Pattern-Recognition Receptors and Gastric Cancer

Chronic inflammation has been associated with an increased risk of several human malignancies, a classic example being gastric adenocarcinoma (GC). Development of GC is known to result from infection of the gastric mucosa by Helicobacter pylori, which initially induces acute inflammation and, in a subset of patients, progresses over time to chronic inflammation, gastric atrophy, intestinal metaplasia, dysplasia, and finally intestinal-type GC. Germ-line encoded receptors known as pattern-recognition receptors (PRRs) are critical for generating mature pro-inflammatory cytokines that are crucial for both Th1 and Th2 responses. Given that H. pylori is initially targeted by PRRs, it is conceivable that dysfunction within genes of this arm of the immune system could modulate the host response against H. pylori infection, and subsequently influence the emergence of GC. Current evidence suggests that Toll-like receptors (TLRs) (TLR2, TLR3, TLR4, TLR5, and TLR9), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (NOD1, NOD2, and NLRP3), a C-type lectin receptor (DC-SIGN), and retinoic acid-inducible gene (RIG)-I-like receptors (RIG-I and MDA-5), are involved in both the recognition of H. pylori and gastric carcinogenesis. In addition, polymorphisms in genes involved in the TLR (TLR1, TLR2, TLR4, TLR5, TLR9, and CD14) and NLR (NOD1, NOD2, NLRP3, NLRP12, NLRX1, CASP1, ASC, and CARD8) signaling pathways have been shown to modulate the risk of H. pylori infection, gastric precancerous lesions, and/or GC. Further, the modulation of PRRs has been suggested to suppress H. pylori-induced inflammation and enhance GC cell apoptosis, highlighting their potential relevance in GC therapeutics. In this review, we present current advances in our understanding of the role of the TLR and NLR signaling pathways in the pathogenesis of GC, address the involvement of other recently identified PRRs in GC, and discuss the potential implications of PRRs in GC immunotherapy.


INTRODUCTION
Of the three main types of stomach cancer, gastric adenocarcinoma (GC), non-Hodgkin's lymphoma, and gastrointestinal stromal tumors, approximately 95% are GC, which remains one of the most commonly diagnosed cancers in the world (1). In 2012, stomach cancer was the fifth most common cancer worldwide, with 952,000 new cases diagnosed, accounting for 6.8% of the total cancer cases (1). Furthermore, it is the third leading cause of cancer-related deaths worldwide, accounting for 8.8% of total deaths from cancer, with 5-year relative survival rates lower than 30%, except in Japan where mass screening has been undertaken for several years (2).
Gastric cancer is a heterogeneous pathology with respect to anatomical location and histological subtypes ( Figure 1A). In relation to location, GC may occur in the cardia or noncardia region of the stomach. Cardia GC has been associated with gastro-esophageal reflux, Helicobacter pylori infection, and atrophic gastritis, male gender, smoking, and diet (3). Epidemiological studies assessing the worldwide incidence of GC by anatomical location have shown an increase in the incidence of cardia GC, however, in high GC risk areas, non-cardia GC remains the most frequent pathology (4). Further, even though cardia and non-cardia GC have been considered etiologically different phenomena, it has been demonstrated that cancer of the cardia among individuals from areas with a high risk of GC represents a subset of cardia GC that is associated with H. pylori-related atrophic gastritis and resembles non-cardia GC pathogenesis (5,6).
According to the Lauren Classification, non-cardia GC has been further subdivided into the two histological variants intestinaltype and diffuse-type. Intestinal-type GC is characterized by the formation of gland-like structures, distal stomach localization, and a predilection for older individuals. It is also more frequent in males (2:1 ratio) and in subjects of lower socioeconomic status (10). This type of GC is often preceded by a precancerous phase that starts with the transition of normal mucosa into multifocal atrophic gastritis. This initial histological alteration is followed by intestinal metaplasia, dysplasia, and finally adenocarcinoma (11). On the other hand, diffuse-type GC is poorly differentiated, affects younger individuals, and has been highly associated with genetic susceptibility (the variant hereditary diffuse GC, which is associated with germ-line mutations in CDH1, a gene encoding E-cadherin) (12,13). Additionally, it is not associated with the formation of precancerous lesions and has been found to affect the entire surface of the stomach. This type of GC is present equally between the two sexes and is associated with a worse prognosis in comparison to intestinal-type GC (10,12).
Most GC cases are sporadic and arise due to the combination of a permissive environment interacting with a susceptible host. Several factors that contribute to the development of GC have been identified; these include bacterial (H. pylori), host, and environmental factors (12).
Helicobacter pylori is a Gram-negative bacterium that infects nearly 50% of the human population (14). In the gastric mucosa, the majority of Helicobacter pylori are found within the mucus layer but they can also be attached to epithelial cells leading to the maintenance, spread, and severity of the infection (15). H. pylori infection has been associated with the development of a range of diseases, including peptic ulcer disease (10%), non-cardia GC (1-3%), and gastric mucosa-associated lymphoid tissue (MALT) lymphoma (<0.1%) (14,(16)(17)(18). Furthermore, this bacterium has been associated with three distinct phenotypes in the infected host: (1) a corpus-predominant gastritis, which has the potential to lead to atrophic gastritis, hypochlorhydria, and to the development of GC; (2) a duodenal ulcer phenotype in which an antrum-predominant gastritis leads to increased gastric acid secretion; and (3) a benign phenotype in which the bacterial infection causes a mild mixed gastritis that has a minor effect on gastric acid production (19).
Helicobacter pylori infection is transmitted by direct humanto-human transmission, via either the oral-oral route, fecal-oral route, or both (14). H. pylori is acquired early in life, the majority of individuals being infected before the age of 10 years with close family members being a common source of infection (20)(21)(22). It has been postulated that early acquisition of infection might be associated with the broad pathological spectrum associated with H. pylori infection and the highly persistent GC incidence rates in genetically susceptible populations who have migrated to Frontiers in Immunology | Tumor Immunity developed countries. In the absence of antibiotic therapy, H. pylori infection generally persists for life (23).
Natural colonization by H. pylori is restricted to humans, primates, and domestic animals such as cats (23)(24)(25). H. pylori is considered to be the dominant microorganism in the human stomach as the majority of bacteria cannot survive in the low gastric pH (26). Several other factors make the human stomach an unfavorable environment for bacterial colonization including peristalsis, poor nutrient availability, and host innate and adaptive immunity (23). The ability of H. pylori to survive and colonize the stomach relates to a number of mechanisms. Most importantly H. pylori, unlike other bacteria, produces large amounts of the enzyme urease, which hydrolyzes urea to ammonia, which subsequently interacts with hydrogen ions in the stomach to form ammonium (27,28). In addition, H. pylori is able to regulate gene expression in response to changes in pH (29). Further, H. pylori expresses multiple paralogous outer membrane proteins, including the blood-group antigen-binding adhesin (BabA), the sialic-acid binding adhesin (SabA), and the outer inflammatory protein (OipA), which appear to bind to receptors on the surface of gastric epithelial cells, which reduces the rate of bacterial elimination as a result of peristalsis (30,31). H. pylori counteracts the lack of nutrients by inducing tissue inflammation and using specific systems that facilitate the transport and uptake of nutritional resources (23). In addition, H. pylori has been reported to produce antibacterial peptides that might decrease competition from other microorganisms (32).
Further, a number of other factors have been shown to help H. pylori evade the host immune system. For example, the vacuolating cytotoxin (VacA) produced by some strains of H. pylori has been shown to inhibit T-cell proliferation as well as antigen presentation by B cells and to alter the normal functions of CD8 + T cells, mast cells, and macrophages (33)(34)(35)(36). In addition, gammaglutamyl transpeptidase, another immunosuppressive factor of H. pylori, has been associated with inhibition of T-cell proliferation by induction of a cell cycle arrest in the G 1 phase (37). Furthermore, H. pylori has been shown to use arginase to downregulate the production of inducible nitric oxide synthase by macrophages (38).
The fact that more than one H. pylori strain can colonize the gastric mucosa provides the opportunity for H. pylori to acquire new genetic sequences and to undergo recombination events (23). One of the most remarkable differences among H. pylori strains is the presence or absence of a 40-kb DNA insertion element known as the cytotoxin-associated gene pathogenicity island (cag PAI) (39). This region contains between 27 and 31 genes flanked by 31-bp repeats and encodes the most widely investigated H. pylori virulence factor, the cytotoxin-associated antigen A (CagA) (40,41). H. pylori strains expressing CagA represent 60-70% of Western strains and approximately 100% of East Asian strains (39,42). CagA is a 120-to 140-kDa protein that is translocated into host cells through a type IV secretion system following attachment to gastric epithelial cells (43). Following translocation, CagA is tyrosine phosphorylated at the EPIYA (glutamate-prolineisoleucine-tyrosine-alanine) motifs by members of the host cell kinase families known as proto-oncogene proteins Abl and Src (18). In Western populations strains, EPIYA-A, EPIYA-B, and varying numbers of EPIYA-C motifs have been reported, while in H. pylori strains from East Asian populations, EPIYA-A and EPIYA-B with EPIYA-D motifs, are found (44). Both phosphorylated and non-phosphorylated CagA result in alterations in the gastric epithelium including: (1) the activation of the protein tyrosine phosphatase, non-receptor type 11 (SHP-2), (2) alterations in cell scattering and proliferation, (3) alterations in cell structure and cell motility, (4) perturbation of epithelial cell differentiation and polarity, (5) alteration of tight junctions, and (6) aberrant activation of β-catenin (45)(46)(47). Furthermore, numerous studies have shown that cag PAI-positive H. pylori strains are associated with an increased risk of gastric diseases including peptic ulcer disease, premalignant gastric lesions and GC (48)(49)(50)(51). Further details of the interplay between H. pylori virulence factors and gastric epithelial cells and GC, can be found in an excellent review by Posselt et al. (44).
In the last two decades, a large number of epidemiological studies have established the association between H. pylori and the subsequent risk of developing both intestinal-type and diffusetype GC (52)(53)(54)(55)(56)(57). This finding has been consistent among different populations. For example, in the study by Parsonnet et al. (57), conducted in Caucasian, African-American, and Asian individuals, subjects infected with H. pylori who had antibodies against CagA were shown to be more likely than uninfected subjects to develop both intestinal-type and diffuse-type GC (OR: 5.1, 95% CI: 2.1-12.2 and OR: 10.1, 95% CI: 2.2-47.4, respectively). Consistently, a further study conducted in a Japanese population showed that, although the association was stronger in cases with intestinal-type GC (OR: 3.2, 95% CI: 1.8-5.8), there was also a positive association between H. pylori infection and diffuse-type GC (OR: 3.0, 95% CI: 1.0-8.8) (53). Further, a study conducted in a Spanish population showed no differences in H. pylori infection between the two GC histological subtypes (58). Similarly, a recent study in German individuals showed that H. pylori prevalence was comparable in patients with intestinal-type (82.1%) and diffuse-type (77.9%) GC (59).
Interestingly, more recent studies, assessing H. pylori infection through Western blot (CagA) for the detection of past infection, have shown an unprecedented association between H. pylori and GC that can be explained by a reduction of the misclassification that might take place when samples are analyzed with the enzymelinked immunosorbent assay (ELISA) alone (60,61 While H. pylori infection has been established as the most important risk factor for GC and was classified as a class 1 carcinogen by the World Health Organization in 1994, the etiology of GC also involves host and environmental factors. This is evidenced by the fact that only 1-3% of H. pylori-infected patients develop GC, and that progression to GC in some subjects occurs even after eradication of the bacterium (18).
Given that H. pylori is initially targeted by germ-line encoded receptors known as pattern-recognition receptors (PRRs), it is conceivable that dysfunction within genes of this arm of the immune system would affect the magnitude and direction of the host inflammatory response against the infection, resulting in an increased risk of GC development. Recent studies clearly show that PRRs are critical for generating mature pro-inflammatory cytokines that are crucial for both Th1 and Th2 responses during H. pylori infection, and these immune responses have been directly associated with gastric immunopathology. In this review, we present current advances in the understanding of the role of PRRs, mainly the Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) signaling pathways, in the pathogenesis of GC, and discuss future directions for continued research in this area. In the first section, we highlight the relevance of inflammation in GC. In subsequent sections, we address new developments in the TLR and NLR signaling pathways in GC, the role of other PRRs in GC, and the new frontier of therapeutic application of these concepts.

INFLAMMATION IN GASTRIC CANCER
It is well established that most cancer cell genotypes are the manifestation of six essential alterations in cell physiology that collectively dictate malignant growth: (1) self-sufficiency in growth signals, (2) insensitivity to growth-inhibitory signals, (3) evasion of programed-cell death (apoptosis), (4) limitless replicative potential, (5) sustained angiogenesis, and (6) tissue invasion/metastasis (8). Recently, inflammation has been considered the seventh hallmark of cancer and an enabling characteristic that facilitates the acquisition of the other hallmarks ( Figure 1B). Inflammation initiated by innate immune cells, mainly macrophage subtypes, mast cells, myeloid progenitors, and neutrophils (62)(63)(64)(65), designed to fight infections and heal wounds, can instead result in unintentional support of multiple cancer hallmark functions, thereby manifesting the widely accepted tumor-promoting consequences of inflammatory responses (9). In addition, active evasion by cancer cells from attack and elimination by immune cells, mainly CD8+ cytotoxic T lymphocytes, CD4+ Type 1 helper T cells, and natural killer (NK) cells, highlights the dual role of an immune system that both antagonizes and promotes cancer development and progression (9).
In the context of tumor enhancement, it has been proposed that once inflammation is initiated, tissue integrity is compromised leading to the multistage process of carcinogenesis by altering targets and pathways that are pivotal for normal tissue homeostasis (66). The mechanisms that are connected to these alterations include production of mutagenic reactive oxygen and nitrogen species as well as synthesis of cytokines and growth factors that favor tumor cell growth (67). In addition, inflammation provides a source of other bioactive molecules to the tumor microenvironment, including survival factors that limit cell death, pro-angiogenic factors, extracellular matrix-modifying enzymes that facilitate angiogenesis, invasion, and metastasis, and inductive signals that lead to activation of the epithelial-mesenchymal transition (a developmental regulatory program that enables epithelial cells to invade, resist apoptosis, and disseminate) (9). Interestingly, inflammation can be considered a "perigenetic alteration" of cancer cells because it may promote growth, expansion, and invasion of tumors even without the involvement of further genetic mutations or epigenetic alterations (68).
In 1988, Correa proposed a human model of intestinal-type gastric carcinogenesis (7). The model hypothesized a sequence of events progressing from acute inflammation to chronic inflammation, to atrophy, to intestinal metaplasia, to dysplasia, to carcinoma in situ, and finally to invasive GC. A subsequent study by Correa evaluated the gastric precancerous process in a Colombian population (7). The results of this cross-sectional study led to the widely accepted conclusion that the severity of atrophy correlates with the prevalence of metaplasia and that the severity of metaplasia correlates with the prevalence of dysplasia, suggesting that the process is indeed a biological continuum (69).
Given that inflammation is a hallmark of gastric carcinogenesis, polymorphisms in genes encoding pro-inflammatory cytokines/chemokines have been the focus of much research in recent years. To date, polymorphisms in the interleukin (IL)-1 family genes have been the most widely studied, including polymorphisms in IL1A, IL1B, and IL1RN that encode IL-1α, IL-1β, and their endogenous receptor antagonist IL-1RA, respectively. In particular, IL-1β, a potent endogenous pyrogen and an important component in the development of Th2-mediated immunity (70,71), has been associated with lipid peroxidation, DNA damage, inhibition of gastric acid secretion, increased H. pylori colonization, and induction of gastric atrophy and dysplasia in the presence or absence of H. pylori (72). Global meta-analyses have shown that the IL1B-511 T allele is significantly associated with an increased risk of developing GC in Caucasians but not Asians or Mestizos (73,74). Furthermore, IL-1 receptor signaling is known to induce the production of genes that not only stimulate tumor growth but are also involved in angiogenesis and metastasis such as matrix metalloproteinases, basic fibroblast growth factor, vascular endothelial growth factor, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, monocytic chemotactic protein 1, and CXCL-2 (75). To date, only one study has addressed the role of IL1R1 (also known as CD121A) in GC and H. pylori infection. The study, conducted in a Caucasian population, showed an increased risk of H. pylori infection in those harboring the IL1R1 Hinfl A allele (OR: 2.01, P-value: 0.009) but failed to show an association with GC (76). In addition, a recent meta-analysis on the endogenous receptor antagonist IL-1RA has shown the IL1RN*22 genotype to increase the risk of gastric precancerous lesions, supporting a role for this polymorphism in the early stages of gastric carcinogenesis (OR: 2.27, 95% CI: 1.40-3.70) (77). A further meta-analysis that included 39 case-control studies, showed statistically significant associations between the IL1RN*22 genotype and both intestinal-type and diffuse-type GC, showing ORs of 1.83 and 1.72, respectively (78). Further examples of polymorphisms in pro-inflammatory cytokines/chemokines that play an essential role promoting inflammation in the context of gastroin- and −857 C/T) (89). In addition to this, a recent comprehensive review on this topic recommended the investigation of other polymorphisms in IL1B (3954 C/T and −1473G/C), IL4 (-168T/C), IL6 (572 G/C and 597 G/A), and IL17 (+7488A/G and −197G/A), given their potential relevance in GC (79).
While extensive evidence supports the important role of proinflammatory cytokines/chemokines in gastric carcinogenesis, given that PRRs, mainly TLRs and NLRs, are important modulators of intestinal epithelial barrier function, epithelial repair, and immune homeostasis in the gastrointestinal tract (90), and that signal transduction from these receptors converges upon a common set of signaling molecules, including the activation of the transcription factors nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB) and the activator protein 1 (AP-1) that lead to the production of pro-inflammatory cytokines/chemokines (e.g., IL-1α, IL-1β, IL-6, IL-8, IL-10, and TNF-α) as well as members of the interferon (IFN) regulatory transcription factor family that mediate type I IFN-dependent responses, defects in PRRs function could be even more important than defects in pro-inflammatory cytokines/chemokines per se in the instauration of an inflammation-related disorder such as GC.

PATTERN-RECOGNITION RECEPTORS IN GASTRIC CANCER
Innate immunity refers to responses that do not require previous exposure to an immune stimulus and represents the first line of host defense in the response to pathogens. PRRs are part of the innate immune system and are pivotal for the detection of invariant microbial motifs. PRRs have been divided into five distinct genetic and functional clades: TLRs, NLRs, C-type lectin receptors (CLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), and absent in melanoma 2 (AIM2)-like receptors (ALRs) (91,92). PRRs are commonly expressed by cells of the innate immune system such as monocytes, macrophages, dendritic cells (DCs), neutrophils, and epithelial cells, as well as cells of the adaptive immune system (93).
Toll-like receptors and CLRs scan the extracellular milieu and endosomal compartments for pathogen-associated molecular patterns (PAMPs), which are highly conserved microbial structures that are essential for microbial survival (94), while intracellular PRRs, including NLRs, RLRs, and ALRs, cooperate to provide cytosolic surveillance (92,93).

TOLL-LIKE RECEPTORS RECOGNITION OF HELICOBACTER PYLORI
The involvement of the TLR signaling pathway in infectious, autoimmune, and inflammatory diseases is well accepted (95). During H. pylori infection, TLRs on gastric epithelial and immune cells recognize diverse PAMPs such as flagellin/unknown PAMP (TLR5), unmethylated CpG motifs (TLR9), and lipopolysaccharide (LPS) (TLR4 and TLR2).
TLR4 was initially identified as the potential signaling receptor for H. pylori LPS on gastric epithelial cells (96)(97)(98)(99). After forming a complex with the LPS-binding protein (LBP), LPS interacts with the monocyte differentiation antigen CD14 (CD14), and subsequently with the myeloid differentiation protein-2 (MD-2) (100). Together with TLR4, this complex induces the TLR4-mediated MyD88-dependent signal transduction pathway, which leads to the rapid activation of transcription factors, mainly NF-κB, and cytokines such as TNF-α, IL-1β, IL-6, and IL-12 (95). On the other hand, stimulation of TLR4 by LPS also facilitates the activation of a MyD88-independent pathway that activates IFN-regulatory factor (IRF) 3 and involves the late phase of NF-κB activation, both of which lead to the production of IFN-β and the expression of IFN-inducible genes (101,102). In addition to LPS, the H. pylori secretory protein HP0175, through its ability to bind to TLR4, was shown to transactivate the epidermal growth factor receptor (EGFR) and stimulate the EGFR-dependent vascular endothelial growth factor production in the GC cell line AGS, which have been linked to H. pylori-associated gastroduodenal diseases, ulcerogenesis, and carcinogenesis (103).
Although early studies concluded that TLR4 is the first innate immune response against H. pylori (104,105), later studies suggested that TLR4 had a limited role, given that H. pylori LPS appeared to bind poorly to LBP, resulting in it being inefficiently transferred to CD14 (106). Consequently, recent studies addressing the role of other TLRs during H. pylori infection, have found TLR2 to be the initial barrier against H. pylori infection (107)(108)(109)(110)(111)(112). A potential explanation for these inter-study differences in relation to the TLRs response to H. pylori might be attributed to cell type (i.e., epithelial versus immune cells), origin of the cell studied (i.e., peritoneal versus bone marrow derived macrophages), and the type of inflammatory response measured (i.e., type of cytokines), and thus, currently any conclusions regarding the role of TLR4 must be treated with caution.
In contrast, there is strong evidence supporting an important role for TLR2 in H. pylori infection, with both animal and cell culture experiments suggesting that TLR2 ligands (LPS or other) exist in H. pylori and related Helicobacter species (112)(113)(114), and that TLR2 may be involved in the innate immune sensing of these bacteria by epithelial cells (113). Furthermore, an interesting publication by Smith et al. (115) showed that H. pylori LPS functions as a classic TLR2 ligand and induces a discrete pattern of chemokine expression in epithelial cells, which involves modulation of the expression of the signaling protein tribbles 3 (TRIB3), a molecule implicated in the regulation of NF-κB.
Yet, the most likely scenario is that both TLR4 and TLR2 are involved in the early immune response against H. pylori as has been demonstrated by a number of investigators (116)(117)(118). For example, Obonyo et al. (116) showed that both TLR2 and TLR4 were crucial signaling receptors for H. pylori activation of the host immune response leading to the secretion of cytokines. Further, Yokota et al. (118) not only showed that H. pylori LPS was initially targeted by TLR2 as described by others, but, for the first time, showed that this TLR2 activation leads to cell proliferation and TLR4 expression via the MEK1/2-ERK1/2 pathway. The final outcome of this signaling pathway is increased proliferation of gastric epithelial cells and the instauration of a strong inflammatory reaction. Once this response is instaurated, H. pylori could then enhance inflammatory reactions mediated by TLR4 agonists such as other bacterial LPS, which would also contribute to gastric www.frontiersin.org FIGURE 2 | Pattern-recognition receptors involvement in Helicobacter pylori infection. H. pylori is recognized by the Toll-like receptors (TLRs) (TLR2, TLR4, TLR5, and TLR9), NOD-like receptors (NLRs) (NOD1, NOD2, NLRP3, and possibly, NLRP12 and NLRX1), RIG-I like receptors (RLRs) (RIG-I and possibly, MDA-5), and C-type lectin receptors (CLRs) (DC-SIGN). TLR4 poorly recognizes H. pylori lipopolysaccharide (LPS) to generate pro-inflammatory cytokines (e.g., IL-1α, IL-1β, IL-6, IL-8, IL-10, and TNF-α) and interferons (IFNs) through the myeloid differentiation primary response gene 88 (MyD88)-dependent and -independent pathways, respectively. TLR2 recognizes H. pylori LPS/peptigoglycan/unknown pathogen-associated molecular pattern (PAMP) while TLR5 poorly recognizes H. pylori flagella and TLR9 recognizes H. pylori DNA (unmethylated CpG motifs). H. pylori recognition by these three TLRs leads to nuclear factor-κB (NF-κB) activation. NOD1 and NOD2 recognize H. pylori peptidoglycan-derived peptides [γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP) and muramyl dipeptide (MDP)], leading to the activation of both transcription factors NF-κB and activator protein (AP)-1. The NLRP3 inflammasome, comprising NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1, recognizes a yet unknown H. pylori PAMP and/or damage-associated molecular pattern (DAMP), and through caspase-1 cleavage, leads to the maturation and secretion of interleukin (IL)-1β and IL-18. NLRX1 and NLRP12, two known negative regulators of NF-κB, appear to be significantly down-regulated during H. pylori infection in vitro, however, their exact role during H. pylori infection remains unclear. RIG-I recognizes H. pylori 5 -triphosphorylated RNA (5 -PRNA) while MDA-5 possibly recognizes H. pylori dsRNA. The dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) recognizes H. pylori fucosylated ligands and this interaction appears to counteract the pro-inflammatory immune response to H. pylori. Only one generic cell type depicting all TLRs, NLRs, RLRs, and CLRs involved in H. pylori recognition is shown here for simplicity. MAL, MyD88 adaptor-like protein, also named TIRAP; TRAM, translocating chain-associating membrane protein; TRIF, TIR domain containing adaptor inducing interferon-beta protein; TBK-1, TANK-binding kinase 1; IRF3, IFN-regulatory factor 3; TRAF6, TNF receptor-associated factor 6; IRAK, interleukin 1 receptor-associated kinase; RAS, proto-oncogene ras; c-RAF, proto-oncogene protein ras; RIP2, receptor-interacting serine/threonine-protein kinase 2, also known as RICK; CARD9, caspase activation and recruitment domain; MD-2, myeloid differentiation protein-2; ILs: interleukins. Names in orange correspond to molecules with a probable but not established role in the host response to H. pylori.
inflammation and subsequent carcinogenesis (118). Further, the heat-shock protein 60, an immune-potent antigen of H. pylori, has been shown to activate NF-κB and induce IL-8 production through TLR2 and TLR4 pathways in gastric epithelial cells, a phenomenon that is likely to contribute to the development of gastric inflammation caused by H. pylori infection (117).
In addition, TLR9 appears to play an important role in H. pylori recognition. Interestingly, Rad et al. (112) identified Frontiers in Immunology | Tumor Immunity TLR9-mediated recognition of H. pylori DNA as a main H. pyloriinduced intracellular TLR signaling pathway in DCs. Further, a study using a murine model of H. pylori infection has suggested that TLR9 signaling is involved in the suppression of H. pylori-induced gastritis in the early phase of infection via downregulation of Th1-type cytokines modulated by IFN-α (119). In addition, a recent study has shown that the gastric epithelia of children respond to H. pylori infection by increasing the expression of TLR2, TLR4, TLR5, and TLR9, as well as the cytokines IL-8, IL-10, and TNF-α (120).
Although TLR5 interaction with H. pylori induces only weak receptor activation (121), TLR5 has been involved in the inflammatory response to H. pylori. An interesting publication by Smith et al. (107), using HEK293 cells transfected with specific TLR expression constructs and MKN45 cells expressing dominant negative versions of TLR2, TLR4, and TLR5, which block the activity of wild-type forms of these receptors, has demonstrated that live H. pylori induces NF-κB activation and chemokine gene expression due to ligation of TLR2 and TLR5. A further study that aimed to explore the involvement of TLR2 and TLR5 in THP-1 cells and HEK293 cell lines (stably transfected with TLR2 or TLR5) during H. pylori infection, has indicated that H. pylori-induced expression of TLR2 and TLR5 can qualitatively shift cag PAI-dependent to cag PAI-independent pro-inflammatory signaling pathways with possible impact on the outcome of H. pylori-associated diseases (122). Given the established TLR5 evasion of α and ε Proteobacteria including H. pylori (123), the TLR5-mediated inflammatory responses during H. pylori infection described by Smith et al. (107) and Kumar Pachathundikandi et al. (122) are likely to be flagellinindependent, and therefore, a still unknown H. pylori factor might be responsible for this.
The importance of TLRs recognition during H. pylori infection and GC development is further supported by the acquired characteristics that enable H. pylori to survive in the human stomach and cause chronic inflammation. For example, H. pylori LPS is characterized by a modification of the lipid A component of LPS that makes it less pro-inflammatory (124) and has been reported to exhibit a 1000-fold reduction in bioactivity as compared to Escherichia coli LPS (125). Also, the flagellin of this bacterium has been shown to be poorly recognized due to modifications in the TLR5 recognition site of the N-terminal D1 domain of flagellin (123).

TOLL-LIKE RECEPTORS AND GASTRIC CARCINOGENESIS
While TLR2, TLR4, TLR5, and TLR9 appear to be important for H. pylori recognition, their role in the evolution of gastritis to more advanced lesions remains unclear. Interestingly, Schmausser et al. (126) showed that TLR9 was not detectable in intestinal metaplasia or dysplasia and was only focally detected in 6 out of 22 gastric carcinomas, while TLR4 and TLR5 were strongly expressed by gastric carcinomas. Consistently, a study by Pimentel-Nunes et al. (127) showed a statistically significant trend for a progressive increase of TLR2, TLR4, and TLR5 expression from normal mucosa to gastric dysplasia (mean expression in normal mucosa: 0.1, gastritis: 1.0, metaplasia: 2.2, and dysplasia: 2.8, Pvalue <0.01), with dysplasia presenting more than 90% positive epithelial cells showing strong expression (2.8, 95% CI: 2.7-3). In addition, these authors showed a significant trend for decrease in TOLLIP and PPARγ, two TLR signaling pathway inhibitors, which was associated with increasing levels of CDX-2, a marker for adenocarcinoma, from normal mucosa to carcinoma (P-value <0.05) (128). Fernandez-Garcia et al. (129) have also reported increased expression of TLR3, TLR4, and TLR9 in GC, and furthermore, these authors noted that TLR3 expression by cancer cells was significantly associated with a poor overall survival in patients with resectable tumors, which lead them to suggest that TLR3 might be an indicator of tumor aggressiveness. Similarly, Yakut et al. (130) investigating the association between serum IL-1β, TLR4 levels, pepsinogen I and II, gastrin 17, vascular endothelial growth factor, and H. pylori CagA status in patients with a range of gastric precancerous lesions, concluded that serum TLR4 levels could be used as a biomarker to differentiate individuals presenting with dysplasia from those with other gastric precancerous lesions, the mean TLR4 level in patients with dysplasia (0.56 ± 0.098 ng/mL) being significantly higher than in patients with H. pylori positive chronic non-atrophic gastritis (0.10 ± 0.15 ng/mL), chronic atrophic gastritis (0.06 ± 0.07 ng/mL), and intestinal metaplasia (0.12 ± 0.18 ng/mL). Furthermore, while TLRs have been shown to be expressed at the apical and basolateral pole of both normal gastric epithelial cells and in H. pylori gastritis, in metaplasia, dysplastic, and neoplastic epithelial cells all TLRs are expressed diffusely and homogeneously throughout the cytoplasm, with no apparent polarization, which may suggest an increased activation of these diffusely over-expressed receptors during gastric carcinogenesis (126,128).
In recent years, TLRs have been associated with tumor development and progression processes including cell proliferation, epithelial-mesenchymal transition, angiogenesis, metastasis, and immunosuppression. Interestingly, Chochi et al. (104) not only showed that H. pylori augmented the growth of GC via the LPS-TLR4 pathway but also found that this bacterium attenuated the antitumor activity and IFN-γ-mediated cellular immunity of human mononuclear cells. In addition, Song et al. (131) have suggested that flagellin-activated TLR5 enhances the proliferation of GC cells through an ERK-dependent pathway. Furthermore, Tye et al. (132) have proposed a novel role for TLR2 in promoting gastric tumorigenesis independent of inflammation, whereby up-regulation of TLR2 within epithelial tumor cells, rather than infiltrating inflammatory cells, by the uncontrolled activation of the oncogenic transcription factor STAT3, promoted gastric tumor cell proliferation, and survival via upregulation of anti-apoptotic genes [e.g., BCL2-related protein A1 (BCL2A1), baculoviral IAP repeat containing 3 (BIRC3), and B-cell CLL/lymphoma 3 (BCL3)]. Further, two processes that facilitate carcinogenesis and involve TLRs have recently been described by Li et al. (133). Using LPS-treated CD14-knockdown GC cells, these authors showed that CD14, an important co-receptor in the TLR4 complex, promotes tumor cell epithelial-mesenchymal transition and invasion through TNF-α (133).
In addition, the expression of tumor-associated molecules known to be important in gastric carcinogenesis has been linked to the activation of the TLR signaling pathway. For example, prostaglandin-endoperoxide synthase 2 (PTGS2), which is also termed cyclooxygenase 2 (COX2), a key enzyme that catalyzes www.frontiersin.org the conversion of arachidonic acid to prostaglandins, has been shown to play a pivotal role in gastric inflammation and carcinogenesis (134). For example, a study by Chang et al. (108), using clinical H. pylori isolates, has shown that H. pylori acts through TLR2/TLR9 to activate both the PI-PLCγ/PKCα/c-Src/IKKα/β and NIK/IKKα/β pathways, resulting in the phosphorylation and degradation of IκBα, which in turn leads to the stimulation of NF-κB and the expression of PTGS2.
Further, as compared with normal cells, cancer cells are more metabolically active and generate more reactive oxygen species (ROS), which affects cell survival. Several studies have suggested that ROS can act as secondary messengers and control a range of signaling cascades, leading to sustained proliferation of cancer cells (135,136). In the context of gastric carcinogenesis, H. pyloriinfected gastric epithelial cells have been shown to generate ROS (137). Interestingly, Yuan et al. (138) recently suggested that TLR4 expression in GC correlated with tumor stage and that activation of TLR4 contributed to GC cell proliferation via mitochondrial ROS production through up-regulation of phosphorylated Akt and NF-κB p65 activation and nuclear translocation.
However, the involvement of TLRs in GC might be more complex than initially suspected as TLRs not only recognize antigenic determinants of viruses, bacteria, protozoa, and fungi, but are also involved in the detection of damage-associated molecular patterns (DAMPs) (e.g., extracellular adenosine triphosphate, hyaluran, extracellular glucose, monosodium urate crystals) (139). Release of DAMPs, which are especially targeted by TLR2 and TLR4 (140)(141)(142)(143)(144)(145) during cancer progression may cause chronic inflammation leading to down-regulation of the ζ chain of the T-cell and NK cell activating receptors [for comprehensive information on this topic see the review by Baniyash et al. (146)], which entails T-cell and NK cell dysfunction, a phenomenon observed in some malignancies such as GC (147,148), colon (149), prostate (150), cervical (151), and pancreatic cancer (152). In addition to immunosuppression, DAMPS appear to facilitate other processes during gastric carcinogenesis. For example, Wu et al. (153) have recently showed that hyaluronan, derived from malignant cells, induced long-lived tumor-associated neutrophils and subsequent malignant cell migration in gastric carcinomas via a TLR4/PI3K interaction.
Collectively, TLRs might be involved in both gastric carcinogenesis mediated by H. pylori infection (a tumor-promoting consequence of inflammatory responses) and in GC perpetuation associated with immunosuppression (active evasion by cancer cells from attack and elimination by immune cells) and increased metastasis.
Given that some authors have failed to show specific associations between polymorphisms in the TLR signaling pathway, especially in TLR2, TLR4, and CD14, and gastric precancerous lesions/GC (157, 160, 162, 164, 172, 174-178, 180-183, 185, 188, 189), we performed the first global meta-analysis to assess the role of TLR2, TLR4, and CD14 polymorphisms in gastric carcinogenesis (155), in an attempt to clarify the limited and current conflicting evidence, and to establish the true impact of the TLR signaling pathway in GC. Our meta-analysis, which included 18 case-control studies conducted in Caucasian, Asian, and Latin American populations, showed that TLR4 Asp299Gly was a definitive risk factor for GC in Western populations (pooled OR: 1.87, 95% CI: 1.31-2.65). In addition, there was a potential association between TLR2 −196 to −174 and GC in Japanese (pooled OR: 1.18, 95% CI: 0.96-1.45) (155). Interestingly, a recent metaanalysis on TLR2 −196 to −174 and the risk of GC, conducted by Cheng et al. (190), failed to reproduce the findings in our metaanalysis, however, their stratification by ethnicity analyses included subjects from both Japan and China, which might explain the different outcomes. A further meta-analysis conducted by Chen et al. (191) (191). Further, Zhao et al. (192) in an updated version of a meta-analysis that was initially conducted by Zhang et al. (193), on the risk of TLR4 polymorphisms and risk of cancer in general, found a significant association with GC after stratifying by cancer type (OR: 2.00, 95% CI: 1.53-2.62). In addition, Zou et al. (194), through a metaanalysis that included 10 case-control studies, not only found that TLR4 Asp299Gly was associated with GC (OR: 1.87, 95% CI: 1.44-2.44), especially non-cardia GC (OR: 2.03, 95% CI: 1.51-2.72), but also gastric precancerous lesions (OR: 2.47, 95% CI: 1.57-3.88), especially in H. pylori-infected individuals (OR: 3.43, 95% CI: 1.92-6.13).
Given limited evidence regarding the association between polymorphisms in other molecules of the TLR signaling pathway and the risk of GC, and the fact that 42% of cases of GC worldwide occur in the Chinese population, we conducted a case-control Frontiers in Immunology | Tumor Immunity The functional relevance of a number of these polymorphisms has already been established. For example, two polymorphisms in TLR4, Asp299Gly, and Thr399Ile, have been shown to disrupt the normal structure of the extracellular domain of TLR4, and thus, as a result, may reduce responsiveness to H. pylori by diminishing the binding affinity of the bacterial ligands (195). In addition, the TLR4 rs11536889 polymorphism, which is located in the center of the 2818-bp TLR4 3 untranslated region (UTR), has recently been shown by Sato et al. (196) to contribute to the translational regulation of TLR4, possibly by binding to microRNAs. Further, these authors elegantly demonstrated that subjects harboring TLR4 rs11536889 exhibited higher levels of TLR4 receptors on monocytes and secreted higher levels of IL-8 in response to LPS (196). In addition, TLR4 rs10759932 has been shown to decrease the expression of forkhead box protein P3 (FOXP3), the most specific marker for natural regulatory T (Treg) cells (197). FOXP3+ Treg cells, which suppress the immune response of antigen-specific T cells, have been demonstrated to play a key role in immunologic tolerance (198). Notably, recent studies have not only shown that in vivo depletion of FOXP3+ Treg cells in H. pylori-infected mice leads to increased gastric inflammation and reduced bacterial colonization (199), but also recruitment of FOXP3+ Treg cells is increased in H. pylori-related human disorders including gastritis (200,201), duodenal ulcer (202), and GC (200,203,204), suggesting that FOXP3+ Treg cells might contribute to lifelong persistence of H. pylori infection. Also, TLR1 rs5743618 appears to impair the surface expression of TLR1 of NK cells and NK cellsderived IFN-γ production (154). Further, TLR2 −196 to −174 has been associated with decreased transcriptional activity of TLR2 (205,206). Similarly, it has been demonstrated that TLR9 rs187084 down-regulates TLR9 expression (207).
Further, CD14 has been shown to activate macrophages/ monocytes to release Th1-type cytokines including IL-12, thus, establishing the chronic inflammation stimulated by H. pylori infection (208)(209)(210). A Th1 predominant response has been extensively associated with the pathogenesis of H. pylori-related gastric disease (211)(212)(213). Currently, however, controversy exists regarding the influence of CD14 −260 on expression of soluble CD14 (sCD14). According to a number of studies, the CD14 −260 T allele is believed to increase sCD14 production and therefore, serum sCD14 levels (214)(215)(216)(217). In contrast, it has been reported that elevated sCD14 levels are associated with H. pylori infection, especially in subjects with the CD14 −260 CC genotype (167). Alternatively, others have argued that this polymorphism has no effect on transcription (218). Since the evidence to date is conflicting, more functional studies are required to clarify this issue.
Overall, it is clear that genetic variability in genes of the TLR signaling pathway plays an important role in GC pathogenesis. Investigations of polymorphisms in different molecules of this pathway among different populations could provide novel insights into targeted treatment in genetically susceptible individuals, and thus, improve primary and secondary prevention of H. pylori-related GC in high risk populations.

NOD-LIKE RECEPTORS RECOGNITION OF HELICOBACTER PYLORI
The NLR family not only recognizes PAMPs but also DAMPs in the cytoplasm (93). The NLRs characteristic structure includes a central nucleotide-binding and oligomerization (NACHT) domain that is present in all NLR family members, a C-terminal LRRs and an N-terminal caspase recruitment (CARD) or pyrin (PYD) domain.
The NACHT domain belongs to a family of P-loop NTPases known as the signal transduction ATPases with numerous domains (STAND) (219). This domain permits activation of the signaling complex via adenosine ATP-dependent oligomerization (94). NACHT domain oligomerization is essential for the activation of NLRs, forming high molecular weight complexes, probably hexamers or heptamers that characterize inflammasomes (molecular complexes involved in the activation of inflammatory caspases for the maturation and secretion of IL-1β, IL-18, and possibly IL-33) and NOD signalosomes (complexes that are assembled upon oligomerization of NOD1 or NOD2 and lead to NF-κB activation through the receptor-interacting protein-2) (94). CARD and PYD are death domains that mediate homotypic protein-protein interactions for down-stream signaling (93,94). These domains are characterized by six α helices that form trimers or dimers with other members of the same subfamily (94). The third domain, the LRR region, has been implicated in ligand sensing and autoregulation of not only NLRs but TLRs (93,94). The LRR is formed by tandem repeats of a structural unit consisting of a β strand and an α helix and is composed of 20-30 amino acids that form a horse-shoe shaped structure rich in the hydrophobic amino acid leucine (220). The NLRPs LRR gene is made up of tandem repeats of exons of exactly 171 nucleotides, which encode one central LRR and two halves of the neighboring LRRs (221). This particular modular organization possibly allows extensive alternative splicing of the LRR region leading to maximum variability in the ligand-sensing unit (94). However, a recent publication by Tenthorey et al. (222) analyzing a panel of 43 chimeric NAIPs, showed that LRR was unnecessary for NAIP/NLRC4 inflammasome ligand specificity, leading them to propose a model in which NAIP activation is instead triggered by ligand binding to NACHTassociated helical domains. This recent evidence suggests that the ligand-sensing function of the LRR domain in NLRs, which has been supported primarily by analogy to the well-established ligand-sensing function of the LRR region in TLRs, needs to be re-examined.
The most widely studied NLRs during H. pylori infection are NOD1 and NOD2, which are expressed in epithelial and antigen-presenting cells, and are known to specifically recognize peptidoglycan-derived peptides (γ-d-glutamyl-mesodiaminopimelic acid and muramyl dipeptide, respectively). An early study, attempting to determine the mechanism whereby H. pylori delivers peptidoglycan to cytosolic host NOD1, demonstrated that H. pylori peptidoglycan is delivered to the host cell via a type IV secretion system (223). More recently, Hutton et al. (224) showed, for the first time, that cholesterol-rich microdomains called lipid rafts, were important for the type IV secretion system-dependent peptidoglycan delivery and subsequent NF-κB activation and IL-8 production, mediated by NOD1. Interestingly, Kaparakis et al. (225) reported a novel mechanism in Gram-negative bacteria, including H. pylori, for the delivery of peptidoglycan to cytosolic NOD1 in host cells that involves outer membrane vesicles that enter epithelial cells through lipid rafts. In addition, Necchi et al. (226) demonstrated the formation of a particle-rich cytoplasmic structure (PaCS) in H. pylori-infected human gastric epithelium having metaplastic or dysplastic foci, where VacA, CagA, urease, outer membrane proteins, NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components, and potentially oncogenic proteins like SHP-2 and ERKs colocalized, inferring that this structure is likely to modulate inflammatory and proliferative responses during H. pylori infection.
The recent finding that NF-κB and AP-1 complexes can be physically translocated to the nucleus in response to NOD1 activation has led to the view that NOD1 is likely to be essential for the induction of both NF-κB and AP-1 activation during H. pylori infection (227). A number of studies have shown up-regulation of NOD1 expression in diverse human cell lines challenged with H. pylori in a cag PAI-dependent manner (228)(229)(230). Further, H. pylori cag PAI-positive strains have recently been shown to activate the NOD1 pathway through two components of the IFN-γ signaling pathway, STAT1 and IRF1 (228). Similarly, expression of NOD2 was shown to significantly sensitize HEK293 cells to H. pyloriinduced NF-κB activation in a cag PAI-dependent manner (231). Further, NOD2, but not NOD1, seems to be required for induction of pro-IL-1β and NLRP3 in H. pylori-infected DCs (232).
A limited number of studies have assessed the interaction between NLRPs and other inflammasome-associated molecules, and H. pylori. NLRPs represent the largest NLR subfamily (14 genes have been identified in humans) and are believed to be the scaffolding proteins of inflammasomes (221,233). NLRPs interact and recruit the adaptor apoptosis-associated speck-like protein (ASC) via PYD-PYD interaction (94). ASC (also known as PYCARD), a key component required for inflammasome formation, is formed by an N-terminal PYD and a C-terminal CARD (234,235). This interaction leads to the recruitment of caspase-1, an intracellular aspartate specific cysteine protease, which subsequently leads to the maturation and release of pro-inflammatory cytokines (236).
An early study by Tomita et al. (237) demonstrated that in H. pylori positive patients antral IL-18 mRNA expression was increased as compared with H. pylori negative patients, however, mature IL-18 protein and active caspase-1 were found to be present in both infected and non-infected gastric mucosa. Interestingly, in the following year, Potthoff et al. (238) reported activation of caspase-3, -8, and -9, but not caspase-1, in AGS cells challenged with H. pylori. However, this finding is in contrast with subsequent studies, which have demonstrated an important role for NLRPs and inflammasome-related molecules in H. pylori infection. For example, Basak et al. (96) demonstrated that H. pylori LPS could activate caspase-1 through Rac1/PAK1 signaling, and that activated caspase-1 played a role in LPS-induced IL-1β maturation (96). Further, ASC-deficient mice challenged with H. pylori have been shown to exhibit higher bacterial loads and significantly lower levels of gastritis, when compared with wild-type mice, and were incapable of producing IL-1β or IL-18 and produced less INF-γ in response to H. pylori infection (239). Later, Hitzler et al. (240) showed in both cultured DCs and in vivo that H. pylori infection activates caspase-1, leading to IL-1β/IL-18 processing and secretion. Consistently, three studies, using human GC cell lines, gastric tissue, and murine models, confirmed increased Frontiers in Immunology | Tumor Immunity expression of caspase-1, IL-1β, and IL-18 in H. pylori-infected cells (171,241,242). Further, Jiang et al. (243), also using a murine model, have reported the expression of NLRP3 inflammasomerelated molecules as well as serum IL-1β, IL-18, and IL-33 levels to be significantly increased in H. pylori-infected mice. More recently, a study by Kim et al. (232) has shown that secretion of IL-1β by DCs infected with H. pylori requires TLR2, NOD2, and the NLRP3 inflammasome.
Given that little is known about the role of NLRPs, inflammasomes, or other molecules involved in the NLR signaling pathways in response to H. pylori infection, we recently assessed the gene expression of 84 different molecules involved in the NLR signaling pathways, through quantitative real-time PCR, using THP-1-derived macrophages infected with two strains of H. pylori, GC026 (GC) and 26695 (gastritis) (173). Our gene expression analyses showed five genes encoding NLRs to be significantly regulated in H. pylori-challenged cells (NLRC4, NLRC5, NLRP9, NLRP12, and NLRX1) (173). Interestingly, NLRP12 and NLRX1, two known NF-κB negative regulators, were markedly downregulated, while NFKB1 and several NF-κB target genes encoding pro-inflammatory cytokines (IFNB1, IL12A, IL-12B, IL6, and TNF ), chemokines (CXCL1, CXCL2, and CCL5) and molecules involved in carcinogenesis (PTGS2 and BIRC3) were markedly upregulated, in THP-1 cells infected with a highly virulent H. pylori strain isolated from a GC patient. These findings highlight the relevance of the NLR signaling pathways in gastric carcinogenesis and its close interaction with NF-κB (173).
Overall, current evidence clearly shows that, in response to H. pylori, members of the NOD and NLRP subfamilies are critical for generating mature pro-inflammatory cytokines/chemokines that are crucial for Th1 responses and lead to H. pylori-related gastric disorders.

NOD-LIKE RECEPTORS AND GASTRIC CARCINOGENESIS
The role of the NLR signaling pathways in the biological continuum that characterizes GC remains relatively unexplored as a very limited number of studies have addressed this issue. For example, Allison et al. (228) have shown that NOD1 expression was significantly increased in human gastric biopsies displaying severe gastritis, when compared with those without gastritis, as well as in gastric tumor tissues, as compared with paired non-tumor tissues. In contrast, Jee et al. (244), who analyzed human GC tissues and GC cell lines, showed that a significant decrease in the expression of caspase-1 was associated with poor survival and was inversely correlated with p53 expression.
Given the reported interaction of H. pylori with NLRs and the importance of this in the development of gastric inflammation and subsequent carcinogenesis, as well as the production of DAMPs during tumor formation (245), further comprehensive studies of the functional relevance of NLRs activation during chronic gastritis, atrophic gastritis, intestinal metaplasia, dysplasia, and GC are clearly warranted.
Given the documented relevance of other NLRs in H. pylori infection and related GC, and that polymorphisms in genes such as NLRP3 (252)(253)(254)(255) and CARD8 (255,256) have been associated with inflammatory gastrointestinal disorders, we addressed, for the first time, the association between 51 polymorphisms in six genes (NLRP3, NLRP12, NLRX1, CASP1, ASC, and CARD8) involved in the NLR signaling pathways and risk of GC in a high risk Chinese population (173). In this study, we found novel associations between CARD8 rs11672725 and the risk of GC, and NLRP12 rs2866112 and the risk of H. pylori infection ( Table 3). Further, we showed that the concomitant presence of polymorphisms involved in the NLR signaling pathways (CARD8, NLRP3, CASP1, and NLRP12) and H. pylori infection dramatically increased the risk of GC in Chinese ( Table 3) (173).
The functional relevance of a number of these polymorphisms has been examined. For example, the introduction of NOD2 R702W, a polymorphism located in the LRR of NOD2, into the HEK293 cell line, resulted in abrogation of H. pylori-induced activation of NF-κB signaling (231). Further, Maeda et al. (257) observed increased NF-κB activation in response to muramyl dipeptide in mice harboring a NOD2 mutation that is homologous to NOD2 rs5743293 (3020insC) in humans. However, it is worth noting that the conclusions described by Maeda et al. (257) must be interpreted with care given that the authors subsequently found a duplication of the 3' end of the wild-type Nod2 locus, including exon 11, which was targeted by the mutation, and therefore, they are currently working to recreate a mutant strain without such a duplication.
Given that investigation of the role of polymorphisms involved in the NLR signaling pathways in GC is a relatively recent field of research, further studies are required to assess the association between these polymorphisms and GC in a range of human populations, especially those at high risk of GC.

OTHER PATTERN-RECOGNITION RECEPTORS AND HELICOBACTER PYLORI -RELATED GASTRIC CANCER
A further two PRR subfamilies, RLRs and CLRs, have been studied in relation to H. pylori infection and gastric carcinogenesis. It is well known that RLRs (RIG-I, MDA-5, and LGP2) induce type I IFN in response to different RNA viruses, however, investigation on the role of RIG-I-like receptors in the recognition of RNA derived from intracellular bacteria is very limited. Interestingly, a study by Rad et al. (112), which used mice lacking simultaneously up to four different TLRs, apart from identifying TLR2 and www.frontiersin.org  Based on this comprehensive literature review, we propose a synergistic interaction between pattern-recognition receptors (PRRs) and Helicobacter pylori in gastric carcinogenesis. The association between PRRs and risk of GC might be a continuum commencing in childhood. Individuals harboring polymorphisms in PRRs could not only be more susceptible to acquisition of H. pylori in childhood but also would present deregulation of NF-κB in gastric epithelial and immune cells, and subsequent uncontrolled production of cytokines/chemokines, due to dysfunctional PRRs. This in turn would impact upon the direction and magnitude of the chronic inflammatory response to H. pylori. As H. pylori, the dominant bacterium in the stomach, gradually disappears upon the development of gastric atrophy, it is plausible that other microbial species might bloom in its absence and perpetuate local inflammation through further PRRs activation. Over time, the combination of these events would facilitate a number of features that promote gastric cancer development including cell proliferation, epithelial-mesenchymal transition, angiogenesis, metastasis, and immunosuppression. adhesion molecule-3-grabbing non-integrin (DC-SIGN) is a CLR expressed on the surface of both macrophages and DCs. Interestingly, it has been shown that H. pylori harbors fucosylated ligands that can be recognized by DC-SIGN (259). Further, H. pylori DC-SIGN ligands appear to actively dissociate the signaling complex down-stream of DC-SIGN (KSR1-CNK-Raf-1) to suppress pro-inflammatory cytokine production (259). In addition, H. pylori LPS Lewis blood-group antigens can bind to DC-SIGN in a fucose or galactose-dependent manner (260,261) and this interaction appears to inhibit a Th1 response in DCs (262). It has also been demonstrated that H. pylori-induced IL-10 production in monocyte-derived DCs is significantly suppressed by the addition of anti-DC-SIGN, TLR2, or TLR4 antibodies, either alone or in combination, before H. pylori stimulation (263). Further, in vitro and in vivo experiments have shown that the expression of DC-SIGN is significantly higher in H. pylori-infected individuals as compared with that in their uninfected counterparts (264,265).
Given the limited but consistent current evidence suggesting a role of RLRs and CLRs in H. pylori infection, and the documented interaction between these signaling pathways and other important PRRs in GC such as TLRs (267,268) and NLRs (269,270), further www.frontiersin.org studies assessing the implications of RLRs and CLRs in H. pylorirelated inflammation and subsequent carcinogenesis need to be conducted.

PATTERN-RECOGNITION RECEPTORS AS THERAPEUTICS TARGETS IN GASTRIC CANCER
Pattern-recognition receptors are increasingly recognized as important players in immunotherapy as PRRs-specific agonists elicit a potent immune response to cancers, allergic diseases, and chronic viral infections, while reducing the risk of an uncontrolled and detrimental systemic inflammatory response (for comprehensive information on this topic refer to the reviews by Hedayat et al. (271) and Paul-Clark et al. (272).
In the context of gastric carcinogenesis, Tye et al. (132), using a GC murine model (gp130 F/F ) displaying elevated gastric TLR2 expression levels, have elegantly shown that genetic and antibodymediated therapeutic targeting of TLR2 leads to a substantial reduction in stomach size and overall tumor burden, including the number of gastric tumors. A further example is presented in the study by Gradisar et al. (273), which suggested that MD-2 is one of the important targets of curcumin (diferuloylmethane), the main component of the spice turmeric (Curcuma longa) that is widely used for gastric disorders in the Indian subcontinent, in its suppression of the innate immune response to bacterial infection. Furthermore, curcumin was recently shown to polarize myeloid-derived suppressor cells, extracted from a human GC xenograft mouse model, toward a M1-like phenotype with an increased expression of CCR7 and decreased expression of the CLR dectin 1, being both observed in vivo (tumor tissue) and in vitro (splenic myeloid-derived suppressor cells from tumorbearing mice) (274). In addition, a study by Yang et al. (171) demonstrated that the combination of catechins and sialic acid is effective in suppressing the inflammatory responses mediated by the inflammasome/caspase-1 signaling pathway in gastric epithelial cells during H. pylori infection. Also, poly(I:C), an agonist of TLR3 and RLRs, has been shown to have a pro-apoptotic effect in vitro, and has significantly inhibited xenograft growth of human GC in a mouse model, through up-regulation of RLRs (RIG-I, MDA-5, and LGP2) as well as an increased expression of Bcl-2 family members, suggesting that it may be a promising chemotherapeutic agent against GC (275).
Given that modulation of PRRs has been proven to be relevant in gastric carcinogenesis through diverse mechanisms, including suppression of H. pylori-induced inflammation and enhancement of cancer cell apoptosis, this approach should be considered a new and promising angle of immunotherapy in GC.

CONCLUSION
In conclusion, abundant evidence supports the pivotal role of PRRs in gastric carcinogenesis as these receptors of the innate immune system, including TLRs, NLRs, CLRs, and RLRs, have been shown to recognize diverse components of H. pylori, the major risk factor of GC. In addition, PRRs are also involved in gastric carcinogenesis per se as these receptors are known to exert tumor-promoting functions (cell proliferation, epithelialmesenchymal transition, angiogenesis, and metastasis) as well as immunosuppression during cancer. Given that host genetic variability in the TLR and NLR signaling pathways are known to be associated with an increased risk of H. pylori infection, the development of gastric precancerous lesions and GC, this knowledge has the potential to allow better prevention of GC through selective treatment and surveillance of individuals harboring high risk genetic profiles. Finally, given that PRRs are increasingly being used as a target for immunotherapy against both cancer and infectious diseases, the established relevance of PRRs in H. pylori infection and GC, could suggest that PRR agonists and/or antagonists may potentially improve the outcome of GC. Based on the extensive evidence presented in the current review, we propose a synergistic interaction between PRRs and H. pylori, which over time, could facilitate the sequence of events that characterizes GC development including inflammation, atrophy, intestinal metaplasia, dysplasia, and finally, GC (Figure 3).

ACKNOWLEDGMENTS
Nadeem O. Kaakoush is supported by an Early Career fellowship from the National Health and Medical Research Council, Australia.