Defining gut microbiome and intestinal epithelial cell development, maturation, and maintenance.
The digestive tract is crucial for our wellbeing. The intestine helps retain our good health via an interplay between the microbiome, intestinal epithelial cells, and immune cells. Intestinal homeostasis is a research topic that is gaining significance outside the traditional gut research field, including gut-lung and gut-brain axis studies with relevance for diseases such as allergic asthma and Alzheimer’s respectively. We lack fundamental knowledge of the establishment of the microbiome and intestinal epithelium.
A large part of intestinal development occurs in the neonatal period. In mice, in the first few weeks after birth, a dramatic switch occurs, especially in the microbial and epithelial compartments. Adult intestinal stem cells and Paneth cells start to appear 14 days after birth (P14). The microbiome changes drastically between P14-P28, seemingly following epithelial maturation, but coinciding with weaning and the introduction of solid foods.
In order to research the establishment of both the intestinal epithelium and the microbiome, we will use three different types of mice, WT mice, LSD1-KO mice, which fully deletes intestinal epithelial Lsd1 and i-LSD1KO mice, which allows us to induce LSD1KO in a later stage. Since LSD1 deletion results in a total lack of Paneth cells, this model will allow us to answer the following questions; Does neonatal maturation of the epithelium, including the appearance of Paneth cells, control the microbiome or vice versa? And, what are determining host factors for this sequence of events?
Microbiome research heavily uses external measures to study the role of gut commensal bacteria. Specifically, the use of germ-free mice or antibiotics to eradicate the microbiome followed by re-colonization are commonly used. We propose to treat pregnant mice with an antibiotic cocktail and will continue antibiotic treatment of pups. This will eradicate most of the microbiome. In addition, to test if adult mature epithelium is actively maintaining the microbiome composition, we will use i-LSD1KO mice. A published protocol using Tamoxifen will be followed to fully delete Lsd1 in the intestinal epithelium of 6 wk old mice. In addition, using an intraperitoneally administered monoclonal antibody, we will study the effect of blocking CXCL16 in innate lymphoid cell recruitment and composition in i-LSD1KO mice.
Then we will assess WT and LSD1KO mice gut microbiome, local intestinal immune cell composition and body growth.
Unfortunately, there are currently no alternatives to animals to analyze neonatal epithelial and microbiome development. This complex, multifactorial process is regulated by different cell types in constant communication with the environment, making it impossible to model this in vitro. However, to limit animal use, we will use in vitro organoid systems when possible. By doing this, we can grow organoids to perform specific analysis derived from a smaller number of mice.
We estimate the total use of 222 mice in this project.
To guarantee the minimum distress level in these mice, animals will be monitored for symptoms of pain,weight loss and dehydration, and will be sacrificed following humane endpoints (as described below and in the attachments). However, mice will be closely monitored to prevent unnecessary suffering.
A large part of intestinal development occurs in the neonatal period. In mice, in the first few weeks after birth, a dramatic switch occurs, especially in the microbial and epithelial compartments. Adult intestinal stem cells and Paneth cells start to appear 14 days after birth (P14). The microbiome changes drastically between P14-P28, seemingly following epithelial maturation, but coinciding with weaning and the introduction of solid foods.
In order to research the establishment of both the intestinal epithelium and the microbiome, we will use three different types of mice, WT mice, LSD1-KO mice, which fully deletes intestinal epithelial Lsd1 and i-LSD1KO mice, which allows us to induce LSD1KO in a later stage. Since LSD1 deletion results in a total lack of Paneth cells, this model will allow us to answer the following questions; Does neonatal maturation of the epithelium, including the appearance of Paneth cells, control the microbiome or vice versa? And, what are determining host factors for this sequence of events?
Microbiome research heavily uses external measures to study the role of gut commensal bacteria. Specifically, the use of germ-free mice or antibiotics to eradicate the microbiome followed by re-colonization are commonly used. We propose to treat pregnant mice with an antibiotic cocktail and will continue antibiotic treatment of pups. This will eradicate most of the microbiome. In addition, to test if adult mature epithelium is actively maintaining the microbiome composition, we will use i-LSD1KO mice. A published protocol using Tamoxifen will be followed to fully delete Lsd1 in the intestinal epithelium of 6 wk old mice. In addition, using an intraperitoneally administered monoclonal antibody, we will study the effect of blocking CXCL16 in innate lymphoid cell recruitment and composition in i-LSD1KO mice.
Then we will assess WT and LSD1KO mice gut microbiome, local intestinal immune cell composition and body growth.
Unfortunately, there are currently no alternatives to animals to analyze neonatal epithelial and microbiome development. This complex, multifactorial process is regulated by different cell types in constant communication with the environment, making it impossible to model this in vitro. However, to limit animal use, we will use in vitro organoid systems when possible. By doing this, we can grow organoids to perform specific analysis derived from a smaller number of mice.
We estimate the total use of 222 mice in this project.
To guarantee the minimum distress level in these mice, animals will be monitored for symptoms of pain,weight loss and dehydration, and will be sacrificed following humane endpoints (as described below and in the attachments). However, mice will be closely monitored to prevent unnecessary suffering.