al., 2007) which are both likely to sustain the high miR-155 biosynthesis induced in LPS-stimulated or H. pylori-infected DCs. An alternative miR-155 activation by bacterial peptidoglycan sensing via the cytoplasmic NOD2 receptor has been reported (Schulte et al., 2013), which may participate to the massive miR-155 induction. Contrary to miR-146, miR-155 was induced via a biphasic profile according to the MOI, suggesting that at a high MOI, the increasing level of some inflammatory cytokines might negatively regulate miR-155 expression. As the anti-inflammatory cytokine IL-10 was found to inhibit LPS-induced miR-155 upregulation without affecting miR-146a (McCoy et al., 2010), it is likely that H. pylori-stimulated IL-10 production is responsible for the decreased miR-155 upregulation at high MOIs.

The role of miR-146 and miR-155 in the innate immune responses was deduced from the identification of their respective targets. As miR-146a targets and silences TNF-receptor-associated factor and IL-1β receptor-associated kinase mRNAs, which are key adapter molecules in the TLR/ NFκ B pathway, the role of miR-146a upregulation in response to pathogens was shown to moderate TLR-triggering of the NFκ B pathway via a negative feedback loop, thus avoiding overproduction of pro-inflammatory IL-1β and TNFα cytokines (Taganov et al., 2006; Nahid et al., 2009; Lu et al., 2010). A similar immune regulatory function was assigned to miR-155 which targets several gene transcripts of the NF-κ B pathway (FADD, Ripk1, IKK, and NIK), as well as the immune cell transcription factor PU, Src homology 2 domain-containing inositol-5-phosphatase (SHIP) and CCAAT enhancer-binding protein beta (C/EBPbeta) (Rodriguez et al., 2007; Tili et al., 2007; Costinean et al., 2009; Schulte et al., 2013). In addition, miR-155 also stabilizes the TNFα mRNA (Tili et al., 2007; Semaan et al., 2011), suggesting that simultaneous anti- and pro-inflammatory effects of miR-155 most likely contribute to the fine-tuning of the TNF-mediated inflammation. We assessed the role of miR-146 and miR-155 in the DC cytokine response to H. pylori using specific antisenses to each miRNA, which inhibit their silencing function by competition with their binding sites on mRNA targets. Thus, the most noticeable effect of miR-155 loss of function in our cell system was an enhanced TNFα production by H. pylori, an effect consistent with the immunomodulatory role assigned to miR-155 upregulation in response to microbial stimuli (O'Connell et al., 2007; Rodriguez et al., 2007; Ceppi et al., 2009; Martinez-Nunez et al., 2009). Enhanced TNFα response to H. pylori in the absence of miR-155 may also be facilitated by the downregulation of miR-125b which we observed in our microarray (Table 1), and which directly targets TNFα (Tili et al., 2007). In our miR-146 loss of function experiment, no cytokine response was markedly altered with the exception of IL-10. The impaired production of this anti-inflammatory cytokine in response to H. pylori is concordant with the previously established immunomodulatory role of miR-146 upon infection, but it was not accompanied by an enhanced production of inflammatory cytokines in anti-miR-146-treated cells as expected. A possible explanation for the decreased IL-10 response to H. pylori in the absence of miR-146 could be that miR-146 loss of function unmasks the regulatory effects of other miRNAs: for instance, let-7e, which directly targets IL-10 (Schulte et al., 2011) was upregulated in H. pylori-activated DCs (Table 1) and may subsequently negatively regulate IL-10. The differences in the effects of miR-146 and miR-155 knockdown, which stress the prominent role of miR-155 in H. pylori-activated DCs, could be related to different functional specializations of these two seemingly co-induced miRNAs, as recently reported in LPS-stimulated murine macrophages (Schulte et al., 2013); miR-146 responded to sub-inflammatory stimuli to prevent TLR activation, and miR-155 responded to pro-inflammatory stimuli as a global limiter of the inflammatory response. Our experimental conditions established a TNFα-filled, pro-inflammatory environment in cultured DCs, and thus preferentially elicited miR-155 functions.

miR-146a and miR-155 expressions may be of special interest in H. pylori-mediated immune pathologies, because they are associated with NFκB activation, a pathway strongly activated during H. pylori infection and in H. pylori-driven MALT lymphoma. High expression of miR-146a was associated with chronic inflammatory diseases (Sonkoly and Pivarcsi, 2009) and miR-155 represents an important element in B cell lymphoma development (Costinean et al., 2006), indicating that alterations in the fine-tuning of innate immune responses by miRNAs may contribute to inflammatory disorders. Other miRNAs, which are differentially regulated in H. pylori-activated DCs, deserve further investigation; among them, miR-187, which was strongly upregulated in the H. pylori-activated DCs, could control IL-10-driven anti-inflammatory responses (Rossato et al., 2012).

In conclusion, the co-culture cell system presented here allowed the identification of pro- and anti-inflammatory effectors in response to H. pylori and revealed the influence of the cagPAI on the immune responses. These important findings pave the way for further investigations on the nature of H. pylori antigens and the subsequently activated signaling pathways involved in the inflammatory response to H. pylori infection.