Overview of the recent results
In 2013, we have identified Akkermansia muciniphila as a key bacterium involved in the control of the gut barrier function and host metabolism (Everard et al PNAS 2013 and patents). We demonstrated that A. muciniphila, a mucin-degrading bacterium that resides in the mucus layer and abundantly colonizes it, negatively correlates with body weight and is decreased under high-fat diet. Moreover, daily administration of A. muciniphila to high-fat-diet-induced obese mice for 4 weeks improves metabolic profile, by decreasing weight gain, restoring mucus layer thickness, antimicrobial peptides production and counteracting metabolic endotoxemia and insulin resistance. We discovered that the fatty acids composition may also strongly contribute to the modulation of the abundance of A. muciniphila. We found that mice fed with a saturated fatty acid diet (lard-enriched diet) exhibited a significant decrease in Akkermansia muciniphila, whereas omega 3 fatty acids (fish oil-enriched diet) dramatically increased Akkermansia muciniphila in the gut. This effect was associated with a better gut barrier function and decreased adipose tissue inflammation, a phenomenon that can be transferred to germ-free recipient mice (Caesar et al. Cell Metabolism 2015).
Besides obesity and diabetes, aging is also linked with A. muciniphila (Schneeberger et al Sci Reports 2015). Indeed, the intestinal levels of this bacterium declined with age upon a normal diet feeding. We found that high-fat diet feeding strongly influenced adipose tissue profile and intestinal microbiota in a way that mimicked aging, or at least older mice. In the same set of experiments, we found by using multifactorial analysis that these changes in A. muciniphila were robustly linked with the expression of lipid metabolism and inflammation markers in adipose tissue, as well as several blood markers (i.e., glucose, insulin, triglycerides, leptin) (Schneeberger et al Sci Reports 2015).
In accordance with the data obtained in rodents, we show in obese humans, that in the basal state, the abundance of Akkermansia muciniphila is inversely related to fasting plasma glucose levels, visceral fat accumulation, and adipocyte diameter in subcutaneous adipose tissue (Dao et al., 2016). More precisely, subjects with higher Akkermansia muciniphila abundance have a lower fasting glucose, triglycerides and lower body composition. In addition, upon caloric restriction, obese individuals with higher baseline Akkermansia muciniphila displayed greater improved insulin sensitivity markers and other cardiometabolic risk factors (Dao et al., GUT 2016), whereas upon gastric bypass Akkermansia muciniphila is dramatically increased (Dao et al. Am J Phyisol Endocrinol Metab. 2019 . Thus, all these data suggest that A. muciniphila is of interest and merits further investigation in humans. However, the classic growth requirements for the culture of Akkermansia muciniphila and its oxygen sensitivity render this bacterium unsuitable for human investigations and putative therapeutic opportunities. Therefore, the sensitivity of Akkermansia muciniphila to oxygen and the presence of animal-derived compounds in its growth medium currently limit the development of translational approaches for human medicine.
The team of Prof. Cani have contributed to solve these critical issues by developing synthetic medium compatible with human administration (collaboration with Prof. Willem de Vos). We demonstrated that Akkermansia muciniphila cultured on this media retains its efficacy (Plovier et al, Nature Medicine 2017 and patent pending). Unexpectedly, pasteurizing Akkermansia muciniphila in order to stabilize the bacterium without destroying it, enhanced its capacity to reduce fat mass development, insulin resistance and dyslipidemia in mice.
These improvements were notably associated with a modulation of the host urinary metabolomics profile and higher intestinal energy excretion upon pasteurized Akkermansia muciniphila treatment. Then we wanted to understand why Akkermansia muciniphila behaved differently when live and pasteurized. By combining genomic and proteomic analyses of Akkermansia muciniphila our collaborators identified proteins encoded by a specific Type IV pili gene cluster in fractions enriched for outer membrane proteins (Plovier et al Nature Medicine 2017). Among these, Amuc_1100 was one of the most abundant. To test this hypothesis, we showed that a His-tagged Amuc_1100 produced in E. coli completely reproduced the beneficial effects of pasteurized Akkermansia muciniphila (see figure) (Plovier et al Nature Medicine 2017 and patents pending). Interestingly, this protein also remains active after heating to 70°C (pasteurization). In a molecular point of view, we demonstrated that Amuc_1100 interacts with TLR-2 and improves the gut barrier (see figure).
Patrice D. Cani and Willem M. de Vos, Frontiers in Microbiology 2017
Finally, we also developed the production of Akkermansia muciniphila at a large scale in order to test the safety and efficacy of the bacterium on parameters associated with cardiometabolic risks factors. The study Microbes4Uc has been published in July 2019. The major aim was to evaluate the safety and tolerability of Akkermansia muciniphila in individuals with excess body weight by supplementing them with different doses of live Akkermansia muciniphila (Akk Synthetic - 1010) or pasteurized Akkermansia muciniphila (Akk Pasteurized - 1010). This study published in Nature Medicine (Depommier et al. 2019), showed that administration of live or pasteurized bacteria grown on the synthetic medium is safe in humans and also improves numerous cardiometabolic risks factors, including insulin sensitivity, insulinemia, inflammation, liver enzymes, cholesterol but also markers of reinforced gut barrier (Depommier et al. Nature Medicine 2019).
These findings provide support for the use of different preparations of A. muciniphila as dietary supplements to target human cardiometabolic risk factors associated with obesity. Based on all these results Prof. Cani has co-founded the spinoff company “A-Manisa Biotech SA” in 2016 and accomplished in 2018 a series A capitalizing A-Mansia at 22 Million euros. The company is devoted to develop a food supplement and a drug based on Akkermansia and on other derived compounds.
Besides Akkermansia muciniphila Prof. Cani and his team have isolated several novel bacteria including one novel genus/species/strain. The bacterium is called Dysosmobacter welbionis in reference to the project WELBIO which is supporting this innovative research since 2012 (Le Roy et al IJSEM 2019, and patent pending). The metabolic effects of these novel bacteria are currently under investigation in the laboratory.
In 2014, we found that a link between the innate immune system from intestinal cells (i.e., the protein MyD88) and energy homeostasis. More precisely, we found that modifying the response of the immune system by deactivating the protein MyD88 in the intestinal cells delay the development of type 2 diabetes induced by a high fat diet, reduces the development of fat mass, reduces the deleterious inflammation observed during obesity and reinforced the gut barrier thereby preventing the leakage of unsuitable bacterial compounds from the intestine to the organism. More importantly, we found that it is experimentally possible, through this modification of the immune system, to induce body weight loss and therefore to have a therapeutic effect despite the fact that the animals were already obese and diabetic. Surprisingly, we found that it is possible to partially protects against obesity and diabetes by transferring (i.e., grafting) the gut microbiota from these mice to axenic mice (i.e., germ free) (Everard et al. Nature Communications 2014). By investigating the role of Myd88 deletion in the hepatocyte and host metabolism, we discovered that hepatic MyD88 is a key factor controlling the onset of glucose intolerance and liver inflammation (Duparc et al, GUT 2017). In a second study, we found that hepatic Myd88 is a key actor controlling the synthesis of different biacotive lipids such as oxysterols and eventually controls the endogenous production of bile acids and related factors (Lefort et al. Am J Physiol Endocrinol Metab 2019).
We have previously identified that the endocannabinoid system links the gut microbiota to adipogenesis in both physiological and pathological situations such as obesity and type 2 diabetes. Our data pointed out that targeting specifically the endocannabinoid system tone in the adipose tissue may contribute to change host-microbiota interactions (for review Cani et al Nature Reviews Endocrinology 2016). In 2015, we published data showing that deleting N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) in the adipose tissue (tissue specific deletion) induces obesity in normal diet-fed mice by promoting fat mass development, insulin resistance and inflammation. This key enzyme is involved in anandamide and NAE biosynthesis. We discovered that the deletion of NAPE-PLD in adipocytes induces also a decreased thermogenic programme (i.e., browning/beiging) in adipose tissue. Importantly, we found that NAPE-PLD deletion in adipose tissue induced a profound shift in the gut microbiota composition and activity. By transferring the microbiota from mice in which the adipose tissue NAPE-PLD was deleted into germ-free recipient mice replicated the overall phenotype (Geurts et al. Nature Communications 2015).
Actually, we discovered that mice harbouring and inducible intestinal epithelial cell (IEC)-specific deletion of NAPE-PLD (Napepld∆IEC) were hyperphagic upon first exposure to a high-fat diet and developed exacerbated diet-induced obesity and hepatic steatosis. Among the mechanisms, we found that these mice displayed a defect in hypothalamic Pomc neurons and alterations in intestinal and plasma NAE and 2-acylglycerols. After long-term HFD exposure, Napepld∆IEC mice presented a lower energy expenditure. The increased hepatic lipid storage was associated with higher lipid absorption in the gut and in the liver. Moreover, Napepld∆IEC mice displayed altered gut microbiota composition. Strikingly, treatment with Akkermansia muciniphila, a bacterium influencing NAE, endocannabinoids and related mediators, partly counteracted the effects of the deletion. These results suggest that intestinal NAPE-PLD is a key sensor in nutritional adaptation to fat intake, gut to brain axis and energy homeostasis and thereby constitute a novel target to tackle obesity and related disorders (Everard*, Plovier*, Rastelli* et al Nature Communications 2019).
Taken together, these findings indicate that bioactive lipids produced by the NAPE-PLD contribute to changes in the gut microbiota even at distance of the organ targeted (e.g., the adipose tissue). These changes then participate in the altered metabolic disorders observed following NAPE-PLD deletion. These results provide strong support for the crosstalk between the gut microbiota and the endocannabinoid system as a potent mediator.
Patrice D. Cani, Hubert Plovier, Matthias Van Hul, Lucie Geurts, Nathalie M. Delzenne, Céline Druart and Amandine Everard. Nature Reviews Endocrinology 2016
Recently, several prebiotics have been tested in original mice models of endothelial dysfunction and gluten-induced obesity. Those data revealed that the improvement of the endothelial dysfunction by fructans and chitin glucans is associated with specific changes in microbiota and increased intestinal production of nitric oxide release. Among microbial metabolites, change in bile acid profiling by inulin-type fructans support their potent contribution to the improvement of gut endocrine and vascular functions (Catry et al Gut 2018, Neyrinck et al Sci Report 2019). Arabinoxylo- and fructo-oligosaccharides were able to improve gluten induced obesity and metabolic disorders, by driving intestinal and microbial gluten cleavage (Olivares et al Mol Nutr Food Res 2019). These data confirm that behind the effect of prebiotics in the caeco-colon, those nutrients are also able to change the digestion of other nutrients in the upper part of the gut, as we have previously shown for dietary lipids and disaccharides (Suriano, Bindels et al Sci Report 2017; Neyrinck et al, J Functional Food 2018; Hiel et al Nutrient 2018). (In the context of a European project (MyNewGut, http://www.mynewgut.eu/), we have also highlighted a potential interest of amino-acid microbial metabolites to counteract hepatic inflammation and of microbial conjugated polyunsaturated fatty acids to improve lipid metabolism (Beaumont et al FASEB J 2018, Pachikian et al Plos One 2018, Delzenne et al Clin Nutr and Proc Nutr Soc 2019), thereby extending the concept of prebiotics and related bioactives. Other classes of food products have been evaluated in term of microbiota modulation in preclinical models of nutritional disorders. Among them, green tea - rhubarb- curcuma- or pomegranate- extracts, as well as spirulina counteract inflammation associated with nutritional disorders. We have also collaborated to the demonstration of the interest of milk polar lipids in obesity (Millard et al Mol Nutr Food Res 2019). Some effects (those of green tea) seem independent on changes in microbiota composition, whereas we could point out interesting bacterial metabolites produced from phenolic compounds, as potential drivers of anti-inflammatory effect (Neyrinck et al Plos One 2013, Mol Nutr Food Res 2017, J Nutr Biochem 2017, Nutrients 2017). Our current projects will evaluate – by using untargeted and targeted metabolomics- the relevance of microbial cometabolites in the changes in behavior (depression, social behavior) in the models of mice transferred with the gut microbiota from obese or alcohol-dependent human volunteers. In 2019, we also published the first data related to inulin-intervention studies programmed in the context of the Food4gut project (https://sites.uclouvain.be/FOOD4GUT/en/). We have demonstrated in a food-based intervention in healthy volunteers that daily eating of locally produced- inulin rich vegetables was able to exert specific and reversible changes in the gut microbiota, and modulates food-related behavior, leading namely to a decrease in envy to eat sugar or fat (Hiel et al Am J Clin Nutr 2019). This fits with the observation of the contribution of inulin to the improvement of sweet taste perception in mice (Bernard et al Nutrients 2019).
Our research team focusses on the development of new therapeutic pharmacological and nutritional tools based on the gut microbiota-host crosstalk in several pathological contexts. Our work highlights the importance of the gut and its microbes to target cancer cachexia, paving the way to new therapeutic opportunities (Genton et al, Front Cell Infect Microbiol 2019; Pötgens et al, Curr Opin Nutr Metab Care 2018). Our work has clearly led to a better consideration of the potential of the gut microbiota in cancer cachexia (Ebner et al, J Cach Sarc Muscle 2020; Argiles et al, Nat Rev Endocrinol 2018; Dzutsev et al, Annu Rev Immunol 2017).
Cancer cachexia is a complex multi-organ syndrome characterized by body weight loss, weakness, muscle atrophy and fat depletion (Fearon et al, Lancet Oncol 2011). Importantly, fat depletion may precede muscle wasting in cancer cachexia and preserving fat mass can spare muscle mass (Das et al, Science 2011). Paradoxically, accumulation of ectopic fat in the liver was found in rodent models of cancer cachexia and in cachectic patients (Berriel et al, Hepatology 2008). Clinically, cachexia results in increased morbidity and mortality rates as well as reduced tolerance to anti-cancer treatments (Farkas et al, J Cach Sarc Muscle 2013). Currently, limited therapeutic options exist for this important medical challenge and new approaches to tackle this syndrome, including innovative and scientifically relevant nutritional and pharmacological tools, are needed (Pötgens et al, Cur Opin Metab Care 2018). In this context, targeting the gut microbiota represents an exciting opportunity for this public health issue.
Links between gut microbiota and cancer have been studied for years (Schwabe & Jobin, Nat Rev Cancer 2013). Our research over the last ten years has evidenced the existence of a crosstalk between the gut, the microbes it harbors and metabolic alterations occurring during cancer.
First, we showed in 2012 that restoring the lactobacilli levels through the administration of lactobacilli counteracted muscle atrophy and decreased systemic inflammation in a mouse model of leukemia and cachexia (Bindels et al, Plos ONE 2012).
Second, we highlighted a common microbial signature (characterized mainly by an increase in Enterobacteriaceae) in preclinical models of cancer cachexia, in strong association with some cachectic features (Bindels et al, Plos ONE 2015; Bindels et al, The ISME J 2016). This microbial signature was not due to the anorexia observed in the last stage of the disease (Bindels et al, Plos ONE 2011; Bindels et al, The ISME J 2016). More recently, we have highlighted that Klebsiella oxytoca was the Enterobacteriaceae species that was fostered in cancer cachexia. We evidenced a mechanism of emergence for this bacterium similar to the one described for the bloom of Enterobacteriaceae during antibiotics consumption. This framework includes a reduction in Treg cells in the intestine, together with a glycolytic switch and a host-derived production of nitrate (Pötgens et al, Sci Rep 2018).
Third, we found drastic changes in the gut permeability and intestinal morphology of cachectic mice. Such changes were strongly correlated with the cachectic features. These alterations occurred independently of anorexia and were driven by interleukin 6. Gut dysfunction was found to be resistant to treatments with an anti-inflammatory bacterium (Faecalibacterium prausnitzii) or with gut peptides involved in intestinal cell renewal (teduglutide, a glucagon-like peptide 2 analogue) (Bindels et al, Oncotarget 2018). We also demonstrated that K. oxytoca behaves as a gut pathobiont contributing to intestinal dysfunction in cachectic mice (Pötgens et al, Sci Rep 2018).
Last but not least, we reported several times that nutritional interventions targeting the microbiota, such as prebiotics or probiotics, decreased cancer progression, reduced morbidity and fat mass loss, and/or increased survival of cachectic mice with leukemia (Bindels et al, the ISME J 2016; Bindels et al, Plos ONE 2015; Bindels et al, Br J Cancer 2013). Our data highlight propionate, a short-chain fatty acid produced through the fermentation of prebiotics, as a potential mediator of this anti-cancer effect observed in leukemic mice with cachexia. Indeed, administration of inulin-type fructans (a well-known prebiotic) increased portal levels of propionate which can control the proliferation of leukemic cells (Bindels et al, Br J Cancer 2013).
Altogether, our studies reveal a previously unexpected link between cancer, cachexia and the gut microbiota. However, the exact mechanisms underlying this crosstalk remain elusive and constitute the topic of research of our team (Prof Bindels' team). To achieve such goal, her team is using targeted and untargeted metabolomics analyses (recent implementation of H1-NMR metabolomics) using the NEST and MASSMETplatforms. These data are integrated with targeted microbial metagenomics and transcriptomics to highlight new pathways involved in this crosstalk. Using such approach, we confirmed several hepatic metabolic alterations previously reported in the literature (such as a reduction in hepatic glycolysis) while revealing new pathways potentially involved in cachectic features.
Specifically, we highlight (i) an activation of the hexosamine pathway in the liver, likely as a consequence of an endoplasmic reticulum stress and an unfold protein response, that may impact the hepatic signaling through O-GlcNAcylation; (ii) a reduction in the carnitine levels and its biosynthesis, and in the phosphatidylinositol pathway as potential contributors to the hepatic steatosis found in these mice; (iii) a reduction in the transformation of carbohydrates and proteins by the gut bacteria, that associates to specific host genic modulations (metabolic and gut barrier functions) (Pötgens et al, J Cach Sarc Muscle 2021).
We further investigated the contribution of bile acids, one of the bacterial co-metabolites identified through this metabolomic study. This led us to highlight a cholestasis in cancer cachexia (2 mouse models and one cohort of 94 patients) and to unequivocally demonstrate that systemic inflammation strongly contributes to the impairment of the hepatobiliary transport system in cancer cachexia. Targeting the enterohepatic circulation, we showed that bile acids contribute to hepatic inflammation and disorders. Altogether, our work highlights a vicious circle between bile acids and inflammation and paves the way to new therapeutic strategies targeting bile acids to control hepatic inflammation and metabolic disturbances in cancer cachexia (Thibaut et al, J Cach Sarc Muscle 2021).
Food intake, appetite and satiety are mainly integrated at the level of hypothalamic neuronal circuits. Importantly, energy balance is also controlled by hedonic/reward brain systems encoded by the neuronal network of the mesolimbic dopaminergic system. Hedonic properties of food stimulate feeding and some food substances (e.g., sugars, sweeteners, salt, and lipids) are more prompt to be involved in these addictive processes. These effects are mediated by abrupt dopamine increases in the brain reward system. This mesocorticolimbic system encodes for the three psychological component of reward: liking, wanting and learning.
During obesity, this gut-to-brain axis is altered at the level of the hedonic responses to food intake, leading to an abnormal increase in energy consumption. Moreover, the concept of the implication of the gut microbiota in the gut-to-brain axis to control food intake emerged over these last years, however the mechanisms still remain incompletely known and the roles of the gut microbiota in the regulation of hedonic/reward aspects of food intake are completely unknown.
Therefore, it is of utmost importance to fill in this gap to better understand the alterations of the gut-to-brain axis to control food intake during obesity and the implication of the gut microbiota in that context.
The originality of this work is to investigate how gut microbes are able to control hedonic and reward system in healthy conditions as well as in the physiopathology of obesity.
In order to proof a causal link between gut microbiota and alterations of hedonic response to food intake associated with obesity, we use gut microbiota transplantation. Preliminary data suggest that transferring the gut microbiota from high-fat diet-induced obese mice into control diet fed mice is enough to alter the dopaminergic signalling in the striatum of the mice in a similar way to alterations observed during obesity such as reduction of D2 receptor. Moreover, these alterations of dopaminergic signalling are associated with alteration of psychological component of reward such as liking. Indeed mice transplanted with the gut microbiota from high-fat diet-induced obese mice present a reduction of the high-fat high-sugar diet consumption in comparison to mice transplanted with the gut microbiota from control fed mice. Altogether these data suggest for the first time the implication of the gut microbiota into the alteration of hedonic regulation of food intake during obesity. These preliminary data need to be confirmed and we will investigate the mechanisms involved in these interactions between the gut microbiota and the hedonic regulation of food intake during obesity.