NOD2/CARD15 genotype influences MDP-induced cytokine release and basal IL-12p40 levels in primary isolated peripheral blood monocytes

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Abstract
The functional link between mutations in NOD2 and Crohn's disease (CD) has not been entirely elucidated. The 1007fs mutation results in loss of NF-kappaB activation in response to muramyl dipeptide (MDP) but has also been linked to an increased IL-1beta processing and IL-12 release. We investigated the basal and MDP-triggered mRNA expression and protein release for TNF-alpha, IL-10, IL-1beta, and IL-12p40 in peripheral blood monocytes from 40 CD patients and 15 healthy individuals with different NOD2 genotypes. Monocytes from individuals with 2 mutated NOD2 alleles (homozygous and compound-heterozygous individuals) displayed an impaired release of TNF-alpha and IL-10 but also of IL-1beta and IL-12p40 in response to MDP. In contrast to other NOD2 variants, the presence of at least 1 1007fs allele in double-mutated individuals completely abrogated NOD2 receptor function. Interestingly, monocytes from CD patients with 2 mutated NOD2 alleles displayed significantly higher basal levels of IL-12p40 in cell culture supernatants compared to wildtype CD patients and control individuals (P = 0.002 and P = 0.008, respectively). This was regardless of the IL23R genotype and was not mirrored by increased IL-12p40 plasma levels in these individuals. The CD-associated NOD2 variants lead, in a dose- and mutation-dependent manner, to an impaired release of TNF-alpha, IL-10, IL-1beta, and IL-12p40 in response to MDP. The finding of increased basal levels for IL-12p40-related cytokines in monocytes with 2 mutated NOD2 alleles is likely to set a new link between NOD2 mutations and the inflammatory mechanisms underlying CD.