Expression of Toll-like receptor-3 is enhanced in active inflammatory bowel disease and mediates the excessive release of lipocalin 2

Abstract

Anti-microbial peptides might influence the pathogenesis and course of inflammatory bowel disease (IBD). We sought to clarify the role of the anti-microbial glycoprotein lipocalin 2 (LCN2) in the colon by determining its localization and regulation in IBD. Following a microarray gene expression study of colonic biopsies from a large IBD population (n = 133), LCN2 was localized using immunohistochemistry and in-situ hybridization. Moreover, we examined the regulation of LCN2 in HT-29 cells with a panel of pattern recognition receptors (PRRs) and sought evidence by immunohistochemistry that the most relevant PRR, the Toll-like receptor (TLR)-3, was indeed expressed in colonic epithelium in IBD. LCN2 was among the 10 most up-regulated genes in both active ulcerative colitis (UCa) and active Crohn's disease (CDa) versus healthy controls. LCN2 protein was found in both epithelial cells and infiltrating neutrophils, while mRNA synthesis was located solely to epithelial cells, indicating that de-novo synthesis and thus regulation of LCN2 as measured in the gene expression analysis takes place in the mucosal epithelial cells. LCN2 is a putative biomarker in faeces for intestinal inflammation, different from calprotectin due to its epithelial site of synthesis. LCN2 release from the colonic epithelial cell line HT-29 was enhanced by both interleukin (IL)-1β and the TLR-3 ligand poly(I:C), and TLR-3 was shown to be expressed constitutively in colonic epithelial cells and markedly increased during inflammation.

Keywords: Crohn's disease; TLR-3; inflammatory bowel disease; lipocalin 2; ulcerative colitis.

Figure 1

The results of microarray-derived gene expression levels of lipocalin 2 (LCN2) mRNA in colonic biopsies are shown as log₂. Mean and individual values plotted. UC: ulcerative colitis; CD: Crohn's disease; a, active; i, inactive. *P < 0·001 versus control, #P < 0·001 versus inactive disease.

Figure 2

(a) Immunohistochemical staining of lipocalin 2 (LCN2) in colonic biopsies from controls, active and inactive ulcerative colitis (UC), active and inactive Crohn's disease (CD). LCN2 is located in the infiltrating neutrophils and the epithelial cells, both with the appearance of enterocytes and goblet cells. We found significantly increased LCN2 expression in epithelium of active ulcerative colitis (UC) and active Crohn's disease (CD) versus controls. (b) In-situ hybridization of LCN2 in the same biopsies as immunohistochemical staining. The LCN2 mRNA is located only in the epithelial cells, indicating that these cells are responsible for de-novo synthesis. Also, we found significant expression of LCN2 mRNA in the epithelium of active disease versus healthy control mucosa. Original magnification ×10 and ×40.

Figure 3

Lipocalin 2 (LCN2) release from HT-29 cells stimulated with pattern recognition receptor (PRR) ligands; from left to right, Toll-like receptors (TLR)-1–9, nucleotide-binding oligomerization domain (NOD)2 and the cytokines interleukin (IL)-10 and IL-1β for 20 h. We found a considerable constitutive release of LCN2. * and #P < 0·05 versus medium. Mean ± standard deviation is shown.

Figure 4

Lipocalin 2 (LCN2) release from HT-29 cells after 20 h stimulation with proinflammatory cytokines with and without poly(I:C). Poly(I:C) and interleukin (IL)-1β both induce LCN2 release, but in an additive pattern. * and #P < 0·05 versus medium control. ¤P < 0·05 versus poly(I:C) control. Mean ± standard deviation.

Figure 5

The poly(I:C) effect on lipocalin 2 (LCN2) release was silenced in HT-29 cells by transfection with Toll-like receptor (TLR)-3 siRNA. The cells were transfected for 24 h using TLR-3 siRNA or control/non-signalling siRNA and then stimulated with poly(I:C) or left unstimulated (medium) for 20 h. * and #P < 0·05. Mean ± s.d. of triplicates.

Figure 6

Immunohistochemical staining of Toll-like receptor (TLR)-3 in colonic biopsies from controls, active and inactive ulcerative colitis (UC), active and inactive Crohn's disease (CD). TLR-3 staining is found in the epithelium and also in various cell types in lamina propria. The TLR-3 expression of the epithelium was enhanced in both active UC and active CD versus healthy controls. Original magnification ×10 and ×40.

Figure 7

(a) Serum lipocalin 2 (LCN2) levels in ulcerative colitis (UC) and Crohn's disease (CD) patients (both active and inactive disease) versus controls. *P < 0·05, two-sided versus controls. (b) Serum LCN2 level in endoscopic verified active UC, n = 22 and inactive UC, n = 16 versus healthy controls, n = 23. *P < 0·05 two-sided, versus inactive UC. For difference between active UC and controls, P = 0·0550. In both figures mean ± standard deviation is shown.