Maria Kaparakis‐Liaskos

The intracellular innate immune receptor NOD1 detects Gram-negative bacterial peptidoglycan (PG) to induce autophagy and inflammatory responses in host cells. To date, the intracellular compartment in which PG is detected by NOD1 and whether NOD1 directly interacts with PG are two questions that remain to be resolved. To address this, we used outer membrane vesicles (OMVs) from pathogenic bacteria as a physiological mechanism to deliver PG into the host cell cytosol. We report that OMVs induced autophagosome formation and inflammatory IL-8 responses in epithelial cells in a NOD1- and RIP2-dependent manner. PG contained within OMVs colocalized with both NOD1 and RIP2 in EEA1-positive early endosomes. Further, we provide evidence for direct interactions between NOD1 and PG. Collectively, these findings demonstrate that NOD1 detects PG within early endosomes, thereby promoting RIP2-dependent autophagy and inflammatory signaling in response to bacterial infection.

Outer membrane vesicles were first described approximately 50 years ago and for many years were considered to be an artifact of bacterial growth. Since that initial discovery, it has become evident that outer membrane vesicles are produced by almost all Gram-negative bacteria as part of their normal growth in addition to driving pathogenesis within the host. More recently, the identification of membrane vesicle (MV) production by some Gram-positive bacteria, parasites, fungi, mycobacteria and infected host cells has significantly broadened the field of MV research and emphasized their importance to pathogenesis. In this review, we will focus on discussing recent advances in the field of bacterial MV biogenesis and the mechanisms whereby they modulate immunity and contribute to pathogenesis. We will highlight findings identifying the contribution of extracellular vesicles produced by Gram-positive bacteria, fungi, parasites, and infected host cells in mediating pathogenesis in addition to the functions of MVs produced by commensal bacteria. Finally, we will discuss recent progress in the development of bacterial MVs as novel vaccines capable of mediating cellular and humoral immune responses.

Gram-negative pathogens ubiquitously shed outer membrane vesicles (OMVs) that play a central role in initiating and regulating pathogenesis in the host. Due to their highly inflammatory nature, OMVs are extensively being examined for their role in mediating disease in addition to their applications in innovative vaccines. A key mechanism whereby OMVs mediate inflammation and disease progression is dependent on their ability to enter host cells. Currently, the role of OMV size on determining their mechanism of cellular entry and their protein composition remains unknown. In this study, we examined the mechanisms whereby OMV size regulates their mode of entry into epithelial cells, in addition to their protein cargo and composition. We identified that a heterogeneous sized population of Helicobacter pylori OMVs entered epithelial cells via macropinocytosis, clathrin, and caveolin-dependent endocytosis. However, smaller OMVs ranging from 20 to 100 nm in size preferentially entered host cells via caveolin-mediated endocytosis. Whereas larger OMVs ranging between 90 and 450 nm in size entered host epithelial cells via macropinocytosis and endocytosis. Most importantly, we identified the previously unknown contribution that OMV size has on determining their protein content, as fewer and less diverse bacterial proteins were contained within small OMVs compared to larger OMVs. Collectively, these findings identify the importance of OMV size in determining the mechanisms of OMV entry into host cells, in addition to regulating their protein cargo, composition, and subsequent immunogenicity. These findings have significant implications in broadening our understanding of the bacterial regulation of virulence determinants and immunogenic proteins associated with OMVs, their role in mediating pathogenesis and in refining the design and development of OMV-based vaccines.

Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host.

Fine-tuning of inflammatory responses by microRNAs (miRNAs) is complex, as they can both enhance and repress expression of pro-inflammatory mediators. In this study, we investigate inflammatory responses following global miRNA depletion, to better define the overall contribution of miRNAs to inflammation. We demonstrate that miRNAs positively regulate Toll-like receptor signaling using inducible Dicer1 deletion and global miRNA depletion. We establish an important contribution of miR-19b in this effect, which potentiates nuclear factor-κB (NF-κB) activity in human and mouse cells. Positive regulation of NF-κB signaling by miR-19b involves the coordinated suppression of a regulon of negative regulators of NF-κB signaling (including A20/Tnfaip3, Rnf11, Fbxl11/Kdm2a and Zbtb16). Transfection of miR-19b mimics exacerbated the inflammatory activation of rheumatoid arthritis primary fibroblast-like synoviocytes, demonstrating its physiological importance in the pathology of this disease. This study constitutes, to our knowledge, the first description of a miR-19 regulon that controls NF-κB signaling, and suggests that targeting this miRNA and linked family members could regulate the activity of NF-κB signaling in inflammation.

The therapeutic potential of extracellular vesicles from eukaryotes has gained strong interest in recent years. However, research into the therapeutic application of their bacterial counterparts, known as bacterial membrane vesicles, is only just beginning to be appreciated. Membrane vesicles (MVs) from both Gram-positive and Gram-negative bacteria offer significant advantages in therapeutic development, including large-scale, cost effective production and ease of molecular manipulation to display foreign antigens. The nanoparticle size of MVs enables their dissemination through numerous tissue types, and their natural immunogenicity and self-adjuvanting capability can be harnessed to induce both cell-mediated and humoral immunity in vaccine design. Moreover, the ability to target MVs to specific tissues through the display of surface receptors raises their potential use as targeted MV-based anti-cancer therapy. This review discusses recent advances in MV research with particular emphasis on exciting new possibilities for the application of MVs in therapeutic design.

Abstract Background Multiple studies have established the importance of the tol‐pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli . In contrast, the functions of Tol‐Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria , remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol‐Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori . Methods H. pylori Δ tolB , Δ pal and Δ tolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme‐linked immunoassay (ELISA), respectively. The protein content and pro‐inflammatory properties of OMVs were determined by mass spectroscopy and interleukin‐8 (IL‐8) ELISA on culture supernatants from OMV‐stimulated cells, respectively. Results H. pylori Δ tolB and Δ pal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H. pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H. pylori Δ tolB and Δ pal bacteria harbored many of the major outer membrane and virulence proteins observed in wild‐type (WT) OMVs. Interestingly, Δ tolB , Δ pal and Δ tolBpal OMVs induced significantly higher levels of IL‐8 production by host cells, compared with WT OMVs. Conclusions This work demonstrates that TolB and Pal are important for membrane integrity in H. pylori . Moreover, it shows how H. pylori tolB ‐ pal genes may be manipulated to develop “hypervesiculating” strains for vaccine purposes.

Bacteria release extracellular vesicles (EVs) known as bacterial membrane vesicles (BMVs) during their normal growth. Gram-negative bacteria produce BMVs termed outer membrane vesicles (OMVs) that are composed of a range of biological cargo and facilitate numerous bacterial functions, including promoting pathogenesis and mediating disease in the host. By contrast, less is understood about BMVs produced by Gram-positive bacteria, which are referred to as membrane vesicles (MVs), however their contribution to mediating bacterial pathogenesis has recently become evident. In this review, we summarise the mechanisms whereby BMVs released by Gram-negative and Gram-positive bacteria are produced, in addition to discussing their key functions in promoting bacterial survival, mediating pathogenesis and modulating host immune responses. Furthermore, we discuss the mechanisms whereby BMVs produced by both commensal and pathogenic organisms can enter host cells and interact with innate immune receptors, in addition to how they modulate host innate and adaptive immunity to promote immunotolerance or drive the onset and progression of disease. Finally, we highlight current and emerging applications of BMVs in vaccine design, biotechnology and cancer therapeutics.

Abstract Extracellular vesicles (EVs) large‐scale production is a crucial point for the translation of EVs from discovery to application of EV‐based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET‐OPEN projects, “The Extracellular Vesicle Foundry” (evFOUNDRY) and “Extracellular vesicles from a natural source for tailor‐made nanomaterials” (VES4US), organized a workshop entitled “massivEVs” to discuss the potential challenges for translation of EV‐based products. This report gives an overview of the topics discussed during “massivEVs”, the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria package various cargo, including DNA that can be transferred to other bacteria or to host cells. OMV-associated DNA has been implicated in mediating horizontal gene transfer (HGT) between bacteria, which includes the dissemination of antibiotic resistance genes within and between bacterial species. Despite the known ability of OMVs to mediate HGT, the mechanisms of DNA packaging into OMVs remain poorly characterized, as does the effect of bacterial growth conditions on the DNA cargo composition of OMVs and their subsequent abilities to mediate HGT. In this study, we examined the DNA content of OMVs produced by the opportunistic pathogen Pseudomonas aeruginosa grown in either planktonic or biofilm conditions. Analysis of planktonic growth-derived OMVs revealed their ability to package and protect plasmid DNA from DNase degradation and to transfer plasmid-encoded antibiotic resistance genes to recipient, antibiotic-sensitive P. aeruginosa bacteria at a greater efficiency than transformation with plasmid alone. Comparisons of planktonic and biofilm-derived P. aeruginosa OMVs demonstrated that biofilm-derived OMVs were smaller but were associated with more plasmid DNA than planktonic-derived OMVs. Additionally, biofilm-derived P. aeruginosa OMVs were more efficient in the transformation of competent P. aeruginosa bacteria, compared to transformations with an equivalent number of planktonic-derived OMVs. The findings of this study highlight the importance of bacterial growth conditions for the packaging of DNA within P. aeruginosa OMVs and their ability to facilitate HGT, thus contributing to the spread of antibiotic resistance genes between P. aeruginosa bacteria. IMPORTANCE Bacterial membrane vesicles (BMVs) mediate interbacterial communication, and their ability to package DNA specifically contributes to biofilm formation, antibiotic resistance, and HGT between bacteria. However, the ability of P. aeruginosa OMVs to mediate HGT has not yet been demonstrated. Here, we reveal that P. aeruginosa planktonic and biofilm-derived OMVs can deliver plasmid-encoded antibiotic resistance to recipient P. aeruginosa. Additionally, we demonstrated that P. aeruginosa biofilm-derived OMVs were associated with more plasmid DNA compared to planktonic-derived OMVs and were more efficient in the transfer of plasmid DNA to recipient bacteria. Overall, this demonstrated the ability of P. aeruginosa OMVs to facilitate the dissemination of antibiotic resistance genes, thereby enabling the survival of susceptible bacteria during antibiotic treatment. Investigating the roles of biofilm-derived BMVs may contribute to furthering our understanding of the role of BMVs in HGT and the spread of antibiotic resistance in the environment.

Histone modifications are critical in regulating gene expression, cell cycle, cell proliferation, and development. Relatively few studies have investigated whether Helicobacter pylori, the major cause of human gastric diseases, affects histone modification. We therefore investigated the effects of H. pylori infection on histone modifications in a global and promoter-specific manner in gastric epithelial cells. Infection of gastric epithelial cells by wild-type H. pylori induced time- and dose-dependent dephosphorylation of histone H3 at serine 10 (H3 Ser10) and decreased acetylation of H3 lysine 23, but had no effects on seven other specific modifications. Different cag pathogenicity island (PAI)-containing-clinical isolates showed similar abilities to induce H3 Ser10 dephosphorylation. Mutation of cagA, vacA, nonphosphorylateable CagA mutant cagA(EPISA), or disruption of the flagella showed no effects, while deletion of the entire cagPAI restored the H3 Ser10 phosphorylation to control levels. Analysis of 27 cagPAI mutants indicated that the genes that caused H3 Ser10 dephosphorylation were similar to those that were previously found to induce interleukin-8, irrespective of CagA translocation. This effect was independent of ERK or p38 pathways and type I interferon signaling. Additionally, c-Jun and hsp70 gene expression was associated with this histone modification. These results demonstrate that H. pylori alters histone modification and host response via a cagA-, vacA-independent, but cagPAI-dependent mechanisms, which contribute to its persistent infection and pathogenesis.

Abstract Gram‐negative bacteria release outer membrane vesicles (OMVs) as part of their normal growth that contain a range of cargo from their parent bacterium, including DNA, RNA, and proteins. The protein content of OMVs is suggested to be similar in composition to various sub‐cellular locations of their parent bacterium. However, very little is known regarding the effect of bacterial growth stage on the size, content, and selective packaging of proteins into OMVs. In this study, the global proteome of Helicobacter pylori and their OMVs throughout bacterial growth are examined to determine if bacterial growth stage affected OMV cargo composition. Analysis of OMVs produced by H. pylori reveals that bacterial growth stage affects the size, composition, and selection of protein cargo into OMVs. Proteomic analysis identifies that the proteome of H. pylori OMVs is vastly different throughout bacterial growth and that OMVs contain a range of proteins compared to their parent bacteria. In addition, bacterial growth stage affects the ability of OMVs to induce the production of IL‐8 by human epithelial cells. Therefore, the findings identify that the size, proteome, and immunogenicity of OMVs produced during various stages of bacterial growth is not comparable. Collectively, these findings highlight the importance of considering the bacterial growth stage from which OMVs are isolated, as this will impact their size, protein composition, and ultimately their biological functions.

Significance Maintaining physiological balance is vital in the primary response to infectious and other stress stimuli to avert damaging inflammation. Delineation of the cell regulatory processes that control inflammatory processes better enable the development of informed strategies to treat associated pathologies. Toward this end, we identify that the promyelocytic leukemia zinc finger (PLZF) transcription factor limits pathogen-induced inflammation. PLZF stabilizes a repressor complex that encompasses histone deacetylase activity, which modifies the state of chromatin. This activity maintains homeostasis by decreasing the scale of induction of select immune response genes. In the absence of PLZF, the chromatin structure is altered, enabling active transcriptional complexes to immediately assemble on gene promoters, resulting in inordinate production of inflammatory cytokines.

Bacterial membrane vesicles (BMVs) are produced by all bacteria and facilitate a range of functions in host-microbe interactions and pathogenesis. Quantification of BMVs is a critical first step in the analysis of their biological and immunological functions. Historically, BMVs have been quantified by protein assay, which remains the preferred method of BMV quantification. However, recent studies have shown that BMV protein content can vary significantly between bacterial strains, growth conditions, and stages of bacterial growth, suggesting that protein concentration may not correlate directly with BMV quantity. Here, we show that the method used to quantify BMVs can alter experimental outcomes. We compared the enumeration of BMVs using different protein assays and nanoparticle tracking analysis (NTA). We show that different protein assays vary significantly in their quantification of BMVs and that their sensitivity varies when quantifying BMVs produced by different species. Moreover, stimulation of epithelial cells with an equivalent amount of BMV protein quantified using different protein assays resulted in significant differences in interleukin 8 (IL-8) responses. Quantification of Helicobacter pylori, Pseudomonas aeruginosa, and Staphylococcus aureus BMVs by NTA and normalization of BMV cargo to particle number revealed that BMV protein, DNA, and RNA contents were variable between strains and species and throughout bacterial growth. Differences in BMV-mediated activation of Toll-like receptors, NF-κB, and IL-8 responses were observed when stimulations were performed with equivalent BMV particle number but not equivalent protein amount. These findings reveal that the method of BMV quantification can significantly affect experimental outcomes, thereby potentially altering the observed biological functions of BMVs. IMPORTANCE Recent years have seen a surge in interest in the roles of BMVs in host-microbe interactions and interbacterial communication. As a result of such rapid growth in the field, there is a lack of uniformity in BMV enumeration. Here, we reveal that the method used to enumerate BMVs can significantly alter experimental outcomes. Specifically, standardization of BMVs by protein amount reduced the ability to distinguish strain differences in the immunological functions of BMVs. In contrast, species-, strain-, and growth stage-dependent differences in BMV cargo content were evident when BMVs were enumerated by particle number, and this was reflected in differences in their ability to induce immune responses. These findings indicate that parameters critical to BMV function, including bacterial species, strain, growth conditions, and sample purity, should form the basis of standard reporting in BMV studies. This will ultimately bring uniformity to the field to advance our understanding of BMV functions.

Gram-negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P. aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.

ABSTRACT Infection with Helicobacter pylori cag pathogenicity island ( cag PAI)-positive strains is associated with more destructive tissue damage and an increased risk of severe disease. The cag PAI encodes a type IV secretion system (TFSS) that delivers the bacterial effector molecules CagA and peptidoglycan into the host cell cytoplasm, thereby inducing responses in host cells. It was previously shown that interactions between CagL, present on the TFSS pilus, and host α 5 β 1 integrin molecules were critical for CagA translocation and the induction of cytoskeletal rearrangements in epithelial cells. As the α 5 β 1 integrin is found in cholesterol-rich microdomains (known as lipid rafts), we hypothesized that these domains may also be involved in the induction of proinflammatory responses mediated by NOD1 recognition of H. pylori peptidoglycan. Indeed, not only did methyl-β-cyclodextrin depletion of cholesterol from cultured epithelial cells have a significant effect on the levels of NF-κB and interleukin-8 (IL-8) responses induced by H. pylori bacteria with an intact TFSS ( P < 0.05), but it also interfered with TFSS-mediated peptidoglycan delivery to cells. Both of these effects could be restored by cholesterol replenishment of the cells. Furthermore, we demonstrated for the first time the involvement of α 5 β 1 integrin in the induction of proinflammatory responses by H. pylori. Taking the results together, we propose that α 5 β 1 integrin, which is associated with cholesterol-rich microdomains at the host cell surface, is required for NOD1 recognition of peptidoglycan and subsequent induction of NF-κB-dependent responses to H. pylori . These data implicate cholesterol-rich microdomains as a novel platform for TFSS-dependent delivery of bacterial products to cytosolic pathogen recognition molecules.

Human Toll-like receptors (TLRs) TLR7, TLR8, and TLR9 are important immune sensors of foreign nucleic acids encountered by phagocytes. Although there is growing evidence implicating TLR7 and TLR9 in the detection of intracellular pathogenic bacteria, characterization of such a role for TLR8 is currently lacking. A recent genetic study has correlated the presence of a TLR8 single nucleotide polymorphism (SNP) (rs3764880:A>G; p.Met1Val) with the development of active tuberculosis, suggesting a role for TLR8 in the detection of phagosomal bacteria. Here we provide the first direct evidence that TLR8 sensing is activated in human monocytic cells following Helicobacter pylori phagocytosis. In addition, we show that rs3764880 fine tunes translation of the two TLR8 main isoforms, without affecting protein function. Although we show that TLR8 variant 2 (TLR8v2) is the prevalent form of TLR8 contributing to TLR8 function, we also uncover a role for the TLR8 long isoform (TLR8v1) in the positive regulation of TLR8 function in CD16+CD14+ differentiated monocytes. Thus, TLR8 sensing can be activated following bacterial phagocytosis, and rs3764880 may play a role in the modulation of TLR8-dependent microbicidal response of infected macrophages. Hum Mutat 31:1069–1079, 2010. © 2010 Wiley-Liss, Inc.

Abstract Virulent Helicobacter pylori strains that specifically activate signaling in epithelial cells via the innate immune molecule, nucleotide oligomerization domain 1 (NOD1), are more frequently associated with IFN-γ–dependent inflammation and with severe clinical outcomes (i.e., gastric cancer and peptic ulceration). In cell culture models, we showed that H. pylori activation of the NOD1 pathway caused enhanced proinflammatory signaling in epithelial cells in response to IFN-γ stimulation through the direct effects of H. pylori on two components of the IFN-γ signaling pathway, STAT1 and IFN regulatory factor 1 (IRF1). Specifically, H. pylori activation of the NOD1 pathway was shown to increase the levels of STAT1-Tyr701/Ser727 phosphorylation and IRF1 expression/synthesis in cells, resulting in enhanced production of the NOD1- and IFN-γ–regulated chemokines, IL-8– and IFN-γ–induced protein 10, respectively. Consistent with the notion that heightened proinflammatory signaling in epithelial cells may have an impact on disease severity, we observed significantly increased expression levels of NOD1, CXCL8, IRF1, and CXCL10 in human gastric biopsies displaying severe gastritis, when compared with those without gastritis (p < 0.05, p < 0.001, p < 0.01, and p < 0.05, respectively). Interestingly, NOD1, CXCL8, and IRF1 expression levels were also significantly upregulated in gastric tumor tissues, when compared with paired nontumor samples (p < 0.0001, p < 0.05, and p < 0.05, respectively). Thus, we propose that cross-talk between NOD1 and IFN-γ signaling pathways contribute to H. pylori–induced inflammatory responses, potentially revealing a novel mechanism whereby virulent H. pylori strains promote more severe disease.

PROTEOMICSVolume 19, Issue 1-2 1970004 Back CoverFree Access Back Cover: Helicobacter pylori Growth Stage Determines the Size, Protein Composition, and Preferential Cargo Packaging of Outer Membrane Vesicles Lauren Zavan, Lauren ZavanSearch for more papers by this authorNatalie J Bitto, Natalie J BittoSearch for more papers by this authorElla L. Johnston, Ella L. JohnstonSearch for more papers by this authorDavid W. Greening, David W. GreeningSearch for more papers by this authorMaria Kaparakis-Liaskos, Maria Kaparakis-LiaskosSearch for more papers by this author Lauren Zavan, Lauren ZavanSearch for more papers by this authorNatalie J Bitto, Natalie J BittoSearch for more papers by this authorElla L. Johnston, Ella L. JohnstonSearch for more papers by this authorDavid W. Greening, David W. GreeningSearch for more papers by this authorMaria Kaparakis-Liaskos, Maria Kaparakis-LiaskosSearch for more papers by this author First published: 24 January 2019 https://doi.org/10.1002/pmic.201970004Citations: 30AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract DOI: 10.1002/pmic.201800209 In article number 1800209, Lauren Zavan et al. examine the proteome of Helicobacter pylori and their outer membrane vesicles (OMVs) produced throughout bacterial growth to determine how growth stage regulates their cargo composition. Analysis of OMVs produced by H. pylori reveal that growth stage affects the size, composition and protein selection into OMVs. The heat abundance map shows the proteome of OMVs differed from that of their parent bacterium, and changes throughout bacterial growth. These findings identify that bacterial growth stage regulates protein cargo selection into OMVs. Citing Literature Volume19, Issue1-2Special Issue: Extracellular Vesicles and ExosomesJanuary 20191970004 RelatedInformation

Bacterial membrane vesicles (BMVs) are nanoparticles produced by both Gram-negative and Gram-positive bacteria that can function to modulate immunity in the host. Both outer membrane vesicles (OMVs) and membrane vesicles (MVs), which are released by Gram-negative and Gram-positive bacteria, respectively, contain cargo derived from their parent bacterium, including immune stimulating molecules such as proteins, lipids and nucleic acids. Of these, peptidoglycan (PG) and lipopolysaccharide (LPS) are able to activate host innate immune pattern recognition receptors (PRRs), known as NOD-like receptors (NLRs), such as nucleotide-binding oligomerisation domain-containing protein (NOD) 1, NOD2 and NLRP3. NLR activation is a key driver of inflammation in the host, and BMVs derived from both pathogenic and commensal bacteria have been shown to package PG and LPS in order to modulate the host immune response using NLR-dependent mechanisms. Here, we discuss the packaging of immunostimulatory cargo within OMVs and MVs, their detection by NLRs and the cytokines produced by host cells in response to their detection. Additionally, commensal derived BMVs are thought to shape immunity and contribute to homeostasis in the gut, therefore we also highlight the interactions of commensal derived BMVs with NLRs and their roles in limiting inflammatory diseases.

Parkinson’s disease (PD) is an increasingly common neurodegenerative disease. It has been suggested that the etiology of idiopathic PD is complex and multifactorial involving environmental contributions, such as viral or bacterial infections and microbial dysbiosis, in genetically predisposed individuals. With advances in our understanding of the gut-brain axis, there is increasing evidence that the intestinal microbiota and the mammalian immune system functionally interact. Recent findings suggest that a shift in the gut microbiome to a pro-inflammatory phenotype may play a role in PD onset and progression. While there are links between gut bacteria, inflammation, and PD, the bacterial products involved and how they traverse the gut lumen and distribute systemically to trigger inflammation are ill-defined. Mechanisms emerging in other research fields point to a role for small, inherently stable vesicles released by Gram-negative bacteria, called outer membrane vesicles in disease pathogenesis. These vesicles facilitate communication between bacteria and the host and can shuttle bacterial toxins and virulence factors around the body to elicit an immune response in local and distant organs. In this perspective article, we hypothesize a role for bacterial outer membrane vesicles in PD pathogenesis. We present evidence suggesting that these outer membrane vesicles specifically from Gram-negative bacteria could potentially contribute to PD by traversing the gut lumen to trigger local, systemic, and neuroinflammation. This perspective aims to facilitate a discussion on outer membrane vesicles in PD and encourage research in the area, with the goal of developing strategies for the prevention and treatment of the disease.

Helicobacter pylori infection has been proposed to be associated with various diseases of the hepatobiliary tract, including cancer of the bile duct epithelial cells (cholangiocarcinoma, CCA). The ability of H. pylori bacteria to cause pathogenic effects in these cells has, however, yet to be investigated. Given that the cag pathogenicity island (cagPAI) is required for H. pylori pathogenesis in gastric epithelial cells, we investigated wild-type and cag mutant strains for their ability to adhere, be internalized and induce pro-inflammatory responses in two bile duct epithelial cell lines derived from cases of CCA. The findings from these experiments were compared to results obtained with the well-characterized AGS gastric cancer cell line. We showed that the cagPAI encodes factors involved in H. pylori internalization in CCA cells, but not for adhesion to these cells. Consistent with previous studies in hepatocytes, actin polymerization and α5β1 integrin may be involved in H. pylori internalization in CCA cells. As for AGS cells, we observed significantly reduced levels of NF-κB activation and IL-8 production in CCA cells stimulated with either cagA, cagL or cagPAI bacteria, when compared with wild-type bacteria. Importantly, these IL-8 responses could be inhibited via either pre-treatment of cells with antibodies to α5β1 integrins, or via siRNA-mediated knockdown of the innate immune signaling molecules, nucleotide oligomerization domain 1 (NOD1) and myeloid differentiation response gene 88 (MyD88). Taken together, the data demonstrate that the cagPAI is critical for H. pylori pathogenesis in bile duct cells, thus providing a potential causal link for H. pylori in biliary tract disease.

The release of bacterial membrane vesicles (BMVs) has become recognized as a key mechanism used by both pathogenic and commensal bacteria to activate innate immune responses in the host and mediate immunity. Outer membrane vesicles (OMVs) produced by Gram-negative bacteria can harbor various immunogenic cargo that includes proteins, nucleic acids and peptidoglycan, and the composition of OMVs strongly influences their ability to activate host innate immune receptors. Although various Gram-negative pathogens can produce OMVs that are enriched in immunogenic cargo compared to their parent bacteria, the ability of OMVs produced by commensal organisms to be enriched with immunostimulatory contents is only recently becoming known. In this study, we investigated the cargo associated with OMVs produced by the intestinal commensal Bacteroides fragilis and determined their ability to activate host innate immune receptors. Analysis of B. fragilis OMVs revealed that they packaged various biological cargo including proteins, DNA, RNA, lipopolysaccharides (LPS) and peptidoglycan, and that this cargo could be enriched in OMVs compared to their parent bacteria. We visualized the entry of B. fragilis OMVs into intestinal epithelial cells, in addition to the ability of B. fragilis OMVs to transport bacterial RNA and peptidoglycan cargo into Caco-2 epithelial cells. Using HEK-Blue reporter cell lines, we identified that B. fragilis OMVs could activate host Toll-like receptors (TLR)-2, TLR4, TLR7 and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), whereas B. fragilis bacteria could only induce the activation of TLR2. Overall, our data demonstrates that B. fragilis OMVs activate a broader range of host innate immune receptors compared to their parent bacteria due to their enrichment of biological cargo and their ability to transport this cargo directly into host epithelial cells. These findings indicate that the secretion of OMVs by B. fragilis may facilitate immune crosstalk with host epithelial cells at the gastrointestinal surface and suggests that OMVs produced by commensal bacteria may preferentially activate host innate immune receptors at the mucosal gastrointestinal tract.

Despite recent advances in our understanding of how Helicobacter pylori causes disease, the factors that allow this pathogen to persist in the stomach have not yet been fully characterized. To identify new virulence factors in H. pylori, we generated low-infectivity variants of a mouse-colonizing H. pylori strain using the classical technique of in vitro attenuation. The resulting variants and their highly infectious progenitor bacteria were then analyzed by global gene expression profiling. The gene expression levels of five open reading frames (ORFs) were significantly reduced in low-infectivity variants, with the most significant changes observed for ORFs HP1583 and HP1582. These ORFs were annotated as encoding homologs of the Escherichia coli vitamin B(6) biosynthesis enzymes PdxA and PdxJ. Functional complementation studies with E. coli confirmed H. pylori PdxA and PdxJ to be bona fide homologs of vitamin B(6) biosynthesis enzymes. Importantly, H. pylori PdxA was required for optimal growth in vitro and was shown to be essential for chronic colonization in mice. In addition to having a well-known metabolic role, vitamin B(6) is necessary for the synthesis of glycosylated flagella and for flagellum-based motility in H. pylori. Thus, for the first time, we identify vitamin B(6) biosynthesis enzymes as novel virulence factors in bacteria. Interestingly, pdxA and pdxJ orthologs are present in a number of human pathogens, but not in mammalian cells. We therefore propose that PdxA/J enzymes may represent ideal candidates for therapeutic targets against bacterial pathogens.

ABSTRACT Helicobacter pylori causes chronic gastritis and avoids elimination by the immune system of the infected host. The commensal bacterium Lactobacillus acidophilus has been suggested to exert beneficial effects as a supplement during H. pylori eradication therapy. In the present study, we applied whole-genome microarray analysis to compare the immune responses induced in murine bone marrow-derived macrophages (BMDMs) stimulated with L. acidophilus , H. pylori , or both bacteria in combination. While L. acidophilus induced a Th1-polarizing response characterized by high expression of interferon beta (IFN-β) and interleukin 12 (IL-12), H. pylori strongly induced the innate cytokines IL-1β and IL-1α. In BMDMs prestimulated with L. acidophilus , H. pylori blocked the expression of L. acidophilus -induced IFN-β and IL-12 and suppressed the expression of key regulators of the Rho, Rac, and Cdc42 GTPases. The inhibition of L. acidophilus -induced IFN-β was independent of H. pylori viability and the virulence factor CagPAI; however, a vacuolating cytotoxin ( vacA ) mutant was unable to block IFN-β. Confocal microscopy demonstrated that the addition of H. pylori to L. acidophilus -stimulated BMDMs redirects intracellular processing, leading to an accumulation of L. acidophilus in the endosomal and lysosomal compartments. Thus, our findings indicate that H. pylori inhibits the development of a strong Th1-polarizing response in BMDMs stimulated with L. acidophilus by blocking the production of IFN-β in a VacA-dependent manner. We suggest that this abrogation is caused by a redirection of the endocytotic pathway in the processing of L. acidophilus . IMPORTANCE Approximately half of the world’s population is infected with Helicobacter pylori . The factors that allow this pathogen to persist in the stomach and cause chronic infections have not yet been fully elucidated. In particular, how H. pylori avoids killing by macrophages, one of the main types of immune cell underlying the epithelium, remains elusive. Here we have shown that the H. pylori virulence factor VacA plays a key role by blocking the activation of innate cytokines induced by the probiotic Lactobacillus acidophilus in macrophages and suppresses the expression of key regulators required for the organization and dynamics of the intracellular cytoskeleton. Our results identify potential targets for the treatment of H. pylori infection and vaccination, since specific inhibition of the toxin VacA possibly allows the activation of an efficient immune response and thereby eradication of H. pylori in the host.

The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as “lipid rafts.” Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248) with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01). HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05), and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05). Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.

The interleukin-1 family members, IL-1β and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1β processing in myeloid cells have been defined, those involved in IL-18 processing, particularly in non-myeloid cells, are still not well understood. Here we report that the host defence molecule NOD1 regulates IL-18 processing in mouse epithelial cells in response to the mucosal pathogen, Helicobacter pylori. Specifically, NOD1 in epithelial cells mediates IL-18 processing and maturation via interactions with caspase-1, instead of the canonical inflammasome pathway involving RIPK2, NF-κB, NLRP3 and ASC. NOD1 activation and IL-18 then help maintain epithelial homoeostasis to mediate protection against pre-neoplastic changes induced by gastric H. pylori infection in vivo. Our findings thus demonstrate a function for NOD1 in epithelial cell production of bioactive IL-18 and protection against H. pylori-induced pathology.

Gram-negative bacteria release outer membrane vesicles (OMVs) that contain cargo derived from their parent bacteria. Helicobacter pylori is a Gram-negative human pathogen that produces urease to increase the pH of the surrounding environment to facilitate colonization of the gastric mucosa. However, the effect of acidic growth conditions on the production and composition of H. pylori OMVs is unknown. In this study, we examined the production, composition, and proteome of H. pylori OMVs produced during acidic and neutral pH growth conditions. H. pylori growth in acidic conditions reduced the quantity and size of OMVs produced. Additionally, OMVs produced during acidic growth conditions had increased protein, DNA, and RNA cargo compared to OMVs produced during neutral conditions. Proteomic analysis comparing the proteomes of OMVs to their parent bacteria demonstrated significant differences in the enrichment of beta-lactamases and outer membrane proteins between bacteria and OMVs, supporting that differing growth conditions impacts OMV composition. We also identified differences in the enrichment of proteins between OMVs produced during different pH growth conditions. Overall, our findings reveal that growth of H. pylori at different pH levels is a factor that alters OMV proteomes, which may affect their subsequent functions.

Bacterial membrane vesicles (BMVs) released by Gram-negative and Gram-positive bacteria are a bona fide secretion system that enable the dissemination of bacterial effector molecules, and can trigger a range of responses in the host. The study of BMV production, composition, and functions can give insights into their roles in mediating bacterial survival, pathogenesis, and disease. Furthermore, BMVs can be harnessed to develop cutting-edge nano-therapeutics including targeted chemotherapy delivery, antimicrobials, and novel vaccines. Here we describe routine methods that can be used for small- or large-scale production, isolation, and purification of outer membrane vesicles produced by Gram-negative bacteria, and membrane vesicles produced by Gram-positive bacteria, which we collectively refer to as BMVs. We discuss methods that can be used to visualize BMVs by electron microscopy, and to quantify their DNA, RNA, and protein cargo. We outline a method for the fluorescent labeling of BMVs that can be applied to examine their ability to interact with and enter host cells using a range of in vitro and in vivo biological assays. Finally, we provide a cell culture-based method that can be used to examine a range of immunogenic properties of BMVs, including their cytotoxicity, ability to activate pathogen-recognition receptors (PRRs), induce autophagy and cytokine responses, and modulate cellular pathways.

Members of the mammalian Nod-like receptor (NLR) protein family are important intracellular sensors for bacteria. Bacteria have evolved under the pressure of detection by host immune sensing systems, leading to adaptive subversion strategies to dampen immune responses for their benefits. These include modification of microbe-associated molecular patterns (MAMPs), interception of innate immune pathways by secreted effector proteins and sophisticated instruction of anti-inflammatory adaptive immune responses. Here, we summarise our current understanding of subversion strategies used by bacterial pathogens to manipulate NLR-mediated responses, focusing on the well-studied members NOD1/2, and the inflammasome forming NLRs NLRC4, and NLRP3. We discuss how bacterial pathogens and their products activate these NLRs to promote inflammation and disease and the range of mechanisms used by bacterial pathogens to evade detection by NLRs and to block or dampen NLR activation to ultimately interfere with the generation of host immunity. Moreover, we discuss how bacteria utilise NLRs to facilitate immunotolerance and persistence in the host and outline how various mechanisms used to attenuate innate immune responses towards bacterial pathogens can also aid the host by reducing immunopathologies. Finally, we describe the therapeutic potential of harnessing immune subversion strategies used by bacteria to treat chronic inflammatory conditions.

Milk is a complex biological fluid that has high-quality proteins including growth factors and also contains extracellular vesicles (EVs). EVs are a lipid bilayer containing vesicles that contain proteins, metabolites and nucleic acids. Several studies have proposed that EVs in cow milk can survive the gut and can illicit cross-species communication in the consuming host organism. In this study, we isolated and characterized extracellular vesicles from the raw milk of the four species of the Bovidae family, namely cow, sheep, goat and buffalo, that contribute 99% of the total milk consumed globally. A comparative proteomic analysis of these vesicles was performed to pinpoint their potential functional role in health and disease. Vesicles sourced from buffalo and cow milk were particularly enriched with proteins implicated in modulating the immune system. Furthermore, functional studies were performed to determine the anti-cancer effects of these vesicles. The data obtained revealed that buffalo-milk-derived EVs induced significantly higher cell death in colon cancer cells. Overall, the results from this study highlight the potent immunoregulatory and anti-cancer nature of EVs derived from the milk of Bovidae family members.

The host has developed an array of systems that enables protection against infection and response to injury, ultimately resulting in the generation of a pro-inflammatory response. The most rapid immune response is mediated via the innate immune system, which is comprised of germ line encoded pathogen recognition receptors (PRRs). This PRR mediated system functions by specifically recognizing conserved structures of microbial molecules or products, known as microbial-associated molecular patterns (MAMPs), ultimately enabling transduction of signaling cascades, gene transcription and the development of a pro-inflammatory innate immune response. The intracellular PRRs nucleotide-binding oligomerization domain protein 1 (NOD1) and NOD2 will be the focus of this review. A brief overview of NOD1 and NOD2 and recent advances in the field regarding the intracellular location and mechanisms of NOD1 signaling will be discussed. These new findings have broadened our understanding of the mechanisms whereby NOD1 signaling results in the induction of the cellular degradation pathway of autophagy and the development of pro-inflammatory responses that activate the adaptive immune system.

EDITORIAL article Front. Immunol., 19 December 2018Sec. Immunological Tolerance and Regulation Volume 9 - 2018 | https://doi.org/10.3389/fimmu.2018.03024

Abstract The sexually transmitted pathogen Neisseria gonorrhoeae releases membrane vesicles including outer membrane vesicles (OMVs) during infections. OMVs traffic outer membrane molecules, such as the porin PorB and lipo‐oligosaccharide (LOS), into host innate immune cells, eliciting programmed cell death pathways, and inflammation. Little is known, however, about the proteome and LOS content of OMVs released by clinical strains isolated from different infection sites, and whether these vesicles similarly activate immune responses. Here, we characterized OMVs from four N. gonorrhoeae isolates and determined their size, abundance, proteome, LOS content, and activation of inflammatory responses in macrophages. The overall proteome of the OMVs was conserved between the four different isolates, which included major outer membrane and periplasm proteins. Despite this, we observed differences in the rate of OMV biogenesis and the relative abundance of membrane proteins and LOS. Consequently, OMVs from clinical isolates induced varying rates of macrophage cell death and the secretion of interleukin‐1 family members, such as IL‐1α and IL‐1β. Overall, these findings demonstrate that clinical isolates of N. gonorrhoeae utilize membrane vesicles to release proteins and lipids, which affects innate immune responses.

The release of extracellular vesicles (EVs) is a process conserved across the three domains of life. Amongst prokaryotes, EVs produced by Gram-negative bacteria, termed outer membrane vesicles (OMVs), were identified more than 50 years ago and a wealth of literature exists regarding their biogenesis, composition and functions. OMVs have been implicated in benefiting numerous metabolic functions of their parent bacterium. Additionally, OMVs produced by pathogenic bacteria have been reported to contribute to pathology within the disease setting. By contrast, the release of EVs from Gram-positive bacteria, known as membrane vesicles (MVs), has only been widely accepted within the last decade. As such, there is a significant disproportion in knowledge regarding MVs compared to OMVs. Here we provide an overview of the literature regarding bacterial membrane vesicles (BMVs) produced by pathogenic and commensal bacteria. We highlight the mechanisms of BMV biogenesis and their roles in assisting bacterial survival, in addition to discussing their functions in promoting disease pathologies and their potential use as novel therapeutic strategies.

Growth of Helicobacter pyloriin vitro depends on supplementation of the medium with blood or serum. However, these supplements often require frozen storage and can show batch-to-batch variation, resulting in differences in bacterial growth. In this study, we introduce the use of a commercially available, lipid-rich supplement called AlbuMAX II(®) (Gibco BRL, Grand Island, NY, USA) for use as a serum/blood replacement for H. pylori culture.The growth of H. pylori on solid and liquid media was examined by comparing growth after supplementation with horse blood, fetal calf serum, β-cyclodextrin or AlbuMAX II(®) (Gibco BRL). Human gastric adenocarcinoma (AGS) cellular responses to H. pylori were measured by NF-κB luciferase assays and IL-8 ELISA.We show that the growth of H. pylori on both solid and liquid media containing AlbuMAX II(®) (Gibco BRL) were comparable to levels obtained on blood agar or liquid media supplemented with serum. Growth was consistently higher in media supplemented with AlbuMAX II(®) (Gibco BRL) than media containing β-cyclodextrin. Furthermore, bacteria grown in AlbuMAX II(®) (Gibco BRL) induced proinflammatory responses in AGS cells.AlbuMAX II(®) (Gibco BRL) can be used as a serum/blood replacement for the cultivation of H. pylori in solid and liquid media. This medium could be useful for an improved understanding of H. pylori metabolism or for antigen production. Furthermore, AlbuMAX II(®) (Gibco BRL) may be suitable for use in remote locations, particularly in areas where frozen storage of serum may be a problem.

EDITORIAL article Front. Immunol., 04 January 2023Sec. Microbial Immunology Volume 13 - 2022 | https://doi.org/10.3389/fimmu.2022.1112427

Cholera toxin (CT) is a mucosal adjuvant capable of inducing strong immune responses to co-administered antigens following oral or intranasal immunization of mice. To date, the direct effect of CT on antigen-specific CD4(+) T cell migration and proliferation profiles in vivo is not well characterized. In this study, the effect of CT on the migration pattern and proliferative responses of adoptively transferred, CD4(+) TCR transgenic T cells in orally or intranasally vaccinated mice, was analyzed by flow cytometry. GFP-expressing or CFSE-labeled OT-II lymphocytes were adoptively transferred to naïve C57BL/6 mice, and mice were subsequently vaccinated with OVA with or without CT via the oral or intranasal route. CT did not alter the migration pattern of antigen-specific T cells, regardless of the route of immunization, but increased the number of transgenic CD4(+) T cells in draining lymphoid tissue. This increase in the number of transgenic CD4(+) T cells was not due to cells undergoing more rounds of cellular division in vivo, suggesting that CT may exert an indirect adjuvant effect on CD4(+) T cells. The findings reported here suggest that CT functions as a mucosal adjuvant by increasing the number of antigen specific CD4(+) T cells independent of their migration pattern or kinetics of cellular division.

Nucleotide oligomerization domain 1 (NOD1) is an intracellular host receptor that senses microbial pathogens by detecting a conserved structure of Gram negative bacterial peptidoglycan (PG). Detection of peptidoglycan by NOD1 ultimately results in a pro-inflammatory cytokine response. However, to date, the intracellular location and the mechanisms whereby NOD1 detects peptidoglycan resulting in the development of pro-inflammatory cytokine responses and autophagy are unknown. We used peptidoglycan-containing bacterial outer membrane vesicles (PG-OMVs) as a tool to elucidate the intracellular location of NOD1 and the mechanisms of NOD1-dependent responses that result in cytokine production and autophagy. Upon entry into host epithelial cells, PG-OMVs from mucosal pathogens induced NOD1-dependent autophagy and IL-8 responses. Fluorescent labelling of peptidoglycan contained within bacterial OMVs revealed that upon entry into host cells, peptidoglycan migrated to early endosomes where it interacted with NOD1 and the NOD1-adaptor protein RIP-2, facilitating the development of an inflammatory response from this location. We showed that migration of PG-OMVs to early endosomes occurred in a NOD1 dependent manner, identifying a previously unknown role for NOD1 in the intracellular migration of peptidoglycan. Most importantly, using fluorescent lifetime imaging microscopy (FLIM)-fluorescence energy transfer (FRET), we were able to show for the first time the direct interaction between bacterial peptidoglycan and NOD1 within host cells. Finally, we found that the NOD1 adaptor protein RIP-2 is essential for the development of NOD1-dependent autophagy and IL-8 production in response to PG-OMVs [1]. This study reveals for the first time the intracellular location and the early recognition events required for the detection of Gram negative bacterial pathogens by NOD1. Moreover, this study is the first to visualise a direct interaction between bacterial peptidoglycan and NOD1. These findings will significantly expand our limited knowledge of the contribution of NOD1 in Gram negative bacterial pathogenesis, innate immunity and inflammatory disorders.

Cancer cachexia is multifactorial and should be targeted using a multimodal form of intervention. The purpose of the present trial was to test the effects of a combined nutrition and physical exercise program on cancer patients with metastatic or locally advanced tumors of the gastrointestinal and lung tracts.Patients were randomized into two groups: One group received a minimum of three standardized individual nutritional counselling sessions and participated in a 60-min exercise program twice a week. The second group received their usual care. The intervention spanned a period of three months. Quality of life (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire version 3.0), physical performance (hand-grip strength, 6-min walk test, timed sit-to-stand test and 1 repetition maximum leg press), nutritional status (body weight, bioelectrical impedance analysis), dietary intake (three-day dietary record) and clinical data (unexpected hospital days, performance status) were tested at baseline and after three and six months.In total, 18 women and 40 men (mean age 63, range 32–81) with metastatic or locally advanced tumors of the gastrointestinal (n = 38) and lung (n = 20) tracts were included. Median adherence to the supervised exercise program was 75%. The median number of individual nutritional counselling sessions was 3.0 (range 0–7 sessions). Post intervention, no difference in global health status/quality of life (overall QoL) was observed. Intervention was superior to UC for the patient-rated symptom scale regarding nausea and vomiting (p = 0.023) and protein intake (p = 0.01). No statistical differences were observed for energy intake, nutritional status and physical performance.The results show good adherence to a combined nutrition and exercise program. The multimodal intervention did not improve overall QoL, but contributed to an adequate protein intake and to the general well-being of the patient by reducing nausea and vomiting.

Outer Membrane Vesicles (OMVs) are bi-layered spherical nanostructures shed by all Gram negative bacteria as part of their normal growth process, and throughout the course of infection, both in vitro and in vivo. OMVs deliver peptidoglycan (PG) from Gram negative pathogens into non-phagocytic epithelial cells. Once internalised, the Pattern Recognition Receptor Nucleotide Oligomerization Domain 1 (NOD1) which detects bacterial PG is essential for the development of OMV-dependent immune-responses in vivo. Here, we characterize the mechanisms whereby PG-containing OMVs initiate innate immune responses, via NOD1-dependent autophagy, as evidenced by the formation of autophagic puncta. Additionally we show this NOD1-response is cell type specific, not occurring in macrophages but occurring in various fibroblast and epithelial lines and primary human epithelial cells. In addition, knockdown of NOD1 or NOD1-/- MEFs are deficient in OMV-induced autophagy and loss of critical components in autophagy results in reduced inflammatory responses to OMVs. We show that NOD1 is triggered by direct association to PG, and this recruits the adaptor RIP2. Inhibition of the RIP2 kinase results in a loss of both autophagy and IL-8 production in response to bacterial OMVs. This work highlights an essential role for NOD1 in sensing bacterial PG, delivered intracellularly through OMVs and activating autophagy, which proves critical for the OMV-induced inflammatory response.

Detection of microbes by the host is essential to restrict microbial colonization, to clear pathogens, and to mount adapted defense reactions, and thus is the key function of the innate immune systems of plants and mammals. Here we provide an introduction into pathogen recognition by the innate immune system of both plants and animals. We will particularly focus on the concept of effector-triggered immunity, and similarities and differences in its function between plants and animals.

Abstract The interleukin-1 family members, IL-1β and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1β processing in myeloid cells have been extensively studied, those involved in IL-18 processing, particularly in non-myeloid cells, are still poorly understood. Here, we have identified the cytosolic sensor NOD1 as a key regulator of IL-18 processing in epithelial cells responding to Helicobacter pylori infection. Importantly, NOD1 processing of IL-18 occurs independently of the canonical inflammasome proteins, NLRP3 and ASC. Instead, NOD1 interacts directly with caspase-1 via homotypic binding of caspase-activation recruitment domains. We show that IL-18 is important in maintaining tissue homeostasis and protecting against pre-neoplastic changes due to gastric H. pylori infection. These findings reveal an unanticipated role for NOD1 in a new type of inflammasome that regulates epithelial cell production of bioactive IL-18 with tissue protective functions.

Abstract The interleukin-1 family members, IL-1β and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1β processing in myeloid cells have been well defined, those involved in IL-18 processing, particularly in non-myeloid cells, are still not well understood. Here, we report that the host defence molecule NOD1 regulates IL-18 processing in epithelial cells to the mucosal pathogens, Helicobacter pylori and Pseudomonas aeruginosa . We show that IL-18 is important in protecting against pre-neoplastic changes induced by gastric H. pylori infection in vivo . NOD1 mediates IL-18 processing via homotypic CARD-CARD interactions with caspase-1, and independently of canonical inflammasome proteins (NLRP3, ASC). These findings reveal an unanticipated role for NOD1 in the formation of bioactive IL-18, thereby underlining the differences in inflammasome functions between haematopoietic and non-haematopoietic cells.

High dietary fibre is fermented by the gut microbiota, resulting in the release of metabolites called short-chain fatty acids (SCFAs). Both fibre and SCFAs can reduce high blood pressure (BP) and its associated cardio-renal complications. However, the underlying mechanisms remain unclear. SCFAs can be detected by metabolite-sensing receptors GPR41 and GPR43, highly expressed by immune cells such as macrophages. We hypothesised that dietary fibre attenuates hypertension by modulating renal macrophage infiltration via metabolite-sensing receptors GPR41 and GPR43. To test this, we developed a novel GPR41/GPR43 double knockout (DKO) mice and characterised the cardiovascular and immune phenotype in both sham and angiotensin-II (Ang-II, 0.5mg/kg/day) treated DKO and wild-type (WT) mice (n=7-12 per group). WT Ang-II mice fed a high-fibre diet had significantly lower renal galectin-3 (p=0.0004), a macrophage marker, compared to WT Ang-II mice fed a low-fibre diet. Sham DKO mice on standard chow diet had no difference in BP or heart function but had higher kidney/tibia length index (p=0.049) and renal fibrosis levels compared to WT mice (p=0.004). Moreover, untreated DKO mice had higher numbers of renal macrophages compared to WT mice (p=0.002). Ang-II infusion of DKO mice resulted in higher BP (p&lt;0.0001), renal fibrosis (p=0.007), and mortality (hazard ratio=5.6) compared to WT mice. In the gut, we found significant inflammatory changes, gut barrier integrity disruption, gut microbiota and metabolome changes (all p&lt;0.05). We also found evidence of lipopolysaccharides (LPS) translocation from the gut into the circulation of DKO mice. In conclusion, we show that a high-fibre diet attenuates hypertension by modulating renal macrophages via the gut microbiota-derived metabolite-sensing GPR41 and GPR43 receptors. These receptors can be targeted as a novel treatment for hypertension.