Tackling the complex regulation of individual intestinal cells
Exploring how individual epithelial cells respond to challenges in the gut.
All animals, including humans, are ecosystems living together with microbial partners which are often essential for the development and function of organs. While we have substantial knowledge of how human cells are communicating with each other to affect cellular processes such as differentiation and metabolism, our understanding is far more limited on how similar signals produced by microbes affect host processes.
Using multidisciplinary approaches based on experimental data and computational tools, we are investigating key microbial influences on epithelial cells in healthy and diseased states of the digestive system.
For that we implement cutting-edge single cell sorting/RNA sequencing approaches on intestinal cell type-specific individual cells challenged with defined microbes. Using specific gene deletion mutants of these microbes allows us to validate the specific impact microbes have on host cellular processes in a cell type-specific manner.
The digestive tract is lined by a single layer of different epithelial cells, each type performing specialised functions important for health such as production of antimicrobials, production of a mucus protective layer and absorption of nutrients etc. All epithelial cell types are derived from stem cells, key undifferentiated cells located at the bottom of the crypts of both small and large intestines (Top left panel figure above). The small population of stem cells replenishes the whole epithelium, which is exposed daily to a wide variety of dietary and microbial compounds, within a few days, yet it maintains an equilibrium (i.e. homeostasis) and functions in a healthy state.
Given the intimate relationships between microbes and human epithelial cells lining the gut (the intestine), microbes are likely to tightly regulate human cells – with changes in the microbial community (microbiota) leading to altered human cell functions (potentially causing disease). When homeostatic barrier functions are breached, dysregulation of many barrier functions occurs, as seen in various digestive pathologies (Inflammatory Bowel Diseases, infections, cancers, etc.).
Despite rapidly accumulating knowledge of microbiota-epithelial cell interactions and disease associations, no detailed mechanisms have been published that could significantly improve prevention and treatment in clinical practice.
Furthermore, until the recent emergence of single cell (and low cell input) RNA sequencing it was impossible to define cell type-specific profiles of the specialised cell types of a tissue (e.g. intestine) nor how these could vary between cells of the same type. Many studies based on the analysis of mixed cell populations failed to clearly identify a response pattern due to the noise inherent to mixed populations.
In recent years established protocols became available for single cell isolation and for interpreting the transcriptome of those cells enabling us to work with rare cell types and to differentiate between individual cells in a population. A working pipeline has been established at EI that now allows us to analyse RNA sequencing data at the single cell level.
We are sorting the cells of interest from samples consisting of different intestinal cell types (Paneth cells, goblet cells, stem cells, etc.) or infected vs. non-infected cells.
Specific cell lineages and surface markers are used to identify the cell populations of interest and physically harvest them using the FACSMelody cell sorter located in EI.
In addition, for microbe-containing host cells, sorting with fluorescently tagged microbial strains is used - allowing us to measure microbial influences in cells directly or indirectly interacting with microbes.
The single cell RNA sequencing is carried out in the Genomics pipeline at EI.
Monitoring how the response of individual intestinal cells varies within each cell type upon challenge with either other host cells or with microbes will define subpopulation cell behaviour and therefore increase the resolution at which we might understand what drives the overall response of intestinal tissue.
The regulatory network we are establishing within cells, between cells, between cell types and in response to microbial influence will contribute to identifying regulatory nodes in cellular pathways where intervention could be tested to restore epithelial homeostasis when it is perturbed or to reduce the impact of these alterations.