Abstract
The origin of the heterogeneous clinical presentation of Crohn's disease (CD) is poorly understood. We therefore aimed to characterise the molecular networks in inflamed tissue of CD patients with ileal and/or colonic disease location, and how they relate to control colon and ileum.
Inflamed colonic (n = 31, 8 L2 + 23 L3) and ileal (n = 31; 17 L1 + 14 L3) biopsies were collected from newly diagnosed CD patients across three Belgian IBD centres (PANTHER study B322201627472/S57662). Patients naïve for biologicals and immunosuppressives, and without previous IBD-related surgery were included within 6 months after diagnosis. Thirty-six colonic and 14 ileal biopsies from non-IBD were used as controls. All biopsies underwent single-end RNA sequencing, and downstream data were corrected for age and gender. Co-expression networks (correlation ≥ 0.55, adj. p ≤ 0.05) were found with weighted gene co-expression network analysis (WGCNA), and pathways with IPA.
WGCNA identified 10 co-expression clusters. The number of genes in these clusters ranged from 180 to 1611. Six clusters were significantly correlated with our clinical traits (colonic CD or ileal CD compared with controls) (Figure 1, left).
Figure 1. Co-expression cluster trait correlations: Weighted Gene Co-expression Network Analysis (WGCNA) was performed on inflamed colonic and ilea( lakpsies of newly diagnosed CD patients and normal biopsies from non-lBD controls_ First, an unsupervised approach clustered similarly expressed genes info groups (termed 'clusters?, which were then tested for correlation with traits of interest (colonic CD vs. normal colon; deal CD vs. normal ileum; control ileum vs. control colon). Only significant (correlation r N155, adip 0.05) are represented (correlation strengths r with adjusted p-values in brackets). Positive correlations represent an up-regulation of the tested trail =1, while negative correlations represent a down-regulation of the tested trait =1). CD, Crohn's disease; r, correlation
Figure 1. Co-expression cluster - trait correlations_2.
More specifically, clusters I and IV were down-regulated in both traits, while clusters II and III were up-regulated in both. Cluster V was down-regulated in ileal CD, and clusters VI and VII were, respectively, down- and up-regulated in colonic CD. IPA analyses showed that cluster I was enriched in xenobiotic metabolism, cluster II in antigen presentation, cluster III in (a)granulocyte adhesion/diapedesis, cluster IV in AMPK signalling, cluster V in melatonin degradation, cluster VI in ERK5 signalling, and cluster VII in Th1 and Th2 activation. Interestingly, clusters I, II, V and VII were up-regulated in ileal vs. colonic location in controls, meaning that in controls, these clusters are more specific to the ileum than to the colon (Fig. 1, right). Cluster VI was down-regulated in colonic vs. ileal location in controls, meaning that these clusters are more specific to colon than to ileum in controls.
In this study, we identified both common and distinct gene expression profiles between inflamed ileum and inflamed colon of CD patients. While some gene networks were in general disease-related (eg. (a)granulocyte adhesion/diapedesis), others reflected an up- or down-regulation of a location-specific signature in disease (eg. melatonin degradation), or even showed a loss of location-specific signatures in disease (eg. xenobiotic metabolism).
