Heterozygous nucleotide-binding oligomerization domain-2 mutations affect monocyte maturation in Crohn's disease

Abstract

Aim: To investigate the function of monocytes in Crohn's disease (CD) patients and to correlate this with disease-associated nucleotide-binding oligomerization domain-2 (NOD2) gene variants.

Methods: Monocytes from 47 consecutively referred CD patients and 9 healthy blood donors were cultured with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and stimulated with lipopolysaccharide (LPS) or muramyldipeptide (MDP), the putative ligand of NOD2.

Results: We found that monocytes from CD patients differentiated in vitro to mature dendritic cells (DCs), as determined by immunophenotype and morphology. NOD2 genotype was assessed in all subjects, and we observed high CD86 expression on immature and LPS-stimulated DCs in NOD2 mutated CD patients, as compared with wtNOD2 CD patients and controls. By contrast, CD86 expression levels of DCs induced to maturity with MDP derived from NOD2-mutated subjects were comparable to those of normal subjects. The amount of IL-12p70 in patient-cell cultures was larger than in controls after LPS treatment, but not after treatment with MDP.

Conclusion: Our results suggest that DCs obtained from patients with mutations in the NOD2 gene display an activated phenotype characterized by high CD86 expression, but have a diminished response to MDP when compared to the terminal differentiation phase. We speculate that the altered differentiation of monocytes might lead to an imbalance between inflammation and the killing ability of monocytes, and may be relevant to the pathogenesis of CD.

Figure 1

Expression of CD80 (A) and CD86 (B) in immature DCs, derived from healthy donors, and mtNOD2 and wtNOD2 patients. ^aP < 0.05, Student’s t test.

Figure 2

Expression of CD83 (A), CD1a (B) and HLADR (C) in DCs, derived from healthy donors, and mtNOD2 and wtNOD2 patients, without stimulus, after LPS or MDP stimulation.

Figure 3

Expression of CD86 in DCs derived from mtNOD2 and wtNOD2 patients and healthy donors without stimulus, after LPS or MDP stimulation. ^aP < 0.05 LPS- vs MDP-stimulated mtNOD2 patients, Student’s t test.

Figure 4

IL-12p70 levels (pg/mL) in monocyte-culture supernatants after stimulation with LPS (■) or MDP (□) in mtNOD2 and wtNOD2 patients and healthy controls. ^aP < 0.05 LPS- vs MDP-stimulated mtNOD2 patients, LPS- vs MDP-stimulated wtNOD2 patients, LPS-stimulated mtNOD2 patients vs LPS-stimulated healthy donors, LPS-stimulated wtNOD2 patients vs LPS-stimulated healthy donors; ^bP < 0.01 LPS- vs MDP-stimulated healthy donors; Student’s t test.