Epithelial expression of interleukin-37b in inflammatory bowel disease

Abstract

Interleukin (IL)-37 is a member of the IL-1 cytokine family. We investigated IL-37b expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, we analysed IL-37b expression in human colonic epithelial cells. The human colonic epithelial cell line T84 and human colonic subepithelial myofibroblasts (SEMFs) were used. IL-37b expression in the IBD mucosa was evaluated by immunohistochemistry. IL-37b mRNA and protein expression were determined by real time-polymerase chain reaction (PCR) and Western blotting, respectively. IL-37b was not detected in the normal colonic mucosa. In the inflamed mucosa of IBD patients, epithelial IL-37b expression was increased markedly. In ulcerative colitis (UC) and Crohn's disease (CD) patients, IL-37b expression was enhanced in the affected mucosa. In the intestinal epithelial cell line T84, the expression of IL-37b mRNA and protein was enhanced by tumour necrosis factor (TNF)-α. This IL-37b induction by TNF-α was mediated by nuclear factor (NF)-κB and activator protein (AP)-1 activation. Furthermore, IL-37b inhibited TNF-α-induced interferon-γ-inducible protein (IP)-10 expression significantly in human colonic SEMFs. Epithelial IL-37b expression was increased in IBD patients, especially UC patients. IL-37b may be involved in the pathophysiology of IBD as an anti-inflammatory cytokine and an inhibitor of both innate and acquired immune responses.

Fig. 1

Immunohistochemical analyses of interleukin (IL)-37b expression in the human colon. (a) Normal mucosa, (b) active ulcerative colitis (UC), (c) inactive UC, (d) active Crohn's disease (CD) and (e) inactive CD. Original magnification ×100.

Fig. 2

Interleukin (IL)-37b mRNA and protein expression in the human intestinal epithelial cell lines. (a) Intestinal epithelial cell line T84 cells were stimulated with cytokines [IL-1β (10 ng/ml), other cytokines (100 ng/ml) and lipopolysaccharide (LPS) (1·0 μg/ml)] for 12 h, and then the IL-37b mRNA expression was determined by real-time polymerase chain reaction (PCR) (left side). Caco-2 cells were also stimulated with tumour necrosis factor (TNF)-α (100 ng/ml) for 12 h, and the IL-37b mRNA expression was determined by real-time PCR (right side). All values are expressed as means ± standard deviation (n = 3). **P < 0·01; a significant difference from the values for medium alone. (b) T84 cells were stimulated with TNF-α (100 ng/ml) for 24 h, and then the intracellular IL-37b was detected by Western blotting.

Fig. 3

Dose- and time-dependent effects of tumour necrosis factor (TNF)-α on interleukin (IL)-37b mRNA expression in T84 cells. (a) Cells were stimulated for 24 h with increasing concentrations of TNF-α, and then the IL-37b mRNA expression was analysed by real-time polymerase chain reaction (PCR) (b) Cells were stimulated with TNF-α (100 ng/ml) for different time-periods, and then the IL-37b mRNA expression was analysed by real-time PCR. All values are expressed as means ± standard deviation (n = 3).**P < 0·01 and *P < 0·05; a significant difference from the values for medium alone.

Fig. 4

Molecular mechanisms underlying interleukin (IL)-37b mRNA expression. (a) Effects of MAP-and PI3-kinase inhibitors on IL-37b mRNA expression in the intestinal epithelial cell line T84. The cells were pretreated with 10 μM mitogen-activated protein (MAP) kinase inhibitors (SB203580, PD098059 or U02016) and 10 μM PI3-kinase inhibitor (LY294002) for 15 min each, and then stimulated with tumour necrosis factor (TNF)-α (100 ng/ml) for 24 h. The IL-37b mRNA expression was then determined by real-time polymerase chain reaction (PCR). All values are expressed as means ± standard deviation (s.d.) (n = 3). **P < 0·01; a significant difference from the values for TNF-α stimulation. (b) Effects of nuclear factor (NF)-κB and/or activator protein (AP)-1 inhibition on IL-37b mRNA expression. T84 cells were infected with an adenovirus expressing IκBΔN or DN-c-Jun, and at 48 h after infection the cells were stimulated with TNF-α (100 ng/ml) for 24 h. IL-37b mRNA expression was then determined by real-time PCR. All values are expressed as means ± s.d. (n = 3). **P < 0·01; a significant difference from the values for TNF-α stimulation.

Fig. 5

Effects of interleukin (IL)-37b on the tumour necrosis factor (TNF)-α-induced interferon-γ-inducible protein (IP)-10 expression in human colonic subepithelial myofibroblasts. Cells were pretreated with or without IL-37b (100 ng/ml) for 6 h, and then incubated further with or without TNF-α (100 ng/ml) for 24 h. The IP-10 secretion was determined by enzyme-linked immunosorbent assay (ELISA). (a) IP-10 mRNA, and (b) IL-10 protein. All values are expressed as means ± standard deviation (n = 3). **P < 0·01, *P < 0·05; a significant difference from the values for TNF-α stimulation.