Outer Membrane Vesicle Production by Helicobacter pylori Represents an Approach for the Delivery of Virulence Factors CagA,VacA and UreA into Human Gastric Adenocarcinoma (AGS) Cells

Helicobacter pylori infection is the etiology of several gastric-related diseases including gastric cancer. Cytotoxin associated gene A (CagA), vacuolating cytotoxin A (VacA) and α-subunit of urease (UreA) are three major virulence factors of H. pylori, and each of them has a distinct entry pathway and pathogenic mechanism during bacterial infection. H. pylori can shed outer membrane vesicles (OMVs). Therefore, it would be interesting to explore the production kinetics of H. pylori OMVs and its connection with the entry of key virulence factors into host cells. Here, we isolated OMVs from H. pylori 26,695 strain and characterized their properties and interaction kinetics with human gastric adenocarcinoma (AGS) cells. We found that the generation of OMVs and the presence of CagA, VacA and UreA in OMVs were a lasting event throughout different phases of bacterial growth. H. pylori OMVs entered AGS cells mainly through macropinocytosis/phagocytosis. Furthermore, CagA, VacA and UreA could enter AGS cells via OMVs and the treatment with H. pylori OMVs would cause cell death. Comparison of H. pylori 26,695 and clinical strains suggested that the production and characteristics of OMVs are not only limited to laboratory strains commonly in use, but a general phenomenon to most H. pylori strains.     

The Innate Immune Glycoprotein Lactoferrin Represses the Helicobacter pylori cag Type IV Secretion System

Chronic infection with Helicobacter pylori increases risk of gastric diseases including gastric cancer. Despite development of a robust immune response, H. pylori persists in the gastric niche. Progression of gastric inflammation to serious disease outcomes is associated with infection with H. pylori strains which encode the cag Type IV Secretion System (cag T4SS). The cag T4SS is responsible for translocating the oncogenic protein CagA into host cells and inducing pro-inflammatory and carcinogenic signaling cascades. Our previous work demonstrated that nutrient iron modulates the activity of the T4SS and biogenesis of T4SS pili. In response to H. pylori infection, the host produces a variety of antimicrobial molecules, including the iron-binding glycoprotein, lactoferrin. Our work shows that apo-lactoferrin exerts antimicrobial activity against H. pylori under iron-limited conditions, while holo-lactoferrin enhances bacterial growth. Culturing H. pylori in the presence of holo-lactoferrin prior to co-culture with gastric epithelial cells, results in repression of the cag T4SS activity. Concomitantly, a decrease in biogenesis of cag T4SS pili at the host-pathogen interface was observed under these culture conditions by high-resolution electron microscopy analyses. Taken together, these results indicate that acquisition of alternate sources of nutrient iron plays a role in regulating the pro-inflammatory activity of a bacterial secretion system and present novel therapeutic targets for the treatment of H. pylori-related disease.     

Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.     

The Helicobacter pylori CagY Protein Drives Gastric Th1 and Th17 Inflammation and B Cell Proliferation in Gastric MALT Lymphoma

Background: the neoplastic B cells of the Helicobacter pylori-related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma proliferate in response to H. pylori, however, the nature of the H. pylori antigen responsible for proliferation is still unknown. The purpose of the study was to dissect whether CagY might be the H. pylori antigen able to drive B cell proliferation. Methods: the B cells and the clonal progeny of T cells from the gastric mucosa of five patients with MALT lymphoma were compared with those of T cell clones obtained from five H. pylori–infected patients with chronic gastritis. The T cell clones were assessed for their specificity to H. pylori CagY, cytokine profile and helper function for B cell proliferation. Results: 22 of 158 CD4⁺ (13.9%) gastric clones from MALT lymphoma and three of 179 CD4⁺ (1.7%) clones from chronic gastritis recognized CagY. CagY predominantly drives Interferon-gamma (IFN-γ) and Interleukin-17 (IL-17) secretion by gastric CD4⁺ T cells from H. pylori-infected patients with low-grade gastric MALT lymphoma. All MALT lymphoma-derived clones dose dependently increased their B cell help, whereas clones from chronic gastritis lost helper activity at T-to-B-cell ratios greater than 1. Conclusion: the results obtained indicate that CagY drives both B cell proliferation and T cell activation in gastric MALT lymphomas.     

In Peripheral Blood Mononuclear Cells Helicobacter pylori Induces the Secretion of Soluble and Exosomal Cytokines Related to Carcinogenesis

Helicobacter pylori promotes the secretion of cytokines that regulate inflammation and carcinogenesis. Immune cells secrete cytokines into the extracellular medium or packaged in exosomes. The objective of this study was to analyze the profile of soluble and exosomal cytokines that were secreted by human peripheral blood mononuclear cells (PBMCs) that were infected with H. pylori and to build a network of interaction between cytokines and cellular proteins. PBMCs were obtained by density gradient centrifugation and infected with H. pylori for 24 h. The infection was verified by immunofluorescence and Western blot for CagA. The exosomes were obtained from culture supernatant by ultracentrifugation and characterized by transmission electron microscopy, particle size analysis, and Western blot for CD9 and CD81. Cytokines were quantified using a multiplex immunoassay in the culture supernatant, intact exosomes, and lysed exosomes. H. pylori adheres to lymphocytes and translocates CagA. In PBMCs, H. pylori induces an increase in the soluble and exosomal IL-1β, IL-6, TNF-α, IL-10, IL-17A, IL-21, and IL-22. The protein–protein interaction (PPI) network shows that soluble and exosomal cytokines interact with proteins that participate in signaling pathways such as NF-κB, MAPK, PI3K-Akt, Jak-STAT, FoxO, and mTOR, that are related to carcinogenesis; moreover, TNF-α had the highest number of interactions. Cytokine-loaded exosomes represent another means of intercellular communication that is activated by H. pylori to stimulate inflammation, carcinogenesis, or cancer progression. Cytokine-loaded exosomes are likely to be associated with extragastrointestinal diseases of inflammatory origin.     

Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells

Helicobacter pylori neutrophil-activating protein (HP-NAP)-induced production of reactive oxygen species (ROS) by neutrophils and monocytes is regulated by pertussis toxin (PTX)-sensitive G proteins, whereas HP-NAP-induced cytokine secretion by monocytes is mediated by Toll-like receptor 2 (TLR2). However, it is unclear whether TLR2 participates in HP-NAP-induced cytokine secretion by neutrophils. Here, all-trans retinoic acid (ATRA)-induced differentiated HL-60 cells were first employed as a neutrophil model to investigate the molecular mechanisms underlying neutrophil responses to HP-NAP. HP-NAP-induced ROS production in ATRA-induced differentiated HL-60 cells is mediated by the PTX-sensitive heterotrimeric G protein-dependent activation of extracellular signal-regulated kinase 1/2 and p38-mitogen-activated protein kinase, which is consistent with the findings reported for human neutrophils. Next, whether TLR2 participated in HP-NAP-induced secretion of interleukin-8 (IL-8) was investigated in neutrophils and ATRA-induced differentiated HL-60 cells. In both cells, TLR2 participated in HP-NAP-induced IL-8 secretion but not HP-NAP-induced ROS production. Interestingly, PTX-sensitive G proteins also contributed to the HP-NAP-induced secretion of IL-8 from neutrophils and the differentiated HL-60 cells. Our ELISA-based binding assay further revealed the competitive binding of Pam₃CSK₄, a TLR2 agonist, and HP-NAP to TLR2, which suggests the presence of specific and direct interactions between HP-NAP and TLR2. Thus, HP-NAP directly interacts with and activates TLR2 to induce IL-8 secretion in neutrophils and ATRA-induced differentiated HL-60 cells.     

The Role of Helicobacter pylori Neutrophil-Activating Protein in the Pathogenesis of H. pylori and Beyond: From a Virulence Factor to Therapeutic Targets and Therapeutic Agents

Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of H. pylori, plays a role in bacterial protection and host inflammation. HP-NAP activates a variety of innate immune cells, including neutrophils, monocytes, and mast cells, to induce their pro-oxidant and pro-inflammatory activities. This protein also induces T-helper type 1 (Th1) immune response and cytotoxic T lymphocyte (CTL) activity, supporting that HP-NAP is able to promote gastric inflammation by activation of adaptive immune responses. Thus, HP-NAP is a potential therapeutic target for the treatment of H. pylori-induced gastric inflammation. The inflammatory responses triggered by HP-NAP are mediated by a PTX-sensitive G protein-coupled receptor and Toll-like receptor 2. Drugs designed to block the interactions between HP-NAP and its receptors could alleviate the inflammation in gastric mucosa caused by H. pylori infection. In addition, HP-NAP acts as a promising therapeutic agent for vaccine development, allergy treatment, and cancer immunotherapy. The high antigenicity of HP-NAP makes this protein a component of vaccines against H. pylori infection. Due to its immunomodulatory activity to stimulate the Th1-inducing ability of dendritic cells, enhance Th1 immune response and CTL activity, and suppress Th2-mediated allergic responses, HP-NAP could also act as an adjuvant in vaccines, a drug candidate against allergic diseases, and an immunotherapeutic agent for cancer. This review highlights the role of HP-NAP in the pathogenesis of H. pylori and the potential for this protein to be a therapeutic target in the treatment of H. pylori infection and therapeutic agents against H. pylori-associated diseases, allergies, and cancer.     

Morphogen Signals Shaping the Gastric Glands in Health and Disease

The adult gastric mucosa is characterised by deep invaginations of the epithelium called glands. These tissue architectural elements are maintained with the contribution of morphogen signals. Morphogens are expressed in specific areas of the tissue, and their diffusion generates gradients in the microenvironment. Cells at different positions in the gland sense a specific combination of signals that instruct them to differentiate, proliferate, regenerate, or migrate. Differentiated cells perform specific functions involved in digestion, such as the production of protective mucus and the secretion of digestive enzymes or gastric acid. Biopsies from gastric precancerous conditions usually display tissue aberrations and change the shape of the glands. Alteration of the morphogen signalling microenvironment is likely to underlie those conditions. Furthermore, genes involved in morphogen signalling pathways are found to be frequently mutated in gastric cancer. We summarise the most recent findings regarding alterations of morphogen signalling during gastric carcinogenesis, and we highlight the new stem cell technologies that are improving our understanding of the regulation of human tissue shape.     

Selective Inhibition of Helicobacter pylori Carbonic Anhydrases by Carvacrol and Thymol Could Impair Biofilm Production and the Release of Outer Membrane Vesicles

Helicobacter pylori, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti-H. pylori activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of H. pylori CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative H. pylori CA inhibitors or to be used in association with current drugs for the management of H. pylori infection and limiting the spread of antibiotic resistance.     

Impact of Exosomes Released by Different Corneal Cell Types on the Wound Healing Properties of Human Corneal Epithelial Cells

Corneal wound healing involves communication between the different cell types that constitute the three cellular layers of the cornea (epithelium, stroma and endothelium), a process ensured in part by a category of extracellular vesicles called exosomes. In the present study, we isolated exosomes released by primary cultured human corneal epithelial cells (hCECs), corneal fibroblasts (hCFs) and corneal endothelial cells (hCEnCs) and determined whether they have wound healing characteristics of their own and to which point they modify the genetic and proteomic pattern of these cell types. Exosomes released by all three cell types significantly accelerated wound closure of scratch-wounded hCECs in vitro compared to controls (without exosomes). Profiling of activated kinases revealed that exosomes from human corneal cells caused the activation of signal transduction mediators that belong to the HSP27, STAT, β-catenin, GSK-3β and p38 pathways. Most of all, data from gene profiling analyses indicated that exosomes, irrespective of their cellular origin, alter a restricted subset of genes that are completely different between each targeted cell type (hCECs, hCFS, hCEnCs). Analysis of the genes specifically differentially regulated for a given cell-type in the microarray data using the Ingenuity Pathway Analysis (IPA) software revealed that the mean gene expression profile of hCECs cultured in the presence of exosomes would likely promote cell proliferation and migration whereas it would reduce differentiation when compared to control cells. Collectively, our findings represent a conceptual advance in understanding the mechanisms of corneal wound repair that may ultimately open new avenues for the development of novel therapeutic approaches to improve closure of corneal wounds.     

Inactivation of the Akt/FOXM1 Signaling Pathway by Panobinostat Suppresses the Proliferation and Metastasis of Gastric Cancer Cells

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related deaths worldwide. Histone deacetylase (HDAC) inhibitors are a new class of cytostatic agents available for the treatment of various cancers and diseases. Although numerous clinical and pre-clinical trials on the anticancer effects of panobinostat have been conducted, only a few reports have investigated its efficacy in gastric cancer. The present study aimed to investigate the effects of panobinostat in gastric cancer cells. Panobinostat significantly inhibited the cell viability and proliferation of the gastric cancer cell lines SNU484 and SNU638 in a dose-dependent manner; it reduced the colony-forming ability of these cells. Moreover, it induced apoptosis as indicated by increased protein levels of cleaved poly ADP-ribose polymerase and cleaved caspase-3. Panobinostat induced the G2/M cell cycle arrest in SNU484 and SNU638 cells and subsequently decreased the G2/M phase regulatory-associated protein expression of p-Wee1, Myt1, and Cdc2. Furthermore, panobinostat significantly inhibited the metastasis of SNU484 and SNU638 cells by regulating the expression of MMP-9 and E-cadherin. Further, it decreased the protein levels of p-Akt and forkhead box protein M1 (FOXM1). These effects were reversed by the Akt agonist SC79 and were accelerated by the Akt inhibitor LY2940002. Moreover, tumor growth in xenograft animal experiments was suppressed by panobinostat. These results indicated that panobinostat inhibits the proliferation, metastasis, and cell cycle progression of gastric cancer cells by promoting apoptosis and inactivating Akt/FOXM1 signaling. Cumulatively, our present study suggests that panobinostat is a potential drug for the treatment of gastric cancer.     

A Role for Human Renal Tubular Epithelial Cells in Direct Allo-Recognition by CD4+ T-Cells and the Effect of Ischemia-Reperfusion

Direct allorecognition is the earliest and most potent immune response against a kidney allograft. Currently, it is thought that passenger donor professional antigen-presenting cells (APCs) are responsible. Further, many studies support that graft ischemia-reperfusion injury increases the probability of acute rejection. We evaluated the possible role of primary human proximal renal tubular epithelial cells (RPTECs) in direct allorecognition by CD4+ T-cells and the effect of anoxia-reoxygenation. In cell culture, we detected that RPTECs express all the required molecules for CD4+ T-cell activation (HLA-DR, CD80, and ICAM-1). Anoxia-reoxygenation decreased HLA-DR and CD80 but increased ICAM-1. Following this, RPTECs were co-cultured with alloreactive CD4+ T-cells. In T-cells, zeta chain phosphorylation and c-Myc increased, indicating activation of T-cell receptor and co-stimulation signal transduction pathways, respectively. T-cell proliferation assessed with bromodeoxyuridine assay and with the marker Ki-67 increased. Previous culture of RPTECs under anoxia raised all the above parameters in T-cells. FOXP3 remained unaffected in all cases, signifying that proliferating T-cells were not differentiated towards a regulatory phenotype. Our results support that direct allorecognition may be mediated by RPTECs even in the absence of donor-derived professional APCs. Also, ischemia-reperfusion injury of the graft may enhance the above capacity of RPTECs, increasing the possibility of acute rejection.     

TGF-β/Smad Signalling Activation by HTRA1 Regulates the Function of Human Lens Epithelial Cells and Its Mechanism in Posterior Subcapsular Congenital Cataract

Congenital cataract is the leading cause of blindness among children worldwide. Patients with posterior subcapsular congenital cataract (PSC) in the central visual axis can result in worsening vision and stimulus deprivation amblyopia. However, the pathogenesis of PSC remains unclear. This study aims to explore the functional regulation and mechanism of HTRA1 in human lens epithelial cells (HLECs). HTRA1 was significantly downregulated in the lens capsules of children with PSC compared to normal controls. HTRA1 is a suppression factor of transforming growth factor-β (TGF-β) signalling pathway, which plays a key role in cataract formation. The results showed that the TGF-β/Smad signalling pathway was activated in the lens tissue of PSC. The effect of HTRA1 on cell proliferation, migration and apoptosis was measured in HLECs. In primary HLECs, the downregulation of HTRA1 can promote the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway and can significantly upregulate the TGF-β/Smad downstream target genes FN1 and α-SMA. HTRA1 was also knocked out in the eyes of C57BL/6J mice via adeno-associated virus-mediated RNA interference. The results showed that HTRA1 knockout can significantly upregulate p-Smad2/3 and activate the TGF-β/Smad signalling pathway, resulting in abnormal proliferation and irregular arrangement of lens epithelial cells and leading to the occurrence of subcapsular cataract. To conclude, HTRA1 was significantly downregulated in children with PSC, and the downregulation of HTRA1 enhanced the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway, which led to the occurrence of PSC.     

Apoptotic Effects of Anthocyanins from Vitis coignetiae Pulliat Are Enhanced by Augmented Enhancer of the Rudimentary Homolog (ERH) in Human Gastric Carcinoma MKN28 Cells

Evidence suggests that augmented expression of a certain gene can influence the efficacy of targeted and conventional chemotherapies. Here, we tested whether the high expression of enhancer of the rudimentary homolog (ERH), which serves as a prognostic factor in some cancers, can influence the efficacy of anthocyanins isolated from fruits of Vitis coignetiae Pulliat, Meoru in Korea (AIMs) on human gastric cancer cells. The anticancer efficacy of AIMs was augmented in ERH-transfected MKN28 cells (E-MKN28 cells). Molecularly, ERH augmented AIM-induced caspase-dependent apoptosis by activating caspase-3 and -9. The ERH-augmented apoptotic effect was related to mitochondrial depolarization and inhibition of antiapoptotic proteins, XIAP, and Bcl-2. In addition, reactive oxygen species (ROS) generation was augmented in AIMs-treated E-MKN28 cells compared to AIMs-treated naïve MKN28 cells. In conclusion, ERH augmented AIM-induced caspase-dependent mitochondrial-related apoptosis in MKN28 cells. A decrease in expression of Bcl-2 and subsequent excessive ROS generation would be the mechanism for ERH-augmented mitochondrial-related apoptosis in AIMs-treated MKN28 cells. A decrease in expression of XIAP would be another mechanism for ERH-augmented caspase-dependent apoptosis in AIMs-treated MKN28 cells.     

Alpha-Lipoic Acid Inhibits Spontaneous Diabetes and Autoimmune Recurrence in Non-Obese Diabetic Mice by Enhancing Differentiation of Regulatory T Cells and Showed Potential for Use in Cell Therapies for the Treatment of Type 1 Diabetes

Type 1 diabetes (T1D) is caused by the destruction of β cells in pancreatic islets by autoimmune T cells. Islet transplantation has been established as an effective treatment for T1D. However, the survival of islet grafts is often disrupted by recurrent autoimmunity. Alpha-lipoic acid (ALA) has been reported to have immunomodulatory effects and, therefore, may have therapeutic potential in the treatment of T1D. In this study, we investigated the therapeutic potential of ALA in autoimmunity inhibition. We treated non-obese diabetic (NOD) mice with spontaneous diabetes and islet-transplantation mice with ALA. The onset of diabetes was decreased and survival of the islet grafts was extended. The populations of Th1 cells decreased, and regulatory T cells (Tregs) increased in ALA-treated mice. The in vitro Treg differentiation was significantly increased by treatment with ALA. The adoptive transfer of ALA-differentiated Tregs into NOD recipients improved the outcome of the islet grafts. Our results showed that in vivo ALA treatment suppressed spontaneous diabetes and autoimmune recurrence in NOD mice by inhibiting the Th1 immune response and inducing the differentiation of Tregs. Our study also demonstrated the therapeutic potential of ALA in Treg-based cell therapies and islet transplantation used in the treatment of T1D.