Visualization and quantification of autophagy in primary gastrointestinal epithelia cell cultures.
Autophagy is an intracellular bulk degradation pathway which can be used during cellular maintenance to encapsulate and target intracellular content such as organelles and proteins to be degraded by the lysosome. The autophagosome is a double membrane vesicle which is used to encapsulate this cellular content. During the cellular response to starvation, autophagy can degrade cytoplasmic organelles and long-lived proteins in lysosomes, to recover amino acids within the cell and form new proteins. Stimulation of autophagy during infection can enhance immune responses by degrading pathogens and increasing presentation of microbial components to the immune system. Genome wide association studies (GWAS) have identified mutations in the autophagy related (Atg) protein Atg16L1, and NOD2 as risk alleles for Crohn’s disease. Atg16L1 is essential for the formation of autophagosomes and autophagosome formation is stimulated following recognition of bacterial cell wall peptide muramyl dipeptide (MDP) by NOD2. Crohn’s disease results from an autoimmune reaction to gut epithelial cells, and this raises the possibility that the risk alleles alter autophagy and/or NOD2-mediated microbial sensing in gut epithelial cells. The aim of this thesis has been to develop methods to quantifying autophagy in primary epithelial cells cultured from human intestinal biopsy material. Atg8/LC3 is the major structural protein of the autophagosome. Images of GFP-Atg8/LC3 puncta generated in response to starvation, or NOD2 signalling, were taken by fluorescence microscopy, and pixel densities were rendered to generate digital datasets that were amenable to statistical analysis. Rendered puncta were displayed graphically to indicate autophagosome diameters, spatial location within the cell, lifetime, expansion and movement. The methods were validated in tissue culture cell lines and then extended to study human colon crypts cultures transduced by adenoviral vectors expressing GFP-LC3. In comparison to cell lines after starvation, there were less GFP LC3 puncta in the smaller epithelial cells from colon crypt tissue (20-30 compared to 5.8+/-2.2). MDP generated significantly more LC3 puncta (9.5+/-4) in colon crypt epithelial cells compared to starvation (5.8+/-2.2). This work provides a method to quantify autophagy in colon crypt cultures, and if applied to biopsies taken from patients will be able to determine if autophagy and NOD2 sensing are altered in patients carrying with Crohn’s disease risk alleles. Keywords: Autophagy, in vitro culture, adenoviral transduction, Imaris quantification.
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