Abstract
Pattern recognition receptors (PRRs) are an integral part of the innate immune system and govern the early control of foreign microorganisms. Single nucleotide polymorphisms (SNPs) in the intracellular pattern recognition receptor nucleotide-binding oligomerization domain-containing protein (NOD2, nucleotide oligomerization domain 2) are associated with Crohn's disease (CD). We investigated the impact of NOD2 polymorphisms on cytokine secretion and proliferation of peripheral blood mononuclear cells (PBMCs) in response to Toll-like receptor (TLR) and NOD2 ligands. Based on NOD2 SNP analyses, 41 CD patients and 12 healthy controls were studied. PBMCs were stimulated with NOD2 and TLR ligands. After 18 h culture supernatants were measured using multiplex assays for the presence of human cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha. In CD patients, TLR-induced GM-CSF secretion was impaired by both NOD2-dependent and -independent mechanisms. Moreover, TNF-alpha production was induced by a TLR-2 ligand, but a down-regulatory function by the NOD2 ligand, muramyl dipeptide, was impaired significantly in CD patients. Intracellular TLR ligands had minimal effect on GM-CSF, TNF-alpha and IL-1beta secretion. CD patients with NOD2 mutations were able to secrete TNF-alpha, but not GM-CSF, upon stimulation with NOD2 and TLR-7 ligands. CD patients have impaired GM-CSF secretion via NOD2-dependent and -independent pathways and display an impaired NOD2-dependent down-regulation of TNF-alpha secretion. The defect in GM-CSF secretion suggests a hitherto unknown role of NOD2 in the pathogenesis of CD and is consistent with the hypothesis that impaired GM-CSF secretion in part constitutes a NOD2-dependent disease risk factor.