Gene expression differences between Crohn's disease aphthous ulcers and healthy Peyer's patches highlight novel therapeutic targets

Abstract

Background and Aim: The earliest macroscopic lesion in Crohn’s disease(CD) is the aphthous ulcer, which overlies Peyer’s patches and lymphoidfollicles. Our aim was to characterize differences in gene expression andthe virome of aphthous ulcers and Peyer’s patches.Methods: Biopsies (n = 24) were obtained from the terminal ileum of 12patients (six with CD and six healthy controls). Aphthous ulcers and adja-cent unaffected mucosa were obtained from patients with CD, and Peyer’spatches and adjacent mucosa from the controls. All patients, except one,were medication free. RNA was extracted using Qiagen kits. NextSeq500 libraries were constructed using NextSeq 500/550 High output kits(Illumina) in a 150 bp paired-end format. Whole genome transcripts wereassessed for quality using FASTQC, trimmed using Trimmomatic, andaligned to the human reference genome using subread mapper. Fragmentcounts were obtained using featureCount, and expression values normal-ized using the trimmed mean of M-values normalization method (TMM).Differential gene expression analyses were performed using generalizedlinear models in edgeR. Cell-specific gene expression was determinedusing ImSig. Reads that did not map to the human genome were used tomine for virus sequences using the VirusSeeker pipeline.Results: We obtained 36 million tags per sample, 87% were retained fordownstream processing, and 93% of these mapped to the human genome.A total of 920 genes were significantly differentially expressed betweenaphthous ulcers and Peyer’s patches (P = <0.001); all were upregulatedin aphthous ulcers. Differential gene expression analysis revealed 34 path-ways that were upregulated in aphthous ulcers relative to Peyer’s patches.Pathways that were over-expressed included those involved in respondingto bacteria, leukocyte chemotaxis, inflammatory response, and creation ofC2 and C4 activators. Receptors for the constant region of IgG were repre-sented in 13 pathways. The cytokine OSM and its receptor (OSMR) werealso over-expressed in aphthous ulcers. ImSig, which is capable of usingtranscriptome data to indicate cell types and their activation state, revealedthat core marker genes for plasma cells were overrepresented in aphthousulcers relative to Peyer’s patches and unaffected or normal mucosa. Therewas no virus common to all aphthous ulcers. We detected human herpesvirus 4 in one aphthous ulcer, human herpes virus 1 in mucosa from apatient with CD, and a novel Totivirus in mucosa of one control patient.Conclusions: A number of gene clusters and immune pathways are over-expressed in aphthous ulcers compared with Peyer’s patches. These bio-logically relevant gene lists highlight a number of new therapeutic targetsand potential biomarkers for early stage disease. Viruses are an unlikelyinitiator of CD Show more