Masking of typical TLR4 and TLR5 ligands modulates inflammation and resolution by Helicobacter pylori.

Helicobacter pylori is a paradigm of chronic bacterial infection and is associated with peptic ulceration and malignancies. H. pylori uses specific masking mechanisms to avoid canonical ligands from activating Toll-like receptors (TLRs), such as lipopolysaccharide (LPS) modification and specific flagellin sequences that are not detected by TLR4 and TLR5, respectively. Thus, it was believed for a long time that H. pylori evades TLR recognition as a crucial strategy for immune escape and bacterial persistence. However, recent data indicate that multiple TLRs are activated by H. pylori and play a role in the pathology. Remarkably, H. pylori LPS, modified through changes in acylation and phosphorylation, is mainly sensed by other TLRs (TLR2 and TLR10) and induces both pro- and anti-inflammatory responses. In addition, two structural components of the cag pathogenicity island-encoded type IV secretion system (T4SS), CagL and CagY, were shown to contain TLR5-activating domains. These domains stimulate TLR5 and enhance immunity, while LPS-driven TLR10 signaling predominantly activates anti-inflammatory reactions. Here, we discuss the specific roles of these TLRs and masking mechanisms during infection. Masking of typical TLR ligands combined with evolutionary shifting to other TLRs is unique for H. pylori and has not yet been described for any other species in the bacterial kingdom. Finally, we highlight the unmasked T4SS-driven activation of TLR9 by H. pylori, which mainly triggers anti-inflammatory responses.

Toll-like Receptor 5 Activation by the CagY Repeat Domains of Helicobacter pylori.

Helicobacter pylori (Hp) is an important human pathogen associated with gastric inflammation and neoplasia. It is commonly believed that this bacterium avoids major immune recognition by Toll-like receptors (TLRs) because of low intrinsic activity of its flagellin and lipopolysaccharides (LPS). In particular, TLR5 specifically detects flagellins in various bacterial pathogens, while Hp evolved mutations in flagellin to evade detection through TLR5. Cancerogenic Hp strains encode a type IV secretion system (T4SS). The T4SS core component and pilus-associated protein CagY, a large VirB10 ortholog, drives effector molecule translocation. Here, we identify CagY as a flagellin-independent TLR5 agonist. We detect five TLR5 interaction sites, promoting binding of CagY-positive Hp to TLR5-expressing cells, TLR5 stimulation, and intracellular signal transduction. Consequently, CagY constitutes a remarkable VirB10 member detected by TLR5, driving crucial innate immune responses by this human pathogen.

Helicobacter pylori Avoids the Critical Activation of NLRP3 Inflammasome-Mediated Production of Oncogenic Mature IL-1ß in Human Immune Cells.

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-1ß (IL-1ß) is implicated in gastric cancer development and certain gene polymorphisms play a role in this scenario. Mature IL-1ß 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-1ß or IL-18, inflammasome activation induces pyroptotic death in cells. We demonstrate that H. pylori infection indeed upregulated the expression of pro-IL-1ß in human immune cells, but secreted only very low amounts of mature IL-1ß. 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-1ß. 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-1ß and signaling-mediated gastric tumorigenesis in humans.

T4SS-dependent TLR5 activation by Helicobacter pylori infection.

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.

Tailor-Made Detection of Individual Phosphorylated and Non-Phosphorylated EPIYA-Motifs of Helicobacter pylori Oncoprotein CagA.

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.

Mechanisms of Inflammasome Signaling, microRNA Induction and Resolution of Inflammation by Helicobacter pylori.

Inflammasome-controlled transcription and subsequent cleavage-mediated activation of mature IL-1ß and IL-18 cytokines exemplify a crucial innate immune mechanism to combat intruding pathogens. Helicobacter pylori represents a predominant persistent infection in humans, affecting approximately half of the population worldwide, and is associated with the development of chronic gastritis, peptic ulcer disease, and gastric cancer. Studies in knockout mice have demonstrated that the pro-inflammatory cytokine IL-1ß plays a central role in gastric tumorigenesis. Infection by H. pylori was recently reported to stimulate the inflammasome both in cells of the mouse and human immune systems. Using mouse models and in vitro cultured cell systems, the bacterial pathogenicity factors and molecular mechanisms of inflammasome activation have been analyzed. On the one hand, it appears that H. pylori-stimulated IL-1ß production is triggered by engagement of the immune receptors TLR2 and NLRP3, and caspase-1. On the other hand, microRNA hsa-miR-223-3p is induced by the bacteria, which controls the expression of NLRP3. This regulating effect by H. pylori on microRNA expression was also described for more than 60 additionally identified microRNAs, indicating a prominent role for inflammatory and other responses. Besides TLR2, TLR9 becomes activated by H. pylori DNA and further TLR10 stimulated by the bacteria induce the secretion of IL-8 and TNF, respectively. Interestingly, TLR-dependent pathways can accelerate both pro- and anti-inflammatory responses during H. pylori infection. Balancing from a pro-inflammation to anti-inflammation phenotype results in a reduction in immune attack, allowing H. pylori to persistently colonize and to survive in the gastric niche. In this chapter, we will pinpoint the role of H. pylori in TLR- and NLRP3 inflammasome-dependent signaling together with the differential functions of pro- and anti-inflammatory cytokines. Moreover, the impact of microRNAs on H. pylori-host interaction will be discussed, and its role in resolution of infection versus chronic infection, as well as in gastric disease development.

Immune Cell Signaling by Helicobacter pylori: Impact on Gastric Pathology.

Helicobacter pylori represents a highly successful colonizer of the human stomach. Infections with this Gram-negative bacterium can persist lifelong, and although in the majority of cases colonization is asymptomatic, it can trigger pathologies ranging from chronic gastritis and peptic ulceration to gastric cancer. The interaction of the bacteria with the human host modulates immune responses in different ways to enable bacterial survival and persistence. H. pylori uses various pathogenicity-associated factors such as VacA, NapA, CGT, GGT, lipopolysaccharide, peptidoglycan, heptose 1,7-bisphosphate, ADP-heptose, cholesterol glucosides, urease and a type IV secretion system for controlling immune signaling and cellular functions. It appears that H. pylori manipulates multiple extracellular immune receptors such as integrin-ß2 (CD18), EGFR, CD74, CD300E, DC-SIGN, MINCLE, TRPM2, T-cell and Toll-like receptors as well as a number of intracellular receptors including NLRP3, NOD1, NOD2, TIFA and ALPK1. Consequently, downstream signaling pathways are hijacked, inducing tolerogenic dendritic cells, inhibiting effector T cell responses and changing the gastrointestinal microbiota. Here, we discuss in detail the interplay of bacterial factors with multiple immuno-regulatory cells and summarize the main immune evasion and persistence strategies employed by H. pylori.

Helicobacter pylori controls NLRP3 expression by regulating hsa-miR-223-3p and IL-10 in cultured and primary human immune cells.

Inflammasome-mediated production of mature IL-1ß 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-1ß in THP-1 monocytes/macrophages but secreted a very low amount of mature IL-1ß. 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-1ß 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.

Inflammasome Activation by Helicobacter pylori and Its Implications for Persistence and Immunity.

Infection with the Gram-negative pathogen Helicobacter pylori is the most prevalent chronic bacterial infection affecting about 50 % of the human world population and is the main risk factor for gastric cancer development. The pro-inflammatory cytokine IL-1ß plays a crucial role in the development of gastric tumors, and polymorphisms in the IL-1 gene cluster resulting in increased IL-1ß production have been associated with increased risk for gastric cancer. Recently, Helicobacter pylori was postulated to activate the inflammasome in human and mouse immune cells, and the molecular mechanisms and the bacterial virulence factors activating the inflammasome were elucidated in cell culture as well as animal models. It appears that H. pylori-induced IL-1ß secretion is mediated by activation of toll-like receptor 2 (TLR-2), Nod-like receptor family member NLRP3 and caspase-1. The cag pathogenicity island-encoded type IV secretion system, lipopolysaccharide, vacuolating cytotoxin, and urease B subunit appear to play a role in inflammasome activation. In addition, recent results indicate that the TLR-2 â†’ NLRP3 â†’ caspase-1 â†’ IL-18 axis is critical to H. pylori-specific immune regulation conferring protection against allergen-induced asthma and inflammatory bowel disease in murine models. The present chapter will review the proposed mechanisms of NLRP3 inflammasome activation during H. pylori infection and discuss the recent progress in this important research field.

Interplay of the Gastric Pathogen Helicobacter pylori with Toll-Like Receptors.

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.

Signal transduction of Helicobacter pylori during interaction with host cell protein receptors of epithelial and immune cells.

Helicobacter pylori infections can induce pathologies ranging from chronic gastritis, peptic ulceration to gastric cancer. Bacterial isolates harbor numerous well-known adhesins, vacuolating cytotoxin VacA, protease HtrA, urease, peptidoglycan, and type IV secretion systems (T4SS). It appears that H. pylori targets more than 40 known host protein receptors on epithelial or immune cells. A series of T4SS components such as CagL, CagI, CagY, and CagA can bind to the integrin α 5ß 1 receptor. Other targeted membrane-based receptors include the integrins αvß 3, αvß 5, and ß 2 (CD18), RPTP-α/ß, GP130, E-cadherin, fibronectin, laminin, CD46, CD74, ICAM1/LFA1, T-cell receptor, Toll-like receptors, and receptor tyrosine kinases EGFR, ErbB2, ErbB3, and c-Met. In addition, H. pylori is able to activate the intracellular receptors NOD1, NOD2, and NLRP3 with important roles in innate immunity. Here we review the interplay of various bacterial factors with host protein receptors. The contribution of these interactions to signal transduction and pathogenesis is discussed.

Blood zinc protoporphyrin, serum total protein, and total cholesterol levels in automobile workshop workers in relation to lead toxicity: Our experience.

Blood zinc protoporphyrin (ZPP), serum total protein (TP), and total cholesterol (TC) levels in automobile workshop workers in relation to lead toxicity were analysed. In the present study, automobile workshop workers (healthy male workers at an age between 28 and 35 from four major automobile workshops in Kottayam, Kerala State, India) and the control (male healthy adults at an age between 28 and 35 residing at Aymanam, a distant village at Kottayam District, Kerala having reduced or no chance of lead exposure) displayed significant difference in blood lead (BPb) and blood ZZP (BZPP) level. The mean value of BPb in automobile workshop workers was 15.76±0.33 µg/dl, while in the control it was 8.20±0.15 µg/dl. In automobile workshop workers, the mean value of BZPP was 34.2±0.62 µg/dl. The control group exhibited a mean of 11.5±0.22 µg/dl. Automobile workshop workers exhibited significant increase in BZPP was corresponding to the increase in BPb level. The total protein levels estimated in automobile workshop workers showed significant decrease compared to control individuals, but was within the reference range of healthy individuals. The mean value of TP level in automobile workshop workers and control was 6.9±0.13 g/dl and 7.71±0.18 g/dl, respectively. There was no significant difference in blood haemoglobin (BHb) level among the automobile workshop workers and control. The serum TC level in automobile workshop workers showed significant decrease compared to the control individuals, but was with in the reference range of healthy individuals. The mean level of serum TC in automobile workshop workers was 162.00±3.44 mg/dl and the same in control was 172.86±4.32 mg/dl. The present study affirms occupational lead toxicity in automobile workshop workers and its effect on serum protein and cholesterol levels.