Synergy between TLR9 and NOD2 innate immune responses is lost in genetic Crohn's disease

Background: Nucleotide binding oligomerisation domain 2 (NOD2; also known as CARD15) mutations are associated with Crohn's disease but how mutations cause disease is poorly understood. Innate immune responses are reportedly enhanced by combined NOD2 ligand (muramyl dipeptide, MDP) and Toll-like receptor 4 ligand (TLR4, lipopolysaccharide) stimulation. Intestinal TLR signalling has a dual role maintaining intestinal homeostasis and protection from injury as well as initiating inflammatory responses. TLR9 is functional in the intestinal epithelium where it is most strongly expressed in Paneth cells. Aims: To study possible interactions between CpG DNA (TLR9 ligand) and MDP using primary human cells of differing NOD2 genotypes. Subjects: NOD2 wild-type healthy controls (n = 7) and NOD2 homozygous Crohn's disease patients (n = 19), age and sex matched. Methods: Peripheral blood mononuclear cells were stimulated with CpG DNA and MDP. Cytokines were measured by enzyme linked immunosorbent assay. Results: Tumour necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8) responses to CpG DNA were similar in NOD2 wild-type and homozygous mutant cells. Concomitant NOD2 stimulation had a marked synergistic effect on CpG DNA induced TNF-alpha responses at 10-100 ng/ml MDP. A mean 2.1-fold increase in CpG DNA induced TNF-alpha responses and a mean 3.7-fold increase in IL-8 responses were observed in NOD2 wild-type cells with 10 ng/ml MDP. This effect was abolished in NOD2 homozygous cells. Conclusions: NOD2 stimulation normally enhances innate immune responses to CpG DNA. This marked synergistic effect is lost in Crohn's disease patients homozygous for NOD2 mutations, with implications for TLR mediated intestinal homeostasis and inflammation.