678 Transcriptomic Changes in Colonic Mucosa in Patients With Campylobacter jejuni Post Infectious Irritable Bowel Syndrome

Abstract

Background: Irritable Bowel Syndrome (IBS) has been associated with changes in the rectosigmoid mucosal expression of immune (TNFSF15, TLRs) and nonimmune (tight junctions, mucus and serotonergic) factors. In a recent microarray based study on Campylobacter jejuni post-infectious IBS (PI-IBS), patients were found to have an increased rectal mucosal expression of CCL11, CCL13, Calpain 8 (pro-inflammatory), GABRE and decreased expression of NR1D1, GPR161. Aim: To determine changes in colonic mucosal RNA expression among patients with C. jejuni PI-IBS in comparison to healthy volunteers using whole transcriptome sequencing. Methods: Sigmoid colonic biopsies were obtained from 5 PI-IBS patients (3 females) and 7 age-matched healthy volunteers (5 females). Biopsies were preserved in RNALater at -80°C. Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA). All samples had RNA Integrity Numbers >7.0. RNA library preparation was performed using the Illumina TruSeq RNA Sample Prep v2 (San Diego, CA) and sequencing was performed as paired-end 51 base reads on an Illumina HiSeq 2000 with 3 samples/ lane using TruSeq SBS Sequencing Kit Version 3. Base calling was performed using Illumina's RTA version 1.12.4.2. Analysis (alignment statistics, in-depth quality control, gene and exon expression, fusion transcripts, and single nucleotide variants) was done using MAP-RSeq v1.2.1.3. Differential expression between samples was done using edgeR algorithm. Results were also compared to our recently published transcriptomic data from rectosigmoid biopsies of IBS-D patients (Am J Physiol 306: G1089-G1098, 2014). Results: Overall, mRNA expressions of 20 genes were changed in patients with C. jejuni PI-IBS as compared to healthy volunteers (P values obtained using false detection rate (FDR) threshold of 5% ranged from 10-5 to 10-9) (Table 1). The genes that stood out as potentially relevant to pathophysiology of PI-IBS included: neurotransmitter-related genes (↑neuropeptide Y receptor type 2, ↑Galanin, ↓nitric oxide synthase 2 (inducible), and ↓indoleamine 2, 3-dioxygenase 1), proteases (↑Secretory leukocyte peptidase inhibitor, ↓Matrix metallopeptidase 3), chemokines (↑chemokine C-C motif ligand 18, ↓chemokine C-X-C motif ligand 10 and 11), carbohydratemetabolism (↑aldolase B, ↑sucrase-isomaltase), ion channels and transporters (↑bestrophin 4, ↑calbindin 2, ↑solute carrier family 3). With the exception of aldolase B expression, there was no overlap with the IBS-D dataset. Conclusion: Transcriptome sequencing of sigmoid colonic mucosa in C. jejuni PI-IBS shows distinct transcriptomic differences from healthy controls and from non-post-infectious IBS-D. These data provide the rationale to explore the role of mucosal factors in the pathophysiology of C. jejuni PI-IBS. Table 1: Gene expression identified by edgeR showing relative expression in C. jejuni PIIBS compared to healthy volunteers