Evaluating the importance of autophagy/xenophagy in protecting the intestinal epithelium in Crohn s Disease

The inflammatory bowel diseases (IBD) are disabling life-long illnesses which are common in the Western world. Typical symptoms common to both illnesses include abdominal pain, weight loss, and diarrhoea. There are two main types of inflammatory bowel disease: Crohn?s disease (CD) and ulcerative colitis (UC). The cause of these illnesses is not certain, but it is now very clear that each condition is influenced by a number of genetic risk factors as well as environmental factors. These include the presence of bacteria in the bowel and smoking habit. Recently, great progress has been made in identifying several new genetic risk factors involved in Crohn?s disease. Two of the most recently identified genes are involved in a process called autophagy, a mechanism by which cells conserve or recycle specific proteins to keep themselves alive in conditions of starvation, or under specific environmental ?stressors?. There is some evidence that autophagy might also be involved in the maintenance of a healthy gut, removing or killing ?bad? bacteria and allowing ?good? bacteria to live there. Changes in the regulation of the autophagy pathway might upset this delicate balance or allow ?good? bacteria to produce unwanted responses from the gut cells, leading to inflammation. In a series of laboratory experiments, we aim to investigate this pathway and to clarify how function is changed in CD patients and the effect of different environmental ?stressors?, and of genetic alterations. Results will indicate whether this pathway is involved in the cause or development of CD and may also indicate the molecular pathways involved which could potentially lead to the development of new drug therapy.

The inflammatory bowel diseases (IBD), Crohn?s disease (CD) and ulcerative colitis (UC) are common causes of gastro-intestinal morbidity in UK, affecting 1 in 250 individuals. Over the last decade there has been striking progress in the understanding of mechanisms of pathogenesis of IBD with many insights from human genetics and genetically-engineered animal models. Focus has moved from the specific immune response towards evaluation of the innate intestinal response in the gastro-intestinal tract with the identification in 2001 of NOD2, an intracellular receptor for muramyl dipeptide, as the first susceptibility gene in CD.
Most recently, several other susceptibility genes for CD have been identified in genome-wide association studies (GWAS) including two genes, ATG16L1 and IRGM, which are involved in autophagy. These discoveries provide the catalyst for the present study; to examine this pathway in CD, and specifically to examine its importance and relevance to the innate immune response in the intestinal epithelium in CD. Autophagy represents a homeostatic mechanism for the removal and turnover of proteins and organelles via the lysosome, as well as an adaptive response to starvation to provide an energy source to maintain cell viability. In addition, autophagy provides defence against specific intracellular bacteria, which are engulfed by autophagosomes and transferred to lysosomes for degradation, a process now known as xenophagy.
In our initial studies, we aim to define the pattern of dysregulation of autophagy/xenophagy characteristic of CD and to identify molecular mechanisms involved. We shall evaluate intestinal biopsies and peripheral blood from CD patients, and controls using available assays.
We shall then explore the relationship between autophagy and NOD2 signalling pathways both in cell lines and in short?term organ culture of intestinal biopsies, thereby building on genetic data implying epistatic interaction and preliminary data that we have generated, which suggest CD-associated NOD2 variation may affect autophagolysosome stability. We shall explore functional consequences of the disease-associated ATG16L1 gene mutation on protein-protein interactions involved in autophagosome formation and stability and on NOD2 gene and protein expression and downstream effects of NFkB activation, defensin, and chemokine synthesis/secretion.
To assess gene-environmental interactions, we shall explore whether pathogen-associated motifs (LPS, MDP or PG) or cigarette smoke constituents affect autophagy pathways and ATG16L1/IRGM gene expression in cell lines and ex vivo organ culture).
Finally we shall address specifically whether dysregulated autophagy allows survival of entero-invasive E. coli strains, now strongly associated with CD, in the intestinal epithelium.