Normal responses to specific NOD1‐activating peptidoglycan agonists in the presence of the NOD2 frameshift and other mutations in Crohn's disease

Abstract

Both NOD2/CARD15 alleles are mutatedin *10% of Crohn's disease patients, causing loss of functional responses to low-dose muropeptide agonists. We hypothesized that NOD2 mutations may also impair NOD1/CARD4 responses, supported by data suggesting NOD2 1007fs/1007fs patients had reduced responses to a putative NOD1 agonist, diaminopimelic acid-containing muramyl tripeptide (M-TriDAP). We measured peripheral blood mononuclear cell (n = 8 NOD2 wild type, n = 4 1007fs/1007fs, n = 6 702Trp/1007fs, n = 5 702Trp/702Trp, n = 3 908Arg/1007fs) responses to NOD1 agonists alone (IL-8/TNF-a), and agonist enhancement of lipopolysaccharide (LPS) responses (IL-1b).Significant responseswereseenwithM-TriDAPat10 nM (as withNOD2 agonists), but only at !100 nM with FK565/TriDAP. M-TriDAP induced IL-8/TNF-a secretion, and enhancement of LPS IL-1b responses was significantly reduced between NOD2 double mutation carriers versus healthy controls, whereas there was no difference with FK565 or TriDAP stimulation, or between 1007fs/1007fs cells and other genotypes. M-TriDAP contains both NOD1 (c-D-Glu-mesoDAP) and NOD2 (MurNAc-L-Ala-D-Glu) minimal structures whereas FK565/TriDAP contain only NOD1 activating structures. M-TriDAP has dual NOD1/NOD2 agonist activity in primary cells, possibly due to different intracellular peptidoglycan processing compared to the HEK293 cell system typically used for agonist specificity studies. Responses to specific NOD1 agonists are unaffected by NOD2 genotype, suggesting independent action of the NOD1 and NOD2 pathways.

To the Editor

“…In conclusion, when data from the study of van Heel et al [1] are correlated with those presented here, the most likely explanation is that NOD2 is necessary for induction of cytokines by NOD1 ligands, whereas synergism between NOD1 and TLR ligands is likely independent of NOD2. …”

“…Using these different NOD1 ligands, as van Heel et al [1] did, could produce results different to those obtained with M-triDAP. In order to test this hypothesis, we stimulated fresh PBMC of NOD2fs patients and HC with the specific NOD1 agonists FK156 and TriDAP.…”

“…It is well known that cytokine production can be induced by these endogenous ligands, and these could synergize with NOD1 stimuli [4]. A strong argument for this hypothesis is represented in the study by van Heel et al [1] by the baseline cytokine production after incubation with culture medium, which was in the range of ng/ml for TNF, which is not seen when fresh PBMC are used. Thus, frozen PBMC in their hands seem to release cytokines at baseline, and when these cells are stimulated with NOD1 ligands the results should be regarded as a synergy experiment.…”

“…We read with great interest the paper by van Heel et al [1] in the June 2006 issue of the European Journal of Immunology. In this study, the authors report that cells of patients with Crohn's disease homozygous for the NOD2 3020insC frameshift mutation have a normal response to specific NOD1 ligands.…”

Genetics of the innate immune response in inflammatory bowel disease

“…224,226,227,313,[315][316][317][318][319][320][321] Conflicting reports have been published on the influence of the CD-associated Leu1007fsinsC NOD2/CARD15 variant on NOD1/CARD4 activation in response to iE-DAP. 322,323 Discerning the precise contribution each individual PAMP receptor makes to the innate immune response has been complicated by the presence of contaminants in the preparations used and by the milieu (e.g., patients versus animal models, cell lines versus mononuclear cells)-specific effects of these receptors. This is illustrated by the conflicting results reported on the interaction between TLR-2 and NOD2/CARD15 signaling by Watanabe et al (who studied splenocytes from NOD2/CARD15 deficient mice, macrophages of mice expressing the Leu1007fsinsC NOD2/ CARD15 variant and performed a luciferase reporter gene experiment in a differentially transfected HT-29 intestinal cell line) and Netea et al (who studied human mononuclear cells, murine peritoneal macrophages and hamster ovary transfected cell lines).…”