The Human Mesenteric Lymph Node Microbiome Differentiates Between Crohn's Disease and Ulcerative Colitis

Miranda G Kiernan, J Calvin Coffey, Kieran McDermott, Paul D Cotter, Raul Cabrera-Rubio, Patrick A Kiely, Colum P Dunne, Miranda G Kiernan, J Calvin Coffey, Kieran McDermott, Paul D Cotter, Raul Cabrera-Rubio, Patrick A Kiely, Colum P Dunne

Abstract

Background and aims: Mesenteric lymph nodes are sites in which translocated bacteria incite and progress immunological responses. For this reason, understanding the microbiome of mesenteric lymph nodes in inflammatory bowel disease is important. The bacterial profile of Crohn's disease mesenteric lymph nodes has been analysed using culture-independent methods in only one previous study. This study aimed to investigate the mesenteric lymph node microbiota from both Crohn's disease and ulcerative colitis patients.

Methods: Mesenteric lymph nodes were collected from Crohn's disease and ulcerative colitis patients undergoing resection. Total DNA was extracted from mesenteric lymph nodes and assessed for the presence of bacterial DNA [16S]. All work was completed in a sterile environment using aseptic techniques. Samples positive for 16S DNA underwent next-generation sequencing, and the identity of bacterial phyla and species were determined.

Results: Crohn's disease mesenteric lymph nodes had a distinctly different microbial profile to that observed in ulcerative colitis. The relative abundance of Firmicutes was greater in nodes from ulcerative colitis patients, whereas Proteobacteria were more abundant in Crohn's disease. Although species diversity was reduced in the mesenteric lymph nodes of patients with Crohn's disease, these lymph nodes contained greater numbers of less dominant phyla, mainly Fusobacteria.

Conclusion: This study confirms that there are distinct differences between the Crohn's disease and ulcerative colitis mesenteric lymph node microbiomes. Such microbial differences could aid in the diagnosis of Crohn's disease or ulcerative colitis, particularly in cases of indeterminate colitis at time of resection, or help explain their mechanisms of development and progression.

Figures

Figure 1.
Figure 1.
Mesenteric lymph node [MLN] mapping. [A] Digital image of the small and large bowel with associated continuous mesentery. Source locations of MLNs are mapped on the mesentery. [B] Proportions of MLNs taken from each location of ulcerative colitis [UC] mesentery. The majority of lymph nodes were taken from the mesorectum [42.9%]. [C] Proportions of MLNs taken from each location of Crohn’s disease [CD] mesentery. The majority of lymph nodes were taken from the ileocolic region [87.5%]. [D] Proportions of 16S PCR–positive MLNs taken from each location of UC mesentery. [E] Proportions of 16S PCR–positive MLNs taken from each location of CD mesentery.
Figure 2.
Figure 2.
Relative abundance of predominant bacterial phyla in mesenteric lymph nodes [MLNs] from inflammatory bowel disease [IBD] patients. There was a distinct difference in the profile of phyla from MLNs of Crohn’s disease [CD] and ulcerative colitis [UC] patients. S = sample number, M = merge of samples of same node.
Figure 3.
Figure 3.
Abundance of phyla in pooled mesenteric lymph nodes [MLNs] of Crohn’s disease [CD] and ulcerative colitis [UC] patients. [A] Quantities [abundance %] of major bacterial phyla Firmicutes, Bacteroides, Proteobacteria, and Actinobacteria in pooled MLNs of UC and CD patients. [B] Statistical analysis of phyla abundance with 95% confidence interval [CI]. MLNs of UC patients had higher proportions of Firmicutes [Mean ± 95% CI: 52.8% ± 6.73% vs 18.6% ± 4.65%; p < 0.0001, independent t-test], whereas MLNs of CD patients had higher proportions of Proteobacteria [36.1% ± 11.06% vs 11.8% ± 3.63%; p = 0.005, Mann–Whitney U test] and other type bacteria, comprising mainly Fusobacteria [7.3% ± 4.98% vs 0.3% ± 0.15%; p < 0.0001, Mann–Whitney U test]. Independent t-tests were used to compare normally distributed [parametric] data, and Mann–Whitney U tests were used to compare non-parametric data. [C] The ratio of Firmicutes to Bacteroidetes in MLNs from UC [1.613] and CD [0.705] patients.
Figure 4.
Figure 4.
Principal coordinate analysis [PCoA] of Unifrac difference. The chart is based on unweighted unifrac distances and displays variation between cohort samples.
Figure 5.
Figure 5.
Microbial diversity of mesenteric lymph nodes [MLNs] from Crohn’s disease [CD] and ulcerative colitis [UC] patients. Shannon Diversity Indices for MLNs of UC and CD patients defined using data from 16S sequencing data at species level. Data are presented as mean ± 95% confidence interval [CI]. MLNs from UC patients demonstrated significantly greater microbial diversity than those of CD patients [1.95 ± 0.0035 vs 0.79 ± 0.0018; p < 0.0001, Mann–Whitney U test]. As data were found to be non-parametric [not-normally distributed], Mann–Whitney U tests were used to compare data.

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