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 (96–99). 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 (107–112). 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 (112–114), 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 (116–118). 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 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 TLR9-mediated recognition of H. pylori DNA as a main H. pylori-induced 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 down-regulation 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 flagellin-independent, 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).

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, P-value <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 up-regulation 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 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. pylori-infected 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 (140–145) 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.

In recent years, a number of investigations have attempted to establish the relationship between polymorphisms in molecules of the TLR signaling pathway and risk of GC. Recent studies, conducted in several populations, have shown associations between the polymorphisms TLR1 rs5743618 (Ile602Ser) (154), TLR2 −196 to −174del (155–158), TLR2 rs3804099 (157), TLR4 rs4986790 (Asp299Gly) (155, 157, 159), TLR4 rs4986791 (Thr399Ile) (160), TLR4 rs10116253 (161), TLR4 rs10983755 (162), TLR4 rs11536889 (+3725G/C) (155), TLR4 rs1927911 (161), TLR5 rs5744174 (158), TLR9 rs187084 (−1486 T/C) (163), and CD14 rs2569190 (−260 C/T) (155, 164–167), and risk of GC development in an ethnic-specific manner (Table 1). In addition, three polymorphisms located in the TLR4 mRNA promoter region (sites −2081, −2026, and −1601) and TLR4 Thr135Ala at the leucine-rich repeat (LRR), have been associated with poorly differentiated GC (168, 169).

Interestingly, some of these polymorphisms including TLR4 Asp299Gly (159, 184), TLR4 Thr399Ile (184, 185), TLR4 rs10759932 (186), CD14-260 C/T (187), and TLR2 −196 to −174del (157), appear to be involved in the biological continuum that results in intestinal-type GC as they have also been associated with gastric precancerous lesions (Table 2).

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 meta-analysis on TLR2 −196 to −174 and the risk of GC, conducted by Cheng et al. (190), failed to reproduce the findings in our meta-analysis, 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) that included 21 case–control studies showed an overall increased risk of GC in individuals harboring TLR4 Asp299Gly (Allele analysis, OR: 1.84, 95% CI: 1.41–2.39) and TLR4 Thr399Ile (Allele analysis, OR: 1.97, 95% CI: 1.22–3.18). Consistently, in stratified analyses by ethnicity, these authors only found an association between TLR4 Asp299Gly (Allele analysis, OR: 1.90, 95% CI: 1.43–2.51) and TLR4 Thr399Ile (Allele analysis, OR: 2.84, 95% CI: 1.56–5.15) in Caucasian individuals (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 meta-analysis 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 study comprising 310 ethnic Chinese individuals (87 non-cardia GC cases and 223 controls with functional dyspepsia), in which 25 polymorphisms involved in the TLR signaling pathway were investigated (170). Seven polymorphisms showed significant associations with GC (TLR4 rs11536889, TLR4 rs10759931, TLR4 rs1927911, TLR4 rs10116253, TLR4 rs10759932, TLR4 rs2149356, and CD14 −260 C/T). In multivariate analyses, TLR4 rs11536889 remained a risk factor for GC even after adjustment (OR: 3.58, 95% CI: 1.20–10.65). Further, TLR4 rs10759932 decreased the risk of H. pylori infection (OR: 0.59, 95% CI: 0.41–0.86) (170). Strikingly, statistical analyses assessing the joint effect of H. pylori and the selected polymorphisms revealed that H. pylori-infected individuals harboring TLR2 rs3804100, TLR2 −196 to −174del, TLR4 rs11536889, MD-2 rs11465996, MD-2 rs16938755, LBP rs2232578, and TIRAP rs7932766 were at most risk of developing GC (Table 1) (170).

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 cells-derived 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 (208–210). A Th1 predominant response has been extensively associated with the pathogenesis of H. pylori-related gastric disease (211–213). 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 (214–217). 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.

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.

Based on phylogenetic analysis of NACHT domains, the NLR family has been shown to comprise three subfamilies: (1) the NOD family which includes NOD1-2, NOD 3 (NLRC3), NOD4 (NLRC5), NOD5 (NLRX1), and CIITA, (2) the NLRPs including NLRP1-14 (also known as NALPs), and (3) the IPAF subfamily, which consists of IPAF (NLRC4) and NAIP (93).

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 NACHT-associated 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-meso-diaminopimelic 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 (228–230). 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. pylori-induced 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 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 inflammasome-related 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 down-regulated, 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 up-regulated, 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.

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.

The majority of studies examining the association between polymorphisms involved in the NLR signaling pathways and the risk of GC have focused on NOD1 and NOD2 polymorphisms. Studies, conducted in a number of populations, have investigated the association between the polymorphisms NOD1 rs2907749 (246), NOD1 rs7789045 (246), NOD1 rs2075820 (E266K) (179, 247), NOD1 rs5743336 (180), NOD2 rs7205423 (246), NOD2 rs7202124 (164), NOD2 rs2111235 (164), NOD2 rs5743289 (164), NOD2 rs2066842 (P268S) (248, 249), NOD2 rs2066844 (R702W) (250), NOD2 rs2066845 (G908R) (184), and NOD2 rs2066847 (L1007insC) (184, 250), and risk of gastric precancerous lesions and GC (Table 3). Further, a recent meta-analysis by Liu et al. (251) that included six case–control studies has shown consistent associations between NOD2 R702W, G908R, and L1007insC, and risk of GC.

Given the documented relevance of other NLRs in H. pylori infection and related GC, and that polymorphisms in genes such as NLRP3 (252–255) 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.

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 TLR9 to be important H. pylori recognizing PRRs, also showed that H. pylori 5′-triphosphorylated RNA can be sensed by RIG-I and can contribute to the TLR-independent type I IFN response to this bacteria in DCs. Further, Tatsuta et al. (258) have recently shown that MDA-5 expression was significantly increased in the human gastric antral mucosa of H. pylori-infected individuals. In addition, these authors showed that increased MDA-5 levels correlated with atrophy and intestinal metaplasia in the corpus of these individuals (258).

C-type lectin receptors bind to carbohydrates (mannose- or fucose-containing glycans) present on pathogens to tailor immune responses to viruses, bacteria, and fungi. DC-specific intercellular 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).

To date, no studies have been conducted to determine the association between genetic polymorphisms involved in the RLR and CLR signaling pathways and GC, however, Kutikhin and Yuzhalin (266) have comprehensively analyzed the oncogenic potential of both RLRs and CLRs, suggesting that future oncogenomic investigations should focus on polymorphisms in MRC1 (rs1926736, rs2478577, rs2437257, and rs691005), CD209 (rs2287886, rs735239, rs4804803, and rs735240), CLEC7A (rs16910526), and RIG-I (rs36055726, rs11795404, and rs10813831).

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 studies assessing the implications of RLRs and CLRs in H. pylori-related inflammation and subsequent carcinogenesis need to be conducted.