J Neurogastroenterol Motil 2018; 24(4): 628-642  https://doi.org/10.5056/jnm18130
Expression of Toll-like Receptors, Pro-, and Anti-inflammatory Cytokines in Relation to Gut Microbiota in Irritable Bowel Syndrome: The Evidence for Its Micro-organic Basis
Ratnakar Shukla,1 Ujjala Ghoshal,1* Prabhat Ranjan,1 and Uday C Ghoshal2
Departments of 1Microbiology and 2Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India
Correspondence to: Ujjala Ghoshal, MD
Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India
Tel: +91-522-2495221, Fax: +91-522-2668129, E-mail: ujjalaghoshal@yahoo.co.in
Received: July 23, 2018; Accepted: August 24, 2018; Published online: October 1, 2018.
© The Korean Society of Neurogastroenterology and Motility. All rights reserved.

cc This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background/Aims
A Subset of patients with irritable bowel syndrome (IBS) may have mild inflammation due to immune activation. Toll-like receptors (TLRs) and cytokines may cause intestinal inflammation. We studied their expression in relation to gut microbiota.
Methods
Expression of TLRs and cytokines was assessed in 47 IBS patients (Rome III) and 25 controls using quantitative real-time polymerase chain reaction. Immunohistochemistry was further performed to confirm the expression of TLR-4 and TLR-5.
Results
Of 47 patients with IBS, 20 had constipation (IBS-C), 20 diarrhea (IBS-D), and 7 unclassified (IBS-U). The mRNA levels of TLR-4 and TLR-5 were up-regulated in IBS patients than controls (P = 0.013 and P < 0.001, respectively). Expression of TLR-4 and TLR-5 at protein level was 4.2-folds and 6.6-folds higher in IBS-D than controls. The mRNA levels of IL-6 (P = 0.003), C-X-C motif chemokine ligand 11 (CXCL-11) (P < 0.001) and C-X-C motif chemokine receptor 3 (CXCR-3) (P < 0.001) were higher among IBS patients than controls. Expression of IL-6 (P = 0.002), CXCL-11 (P < 0.001), and CXCR-3 (P < 0.001) were up-regulated and IL-10 (P = 0.012) was down-regulated in IBS-D patients than controls. Positive correlation was seen between TLR-4 and IL-6 (P = 0.043), CXCR-3, and CXCL-11 (P = 0.047), and IL-6 and CXCR-3 (P = 0.003). Stool frequency per week showed positive correlation with mRNA levels of TLR-4 (P = 0.016) and CXCR-3 (P = 0.005), but inversely correlated with IL-10 (P = 0.002). Copy number of Lactobacillus (P = 0.045) and Bifidobacterium (P = 0.011) showed correlation with IL-10 in IBS-C, while Gram-positive (P = 0.031) and Gram-negative bacteria (P = 0.010) showed correlation with CXCL-11 in IBS-D patients.
Conclusions
Altered immune activation in response to dysbiotic microbiota may promote intestinal inflammation in a subset of patients with IBS.
Keywords: Cytokines; Immunohistochemistry; Lipopolysaccharide; Peptidoglycan; Toll-like receptors
Figures
Fig. 1. Immunohistochemical analysis of Toll-like receptors (TLRs) 4 and 5 among different subtypes of patients with irritable bowel syndrome (IBS) and controls. Figures (A) and (B) show protein expression of TLR-4 and TLR-5 in colonic biopsies among different subtypes of IBS patients and controls. Protein expression of TLR-4 (C) and TLR-5 (D) was quantified using ImageJ software plugins. Figures (C) and (D) show the percentage contribution of positive cells for TLR-4 and TLR-5. IBS-C, constipation-predominant IBS; IBS-D, diarrhea-predominant IBS.



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