Abstract
Crohn’s disease is associated with an altered innate immune response of pathogenic importance. This altered response can be associated to loss-of-function polymorphisms in the NOD2 (nucleotide-binding oligomerization domain-containing protein 2) gene, but also changes in transcriptional and post-transcriptional regulatory layers, including microRNA activity. Here, we characterized the link between NOD2 genotype and inflammatory-mediated changes in innate signaling by studying transcriptional and post-transcriptional activity in response to NOD2-agonist muramyl dipeptide in monocytes from healthy controls, and Crohn’s disease patients with and without NOD2 loss-of-function polymorphisms. We measured the expression of genes and microRNAs in monocytes from these subjects after stimulation with muramyl dipeptide. Gene expression profiles mainly distinguished the actual muramyl dipeptide response, but not the genotype. A hyper-responsive phenotype was found in Crohn’s disease patients without NOD2 mutations, characterized by upregulated cytokine receptors and general downregulation of microRNA expression. Conversely, microRNA expression could identify genotype-specific differences between subject groups but exhibited little change upon muramyl dipeptide treatment. Only two microRNAs showed muramyl dipeptide-induced response, including miR-155, which was found to regulate multiple genes and whose host gene was one of the highest muramyl dipeptide responders. miR-155 was upregulated in Crohn’s disease patients with NOD2 mutations following lipopolysaccharide and Escherichia coli treatment, but the upregulation was substantially reduced upon muramyl dipeptide treatment. While Crohn’s disease patients with NOD2 mutations on average showed a reduced muramyl dipeptide response, the cohort exhibited large individual variance: a small subset had inflammatory responses almost comparable to wild-type patients on both gene and miR-155 regulatory levels.
Highlights
Crohn’s disease (CD)[1] constitutes together with ulcerative colitis (UC)[2] the two most prevailing disorders under the umbrella term inflammatory bowel disease (IBD).[3]
One of the key genes identified through these studies is NOD2,8, 9 encoding a cytosolic pathogen recognition receptor that binds to muramyl dipeptide (MDP), the smallest bioactive component of peptidoglycans present in most bacteria.[10]
Activation of NOD2 leads to induction of key immune signaling pathways involved in CD pathogenesis, including the upregulation of various pro-inflammatory cytokines.[11]
Summary
Crohn’s disease (CD)[1] constitutes together with ulcerative colitis (UC)[2] the two most prevailing disorders under the umbrella term inflammatory bowel disease (IBD).[3]. Quantitative PCR (qPCR) analysis for two selected chemokines, CXCL1 and CCL2, in a larger cohort confirmed upregulation after MDP treatment in healthy controls and CDWT, but not in CDNOD2 patients (Fig. 3c,d; Supplementary Fig. 3c, d show corresponding RNA-Seq data) This is consistent with the established link between NOD2 and cytokine induction through the NF-κB pathway.[11] responses ranging from a total loss to a nearly intact inflammatory response. Consistent with the analysis above, only two miRNAs (miR-155 and miR-190A) were significantly changed as a response
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