Background and Aim Accurate, noninvasive biomarkers are needed to diagnose and monitor inflammatory bowel disease (IBD). Neutrophil gelatinase‐associated lipocalin (NGAL), also known as lipocalin 2, is expressed in inflamed colonic epithelium and neutrophilic granulocytes. This study explores its properties as a biomarker in feces and plasma and, for the first time, compares fecal NGAL systematically with the existing fecal biomarker calprotectin. Methods Neutrophil gelatinase‐associated lipocalin was measured in feces from 73 patients with IBD, 21 patients with infectious enterocolitis, 21 patients with irritable bowel syndrome, and 23 healthy subjects using ELISA. The results were correlated to calprotectin, clinical score, endoscopic score, and high‐sensitive C‐reactive protein. Plasma from 119 patients with IBD and 28 healthy controls was analyzed for NGAL. Results Fecal NGAL levels (median and interquartile range) were significantly elevated in active ulcerative colitis (UC) 6.05 (3.6–15.1) mg/kg and Crohn's disease (CD) 4.9 (1.5–7.7) mg/kg, compared with patients with inactive UC 1.3 (0.4–2.6) mg/kg, inactive CD 1.5 (0.5–1.7) mg/kg, irritable bowel syndrome 0.4 (0.2–0.6) mg/kg, and healthy controls (HC) 0.3 (0.1–0.4) mg/kg. Patients with infectious enterocolitis had significantly higher fecal‐NGAL levels, 2.7 (1.4–5.6) mg/kg than HC. Sensitivity and specificity was 94.7% and 95.7%, respectively, for distinguishing between active IBD and HC. Stability of NGAL in stool was excellent for 7 days in room temperature. Plasma NGAL was significantly elevated in UC and CD compared with HC. Conclusions Fecal NGAL is a promising biomarker for IBD. As existing biomarkers are expressed mainly in granulocytes, NGAL's epithelial localization may give supplementary diagnostic information.

Background and Aims Intestinal epithelial cells [IECs] secrete cytokines that recruit immune cells to the mucosa and regulate immune responses that drive inflammation in inflammatory bowel disease [IBD]. However, experiments in patient-derived IEC models are still scarce. Here, we aimed to investigate how innate immunity and IEC-specific pattern recognition receptor [PRR] signalling can be involved in an enhanced type I interferon [IFN] gene signature observed in colon epithelium of patients with active IBD, with a special focus on secreted ubiquitin-like protein ISG15. Methods Gene and protein expression in whole mucosa biopsies and in microdissected human colonic epithelial lining, in HT29 human intestinal epithelial cells and primary 3D colonoids treated with PRR-ligands and cytokines, were detected by transcriptomics, in situ hybridisation, immunohistochemistry, western blots, and enzyme-linked immunosorbent assay [ELISA]. Effects of IEC-secreted cytokines were examined in human peripheral blood mononuclear cells [PBMCs] by multiplex chemokine profiling and ELISA. Results The type I IFN gene signature in human mucosal biopsies was mimicked in Toll-like receptor TLR3 and to some extent tumour necrosis factor [TNF]-treated human IECs. In intestinal biopsies, ISG15 expression correlated with expression of the newly identified receptor for extracellular ISG15, LFA-1 integrin. ISG15 was expressed and secreted from HT29 cells and primary 3D colonoids through both JAK1-pSTAT-IRF9-dependent and independent pathways. In experiments using PBMCs, we show that ISG15 releases IBD-relevant proinflammatory cytokines such as CXCL1, CXCL5, CXCL8, CCL20, IL1, IL6, TNF, and IFNγ. Conclusions ISG15 is secreted from primary IECs upon extracellular stimulation, and mucosal ISG15 emerges as an intriguing candidate for immunotherapy in IBD.

Serotonin is a highly conserved and ubiquitous signalling molecule involved in a vast variety of biological processes. Approximately 90% of serotonin is produced in the gastrointestinal tract, where it is suggested to act as a prominent regulatory molecule in the inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC). Extracellular and circulating serotonin levels are thought to be elevated during intestinal inflammation, but the underlying mechanisms has been poorly understood. The data on human material is limited, contradictory and in need of further investigation and substantiating. In this study we show a potent and significant downregulation of the dominant serotonin reuptake transporter (SERT) mRNA (SLC6A4) in active CD ileitis, CD colitis and UC colitis, compared to healthy controls. The mRNA of the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase 1 was unregulated. Immunohistochemistry showed expression of SERT protein in both the epithelium and the lamina propria and localised the downregulation to the epithelial monolayer. Laser capture microdissection followed by RNA sequencing confirmed downregulation of SLC6A4 in the epithelial monolayer during intestinal inflammation. Patient-derived colon epithelial cell lines (colonoids) incubated with the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) reduced SERT expression. In summary, these results show that intestinal inflammation potently reduces the expression of SERT in both CD and UC, and that TNFα alone is sufficient to induce a similar reduction in colonoids. The reduced serotonin reuptake capacity may contribute to the increased interstitial serotonin level associated with intestinal inflammation.

The antimicrobial glycoprotein neutrophil gelatinase-associated lipocalin (NGAL) is strongly expressed in several infectious, inflammatory and malignant disorders, among these inflammatory bowel disease (IBD). Fecal and serum NGAL is elevated during active IBD and we have recently shown that fecal NGAL is a novel biomarker for IBD with a test performance comparable to the established fecal biomarker calprotectin. This study examines expression of NGAL in the healthy gut and in Crohn’s disease (CD), with emphasis on the previously unexplored small intestine. Pinch biopsies were taken from active and inactive CD in jejunum, ileum and colon and from the same sites in healthy controls. Microarray gene expression showed that the NGAL gene, LCN2, was the second most upregulated among 1820 differentially expressed genes in terminal ileum comparing active CD and controls (FC 5.86, p = 0.027). Based on immunohistochemistry and in situ hybridization findings, this upregulation most likely represented increased expression in epithelial cells. Double immunofluorescence showed NGAL expression in 49% (range 19–70) of Paneth cells (PCs) in control ileum with no change during inflammation. In healthy jejunum, the NGAL expression in PCs was weak to none but markedly increased during active CD. We further found NGAL also in metaplastic PCs in colon. Finally, we show for the first time that NGAL is expressed in enteroendocrine cells in small intestine as well as in colon.