Abstract
Gut microbiota comprise important environmental exposures that influence human immune systems and may alter the clinical course of inflammatory bowel disease (IBD). Little is known about the role of gut bacteriophages (viral components that infect prokaryotic bacteria) and their interactions with the host’s immune responses. We tested the hypotheses that (1) immune responses of individuals with IBD to phages differ from those without IBD and (2) immune responses to phages are associated with disease type (i.e. those with Crohn’s disease have different responses than those with ulcerative colitis).
We have constructed the first bacteriophage peptidome library (“phageome”), based on sequencing of environmental phages and large-scale metagenomic sequencing of virus-like particles isolated from stool samples from IBD patients and their non-IBD household contacts. Using Phage ImmunoPrecipitation Sequencing (PhIP-Seq) technology, we generated complete serum antibody binding profiles of 48 IBD patients (16 ulcerative colitis, 11 Crohn’s, and 11 indeterminate), 9 of their non-IBD household contacts, and an independent non-IBD cohort of 674 volunteers collected by the Vaccine Research Center (VRC) at the National Institutes of Health. Antibody binding profiles were compared among groups using nonparametric statistics.
IBD patients as a group had lower antibody responses to specific phages compared to both non-IBD household contacts and the non-IBD VRC controls; this difference was significant and remained after control for unequal sample sizes [Figure 1]. Patients with Crohn’s disease compared to those with ulcerative colitis had similar antibody responses. Particularly for phages of the genera Phifelvirus, the immune responses of Crohn’s patients were significantly reduced compared to their non-IBD household contacts, while the immune responses of patients with ulcerative colitis did not significantly differ from non-IBD household contacts [Figure 2]. IBD disease type comparisons to the VRC controls yielded similar results.
Violin plots showing antibody enrichments (hits) to four phage genera for each individual in the healthy control (VRC) cohort (n=674) compared to enrichments for each individual in the IBD cohort (n=48). Hits to these four phage genera showed the most statistically significant differences in immune responses between the VRC and IBD cohorts. Mann-Whitney statistics used for comparisons.
Violin plots showing antibody enrichments (hits) to three phage genera for each individual with Crohn’s disease (n=11) compared to each individual with ulcerative colitis (n=16) and each non-IBD household control (n=9). Hits to these three genera showed the most statistically significant differences in immune responses between these three cohorts. Kruskal-Wallis and Mann-Whitney statistics used for comparisons. * denotes p ≤ 0.05, while ** denotes p ≤ 0.01.
PhIP-Seq with a phageome library can be used to study the relationship between immune responses and gut bacteriophages in IBD. Our results suggest that IBD patients may have lower antibody responses to specific phages compared to non-IBD individuals. Differential antibody reactivities in Crohn’s disease vs. ulcerative colitis compared to their household contacts and VRC controls suggest disease-specific response to the gut phageome that warrant further study.
