ORIGINAL ARTICLE Mucosal NOD2 Expression and NF-B Activation in Pediatric Crohn’s Disease Laura Stronati, PhD,* Anna Negroni, PhD,* Paola Merola, DSc,* Veronica Pannone, MD, † Osvaldo Borrelli, MD, † Manuela Cirulli, MD, † Vito Annese, MD, ‡ and Salvatore Cucchiara, MD, PhD † Background: Recent advances in the pathogenesis of Crohn’s disease (CD) have suggested that an aberrant innate immune re- sponse initiates the cascade of events leading to T-cell activation and to disease development. NOD2 protein, which is mainly expressed by innate immunity cells, appears to play a key role against bacteria by triggering a host defense response through the activation of the transcriptor factor NF-B and a consequent proinflammatory cyto- kine production. The present study was aimed at investigating the expression and activity of NOD2, NF-B, and of 2 proinflammatory cytokines, TNF and IL-1, in mucosal biopsies of CD affected children compared to healthy controls. Methods: In all, 22 children with active CD and 10 matched controls were entered in the study. mRNA and protein expressions were detected using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot; NF-B binding activity was assessed by electromobility gel shift assay (EMSA). Results: NOD2 and IL-1 mRNAs were upregulated in CD chil- dren. Protein levels of NOD2, TNF, and nuclear NF-B, as well as the binding activity of NF-B to a consensus DNA sequence, were significantly increased in inflamed mucosa of patients as compared to controls. Moreover, NF-B activity was strongly upregulated in patients also when bound to the NOD2 promoter site. No difference was seen between patients and controls when NF-B binding activ- ity was determined in the uninflamed tissue. Conclusions: This study suggests that altered mechanisms regu- lating NOD2 induction, NF-B activation and cytokine production may contribute to dysregulate the innate immune response underly- ing pediatric CD. (Inflamm Bowel Dis 2008;14:295–302) Key Words: Crohn’s disease, innate immunity, inflammation, intestinal mucosa, children C rohn’s disease (CD) is a disorder characterized by chronic inflammation of the gastrointestinal tract. Al- though the molecular basis of the pathogenesis is not yet clear, it has been suggested that the disease occurs in genet- ically susceptible hosts as a consequence of a dysregulated response of the mucosal immune system toward the commen- sal enteric flora. 1–3 Although CD has been classically thought to be sustained by an altered adaptive immune response, it has recently been hypothesized that the primary defect could be an impaired innate immune system, which relies on spe- cific sensing of conserved pathogen-associated molecular pat- terns (PAMPs). 4,5 Several classes of proteins, termed pattern recognition molecules (PRMs), have been assigned to partic- ipate in these recognition events. 6 Among them, nucleotide- binding domain (NOD) proteins, a new class of intracellular receptors, in particular NOD1 and NOD2, seem to have a pivotal role in regulating inflammatory cytokine release and in controlling the development of inflammatory reactions. 7–9 NOD2 is expressed in monocytes, macrophages, den- dritic cells, epithelial cells, and Paneth cells. This protein consists of 2 N-terminal caspase-activation recruitment do- main (CARD) regions, a NOD, and a C-terminal leucin-rich- repeat region (LRR) and is involved in the recognition of the bacterial peptidoglycan-derived muramyl dipeptide (MDP) through the LRR region leading to the induction of immune response. Current evidence suggests that NOD2 interacts with a serine threonine kinase RICK (or RIP2), leading to the activation of NF-B, a major transcriptional regulator of proinflammatory cytokines involved in intestinal inflamma- tion, such as TNF and IL-1. 10 –13 Evidence of the role of the innate response in inflam- matory bowel disease (IBD) comes from the recent associa- tion between susceptibility to CD and NOD2 leucine-rich repeat variants, in particular a frameshift mutation that leads to a partial truncation of the protein in the LRR and is associated with a defect in the MDP response. 14,15 Most studies on NOD2 function in the immune re- sponse in CD have been mainly based on plasmid transfected cell lines or mouse models and very few have used primary Received for publication September 10, 2007; Accepted October 8, 2007. From the *Section of Toxicology and Biomedical Sciences, Enea, Rome, Italy; † Department of Paediatrics, University of Rome, La Sapienza, University Hospital Umberto I, Rome, Italy, ‡ Unita ` di Gastroenterologia, Ospedale IRCCS Casa Sollievo della Sofferenza San Giovanni Rotondo (Fg), Italy. Reprints: Salvatore Cucchiara, MD, PhD, Professor of Pediatrics, Division of Pediatric Gastroenterology, Head, Department of Pediatrics, University of Rome “La Sapienza,” Viale Regina Elena, 324, 00161 Rome, Italy (e-mail: salvatore.cucchiara@uniroma1.it). Copyright © 2007 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1002/ibd.20332 Published online 18 December 2007 in Wiley InterScience (www. interscience.wiley.com). Inflamm Bowel Dis ● Volume 14, Number 3, March 2008 295