Multidimensional analysis of the microbial and genetic links in inflammatory bowel disease

Abstract

Over 200 genes have been associated with IBD. Most of these are responsible for bacterial sensing and immune processing. Little is known about the dynamics of the gut microbiome over time, in the early Crohn's lesion and in mesenteric lymph nodes. Aims of the studies To detect differences in the IBD microbiome over time (longitudinal study), space (lymph node study) and in the early lesion (aphthous ulcers versus normal tissue). To use whole genome sequencing (WGS) to find a rare gene in a subset of IBD patients with PSC-IBD.

Study 1: The role of inflammation in temporal shifts in the IBD mucosal microbiome. We studied the mucosal microbiome of patients who underwent multiple procedures over time (average 2.4 years between each procedure). We found significantly lower alpha diversity (within sample variation) in patients who had undergone previous abdominal surgery. There were no significant associations with antibiotics, smoking, IBD subtype, inflammation or medication class. Significantly, we found great changes in beta diversity (between-sample variation) in patients with active inflammation at both time-points. This suggests that inflammation is closely associated with shifts in the microbiome over time.

Study 2: The microbiome of translocated bacterial populations in patients with and without IBD. We interrogated the mesenteric lymph nodes of patients (and controls) who had undergone intestinal resection, using the presence of bacterial RNA as a surrogate for viability. We found large numbers of viable bacteria present in patients with IBD and surgical controls. The bacterial populations were similar to those at the mucosal surface, however Escherichia/Shigella were significantly more abundant in lymph nodes compared with mucosal sites, which gives further evidence of involvement of these bacteria in IBD pathogenesis.

Study 3: The microbiome of Crohn's disease aphthous ulcers. In this study, we compared the microbiome of the early Crohn's lesion with that of adjacent mucosa, and healthy controls. We found that the microbiome did not significantly differ, and that changes that had previously been associated with IBD were not present in the early Crohn's lesion. This was also true for the three patients with new-onset disease. This provides further evidence to the hypothesis that inflammation causes dysbiosis. A separate study demonstrated that the transcriptome of aphthous ulcers is very different to that of healthy mucosa and Peyers patches.

Study 4: Whole genome sequencing in primary sclerosing cholangitis (PSC) and IBD. Genome-wide association studies (GWAS) have previously identified 22 genes associated with PSC. WGS has been performed before in PSC. This allows the discovery of rare mutations. We performed WGS on 10 patients with PSC/IBD and 16 of their parents, to identify rare alleles or novel mutations. We also analysed the 22 previously identified loci from GWAS. While we did not find any novel loci, we did find that our patient population had a higher-than-expected frequency of a PTPN2 mutation compared with the broader population (63% versus 38%, p=0.015), further implicating this gene.

Overall summary of findings The dramatic shifts that have been consistently observed in the IBD microbiome correlate with the presence of ongoing inflammation. We have demonstrated that these changes do not appear to be present in the early Crohn's lesion. This implicates inflammation itself as the cause of these changes. The altered microbiome therefore may not be relevant in the causation of ongoing immune activation. Moreover, bacteria that are able to evade the initial immune defences appear to be very important in IBD. These findings may help to explain the tepid clinical response that treatments directed at altering the microbiome in IBD have had in obtaining and maintaining remission (antibiotics and faecal microbial transplant).