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BestTreat – Building a Gut Microbiome Engineering Toolbox for In-Situ Therapeutic Treatments for Non-alcoholic Fatty Liver Disease

Periodic Reporting for period 1 - BestTreat (BestTreat – Building a Gut Microbiome Engineering Toolbox for In-Situ TherapeuticTreatments for Non-alcoholic Fatty Liver Disease)

Reporting period: 2018-09-01 to 2020-08-31

In recent years, the human gut microbiome imbalance has been found to affect a myriad of diseases such as cardiovascular disease and metabolic syndrome, with its components type-2 diabetes and obesity. Even though the interaction of gut microbiota and non-alcoholic fatty liver disease (NAFLD) is still poorly understood, recent research has shown that gut microbiome imbalance and intestinal permeability can play a role in NAFLD progression.
Non-alcoholic fatty liver disease (NAFLD) describes a range of conditions caused by the accumulation of excess fat in the liver. An estimation of 24% global occurrence is reported. It affects predominantly overweight and obese individuals, who drink little or no alcohol. In its early stage, simple steatosis, it is usually not causing any harm but can develop into severe liver damage, like steatohepatitis and liver cirrhosis. It is also believed to be a major cause for liver cancer.
The focus of research in BestTreat is to analyze the difference between microbiome of healthy people vs people with NAFLD. As it is a disease estimated to affect around one fourth of the world population, this research is of high importance for society.
The overall objectives of the program are:
i) To combine data- and hypothesis-driven approaches with emerging experimental technologies,
ii) To investigate the complex interactions between the gut microbiome and its host as well as,
iii) To understand the role of microbiome imbalance on NAFLD state and progression and to identify new therapeutic avenues for NAFLD.
A complete analysis of the metagenomic and metabolomic data of human subjects with NAFLD after a dietary intervention was performed. Research tasks completed include analysing the significantly changing species, performing correlation analysis between species, clinical data and metabolites, functional analysis, as well as integrating data from literature. All this data gathered together allowed us to short list the investigated species to 5 that have been pointed out as to play a role in NAFLD progression. The selected strains are currently used in follow up projects from the preclinical and engineering studies beneficiary teams for validation and mechanistic investigations. Furthermore, the following results were obtained:

Comparisons of the clinical effects to NAFLD under different intensity of exercise
The evaluation of the clinical effects to NAFLD under different intensity of exercise was assessed. A significant difference between the groups in WC (waist circumference), cardiorespiratory fitness level, achieved workload and fasting glucose concentrations was observed. However, other measured parameters such as the IHL (intrahepatic lipid content) content and two liver enzymes did not show a significant change for all participants. Therefore, we are now trying to understand whether the initial microbiome composition can be used to predict the improvement in the values of the liver function related parameters.

Protocol to culture strains that could be used as microbiome therapeutics
An efficient process allowing to target and selectively isolate gut microbes carrying genetic markers of interests was designed. Furthermore, this protocol was applied to isolate bacterial strains which are predicted to preferentially grow in specific regions of the gut. This approach allows strengthening the delivery of therapeutics. This key milestone provides strong foundations to progress towards the development of NAFLD therapies. Further developments are required on these lead candidates strains, in order to fully characterize their safety and capabilities, before the deployment in mouse and human trials. This protocol could as well be used to target other genomic markers of interest, significantly improving the scope of culture-based microbiome studies and therapies.

Association of gut microbiota and NAFLD/predictive role of microbiota to diagnosis
After 4 months of RS (resistant starch) intervention, compared with the CS (control starch) group, RS significantly lowered IHTC (intrahepatic triglyceride content) with a mean reduction of -9.08% after adjusting for baseline IHTC values. RS also reduced bodyweight, body fat, abdominal fat, liver enzymes, lipid profiles, fasting insulin, insulin resistance and serum FGF21. We identified metabolites and microbial species associated to RS intervention, some of which are also significantly correlated with the improvement of crucial clinical measurements for NAFLD. More importantly, these correlations and the beneficial effects of RS intervention on NAFLD were independent of body weight or other obesity-related phenotypes. Further multi-omics integration analyses of metabolome, microbial species and functions shed light on possible mechanistic links and revealed a panel of metabolites and gut microbiota species that are potentially related to the pathogenesis or amelioration of NAFLD. Through in vitro and in vivo experimental models, we were able to verify the role of multiple metabolites and one specific gut microbial species in NAFLD.
Progress beyond the state of art: The recent explosion of metagenomic data generation calls for new computational approaches that provide a more dynamic description of the host-microbe interactions. Applying microbiome systems biology approaches in BestTreat had allowed us to go beyond statistical and comparative studies and approach microbiome research across multiple levels. We are moving from correlative to causative evidence between shifts in the microbiome and pathophysiology of NAFLD, by cross-sectional studies, prospective studies and randomized controlled intervention studies in disease subjects and a combination of emerging new technologies, including transplantation of human fecal microbiota to germ free and evaluation of single species and microbial consortia in stem cell lines and artificial gut systems. By conducting a comprehensive bed-to-bench-to-bed study we expect to offer novel therapy solutions in the form of live biotherapeutics for NAFLD.
Expected results until the end of the project: BestTreat is working on researching and developing novel diagnostic and therapeutic approaches involving gut microbiome and this will be achieved by discovering bacterial strains that protect from NAFLD or contribute to slow down NAFLD progression.
Potential impacts: The BestTreat research program aims at meaningful research as well as integration and training to tackle challenges with high industrial and socio-economic impact for which no solutions are currently available. BestTreat will be of high importance to the future of European research within the field of gut microbiota, with direct applications on novel therapeutics, which will be beneficial to European industrial competitiveness.
BestTreat will train young researchers with use the most advanced methodologies to gain expertise in the most promising bacterial strains for live biotherapeutics, and that will be guided by internationally renowned experts from both academic and non-academic sectors towards innovative solutions with major impact for the European economy.
BestTreat focus, issue and outcome