Crohn's disease patients homozygous for the 3020insC NOD2 mutation have a defective NOD2/TLR4 cross-tolerance to intestinal stimuli

Abstract

Mutations in nucleotide-binding oligomerization domain-2 (NOD2), leading to defective recognition of bacterial peptidoglycans, are associated with Crohn's disease. The underlying mechanism that results in increased inflammation in the guts of the patients bearing NOD2 mutations is still unclear. We hypothesized that NOD2 engagement leads to cross-tolerance to stimulation of Toll-like receptors (TLR), and we investigated whether patients with Crohn's disease who bear NOD2 mutations display a disturbed NOD2/TLR cross-tolerance. Peripheral blood mononuclear cells preincubated with NOD2 ligands were specifically down-regulated for the production of tumour necrosis factor-alpha (TNF-alpha) induced by the TLR4 ligand lipopolysaccharide, as well as by intestinal microorganisms, whereas the production of anti-inflammatory cytokines was not modulated. While in cells isolated from patients with Crohn's disease with the wild-type NOD2 allele, the NOD2 engagement led to a similar cross-tolerance to TLR4-dependent stimulation of TNF-alpha, the cross-tolerance between NOD2 and TLR4 was absent in the cells of five patients homozygous for the 3020insC NOD2 mutation, leading to uninhibited release of TNF-alpha by TLR4 ligands and intestinal bacteria. In conclusion, we propose the absence of NOD2/TLR4 cross-tolerance as a central mechanism for the increased susceptibility to Crohn's disease in individuals with NOD2 mutations.

Figure 1

NOD2 induces cross-tolerance for TLR4-induced TNF-α. (a) Mononuclear cells isolated from five healthy volunteers were preincubated for 24 hr with various concentrations of MDP (0·1, 1 and 10 μg/ml), and subsequently stimulated for 24 hr with LPS (1 ng/ml), Pam3Cys (10 μg/ml) or MALP (10 μg/ml). (b) To investigate whether MDP can also induce tolerance to stimulation with intestinal pathogens, mononuclear cells tolerized with MDP in various concentrations (see above) were subsequently stimulated with heat-killed S. typhimurium or B. fragilis (both at a concentration of 1 × 10⁶ microorganisms/ml). Concentrations of TNF-α were measured by enzyme-linked immunosorbent assay. Data are presented as mean ± SD, and compared by Wilcoxon paired test (n = 5, *P < 0·05).

Figure 2

NOD2 does not influence the induction of IL-6 and IL-10 stimulated by TLRs. MNC isolated from five healthy volunteers were preincubated for 24 hr with MDP (10 μg/ml), and subsequently stimulated for another 24 hr with LPS (1 ng/ml) or Pam3Cys (10 μg/ml). Concentrations of IL-6 (a) and IL-10 (b) were measured by enzyme-linked immunosorbent assay. Data are presented as mean ± SD, and compared by Wilcoxon paired test (n = 5, all differences not significant).

Figure 3

Peptidoglycan is present in the intestinal mucosa. Immunostaining using a monoclonal mouse anti-MDP antibody (0·3 μg/ml) was performed in the colonic mucosa of healthy individuals (a) and patients with Crohn’s disease (b). The secondary antibody, biotinylated rabbit anti-mouse immunoglobulin G, was added to the incubation for 30 min. Slides were stained with streptavidin peroxidase, developed with DAB, and counterstained with haematoxylin for 30 seconds (magnification 400×).

Figure 4

Cross-tolerance induced by NOD2 stimulation is absent in patients with the 3020insC mutation. Cross-tolerance between NOD2 and TLR4, as measured by the effect of MDP (10 μg/ml) preincubation on LPS-induced (1 ng/ml) TNF-α production, was assessed in five healthy controls, five Crohn’s patients bearing the wild-type allele (NOD2wt), and five patients with the 3020insC NOD2 mutation (NOD2fs). Data are presented as mean ± SD, and compared by Mann–Whitney test (n = 5, *P < 0·05).