Abstract
The first and most consistently replicated risk gene linked to Crohn’s Disease (CD) is nucleotide binding oligomerization domain 2 (NOD2). NOD2 plays multifaceted roles in maintaining gut homeostasis, and impairment of NOD2 function contributes to CD pathogenesis by altering gut immunity. However, therapies specifically enhancing NOD2 function do not currently exist. NOD2 is negatively regulated by carbamoyl phosphate synthetase/ aspartate transcarbamylase/ dihydroorotase (CAD), an enzyme involved in nucleotide synthesis. In vitro studies suggest that pharmacologic inhibition of CAD activity enhances NOD2-dependent signaling and anti-bacterial activity. This study examined the specific effects of CAD inhibition on NOD2-dependent immune responses and investigated whether enhancement of NOD2 function in vivo using a CAD enzyme inhibitor could be a novel therapeutic target for CD. NOD2-dependent signal transduction, target gene expression, cytokine secretion and Salmonella killing was assessed in vitro using HCT116 human epithelial cells, mouse bone marrow-derived macrophages (BMDM) and human peripheral blood monocyte-derived macrophages from healthy individuals (n = 6) and CD patients (n = 7) by immunoblot, qPCR, ELISA and gentamicin protection assay. The effect of enhanced NOD2 function by CAD inhibition was assessed in vivo using the 2.5% dextran sulfate sodium (DSS)-induced colitis model in C56BL/6 and Nod2−/− mice. Disease activity index, CAD enzyme activity, serum amyloid A levels (systemic inflammation), colonic cytokine production (local inflammation), antimicrobial peptide expression and a 12 point histologic colitis score were evaluated. Inhibition of CAD activity specifically enhanced intracellular Salmonella killing by macrophages from both healthy individuals (37.4% ± 7.0% enhancement, P = 0.006) and CD patients (29.4% ± 6.1% enhancement, P = 0.008). Enhanced bactericidal activity in response to CAD inhibition was dependent on NOD2 expression, as it was not observed in HCT116 cells treated with NOD2 RNAi or in Nod2−/− BMDM treated with CAD inhibitors. CAD inhibition stimulated autophagy induction and antimicrobial peptide expression without altering TNFα, IL-6, or IL-1β secretion in vitro. In experimental colitis models using a pharmacologic CAD inhibitor, enzyme activity was significantly inhibited (29.8 ± 5.4 versus 52.7 ± 2.9 nM, P < 0.001) and resulted in improved colitis scores (5.7 ± 0.5 versus 9.5 ± 0.9, P = 0.003) and reduced serum amyloid A levels, (489.6 ± 237 versus 1315.1 ± 326.5 ng/mL, P < 0.01). These effects were not observed in Nod2−/− mice. Systemic CAD inhibition also enhanced colonic antimicrobial peptide expression and reduced colonic cytokine secretion. Inhibition of CAD enzyme activity preferentially activates NOD2-dependent antimicrobial responses without inducing pro-inflammatory cytokine production. Stimulation of NOD2-dependent antimicrobial defenses protects against experimental colitis and reduces systemic inflammation. These findings suggest that CAD may be a future therapeutic target for CD.
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