Ulcer-associated cell lineage expresses genes involved in regeneration and is hallmarked by high neutrophil gelatinase-associated lipocalin (NGAL) levels.

Neutrophil gelatinase-associated lipocalin (NGAL), also known as Lipocalin 2, is an antimicrobial protein, encoded by the gene LCN2, strongly upregulated in inflammatory bowel disease (IBD) and a promising biomarker for IBD. Here we demonstrate that NGAL is highly expressed in all parts of pyloric metaplasia, also known as the ulcer-associated cell lineage (UACL), a metaplastic cell lineage suggested to play a role in wound healing in Crohn's disease (CD). We further show NGAL expression in regenerative intestinal crypts and in undifferentiated patient-derived colonoids. This indicates that NGAL is important in the tissue regeneration process. The remarkable overexpression of NGAL in UACL led us to explore the pathobiology of these cells by transcriptome-wide RNA sequencing. This study is, to our knowledge, the first to characterize the UACL at this level. Biopsies with UACL and inflamed non-UACL epithelium from the terminal ileum of CD patients and epithelium from healthy controls were laser capture microdissected for RNA sequencing. Among the 180 genes differentially expressed between UACL and control epithelium, the ten most-upregulated genes specific for UACL were MUC5AC, PGC, MUC6, MUC5B, LCN2, POU2AF1, MUC1, SDC3, IGFBP5, and SLC7A5. PDX1 was among the most upregulated in both UACL and inflamed non-UACL epithelium. Immunohistochemistry and iDisco 3D visualization was used to characterize UACL histo-morphologically, and to validate protein expression of 11 selected differentially expressed genes. Among these genes, LCN2, NOTCH2, PHLDA1, IGFBP5, SDC3, BPIFB1, and RCN1 have previously not been linked to UACL. Gene expression results were analyzed for functional implications using MetaCore, showing that differentially expressed genes are enriched for genes involved in cell migration and motility, and for biomarkers of gastrointestinal neoplasia. These results support a role for UACL as part of the reepithelialization process during and after destructive intestinal inflammation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Mucosal toll-like receptor 3-dependent synthesis of complement factor B and systemic complement activation in inflammatory bowel disease.

BACKGROUND: Recent studies link Toll-like receptor 3 (TLR3) to the pathogenesis of inflammatory bowel disease (IBD). Screening TLR3-agonist response in an intestinal epithelial cell line, we found complement factor B mRNA (CFB) potently upregulated and went on to further study localization of complement factor B synthesis and systemic activation of complement in ulcerative colitis and Crohn's disease. METHODS: In a transcriptome analysis of poly (I:C) stimulated HT-29 cells, we found CFB highly upregulated downstream of TLR3. We sought to confirm CFB upregulation in a microarray gene expression analysis on colonic biopsies from an IBD population (n = 133). Immunohistochemical staining and in situ hybridization were done to identify cellular sources of factor B and CFB. Systemic complement activation was assessed in plasma (n = 18) using neoepitope-based enzyme linked immunosorbent assay. RESULTS: CFB mRNA and protein were abundantly expressed in the colonic epithelial cell line, and synthesis enhanced by the poly (I:C) TLR3 ligand. In inflamed versus normal colonic mucosa of ulcerative colitis and Crohn's disease, CFB mRNA was the most significantly overexpressed gene and the mRNA abundance ratio was among the 50 highest. Epithelial cells were the dominating site of factor B expression. Systemic complement activation was significantly higher in active than in nonactive IBD. CONCLUSIONS: This study is the first to link TLR3 to activation of the alternative complement pathway. Complement factor B is potently upregulated locally in IBD in addition to having a possible central role in systemic complement activation. This suggests a prominent role for complement in IBD pathogenesis.

Enhanced expression of CXCL10 in inflammatory bowel disease: potential role of mucosal Toll-like receptor 3 stimulation.

BACKGROUND: We explored the gene expression in colonic biopsies of active and inactive inflammatory bowel disease (IBD) in an extensive material of ulcerative colitis (UC) and Crohn's disease (CD). The chemokine CXCL10 and its receptor CXCR3 were among the upregulated genes. This study examined the expression of CXCL10 and the mechanisms for its release in patients with UC or CD and in intestinal epithelial cell (IEC) lines. METHODS: A microarray gene expression analysis was done on colonic biopsies (n = 133) from patients with IBD. Biopsies were studied with immunohistochemistry for CXCL10 and CXCR3 expression. Mechanisms for CXCL10 release in peripheral blood mononuclear cells (PBMCs) and in the colonic epithelial cell lines HT-29 and SW620 were studied upon pattern recognition receptor (PRR) stimulation. RESULTS: CXCL10 and CXCR3 mRNA abundances were increased in biopsies from active UC and CD compared to inactive disease and controls. CXCL10 was mainly localized to mucosal epithelial cells, with increased immunostaining in active IBD. CXCR3-positive cells were scattered in the lamina propria. CXCL10 was secreted from the colonic epithelial cell lines in response to the Toll-like receptor 3 (TLR3) ligand polyinosinic: polycytidylic acid (poly(I:C)). This ligand also induced a marked release of CXCL10 in PBMCs from IBD patients and controls. CONCLUSIONS: We identified CXCL10 and CXCR3 as upregulated genes in colonic mucosa in active IBD. The TLR3-ligand poly(I:C) markedly increased release of CXCL10 in colonic epithelial cell lines, suggesting a TLR3-mediated CXCL10 release from mucosal epithelial cells in IBD patients.

Whole genome gene expression meta-analysis of inflammatory bowel disease colon mucosa demonstrates lack of major differences between Crohn's disease and ulcerative colitis.

BACKGROUND: In inflammatory bowel disease (IBD), genetic susceptibility together with environmental factors disturbs gut homeostasis producing chronic inflammation. The two main IBD subtypes are Ulcerative colitis (UC) and Crohn's disease (CD). We present the to-date largest microarray gene expression study on IBD encompassing both inflamed and un-inflamed colonic tissue. A meta-analysis including all available, comparable data was used to explore important aspects of IBD inflammation, thereby validating consistent gene expression patterns. METHODS: Colon pinch biopsies from IBD patients were analysed using Illumina whole genome gene expression technology. Differential expression (DE) was identified using LIMMA linear model in the R statistical computing environment. Results were enriched for gene ontology (GO) categories. Sets of genes encoding antimicrobial proteins (AMP) and proteins involved in T helper (Th) cell differentiation were used in the interpretation of the results. All available data sets were analysed using the same methods, and results were compared on a global and focused level as t-scores. RESULTS: Gene expression in inflamed mucosa from UC and CD are remarkably similar. The meta-analysis confirmed this. The patterns of AMP and Th cell-related gene expression were also very similar, except for IL23A which was consistently higher expressed in UC than in CD. Un-inflamed tissue from patients demonstrated minimal differences from healthy controls. CONCLUSIONS: There is no difference in the Th subgroup involvement between UC and CD. Th1/Th17 related expression, with little Th2 differentiation, dominated both diseases. The different IL23A expression between UC and CD suggests an IBD subtype specific role. AMPs, previously little studied, are strongly overexpressed in IBD. The presented meta-analysis provides a sound background for further research on IBD pathobiology.