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

Abstract

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.

Keywords: CD; IBD; NGAL; UACL; pyloric metaplasia; regeneration; wound healing.

Figure 1

NGAL/LCN2 is expressed in all stages of small intestinal UACL and in colonic regenerative crypts. (A) Serial sections (from left to right) showing H&E, AB‐PAS, IHC for NGAL and ISH for LCN2 with positivity in UACL‐crypts. (B) IHC NGAL (left) and AB‐PAS (right) showing scattered NGAL and AB/PAS positive cells possibly part of the early formation of UACL buds. (C) Serial sections with ISH LCN2, ISH LGR5 and IHC Ki‐67 showing LCN2 in both proliferating (black arrows) and nonproliferating (white arrows) parts of the UACL. LGR5‐positive stem cell area in adjunction with UACL marked with white arrow‐head. (D) Serial sections with IHC for NGAL (top) and MUC5AC (bottom) showing NGAL expression in all parts of UACL, while MUC5AC was expressed mainly toward the lumen of the bowel. (E) IHC for NGAL in colonic non‐UACL inflamed epithelium with crypt abscesses and branching. NGAL expression is intense in the reactive and branching epithelium (arrows). NGAL is also expressed in granulocytes and in cell debris.

Figure 2

IHC for selected proteins encoded by genes differentially expressed in UACL confirms the RNA‐sequencing data from UACL‐crypts. All genes were upregulated except MUC2 which was significantly downregulated in the gene expression analysis. Serial sections from inflamed mucosa of active CD with UACL‐crypts (left) and normal control mucosa in comparison (narrow, right). (A) H&E. UACL marked with arrows. (B) Insulin‐like growth factor‐binding protein 5. (C) Mucin 5ac. (D) Mucin 2. (E) Reticulocalbin 1. (F) Pleckstrin homology‐like domain family A member 1. (G) Pepsinogen C. (H) Lysozyme. (I) BPI fold‐containing family B member 1. (J) Neutrophil gelatinase‐associated lipocalin. K Notch 2. L syndecan 3. ×10 objective magnification.

Figure 3

IDisco 3D surface visualization of NGAL‐positive (green) UACL‐crypts with buds and neutrophils in inflamed ileal mucosa from CD. Hoechst nuclear counterstain (blue).

Figure 4

NGAL is expressed in undifferentiated human colonoids. (A) ISH for LCN2 in colonoids (from left to right) on day 0, day 6 and day 12. Scale bar 50 μm. (B) ELISA for NGAL in supernatants from cultures of colonoids kept undifferentiated, or being differentiated (Representative of n = 7. Start of differentiation on day 8.) (C) ELISA for NGAL in supernatants from cultures of undifferentiated and differentiated colonoids (first two columns), and colonoids differentiated for 2 days before restimulation with Wnt3a, Notch (by removing the notch‐inhibitor DAPT), MAP kinase inhibitor (SB202190), nicotinamide, or all four factors for 2 days (last five columns). Mean and SEM from n = 3 biological replicates. (D) Serial sections of undifferentiated (left) and differentiated (right) colonoids. ISH for LCN2 (top) and IHC for CK20 (bottom) shows LCN2 expression mainly in CK20‐negative, undifferentiated colonoids. Scale bar 100 μm.