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.

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.

Identification of a muropeptide precursor transporter from gut microbiota and its role in preventing intestinal inflammation.

The gut microbiota is a considerable source of biologically active compounds that can promote intestinal homeostasis and improve immune responses. Here, we used large expression libraries of cloned metagenomic DNA to identify compounds able to sustain an anti-inflammatory reaction on host cells. Starting with a screen for NF-κB activation, we have identified overlapping clones harbouring a heterodimeric ATP-binding cassette (ABC)-transporter from a Firmicutes. Extensive purification of the clone's supernatant demonstrates that the ABC-transporter allows for the efficient extracellular accumulation of three muropeptide precursor, with anti-inflammatory properties. They induce IL-10 secretion from human monocyte-derived dendritic cells and proved effective in reducing AIEC LF82 epithelial damage and IL-8 secretion in human intestinal resections. In addition, treatment with supernatants containing the muropeptide precursor reduces body weight loss and improves histological parameters in Dextran Sulfate Sodium (DSS)-treated mice. Until now, the source of peptidoglycan fragments was shown to come from the natural turnover of the peptidoglycan layer by endogenous peptidoglycan hydrolases. This is a report showing an ABC-transporter as a natural source of secreted muropeptide precursor and as an indirect player in epithelial barrier strengthening. The mechanism described here might represent an important component of the host immune homeostasis.

α-Synuclein expression in response to bacterial ligands and metabolites in gut enteroendocrine cells: an in vitro proof of concept study.

Caudo-rostral migration of pathological forms of α-synuclein from the gut to the brain is proposed as an early feature in Parkinson's disease pathogenesis, but the underlying mechanisms remain unknown. Intestinal epithelial enteroendocrine cells sense and respond to numerous luminal signals, including bacterial factors, and transmit this information to the brain via the enteric nervous system and vagus nerve. There is evidence that gut bacteria composition and their metabolites change in Parkinson's disease patients, and these alterations can trigger α-synuclein pathology in animal models of the disorder. Here, we investigated the effect of toll-like receptor and free fatty acid receptor agonists on the intracellular level of α-synuclein and its release using mouse secretin tumour cell line 1 enteroendocrine cells. Secretin tumour cell line 1 enteroendocrine cells were treated for 24 or 48 h with toll-like receptor agonists (toll-like receptor 4 selective lipopolysaccharide; toll-like receptor 2 selective Pam3CysSerLys4) and the free fatty acid receptor 2/3 agonists butyrate, propionate and acetate. The effect of selective receptor antagonists on the agonists' effects after 24 hours was also investigated. The level of α-synuclein protein was measured in cell lysates and cell culture media by western blot and enzyme-linked immunosorbent assay. The level of α-synuclein and tumour necrosis factor messenger RNA was measured by quantitative reverse transcription real-time polymerase chain reaction. Stimulation of secretin tumour cell line 1 enteroendocrine cells for 24 and 48 hours with toll-like receptor and free fatty acid receptor agonists significantly increased the amount of intracellular α-synuclein and the release of α-synuclein from the cells into the culture medium. Both effects were significantly reduced by antagonists selective for each receptor. Toll-like receptor and free fatty acid receptor agonists also significantly increased tumour necrosis factor transcription, and this was effectively inhibited by corresponding antagonists. Elevated intracellular α-synuclein increases the likelihood of aggregation and conversion to toxic forms. Factors derived from bacteria induce α-synuclein accumulation in secretin tumour cell line 1 enteroendocrine cells. Here, we provide support for a mechanism by which exposure of enteroendocrine cells to specific bacterial factors found in Parkinson's disease gut dysbiosis might facilitate accumulation of α-synuclein pathology in the gut.

The bacterial species profiles of the lingual and salivary microbiota differ with basic tastes sensitivity in human.

Taste perception is crucial and impairments, which can be linked to pathologies, can lead to eating disorders. It is triggered by taste compounds stimulating receptors located on the tongue. However, the tongue is covered by a film containing saliva and microorganisms suspected to modulate the taste receptor environment. The present study aimed to elucidate the links between taste sensitivity (sweetness, sourness, bitterness, saltiness, umami) and the salivary as well as the tongue microbiota using shotgun metagenomics. 109 bacterial species were correlated with at least one taste. Interestingly, when a species was correlated with at least two tastes, the correlations were unidirectional, indicating a putative global implication. Some Streptococcus, SR1 and Rickenellaceae species correlated with five tastes. When comparing both ecosystems, saliva appears to be a better taste predictor than tongue. This work shows the implication of the oral microbiota in taste and exhibits specificities depending on the ecosystem considered.

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.

Cross talk between Paneth and tuft cells drives dysbiosis and inflammation in the gut mucosa.

Gut microbiota imbalance (dysbiosis) is increasingly associated with pathological conditions, both within and outside the gastrointestinal tract. Intestinal Paneth cells are considered to be guardians of the gut microbiota, but the events linking Paneth cell dysfunction with dysbiosis remain unclear. We report a three-step mechanism for dysbiosis initiation. Initial alterations in Paneth cells, as frequently observed in obese and inflammatorybowel diseases patients, cause a mild remodeling of microbiota, with amplification of succinate-producing species. SucnR1-dependent activation of epithelial tuft cells triggers a type 2 immune response that, in turn, aggravates the Paneth cell defaults, promoting dysbiosis and chronic inflammation. We thus reveal a function of tuft cells in promoting dysbiosis following Paneth cell deficiency and an unappreciated essential role of Paneth cells in maintaining a balanced microbiota to prevent inappropriate activation of tuft cells and deleterious dysbiosis. This succinate-tuft cell inflammation circuit may also contribute to the chronic dysbiosis observed in patients.

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.

Neonatal Programming of Microbiota Composition: A Plausible Idea That Is Not Supported by the Evidence.

Underpinning the theory "developmental origins of health and disease" (DOHaD), evidence is accumulating to suggest that the risks of adult disease are in part programmed by exposure to environmental factors during the highly plastic "first 1,000 days of life" period. An elucidation of the mechanisms involved in this programming is challenging as it would help developing new strategies to promote adult health. The intestinal microbiome is proposed as a long-lasting memory of the neonatal environment. This proposal is supported by indisputable findings such as the concomitance of microbiota assembly and the first 1,000-day period, the influence of perinatal conditions on microbiota composition, and the impact of microbiota composition on host physiology, and is based on the widely held but unconfirmed view that the microbiota is long-lastingly shaped early in life. In this review, we examine the plausibility of the gut microbiota being programmed by the neonatal environment and evaluate the evidence for its validity. We highlight that the capacity of the pioneer bacteria to control the implantation of subsequent bacteria is supported by both theoretical principles and statistical associations, but remains to be demonstrated experimentally. In addition, our critical review of the literature on the long-term repercussions of selected neonatal modulations of the gut microbiota indicates that sustained programming of the microbiota composition by neonatal events is unlikely. This does not exclude the microbiota having a role in DOHaD due to a possible interaction with tissue and organ development during the critical windows of neonatal life.

Roseburia, a decreased bacterial taxon in the gut microbiota of patients suffering from anorexia nervosa.

Anorexia nervosa (AN) is a severe eating disorder which can lead to malnutrition and life threatening complications with high mortality rates. We designed our analysis to identify gut microbial taxa differentially abundant between AN and HC across different 16S rRNA gene datasets. We identified a reduced abundance, diversity and richness of Roseburia genus in the microbiota of patients with AN. Cares leading to partial recovery of patients with AN during hospitalization did not restore Roseburia to the levels of HC. AN dietary habit, either purgative or restrictive, did not affect Roseburia abundance. Roseburia genus and related species abundance were correlated with different health host metabolic markers. Roseburia species are key functional taxa in the human gut microbiome. Low gut Roseburia levels have been linked with other human pathologies. Our study highlights Roseburia species as a major decreased component in the gut of patients with AN.

Fecal Microbiota Transplant from Human to Mice Gives Insights into the Role of the Gut Microbiota in Non-Alcoholic Fatty Liver Disease (NAFLD).

Non-alcoholic fatty liver diseases (NAFLD) are associated with changes in the composition and metabolic activities of the gut microbiota. However, the causal role played by the gut microbiota in individual susceptibility to NAFLD and particularly at its early stage is still unclear. In this context, we transplanted the microbiota from a patient with fatty liver (NAFL) and from a healthy individual to two groups of mice. We first showed that the microbiota composition in recipient mice resembled the microbiota composition of their respective human donor. Following administration of a high-fructose, high-fat diet, mice that received the human NAFL microbiota (NAFLR) gained more weight and had a higher liver triglycerides level and higher plasma LDL cholesterol than mice that received the human healthy microbiota (HR). Metabolomic analyses revealed that it was associated with lower and higher plasma levels of glycine and 3-Indolepropionic acid in NAFLR mice, respectively. Moreover, several bacterial genera and OTUs were identified as differently represented in the NAFLR and HR microbiota and therefore potentially responsible for the different phenotypes observed. Altogether, our results confirm that the gut bacteria play a role in obesity and steatosis development and that targeting the gut microbiota may be a preventive or therapeutic strategy in NAFLD management.

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.

Alternative stable states in the intestinal ecosystem: proof of concept in a rat model and a perspective of therapeutic implications.

BACKGROUND: Chronic immune-mediated diseases are rapidly expanding and notoriously difficult to cure. Altered relatively stable intestinal microbiota configurations are associated with several of these diseases, and with a possible pre-disease condition (more susceptible to disease development) of the host-microbiota ecosystem. These observations are reminiscent of the behavior of an ecosystem with alternative stable states (different stable configurations that can exist under identical external conditions), and we recently postulated that health, pre-disease and disease represent such alternative states. Here, our aim was to examine if alternative stable states indeed exist in the intestinal ecosystem. RESULTS: Rats were exposed to varying concentrations of DSS in order to create a wide range of mildly inflammatory conditions, in a context of diet-induced low microbiota diversity. The consequences for the intestinal microbiota were traced by 16S rRNA gene profiling over time, and inflammation of the distal colon was evaluated at sacrifice, 45 days after the last DSS treatment. The results provide the first formal experimental proof for the existence of alternative stable states in the rat intestinal ecosystem, taking both microbiota and host inflammatory status into consideration. The alternative states are host-microbiota ecosystem states rather than independent and dissociated microbiota and host states, and inflammation can prompt stable state-transition. Based on these results, we propose a conceptual model providing new insights in the interplay between host inflammatory status and microbiota status. These new insights call for innovative therapeutic strategies to cure (pre-)disease. CONCLUSIONS: We provide proof of concept showing the existence of alternative stable states in the rat intestinal ecosystem. We further propose a model which, if validated in humans, will support innovative diagnosis, therapeutic strategy, and monitoring in the treatment of chronic inflammatory conditions. This model provides a strong rationale for the application of combinatorial therapeutic strategies, targeting host and microbiota rather than only one of the two in chronic immune-mediated diseases. Video Abstract.

Impact of pemetrexed chemotherapy on the gut microbiota and intestinal inflammation of patient-lung-derived tumor xenograft (PDX) mouse models.

Chemotherapy remains the gold standard for advanced cancer. Pemetrexed, a chemotherapeutic agent used in non-small cell lung cancer, can induce significant side effects in patients. Although microbiota's role in the efficacy and/or toxicity of chemotherapy agents has been demonstrated, the impacts of pemetrexed on the gut microbiota and on gastrointestinal inflammation remain unknown. The objective of this study was to evaluate the impact of pemetrexed and the tumor graft on the gut microbiota composition in immunodeficient mice. The faecal microbiota composition was studied with metabarcoding before, 24-h and one week after treatment. The colon epithelial barrier integrity was evaluated by histological examination, intestinal permeability measurement, and selected cytokines quantification. The tumor graft induced some variations in the microbiota composition. Pemetrexed further increased the relative abundance of Enterobacteriaceae and 3 families from the Firmicutes phylum: Enterococcaceae, Lactobacillaceae and Streptococcaceae. Pemetrexed also significantly altered the epithelial barrier integrity, which was associated with early inflammation. This pilot study shows that the association of a lung tumor graft with pemetrexed causes an alteration in the microbiota composition. Such information increases our knowledge about the impact of chemotherapy on the microbiota, which could help to minimize side effects and improve therapeutic effectiveness in the future.

Microbiota-gut brain axis involvement in neuropsychiatric disorders.

Introduction: The microbiota-gut brain (MGB) axis is the bidirectional communication between the intestinal microbiota and the brain. An increasing body of preclinical and clinical evidence has revealed that the gut microbial ecosystem can affect neuropsychiatric health. However, there is still a need of further studies to elucidate the complex gene-environment interactions and the role of the MGB axis in neuropsychiatric diseases, with the aim of identifying biomarkers and new therapeutic targets, to allow early diagnosis and improving treatments. Areas covered: To review the role of MGB axis in neuropsychiatric disorders, prediction and prevention of disease through exploitation, integration, and combination of data from existing gut microbiome/microbiota projects and appropriate other International '-Omics' studies. The authors also evaluated the new technological advances to investigate and modulate, through nutritional and other interventions, the gut microbiota. Expert opinion: The clinical studies have documented an association between alterations in gut microbiota composition and/or function, whereas the preclinical studies support a role for the gut microbiota in impacting behaviors which are of relevance to psychiatry and other central nervous system (CNS) disorders. Targeting MGB axis could be an additional approach for treating CNS disorders and all conditions in which alterations of the gut microbiota are involved.

A Guide for Ex Vivo Handling and Storage of Stool Samples Intended for Fecal Microbiota Transplantation.

Owing to the growing recognition of the gut microbiota as a main partner of human health, we are expecting that the number of indications for fecal microbiota transplantation (FMT) will increase. Thus, there is an urgent need for standardization of the entire process of fecal transplant production. This study provides a complete standardized procedure to prepare and store live and ready-to-use transplants that meet the standard requirements of good practices to applied use in pharmaceutical industry. We show that, if time before transformation to transplants would exceed 24 hours, fresh samples should not be exposed to temperatures above 20 °C, and refrigeration at 4 °C can be a safe solution. Oxygen-free atmosphere was not necessary and simply removing air above collected samples was sufficient to preserve viability. Transplants prepared in maltodextrin-trehalose solutions, stored in a -80 °C standard freezer and then rapidly thawed at 37 °C, retained the best revivification potential as  proven by 16S rRNA profiles, metabolomic fingerprints, and flow cytometry assays over a 3-month observation period. Maltodextrin-trehalose containing cryoprotectants were also efficient in preserving viability of lyophilized transplants, either in their crude or purified form, an option that can be attractive for fecal transplant biobanking and oral formulation.

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.

Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells.

The ligand activated transcription factor, aryl hydrocarbon receptor (AhR) emerged as a critical regulator of immune and metabolic processes in the gastrointestinal tract. In the gut, a main source of AhR ligands derives from commensal bacteria. However, many of the reported microbiota-derived ligands have been restricted to indolyl metabolites. Here, by screening commensal bacteria supernatants on an AhR reporter system expressed in human intestinal epithelial cell line (IEC), we found that the short chain fatty acid (SCFA) butyrate induced AhR activity and the transcription of AhR-dependent genes in IECs. We showed that AhR ligand antagonists reduced the effects of butyrate on IEC suggesting that butyrate could act as a ligand of AhR, which was supported by the nuclear translocation of AhR induced by butyrate and in silico structural modelling. In conclusion, our findings suggest that (i) butyrate activates AhR pathway and AhR-dependent genes in human intestinal epithelial cell-lines (ii) butyrate is a potential ligand for AhR which is an original mechanism of gene regulation by SCFA.

Prediction of the intestinal resistome by a three-dimensional structure-based method.

The intestinal microbiota is considered to be a major reservoir of antibiotic resistance determinants (ARDs) that could potentially be transferred to bacterial pathogens via mobile genetic elements. Yet, this assumption is poorly supported by empirical evidence due to the distant homologies between known ARDs (mostly from culturable bacteria) and ARDs from the intestinal microbiota. Consequently, an accurate census of intestinal ARDs (that is, the intestinal resistome) has not yet been fully determined. For this purpose, we developed and validated an annotation method (called pairwise comparative modelling) on the basis of a three-dimensional structure (homology comparative modelling), leading to the prediction of 6,095 ARDs in a catalogue of 3.9 million proteins from the human intestinal microbiota. We found that the majority of predicted ARDs (pdARDs) were distantly related to known ARDs (mean amino acid identity 29.8%) and found little evidence supporting their transfer between species. According to the composition of their resistome, we were able to cluster subjects from the MetaHIT cohort (n = 663) into six resistotypes that were connected to the previously described enterotypes. Finally, we found that the relative abundance of pdARDs was positively associated with gene richness, but not when subjects were exposed to antibiotics. Altogether, our results indicate that the majority of intestinal microbiota ARDs can be considered intrinsic to the dominant commensal microbiota and that these genes are rarely shared with bacterial pathogens.