Autophagy in Helicobacter pylori-Induced ...

REVIEW Immuno-Gastroenterology 2:3, 132-145; July/August/September 2013; © 2013 STM Publishing

Autophagy in Helicobacter pylori-Induced Inflammation and Disease Evangelos Kazakos1, Nick Dorrell2, Jannis Kountouras1 ¹Department of Medicine, Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece; 2Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK

Summary

Co-adaptation of Helicobacter pylori (Hp) with its human host requires the bacterium’s ingenious ability to orchestrate epithelial cell signaling to maintain a state of mucosal immune activation ultimately, dictating infection outcomes. Autophagy has been intimately involved in tailoring the inflammatory response upon intracellular sensing of bacterial constituents. Highly adapted microorganisms have evolved strategies to subvert autophagy by persisting inside autophagosomes whereas, loss of autophagy delays global turnover of ubiquitylated cargos leading to the accumulation of misfolded proteins and p62/SQSTM1 and NBR1 complexes linked to tumorigenesis. Genome-wide association screens have identified the existence of genetic predisposition loci related to polymorphisms affecting the autophagic modulator ATG16L1 and the autophagy-related factor IRGM. The present review aims at: i. presenting recent advances regarding the role of autophagy in the pathophysiology of Hp infection; and ii. offering possible projections of data related to other pathogens in the context of studying Hp-induced pathologies, aiding future research. Immunogastroenterology 2013; 2:132-145

Key words

autophagy; Helicobacter pylori; CagA; inflammation; oncogenesis; p62/SQSTM1; ubiquitylation; VacA

Introduction Autophagy is a fundamental, highly conserved process in all eukaryotes, for maintaining cytoplasmic homeostasis as a response to various nutritional, stress-related and immunological stimuli. There are three types of autophagy: (1) macroautophagy, (2) microautophagy and (3) chaperone-mediated autophagy, which differ in the process of cargo delivery to the lysosome and the nature of the cargo/substrate. Specifically, autophagy is a reparative, life-sustaining process by which cytoplasmic components are sequestered in the double-membrane structured autophagosomes and then degraded after autophagosomes fuse with lysosomes; it plays a critical role in nutrient recycling, development, cell homeostasis, and defense against pathogens and toxic products.1,2 It serves as a critical protective response during conditions of endoplasmic reticulum (ER) stress through the bulk removal and degradation of unfolded proteins and damaged organelles; in particular, mitochondria (mitophagy) and ER (reticulophagy). On the other hand, autophagy appears to be involved in many pathologies including cancer pathogenesis and progression. In complex metazoan organisms, autophagy is now considered as a bona fide immunological process with a wide, functionally diverse array of effector mechanisms that span both innate and adaptive Correspondence to: Evangelos Kazakos, Department of Medicine, Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece; Email: [email protected] Submitted: 09/05/2013; Revised: 16/06/2013; Accepted: 17/06/2013 DOI: 10.7178/ig.41

132

immunity.3,4 These roles include the following: i. Recognition, capture and direct elimination of invading microorganisms - a process termed as xenophagy; ii. Regulation of pathogenrecognition receptor (PRR) signaling and effector functions; iii. Regulation of inflammasome output and ‘unconventional secretion’ of alarmins; and iv. Cytosolic processing of endogenous antigens for Major Histocompatibility Complex (MHC)-II presentation by acting as a topological inverter.5 Autophagy is genetically regulated and currently more than 30 human Atg genes have been reported to participate in autophagosome formation;6 ATG products assembled by ubiquitin-like coupling, contribute to autophagosome formation and degradation of damaged cytosolic organelles, bulk protein aggregates and microorganisms.7 In principle, autophagy involves three morphologically detectable execution stages subdivided into: i. Initiation - formation of phagophores; ii. Elongation - engulfment of sequestered cargo by autophagosomes; and iii. Maturation - autophagosome fusion with endo-lysomal compartments.3 Autophagy is regulated by a core-signaling pathway that encompasses two major complexes, the Ulk1/Tor complex 1 and the phosphatidylinositol 3 kinase (PI3K) hVPS34beclin1 complex along with associated interacting partners.8 Helicobacter pylori (Hp) is a well-adapted pathobiont, that selectively colonizes the human gastric epithelium affecting at least half of the world’s population; Hp is unique in that it resides within the gastric mucosal environment despite this being a highly acidic environment, which normally serves as a means of protection from bacterial pathogens. Once it has become established within this ecological niche, colonization results

Immuno-Gastroenterology

Volume 2 Issue 3

in chronic inflammatory process of the gastric mucosa and the induction of histological gastritis. Many people infected with Hp remain asymptomatic until the bacterium evades the immune system to achieve persistent colonization. Long-term Hp infection leads to the more severe gastric disorders including peptic ulcer, gastric cancer and mucosal-associated lymphoid tissue (MALT) lymphoma.9,10,11 Moreover, Hp infection has been associated with a diverse array of extragastric disorders potentially related with defective autophagy. Subsequent clinical outcomes are dependent on a complex interaction of selected, hypervirulent strains and host predisposing factors, notably gene promoter polymorphisms that determine host immune responses. Increasing evidence suggests that Hp represents a facultative intracellular bacterium, able to reside inside epithelial cells and professional phagocytes.12 Bacterial entry, dictated by a series of host signaling events, occurs via endocytosis whereas a novel Hp invasin, NudA, influences this process in vitro.13 Since autophagy is a mechanism by which host cells can eliminate intracellular pathogens, its implication as an emerging aspect of Hp pathophysiology through a wide impact on the mounting of an adequate host immune response is only beginning to be revealed. Therefore, this review is an attempt to summarize current knowledge and provide a platform for future research based on the parallel citation of data derived from the study of other pathogens. Role of Hp virulence agents in autophagy Vacuolating cytotoxin A (VacA) (Fig. 1) VacA, one of the major virulence factors released by Hp, is a hetero-dimeric protein of about 88 kDa, comprising the subunits p58 and p34 associated by non-covalent interactions.14 Initially identified as an inducer of cytosolic, epinuclear-located vacuoles of both late endosomal and early lysosomal origin,15 Hp VacA also promotes a plethora of cellular activities in the gastric mucosa to which, engagement of the host’s apoptotic machinery serves as a common denominator that favors Hp colonization and persistence; VacA plays a role in the pathogenesis of both peptic ulcer and gastric cancer. Subsequent to binding to receptor protein tyrosine phosphatase (RPTP) α and β, epidermal growth factor (EGF) receptor (EGFR) and a number of other lipid raft associated receptors such as sphingomyelin,16,17 VacA is endocytosed and selectively targeted to the mitochondria where it induces activation of dynamin-related protein I (DrpI) – a critical regulator of the mitochondrial fission within cells18 - several proapoptotic effectors such as Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak) proteins, and finally downstream executioner caspases 8 and 919 resulting in mitochondrial outer membrane permeabilization, formation of invasive chloride channels20 and ultimately apoptosis. Central to the toxin’s pleiotropic role lies the formation of membrane anion-selective channels that facilitate the transport of chloride ions, resulting in an increase in intra-luminal chloride concentrations19 with unknown as yet consequences.21 Vac A-mediated mitochondrial fission requires the activation of Drp1, a GTPase, transcriptionally regulated by p53 that causes scission of the mitochondrial outer membrane, resulting in the initiation of apoptosis (Fig. 1). In particular, p34 subunit carries www.stmconnect.com/ig

a unique targeting signal sequence different from all targeting signatures previously identified in endogenous mitochondrial proteins or bacterial effector proteins, acting essentially as a small pore-forming toxin targeting the mitochondrial inner membrane.21 Pore-forming agents from different bacterial species such as Streptococcus pyogenes, Listeria monocytogenes, Shigella flexneri, Salmonella enterica and Staphylococcus aureus, membrane damage appear to disrupt protein translation of cytokines and antimicrobial peptides and modulate autophagy.22 Those bacterial effectors elicit a specific immune response, of particular relevance to epithelial cells, termed as effector-triggered immunity (ETI) that enables host to distinguish pathogens from non-pathogens.23 The outputs of this process, mediated by the p38 MAPK pathway and nucleotide oligomerization domain (NOD), LRR - and pyrin domain containing 3 (NLRP3) inflammasome, are the induction of a stress response, the secretion of the antibacterial interleukin (IL)-1β, IL-18 and the orchestration of membrane repair either through the sterol regulatory element binding – proteins (SREBPs) or the endocytosis/exocytosis of damaged membrane elements and embedded toxins.23 This cascade of events that appears to be intimately associated with autophagy, despite being a mechanism of immune surveillance, has been proposed also to promote bacterial colonization and dissemination. This latter perspective is of paramount importance in the context of Hp co-evolution with its human host. Programmed VacA-mediated cell necrosis coincides with the release of the proinflammatory high morbidity group box 1 protein (HMGB1),24 a damage-molecular pattern (DAMP) – alarmin that undergoes a stepwise displacement from the nucleus into the cytoplasm and ultimately to the extracellular space either by ‘unconventional secretion’ or, as aforementioned, cell death release. Each stage of translocation results in inducing autophagy; intracytoplasmically through dissociation of the Beclin1/Bcl-2 complexes, akin to the action of tumor necrosis factor receptorassociated factor (TRAF) 6 and the c-Jun N-terminal Kinases (JNK), described later on, or extracellularly by receptors for advanced glycation end-products (RAGE) signaling.25 DAMPs including ATP, IL-1β as well as DNA and RNA in complexes with the antimicrobial peptide LL-37 (cathelicidin), heat shock proteins (Hsps), and calreticulin,26 are known inducers of autophagy in an attempt to contain cell or tissue injury under sterile conditions. Although, this effect appears to be cytoprotective, the progression of tissue injury results in the formation of proinflammatory complexes between HMGB1 and microorganism-associated molecular patterns (MAMPs) e.g., lipopolysaccharide (LPS) and IL-1β.27 Recent data also indicates that autophagosomes also package DAMP molecules that activate innate immune response and enhance dendritic cell (DC) or T-cell function. Despite profound progress in VacA research, the significance of vacuolation in the pathophysiology of Hp infection remains elusive although it is plausible that this may disrupt protein trafficking pathways to and from the plasma membrane.28 VacA also exerts local immunosuppressive activities by inhibiting antigenic peptide processing and presentation to CD4 T cells.29 Furthermore, VacA inhibits antigen-mediated clonal expansion of CD4+ and CD8+T-cells, by interfering with IL-2mediated signaling and proliferation of B-lymphocytes thereby


133

Research paper

Figure 1. Main host cell remodeling actions of vacuolating cytotoxin A (VacA) in relation to autophagy. Autophagy and vacuole formation. VacA enters the epithelial cell by pinocytosis and initially resides into Glycosylphosphatidylinositol (GPI)-anchored protein-enriched early endosomal (GEECs) from which it is later transported by an F-actin-dependent mechanism into early and late endosomal compartments (EE and LE). LE fusion with cathepsin D-containing lysosomes (L) induces the characteristic vacuole biogenesis.VacA promotes autophagic signaling within intoxicated epithelial cells resulting in the formation of autophagosomes (AP), and autophagolysosomes (AL), that mediate intracellular persistence. Mitochondrial dysfunction and apoptosis.Vac A-mediated mitochondrial fission requires the activation of dynamin-related protein (Drp)1, a GTPase, transcriptionally regulated by p53 that causes scission of the mitochondrial outer membrane, resulting in the initiation of apoptosis. p53 is found exclusively in mitochondria and can bind to Bcl-2/Bcl-xl complexes, thereby inducing cytochrome c release and the consequent activation of caspases 9 and 3. Interestingly, miRNA MIR30B, recently reported to suppress autophagy, disrupts mitochondrial fission by targeting p53. High-mobility group protein (HMGB)1 is a pro-autophagic protein that upon translocation into the cytoplasm leads to the dissociation of the Bcl-2/Bcl-xl complexes, thus limiting programmed cell death and enhancing cell survival. Hyperproliferation. VacA, possibly in synergy with CagA, activates the PI3K cascade, leading to the phosphorylation (P) and inactivation of GSK3β. As a result, β-catenin is being translocated to the nucleus where it upregulates several genes leading to increased proliferation and aberrant differentiation. p62/SQSTM1 binding to the DSH protein of the Wnt pathway and its subsequent degradation by the autophagic machinery, limits the excessive activation of β-catenin.

subverting adaptive immune defenses.30,31 During the course of infection, internalized VacA exhibits a series of effects that alter gastric epithelial architecture by disrupting cell-cell junctional complexes-tight junctions and adherens junctions otherwise maintained via calcium-dependent interactions between E-cadherin and α- and β-catenins.17 Aberrant accumulation of β-catenin by Hp requires the aforementioned inactivation of the kinase activity of GSK3β, as a result of Wnt binding to its receptor, Frizzled and activation of Dishevelled and Wnt co-receptors, Low-density Lipoprotein Receptor-related Protein (LRP)-5 and LRP-6.32 Recent studies33 have now demonstrated that autophagy leads to the down-regulation of the Wnt signaling pathway via autophagic degradation of Dvl2. These findings invoke an elegant crosstalk between chronic, microbial-induced inflammation and virulence factor-mediated protection of host cells. However, prolonged incubation with VacA results in inactivation of Akt and activation of GSK3β, which then down134

regulates β-catenin activity.34 Recent insights into the mechanisms that trigger this selective permeabilization of the paracellular ion barriers, have revealed that VacA induces aberrant β-catenin nucleus translocation, a downstream component of the Wnt pathway in a PI3K-dependent manner, thus promoting transcription of target genes including the proto-oncogenes c-myc and cyclin D1 as well as caudal type homeobox I (CDX1), that mediate intestinal metaplasia.34,35 Research for specific cell surface receptors that mediate VacA-induced regulation of autophagy, recently identified that LRP-1 binding of VacA is an absolute prerequisite for autophagosome biogenesis and apoptosis.36 Moreover, soluble VacA present in culture supernatants can disrupt the degradative potential of the endocytic pathway providing sufficient stress signals to initiate the formation of autophagosomes in a process that requires the presence of an intact anion channel-forming capacity.37 VacA-LC3 co-localisation studies, revealed the presence


Volume 2 Issue 3

Research paper

of the VacA toxin within autophagosomes. Autophagosomal compartments despite being - to a large extent - dependent on VacA delivery and sharing a common origin from late endosomal - lysosomal compartments fusion, appear to be different in size, morphology and occurrence, as assessed by EM and siRNA data, from the large vesicles generated as a direct result of VacA intoxication.37 However, short-term (6 h) VacA exposure was shown to modulate intracellular toxin stability, which in turn decreases the extent of large vacuole formation indicating that VacA-induced autophagy might represent a host mechanism to limit toxin-induced cellular damage.37 Prolonged, mimicking chronic infection, VacA-mediated induction of autophagy appears to facilitate the host cell’s cytosol remodeling into a suitable replicative niche for intracellular Hp. However, as experiments in which wildtype and autophagy deficient Atg5 -/- mouse embryonic fibroblasts (MEFs) were employed showed that, similar to Shigella mutants - whose growth is restricted by autophagy,38 survival of VacA+ Hp was increased in autophagy disrupted Atg5 -/- MEFs in comparison to wildtype MEFs. Whether this observation should be considered as a doseand invasion time- dependent epiphenomenon, remains elusive. Subversion of autophagy by MIR30B through down-regulation of Becn1 and Atg12 expression allows intracellular Hp to evade autophagic clearance, thereby contributing to the persistence of Hp infection.39 Noteworthy, the latter event appears to be unique to Hp infection since other bacteria such as Escherichia coli (E. coli) or autophagy modulators such as rapamycin fail to induce a similar effect.35 Furthermore, overexpression of VacA treatment of AGS cells, independent of bacteria, induces autophagosome-lysosomal fusion and acquisition of an acidic pH important in maturation of pro-cathepsin D; however, cathepsin D levels remain insignificant disrupting lysosomal degradation and promoting intracellular persistence.40 Interestingly, similar autophagosome-like compartments harbouring early endosomal and ER markers but lacking the presence of lysosomal protease cathepsin D have been observed in Porphyromonas gingivalis and Brucella abortus experimental infections.41 Maintenance of those intracellular vacuoles in Brucella abortus, has been shown to be dependent on the virB operon encoding genes homologous to those linked to T4SS effectors in other species.42 Since virulent cytotoxin-cagPAI (+) Hp strains possess a highly functional T4SS it would be interesting to investigate whether other than CagA bacterial effector components of the cagPAI or a novel undefined, as recent genomic data suggests, T4SS may contribute to lysosomal evasion. Despite its rare occurrence, Hp cagPAI (-) driven inflammation can lead to gastritis and severe Hp-related disease outcomes.43 In those cases, delivery of bacterial effectors occurs via the release of outer membrane vesicles (OMVs), shown to contain cell wall components and biologically active VacA that enter epithelial cells via membrane lipid rafts ultimately inducing proinflammatory cytokine responses (IL-8) and apoptosis.44 Subversion of the autophagic pathway is further demonstrated in vivo by the accumulation of p62/SQSTM1 aggregates and reactive oxygen species (ROS) in AGS cells and human gastric biopsies infected with s1m1 VacA (+) Hp.40 The latter events, support the notion that impaired autophagy promotes gastric inflammation, tissue damage and ultimately carcinogenesis. www.stmconnect.com/ig

As already discussed, assembly of the autophagic machinery, involves a number of proteinaceous components among which autophagy related 16-like 1 (ATG16L1) plays a central role in regulating cellular membrane recycling and homeostasis.45 Previous genome-wide association studies have established a causal link between a non-synonymous SNP (T300A) in the Atg16L1 resulting in the production of an unstable variant of the corresponding protein and Crohn’s disease (CD).46,47 Extensive genotyping of Hp-infected individuals from two ethnically distinct populations,40 identified a similar positive correlation between the same hypomorphic Atg16L1 allele and susceptibility to Hp infection. Using previously extracted data from a mouse model of CD, it would be plausible to suggest that a defective ATG16L1 variant would account for both autophagosome dysfunction and impairment in antimicrobial peptide release from the gastric epithelium.45 Moreover, peripheral blood monocytes from individuals with the Atg16L1 risk variant show impaired autophagic responses to VacA exposure. Recently, a group of researchers reported a possible association between a certain polymorphism in the autophagy Irgm gene – implicated in CD and mycobacterial infections - and susceptibility to gastric cancer, though such data remains to be further ascertained.48 Cytotoxin-associated gene A (CagA) CagA is a major virulence constituent of Hp that translocates into the host cell by the cagPAI-encoded Type IV secretion system (T4SS) through integrin α5β1 receptor binding.49 At the site of physical attachment, CagA directly interacts with cellular phospholipids causing a rapid and transient externalisation of phosphatidylserine (PS) that, presumably, creates a membrane pore or channel hence allowing CagA translocation across the membrane through cell surface remodeling.50 After delivery into the host cells, CagA localization is strongly dependent on the status of epithelial polarity that ultimately dictates the degree of CagA/PS interaction.50 Upon entry into non-polarized cells, CagA undergoes targeted tyrosine phosphorylation by host Src and Abl kinases at multiple amino acid residues – termed EPIYA motifs – encoded within the 3’ terminus of CagA.51,52 CagA phosphorylation facilitates activation of the cellular Src homology 2 (SH2)-containing tyrosine phosphatase-2 (SHP2) leading to cytoskeletal rearrangements and cellular elongation, termed the ‘’hummingbird phenotype’’, by stimulating the RAP1A-BRAF-ERK signaling pathway.53 CagA phosphorylation-independent events occurring in polarized epithelial cells, include the interaction of translocated but unphosphorylated CagA with a number of cytoplasmic proteins, mainly phosphatidylserine, which leads to the disruption of apical junction complexes, loss of cellular polarity, IL-8 mediated, pro-inflammatory and mitogenic responses.54 These findings, supported by epidemiological data, confirmed that CagA functions as an oncoprotein; the phosphorylated form of CagA has been shown to induce gastric epithelial cell proliferation and carcinoma and is therefore considered a potential oncoprotein.53 An investigated feature of CagA-induced carcinogenesis is the down-regulation of the tumor suppressor activities of the apoptosis stimulating protein of p53 (ASPP2), controlling host responses on DNA damage and oncogenic stimuli.55 The transcriptional


135

Research paper

activator p53 has been shown to be inactivated by mutations in approximately 40% of gastric tumors from individuals harbouring cag (+) Hp strains.56 Non-mutational inhibition mechanisms of p53 are also evident, in a manner similar to that used by oncogenic DNA viruses.57 More concrete, CagA was shown to associate with ASPP2, an event leading to the recruitment of cytosolic p53 that is subsequently degraded by the proteasome.58 Following binding to CagA, ASPP2 is being redistributed in proximity to the plasma membrane thereby altering its function.29 Indeed, cytoplasmic localisation is linked to pro-oncogenic function, whereas nuclearly located ASPP2/p53 complex acts as a tumor suppressor.59 Cellular localisation of p53 is decisive, as it does not solely dictate ASPP2 function, but also modulates autophagy. Nuclear p53 transactivates a number of autophagy inducers such as damage-regulated autophagy modulator 1 (DRAM1) and sestrin2 while cytoplasmic p53 inhibits autophagy by a yet, unknown mechanism.60 However emerging data regarding p53 expression suggests, that it may act in an isoform-dependent manner. More specifically, the human ∆133p53 isoform, which is produced from an alternative intragenic promoter, has been suggested to promote tumorigenesis.61 Similarly, cagPAI-mediated interaction of Hp with gastric epithelial cells, induces ∆133p53 and ∆160p53 isoforms that hamper p53-dependent transcription and promote epithelial cell survival.62 Further to that, increased expression of ∆133p53 was found to increase nuclear factor (NF)-κB transcriptional activity of NF-κB target genes, such as pro-inflammatory cytokines and anti-apoptotic Bcl-2 family proteins, thereby promoting prosurvival signals that may further inhibit apoptosis. In contrast, blocking NF-κB translocation using the nuclear import inhibitor SN50, promotes p53-related autophagy activation and tumor cell apoptosis.63 Taken together, these findings establish a role for CagA modulation of autophagy as an additional parameter in gastric oncogenesis. Beginning with the observation that intracellular CagA levels decrease over time, Tsugawa et al.64 reported a novel, VacA dependent mechanism by which, CagA degradation is mediated by the autophagic pathway. More specifically, m1VacA binding to LRP1 depletes intracellular glutathione (GSH), causing ROS accumulation and enhanced Akt phosphorylation. Activation of Akt induces murine double minute 2 (MDM2)-mediated p53 degradation that ultimately activates autophagy. These findings are suggestive of a protective role of ROS to epithelial cells, forcing also researchers to re-examine the biological consequences of CagA translocation.65 Interestingly, CagA specifically accumulates in gastric cells expressing CD44 variant 9 (CD44v9), a cell-surface marker that exhibits enhanced expression in chronic inflammation, associated with cancer stem cells (CSCs) as well as hematopoietic stem cells. The autophagic pathway in CD44positive gastric CSCs is repressed because of their relevant resistance to ROS, which is supported by augmented intracellular GSH levels. These findings also provide a novel molecular link between Hp and gastric oncogenesis via the specific accumulation of CagA in gastric progenitor-like cells. Accumulating evidence supports the presence of a functional antagonism at multiple levels, between CagA and VacA. This notion first became evident by Yokoyama et al.66 who demonstrated 136

that VacA counteracted the stimulatory effect of CagA on the transcriptional activity of host nuclear transcription factor of T-activated cells (NFAT). Later on, Argent et al.67 found that VacAmediated vacuolation attenuated CagA-induced cell elongation and vice versa by a mechanism engaging EGFR- and Erk1/2 signaling pathways.68 Furthermore, VacA also inhibits CagAinduced epithelial scattering via inhibition of CagA-dependent mitogen activated protein kinase (MAPK) activation. In further support of this delicate interplay, recent work showed that CagA counteracts, by two distinct non-overlapping mechanisms, the apoptotic effect of VacA on gastric epithelial cells.69 Acting in a stepwise manner, phosphorylated CagA blocks trafficking, by dynamic actin structures, of pinocytosed VacA towards the late endosomal, LAMP1 (+), compartments and mitochondria, while preventing vacuolation. Via a complementary mechanism, the unphosphorylated form is preventing the activation of the intrinsic apoptotic pathway by activating the NF-κB pathway - IκBα degradation resulting in NF-κB release and nuclear translocation - allowing the up-regulation of anti-apoptotic factors and IL-8 production at 24 h post infection. Those events demonstrate that Hp virulence factors counteract one another to minimise host damage once infection is established and finetune their virulence while promoting their propagation. Bone marrow-derived cells in Hp infection Professional phagocytes such as monocytes, macrophages and bone marrow-derived dendritic cells were among the first cell types in which Hp was shown to replicate inside autophagosomes.70 Infection of wild-type, but not TLR2 and TLR4 deficient, bone marrow-derived dendritic cells, which appears to be dependent on both VacA and CagA, disrupt the transport of MHC II molecules from the cytoplasm to the cell membrane and the proliferation of antigen-presenting cells. Bacteria are eventually being eliminated at 24h post infection.69 These events further support current experimental data indicating that Hp infection leads to the development of chronic inflammation, hyperplasia, metaplasia, dysplasia and recruitment and accumulation of bone marrow-derived stem cells (BMDSCs) which may contribute to tumor formation in animal models with Hp-induced chronic gastric inflammatory process.9,71 Because Hp similarly induces inflammatory changes in colonic mucosa,72 it would be reasonable to further speculate that chronic Hp infection in humans also induces repopulation of the colon with BMDSCs that might facilitate colon adenoma and cancer development and progression. In this respect, our own preliminary studies indicated increased expression of the mentioned CD44 (a marker of CSCs and human hematopoietic stem and progenitor cells) in malignant colonic tissue in 75.6% patients with colorectal cancer.73 Extending these preliminary data, increased expression of CD44 was observed in 78% and 16% of patients with cancers and polyps, respectively.74 We also obtained comparable data with gastric cancer.9,71 Therefore, these findings suggest the possible involvement of BMDSCs and/or CSCs in Hp-associated gastric cancer development and colon adenoma and cancer growth and/ or progression75 possibly associated with suppressed autophagic process. However, future large-scale relative studies are warranted


Volume 2 Issue 3

Research paper

to elucidate in depth these findings. Ubiquitin regulation of autophagic host cell response to Hp infection Ubiquitylation is a core, eukaryotic cell quality control process that regulates protein degradation, translocation and function;76 the ubiquitin/proteasome pathway (UPS), a highly complex, mediated by ubiquitin, contributes to intracellular protein degradation in a specific manner thereby involved in the regulation of a series of cellular processes, including cell-cycle division, DNA repair, cell growth and differentiation, quality control, bacterial infections, and apoptosis. Abnormalities in protein degradation systems are involved in the pathogenesis of several serious human diseases.77 In particular, the UPS contributes to precise connectivity throughout development, while later assures functionality by regulating a broad spectrum of neuron-specific cellular processes. Aberrations in this system have been involved in the pathogenesis of neurodevelopmental and neurodegenerative disorders;78 evidence linking ubiquitylation and autophagy first emerged in neurodegenerative disease studies that demonstrated the intracellular accumulation of ubiquitin-tagged proteins in Atg5 and Atg7 deficient mice.79 Both ubiquitylation and autophagy also constitute key signaling pathways in the host response to infection80 as a growing body of evidence suggests that those pathways are intertwined in controlling intracellular trafficking of pathogens. During infection, a diverse array of bacterial effectors delivered to the cytosol, are subjected to ubiquitin conjugation. Those substrates trigger the formation of protein cargoes, aggresome-like induced structures (ALIS) similar to those observed in Legionella pneumophila or Salmonella - infected macrophages,81,82 subsequently targeted by ubiquitin receptor proteins such as p62/sequestosome (p62/ SQSTM1) to selective autophagy.83 Invasive bacterial pathogens such as Salmonella typhimurium, Shigella flexneri and Listeria monocytogenes, have been identified as ubiquitinated substrates that recruit the adaptor protein p62 destined for autophagic degradation.84 Mutations in either binding domains – UbA or LiR - of p62 resulted in increased bacterial replication.59 Similarly, increased numbers of p62-associated bacteria were observed in Atg5-/- MEFs.85 Nevertheless, as subsequent studies showed, siRNA treatment of p-62 bound cells did not prevent autophagy pointing thus towards an alternative autophagy-regulating pathway in intracellular S. typhimurium infection, that utilises nuclear dot protein 52 (NDP52), as a novel adaptor protein able to recruit TBK-1 (Tank-binding kinase-1). The latter appears to affect the integrity of Salmonella-containing vacuoles and is also involved in type I IFN production.86 Despite intensive research, the intracellular fate of Hp and its products remains largely unknown. Recently, a breakthrough study provided in vivo evidence of a direct interaction between Hp virulence factors, cytosolic receptors and ubiquitin-proteasome complexes, co-localised inside distinct, cytoplasmic compartments named as particle-rich cytoplasmic structures (PaCS).87 More specifically, immunocytochemical characterisation of PaCS contents revealed the presence of both VacA and CagA, urease and outer membrane vesicles occasionally coupled with Hp, www.stmconnect.com/ig

peptidoglycan-sensing NOD1 receptor, E1 A/B ubiquitinactivating ligases and polyubiquitinated proteins and finally, several proteasome subunits including LMP7, characterising IFN-γ immunoproteasome.87 Indeed, gene expression profiling in Hp-infected antral mucosa demonstrated a marked induction in the production of ribosomal proteins RPS14 and RPS27 and proteasome components – PSMD5 and PSME2 as opposed to the down-regulation of the ubiquitylation modulators ASB15, RNF138 and RWDD4A.88 The former are E3 ubiquitin ligases that display their regulatory roles by suppressing NF-κB activation and Wnt/β-catenin signaling whereas RWDD4SA’s function is yet to be annotated.85 What is more intriguing, selective PaCS reactivity was also evident for certain oncogenic CagA-related mediators, such as SHP2 phosphatase and ERK/MAPK kinases. These findings together with the distribution of PaCS inside dysplastic foci clearly suggest a role in the regulation of epithelial cell proliferation and oncogenic transformation also with respect to the putative role of ubiquitylation in neoplastic development.89 Since NF-κB, ubiquitin and proteasome have been shown be important factors in carcinogenesis, NF-κB positive expression in gastric malignancy, frequently caused by Hp infection, is found to be significant higher than that in adjacent gastric mucosal tissues; the increase of NF-κB activation is accompanied by the increases of ubiqutin, 26S proteasome activation and a degradation of IκBalpha but not the ubiquitin-conjugated IκB-alpha/NFκB complex in gastric carcinoma. Besides, NF-κB expression is significantly increased in patients with lymph node metastasis, indicating that the constitutive activation of NF-κB is likely due to the activation of UPS, and NF-κB can be used as a prognostic marker in gastric carcinoma patients.90 Moreover, the UPS in involved in the pathogenesis of muscle protein hypercatabolism and gastric cancer cachexia.91 On the other hand, inhibition of proteasome function in gastric cancer cells induces apoptosis and proteasomal inhibitors appear to have potential use as novel anticancer agents in gastric malignancy.92 p62/SQSTM1: linking autophagy to oncogenesis Encoded by SQSTM1, the ubiquitin-binding protein p62 or sequestosome 1 is a scaffold, adaptor protein that modulates proteinprotein interactions, and as a key component of multiprotein complexes, it mediates various cell functions, including cell signaling, receptor internalization selective autophagy, and tumorigenesis.93,94 As already stated, defective autophagy leads to marked accumulation of p62/SQSTM1 aggregates inside the cytoplasm of Hp-infected epithelial cells. In this regard, disrupted autophagy leads to accumulation of SQSTM1/p62 and ROS both in vitro and in vivo in biopsy samples from patients with Crohn’s disease infected with VacA(+) but not VacA(-) strains with the potential for subsequent malignant susceptibility.95 The sequestosome 1 represents a multifunctional protein containing multiple domains that confer the ability to interact with components in essential oncogenic signaling pathways such as the Ras/Raf/MAPK and NF-κB pathways. More concrete, UBA and LIR domains bridge, as described above, polyubiquitinated protein complexes with LC3 ridding the cytoplasm of defective


137

Research paper

organelles or bacteria. The N-terminal PB1 (Phox and Bpem1) domain is involved in the binding of p62 to atypical PKC (aPKC) or ERK1. Two neighbouring domains, ZZ and TRAF6, regulate the activation of the NF-κB pathway through binding to RIP and TRAF6, respectively, while between those regions the binding site for the Raptor protein that acts as an mTOR activator is situated.96 Finally, the Keap-interacting region binds Keap1 to induce the nuclear translocation of the NRF2 transcription factor engaged in the antioxidant response. Therefore, p62/SQSTM1 acts intrinsically as a pro-oncogene that mediates inflammationdriven oncogenesis (NF-κB/MAPK) and cell transformation (mTOR). Strikingly, transcriptional regulation of p62 intracellular levels is not only dependent on NRF2 activation by oxidative stimuli. Analysis of the protein’s promoter binding sites led to the conclusion that the constitutive expression of the Ras/MEK/ERK and NF-κB directly regulate p62 mRNA expression triggering thus Ras signaling amplification in a positive feedback loop.97,98 Recent studies have also demonstrated the involvement of alternative signaling events in the regulation of p62 transciption such as the c-Jun (JNK) pathway99,100 and KrasG12D-induced AP-1 activation98 both of which, converge to the NF-κΒ translocation. Importantly, constant p62 expression resulting from autophagy defects is sufficient to alter NF-κB regulation and gene expression and to promote oncogenesis. Accumulation of p62 in response to metabolic stress is a striking phenotype of autophagy-defective cancer cells, suggesting that defective protein quality control may contribute to carcinogenesis and that autophagy is the main mechanism by which cancer cells turnover p62. Moreover, failure of autophagy-defective tumor cells to eliminate p62 is sufficient for tumorigenesis. Therefore, defective autophagy is a mechanism for p62 upregulation frequently observed in human tumors that contributes directly to oncogenesis likely by perturbing the signal transduction adaptor function of p62-controlling pathways critical for oncogenesis.101 However, as already mentioned, p62 can also exert tumor-suppressive activity by promoting the inactivation of the Wnt pathway through autophagy.102 Regulation of bacterial sensing via autophagy: Pathogen-recognition receptors (PRRs) and autophagy in Hp infection Bacterial sensing is accomplished via specific PRRs, which in principle consist of a recognition domain and a protein-proteininteracting domain for downstream signaling. There are three major classes of PRRs: Toll-like receptors (TLRs), retinoic acidinducible gene I (RIG I)-like helicase receptors (RLRs), and (NOD-like receptors (NLRs).103 To initiate immune responses, PRRs recognize MAMPs and induce a number of proinflammatory cytokines as a widely known output. A new addition to the repertoire of PRR stimulation outputs is the recently described induction of autophagy downstream of TLR stimulation.104,105 Specifically, during Hp infection the host develops a robust innate immune response initiated by PRRs that function as receptors of specific ligands MAMPs, such as flagellin, peptidoglycan, lipopolysacchade and formulated peptides.106 PRRs are expressed in non-lymphoid epithelial cells, neutrophils and DCs and include cell-surface and endosomal- TLR protein 138

family as well as the cytosolic NOD proteins 1 and 2. The key PRR signaling cascades converge on the activation of NF-κΒ that results in the production of proiflammatory cytokines and antimicrobial peptides.107,108 Autophagy pathway components show physical, signaling and regulatory interactions with conventional PRRs including the aforementioned TLRs- historically the first class of PRRs to be connected to autophagy - NODs and RLRs.5 TLRs Autophagy is a downstream effector of TLR signaling and TLRs are among the best-characterized PRRs. TLR1, TLR2, TLR4, TLR5, and TLR6 are positioned mostly on the cell surface and primarily recognize bacterial components, and TLR3, TLR7, TLR8, and TLR9 are principally in the endocytic compartments and mainly recognize viral products.8,109 After recognition of pathogen-derived components, individual TLRs trigger distinctive responses by recruiting a different combination of four Toll-IL-1 receptor (TIR) domain-containing adapter molecules: myeloid differentiation primary response protein 88 (MyD88), used by all TLRs except TLR3; TIR domain-containing adapter protein (TIRAP) or MyD88 adapter-like (MAL), used by TLR2 and TLR4 as a bridge to recruit MyD88; TIR domain-containing adapter-inducing IFN-β (TRIF) or TIR domain-containing adapter molecule 1 (TICAM-1), used by TLR3 and TLR4; and TRIF-related adapter molecule (TRAM) or TICAM-2, used only by TLR4 for interactions with TRIF.110 TLR signaling cascades activate the transcription factors NF-κB and activator protein-1 (AP-1), which are common to all TLRs, resulting in the production of inflammatory cytokines and chemokines. TLR3, TLR4, TLR7, TLR8, and TLR9 also activate IFN regulatory factor 3 (IRF3) and/or IRF7, leading to the production of type I IFN (IFN-α and IFN-β).109 Type I IFN can induce antiviral state in most cells. Other cytokines and chemokines initiate and amplify inflammatory responses by recruiting and activating various innate immune cells such as monocytes, neutrophils, and natural killer cells.109 Four TLRs (2,4,5,9) have been detected on the apical and basolateral surface of epithelial cells in the inflamed gastric mucosa and are important for Hp recognition. TLR2 recognises diverse MAMPs including lipoproteins, lipoteichoic acid and peptidoglycan.111 An essential role for TLR2 signaling in recognition of live Hp has been supported by several authors, possibly mediated by Hp-neutrophil-activating protein (NAP) and/or LPS and Hsp-60112,113,114 while others concluded that TLR2 and to as lesser extent TLR4, may be the predominant surface receptors in DCs recruited in response to NF-κΒ signaling at the site of infection.115 Further relative data indicate that functional urease expressed on the surface of Hp directly stimulates autoantibody-inducing B cells through innate TLR2 to produce diverse autoantibodies and might induce autoimmune disorders.116 Despite conflicting data regarding the role of TLR4 in epithelial and macrophage responses to Hp stimulation, it has been demonstrated that TLR4 is involved in the generation of pro-inflammatory responses by Hp LPS in animal models and


Volume 2 Issue 3

Research paper

transfection experiments.111,112 Notably, exclusive TLR4 expression may enable extranodal marginal zone B-cell lymphomas of MALT type to interact with Hp and autoantigens.117 TLR5 had been identified as a bacterial flagellin receptor yet - it is now established that Hp flagellin molecules lack the conserved sequences required for full TLR5 agonist activity and therefore escape recognition.118 Recent evidence indicates that Hp induces expression of TLR5 and can qualitatively shift cagPAI-dependent to cagPAI-independent proinflammatory signaling pathways with potential impact on the outcome of Hp-associated diseases.119 Scarce data exists in relation to TLR9-dependent responses in Hp stimulation. TLR9, located at the endoplasmic reticulum, recognizes bacterial unmethylated CpG DNA sites and expression of TLR9 mRNA is induced in macrophages, DCs, and CD3+ cells in Hp-infected gastric mucosa. Signal transduction of TLR9 suppresses Hp-induced gastritis in the early stages through downregulation of T helper (Th) 1-type cytokines modulated by IFN-α/β.120 Furthermore, cyclooxygenase (COX)-2 expression and the subsequent angiogenic responses by gastric epithelial cells induced by Hp occur via TLR2/TLR9 dependent pathways that required NF-κΒ activation.121 In addition to in vivo studies, genetic analyses have provided evidence that certain sequence polymorphisms in TLR2/4 and 9 influence the response phenotype during Hp infection associated with higher rates of gastric atrophy and cancer.122,123 Several studies in different populations, however, failed to identify uniform associations possibly due to non-informative haplotype distributions.29 Consequently, interpretation of TLR mediated responses to Hp remains largely inconclusive, as they appear to be host – and/ or strain-specific. Recent evidence examining PRRs as environmental sensors of autophagy, may provide a more comprehensive context for the understanding of host response to Hp infection. TLR signaling intersects with autophagy in two ways: a. as an effector mechanism of microbial elimination downstream of TLR activation; and b. as a ‘topological inverter device’ that assists TLRs in meeting their cognate ligands.3 Positive regulation of autophagy by TLR4 occurs via the E3 ligase TRAF6 which ubiquitinates Beclin 1 leading, through the TLR adaptors MyD88 and TRIF, to its dissociation from inhibitory complexes with Bcl-2 that suppress autophagy.124 As mentioned earlier, Hp co-localises with several host cell effectors inside large vesicular compartments – PaCS, similar to the Salmonellacontaining vacuoles (SCVs) that are targeted for xenophagic sequestration. However, a number of bacteria escape into the cytosol where they are ubiquitinated leading to the recruitment of the autophagic receptors NDP52, p62/SQSTM1, and the NF-κBactivating kinase, TANK-binding kinase 1 (TBK1).86 In response to LPS, TLR4 activates TBK1 leading to the phosphorylation of the LC3-binding protein optineurin (OPTN), thereby guiding ubiquitin-coated bacteria to nascent autophagosomes.125 MHC class II-restricted endogenous antigen presentation is accomplished by the fusion of MHC II loading compartments and autophagosomes where cytosolic proteins are sequestered. Therefore, autophagy acts as a topological inversion device that www.stmconnect.com/ig

facilitates delivery to the endomembranous compartments facing the lumen.8 Acting in a similar manner, autophagy assists cytosolic MAMPs fusion with endosomally-located PRRs such as TLR7.126 Finally, recent data indicate that gastric oncogenesis is associated with decreasing levels of TLRs inhibitors and elevated TLRs levels throughout all spectrum of Hp-related lesions. Therefore future relative studies are required to investigate whether modulation of those receptors activity might influence gastric oncogenesis and cancer progression.127 NODs NOD-NLR proteins are a family of receptors that play a crucial role in microbial sensing, leading to the initiation of antimicrobial immune responses; with over 20 members identified in humans, NLRs represent important components of the mammalian innate immune system, serving as intracellular receptors for pathogens and for endogenous molecules developed by tissue injury. NOD1 and NOD2 are characterized by a central NOD, an N-terminal effector-binding domain (CARD) and a C-terminal ligand recognition domain that is comprised of leucine-rich repeats (LRR).128 NOD1 senses a muropeptide found mostly in Gram-negative bacterial peptidoglycans (PGs), whereas NOD2 senses bacterial molecules produced during PG synthesis or degradation.108 NOD1 and NOD2 proteins operate as microbial sensors through the recognition of specific PG constituents of bacteria. Upon activation, these NLR family members initiate signal transduction mechanisms that include stimulation of NF-κB, stress kinases, IRFs (interferon regulatory factors) and autophagy. Dysregulation of the function of multiple NLR family members has been associated, both in mice and humans, with a predisposition for infection and inflammatory disease; hereditary polymorphisms in the genes encoding NOD1 and NOD2 have been linked to an increasing number of chronic inflammatory disorders.129 Despite our increased understanding of NLR function and interactions, however, several aspects related to mechanisms of sensing, downstream signaling, and in vivo functions remain elusive.130 Both proteins are mainly expressed by two, regularly exposed to PG products, cell types antigen-presenting cells (APCs) and epithelial cells while their expression is governed by proinflammatory cytokines albeit in distinct ways. Constitutive expression of NOD1 in epithelial cell lines is upregulated by IFN-γ acting through the transcription factor IFN-regulatory factor 1 (IRF1) at the CARD4 promoter, whereas NF-κB activation by TNF does not alter NOD1 basal expression.131,132 On the contrary, NOD2 is upregulated by TNF whereas this effect is augmented by IFN-γ.108 NOD1 and NOD2 are becoming known as key regulators of the mentioned chronic inﬂammatory conditions and several reports demonstrated that the polymorphisms of the NOD1 and NOD2 genes in different populations are related to variant clinical outcomes of Hp infection.133 Activation of NOD1 by Hp is achieved either by cellular invasion, cag-PAI dependent delivery of PG into the cytoplasm107 or as recently proposed a novel mechanism of PG delivery inside the cytosol that utilizes OMVs entering epithelial cells via membrane-bound lipid rafts.44


139

Research paper

NOD1 sensing initiates a signaling cascade that culminates in the induction of NF-κΒ activation and pro-inflammatory cytokine, including CXCL8, responses in epithelial cells.107 NOD2 was also reported to mediate responses to cag-PAI positive Hp in NOD2-trasfected epithelial cells134,135 but data is currently limited. Both NOD1 and NOD2 activation is accompanied by the recruitment, through homotypic CARD-CARD interactions of the serine threonine kinase RICK (also known as RIP-2) that undergoes K63-linked ubiquitylation leading to the creation of a scaffold that ultimately results in the activation of the NF-κΒ and p38MAPK signaling pathways.135 As already stated, stimulation of epithelial cells with NOD1 ligands leads to a robust cytokine response comprised of Th1 chemokines notably IFN-γ-induced protein of 10kDa, IP-10.136 This induction was not accompanied by NF-κΒ activation but rather the interaction of RICK and TRAF3 that results in the production of IFN-β ultimately IP10 pointing, thus towards a unique NOD1 signaling pathway more commonly identified in viruses.132 Although the advantage of using this mechanism of host response instead of relying totally on the NF-κB pathway is not yet fully understood, it has been speculated that since type I IFN has both pro- and antiinflammatory properties it may be useful in limiting the infection and control inflammation. In addition, NOD1 was shown to upregulate the expression of DEFB4 encoding for the antimicrobial peptide β-defensin-2 (hBD2), in response to cagPAI(+) Hp in AGS cells.137 In contrast, DEFB103 – encoding for hbD3 – promoter activation was mediated by a NOD1/cagPAI independent but ERK-dependent mechanism.134,138 Works by Travassos et al.139 have concentrated on the role of NOD signaling in autophagy manifesting that both NOD proteins can initiate autophagy in response to bacterial PG sensing.140 As shown, in NOD-mediated autophagy both RIP-2 recruitment and NF-κΒ activation were dispensable as it rather involved a direct interaction between NODs and the autophagic protein ATG16L1 at the site of bacterial entry, as in Shigella flexneri infection.141 Of significance, they further demonstrated that while the most prevalent Nod2 (R702W) mutation implicated in patients with CD failed to localise to the membrane and retained ATG16L to the cytosol resulting in impaired autophagosome maturation, the corresponding CD risk-associated ATG16L1 variant co-localised normally with NODs but did not result in intense autophagosome formation.140 This important finding provided further support towards the existence of a crucial NOD/ATG16L1 axis involved in bacterial handling and presentation. Despite that during Hp invasion of host cells, the NOD2 R702W variant fails to mediate a similar response, it has been associated strongly with gastric MALT lymphoma.142 In light of the sparse and often conflicting data regarding the role of Nod1 and Nod2 polymorphisms as host predisposing factors in Hp disease and gastric cancer,133,143 it would be interesting to investigate the existence and consequences of similar NOD2 interactions with ATG16L1 in Hp infection. Inflammasomes Inflammasomes, assemble upon recognition of a variety of MAMPs or DAMPs by members of the NLR family, regulate the activation 140

of inflammatory caspases, mainly caspase-1, which cleave IL1β and IL-18 and stimulate their release.130 Consequently, they provoke inflammatory responses and eventually apoptotic events. NLR proteins that participate in inflammasome structure include NLRP1, NLRP3, NLRC4, and AIM2 that associate via homophilic CARD-CARD and PYD-PYD interactions with each other and with the apoptosis-associated speck-like protein (ASC).140 Despite limited evidence regarding the role of inflammasome activation in Hp-mediated innate immune responses, limited evidence suggests that asc-deficient mice challenged with Hp, exhibited higher bacterial loads though lower levels of gastritis, partially attributed to diminished concentrations of IL-1β, IL18 and IFN-γ.144 In this respect, recent studies show that the processing and release of a regulatory caspase-1 substrate, IL-18, counteracts the proinflammatory activities of the another caspase-1 substrate, IL-1β, thereby balancing the control of Hp infection with the prevention of excessive gastric immunopathology.145 NLRC4 is activated by Gram-negative bacteria possessing type III (T3SS) or type IV (T4SS) secretion systems that enable delivery of flagellin to the cytosol despite the presence of other candidate proteins containing similar motifs proposed.130,146 However, its role in Hp infection remains elusive. Functional interrelations between inflammasome activation and autophagy appear to be crucial for host cell and tissue homeostasis. Housekeeping levels of autophagy prevents spurious activation of inflammasome by endogenous agonists such as ROS or mitochondrial DNA released by obsolete mitochondria.147 There is emerging evidence of significant crosstalk between apoptosis and autophagy, which can be linked to inflammasome activation.148 Inflammasomes are cytosolic multi-protein platforms that sense both microbial and damage-associated molecular patterns and introduce a potent innate immune anti-microbial response; they play a role in the orchestration and regulation of the intestinal immune response, regarding the maintenance of intestinal homeostasis, enteric infection, auto-inflammation, and tumorigenesis.149 In case of autophagy impairment, endogenous sources may lead to aberrant IL-1β release, resulting in sterile inflammation. This is of particular relevance to the role of autophagy in CD, as loss of Atg16L in a murine model of the disease resulted in elevated IL-1β levels.126 Recent data related to the negative regulation of inflammasome activation by autophagy, suggests that AIM2 and NLRP3 stimulatory signals also induce autophagosome formation either by ubiquitin-tagging of ASC component or RALB activation resulting in autophagosomal engulfment of the inflammasome, thereby limiting production of mature IL-1β.150 In the quest for additional associations between known diseases and autophagy control of inflammasome output, it has been suggested that excess IL-1β levels observed in Altzheimer’s disease patients combined with the ability of neuronal cells to promote inflammasome activation, may implicate a role for autophagy.5 This hypothesis warranted further investigation in the context of Hp infection, given the existing strong association between Hp inflammatory response and neurodegenerative diseases.151 In contrast to the inhibitory regulation of basal autophagy in unscheduled inflammasome activation, induced autophagy


Volume 2 Issue 3

Research paper

Inflammasome

Virulence factors

· AIM2, NLRP3: ubiquitination of ASC adapter · NLRP4, NLRC4: Beclin 1 binding

· · · ·

VacA VacA: PL3K/Akt, NF-κB CagA: nuclear p53 CagA: cytosolic p53,NF-κB

Danger signalling (DAMPs)

Pattern recognition receptors · TLR4: i. Beclin1/Bcl-2 dissociation ii. TBK1 activation · TLR2,5,9 · NOD1: NOD1/ATG16L1 complex

Autophagy Inducers/ Repressors

· HMGB1: Beclin1/Bcl-2 dissociation · ROS: i. LC1 LC3 ii. ER stress iii. MIR30B down-regulation · INOS: S- nitrosylation of IKK-β (NF-κB independent)

Figure 2. Pathogen and cellular modulators of autophagy during Helicobacter pylori (Hp) infection. Although pathogen-recognition receptors (PRRs) are being considered as potent activators of autophagy, however the exact biochemical mechanisms that underlie this effect are not fully understood. Toll-like receptor (TLR) adaptors, RIF and MyD88, interact with the autophagy protein, Beclin 1, resulting in the disruption of its complex interaction with Bcl-2. Nucleotide -binding and oligomerization domain (NOD)1 activation leads to autophagy related 16-like 1 (ATG16L1) protein recruitment to the sites of bacterial entry. Nevertheless whether such recruitment is sufficient for the initiation of autophagy or other as of yet unidentified signals are required is still unclear. Both VacA and CagA, display a Janus-faced role in autophagy regulation that may be reconcilable in the setting of acute versus chronic infection, a postulation that warrants further investigation. Assembled inflammasomes are potent activators of autophagy as they undergo ubiquitination and recruit the autophagic adaptor p62/SQSTM1, which enables their delivery to autophagosomes to temper inflammation. NLRC4 role in Hp infection still remains elusive. Generally, damage-molecular patterns (DAMPs) are well-known inducers of autophagy; however recent studies indicate that cellular stress can repress autophagy via the reactive nitrogen species by S-nitrosylation of the inhibitor of kappa B kinase (IKKβ) in an nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κΒ) independent pathway.

enhances, in the short-term acute phase, IL1β and IL-18 output by supplementing their release via the ‘unconventional secretion’ pathway.5 Overall, inflammatory modulation by IL-1β, consists of Tand B-cell activation and downstream cytokine production. Of paramount importance in Hp pathogenesis, is IL-1β’s potent induction of hypochlohydria, a significant co-factor in the modulation of the gastric microenvironment that contributes to the development of precancerous lesions and ultimately, gastric adenocarcinoma.152 IL-1β is encoded by the corresponding IL1-β gene in which three polymorphisms have been reported, all representing C-T base transitions at positions -511, -31 and +3954 base pairs from the transcriptional start site.153 Studies in different populations of European and African origin have identified significant correlations between IL-1β polymorphisms and gastric cancer; 143 polymorphisms in IL-1β and its endogenous receptor antagonist are associated with risk of Hp-related gastric cancer.9 In contrast, relevant data extracted from extensive studies in Asia, failed, with the exception of the Chinese, to verify a causal link, thereby indicating a strong epigenetic effect on disease progression (Fig. www.stmconnect.com/ig

2).143 Clinical consequences of autophagy in Hp infection Because, Hp is able to persistently colonize its host despite inducing expression of several antimicrobial peptides, including defensins, and moreover, inherited variation in defensin gene expression may contribute to susceptibility to several diseases, including CD, recent data indicate that small intestinal CD appears to be a complex disease of the Paneth cells (PC) (i.e., Paneth’s disease). These cells produce different broad-spectrum antimicrobial peptides most abundantly the α-defensins HD-5 and -6 (DEFA5 and DEFA6). In small intestinal CD both these PC products are specifically reduced. As a functional consequence, ileal extracts from CD patients are compromised in clearing bacteria and enteroadherent E. coli colonize the mucosa. Mechanisms for defective antimicrobial PC function are complex and include an association with NOD-2 loss of function mutation, a disturbance of the Wnt pathway transcription factor TCF7L2 (also known as TCF4), the autophagy factor ATG16L1, the endosomal stress protein XBP1, the TLR-9, the calcium mediated


141

Research paper

potassium channel KCNN4 as well as mutations or inactivation of HD5.154 In this context, the prevalence of Hp infection in the inflammatory bowel disease patients appears to be common (38.2%-47%) in Europe. Moreover, enterohepatic and gastric Hp species have been documented in fecal specimens from children with CD using PCR, and Hp was recently found in the intestinal mucosa of a patient affected by CD;74 these findings may suggest that Hp infected patients with defective PC-related autophagic machinery might lead to defective microbial killing, increased exposure to commensal and pathogenic intestinal bacteria and T cell activation155 resulting in CD development and/or progression. However, further relative studies are needed to elucidate this speculation. Finally, autophagy may be involved in blood-brain barrier (BBB) disruption;156,157 BBB disruption plays a key role in a number of central nervous system diseases, particularly those associated with neurodegeneration including Hp-related neurodegenerative disorders.151 In this regard, Hp infection, by releasing several inflammatory mediators, could induce BBB breakdown, thereby being involved in the pathogenesis of neuropathies such as Alzheimer’s disease and glaucoma.158 For instance, Hp could indirectly affect the brain through the release TNF-α acting at a distance; TNF-α is involved in BBB disruption through matrix metalloproteinases’ up-regulation. Furthermore, Hp circulating antibodies might also access brain due to BBB disruption possibly contributing to glaucoma pathophysiology; when serum-specific antibodies access the brain, they are capable of killing retinal cells. Likewise, an influx of Hp-infected monocytes, owing to defective autophagy resulting in Hp replication inside autophagic vesicles, through the disrupted BBB, might lead to neuropathies. Hp VacA also exhibits chemotactic activities to bone marrow-derived mast cells inducing the production of proinflammatory cytokines, which disturb the BBB contributing to neurodegeneration.158,159,160 Concluding remarks In recent years data deriving from genome-wide association studies acknowledged autophagy as a significant pathway in immunologically mediated inflammation and oncogenesis. On the other hand, as an intimately intertwined phenomenon with all facets of immunity, it appears acting as a central regulator of host innate immune responses. Hp has been an ancient coloniser of the human gastrointestinal tract161 meaning that, like many human commensal bacteria, has evolved specific strategies to effectively attenuate host immune responses. Furthermore, it has been shown to replicate inside autophagosome structures of dendritic, macrophage and epithelial cells evading lysosomalinduced degradation - a process that is centrally important to the bacteriums’ near perfect ability to persist for life. This latter event relates directly to the high prevalence of antimicrobial resistance among Hp strains and points to alternative therapeutic schemes that may target intracellular Hp populations. Current evidence indicates that, autophagy is being implicated in innate recognition and control of Hp. In the acute stage of Hp infection in naïve hosts, autophagy limits bacterial load and VacA induced cell damage. However, as recent transcriptome and proteomic analysis 142

suggests, persistent challenge with Hp, compromises autophagy either as a VacA-dependent response40 or by MIR30B - mediated targeting of Bcl2 interacting coiled-coil protein (BECN1) and ATG12 that participate in autophagosome formation.39 Remarkably, Hp persistence induces a potent pro-inflammatory cytokine production as well as strong humoral and cell-mediated immune responses, associated with the development of severe gastritis and, subject to several, as yet enigmatic factors, gastric cancer. Mainstream theory of Hp-induced oncogenesis supports the development of adenocarcinoma as the end consequence of a progressive metaplastic process in which atrophy takes central stage.162 In addition to the fact that the exact cascades of events that follow atrophic changes in the gastric mucosa remain largely unidentified, this notion has been further challenged by experiments involving cagA-transgenic mice that develop adenocarcinoma without preceded inflammation.163 Numerous lines of evidence suggest that, similarly to other types of cancer, autophagy may be of particular biological importance in gastric cancer development. Defective autophagy is usually accompanied by extensive accumulation of p62/ SQSTM1-containing aggregates, - an excellently -characterized disease-related autophagy receptor, which enhances its function as a scaffold protein in several signaling cascades such as the Ras/Raf/MAPK - NF-κΒ and PI3K/Akt/mTOR that are often modified during oncogenesis to accelerate growth through nutrient availability as well as proliferation and invasion of malignant cells.96 Persistence of damaged cytosolic organelles (e.g., mitochondria) and expression of mutated, misfolded proteins in autophagy- deficient cells evokes the activation of oxidative stressrelated DNA-damaging response164 that may ultimately enhance genomic instability leading to oncogenesis.165 Wnt-β-catenin pathway provides a strong pro-proliferative signal, essential in progenitor stem cell maintenance, a key effector mechanism that augments carcinogenesis in human Hp-related gastric and colorectal tumors.71, 166,167,168 Studies focusing on the interaction of Hp with cellular subpopulations inside the inflamed mucosa, revealed an intimate relationship between invading bacteria and gastric progenitor stem cells that result in aberrant activation of the oncogenic Wnt pathway169 and defective autophagy which may lead to rapidly evolving precancerous lesions, similar to those observed in Mongolian gerbils, in the absence of preceded atrophy.170 Recent reports suggest that autophagy modification by known antioxidant (catechins) and antiadhesive agents (sialic acid) appears to improve Hp eradication rates.171,172 More specifically, the application of a combination of catechins and sialic acid in AGS cells and BALB/c mice, significantly reduced ROS production and Bax/Bcl2-mediated apoptosis but enhanced Beclin-1 - mediated autophagy in a dose-dependent manner. Pharmacologic modulation of autophagy to tackle certain infectious diseases has been strongly proposed in recent years,173 supported by evidence indicating that antimycobacterial drugs - isoniazid and pyrazinamide, promote the activation of the autophagic pathway to achieve a successful bactericidal effect and reduce inflammation.174 1,25 dihydroxyvitamin D was shown also to prevent human immunodeficiency virus (HIV)


Volume 2 Issue 3

Research paper

type I replication by triggering autophagy.175 Notably, vitamin D appears to play an important role in controlling Hp-related inflammation and inducing the epithelial expression of defensins and cathelicidin176,177 by yet unexplored mechanisms that may involve the induction of autophagy. Furthermore, loss of vitamin D3 upregulated protein 1 (VDUP1) stimulates expression of NFκB signaling pathway thus promoting cell proliferation, which in turn accelerates the development of gastric cancer.178 Therefore, it is becoming evident that autophagy-related events are critical in the evolution of Hp infection. Despite that several strides have been made, a deeper understanding of the precise mechanisms through which autophagy is implicated in the pathophysiology of Hp disease is an absolute necessity to achieve substantial progress in prevention and patient care. The latter is gaining increasing attention especially in view of new anticancer therapies that stimulate autophagy to maximise their efficacy.164 Conflicts of interest The authors declared no conflicts of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16.

17. 18.

19. 20. 21.

22. 23.

Kim SH, Kwon C, Lee JH, et al. Genes for plant Autophagy: Functions and interactions. Mol Cells 2012; 34(5):413-23. Martinet W, De Meyer I, Verheye S, et al. Drug-induced macrophage autophagy in atherosclerosis: for better or worse? Basic Res Cardiol 2013; 108(1):321. Deretic V. Autophagy in immunity and cell-autonomous defense against intracellular microbes. Immunol Rev 2011; 240(1):92-104. Valdor R, Macian F. Autophagy and the regulation of the immune response. Pharmacol Res 2012; 66(6):475-83. Deretic V. Autophagy as an innate immunity paradigm: expanding the scope and repertoire of pattern recognition receptors. Curr Opin Immunol 2012; 24(1):21-31. Morita E, Yoshimori T. Membrane recruitment of autophagy proteins in selective autophagy. Hepatol Res 2012; 42(5):435-41. Romao S, Munz C. Autophagy of pathogens alarms the immune system and participates in its effector functions. Swiss Med Wkly 2011; 141:w13198. Delgado M, Singh S, De Haro S, et al. Autophagy and pattern recognition receptors in innate immunity. Immunol Rev 2009; 227(1):189-202. Kountouras J, Zavos C, Chatzopoulos D, et al. New aspects of Helicobacter pylori infection involvement in gastric oncogenesis. J Surg Res 2008; 146(1):149-58. Tan VP, Wong BC. Helicobacter pylori and gastritis: Untangling a complex relationship 27 years on. J Gastroenterol Hepatol 2011; 26 Suppl 1:42-5. Wotherspoon AC, Ortiz-Hidalgo C, Falzon MR, et al. Helicobacter pylori-associated gastritis and primary B-cell gastric lymphoma. Lancet 1991; 338(8776):1175-6. Dubois A, Boren T. Helicobacter pylori is invasive and it may be a facultative intracellular organism. Cell Microbiol 2007; 9(5):1108-16. Liu H, Semino-Mora C, Dubois A. Mechanism of H. pylori intracellular entry: an in vitro study. Front Cell Infect Microbiol 2012; 2:13. Lupetti P, Heuser JE, Manetti R, et al. Oligomeric and subunit structure of the Helicobacter pylori vacuolating cytotoxin. J Cell Biol 1996; 133(4):801-7. Papini E, de Bernard M, Milia E, et al. Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments. Proc Natl Acad Sci U S A 1994; 91(21):9720-4. Allison CC, Ferrero RL. Role of virulence factors and host cell signaling in the recognition of Helicobacter pylori and the generation of immune responses. Future Microbiol 2010; 5(8):1233-55. Wessler S, Backert S. Molecular mechanisms of epithelial-barrier disruption by Helicobacter pylori. Trends Microbiol 2008; 16(8):397-405. Jain P, Luo ZQ, Blanke SR. Helicobacter pylori vacuolating cytotoxin A (VacA) engages the mitochondrial fission machinery to induce host cell death. Proc Natl Acad Sci U S A 2011; 108(38):16032-7. Cover TL, Blanke SR. Helicobacter pylori VacA, a paradigm for toxin multifunctionality. Nat Rev Microbiol 2005; 3(4):320-32. Rassow J, Meinecke M. Helicobacter pylori VacA: a new perspective on an invasive chloride channel. Microbes Infect 2012; 14(12):1026-33. Domanska G, Motz C, Meinecke M, et al. Helicobacter pylori VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane. PLoS Pathog 2010; 6(4):e1000878. von Hoven G, Kloft N, Neukirch C, et al. Modulation of translation and induction of autophagy by bacterial exoproducts. Med Microbiol Immunol 2012; 201(4):409-18. Stuart LM, Paquette N, Boyer L. Effector-triggered versus pattern-triggered immunity:

www.stmconnect.com/ig

how animals sense pathogens. Nat Rev Immunol 2013; 13(3):199-206. 24. Radin JN, Gonzalez-Rivera C, Ivie SE, et al. Helicobacter pylori VacA induces programmed necrosis in gastric epithelial cells. Infect Immun 2011; 79(7):2535-43. 25. Shi CS, Kehrl JH. TRAF6 and A20 regulate lysine 63-linked ubiquitination of Beclin-1 to control TLR4-induced autophagy. Sci Signal 2010; 3(123):ra42. 26. Yi Y, Zhou Z, Shu S, et al. Autophagy-assisted antigen cross-presentation: Autophagosome as the argo of shared tumor-specific antigens and DAMPs. Oncoimmunology 2012; 1(6):976-8. 27. Bianchi ME. HMGB1 loves company. J Leukoc Biol 2009; 86(3):573-6. 28. Palframan SL, Kwok T, Gabriel K. Vacuolating cytotoxin A (VacA), a key toxin for Helicobacter pylori pathogenesis. Front Cell Infect Microbiol 2012; 2:92. 29. Peek RM Jr, Fiske C, Wilson KT. Role of innate immunity in Helicobacter pylori-induced gastric malignancy. Physiol Rev 2010; 90(3):831-58. 30. Boncristiano M, Paccani SR, Barone S, et al. The Helicobacter pylori vacuolating toxin inhibits T cell activation by two independent mechanisms. J Exp Med 2003; 198(12):1887-97. 31. Torres VJ, VanCompernolle SE, Sundrud MS, et al. Helicobacter pylori vacuolating cytotoxin inhibits activation-induced proliferation of human T and B lymphocyte subsets. J Immunol 2007; 179(8):5433-40. 32. Tolwinski NS, Wieschaus E. Rethinking WNT signaling. Trends Genet 2004; 20(4):17781. 33. Gao C, Cao W, Bao L, et al. Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation. Nat Cell Biol 2010; 12(8):781-90. 34. Nakayama M, Hisatsune J, Yamasaki E, et al. Helicobacter pylori VacA-induced inhibition of GSK3 through the PI3K/Akt signaling pathway. J Biol Chem 2009; 284(3):1612-9. 35. Peek RM Jr, Crabtree JE. Helicobacter infection and gastric neoplasia. J Pathol 2006; 208(2):233-48. 36. Yahiro K, Satoh M, Nakano M, et al. Low-density lipoprotein receptor-related protein-1 (LRP1) mediates autophagy and apoptosis caused by Helicobacter pylori VacA. J Biol Chem 2012; 287(37):31104-15. 37. Terebiznik MR, Raju D, Vazquez CL, et al. Effect of Helicobacter pylori’s vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells. Autophagy 2009; 5(3):370-9. 38. Ogawa M, Yoshimori T, Suzuki T, et al. Escape of intracellular Shigella from autophagy. Science 2005; 307(5710):727-31. 39. Tang B, Li N, Gu J, et al. Compromised autophagy by MIR30B benefits the intracellular survival of Helicobacter pylori. Autophagy 2012; 8(7):1045-57. 40. Raju D, Hussey S, Ang M, et al. Vacuolating cytotoxin and variants in Atg16L1 that disrupt autophagy promote Helicobacter pylori infection in humans. Gastroenterology 2012; 142(5):1160-71. 41. Ham H, Sreelatha A, Orth K. Manipulation of host membranes by bacterial effectors. Nat Rev Microbiol 2011; 9(9):635-46. 42. Comerci DJ, Martinez-Lorenzo MJ, Sieira R, et al. Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole. Cell Microbiol 2001; 3(3):159-68. 43. Kuipers EJ, Perez-Perez GI, Meuwissen SG, et al. Helicobacter pylori and atrophic gastritis: importance of the cagA status. J Natl Cancer Inst 1995; 87(23):1777-80. 44. Kaparakis M, Turnbull L, Carneiro L, et al. Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells. Cell Microbiol 2010; 12(3):372-85. 45. Cadwell K, Liu JY, Brown SL, et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature 2008; 456(7219):259-63. 46. Baldassano RN, Bradfield JP, Monos DS, et al. Association of the T300A nonsynonymous variant of the ATG16L1 gene with susceptibility to paediatric Crohn’s disease. Gut 2007; 56(8):1171-3. 47. Prescott NJ, Fisher SA, Franke A, et al. A nonsynonymous SNP in ATG16L1 predisposes to ileal Crohn’s disease and is independent of CARD15 and IBD5. Gastroenterology 2007; 132(5):1665-71. 48. Burada F, Plantinga TS, Ioana M, et al. IRGM gene polymorphisms and risk of gastric cancer. J Dig Dis 2012; 13(7):360-5. 49. Kwok T, Zabler D, Urman S, et al. Helicobacter exploits integrin for type IV secretion and kinase activation. Nature 2007; 449(7164):862-6. 50. Murata-Kamiya N, Kikuchi K, Hayashi T, et al. Helicobacter pylori exploits host membrane phosphatidylserine for delivery, localization, and pathophysiological action of the CagA oncoprotein. Cell Host Microbe 2010; 7(5):399-411. 51. Selbach M, Moese S, Hauck CR, et al. Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo. J Biol Chem 2002; 277(9):6775-8. 52. Stein M, Bagnoli F, Halenbeck R, et al. c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs. Mol Microbiol 2002; 43(4):971-80. 53. Higashi H, Nakaya A, Tsutsumi R, et al. Helicobacter pylori CagA induces Rasindependent morphogenetic response through SHP-2 recruitment and activation. J Biol Chem 2004; 279(17):17205-16. 54. Wroblewski LE, Peek RM, Jr., Wilson KT. Helicobacter pylori and gastric cancer: factors that modulate disease risk. Clin Microbiol Rev 2010; 23(4):713-39. 55. Wei J, O’Brien D, Vilgelm A, et al. Interaction of Helicobacter pylori with gastric epithelial cells is mediated by the p53 protein family. Gastroenterology 2008; 134(5):1412-23. 56. Shibata A, Parsonnet J, Longacre TA, et al. CagA status of Helicobacter pylori infection and p53 gene mutations in gastric adenocarcinoma. Carcinogenesis 2002; 23(3):419-24. 57. Mauser A, Saito S, Appella E, et al. The Epstein-Barr virus immediate-early protein BZLF1 regulates p53 function through multiple mechanisms. J Virol 2002; 76(24):12503-12.


143

Research paper

58. Buti L, Spooner E, Van der Veen AG, et al. Helicobacter pylori cytotoxin-associated gene A (CagA) subverts the apoptosis-stimulating protein of p53 (ASPP2) tumor suppressor pathway of the host. Proc Natl Acad Sci U S A 2011; 108(22):9238-43. 59. Vigneron AM, Ludwig RL, Vousden KH. Cytoplasmic ASPP1 inhibits apoptosis through the control of YAP. Genes Dev 2010; 24(21):2430-9. 60. Yang ZJ, Chee CE, Huang S, et al. The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther 2011; 10(9):1533-41. 61. Khoury MP, Bourdon JC. The isoforms of the p53 protein. Cold Spring Harb Perspect Biol 2010; 2(3):a000927. 62. Wei J, Noto J, Zaika E, et al. Pathogenic bacterium Helicobacter pylori alters the expression profile of p53 protein isoforms and p53 response to cellular stresses. Proc Natl Acad Sci U S A 2012; 109(38):E2543-50. 63. Zhu BS, Xing CG, Lin F, et al. Blocking NF-kappaB nuclear translocation leads to p53related autophagy activation and cell apoptosis. World J Gastroenterol 2011; 17(4):47887. 64. Tsugawa H, Suzuki H, Saya H, et al. Reactive Oxygen Species-Induced Autophagic Degradation of Helicobacter pylori CagA Is Specifically Suppressed in Cancer Stem-like Cells. Cell Host Microbe 2012; 12(6):764-77. 65. Wroblewski LE, Peek RM, Jr. When guests simply will not leave. Cell Host Microbe 2012; 12(6):733-4. 66. Yokoyama K, Higashi H, Ishikawa S, et al. Functional antagonism between Helicobacter pylori CagA and vacuolating toxin VacA in control of the NFAT signaling pathway in gastric epithelial cells. Proc Natl Acad Sci U S A 2005; 102(27):9661-6. 67. Argent RH, Thomas RJ, Letley DP, et al. Functional association between the Helicobacter pylori virulence factors VacA and CagA. J Med Microbiol 2008; 57(Pt 2):145-50. 68. Tegtmeyer N, Zabler D, Schmidt D, et al. Importance of EGF receptor, HER2/Neu and Erk1/2 kinase signalling for host cell elongation and scattering induced by the Helicobacter pylori CagA protein: antagonistic effects of the vacuolating cytotoxin VacA. Cell Microbiol 2009; 11(3):488-505. 69. Oldani A, Cormont M, Hofman V, et al. Helicobacter pylori counteracts the apoptotic action of its VacA toxin by injecting the CagA protein into gastric epithelial cells. PLoS Pathog 2009; 5(10):e1000603. 70. Wang YH, Gorvel JP, Chu YT, et al. Helicobacter pylori impairs murine dendritic cell responses to infection. PLoS One 2010; 5(5):e10844. 71. Pilpilidis I, Kountouras J, Zavos C, et al. Upper gastrointestinal carcinogenesis: H. pylori and stem cell cross-talk. J Surg Res 2011; 166(2):255-64. 72. Konturek PC, Kania J, Burnat G, et al. Prostaglandins as mediators of COX-2 derived carcinogenesis in gastrointestinal tract. J Physiol Pharmacol 2005; 56 Suppl 5:57-73. 73. Kapetanakis N, Kountouras J, Venizelos I, et al. Correlation between H. pylori infection and glycoprotein CD44 in colorectal carcinoma. Ann Gastroenterol 2008; 21:25. 74. Kapetanakis N, Kountouras J, Zavos C, et al. Potential oncogenic properties of mobilized stem cells in a subpopulation of inflammatory bowel disease patients infected with Helicobacter pylori. Inflamm Bowel Dis 2013; 19(2):E27-9. 75. Kapetanakis N KJ, Zavos C, et al. Helicobacter pylori infection and colorectal carcinoma: pathologic aspects. Gastrointest Oncol 2012; 3:377-9. 76. Dupont N, Temime-Smaali N, Lafont F. How ubiquitination and autophagy participate in the regulation of the cell response to bacterial infection. Biol Cell 2010; 102(12):62134. 77. Chen K CH, Zhou RJ. Molecular mechanisms and functions of autophagy and the ubiquitin-proteasome pathway. Yi Chuan 2012; 34(1):5-18. 78. Baptista MS, Duarte CB, Maciel P. Role of the ubiquitin-proteasome system in nervous system function and disease: using C. elegans as a dissecting tool. Cell Mol Life Sci 2012; 69(16):2691-715. 79. Hara T, Nakamura K, Matsui M, et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 2006; 441(7095):885-9. 80. Ligeon LA, Temime-Smaali N, Lafont F. Ubiquitylation and autophagy in the control of bacterial infections and related inflammatory responses. Cell Microbiol 2011; 13(9):1303-11. 81. Fujita K, Srinivasula SM. TLR4-mediated autophagy in macrophages is a p62-dependent type of selective autophagy of aggresome-like induced structures (ALIS). Autophagy 2011; 7(5):552-4. 82. Szeto J, Kaniuk NA, Canadien V, et al. ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy. Autophagy 2006; 2(3):189-99. 83. Mesquita FS, Thomas M, Sachse M, et al. The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. PLoS Pathog 2012; 8(6):e1002743. 84. Zheng YT, Shahnazari S, Brech A, et al. The adaptor protein p62/SQSTM1 targets invading bacteria to the autophagy pathway. J Immunol 2009; 183(9):5909-16. 85. Birmingham CL, Smith AC, Bakowski MA, et al. Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole. J Biol Chem 2006; 281(16):11374-83. 86. Thurston TL, Ryzhakov G, Bloor S, et al. The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria. Nat Immunol 2009; 10(11):1215-21. 87. Necchi V, Sommi P, Ricci V, et al. In vivo accumulation of Helicobacter pylori products, NOD1, ubiquitinated proteins and proteasome in a novel cytoplasmic structure. PLoS One 2010; 5(3):e9716. 88. Yang ZM, Chen WW, Wang YF. Gene expression profiling in gastric mucosa from Helicobacter pylori-infected and uninfected patients undergoing chronic superficial gastritis. PLoS One 2012; 7(3):e33030.

144

89. Mani A, Gelmann EP. The ubiquitin-proteasome pathway and its role in cancer. J Clin Oncol 2005; 23(21):4776-89. 90. Wu L, Pu Z, Feng J, et al. The ubiquitin-proteasome pathway and enhanced activity of NF-kappaB in gastric carcinoma. J Surg Oncol 2008; 97(5):439-44. 91. Bossola M, Muscaritoli M, Costelli P, et al. Increased muscle proteasome activity correlates with disease severity in gastric cancer patients. Ann Surg 2003; 237(3):384-9. 92. Fan XM, Wong BC, Wang WP, et al. Inhibition of proteasome function induced apoptosis in gastric cancer. Int J Cancer 2001; 93(4):481-8. 93. Moscat J, Diaz-Meco MT, Wooten MW. Signal integration and diversification through the p62 scaffold protein. Trends Biochem Sci 2007; 32(2):95-100. 94. Nezis IP, Stenmark H. p62 at the interface of autophagy, oxidative stress signaling, and cancer. Antioxid Redox Signal 2012; 17(5):786-93. 95. Raju D, Hussey S, Jones NL. Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans. Autophagy 2012; 8(9):1387-8. 96. Puissant A, Fenouille N, Auberger P. When autophagy meets cancer through p62/ SQSTM1. Am J Cancer Res 2012; 2(4):397-413. 97. Duran A, Amanchy R, Linares JF, et al. p62 is a key regulator of nutrient sensing in the mTORC1 pathway. Mol Cell 2011; 44(1):134-46. 98. Ling J, Kang Y, Zhao R, et al. KrasG12D-induced IKK2/beta/NF-kappaB activation by IL-1alpha and p62 feedforward loops is required for development of pancreatic ductal adenocarcinoma. Cancer Cell 2012; 21(1):105-20. 99. Puissant A, Auberger P. AMPK- and p62/SQSTM1-dependent autophagy mediate Resveratrol-induced cell death in chronic myelogenous leukemia. Autophagy 2010; 6(5):655-7. 100. Puissant A, Robert G, Fenouille N, et al. Resveratrol promotes autophagic cell death in chronic myelogenous leukemia cells via JNK-mediated p62/SQSTM1 expression and AMPK activation. Cancer Res 2010; 70(3):1042-52. 101. Mathew R, Karp CM, Beaudoin B, et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell 2009; 137(6):1062-75. 102. Gao C, Chen YG. Selective removal of dishevelled by autophagy: a role of p62. Autophagy 2011; 7(3):334-5. 103. Takeuchi O, Akira S. MDA5/RIG-I and virus recognition. Curr Opin Immunol 2008; 20(1):17-22. 104. Xu Y, Jagannath C, Liu XD, et al. Toll-like receptor 4 is a sensor for autophagy associated with innate immunity. Immunity 2007; 27(1):135-44. 105. Delgado MA, Elmaoued RA, Davis AS, et al. Toll-like receptors control autophagy. EMBO J 2008; 27(7):1110-21. 106. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004; 4(7):499-511. 107. Viala J, Chaput C, Boneca IG, et al. Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat Immunol 2004; 5(11):1166-74. 108. Strober W, Murray PJ, Kitani A, et al. Signalling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol 2006; 6(1):9-20. 109. Lee MS, Kim YJ. Signaling pathways downstream of pattern-recognition receptors and their cross talk. Annu Rev Biochem 2007; 76:447-80. 110. O’Neill LA, Bowie AG. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 2007; 7(5):353-64. 111. Akira S. TLR signaling. Curr Top Microbiol Immunol 2006; 311:1-16. 112. Mandell L, Moran AP, Cocchiarella A, et al. Intact gram-negative Helicobacter pylori, Helicobacter felis, and Helicobacter hepaticus bacteria activate innate immunity via tolllike receptor 2 but not toll-like receptor 4. Infect Immun 2004; 72(11):6446-54. 113. Torok AM, Bouton AH, Goldberg JB. Helicobacter pylori induces interleukin-8 secretion by Toll-like receptor 2- and Toll-like receptor 5-dependent and -independent pathways. Infect Immun 2005; 73(3):1523-31. 114. Gobert AP, Bambou JC, Werts C, et al. Helicobacter pylori heat shock protein 60 mediates interleukin-6 production by macrophages via a toll-like receptor (TLR)-2-, TLR-4, and myeloid differentiation factor 88-independent mechanism. J Biol Chem 2004; 279(1):245-50. 115. Rad R, Ballhorn W, Voland P, et al. Extracellular and intracellular pattern recognition receptors cooperate in the recognition of Helicobacter pylori. Gastroenterology 2009; 136(7):2247-57. 116. Kobayashi F, Watanabe E, Nakagawa Y, et al. Production of autoantibodies by murine B-1a cells stimulated with Helicobacter pylori urease through toll-like receptor 2 signaling. Infect Immun 2011; 79(12):4791-801. 117. Adam P, Schmausser B, Gobeler-Kolve M, et al. Gastric extranodal marginal zone B-cell lymphomas of MALT type exclusively express Toll-like receptor 4 in contrast to other lymphomas infiltrating the stomach. Ann Oncol 2008; 19(3):566-9. 118. Andersen-Nissen E, Smith KD, Strobe KL, et al. Evasion of Toll-like receptor 5 by flagellated bacteria. Proc Natl Acad Sci U S A 2005; 102(26):9247-52. 119. Kumar Pachathundikandi S, Brandt S, Madassery J, et al. Induction of TLR-2 and TLR5 expression by Helicobacter pylori switches cagPAI-dependent signalling leading to the secretion of IL-8 and TNF-alpha. PLoS One 2011; 6(5):e19614. 120. Otani K, Tanigawa T, Watanabe T, et al. Toll-like receptor 9 signaling has antiinflammatory effects on the early phase of Helicobacter pylori-induced gastritis. Biochem Biophys Res Commun 2012; 426(3):342-9. 121. Chang YJ, Wu MS, Lin JT, et al. Helicobacter pylori-Induced invasion and angiogenesis of gastric cells is mediated by cyclooxygenase-2 induction through TLR2/TLR9 and promoter regulation. J Immunol 2005; 175(12):8242-52. 122. Tahara T, Arisawa T, Wang F, et al. Toll-like receptor 2 -196 to 174del polymorphism influences the susceptibility of Japanese people to gastric cancer. Cancer Sci 2007;


Volume 2 Issue 3

Research paper

98(11):1790-4. 123. Anderson AE, Worku ML, Khamri W, et al. TLR9 polymorphisms determine murine lymphocyte responses to Helicobacter: results from a genome-wide scan. Eur J Immunol 2007; 37(6):1548-61. 124. Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. Nature 2011; 469(7330):323-35. 125. Wild P, Farhan H, McEwan DG, et al. Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science 2011; 333(6039):228-33. 126. Saitoh T, Fujita N, Jang MH, et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature 2008; 456(7219):264-8. 127. Pimentel-Nunes P, Goncalves N, Boal-Carvalho I, et al. Helicobacter pylori induces increased expression of Toll-like receptors and decreased Toll-interacting protein in gastric mucosa that persists throughout gastric carcinogenesis. Helicobacter 2013; 18(1):22-32. 128. Inohara N, Nunez G. NODs: intracellular proteins involved in inflammation and apoptosis. Nat Rev Immunol 2003; 3(5):371-82. 129. Correa RG, Milutinovic S, Reed JC. Roles of NOD1 (NLRC1) and NOD2 (NLRC2) in innate immunity and inflammatory diseases. Biosci Rep 2012; 32(6):597-608. 130. Elinav E, Strowig T, Henao-Mejia J, et al. Regulation of the antimicrobial response by NLR proteins. Immunity 2011; 34(5):665-79. 131. Hisamatsu T, Suzuki M, Podolsky DK. Interferon-gamma augments CARD4/NOD1 gene and protein expression through interferon regulatory factor-1 in intestinal epithelial cells. J Biol Chem 2003; 278(35):32962-8. 132. Watanabe T, Asano N, Kitani A, et al. NOD1-Mediated Mucosal Host Defense against Helicobacter pylori. Int J Inflam 2010; 2010:476482. 133. Wang P, Zhang L, Jiang JM, et al. Association of NOD1 and NOD2 genes polymorphisms with Helicobacter pylori related gastric cancer in a Chinese population. World J Gastroenterol 2012; 18(17):2112-20. 134. Rosenstiel P, Huse K, Till A, et al. A short isoform of NOD2/CARD15, NOD2-S, is an endogenous inhibitor of NOD2/receptor-interacting protein kinase 2-induced signaling pathways. Proc Natl Acad Sci U S A 2006; 103(9):3280-5. 135. Hasegawa M, Fujimoto Y, Lucas PC, et al. A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB activation. EMBO J 2008; 27(2):373-83. 136. Watanabe T, Asano N, Fichtner-Feigl S, et al. NOD1 contributes to mouse host defense against Helicobacter pylori via induction of type I IFN and activation of the ISGF3 signaling pathway. J Clin Invest 2010; 120(5):1645-62. 137. Boughan PK, Argent RH, Body-Malapel M, et al. Nucleotide-binding oligomerization domain-1 and epidermal growth factor receptor: critical regulators of beta-defensins during Helicobacter pylori infection. J Biol Chem 2006; 281(17):11637-48. 138. Grubman A, Kaparakis M, Viala J, et al. The innate immune molecule, NOD1, regulates direct killing of Helicobacter pylori by antimicrobial peptides. Cell Microbiol 2010; 12(5):626-39. 139. Travassos LH, Carneiro LA, Ramjeet M, et al. Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry. Nat Immunol 2010; 11(1):55-62. 140. Ramjeet M, Hussey S, Philpott DJ, et al. ‘Nodophagy’: New crossroads in Crohn disease pathogenesis. Gut Microbes 2010; 1(5):307-15. 141. Galluzzi L, Kepp O, Zitvogel L, et al. Bacterial invasion: linking autophagy and innate immunity. Curr Biol 2010; 20(3):R106-8. 142. Rosenstiel P, Hellmig S, Hampe J, et al. Influence of polymorphisms in the NOD1/ CARD4 and NOD2/CARD15 genes on the clinical outcome of Helicobacter pylori infection. Cell Microbiol 2006; 8(7):1188-98. 143. Sutton P, Mitchell HM. Helicobacter pylori in the 21st century. Wallingford, Oxfordshire, UK ; Cambridge, MA: CABI; 2010. 144. Benoit BN, Kobayashi M, Kawakubo M, et al. Role of ASC in the mouse model of Helicobacter pylori infection. J Histochem Cytochem 2009; 57(4):327-38. 145. Hitzler I, Sayi A, Kohler E, et al. Caspase-1 has both proinflammatory and regulatory properties in Helicobacter infections, which are differentially mediated by its substrates IL-1beta and IL-18. J Immunol 2012; 188(8):3594-602. 146. Miao EA, Mao DP, Yudkovsky N, et al. Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome. Proc Natl Acad Sci U S A 2010; 107(7):3076-80. 147. Nakahira K, Haspel JA, Rathinam VA, et al. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol 2011; 12(3):222-30. 148. Salminen A, Kaarniranta K, Kauppinen A. Beclin 1 interactome controls the crosstalk between apoptosis, autophagy and inflammasome activation: Impact on the aging process. Ageing Res Rev 2013; 12(2):520-34. 149. Elinav E, Henao-Mejia J, Flavell RA. Integrative inflammasome activity in the regulation of intestinal mucosal immune responses. Mucosal Immunol 2013; 6(1):4-13.

www.stmconnect.com/ig

150. Shi CS, Shenderov K, Huang NN, et al. Activation of autophagy by inflammatory signals limits IL-1beta production by targeting ubiquitinated inflammasomes for destruction. Nat Immunol 2012; 13(3):255-63. 151. Kountouras J, Zavos C, Gavalas E, et al. Helicobacter pylori may be a common denominator associated with systemic and multiple sclerosis. Joint Bone Spine 2011; 78(2):222-3; author reply 223. 152. Wolfe MM, Nompleggi DJ. Cytokine inhibition of gastric acid secretion--a little goes a long way. Gastroenterology 1992; 102(6):2177-8. 153. El-Omar EM, Carrington M, Chow WH, et al. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 2000; 404(6776):398-402. 154. Wehkamp J, Stange EF. Paneth’s disease. J Crohns Colitis 2010; 4(5):523-31. 155. Caprilli R, Lapaquette P, Darfeuille-Michaud A. Eating the enemy in Crohn’s disease: an old theory revisited. J Crohns Colitis 2010; 4(4):377-83. 156. Li L, Khatibi NH, Hu Q, et al. Transmembrane protein 166 regulates autophagic and apoptotic activities following focal cerebral ischemic injury in rats. Exp Neurol 2012; 234(1):181-90. 157. Chen L, Zhang B, Toborek M. Autophagy is involved in nanoalumina-induced cerebrovascular toxicity. Nanomedicine 2013; 9(2):212-21. 158. Kountouras J, Boziki M, Zavos C, et al. A potential impact of chronic Helicobacter pylori infection on Alzheimer’s disease pathobiology and course. Neurobiol Aging 2012; 33(7):e3-4. 159. Kountouras J, Zavos C, Deretzi G, et al. Helicobacter pylori might be a potential therapeutic target in epilepsy. Med Hypotheses 2011; 76(5):763. 160. Kountouras J, Zavos C, Katsinelos P, et al. Glaucoma and Helicobacter pylori: eyes “wide open”! Dig Liver Dis 2012; 44(11):962-3; author reply 963. 161. Atherton JC, Blaser MJ. Coadaptation of Helicobacter pylori and humans: ancient history, modern implications. J Clin Invest 2009; 119(9):2475-87. 162. Correa P. Helicobacter pylori and gastric cancer: state of the art. Cancer Epidemiol Biomarkers Prev 1996; 5(6):477-81. 163. Ohnishi N, Yuasa H, Tanaka S, et al. Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in mouse. Proc Natl Acad Sci U S A 2008; 105(3):1003-8. 164. Janku F, McConkey DJ, Hong DS, et al. Autophagy as a target for anticancer therapy. Nat Rev Clin Oncol 2011; 8(9):528-39. 165. White E, DiPaola RS. The double-edged sword of autophagy modulation in cancer. Clin Cancer Res 2009; 15(17):5308-16. 166. Kapetanakis N KJ, Zavos C, et al. Association of Helicobacter pylori infection with colorectal cancer. Immuno-Gastroenterology 2013; 2(1):47-56. 167. Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature 2005; 434(7035):84350. 168. Backues SK, Klionsky DJ. Autophagy gets in on the regulatory act. J Mol Cell Biol 2011; 3(2):76-7. 169. Polk DB, Peek RM, Jr. Helicobacter pylori: gastric cancer and beyond. Nat Rev Cancer 2010; 10(6):403-14. 170. Franco AT, Israel DA, Washington MK, et al. Activation of beta-catenin by carcinogenic Helicobacter pylori. Proc Natl Acad Sci U S A 2005; 102(30):10646-51. 171. Yang JC, Shun CT, Chien CT, et al. Effective prevention and treatment of Helicobacter pylori infection using a combination of catechins and sialic acid in AGS cells and BALB/c mice. J Nutr 2008; 138(11):2084-90. 172. Yang JC, Chien CT. A new approach for the prevention and treatment of Helicobacter pylori infection via upregulation of autophagy and downregulation of apoptosis. Autophagy 2009; 5(3):413-4. 173. Rubinsztein DC, Codogno P, Levine B. Autophagy modulation as a potential therapeutic target for diverse diseases. Nat Rev Drug Discov 2012; 11(9):709-30. 174. Kim JJ, Lee HM, Shin DM, et al. Host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action. Cell Host Microbe 2012; 11(5):457-68. 175. Campbell GR, Spector SA. Hormonally active vitamin D3 (1alpha,25dihydroxycholecalciferol) triggers autophagy in human macrophages that inhibits HIV-1 infection. J Biol Chem 2011; 286(21):18890-902. 176. Schwalfenberg GK. A review of the critical role of vitamin D in the functioning of the immune system and the clinical implications of vitamin D deficiency. Mol Nutr Food Res 2011; 55(1):96-108. 177. Antico A, Tozzoli R, Giavarina D, et al. Hypovitaminosis D as predisposing factor for atrophic type A gastritis: a case-control study and review of the literature on the interaction of Vitamin D with the immune system. Clin Rev Allergy Immunol 2012; 42(3):355-64. 178. Kwon HJ, Won YS, Nam KT, et al. Vitamin D(3) upregulated protein 1 deficiency promotes N-methyl-N-nitrosourea and Helicobacter pylori-induced gastric carcinogenesis in mice. Gut 2012; 61(1):53-63.


145