Chronic colitis induces expression of β-defensins in murine intestinal epithelial cells

Abstract

Anti-microbial peptides are important effectors in innate immunity. In the gut they defend against pathogens, shape the commensal microbiota and probably control intestinal homeostasis. Ulcerative colitis (UC), but not Crohn's disease, shows increased expression of inducible β-defensins (hBD-2, hBD-3 and hBD-4) in colonic epithelial cells. Does inducible defensin production precede the chronic intestinal inflammation characteristic of UC, or is it a consequence of the T cell-driven chronic inflammation? The aim was to analyse defensin mRNA and protein expression in colonic epithelial cells in two colitis mouse models resembling UC, the interleukin (IL)-2(-/-) mouse and the dextran sulphate sodium (DSS)-induced colitis mouse. Defensin mRNA was assayed by in situ hybridization and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Defensin peptide was assayed by immunohistochemistry. Mouse β-defensin 3 (mBD-3, orthologue to hBD-2) was up-regulated strongly in colonic epithelium of 15-week-old IL-2(-/-) mice and DSS-induced colitis mice with chronic bowel inflammation, but not in apparently healthy IL-2(-/-) 5-week-old mice, IL-2(+/-) 15-week-old mice or in acute stage DSS mice. Up-regulation was seen both at the mRNA- and at the protein level (only mBD-3 investigated). IL-17, but not several other cytokines, including interferon (IFN)-γ, induced mBD-3 mRNA expression in mouse colon carcinoma cells. The mRNA expression level of the constitutively expressed α-defensin, cryptdin-4, was up-regulated marginally in acute stage DSS-colitis mice and in IL-2(-/-) mice before signs of colitis. Inducible β-defensin expression in colonic epithelium is the consequence of the chronic bowel inflammation caused by activated T cells releasing cytokines including IL-17.

Fig. 1

Mouse β-defensin 3 (mBD-3) mRNA (a, b) and peptide (c–f) is expressed in the chronically inflamed colonic mucosa of interleukin (IL)-2^−/− 15-week-old mice (a–d) and chronic dextran sulphate sodium (DSS)-induced colitis mice (e, f) as revealed by in situ hybridization and immunohistochemistry, respectively. (a) In situ hybridization with mBD-3 anti-sense probe; arrowheads indicate epithelial cell staining. Arrows indicate stained cells in the lamina propria. (b) In situ hybridization with negative control mBD-3 sense probe; (c) anti-mBD-3 peptide immunoperoxidase staining of IL-2^−/− 15-week-old mice colon; (d) anti-mBD-3 immunoperoxidase staining of IL-2^+/+ mouse colon; (e) anti-mBD-3 immunoperoxidase staining of colon from a mouse with chronic DSS-induced colitis; (f) negative immunohistochemistry control, colon from mouse with chronic DSS-induced colitis incubated with immunoglobulin G fraction of normal rabbit serum. Arrows in (c) and (e) indicate epithelial staining of colonic tissues. Original magnification: (a,b,c) × 252; (d,e,f) × 126.

Fig. 2

Mouse β-defensin 3 (mBD-3) (a,b,c) and cryptdin-4 (Crp-4) (d,e,f) mRNA levels in isolated epithelial cells from colonic (a,b,d,e) and small intestinal (c,f) mucosa of interleukin (IL)-2^−/− 15-week-old mice, IL-2^−/− 5-week-old mice and IL-2^+/− 15-week-old mice (a,c,d,f) and acute and chronic DSS-induced colitis mice (b,e) and 5- and 15-week-old control mice (indicated by IL-2^+/+ 5-week-old mice and IL-2^+/+ 15-week-old mice or control, respectively; a–f), as determined by real-time quantitative reverse transcription–polymerase chain reaction. Each dot represents one mouse. Horizontal bars depict median values. Statistically significant differences are indicated.

Fig. 3

Interleukin (IL)-17 but not tumour necrosis factor (TNF)-α or interferon (IFN)-γ induces significant increase in mouse β-defensin 3 (mBD-3) mRNA levels in the mouse colon carcinoma cell line, CMT93. CMT93 cells were incubated with serial dilutions of the three cytokines, Escherichia coli-derived lipopolysaccharide (3 µg/ml) or medium alone (–) and mBD-3 mRNA levels were determined by real-time quantitative reverse transcription–polymerase chain reaction. Bars indicate mean values of three experiments and whiskers, 1 standard deviation.