Differential mRNA expression in ileal and colonic biopsies in irritable bowel syndrome with diarrhea or constipation

Abstract

Altered mucosal functions are documented in jejunal or colorectal mucosa from patients with irritable bowel syndrome (IBS). Our aim was to quantify ileal, ascending, and rectosigmoid colon mucosal expression of genes in IBS-diarrhea (D) and IBS-constipation (C). Forty-four patients with IBS-D, 30 with IBS-C, and 30 healthy volunteers underwent colonoscopic ileal, ascending, and rectosigmoid colon biopsies. Biopsies were stored in RNA_later at −80 °C, purified with on-column DNase, cDNA libraries prepared from 100–200 ng of total RNA, sequenced on Illumina NovaSeq 6000, and analyzed on Illumina’s RTA version 3.4.4. Normalized mRNA expression was obtained using MAP-RSeq bioinformatics pipeline. Differential expressions in the groups (Log2-fold change) were measured using the bioinformatics package edgeR 2.6.2, corrected for false discovery rate (P_ADJ <0.05). There were 30 females with IBS-C and 31 females and 13 males with IBS-D. In IBS-D and IBS-C groups, there were differential expressions of 181 genes in ascending colon and 199 genes in rectosigmoid colon. The majority were gene upregulations in IBS-D with functions reflecting activation of inflammation genes, TRPV1 (visceral hypersensitivity) and neurotransmitters/receptors (specifically purinergic, GABA, and cannabinoid). Although gene differential expressions in the ascending and rectosigmoid colon mucosa of the two groups were different, the diverse upregulated genes involved immune functions, receptors, transmitters, ion channels, and transporters. Conversely, there was reduced expression of PI15 and PI16 genes that inhibit proteases. In patients with IBS-D and IBS-C, differential expressions of genes related to immune, transmitter, nociceptive, protease inhibition, channel, and transporter functions suggest opportunities to reverse the pathobiology and treat patients with IBS.

NEW & NOTEWORTHY This study compares gene expression in mucosa of the terminal ileum, right colon, and left colon in patients with diarrhea- or constipation-predominant irritable bowel syndrome (IBS) and contrasts expression between these two disease entities and also between each entity and mucosa from healthy controls. The study shows there is differential expression of genes related to immune, transmitter, nociceptive, ion channel, and transporter functions, as well as reduced serine protease inhibition, in patients with IBS.

INTRODUCTION

Irritable bowel syndrome (IBS) is a chronic functional bowel disease with a prevalence of up to 20% in the US population characterized by the presence of abdominal pain or discomfort, bloating, and a change in bowel habits in the absence of any other organic cause. Although the pathogenesis has not been fully elucidated, different possible pathophysiological mechanisms have been implicated in the development of IBS including inflammation, changes in intestinal barrier function, ion transport, inappropriate neurotransmission, intestinal hypersensitivity, and mast cell products such as histamine and serine proteases (1, 2).

A summary of the literature on alterations in mucosal expression of genes in rectocolonic mucosa (with a few studies of jejunal or ileal mucosa) in IBS is shown in Table A1 (3–43). This includes three studies from our laboratory. First, in patients with IBS-D (n = 47) and healthy controls (n = 17), rectosigmoid mucosal mRNA expression of secretory genes (GUC2AB, PDZD3), and neurotransmitter (PR2Y4) was increased, whereas expression of barrier-related genes (CLDN1 and FN1) was decreased. One immune-related gene was upregulated (C4BP4) and one downregulated (CCL20). In addition, expression of a selected ion transport protein (PDZD3) in 10 patients with IBS-D was higher compared with mucosa of 4 healthy controls (44).

Second, a study using RNA-sequencing of rectosigmoid mucosal biopsies in 9 female patients with IBS-D with rapid colonic transit and 9 female controls showed up- or downregulated mRNA expressions of 21 genes in these classes: neurotransmitters (P2RY4, VIP); cytokines (CCL20); immune function [C4BPA complement cascade, interferon-related (IFIT3)]; mucosal repair and cell adhesion [trefoil protein (TFF1), retinol-binding protein (RBP2); fibronectin (FN1)]; and ion channel functions [guanylate cyclase (GUCA2B), PDZ domain-containing protein 3 (PDZD3)] (45).

Third, in a pilot study of ileal biopsies from 11 patients with IBS-D, 17 IBS-C, and 14 healthy controls, there were differencesinf mRNA expression in IBS-D relative to IBS-C with upregulation of barrier proteins (TJP1, FN1, CLDN1, and CLDN12), repair function (TFF1), and cellular functions. In ileal mucosal biopsies from IBS-C relative to healthy controls, there was reduced GPBAR1 (bile acid) receptor, myosin light chain kinase (MYLK in barrier function), and innate immunity (TLR3), but increased mRNA expression of cadherin cell adhesion mechanisms (CTNNB1) and transport genes SLC9A1 [Na-H exchanger (NHE1)] and INADL (indirect effect on ion transport) (46). As documented, these studies involved a relatively small number of patients and controls, a discrete selection of genes of interest, and to date, RNA-Seq in our studies was conducted only in 9 patients.

A separate analysis of the literature documented activation of the ATP ion-gated channels, voltage-gated sodium (Nav) and calcium (Cav) channels, as well as the activation of protease-activated receptors (PAR2), transient receptor potential vanilloide-1, serotonin, cannabinoids, and cholecystokinin are involved in the genesis of visceral hypersensitivity in IBS (47).

Given the altered mucosal functions suggested by differential gene expression in reports of jejunal, ileal, or colorectal mucosa usually acquired at a single region in patients with IBS, our aim was to quantify ileal, ascending (right), and rectosigmoid (left) colon mucosal expression of genes in IBS-diarrhea (IBS-D) and IBS-constipation (IBS-C).

METHODS

Regulatory

The protocol was approved by the Mayo Clinic Institutional Review Board (IRB No. 16–001445). Participants had given consent for their electronic medical records to be used for research purposes.

Participants and Design

We screened 1,744 patients with IBS-D [based on Rome III criteria (48), which were standard at the time of commencement of the study] for eligibility to participate in the studies. Participants in the colonoscopy study are summarized in Fig. 1. Screen failures and reasons for not completing the studies in the two aims are provided in Fig. 1.

Figure 1.

Participants in the colonoscopy study. Numbers represent number of individual participants.

Patient and Public Involvement

The public is involved in the discussion of the approval of the protocol by the Institutional Review Board since, by law, there must be public representation on the IRB.

In addition, in accordance with the requirements of the National Institutes of Health (NIH) for sharing information acquired through NIH funding, the anonymized information will be submitted in accordance with the Guidance on the NIH Genomic Data Sharing Policy. As the information from this research study does not have immediate clinical application, the information is not included in the patients’ medical records or communicated to the patients.

Ileocolonoscopy, Mucosal Biopsies, and RNA Sequencing

All patients underwent bowel preparation using a standard PEG3350-electrolyte solution on the evening before the procedure. The patients presented in the fasting state and underwent colonoscopy with or without ileoscopy under intravenous sedation with midazolam and analgesia provided by fentanyl. Biopsies were obtained from the terminal ileum, ascending, and rectosigmoid colon. Biopsies were stored in RNA_later at −80°C. RNA was purified from biopsies with on-column DNase to remove genomic DNA. cDNA libraries were prepared from 100 to 200 ng of total RNA according to the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA). The completed libraries were sequenced on Illumina NovaSeq 6000 as 100 × 2 paired-end reads. Base-calling was performed using Illumina’s RTA version 3.4.4.

Selection of Genes of Biological Interest

All genes meeting the predefined criteria for differential expression and statistical significance were reviewed for their relevance with IBS or any of the predefined pathophysiological mechanisms or associated intestinal diseases. The used databases include MEDLINE, Embase, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and Web of Science. The HGNC Gene symbol and, if available, the alias symbol were used as the primary keywords combined with selected terms of biological interest.

Selected terms include “Permeability,” “intestinal barrier,” “Tight junction,” “Motility,” “mRNA decay,” “MiRNA,” “Neurotransmission,” “neurotransmitter signaling,” “cAMP-PKA signaling,” “Neurite outgrowth”, “Tryptase,” “Serine protease,” “inflammation,” “immune function,” “cytokine,” “T cell,” “B cell” “mast-cell,” “hypersensitivity,” “visceral sensitivity,” “Ion channel,” “Secretion,” “Ion exchange,” “Bile acid,” “ubiquitin,” “Purine,” “Intestine,” “colon,” “ileum,” “bowel’ “IBS,” “Bowel Syndrome,”’ “FGD,” “IBD,” “Crohn’s disease,” “colitis ulcerosa,” “ulcerative colitis,” “diarrhea,” “constipation,” “abdominal pain,” “colitis,” “bile acid diarrhea,” “bile acid malabsorption,” and “ileitis.”

Data Analysis

RNA-sequencing analysis.

The raw paired-end reads for all RNA-Seq samples were processed through the Mayo RNA-sequencing (RNA-Seq) bioinformatics pipeline, MAP-RSeq version 3.1.4 (49). Briefly, MAP-RSeq employs the very fast, accurate, and splice-aware aligner, STAR (50), to align reads to the reference human genome build hg38. The aligned reads were then used to quantify gene and exon expression using the Subread package (51) to obtain both raw and normalized (FPKM – fragments per kilobase per million mapped reads) reads. Comprehensive analyses were also run on the aligned reads to assess quality of the sequenced libraries.

Differential expression.

Using the raw gene counts report from MAP-RSeq, genes that are differentially expressed between the groups were assessed using the bioinformatics package edgeR 2.6.2 (52).

Pathway analysis.

With the use of differentially expressed genes identified by edgeR [adjusted P < 0.05, canonical pathway analysis was performed using the Enrichr (https://maayanlab.cloud/Enrichr/)] database (53). Pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (54) and biological processes from Gene ontology (http://geneontology.org/) were used to identify pathways altered due to the differential genes between groups IBS-C and IBS-D or AA versus BB.

As a complementary approach, gene set enrichment analysis (GSEA) (55) was also used to determine whether an a priori defined set of genes shows statistically significant, concordant differences between the groups being compared. The expression profile of all protein-coding genes for samples in the groups was used for GSEA. The molecular signature database (56) was chosen to identify enriched pathways between groups, that is IBS-D compared with IBS-C and separately IBS-D compared with healthy controls and IBS-C compared with healthy controls.

Statistical Analysis

Genes found different between groups were reported along with their magnitude of fold change (log2 scale) and their level of significance (false discovery rate, FDR < 5%).

All authors had access to the study data and reviewed and approved the final manuscript for submission for consideration of publication.

RESULTS

Demographics of Participants

There were 30 females with IBS-C, 31 females and 13 males with IBS-D, and 14 female and 16 male healthy controls. The race of participants was 96 White, 3 Latino, 1 Black, 1 Asian/Indian, and 3 of unknown race. The ages were different in the IBS groups [IBS-C: 51.2 ± 17.4 (SD) yr; IBS-D: 40.0 ± 12.8 yr; P < 0.001] and the age of healthy controls was 45.6 ± 13.3 yr. With reference to biopsy acquisition from participants shown in Fig. 1, biopsies were obtained at the three sites in all, except 2 patients with IBS-C and 9 healthy controls (no right colon or terminal ileal biopsies in these participants).

Differential Gene Expression between IBS-C and IBS-D

Terminal ileal mucosa.

There were 33 genes that were upregulated in ileal mucosa in IBS-D compared with IBS-C; 32 genes were expressed in the Y chromosome and therefore deemed noncontributory to the goal of understanding potential functional differences, given the preponderance of females in our study. The gene TYW1B located in chromosome 7 (log2FoldChange 1.708; P_ADJ 0.0005) is involved in oxidoreductase activity and iron and sulfur cluster binding and it is therefore of unclear significance in IBS.

Colonic mucosae.

There were 181 genes (P_adj < 0.05) that were differentially expressed in IBS-D and IBS-C in the right colon biopsies and 199 (P_adj < 0.05) genes in the left colon. We observed differential expression of genes in mucosa that were present in both regions of several classes and individual genes in IBS-D compared with IBS-C as summarized in Table 1. Most genes with altered expression were upregulations in IBS-D. The functions of these genes reflect activation of inflammation genes (lymphocyte development, B or T lymphocyte function and differentiation, and activation of chemokines and inflammatory pathway activation), TRPV1 (visceral hypersensitivity), and neurotransmitters/receptors (specifically purinergic, GABA, and cannabinoid) and the potassium channel (KCNH2).

Table 1.

Differential expressions in IBS-D compared with IBS-C of the same genes that were upregulated in both right and left colonic mucosa

		Right Colon			Left Colon
Mucosal Function	Gene Symbol	Gene function	Log2-fold change	P adj	Log2-fold change	P adj
Inflammation or immune function
Lymphocyte development	CD19	B cell development	1.021	0.008	1.137	6.70E-05
	IKZF3	Lymphocyte differentiation	1.411	9.87E-06	1.414	2.17E-08
	CXCR5	Lymphocyte differentiation	1.479	0.006	1.185	0.003
B or T lymphocyte function and differentiation	CD79B	B lymphocyte receptor	1.078	0.002	1.107	2.33E-05
	CD22	B lymphocyte inhibition	1.533	0.0001	1.465	9.13E-07
	CHI3L2	Stimulation of type 2 T helper lymphocytes	1.237	0.005	1.176	0.0003
	CR2	Participates in B lymphocyte activation	1.624	0.002	1.697	3.54E-05
	BANK1	B lymphocyte function	1.112	0.002	1.109	0.0002
	FCRL3	Fc receptor-like genes expressed in B lymphocytes	1.535	0.0004	1.349	0.0001
	FCRL1		1.415	0.01	2.005	6.25E-07
	MS4A1 (CD20)	Differentiation of B-cell to plasma cell	1.514	0.006	1.288	0.006
Chemokines and immune activation	CCL23	Chemokine	1.739	2.52E-05	1.176	0.001
	CXCL13	Chemokine	1.742	0.006	1.368	0.0143
	CTLA4	Immune checkpoint	1.486	0.0001	1.039	0.010
	PILRB	Immune activation	1.201	0.0001	1.645	1.53E-06
	MAP3K14	NFκB-inducing kinase	1.011	1.79E-06	1.005	5.6E-08
	TCL1A	Associated with NFκB	2.024	0.002	1.752	0.002
	TLR10	Pathogen recognition	1.103	0.006	1.343	1.39E-05
	TNFSF11	Encodes a member of the TNF cytokine family	1.312	0.007	1.170	0.0002
Receptors and ion channels
	P2RX5	Purinoceptors for ATP	1.284	0.0002	1.433	9.33E-08
	CNR2	Cannabinoid receptor 2	1.064	0.009	1.096	0.0002
	GABRE	GABA-A receptor subunit epsilon	1.244	0.0002	1.105	0.002
	RNF207	Expression of K+ channel KCNH2	1.254	0.0003	1.099	0.001
Visceral hypersensitivity
	TRPV1	TRPV1 channel: nociception	1.384	0.0002	1.373	2.41E-06
Miscellaneous
	DNHD1	Aka C11orf47: microtubule motor activity	1.154	0.0006	1.047	0.0001
	COL27A1	Fibrillar collagens	1.261	0.0003	1.241	0.00001

IBS, irritable bowel syndrome.

Table 2 lists differential expressions of genes in the right and left colon mucosa in IBS-D and IBS-C. Nevertheless, there were similar functions of many of the diverse genes that were differentially expressed since they also involved immune functions, receptors, and membrane channels all of which were upregulated, and protease genes which were downregulated. The upregulated immune function genes in the right colon mucosa appeared to be associated with neutrophil function, whereas those in the left colon were possibly more closely aligned to lymphocyte function. The left colon mucosal also demonstrated upregulation of genes involved in cellular transcription or migration.

Table 2.

Differential expressions of genes (up- or downregulated) in IBS-D compared with IBS-C that were different in right and left colonic mucosa

	Right Colon				Left Colon
Mucosal Function	Gene symbol	Gene function	Log2-fold change	P adj	Gene symbol	Gene function	Log2-fold change	P adj
Upregulated
Immune function	LTF	Neutrophil lactotransferrin	1.412	0.004	BLK	B lymphocyte development, differentiation, signaling	1.161	1.39E-05
					IL21R	Interleukin receptor	1.018	1.76E-05
					TRDV1	Antigen recognition by T lymphocytes	1.002	0.048
					LY6G5B	Leukocyte antigen-6 gene in MHC class III	1.260	0.0008
Receptor	GABBR1	GABA-B receptor subunit 1	1.012	0.003
membrane channels	SLC6A7	Terminates action of proline, Na+ re-uptake, l-proline transporter	1.221	0.003	ABCA10	Translocation of substrates across cellular membranes	1.085	0.013
					YJEFN3	Cholesterol efflux	1.576	0.001
Cellular transcription or migration					RLTPR	aka CARMIL2B: controls cell migration	1.205	1.76E-05
					PARP15	Transcription repressor	1.144	0.0002
Downregulated
Protease activity	PI15	Peptidase inhibitor	−1.046	0.008	PI16	Protease inhibitor	−1.995	2.62E-05
Receptors	GABRB3	GABA-B receptor subunit 3	−1.08	0.001
	VIP	VIP receptor	−1.35	0.018
Structural					OLFM4	Facilitates cell adhesion	−1.177	0.008
					DES	Desmin (DES): a muscle scaffolding protein that also anchors mitochondria	−1.149	0.0001

IBS, irritable bowel syndrome. Overlap represents the number of genes in specific pathways (metabolism, synthesis, activation) showing differential expression among total number of genes in that pathway.

Conversely, there were a few genes that were differentially expressed in one, not both regions. For example, in the right colon, there was selectively upregulated GABA-B receptor subunit 1 and downregulated GABA-B receptor subunit 3 and VIP receptor, whereas in the left colon there was selective downregulation of structural genes involved in cell adhesion (OLFM4) and cellular or organelle scaffolding [DES (desmin)].

The pathway EnrichR analysis identified three pathways identified by the differential expression of individual genes in the left colon mucosa that differentiated IBS-D from IBS-C (Table 3). In left colon mucosa of IBS-D compared with IBS-C, there was downregulation of tyrosine metabolism and glycosaminoglycan (mucopolysaccharide) biosynthesis, and upregulation of B lymphocyte activation.

Table 3.

Pathway analyses identified as different, based on differential expressions in left colon mucosa in IBS-D compared with IBS-C

Pathway Term	Overlap	P adj	Odds Ratio	Combined Score	Genes	DE	Confirmation of Category
Tyrosine metabolism	1/36	0.041	95.0	416.2	TYRP1	Down	KEGG_2019_ Human
Glycosaminoglycan (mucopolysaccharide) biosynthesis	1/53	0.041	63.9	255.3	HS3ST2	Down	KEGG_2019_ Human
B cell activation	6/73	0.021	10.7	108.2	FCRL1; CD79B; CR2; BANK1; JAK3; MS4A1	Up	Gene Ontology _Biological Process_2018

IBS, irritable bowel syndrome; DE, differential expressions; up, upregulated; down, downregulated.

A summary of the differential expression in colonic mucosa between IBS-D and IBS-C is shown in Fig. 2.

Figure 2.

Conceptual summary of the overall (A), right colon (B), and left colon (C) cellular mechanisms that are similarly or differentially expressed in colonic mucosa of patients with IBS-D compared with mucosa from patients with IBS-C. Mechanisms that appear in green boxes showed increased differential expression, those in orange boxes showed decreased expression, and those in blue boxes showed similar expression (separately for right colon and left colon) in mucosa from IBS-D compared with mucosa from IBS-C. IBS, irritable bowel syndrome.

Differential Gene Expression between IBS-D or IBS-C and Healthy Controls

Ileal mucosa.

There were 40 genes that were differentially expressed in ileal mucosa between IBS-C and normal healthy volunteers; 35 were expressed on the Y chromosome. Five other genes located in chromosomes 6, 12, or 13 were downregulated (RERGL, MFAP5, MEDAG), SDS and PLA2G7 were upregulated (IBSC vs. NHV TI); one gene, MEDAG, is involved in adipocyte regulation and the other genes and their functions are shown in Table 4. There were 14 genes that were differentially expressed in ileal mucosa from patients with IBS-D compared with healthy volunteers. Among these, eight wereprotein-coding, with DNAH1 (dynein axonemal heavy chain 1) related to sperm motility and LDHC (l-lactate dehydrogenase C chain) catalyzes the conversion of l-lactate and NAD to pyruvate and NADH in the final step of anaerobic glycolysis and COL27A1 [collagen α-1(XXVII) chain] involved in cartilage calcification. The remaining functionally relevant genes are listed in Table 4. It is worth noting that both RERGL and MFAP5 are downregulated in both IBS-C and IBS-D relative to normal volunteers.

Table 4.

Differential gene expressions in terminal ileal mucosa in patients with IBS-C or IBS-D compared with normal healthy volunteers

Gene Symbol	Gene Function	Log2-Fold Change	P adj	DE
IBS-C compared with health controls
RERGL	G Protein activity	−1.853	0.042	Down
MFAP5	Vascular growth factor	−1.128	0.037	Down
SDS	Represses transcription and augments histone deacetylase activity	1.057	0.030	Up
PLA2G7	Inflammatory and oxidative stress response	1.024	0.022	Up
IBS-D compared with healthy controls
RERGL	G Protein activity	−1.698	0.028	Down
MFAP5	Vascular growth factor	−1.146	0.028	Down
PILRB	Paired immunoglobulin-like type 2 receptor β, in immune function	1.458	0.044	Up
EML6	Microtubular assembly	1.303	0.028	Up
NBPF12	Neuroblastoma breakpoint family member 12; possibly neural development	1.290	0.041	Up

DE, differential expressions; up, upregulated; down, downregulated.

Colonic mucosae.

Table 5 shows the differentially expressed genes between patients with IBS-D and normal healthy volunteers. There were several differentially expressed genes in both right and left colon: 154 genes (of which 39 were pseudogenes) in the right colon and 184 genes (of which 42 were pseudogenes) in the left colon. The genes with clearest functional potential in the right or left colon mucosa are summarized and grouped according to function in Table 5, that is inflammation/immune function, differentiation/cell development, neurotransmitters, and receptors (including the sensory transmission receptor TRPV1), and nutrient channels. Most of these genes are upregulated in IBS-D relative to healthy controls. However, it is worth noting that the serine protease-activating gene TMPRSS15 and the trypsin inhibitor PI15 are both downregulated in IBS-D and these functions would be expected to reduce proteolytic effects of endogenous pancreatic and bacterial proteases or peptidases. Another downregulated gene is SULT2A1, a sulfotransferase; within the colon, this may actually reduce the detoxification of the intraluminal bile acids.

Table 5.

Differential expressions in colonic mucosa for IBS-D compared with healthy controls

Mucosal Function	Gene Symbol	Gene Function	Log2-Fold Change	P adj	DE
Right colon mucosa
Inflammation/immune	PILRB	Immune regulator	1.444	6.08E-05	Up
	IKZF3	Lymphocyte differentiation	1.082	0.002777	Up
	LY6G5B	Lymphocyte antigen-6 gene in MHC class III	1.545	0.00031	Up
	CCL21	Chemokine	−1.051	3.39E-02	Down
	CXCL1	Antimicrobial chemokine	1.778	1.49E-05	Up
	CXCL2	Chemokine	1.515	2.87E-04	Up
	CXCL3	Chemokine	1.264	0.001805	Up
	NFKBIZ	Transcriptional regulator for NFκB	1.249	5.11E-06	Up
	TNFRSF25	TNF receptor; immune	1.005	0.008753	Up
	ZC3H12A	Inflammation, immune homeostasis	1.243	7.02E-07	Up
Cell development and transcription	ZNF713	Transcriptional regulation	1.224	0.000548	Up
	ZNF785	Transcriptional regulation	1.156	1.16E-05	Up
	EGR1	Transcription	1.535	6.93E-05	Up
	FOS	Cell development	1.367	0.000306	Up
	HBEGF	Growth factor	1.020	8.58E-05	Up
	TLCD2	Plasma membrane composition	1.244	1.10E-06	Up
	SOAT2	Formation of fatty acid-cholesterol esters (less soluble in membranes than cholesterol	−2.434	0.000235	Down
	CALB2	Intracellular Ca-binding protein	−1.605	9.98E-05	Down
	MS4A10	Signal transduction	−1.370	0.031217	Down
Serine proteases	PI15	Trypsin inhibitor	−1.241	2.19E-03	Down
	TMPRSS15	Serine protease	−3.439	2.51E-04	Down
Receptors and Ion/nutrient channels	GABRE	GABA receptor	1.087	1.25E-02	Up
	RNF207	Potassium channel KCNH2	1.223	4.25E-03	Up
	TRPV1	Receptor, sensation	1.306	0.000694	Up
	VIP	Neurotransmitter	−2.415	2.40E-05	Down
	SCNN1G	Na+ permeable non-voltage-sensitive ion channel	2.361	1.69E-04	Up
	SLC6A7	Na+ reuptake l-proline transporter	1.299	4.35E-03	Up
	SLC6A19	Epithelial resorption of neutral amino acids	−1.243	0.008506	Down
	SULT2A1	Sulfonation of bile acids	−3.074	2.49E-03	Down
Left colon mucosa
Inflammation/immune	CCL3L3	Chemokine ligand	1.286	0.002	Up
	CCR4	Chemokine receptor	1.030	0.002	Up
	CXCR5	Chemokine receptor	1.020	0.031	Up
	CD22	B cell receptor signaling	1.095	0.001	Up
	CR2	Complement receptor	1.043	0.016	Up
	FCRL1	Fc receptor	1.592	8.5E-05	Up
	IL1B	Interleukin	1.003	0.0008	Up
	LY6G5B	Lymphocyte antigen-6 gene in MHC class III	1.270	0.003	Up
	IKZF3	Lymphocyte differentiation	1.277	7.66E-05	Up
	PILRB	Immunoglobulin-like receptor	1.433	0.000126	Up
	TNFRSF9	Cytokine-related	1.097	0.003524	Up
	TNFSF11	Cytokine-related	1.219	0.000506	Up
	NFKBIZ	Inhibitor of NFκB	1.062	4.88E-06	Up
Differentiation/ cell development	EGR1	Early growth factor	1.042	0.018849	Up
	EGR2		1.070	0.00621	Up
	EGR3		1.245	0.001438	Up
	HBEGF	EGF-like growth factor	1.254	0.000223	Up
	TLCD2	Composition and fluidity of the plasma membrane	1.081	5.07E-06	Up
	MUC17	Cell surface mucin	1.317	0.00088	Up
Neurotransmitters	GABRE	GABA receptor	1.147	0.00195	Up
	TRPV1	Visceral hypersensitivity	1.572	1.04E-05	Up
	VIP	Neurotransmitter	−1.552	0.000213	Down
Receptors/channels	GPR132	Cellular proliferation	1.030	0.000127	Up
	GPR157	Secretin and VIP receptor	1.009	4.01E-06	Up
	RNF207	Potassium channel KCNH2	1.056	0.005411	Up
	SCNN1G	Sodium channel	1.068	0.020921	Up
	SLC6A7	Na+ reuptake l-proline transporter	1.054	0.001661	Up
	NR4A2	Receptor protects dopaminergic neurons	1.098	0.000671	Up
	P2RX5	Purinergic receptor	1.045	0.000106	Up
Protease	PI16	Protease inhibitor	−2.044	1.2E-06	Down

DE, differential expressions; up, upregulated; down, downregulated.

The differential expression in the left colon mucosa in IBS-D compared with healthy controls is remarkably consistent with the observations in the right colon, particularly in the functions related to inflammation/immune function, differentiation/cell development, neurotransmitters and receptors, and downregulation of a peptidase inhibitor (PI16).

Table 6 shows the differential expression between patients with IBS-C and normal controls. There were 42 RNA genes differentially expressed in the right colon and 48 genes differentially expressed in the left colon. Although the specific genes with differential expression in right and left colon differed, the functions of genes with clear significance are related to inflammation, ion-binding, or carrier-related genes and early growth and cellular differentiation.

Table 6.

Differential expressions in colonic mucosa for IBS-C vs. healthy controls

Mucosal Function	Gene Name (Symbol)	Gene Function	Log2-Fold Change	P adj	DE
Right colon mucosa
Inflammation-related genes	C-X-C Motif Chemokine Ligand 1 (CXCL1)	Chemotaxis for neutrophils and inflammation, and affects endothelial cells in an autocrine fashion	1.392443	0.014487	Up
	C-C Motif Chemokine Ligand 21 (CCL21)	Inhibits hemopoiesis and stimulates chemotaxis	−1.61646	0.018378	Down
Ion-binding or carrier-related genes	S100 Calcium Binding Protein P (S100P)	Binds calcium ions	1.030017	0.000677	Up
	Solute Carrier Family 6 Member 19 (SLC6A19)	Apical membrane epithelial transporter mediating absorption of neutral amino acids (e.g., leucine) in intestine. Defects in SLC6A19 are a cause of Hartnup disease.	−1.68011	0.018721	Down
Left colon mucosa
Inflammation-related genes	Prostaglandin-Endoperoxide Synthase 2 (PTGS2)	Mediator of inflammation and/or prostanoid signaling; induction by cytokines and mitogens	1.193292	0.00539	Up
	Cellular Communication Network Factor 1 (CYR61)	Promotes cell proliferation, chemotaxis, angiogenesis and cell adhesion	1.367793	0.018244	Up
	Activating Transcription Factor 3 (ATF3)	Binds the cAMP response element (CRE), a sequence which is present in many viral and cellular promoters. ATF3 represses transcription from viral and cellular promoters with ATF sites	1.234673	0.014649	Up
	C-X-C Motif Chemokine Ligand 8 (CXCL8)	Major mediator of the inflammatory response, encoding interleukin-8, a chemotactic factor that attracts neutrophils, basophils, and T-cells, and activates neutrophils	1.426137	0.043853	Up
Genes related to early growth and cellular differentiation	Early Growth Response 1 (EGR1)	Transcriptional regulator that activates the transcription of target genes whose products are required for mitogenesis and differentiation.	1.717686	0.000809	Up
	Early Growth Response 2 (EGR2)	DNA-binding transcription factor activating 2 specific DNA sites located in the promoter region of HOXA4	1.504452	0.008922	Up
	Early Growth Response 3 (EGR3)	Transcription factor involved in muscle spindle development	1.912399	0.000297	Up

IBS, irritable bowel syndrome; DE, differential expressions; up, upregulated; down, downregulated.

DISCUSSION

Our current study builds upon a significant body of literature that had documented, predominantly in the rectosigmoid colon, the differences in mucosal gene expressions of targeted genes in variable numbers of patients with IBS-C or IBS-D, and healthy controls. Our study involved biopsies from both the right and left colon in 45 patients with IBS-D, 30 patients with IBS-C, and 30 healthy controls. In addition, we embarked upon RNA sequencing to encompass all potential mechanisms that would differentially be expressed in the three groups in two colonic regions.

We document important insights into the differential gene expressions between IBS-C and IBS-D (Fig. 2). Importantly, many differential expressions of genes were identified in both right and left colon biopsies, generally with upregulation in expression in IBS-D compared with IBS-C. The classes of gene functions with upregulated expression included inflammation genes, particularly related to B or T lymphocyte function and differentiation or activation of inflammatory pathways, and importantly upregulation of receptors involved in both visceral hypersensitivity, that is TRPV1, and neurotransmitters or ion channels.

The importance of the observed upregulation of immune activation or inflammatory mechanisms in our study of patients with IBS is supported by several published reports at the mucosal level in patients with IBS demonstrating increased expression of IL-1β (40) and TNF-α (57) and decreased levels of IL-10 (35, 37) and IL-8 (58). These changes can lead to activation of the immune response, cytokine imbalances, and low-grade mucosal inflammation. It is intriguing to note that there is recent evidence that long-term dietary patterns, possibly with modification of the gut microbiome, may be contributing to some of these inflammatory changes in diverse cohorts with digestive tract diseases including Crohn’s disease, ulcerative colitis, irritable bowel syndrome, and in the general population (59). These observations also support the notion that mucosal protection (60) or probiotics (61) may provide novel strategies to reverse chronic diarrheal disorders including IBS-D and functional diarrhea.

The importance of TRPV1 in the context of IBS has recently been the subject of studies that show decreased TRPA1 and/or TRPV1 receptor expression with aging in health and IBS associated with evidence of decreased abdominal pain (62), as well as by the demonstration that TRPV-mediated small intestinal chemosensitivity may mediate postmeal symptoms in IBS (63). It is also of particular interest that there was upregulation of the potassium channel KCNH2, which is very relevant to motor function in the colon. Specifically, it has been demonstrated that the expression of this channel is reduced in the aganglionic segment of the colon in Hirschsprung disease (64).

We have identified differentially expressed genes in the right and left colon mucosa in IBS-D and IBS-C, but there was consistency in the regional differences since the differential expression also involved the same functions pertaining to immune mechanisms, receptors, and membrane channels which were upregulated, and protease genes, which were downregulated. The significance of the differences in genes expressed in the two regions, as tabulated in Table 3, will require further investigation to explore the potential relevance of altered regulation of GABA_B receptor subunits and VIP, as well as structural genes that were downregulated in left colonic mucosa of patients with IBS-D.

In addition to the analysis of the individual genes and the overall functions expressed by those genes, the pathway analysis identified three specific functions that were differentiated in the left colon mucosa of IBS-D relative to IBS-C. These functions were: first, downregulation of tyrosine metabolism that could be relevant as a precursor of catecholamine neurotransmitters; second, downregulation of glycosaminoglycan (mucopolysaccharide) biosynthesis that is relevant to the synthesis of colonic mucins; and third, upregulation of B lymphocyte activation that has implications for the low-level inflammatory processes observed in patients with diarrheal diseases including the frequently overlapping lymphocytic colitis.

The comparison of mucosal expressions of the right and left colon in IBS-D compared with healthy controls also documents the important biological differences manifesting as upregulation of immune activation, cell development and transcription, and diverse receptors and ion or nutrient channels. Although the specific details of the genes may differ in the right and left colon, there is commonality in the functions mentioned above that differentiates IBS-D colon mucosa from that of healthy controls. In addition, both right and left colon mucosa document decreased serine protease inhibition, which is consistent with propensity to proteolytic damage of the mucosa in IBS-D as a result of reduced protection from the downregulated serine protease inhibitors, PI15 and PI16.

Our final comparison between mucosal expressions of the right and left colon between IBS-C and healthy controls has documented alterations in the expressions of inflammation-induced genes, ion binding and solute carrier-related genes in the right colon, in contrast to the consistent upregulation of inflammation-related genes, as well as genes associated with early growth and cellular differentiation in the left colon mucosa of patients with IBS-C compared with healthy controls. The upregulation of immune mechanisms in the colonic mucosa in patients with IBS-C relative to mucosa from healthy controls has not been extensively studied in the prior literature (summarized in Table 1).

Summary of Findings, Their Potential Mechanisms, and Possible Clinical Relevance

This large study involving RNA-Seq of mucosal biopsies from right and left colon in patients with IBS-D and IBS-C has demonstrated the differential expressions of genes related to diverse functions involving immune, transmitter, nociceptive, ion channel, and transporter activities, and protease inhibition. There were 181 genes in the right colon and 199 genes in the left colon that demonstrated differences in expression. These observed functional differences at the level of the mucosa reinforce the relevance of peripheral mechanisms in the pathobiology of IBS (1, 2) and the potential for injurious substances such as bile acids with their detergent properties (65) or intraluminal serine proteases to activate immune mechanisms or stimulate visceral sensation, as documented by the increased expression of TRPV1. These observations also suggest options for treatment of IBS. These include established approaches such as neutralizing bile acids with sequestrants in patients with increased synthesis or excretion of bile acids or reducing intraluminal proteases with mast cell stabilizers. It also introduces novel therapeutic opportunities as recently demonstrated by the use of resolvins (which are endogenous anti-inflammatory lipid mediators) to reduce TRPV1-mediated visceral hypersensitivity (66) or treatment with divertin, which targets long myosin light chain kinase (MLCK)-1 to act on the perijunctional actomyosin ring and prevent cytokine (such as TNF)-induced barrier loss, as has been demonstrated in models of immune colitis (67).

In conclusion, our study has demonstrated that, although the specific genes in the right and left colon mucosa may be different, patients with IBS-C or IBS-D show upregulations of genes related to inflammation or immune activation, cellular transporters, and visceral hypersensitivity, as well as receptors that respond to ATP, GABA, and cannabinoid molecules, or an ion (potassium) channel. Notable mechanisms that are downregulated are genes related to protease inhibitors, and immune dysregulation or inflammation may result from effects of either endogenous proteases, such as those originating in the pancreas or mast cells, or from intraluminal microorganisms. These findings also suggest that searching for biomarkers of these biological processes may constitute novel targets for treatment in IBS.

GRANTS

M. Camilleri is supported by National Institutes of Health Grant R01-DK115950. The study was facilitated by the CCaTS Clinical Research Trials Unit at Mayo Clinic Grant UL1-TR002377 from National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHORS CONTRIBUTIONS

M.C. conceived and designed research; P.C., X.J.W., V.C., D.M., A.T., S.M., and I.B. performed experiments; M.C., Y.M., N.K.N., and A.N. analyzed data; M.C. interpreted results of experiments; M.C. prepared figures; M.C. drafted manuscript; M.C., Y.M., P.C., X.J.W., V.C., D.M., A.T., S.M., N.K.N., I.B., and A.N. edited and revised manuscript; M.C., Y.M., P.C., X.J.W., V.C., D.M., A.T., S.M., N.K.N., I.B., and A.N. approved final version of manuscript.

APPENDIX

Table A1 presents a summary of the literature reporting differences in mucosal expression of genes in rectocolonic mucosa (with a few studies of jejunal or ileal mucosa) in IBS relative to healthy controls.

Table A1.

Literature review of reported differences in mucosal gene expression in IBS relative to healthy controls

Function	No. IBS or Controls Studied	Expression in Colonic (Predominantly Rectosigmoid) Mucosal Biopsies in IBS	Reference in Main Manuscript
Barrier function, TJ protein mRNA expression	36 IBS (15 IBS-C, 21 IBS-D), 25 HC	Upregulation of mucin gene (MUC20) and dual oxidase 2 (DUOX2); reduced caspase-1 (CASP1) and lysozyme (LYZ)	Aerssens et al. (3)
	21 IBS, 12 HC	Lower expression of ZO-1 mRNA in biopsies of IBS as compared with healthy subjects	Piche et al. (4)
	45 IBS-D; 22 + gluten; 23 gluten-free diet;	Lower expression of ZO-1, claudin-1, and occludin in rectosigmoid mucosa of IBS-D with gluten containing diet	Vazquez et al. (5)
	Among IBS-D: 13 GFD and 14 GCD	Increased small intestine epithelial MLC phosphorylation and increased colonocyte expression of the paracellular Na+ channel claudin-15 by diet with gluten	Wu et al. (6)
	19 IBS-D, 14 IBS-C, 15 IBS-M, 2 IBS-U, 31 HC	Downregulation of ZO-1, claudin, and occludin protein expression in IBS-C and IBS-D; no changes in mRNA	Bertiaux-Vandaele et al. (7)
	109 IBS-D, or IBS-C and 36 HC	Reduced levels of Claudin-1 and nuclear factor-κB-repressing factor I n IBS-D (targeted by microRNA29)	Zhou et al. (8)
	40 IBS, 10 HC; patients stratified by increased mucosal permeability (L:M ratio)	RNA-seq of ileal mucosa: 185 genes differentially expressed, many related to mucosal inflammation and immunity; expression of claudin 4 significantly higher in those with increased small intestinal permeability	Li et al. (9)
Trypsin-like activity	Supernatants from colonic mucosal biopsies from patients with IBS	Increased trypsin-like activity associated with colonic epithelium; trypsin-3 upregulated in tissues from patients with IBS. Trypsin-3 was able to signal to human submucosal enteric neurons	Rolland-Fourcade et al. (10)
Tryptase	20 IBS-D, 8 IBS-C, 10 HC	Increased mRNA and protein expression of tryptase and increased mRNA PAR-2 in combined IBS vs. controls	Liang et al. (11)
Protease-activated receptor	16 IBS-C, 18 IBS-D, 18 HC	Expression of PAR4 (mRNA or protein levels) was lower in IBS than the HC	Zhao et al. (12)
	23 PI-IBS, 17 HC	Reduced PAR4 expression in mast cells of PI-IBS	Han et al. (13)
Serotonergic functions	14 IBS-C, 26 IBS-D, 25 HC	Reduced SERT-P expression (normal in some studies) Increased p11 (S-100A10, or calpactin I light chain) expressionSERT mRNA and protein expression related to 5-HTTLPR genotype	Coates et al. (14),Camilleri et al. (15)
	254 IBS, 120 HC	Reduced SERT and normal TpH1 expression	Wang et al. (16)
	23 IBS, 15 HC	Lower TPH-1 and SERT mRNA levels in the rectum associated with rectal hypersensitivity. No significant differences in PAR-2 and trypsinogen IV expression. Rectal substance P content was increased in IBS.	Kerckhoffs et al. (17)
	12 IBS (11-16 yr), and colon diseases (8-18 yr)	Baseline S100A10 and TPH1 expression were higher in ramosetron responders than in nonresponders.	Faure et al. (18)
	42 IBS-D in RCT of ramosetron	Baseline S100A10 and TPH1 expression were higher in ramosetron responders than in nonresponders.	Shiotani et al. (19)
Receptor expression	9 IBS-C, 10 IBS-D, 12 IBS-M and 32 HC	Increased expression of µ-opioid receptor, β-endorphin, and cannabinoid CB2 receptors in the mucosa of IBS patients, where they are localized to immune cells	Dothel et al. (20)
	3 IBS-D, 3 IBS-C and 10 HC	FAAH mRNA levels lower in patients with IBS-C	Fichna et al. (21)
	36 IBS-D, 15 HC	Increased mRNA and protein expression of P2Y1, P2Y2, and TRPV1 receptors in IBS-D compared with controls	Luo et al. (22)
Other transmitters	20 IBS-D, 8 IBS-C, 10 HC	Increased CGRP, VIP and SP protein expression in IBS-D compared with both IBS-C and HC	Liang et al. (11)
	23 IBS with VH, 25 IBS with NS, 25 HC	Rectal TRPV1 expression similar in VH-IBS, NS-IBS, and HVs on both mRNA and protein expression	van Wanrooij et al. (23)
	42 IBS-D, 20 HC	Increased leptin mRNA and protein expression (on mast cells and nerves)	Liu et al. (24)
	31 IBS-D, 20 HC	Increased BDNF mRNA and protein	Zhang et al. (25)
	11 IBS-D, 15 IBS-C, 13 HC	Higher mRNA expression of G protein-coupled estrogen receptor in IBS-D than in IBS-C and healthy subjects	Qin et al. (26)
	19 IBS-D, 16 HC	iNOS mRNA and protein levels upregulated in IBS-D compared with health	An et al. (27)
Enteric glial cells	13 IBS-C; 10 IBS-D; 11 IBS-M, 24 HC	Mucosal area immunoreactive for S100β was significantly reduced in biopsies of patients with IBS (all subtypes)	Lilli et al. (28)
Ion or water channels	14 IBS, 16 HC	Increased Cav 3.2 calcium channel expression	Scanzi et al. (29)
	44 IBS-D, 10 IBS-C, 17 HC	Increased AQP7 and 8 and decreased AQP3 expressions in rectosigmoid mucosa in IBS-D	Camilleri et al. (30)
	26 IBS-D, 30 controls	Decreased AQP 8 in ascending or descending colon mucosa	Wang et al. (31)
Immune activation	22 IBS patients, 29 controls	Increased IL13 mRNA that correlated with genetic SNP rs1881457-IL13	Wouters et al. (32)
Mucosal immune anti-inflammatory	SRMA of 3 studies of IBS including PI-IBS	Colonic IL-10 mRNA expression significantly lower in IBS than in controls	Bashashati et al. (33)
	20 PI-IBS, 18 IBS non-PI, 20 controls	IL-10 mRNA and protein levels in PI-IBS lower than in non-PI-IBS or controls	Chen et al. (34)
	16 IBS-C, 15 IBS-D, 14 IBS-M, 41 controls	Reduced IL-10 and NK1 expression in IBS-D only in females	Chang et al. (35)
Mucosal immune activation: proinflammatory IL-8, IL-1 β and TLRs	20 IBS-D, 14 IBS-C, 14 IBS-M, 31 controls	Upregulation of TLR2 and TLR4 mRNA in epithelial cells in IBS-M	Belmonte (36)
	109 IBS, 36 controls	mRNA expression of IL-10 and FOXP3 tended to be decreased in patients with IBS	Bennet et al. (37)
	43 IBS-D, 24 controls	Increased TLR4 (but not other TLRs or TNF-α, IL-1, and IL-6) mRNA expression in IBS-D; TLR4 expression also correlated with increased expressions of diverse receptors; e.g., TRPA1, BDKRB2, CMKLR1	Jalanka et al. (38)
	8 IBS-C, 7 IBS-D, IBS-M, 14 controls	Increased mRNA expression of TLR4, TLR5 and TLR9 in jejunal mucosa	Dlugosz et al. (39)
	20 PI-IBS, 18 IBS non-PI, 20 controls	Higher IFN-γ mRNA in ascending, descending and rectal mucosa in PI-IBS group than non-PI-IBS or controls	Chen et al. (34)
	8 PI-IBS, 7 postenteritis, 18 controls	Greater expression of IL-1β mRNA in rectal biopsies than INF-CON pts both during and 3 months postenteritis.	Gwee et al. (40)
	80 IBS-D + Blastocystis (type 1 or 3), 80 IBS-D without Blastocystis	With Blastocystis type 1, IL-8 mRNA was increased compared with type 3 Blastocystis and control (no Blastocystis)	Yakoob et al. (41)
Mono-carboxylate transporters	15 IBS-C,15 IBS-D. 15 HC	Reduced MCT1 (SLC16A1) and SMCT1 (SLC5A8) in both IBS groups, with reduced acetate, propionate, and butyrate in IBS-C and acetate only in IBD	Fredericks et al. (42)
Intracellular mediators	10 IBS-C, 10 IBS-D, 5 HC	Weighted gene coexpression network analysis identified activation of the cAMP/protein kinase A signaling pathway	Videlock et al. (43)

HC, healthy controls; IBS, irritable bowel syndrome.