Oversecretion and Overexpression of Nicotinamide Phosphoribosyltransferase/Pre-B Colony-Enhancing Factor/Visfatin in Inflammatory Bowel Disease Reflects the Disease Activity, Severity of Inflammatory Response and Hypoxia

Abstract

Nicotinamide phosphoribosyltransferase's (Nampt) association with inflammatory bowel disease (IBD) is unclear. The study was aimed at unraveling Nampt's clinical and diagnostic relevance. The serum concentration (Luminex-xMAP® technology) was measured in 113 patients with Crohn's disease (CD), 127 with ulcerative colitis (UC) and 60 non-IBD controls: 40 healthy individuals and 20 with irritable bowel syndrome (IBS). The leukocyte (44 CD/37 UC/19 IBS) and bowel expression (186 samples) was also evaluated (RT-qPCR). All were referred to IBD phenotype, activity, treatment, and inflammatory/nutritional/angiogenic/hypoxia indices. Serum-Nampt and leukocyte-Nampt were positively correlated and were more elevated in active-IBD than in IBS, with leukocyte-Nampt being a fair differential marker. Serum-Nampt in UC positively correlated with its clinical and endoscopic activity as well as with pro-inflammatory cytokines. Serum-Nampt ≤1.54 ng/mL was a good indicator of mucosal healing. The expression of Nampt was up-regulated both in inflamed and quiescent colon and reflected, similarly to leukocyte-Nampt, the clinical activity of IBD. Bowel-Nampt was independently associated with IL1B and hypoxia-inducible factor 1α (HIF1A) expression in inflamed bowel but with FGF2 expression in quiescent bowel. In summary, Nampt's elevation in IBD at local and systemic levels, and protein and mRNA levels, reflects IBD activity and is associated with inflammation, hypoxia (active) and tissue repair (inactive disease).

Keywords: Crohn’s disease; Nicotinamide phosphoribosyltransferase (Nampt); biomarker; epithelial-to-mesenchymal transition; hypoxia; inflammatory bowel disease (IBD); mucosal healing; pre-B factor (PBEF); ulcerative colitis; visfatin.

Figure 1

Nampt in IBD. (A) Comparison of S-Nampt in IBD, IBS- and healthy controls; (B) S-Nampt association with IBD phenotype and activity; (C) comparison of leukocyte expression of Nampt in active and non-active IBD and IBS-controls; (D) the association of leukocyte expression of Nampt with the disease phenotype and activity. Data are presented as medians with 95% confidence interval and analyzed using Kruskal–Wallis H test. Bars are used in panels A and B due to the presence of several cases with extremely high concentrations of S-Nampt, which, in the case of a dot-plot, make medians unreadable. S-Nampt, serum Nampt; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; CDa, active Crohn’s disease; CDi, inactive Crohn’s disease; UCa, active ulcerative colitis; UCi, inactive ulcerative colitis; L-Nampt, Nampt expression in leukocytes; NRQ, normalized relative quantities (fold-change against geometric mean across all investigated samples); IBDa/c, expression ratio of L-Nampt in active IBD to IBS-controls; IBDa/i, expression ratio of L-Nampt in active to inactive IBD; CDa/i, expression ratio of L-Nampt in active to inactive CD; UCa/i, expression ratio of L-Nampt in active to inactive UC; CDa/UCa, expression ratio of L-Nampt in active CD to active UC. a, significantly different from others; b, significantly different from CDi; c, significantly different from UCi; d, significantly different from CDa; e, significantly different from UCa.

Figure 2

Nampt as an IBD marker. (A) S-Nampt as a marker differentiating patients with active and non-active disease; (B) S-Nampt as a marker differentiating patients with active UC from those with active CD and IBS; (C) S-Nampt as a marker differentiating UC patients with mucosal inflammation (Mayo endoscopic scores 2 and 3) from those without (scores 0 and 1); (D) L-Nampt as a marker differentiating IBD patients with active disease from IBS; (E) L-Nampt as a marker differentiating patients with active CD from active UC. Data are presented as ROC curves (straight line) with 95% confidence interval (CI) (dashed lines). Diagonal line presents the performance of a chance marker for which AUC = 0.5. Grey circle indicates optimal cut-off. Boxes contain data on the AUCs with 95%CI and probabilities of them being significantly different from a chance marker, optimal cut-offs with corresponding sensitivities and specificities. IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis; IBS, irritable bowel syndrome; inflam., inflammation; AUC, area under receiver operating characteristic (ROC) curve; sens., sensitivity; spec., specificity.

Figure 2

Nampt as an IBD marker. (A) S-Nampt as a marker differentiating patients with active and non-active disease; (B) S-Nampt as a marker differentiating patients with active UC from those with active CD and IBS; (C) S-Nampt as a marker differentiating UC patients with mucosal inflammation (Mayo endoscopic scores 2 and 3) from those without (scores 0 and 1); (D) L-Nampt as a marker differentiating IBD patients with active disease from IBS; (E) L-Nampt as a marker differentiating patients with active CD from active UC. Data are presented as ROC curves (straight line) with 95% confidence interval (CI) (dashed lines). Diagonal line presents the performance of a chance marker for which AUC = 0.5. Grey circle indicates optimal cut-off. Boxes contain data on the AUCs with 95%CI and probabilities of them being significantly different from a chance marker, optimal cut-offs with corresponding sensitivities and specificities. IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis; IBS, irritable bowel syndrome; inflam., inflammation; AUC, area under receiver operating characteristic (ROC) curve; sens., sensitivity; spec., specificity.

Figure 3

Correlation of Nampt with the disease activity. (A) L-Nampt with CDAI; (B) L-Nampt with RI; (C) B-Nampt in small intestine with CDAI; (D) B-Nampt in large intestine with RI. Data analyzed using Spearman rank correlation test. L-Nampt, Nampt expression in leukocytes; B-Nampt, Nampt expression in the intestine; NRQ, normalized relative quantities (fold change against geometric mean across all investigated samples); CDAI, Crohn’s disease activity index; RI, Rachmilewitz index.

Figure 4

Nampt expression in the bowel. (A) B-Nampt in small bowel; (B) B-Nampt in large bowel; (C) B-Nampt in paired samples from the same patient; (D) B-Nampt in large bowel: comparison with normal tissues derived from patients with polyps or colorectal adenocarcinomas. B-Nampt, Nampt expression in the bowel; NRQ, normalized relative quantities (fold change against geometric mean across all investigated samples); norm_P, samples of macroscopically normal colonic tissue obtained from patients with polyps during endoscopy; norm_T, samples of macroscopically normal colonic tissue (resection margins) obtained from patients with colorectal adenocarinomas during curative resection of tumors; q, quiescent; i, inflamed; i/q, ratio of expression in inflamed to quiescent tissue; nP, normal tissue derived from patients with polyps; nT, normal tissue derived from patients with tumors; i/nP, ratio of expression in inflamed to normal tissue from patients with polyps; i/nT, ratio of expression in inflamed to normal tissue from patients with tumors; q/nP, ratio of expression in quiescent to normal tissue from patients with polyps; q/nT, ratio of expression in quiescent to normal tissue from patients with tumors. Data are presented as geometric means with 95% confidence interval (CI) and analyzed using t-test for unpaired samples (panels A and B) or t-test for paired samples (panel C) or as medians with 95%CI and analyzed with Kruskal–Wallis H test (panel D). a, significantly different from norm_P; b, significantly different from norm_T; c, significantly different from inflamed; d, significantly different from quiescent.