Periodic Reporting for period 1 - BESIDE (Bile acids and epithelial stem cells in intestinal development)
Berichtszeitraum: 2018-04-01 bis 2020-03-31
BESIDE investigated the effect of secondary bile acids on gut maturation during the first stages of life at APC Microbiome Ireland. The preliminary data from this project showed exciting and potentially groundbreaking results. The gut microbiota, which is a complex community of microorganisms (bacteria, viruses, and fungi) that inhabit our gut, have key functions in our body including the development of the immune system, metabolisms and maturation of the gastrointestinal tract (GIT). The establishment of the gut microbiota starts at birth and develops over time coinciding with the maturation of the GIT suggesting that these early microorganisms and the compounds they produce may have an impact on gut development. Bile acids, the principal components of bile are released into the intestine to aid in the digestion of fats and vitamins. These bile acids are released conjugated to amino acids (primary bile acids), which are known to be toxic to bacteria. Some microorganisms of the gut are able to break the bile acids down through activity of a bacterial enzyme called bile salt hydrolase (BSH). These BSH positive bacteria have an impact on physiological processes and they also belong to some of the first colonizers of the GIT suggesting that BSH activity may have a crucial effect in the gut development and maturation. This project investigated how bacteria with this BSH activity influence the development of the GIT. The presence of microorganisms in the gut during the early stages of life is a critical event in the normal development of the GIT. Imbalances in the microbiota in early life have been linked to the development of long-term disorders (including asthma, atopic disease, and necrotising enterocolitis). Given the huge cost of treating these diseases, the preliminary results could have major implications for public health strategies because correcting imbalances in early development of the microbiota (through bacterial or biochemical interventions) could provide new modalities to treat or prevent a range of disorders. Some highlights from the BESIDE project included being nominated and selected as a representative of the EU MSCA programme to attend the 70th Lindau Nobel Laureate Meeting and being hired immediate afterwards by the Centre for Research in Vascular Biology.
The BESIDE project had 6 work packages (WPs) and the following work has been conducted and main results achieved:
WP1. Effects of BSH activity on proliferation of intestinal epithelial stem cells (IESCs): During this WP the fellow was trained in state-of-the-art flow cytometry techniques and she developed optimized and reproducible protocols for the isolation of IESCs from the murine small intestine and colon. Once isolated, the IESCs were successfully grown as 3D in vitro models. To determine the effects of BSH, different concentrations of bile acids at different times were added to these cells and the toxic effect was evaluated with different viability tests. Once the optimal concentration and exposure time was established, a screening of over 50 genes involved in differentiation and proliferation was carried out. This led to the selection of target genes to be analysed in the rest of the WP.
WP2. Effect of BSH activity impact on IESCs differentiation and crypt development: Isolated IESCs were grown in different media in order to promote differentiation. However, the IESCs failed to differentiate in all the media tested. Thus, crypts isolated from the small intestine and the colon, were used instead of IESCs. This gave us a robust protocol to grow both types of crypts, which were differentiated into organoids. Protocols for determination of proliferation and differentiation rates using flow cytometry were established. The RT-qPCR analyses for small intestine organoids were completed using the gene panel selected in WP1. Results showed that BSH led to a modulation in the expression of the differentiation and proliferation markers. The protocol for the fixation of organoids and staining with differentiation markers was optimized for analysis by confocal microscopy in University College Dublin. The RT-qPCR analyses of colon organoids and the confocal microscopy had to take place in March 2020 but, due to the COVID-19 outbreak, Universities in the Republic of Ireland are closed so they had to be postponed.
WP3. In vivo impact of BSH activity on GIT development in a murine germ-free model: Two separate animal trials were performed, one using a bacteria expressing BSH and one using individual bile acids to evaluate the effect in the GIT development and maturation. A panel of 25 genes was evaluated in both experiments. Results showed that BSH activity is able to modulate key genes involved in maturation. Histological and immunohistochemistry assays showed that BSH expression stimulated an increase in a number of differentiation parameters as well as for molecular markers. In addition, an extra deliverable was included in this WP. The analysis of metabolites was performed at the Centre for Omic Sciences (Tarragona), which allowed to strength the results obtained from these experiments.
WP4. Communication and Dissemination: The dissemination plan was discussed on regular meetings. There is a draft review paper and journal article in preparation. Also, there is a second paper expected to be released by 2021, as some of the experiments had to be postponed. The work carried out has been also shown in different workshops and to non-scientific audiences as a result of the participation in different outreach activities.
WP5 and WP6 were related to Training and Career Development and Transfer of Knowledge and Management. All deliverables and milestones included in these WPs have been achieved.
Regular meetings with the group facilitated the evaluation of the project’s progress and to troubleshoot any issue related to all WP progression.
WP1. Effects of BSH activity on proliferation of intestinal epithelial stem cells (IESCs): During this WP the fellow was trained in state-of-the-art flow cytometry techniques and she developed optimized and reproducible protocols for the isolation of IESCs from the murine small intestine and colon. Once isolated, the IESCs were successfully grown as 3D in vitro models. To determine the effects of BSH, different concentrations of bile acids at different times were added to these cells and the toxic effect was evaluated with different viability tests. Once the optimal concentration and exposure time was established, a screening of over 50 genes involved in differentiation and proliferation was carried out. This led to the selection of target genes to be analysed in the rest of the WP.
WP2. Effect of BSH activity impact on IESCs differentiation and crypt development: Isolated IESCs were grown in different media in order to promote differentiation. However, the IESCs failed to differentiate in all the media tested. Thus, crypts isolated from the small intestine and the colon, were used instead of IESCs. This gave us a robust protocol to grow both types of crypts, which were differentiated into organoids. Protocols for determination of proliferation and differentiation rates using flow cytometry were established. The RT-qPCR analyses for small intestine organoids were completed using the gene panel selected in WP1. Results showed that BSH led to a modulation in the expression of the differentiation and proliferation markers. The protocol for the fixation of organoids and staining with differentiation markers was optimized for analysis by confocal microscopy in University College Dublin. The RT-qPCR analyses of colon organoids and the confocal microscopy had to take place in March 2020 but, due to the COVID-19 outbreak, Universities in the Republic of Ireland are closed so they had to be postponed.
WP3. In vivo impact of BSH activity on GIT development in a murine germ-free model: Two separate animal trials were performed, one using a bacteria expressing BSH and one using individual bile acids to evaluate the effect in the GIT development and maturation. A panel of 25 genes was evaluated in both experiments. Results showed that BSH activity is able to modulate key genes involved in maturation. Histological and immunohistochemistry assays showed that BSH expression stimulated an increase in a number of differentiation parameters as well as for molecular markers. In addition, an extra deliverable was included in this WP. The analysis of metabolites was performed at the Centre for Omic Sciences (Tarragona), which allowed to strength the results obtained from these experiments.
WP4. Communication and Dissemination: The dissemination plan was discussed on regular meetings. There is a draft review paper and journal article in preparation. Also, there is a second paper expected to be released by 2021, as some of the experiments had to be postponed. The work carried out has been also shown in different workshops and to non-scientific audiences as a result of the participation in different outreach activities.
WP5 and WP6 were related to Training and Career Development and Transfer of Knowledge and Management. All deliverables and milestones included in these WPs have been achieved.
Regular meetings with the group facilitated the evaluation of the project’s progress and to troubleshoot any issue related to all WP progression.
This project has exciting preliminary results that go beyond the current state-of-the-art and could potentially form the basis of an ERC Starter application. Colonization of the gut in the early stages of life is a critical event in the normal development of the GIT barrier and imbalances in this association have been linked to the development of long-term disorders (including asthma, ectopic disease, and necrotising entercolitis). The underlying premise of BESIDE is that understanding the effect of the early colonizers in the GIT maturation and development as well as the molecular events that take place from such microbe-host association is fundamental in order to provide new modalities to treat or prevent a range of such disorders. My research showed that BSH activity helps for the maturation of the GIT. This has wide reaching implications given the huge cost of these diseases for the European economy. Therefore, the preliminary results within BESIDE could have major implications for public health strategies most notably through correcting the imbalances in early development of the microbiota (through bacterial or biochemical interventions). This cross-cutting interdisciplinary research directly relates the objective of Horizon 2020 Work Programme (i.e. Social Challenge 1: Health, Demographic Change and Wellbeing) which aims to promote an effective health campaign towards disease prevention. It also could also potentially contribute to finding therapies or preventing some non-communicable diseases or mitigating their symptoms.