An Insight into Functional Metagenomics: A High-Throughput Approach to Decipher Food-Microbiota-Host Interactions in the Human Gut.

Our understanding of the symbiotic relationship between the microbiota and its host has constantly evolved since our understanding that the "self" was not only defined by our genetic patrimony but also by the genomes of bugs living in us. The first culture-based methods highlighted the important functions of the microbiota. However, these methods had strong limitations and did not allow for a full understanding of the complex relationships that occur at the interface between the microbiota and the host. The recent development of metagenomic approaches has been a groundbreaking step towards this understanding. Its use has provided new insights and perspectives. In the present chapter, we will describe the advances of functional metagenomics to decipher food-microbiota and host-microbiota interactions. This powerful high-throughput approach allows for the assessment of the microbiota as a whole (including non-cultured bacteria) and enabled the discovery of new signaling pathways and functions involved in the crosstalk between food, the gut microbiota and its host. We will present the pipeline and highlight the most important studies that helped to develop the field. To conclude, we will emphasize the most recent developments and hot topics in functional metagenomics.

Adlercreutzia equolifaciens Is an Anti-Inflammatory Commensal Bacterium with Decreased Abundance in Gut Microbiota of Patients with Metabolic Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) affects about 20-40% of the adult population in high-income countries and is now a leading indication for liver transplantation and can lead to hepatocellular carcinoma. The link between gut microbiota dysbiosis and NAFLD is now clearly established. Through analyses of the gut microbiota with shotgun metagenomics, we observe that compared to healthy controls, Adlercreutzia equolifaciens is depleted in patients with liver diseases such as NAFLD. Its abundance also decreases as the disease progresses and eventually disappears in the last stages indicating a strong association with disease severity. Moreover, we show that A. equolifaciens possesses anti-inflammatory properties, both in vitro and in vivo in a humanized mouse model of NAFLD. Therefore, our results demonstrate a link between NAFLD and the severity of liver disease and the presence of A. equolifaciens and its anti-inflammatory actions. Counterbalancing dysbiosis with this bacterium may be a promising live biotherapeutic strategy for liver diseases.

Exposure to anti-seizure medications impact growth of gut bacterial species and subsequent host response.

Anti-seizure medications (ASMs) are the first line of treatment for seizure control in children with epilepsy. Cumulative evidence suggests an imbalanced gut microbiota in refractory epilepsy patients. We systematically investigated the differential antimicrobial impacts of nine ASM active ingredients, seven common excipients of ASMs, and four syrup formulations on core early-life gut microbiota strains. Additionally, we evaluated the toxicity and gene expression profiles of HT-29 colon epithelial cells when exposed to active ingredients with or without bacterial supernatants. The physicochemical structure of ASM active ingredients and bacterial phylogeny were found to be related to ASM toxicity. Carbamazepine, lamotrigine, and topiramate reduced the growth of more than ten strains along with syrup excipient propyl-paraben. Various artificial sweeteners present in ASM formulations stimulated the growth of gut bacterial strains. The active ingredients that were more toxic to bacterial strains also exhibited toxicity towards HT-29 cells, yet Bifidobacterium longum supernatant reduced cytotoxic effects of carbamazepine and lamotrigine. Akkermansia muciniphila or mixed community supernatants reduced the expression of drug resistance genes in HT-29 cell lines. In summary, our results indicate that several ASM active ingredients and their excipients regulate the growth of gut bacterial strains in a species-specific manner. Interactions between ASMs and gut epithelial cells might be modulated by gut microbial metabolites.

Fructose malabsorption induces cholecystokinin expression in the ileum and cecum by changing microbiota composition and metabolism.

Current fructose consumption levels often overwhelm the intestinal capacity to absorb fructose. We investigated the impact of fructose malabsorption on intestinal endocrine function and addressed the role of the microbiota in this process. To answer this question, a mouse model of moderate fructose malabsorption [ketohexokinase mutant (KHK)-/-] and wild-type (WT) littermate mice were used and received a 20%-fructose (KHK-F and WT-F) or 20%-glucose diet. Cholecystokinin (Cck) mRNA and protein expression in the ileum and cecum, as well as preproglucagon (Gcg) and neurotensin (Nts) mRNA expression in the cecum, increased in KHK-F mice. In KHK-F mice, triple-label immunohistochemistry showed major up-regulation of CCK in enteroendocrine cells (EECs) that were glucagon-like peptide-1 (GLP-1)+/Peptide YY (PYY-) in the ileum and colon and GLP-1-/PYY- in the cecum. The cecal microbiota composition was drastically modified in the KHK-F in association with an increase in glucose, propionate, succinate, and lactate concentrations. Antibiotic treatment abolished fructose malabsorption-dependent induction of cecal Cck mRNA expression and, in mouse GLUTag and human NCI-H716 cells, Cck mRNA expression levels increased in response to propionate, both suggesting a microbiota-dependent process. Fructose reaching the lower intestine can modify the composition and metabolism of the microbiota, thereby stimulating the production of CCK from the EECs possibly in response to propionate.-Zhang, X., Grosfeld, A., Williams, E., Vasiliauskas, D., Barretto, S., Smith, L., Mariadassou, M., Philippe, C., Devime, F., Melchior, C., Gourcerol, G., Dourmap, N., Lapaque, N., Larraufie, P., Blottière, H. M., Herberden, C., Gerard, P., Rehfeld, J. F., Ferraris, R. P., Fritton, J. C., Ellero-Simatos, S., Douard, V. Fructose malabsorption induces cholecystokinin expression in the ileum and cecum by changing microbiota composition and metabolism.

TLR ligands and butyrate increase Pyy expression through two distinct but inter-regulated pathways.

The intestinal epithelium is an active barrier separating the host from its microbiota. It senses microbial compounds through expression of a wide range of receptors including the Toll-like receptors (TLRs). TLRs have been shown to regulate epithelium permeability or secretion of defensin by Paneth cells. However, the expression and function of TLRs in enteroendocrine L-cells, a specific subtype of intestinal cells secreting PYY and GLP-1, have not yet been assessed. PYY and GLP-1 are implicated in regulation of gut motility, food intake and insulin secretion, and are of great interest regarding obesity and type 2 diabetes. Using a cellular model of human L-cells and a reporter system for NF-κB activation pathway, we reported functional expression of TLRs in these cells. Stimulation with specific TLR-agonists increased expression of Pyy but not Proglucagon in an NF-κB-dependent manner. Moreover, the effect of TLR stimulation was additive to butyrate, a product of bacterial fermentation, on Pyy expression. Additionally, butyrate also increased Tlr expression, including Tlr4, and the NF-κB response to TLR stimulation. Altogether, our results demonstrated a role of TLRs in the modulation of Pyy expression and the importance of butyrate, a product of bacterial fermentation in regulation of microbial TLR-dependent sensing.

Three glycosylated serine-rich repeat proteins play a pivotal role in adhesion and colonization of the pioneer commensal bacterium, Streptococcus salivarius.

Bacterial adhesion is a critical step for colonization of the host. The pioneer colonizer and commensal bacterium of the human gastrointestinal tract, Streptococcus salivarius, has strong adhesive properties but the molecular determinants of this adhesion remain uncharacterized. Serine-rich repeat (SRR) glycoproteins are a family of adhesins that fulfil an important role in adhesion. In general, Gram-positive bacterial genomes have a unique SRR glycoprotein-encoding gene. We demonstrate that S. salivarius expresses three large and glycosylated surface-exposed proteins - SrpA, SrpB and SrpC - that show characteristics of SRR glycoproteins and are secreted through the accessory SecA2/Y2 system. Two glycosyltransferases - GtfE/F - encoded outside of the secA2/Y2 locus, unusually, perform the first step of the sequential glycosylation process, which is crucial for SRR activity. We show that SrpB and SrpC play complementary adhesive roles involved in several steps of the colonization process: auto-aggregation, biofilm formation and adhesion to a variety of host epithelial cells and components. We also show that at least one of the S. salivarius SRR glycoproteins is important for colonization in mice. SrpA, SrpB and SrpC are the main factors underlying the multifaceted adhesion of S. salivarius and, therefore, play a major role in host colonization.

Enterococcus faecalis prophage dynamics and contributions to pathogenic traits.

Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.

Salmonella polarises peptide-MHC-II presentation towards an unconventional Type B CD4+ T-cell response.

Distinct peptide-MHC-II complexes, recognised by Type A and B CD4(+) T-cell subsets, are generated when antigen is loaded in different intracellular compartments. Conventional Type A T cells recognize their peptide epitope regardless of the route of processing, whereas unconventional Type B T cells only recognise exogenously supplied peptide. Type B T cells are implicated in autoimmune conditions and may break tolerance by escaping negative selection. Here we show that Salmonella differentially influences presentation of antigen to Type A and B T cells. Infection of bone marrow-derived dendritic cells (BMDCs) with Salmonella enterica serovar Typhimurium (S. Typhimurium) reduced presentation of antigen to Type A T cells but enhanced presentation of exogenous peptide to Type B T cells. Exposure to S. Typhimurium was sufficient to enhance Type B T-cell activation. Salmonella Typhimurium infection reduced surface expression of MHC-II, by an invariant chain-independent trafficking mechanism, resulting in accumulation of MHC-II in multi-vesicular bodies. Reduced MHC-II surface expression in S. Typhimurium-infected BMDCs correlated with reduced antigen presentation to Type A T cells. Salmonella infection is implicated in reactive arthritis. Therefore, polarisation of antigen presentation towards a Type B response by Salmonella may be a predisposing factor in autoimmune conditions such as reactive arthritis.

The NF-κB binding site located in the proximal region of the TSLP promoter is critical for TSLP modulation in human intestinal epithelial cells.

Thymic stromal lymphopoietin (TSLP) is constitutively secreted by intestinal epithelial cells. It regulates gut DCs, therefore, contributing to the maintenance of immune tolerance. In the present report, we describe the regulation of TSLP expression in intestinal epithelial cells and characterize the role of several NF-κB binding sites present on the TSLP promoter. TSLP expression can be stimulated by different compounds through activation of p38, protein kinase A, and finally the NF-κB pathway. We describe a new NF-κB binding element located at position -0.37 kb of the promoter that is crucial for the NF-κB-dependent regulation of TSLP. We showed that mutation of this proximal NF-κB site abrogates the IL-1ß-mediated transcriptional activation of human TSLP in several epithelial cell lines. We also demonstrated that both p65 and p50 subunits are able to bind this new NF-κB binding site. The present work provides new insight into epithelial cell-specific TSLP regulation.

Fusobacterium nucleatum promotes inflammatory and anti-apoptotic responses in colorectal cancer cells via ADP-heptose release and ALPK1/TIFA axis activation.

The anaerobic bacterium Fusobacterium nucleatum is significantly associated with human colorectal cancer (CRC) and is considered a significant contributor to the disease. The mechanisms underlying the promotion of intestinal tumor formation by F. nucleatum have only been partially uncovered. Here, we showed that F. nucleatum releases a metabolite into the microenvironment that strongly activates NF-κB in intestinal epithelial cells via the ALPK1/TIFA/TRAF6 pathway. Furthermore, we showed that the released molecule had the biological characteristics of ADP-heptose. We observed that F. nucleatum induction of this pathway increased the expression of the inflammatory cytokine IL-8 and two anti-apoptotic genes known to be implicated in CRC, BIRC3 and TNFAIP3. Finally, it promoted the survival of CRC cells and reduced 5-fluorouracil chemosensitivity in vitro. Taken together, our results emphasize the importance of the ALPK1/TIFA pathway in Fusobacterium induced-CRC pathogenesis, and identify the role of ADP-H in this process.

ANGPTL4 expression induced by butyrate and rosiglitazone in human intestinal epithelial cells utilizes independent pathways.

Short-chain fatty acids (SCFAs), such as butyrate and propionate, are metabolic products of carbohydrate fermentation by the microbiota and constitute the main source of energy for host colonocytes. SCFAs are also important for gastrointestinal health, immunity, and host metabolism. Intestinally produced angiopoietin-like protein 4 (ANGPTL4) is a secreted protein with metabolism-altering properties and may offer a route by which microbiota can regulate host metabolism. Peroxisome proliferator-activated receptor (PPAR)-γ has previously been shown to be involved in microbiota-induced expression of intestinal ANGPTL4, but the role of bacterial metabolites in this process has remained elusive. Here, we show that the SCFA butyrate regulates intestinal ANGPTL4 expression in a PPAR-γ-independent manner. Although PPAR-γ is not required for butyrate-driven intestinal ANGPTL4 expression, costimulating with PPAR-γ ligands and SCFAs leads to additive increases in ANGPTL4 levels. We suggest that PPAR-γ and butyrate rely on two separate regulatory sites, a PPAR-responsive element downstream the transcription start site and a butyrate-responsive element(s) within the promoter region, 0.5 kb upstream of the transcription start site. Furthermore, butyrate gavage and colonization with Clostridium tyrobutyricum, a SCFA producer, can independently induce expression of intestinal ANGPTL4 in germ-free mice. Thus, oral administration of SCFA or use of SCFA-producing bacteria may be additional routes to maintain intestinal ANGPTL4 levels for preventive nutrition or therapeutic purposes.

HLA class I allelic sequence and conformation regulate leukocyte Ig-like receptor binding.

Leukocyte Ig-like receptors (LILRs) are a family of innate immune receptors predominantly expressed by myeloid cells that can alter the Ag presentation properties of macrophages and dendritic cells. Several LILRs bind HLA class I. Altered LILR recognition due to HLA allelic variation could be a contributing factor in disease. We comprehensively assessed LILR binding to >90 HLA class I alleles. The inhibitory receptors LILRB1 and LILRB2 varied in their level of binding to different HLA alleles, correlating in some cases with specific amino acid motifs. LILRB2 displayed the weakest binding to HLA-B*2705, an allele genetically associated with several autoimmune conditions and delayed progression of HIV infection. We also assessed the effect of HLA class I conformation on LILR binding. LILRB1 exclusively bound folded ß(2)-microglobulin-associated class I, whereas LILRB2 bound both folded and free H chain forms. In contrast, the activating receptor LILRA1 and the soluble LILRA3 protein displayed a preference for binding to HLA-C free H chain. To our knowledge, this is the first study to identify the ligand of LILRA3. These findings support the hypothesis that LILR-mediated detection of unfolded versus folded MHC modulates immune responses during infection or inflammation.

Salmonella regulates polyubiquitination and surface expression of MHC class II antigens.

Salmonella typhimurium is a facultative pathogen capable of entering and replicating in both professional and non-professional antigen presenting cells. Control of infection requires MHC class II restricted CD4 T-helper cell responses. Here we show that Salmonella infection induced polyubiquitination of HLA-DR, a post-translational modification that led to removal of mature, peptide loaded, alphabeta dimers from the cell surface. Immature alphabetaIi complexes were unaffected. Surface expression of all class II isotypes, HLA-DP, -DQ, and -DR, was reduced in infected cells, but other cell-surface molecules that traffic through class II peptide loading compartments were unaffected. A Salmonella strain carrying a mutation in ssaV did not induce ubiquitination of class II, implicating Salmonella T3SS-2 effector proteins in the process. T3SS-2 effectors, with established or proposed roles in ubiquitination, were not required for class II down-regulation, suggesting that an additional T3SS-2 effector is involved in regulating MHC class II ubiquitination. Although recognized as a viral immune evasion strategy, here, we demonstrate that bacteria can control surface MHC expression through ubiquitination.

Akkermansia muciniphila upregulates genes involved in maintaining the intestinal barrier function via ADP-heptose-dependent activation of the ALPK1/TIFA pathway.

The commensal bacteria that make up the gut microbiota impact the health of their host on multiple levels. In particular, the interactions taking place between the microbe-associated molecule patterns (MAMPs) and pattern recognition receptors (PRRs), expressed by intestinal epithelial cells (IECs), are crucial for maintaining intestinal homeostasis. While numerous studies showed that TLRs and NLRs are involved in the control of gut homeostasis by commensal bacteria, the role of additional innate immune receptors remains unclear. Here, we seek for novel MAMP-PRR interactions involved in the beneficial effect of the commensal bacterium Akkermansia muciniphila on intestinal homeostasis. We show that A. muciniphila strongly activates NF-κB in IECs by releasing one or more potent activating metabolites into the microenvironment. By using drugs, chemical and gene-editing tools, we found that the released metabolite(s) enter(s) epithelial cells and activate(s) NF-κB via an ALPK1, TIFA and TRAF6-dependent pathway. Furthermore, we show that the released molecule has the biological characteristics of the ALPK1 ligand ADP-heptose. Finally, we show that A. muciniphila induces the expression of the MUC2, BIRC3 and TNFAIP3 genes involved in the maintenance of the intestinal barrier function and that this process is dependent on TIFA. Altogether, our data strongly suggest that the commensal A. muciniphila promotes intestinal homeostasis by activating the ALPK1/TIFA/TRAF6 axis, an innate immune pathway exclusively described so far in the context of Gram-negative bacterial infections.

Interactions between human NK cells and macrophages in response to Salmonella infection.

NK cells play a key role in host resistance to a range of pathogenic microorganisms, particularly during the initial stages of infection. NK cell interactions with cells infected with viruses and parasites have been studied extensively, but human bacterial infections have not been given the same attention. We studied crosstalk between human NK cells and macrophages infected with intracellular Salmonella. These macrophages activated NK cells, resulting in secretion of IFN-gamma and degranulation. Reciprocally, NK cell activation led to a dramatic reduction in numbers of intramacrophagic live bacteria. We identified three elements in the interaction of NK cells with infected macrophages. First, communication between NK cells and infected macrophages was contact-dependent. The second requirement was IL-2- and/or IL-15-dependent priming of NK cells to produce IFN-gamma. The third was activation of NK cells by IL-12 and IL-18, which were secreted by the Salmonella-infected macrophages. Adhesion molecules and IL-12Rbeta2 were enriched in the contact zone between NK cells and macrophages, consistent with contact- and IL-12/IL-18-dependent NK activation. Our results suggest that, in humans, bacterial clearance is consistent with a model invoking a "ménage à trois" involving NK cells, IL-2/IL-15-secreting cells, and infected macrophages.

SCFA: mechanisms and functional importance in the gut.

In recent years, the importance of the gut microbiota in human health has been revealed and many publications have highlighted its role as a key component of human physiology. Owing to the use of modern sequencing approaches, the characterisation of the microbiome in healthy individuals and in disease has demonstrated a disturbance of the microbiota, or dysbiosis, associated with pathological conditions. The microbiota establishes a symbiotic crosstalk with their host: commensal microbes benefit from the nutrient-rich environment provided by the gut and the microbiota produces hundreds of proteins and metabolites that modulate key functions of the host, including nutrient processing, maintenance of energy homoeostasis and immune system development. Many bacteria-derived metabolites originate from dietary sources. Among them, an important role has been attributed to the metabolites derived from the bacterial fermentation of dietary fibres, namely SCFA linking host nutrition to intestinal homoeostasis maintenance. SCFA are important fuels for intestinal epithelial cells (IEC) and regulate IEC functions through different mechanisms to modulate their proliferation, differentiation as well as functions of subpopulations such as enteroendocrine cells, to impact gut motility and to strengthen the gut barrier functions as well as host metabolism. Recent findings show that SCFA, and in particular butyrate, also have important intestinal and immuno-modulatory functions. In this review, we discuss the mechanisms and the impact of SCFA on gut functions and host immunity and consequently on human health.

Glucose but Not Fructose Alters the Intestinal Paracellular Permeability in Association With Gut Inflammation and Dysbiosis in Mice.

A causal correlation between the metabolic disorders associated with sugar intake and disruption of the gastrointestinal (GI) homeostasis has been suggested, but the underlying mechanisms remain unclear. To unravel these mechanisms, we investigated the effect of physiological amounts of fructose and glucose on barrier functions and inflammatory status in various regions of the GI tract and on the cecal microbiota composition. C57BL/6 mice were fed chow diet and given 15% glucose or 15% fructose in drinking water for 9 weeks. We monitored caloric intake, body weight, glucose intolerance, and adiposity. The intestinal paracellular permeability, cytokine, and tight junction protein expression were assessed in the jejunum, cecum, and colon. In the cecum, the microbiota composition was determined. Glucose-fed mice developed a marked increase in total adiposity, glucose intolerance, and paracellular permeability in the jejunum and cecum while fructose absorption did not affect any of these parameters. Fructose-fed mice displayed increased circulation levels of IL6. In the cecum, both glucose and fructose intake were associated with an increase in Il13, Ifnγ, and Tnfα mRNA and MLCK protein levels. To clarify the relationships between monosaccharides and barrier function, we measured the permeability of Caco-2 cell monolayers in response to IFNγ+TNFα in the presence of glucose or fructose. In vitro, IFNγ+TNFα-induced intestinal permeability increase was less pronounced in response to fructose than glucose. Mice treated with glucose showed an enrichment of Lachnospiracae and Desulfovibrionaceae while the fructose increased relative abundance of Lactobacillaceae. Correlations between pro-inflammatory cytokine gene expression and bacterial abundance highlighted the potential role of members of Desulfovibrio and Lachnospiraceae NK4A136 group genera in the inflammation observed in response to glucose intake. The increase in intestinal inflammation and circulating levels of IL6 in response to fructose was observed in the absence of intestinal permeability modification, suggesting that the intestinal permeability alteration does not precede the onset of metabolic outcome (low-grade inflammation, hyperglycemia) associated with chronic fructose consumption. The data also highlight the deleterious effects of glucose on gut barrier function along the GI tract and suggest that Desulfovibrionaceae and Lachnospiraceae play a key role in the onset of GI inflammation in response to glucose.

Brucella control of dendritic cell maturation is dependent on the TIR-containing protein Btp1.

Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.

Extracellular Vesicles Produced by the Probiotic Propionibacterium freudenreichii CIRM-BIA 129 Mitigate Inflammation by Modulating the NF-κB Pathway.

Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, whose production is considered to be a widespread phenomenon in living organisms. Bacterial EVs are associated with several processes that include survival, competition, pathogenesis, and immunomodulation. Among probiotic Gram-positive bacteria, some Propionibacterium freudenreichii strains exhibit anti-inflammatory activity, notably via surface proteins such as the surface-layer protein B (SlpB). We have hypothesized that, in addition to surface exposure and secretion of proteins, P. freudenreichii may produce EVs and thus export immunomodulatory proteins to interact with the host. In order to demonstrate their production in this species, EVs were purified from cell-free culture supernatants of the probiotic strain P. freudenreichii CIRM-BIA 129, and their physicochemical characterization, using transmission electron microscopy and nanoparticle tracking analysis (NTA), revealed shapes and sizes typical of EVs. Proteomic characterization showed that EVs contain a broad range of proteins, including immunomodulatory proteins such as SlpB. In silico protein-protein interaction predictions indicated that EV proteins could interact with host proteins, including the immunomodulatory transcription factor NF-κB. This potential interaction has a functional significance because EVs modulate inflammatory responses, as shown by IL-8 release and NF-κB activity, in HT-29 human intestinal epithelial cells. Indeed, EVs displayed an anti-inflammatory effect by modulating the NF-κB pathway; this was dependent on their concentration and on the proinflammatory inducer (LPS-specific). Moreover, while this anti-inflammatory effect partly depended on SlpB, it was not abolished by EV surface proteolysis, suggesting possible intracellular sites of action for EVs. This is the first report on identification of P. freudenreichii-derived EVs, alongside their physicochemical, biochemical and functional characterization. This study has enhanced our understanding of the mechanisms associated with the probiotic activity of P. freudenreichii and identified opportunities to employ bacterial-derived EVs for the development of bioactive products with therapeutic effects.

The inhibitory receptor LILRB4 (ILT3) modulates antigen presenting cell phenotype and, along with LILRB2 (ILT4), is upregulated in response to Salmonella infection.

BACKGROUND: Leukocyte Ig-like receptors (LILR) are a family of innate immune receptors with immunomodulatory functions. High-level expression of the receptors LILRB2 (ILT4) and LILRB4 (ILT3) is a feature of tolerogenic antigen presenting cells and has been observed in cancer and transplant situations. There are relatively few studies regarding these receptors in the context of infection and it is not yet clear how LILRB4 exerts its inhibitory effects. RESULTS: We studied the effects of LILRB4 ligation on antigen presenting cell phenotype, and the expression of LILRB2 and LILRB4 on Salmonella-infected antigen presenting cells. Ligation of LILRB4 throughout in vitro culture of dendritic cells led to an upregulation of the co-stimulatory protein CD86. Alterations in the production of IL-8 and IL-10 by LILRB4-ligated macrophages were also observed. Infection with Salmonella typhimurium or TLR stimulation with Salmonella components led to an upregulation of LILRB2 and LILRB4. CONCLUSION: Our results indicate that the inhibitory effects of LILRB4 do not result from a failure to upregulate co-stimulatory proteins. In addition to the high level expression that can render antigen presenting cells tolerogenic, there may be a role for lower level expression and activity of LILRB2 and LILRB4 in response to TLR signalling during an immune response to bacterial infection.