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The relationship between the intestinal mucosal barrier and gut microbiota in metabolic diseases

Final Report Summary - MUCUS AND METABOLISM (The relationship between the intestinal mucosal barrier and gut microbiota in metabolic diseases)

1.1 Summary description of the project objectives
Obesity is a world-wide epidemic and currently about 350 million people suffer from diabetes, resulting in more than 3 million deaths per year. While the gut microbiota has been identified as a contributing factor to obesity and insulin resistance in mice and humans, the underlying disease-causing mechanisms are yet to be defined. The aim of this proposal was to investigate the interactions between the gut microbiota and the intestinal mucosal barrier in metabolic disease.
A complex microbial community with up to 1000 different species colonizes the human intestine. While its composition is specific for each individual, a core gut microbiota is shared between humans. However, the composition of gut microbiota is altered in individuals who are obese, diabetic or suffer from cardiovascular disease.
The high density of the gut microbiota provides a continuous challenge and potential threat to the gut epithelium. As only one single, mucus-covered cell layer separates microbes from human tissue the mucosal barrier is essential in preventing microbial translocation. One epithelial defense mechanism is the secretion of antimicrobial peptides (AMPs). These endogenous peptide antibiotics have broad-spectrum antimicrobial activity and can also modulate the composition of gut microbiota.
The intestinal mucus layer itself is an additional mechanism to protect the epithelium from the luminal microbiota. This is especially important in the colon, where mucus forms two distinct layers: the outer layer is heavily colonized by bacteria, which can degrade mucus glycans as energy source. The inner layer, in contrast, is sterile and prevents microbes from reaching and translocating the intestinal epithelium.
In this project I focused on the intestinal barrier between host and microbiota. I tested if and how the shifted microbial composition in metabolic diseases is associated with alterations in mucosal AMP production and mucus function by using mouse models and samples from human patients. The planned investigations will help to better understand the relationship between the mucosal host barrier and metabolic disease.

1.2 Description of the work performed
At the beginning of the project I obtained training in how to perform mouse experiments, which includes theoretical experiment planning as well as handling laboratory mice. Different mouse models (diet-induced obesity and genetic obesity) were then used to measure expression of intestinal AMPs and to study mucus properties of the gut. Furthermore, I got trained in cultivating strictly anaerobic gut bacteria in an oxygen-free Coy chamber and how to apply such bacteria to mice. In the host laboratory I also learned how to characterize microbial communities by state-of-the-art 16S rDNA microbiota analyses and identified critical aspects of the experimental setup. During the project I applied and expanded my newly gained knowledge by performing and analyzing microbiota analyses for collaboration partners. In addition, in the laboratory of our collaborator I learned how to analyze intestinal mucus properties on live tissue explants, a method that can be performed by only a few groups in the world. Thus, during my project I acquired several new techniques that expanded my methodological skills spectrum.
In fruitful discussions within the host laboratory and the group of our collaborator I obtained knowledge in the fields of gut microbiota, metabolism and mucus biology. Moreover, together with my mentor I composed a review on molecular signals from the gut microbiota, which was published in Nature Medicine in October 2016.
During my project I had the chance to attend several scientific conferences and meetings, where I could discuss my work with researchers in the field. This included an invited presentation during an international conference on antimicrobial activity and defense strategies at the mucosal surface in Portugal in April 2016.

1.3 Description of the main results
In two different mouse models of obesity we detected altered expression of intestinal AMPs in the small intestine. Analysis of germ-free (GF) mice and mice colonized with a consortium of 10 defined human gut bacteria also revealed altered expression of distinct AMPs. This indicates that the gut bacteria play an important role in modulating AMP expression, which is possibly important in obesity and metabolic disease.
AMP expression was also analyzed in a test-cohort of diabetes patients. From the same individuals intestinal biopsies and stool samples were obtained in order to analyze gut microbiota composition. This small cohort was used to test feasibility of the analyses and to review the challenging sampling technique. While feasibility was confirmed, the sampling technique needed to be optimized, which delayed the analysis of a larger human patient cohort.
The properties of the intestinal mucus layer were also studied in two different mouse models of obesity. While genetically obese mice exhibited only a weak effect on mucus function, mice with diet-induced obesity had a defective colonic mucus layer. Microbiota analyses suggested a causative effect of the gut bacteria, a hypothesis that we could prove experimentally. Currently we are performing the last experiments for a publication on these results.
In summary, in this project we found a weakened intestinal barrier in diet-induced obesity: In the small intestine AMP expression is reduced while the large intestine has a defective inner mucus layer.

1.4 Expected final results and their potential impact and use
In my project we found a weakened intestinal barrier in mouse models of obesity. Experiments with samples obtained from humans with obesity and diabetes are under way to confirm whether similar intestinal barrier defects exist in human obesity.
As a continuation of this project we are currently investigating whether dietary interventions can strengthen the intestinal barrier via modulation of the gut bacteria in mice. Once we identify whether the defective barrier function is cause or consequence of metabolic disease, these findings can be employed as a therapeutic strategy. As such, strategies involving probiotic, prebiotic or symbiotic approaches may open up novel non-invasive possibilities of interventions. In our hope, this would eventually help to treat the high number of individuals with metabolic disease.