title: Synergy between TLR9 and NOD2 innate immune responses is lost in genetic Crohn's disease.
abstract: 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.
  
  
  OBJECTIVE
  To study possible interactions between CpG DNA (TLR9 ligand) and MDP using primary human cells of differing NOD2 genotypes.
  
  
  METHODS
  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.
authors.affiliation: Intestinal Inflammation and Repair Group, Department of Gastroenterology, Imperial College London, Hammersmith Campus, London W12 0NN, UK. d.vanheel@imperial.ac.uk
authors.forename: D A
authors.initials: DA
authors.lastname: van Heel
authors.forename: S
authors.initials: S
authors.lastname: Ghosh
authors.forename: K A
authors.initials: KA
authors.lastname: Hunt
authors.forename: C G
authors.initials: CG
authors.lastname: Mathew
authors.forename: A
authors.initials: A
authors.lastname: Forbes
authors.forename: D P
authors.initials: DP
authors.lastname: Jewell
authors.forename: R J
authors.initials: RJ
authors.lastname: Playford
mesh_terms: D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000276:immunology; D000328:Adult; D015153:Blotting, Western; D002478:Cells, Cultured; D018899:CpG Islands / Q000276:immunology; D003424:Crohn Disease / Q000235:genetics* / Q000276:immunology*; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D018928:Immunity, Mucosal; D016209:Interleukin-8 / Q000096:biosynthesis; D007413:Intestinal Mucosa / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000276:immunology*; D008024:Ligands; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology*; D008875:Middle Aged; D009154:Mutation; D053473:Nod2 Signaling Adaptor Protein; D011956:Receptors, Cell Surface / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D051197:Toll-Like Receptor 4; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D016209:Interleukin-8; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008562:Membrane Glycoproteins; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; C495371:TLR9 protein, human; D051197:Toll-Like Receptor 4; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D000119:Acetylmuramyl-Alanyl-Isoglutamine