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Inflammasome-mediated production of mature IL-1β and IL-18 cytokines represents an important innate immune response against infecting pathogens. Helicobacter pylori, one of the most successful and persistent human pathogens, induces severe inflammation leading to gastritis and more serious gastric diseases. H. pylori modulates different immune responses for its survival and inflammasome signaling is manipulated by the cag pathogenicity island (cagPAI), urease and VacA cytotoxin. Here we report that H. pylori regulates NLRP3 expression, an inflammasome forming regulator, in infected THP-1 monocytes. This response was independent of the major H. pylori pathogenicity-associated factors CagA, VacA, Cgt, FlaA and cagPAI. Two NLRP3 expression controlling factors, the NLRP3 mRNA targeting microRNA hsa-miR-223-3p and cytokine IL-10, were found to work in tandem for its regulation. H. pylori infection also induced copious amount of pro-IL-1β in THP-1 monocytes/macrophages but secreted a very low amount of mature IL-1β. Moreover, secreted IL-10 correlated with the down-regulation of nigericin-induced NLRP3 inflammasome activation of LPS-primed THP-1 monocytes and human PBMCs from volunteers. However, H. pylori-treated PBMCs secreted significantly more mature IL-1β throughout the infection period, which suggests a different mode of activation. Taken together, this study demonstrates targeting of inflammasome-forming NLRP3, an important innate immunity component, and crucial manipulation of pro- and anti-inflammatory cytokines in H. pylori infection.

Helicobacter pylori is the causative agent for developing gastritis, gastric ulcer, and even gastric cancer. Virulent strains carry the cag pathogenicity island (cagPAI) encoding a type-IV secretion system (T4SS) for injecting the CagA protein. However, mechanisms of sensing this pathogen through Toll-like receptors (TLRs) and downstream signalling pathways in the development of different pathologies are widely unclear. Here, we explored the involvement of TLR-2 and TLR-5 in THP-1 cells and HEK293 cell lines (stably transfected with TLR-2 or TLR-5) during infection with wild-type H. pylori and isogenic cagPAI mutants. H. pylori triggered enhanced TLR-2 and TLR-5 expression in THP-1, HEK293-TLR2 and HEK293-TLR5 cells, but not in the HEK293 control. In addition, IL-8 and TNF-α cytokine secretion in THP-1 cells was induced in a cagPAI-dependent manner. Furthermore, we show that HEK293 cells are not competent for the uptake of T4SS-delivered CagA, and are therefore ideally suited for studying TLR signalling in the absence of T4SS functions. HEK293 control cells, which do not induce TLR-2 and TLR-5 expression during infection, only secreted cytokines in small amounts, in agreement with T4SS functions being absent. In contrast, HEK293-TLR2 and HEK293-TLR5 cells were highly competent for inducing the secretion of IL-8 and TNF-α cytokines in a cagPAI-independent manner, suggesting that the expression of TLR-2 or TLR-5 has profoundly changed the capability to trigger pro-inflammatory signalling upon infection. Using phospho-specific antibodies and luciferase reporter assays, we further demonstrate that H. pylori induces IRAK-1 and IκB phosphorylation in a TLR-dependent manner, and this was required for activation of transcription factor NF-κB. Finally, NF-κB activation in HEK293-TLR2 and HEK293-TLR5 cells was confirmed by expressing p65-GFP which was translocated from the cytoplasm into the nucleus. These data indicate that H. pylori-induced expression of TLR-2 and TLR-5 can qualitatively shift cagPAI-dependent to cagPAI-independent pro-inflammatory signalling pathways with possible impact on the outcome of H. pylori-associated diseases.

Toll-like receptor TLR5 recognizes a conserved domain, termed D1, that is present in flagellins of several pathogenic bacteria but not in Helicobacter pylori . Highly virulent H. pylori strains possess a type IV secretion system (T4SS) for delivery of virulence factors into gastric epithelial cells. Here, we show that one of the H. pylori T4SS components, protein CagL, can act as a flagellin-independent TLR5 activator. CagL contains a D1-like motif that mediates adherence to TLR5 + epithelial cells, TLR5 activation, and downstream signaling in vitro. TLR5 expression is associated with H. pylori infection and gastric lesions in human biopsies. Using Tlr5 -knockout and wild-type mice, we show that TLR5 is important for efficient control of H. pylori infection. Our results indicate that CagL, by activating TLR5, may modulate immune responses to H. pylori . Toll-like receptor TLR5 recognizes a domain, D1, that is present in flagellins of several pathogenic bacteria but not in Helicobacter pylori . Here, the authors show that TLR5 can be activated independently of flagellin by a component of the H. pylori type IV secretion system that contains a D1-like motif.

The role of microbiota in human health and disease is becoming increasingly clear as a result of modern microbiome studies in recent decades. The gastrointestinal tract is the major habitat for microbiota in the human body. This microbiota comprises several trillion microorganisms, which is equivalent to almost ten times the total number of cells of the human host. Helicobacter pylori is a known pathogen that colonizes the gastric mucosa of almost half of the world population. H. pylori is associated with several gastric diseases, including gastric cancer (GC) development. However, the impact of the gastric microbiota in the colonization, chronic infection, and pathogenesis is still not fully understood. Several studies have documented qualitative and quantitative changes in the microbiota’s composition in the presence or absence of this pathogen. Among the diverse microflora in the stomach, the Firmicutes represent the most notable. Bacteria such as Prevotella sp., Clostridium sp., Lactobacillus sp., and Veillonella sp. were frequently found in the healthy human stomach. In contrast, H.pylori is very dominant during chronic gastritis, increasing the proportion of Proteobacteria in the total microbiota to almost 80%, with decreasing relative proportions of Firmicutes. Likewise, H. pylori and Streptococcus are the most abundant bacteria during peptic ulcer disease. While the development of H. pylori-associated intestinal metaplasia is accompanied by an increase in Bacteroides, the stomachs of GC patients are dominated by Firmicutes such as Lactobacillus and Veillonella, constituting up to 40% of the total microbiota, and by Bacteroidetes such as Prevotella, whereas the numbers of H. pylori are decreasing. This review focuses on some of the consequences of changes in the gastric microbiota and the function of probiotics to modulate H. pylori infection and dysbiosis in general.

The gastric pathogen and carcinogen Helicobacter pylori (H. pylori) encodes a type IV secretion system for translocation of the effector protein CagA into host cells. Injected CagA becomes tyrosine-phosphorylated at the five amino acid residue Glutamate-Proline- Isoleucine-Tyrosine-Alanine (EPIYA)-sequence motifs. These phosphorylated EPIYA-sites represent recognition motifs for binding of multiple host factors, which then manipulate signaling pathways to trigger gastric disease. Thus, efficient detection of single phosphorylated EPIYA-motifs in CagA is required. Detection of phospho-CagA is primarily performed using commercial pan-phosphotyrosine antibodies. However, those antibodies were originally generated to recognize many phosphotyrosines in various mammalian proteins and are not optimized for use in bacteria. To address this important limitation, we synthesized 11-mer phospho- and non-phospho-peptides from EPIYA-motifs A, B, and C, and produced three phospho-specific and three non-phospho-specific rabbit polyclonal CagA antibodies. These antibodies specifically recognized the corresponding phosphorylated and non-phosphorylated EPIYA-motifs, while the EPIYA-C antibodies also recognized the related East-Asian EPIYA-D motif. Otherwise, no cross-reactivity of the antibodies among EPIYAs was observed. Western blotting demonstrated that each EPIYA-motif can be predominantly phosphorylated during H. pylori infection. This represents the first complete set of phospho-specific antibodies for an effector protein in bacteria, providing useful tools to gather information for the categorization of CagA phosphorylation, cancer signaling, and gastric disease progression.

Helicobacter pylori persistently colonizes the human stomach, and is associated with inflammation-induced gastric cancer. Bacterial crosstalk with the host immune system produces various inflammatory mediators and subsequent reactions in the host, but not bacterial clearance. Interleukin-1β (IL-1β) is implicated in gastric cancer development and certain gene polymorphisms play a role in this scenario. Mature IL-1β production depends on inflammasome activation, and the NLRP3 inflammasome is a major driver in H. pylori-infected mice, while recent studies demonstrated the down-regulation of NLRP3 expression in human immune cells, indicating a differential NLRP3 regulation in human vs. mice. In addition to the formation of mature IL-1β or IL-18, inflammasome activation induces pyroptotic death in cells. We demonstrate that H. pylori infection indeed upregulated the expression of pro-IL-1β in human immune cells, but secreted only very low amounts of mature IL-1β. However, application of exogenous control activators such as Nigericin or ATP to infected cells readily induced NLRP3 inflammasome formation and secretion of high amounts of mature IL-1β. This suggests that chronic H. pylori infection in humans manipulates inflammasome activation and pyroptosis for bacterial persistence. This inflammasome deregulation during H. pylori infection, however, is prone to external stimulation by microbial, environmental or host molecules of inflammasome activators for the production of high amounts of mature IL-1β and signaling-mediated gastric tumorigenesis in humans.

Toll-like receptors (TLRs) are crucial for pathogen recognition and downstream signaling to induce effective immunity. The gastric pathogen Helicobacter pylori is a paradigm of persistent bacterial infections and chronic inflammation in humans. The chronicity of inflammation during H. pylori infection is related to the manipulation of regulatory cytokines. In general, the early detection of H. pylori by TLRs and other pattern recognition receptors (PRRs) is believed to induce a regulatory cytokine or chemokine profile that eventually blocks the resolution of inflammation. H. pylori factors such as LPS, HSP-60, NapA, DNA, and RNA are reported in various studies to be recognized by specific TLRs. However, H. pylori flagellin evades the recognition of TLR5 by possessing a conserved N-terminal motif. Activation of TLRs and resulting signal transduction events lead to the production of pro- and anti-inflammatory mediators through activation of NF-κB, MAP kinases, and IRF signaling pathways. The genetic polymorphisms of these important PRRs are also implicated in the varied outcome and disease progression. Hence, the interplay of TLRs and bacterial factors highlight the complexity of innate immune recognition and immune evasion as well as regulated processes in the progression of associated pathologies. Here we will review this important aspect of H. pylori infection.