Vassilia Theodorou

Homeostasis of the gut barrier and potential biomarkers

The gut barrier plays a crucial role by spatially compartmentalizing bacteria to the lumen through the production of secreted mucus and is fortified by the production of secretory IgA (sIgA) and antimicrobial peptides and proteins. With the exception of sIgA, expression of these protective barrier factors is largely controlled by innate immune recognition of microbial molecular ligands. Several specialized adaptations and checkpoints are operating in the mucosa to scale the immune response according to the threat and prevent overreaction to the trillions of symbionts inhabiting the human intestine. A healthy microbiota plays a key role influencing epithelial barrier functions through the production of short-chain fatty acids (SCFAs) and interactions with innate pattern recognition receptors in the mucosa, driving the steady-state expression of mucus and antimicrobial factors. However, perturbation of gut barrier homeostasis can lead to increased inflammatory signaling, increased epithelial permeability, and dysbiosis of the microbiota, which are recognized to play a role in the pathophysiology of a variety of gastrointestinal disorders. Additionally, gut-brain signaling may be affected by prolonged mucosal immune activation, leading to increased afferent sensory signaling and abdominal symptoms. In turn, neuronal mechanisms can affect the intestinal barrier partly by activation of the hypothalamus-pituitary-adrenal axis and both mast cell-dependent and mast cell-independent mechanisms. The modulation of gut barrier function through nutritional interventions, including strategies to manipulate the microbiota, is considered a relevant target for novel therapeutic and preventive treatments against a range of diseases. Several biomarkers have been used to measure gut permeability and loss of barrier integrity in intestinal diseases, but there remains a need to explore their use in assessing the effect of nutritional factors on gut barrier function. Future studies should aim to establish normal ranges of available biomarkers and their predictive value for gut health in human cohorts.

The liver X receptor: A master regulator of the gut–liver axis and a target for non alcoholic fatty liver disease

Since it is associated to the obesity epidemic, non alcoholic fatty liver disease (NAFLD) has become a major public health issue. NAFLD ranges from benign hepatic steatosis, i.e. abnormally elevated triglyceride accumulation, to non alcoholic steatohepatitis (NASH) that can lead to irreversible liver damages. The search for pharmacological and dietary approaches to treat or prevent NAFLD has pointed at nuclear receptors as sensible targets. Indeed, nuclear receptors are ligand-sensitive transcription factors that play a central role in hepatic lipid metabolism. Among nuclear receptors, the liver X receptor has been identified as an oxysterol receptor. It is involved in the control of various aspects of lipid metabolism that are reviewed in this manuscript. We highlight the role of LXR in the gut-liver axis and the studies that have provided a rationale for strategies specifically targeting the hepatic activity of LXR in NAFLD.

Luminal Cathepsin G and Protease-Activated Receptor 4 A Duet Involved in Alterations of the Colonic Epithelial Barrier in Ulcerative Colitis

Impairment of the colonic epithelial barrier and neutrophil infiltration are common features of inflammatory bowel disease. Luminal proteases affect colonic permeability through protease-activated receptors (PARs). We evaluated: (i) whether fecal supernatants from patients with ulcerative colitis (UC) trigger alterations of colonic paracellular permeability and inflammation, and (ii) the roles of cathepsin G (Cat-G), a neutrophil serine protease, and its selective receptor, PAR(4), in these processes. Expression levels of both PAR(4) and Cat-G were determined in colonic biopsies from UC and healthy subjects. The effects of UC fecal supernatants on colonic paracellular permeability were measured in murine colonic strips. Involvement of Cat-G and PAR(4) was evaluated using pepducin P4pal-10 and specific Cat-G inhibitor (SCGI), respectively. In addition, the effect of PAR(4)-activating peptide was assessed. UC fecal supernatants, either untreated or pretreated with SCGI, were infused into mice, and myeloperoxidase activity was determined. PAR(4) was found to be overexpressed in UC colonic biopsies. Increased colonic paracellular permeability that was triggered by UC fecal supernatants was blocked by both SCGI (77%) and P4pal-10 (85%). Intracolonic infusion of UC fecal supernatants into mice increased myeloperoxidase activity. This effect was abolished by SCGI. These observations support that both Cat-G and PAR(4) play key roles in generating and/or amplifying relapses in UC and provide a rationale for the development of new therapeutic agents in the treatment of this disease.

Is Crohn's creeping fat an adipose tissue?

Background: In human pathology, the “creeping fat” (CF) of the mesentery is unique to Crohns disease (CD). CF is usually referred to as an ectopic extension of mesenteric adipose tissue (MAT). However, since no animal model developing CF has ever been established, very little is known about this type of fat‐depot expansion and its role in the development of the disease. Methods: We developed and standardized an experimental protocol in mice that reproducibly induces CF development when a severe colonic inflammation is obtained by intracolonic instillation of DNBS. Results: Macro‐microscopic observations revealed a fatty appearance of CF. Yet when compared to MAT from the same animals, CF contains very little triglycerides, few adipocytes, and we observed a very low expression and protein levels of both adipose markers (hormone‐sensitive lipase, perilipin) and adipocytokines (leptin, adiponectin). The decreased expression of perilipin in CF was also observed by immunohistochemistry. Conversely, the expression of proinflammatory and fibrous markers (Pref‐1) was much higher in CF than in MAT. These observations were fully consistent with those made on CF recovered from five CD patients and compared with subcutaneous and mesenteric fat from the same patients. Conclusions: Altogether, this work reports an original experimental mice model of CF. In this model we establish for the first time that CF only occurs in severe colonic inflammation and shows an inflammatory, fibrous but not an adipose pattern. (Inflamm Bowel Dis 2011)

Essential fatty acids deficiency promotes lipogenic gene expression and hepatic steatosis through the liver X receptor.

BACKGROUND & AIMSnNutrients influence non-alcoholic fatty liver disease. Essential fatty acids deficiency promotes various syndromes, including hepatic steatosis, through increased de novo lipogenesis. The mechanisms underlying such increased lipogenic response remain unidentified.nnnMETHODSnWe used wild type mice and mice lacking Liver X Receptors to perform a nutrigenomic study that aimed at examining the role of these transcription factors.nnnRESULTSnWe showed that, in the absence of Liver X Receptors, essential fatty acids deficiency does not promote steatosis. Consistent with this, Liver X Receptors are required for the elevated expression of genes involved in lipogenesis in response to essential fatty acids deficiency.nnnCONCLUSIONSnThis work identifies, for the first time, the central role of Liver X Receptors in steatosis induced by essential fatty acids deficiency.

Impact of mycotoxins on the intestine: are mucus and microbiota new targets?

ABSTRACT There is an increasing awareness of the deleterious effects attributed to mycotoxins during their fate within the gut, particularly for deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA), fumonisin B1 (FB1), aflatoxin B1 (AFB1), and patulin (PAT). Evidence indicates that disruption of the epithelial barrier is well established. However, intestinal barrier function on its luminal side involves two other partners, mucus and microbiota, which have rarely been considered in the context of mycotoxin exposure. The current review aimed at providing a summary of DON, ZEN, OTA, FB1, AFB1, and PAT effects on intestinal barrier function, with special focus on mucus and microbiota. DON, ZEN, OTA, FB1, AFB1, and PAT are known to markedly affect epithelial cell integrity and functions. Regarding mucus, DON is the most documentated mycotoxin. In vivo, toxicological impact of DON generally has only been assessed through goblet cell number. Evaluation of the mycotoxins/mucus interplay considering other indicators such as composition, thickness, and penetrability of mucus, mucin O-glycosylation thus warrants further attention. With respect to microbiota, few short-term studies to date have been reported indicating deleterious effects. However, long-term exposure to mycotoxins may also produce significant changes in microbiota composition and metabolic activity, which requires further experimentation. In conclusion, mucus and microbiota are key targets for dietary mycotoxins although assessment of induced effects is preliminary. A significant research effort is now underway to determine the adverse consequences of mycotoxins on mucus and microbiota considered as individual but also as tightly connected gut players.

Mucus organisation is shaped by colonic content; a new view

The colonic mucus barrier is commonly described as a continuous double layer covering the epithelium, separating the microbiota from the intestinal tissue. This model is currently considered valid throughout the colon. The colon is characterised by regional anatomo-functional specificities such as presence and consistency of contents and location. In this study, we characterised the organisation of the colonic mucus barrier in proximal and distal colon of rodents by histological and FISH staining, taking into account aforementioned specificities. By using longitudinal sections and imaging extensive areas of tissue with and without colonic contents, we have obtained a spatiotemporal overview of mucus organisation in the colon. We describe for the first time that the colonic mucus layer covers the faeces instead of the epithelium in the distal colon. This faecal mucus layer confines the microbiota to the faeces and prevents it from remaining in empty distal colon. In the proximal colon, the mucus did not form a separating layer between bacteria and epithelium. We conclude that the organisation of colonic mucus is reliant on the presence of the colonic content, and the location within the colon. Our findings reopen the discussion on the nature of the colonic mucus barrier.

Intracolonic infusion of fecal supernatants from ulcerative colitis patients triggers altered permeability and inflammation in mice: Role of cathepsin G and protease‐activated receptor‐4

Background: Cathepsin G (Cat‐G) is a neutrophil serine‐protease found in the colonic lumen of ulcerative colitis (UC) patients. Cat‐G is able to activate protease‐activated receptor‐4 (PAR4) located at the apical side of enterocytes, leading to epithelial barrier disruption. However, the mechanisms through which Cat‐G triggers inflammation are not fully elucidated. The aims of our study were to evaluate in vivo the effects of UC fecal supernatants and Cat‐G on epithelial barrier function and inflammation, and the connection between these two parameters. Methods: Male balb/c mice were used in this study. We evaluated the effect of a 2‐hour intracolonic infusion of 1) fecal supernatants from UC patients pretreated or not with specific Cat‐G inhibitor (SCGI); 2) PAR4‐activating peptide (PAR4‐AP); and 3) Cat‐G on colonic myeloperoxidase (MPO) activity and paracellular permeability (CPP). The involvement of PAR4 was assessed by pretreating animals with pepducin P4pal‐10, which blocks PAR4 signaling. We investigated the role of myosin light chain (MLC) kinase by using its inhibitor, ML‐7, and we determined phosphorylated MLC (pMLC) levels in mice colonic mucosa. Results: UC fecal supernatants, Cat‐G, and PAR4 agonist increased both CPP and MPO activity in comparison with healthy subjects fecal supernatants. ML‐7 inhibited the CPP increase triggered by Cat‐G by 92.3%, and the enhanced MPO activity by 43.8%. Intracolonic infusion of UC fecal supernatant determined an increased phosphorylation level of MLC. Conclusions: These observations support that luminal factors such as Cat‐G play an important proinflammatory role in the pathogenesis of colitis, mainly depending on CPP increase by MLC phosphorylation. (Inflamm Bowel Dis 2011)

Paneth Cell Defects Induce Microbiota Dysbiosis in Mice and Promote Visceral Hypersensitivity

BACKGROUND & AIMSnSeparation of newborn rats from theirxa0mothers induces visceral hypersensitivity and impaired epithelial secretory cell lineages when they are adults. Little is known about the mechanisms by which maternal separation causes visceral hypersensitivity or its relationship with defects in epithelial secretory cell lineages.nnnMETHODSnWe performed studies with C3H/HeN mice separated from their mothers as newborns and mice genetically engineered (Sox9flox/flox-vil-cre on C57BL/6 background) to have deficiencies in Paneth cells. Paneth cell deficiency was assessed by lysozyme staining of ileum tissues and lysozyme activity in fecal samples. When mice were 50 days old, their abdominal response to colorectal distension was assessed by electromyography. Fecal samples were collected and microbiota were analyzed using Gut Low-Density Array quantitative polymerase chain reaction.nnnRESULTSnMice with maternal separation developed visceral hypersensitivity and defects in Paneth cells, as reported fromxa0rats, compared with mice without maternal separation. Sox9flox/flox-vil-Cre mice also had increased visceral hypersensitivity compared with control littermate Sox9flox/flox mice. Fecal samples from mice with maternal separation and from Sox9flox/flox-vil-cre mice had evidence for intestinal dysbiosis of the microbiota, characterized by expansion of Escherichia coli. Daily gavage of conventional C3H/HeN adult mice with 109 commensal E coli induced visceral hypersensitivity. Conversely, daily oral administration of lysozyme prevented expansion of Exa0coli during maternal separation and visceral hypersensitivity.nnnCONCLUSIONSnMice with defects in Paneth cells (induced byxa0maternal separation or genetically engineered) have intestinal expansion of E coli leading to visceral hypersensitivity. These findings provide evidence that Paneth cell function and intestinal dysbiosis are involved in visceral sensitivity.

Modifications of mesenteric adipose tissue during moderate experimental colitis in mice

AIMSnAdipose tissue secretes various proteins referred to as adipokines, being involved in inflammation. It was recognized that mesenteric adipose tissue (MAT) is altered by inflammation, and pathologies such as inflammatory bowel disease (IBD). The aim of this study was to investigate the alterations of the mesenteric adipose tissue in two experimental colitis models in mice adapted to obtain moderate colonic inflammation.nnnMAIN METHODSnColonic inflammation was obtained using two models, either DSS dissolved in drinking water or intra-colonic instillation of DNBS. The expression of adipokines (leptin and adiponectin) and inflammatory markers (IL-6, MCP-1, F4/80) was studied by qRT-PCR in the MAT of treated and control mice.nnnKEY FINDINGSnObservations of the colon and IL-6 plasma level determination demonstrated that DNBS treatment led to stronger inflammation. Colitis induced a decrease of mRNA encoding to leptin and adiponectin in MAT. In contrast, colonic inflammation led to an increase of mRNA encoding to IL-6, MCP-1 and F4/80, a specific marker of macrophages.nnnSIGNIFICANCEnThe mesenteric adipose tissue, in two models of moderate colitis, shows a loss of adipose profile and a strong increase of inflammatory pattern, close to the observations made in MAT of IBD patients. These data suggest that these pro-inflammatory modifications of MAT have to be taken into account in the pathophysiology of IBD.