The role of autophagy in the innate immune response in primary cells and organoids

Buck, Jasmine (2015) The role of autophagy in the innate immune response in primary cells and organoids. Doctoral thesis, University of East Anglia.

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Abstract

Crohn’s disease is an inflammatory condition that can affect any area of the gastrointestinal tract, mainly the distal small intestine (ileum). Genome wide association studies (GWAS) have identified a small nucleotide polymorphism (SNP) in the autophagy gene ATG16L1, conferring a T300A mutation, as a susceptibility allele for Crohn’s disease. Autophagy is an intracellular degradation pathway that captures damaged or long-lived proteins and organelles within a double-membrane autophagosome and delivers its cargo to lysosomes. Autophagy can degrade intracellular pathogens, such as Salmonella and Sindbis virus (SINV), and can regulate immune signalling during infection. ATG16L1 is essential protein in the autophagy pathway.
Mice deficient in ATG16L1 (ATG16L1-/- mice) and mice deficient in ATG16L1 specifically in intestinal epithelial cells (ATG16L1ΔIEC mice) were generated in the lab. Electron microscopy showed swollen and damaged mitochondria in the ileal epithelial cells of ATG16L1ΔIEC mice. ATG16L1-deficient mouse embryonic fibroblasts (MEFs) were also generated and ATG16L1ΔIEC mice were used to establish small intestinal organoid cultures lacking ATG16L1. The aim of this thesis was to use these primary cells and organoid cultures to investigate the role of ATG16L1 in the innate immune response and mitochondrial function at a cellular level.
Crypt isolation and organoid culture procedures were optimised using control mice. Growth and differentiation of organoid cultures generated from control and ATG16L1ΔIEC mice were not found to be significantly different and proteomic mass spectrometry analysis showed similar protein expression levels.
Loss of ATG16L1 had no significant effect on the innate response to Salmonella typhimurium in mice in vivo, determined by histological staining to compare levels of inflammation and immunostaining of Paneth cell anti-microbial granule morphology.
Interestingly, following infection with a model RNA virus, Sindbis virus (SINV), loss of autophagy in transformed MEF cells led to an increase in transcription of interferon-beta (IFN-β) and interferon stimulated genes (ISG), compared to wild-type (WT) MEFs. Primary ATG16L1 knock-out MEF cells also showed increased ISG mRNA transcription upon SINV infection however there was no change in type 1 interferon transcription. In small intestinal 3 organoids however, there was a greater transcriptional induction of ISG mRNA following SINV infection of wild-type organoids compared to ATG16L1-deficient organoids.
In contrast to investigations with mouse intestinal tissue, there was no significant difference in mitochondrial morphology, mitochondrial membrane potential and reactive oxygen species levels between wild-type and ATG16L1 KO MEF cells or organoid cultures. Significantly, however, caspase-3 activation levels were found to be elevated in ATG16L1 KO MEF cells. This implies that ATG16L1 plays a role in controlling apoptosis or mitochondrial function through caspase-3 activation.
The findings of this study will help aid our understanding of the aetiology of Crohn’s disease with regards to the role of autophagy and pathogenic challenge.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User:	Megan Ruddock
Date Deposited:	15 May 2018 14:44
Last Modified:	15 May 2018 14:44
URI:	https://ueaeprints.uea.ac.uk/id/eprint/67071
DOI:

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