Scientific Abstract

Proposal No.  IBD-0126R2
Principal Investigator:  Helga-Paula Török, M.D. (replacement PI); Christian Folwaczny, M.D. (original PI)
Applicant Organization:  Klinikum der Universität München (Germany)
Project Title:  NOD2/CARD15 mutations and apoptosis in Crohn's disease
Period of Award:  July 1, 2005 – March 31, 2007

NOD2 (CARD15) mutations are associated with Crohn’s disease (CD) in Caucasians. Abrogated or decreased NF-κB activation and cytokine release following stimulation with muramyl dipeptide (MDP) in NOD2-overexpressing cells and in monocytes bearing CD-associated NOD2 mutations was observed.  These results are counterintuitive to the clinical situation in CD characterized by a systemic and mucosal inflammatory process with an excessive cytokine production.  Reduced apoptosis of monocytes and other immunocompetent cells is of paramount importance in CD.  To date, little is known about the role of the NOD2 protein in apoptosis.  However, the presence of two CARD domains (caspase activation and recruitment domains) at the N-terminus is compatible with a role of NOD2 in the programmed cell death.  Differences in apoptosis of immunocompetent cells caused by mutations in NOD2 could be a possible explanation linking NOD2 mutations to CD.

We propose to study the role of NOD2 and of CD-associated variants in apoptosis by using cells constitutively expressing these proteins, e.g., immunocompetent cells from individuals with different NOD2 genotypes.  Initially, hallmarks of apoptosis such as DNA laddering, Annexin V binding and ICE protease activity in peripheral blood monocytes and lymphocytes will be monitored after addition of cell death-inducers to whole blood preparations from patients displaying wild type NOD2 and CD-associated NOD2 variants.  Subsequently, a dissection of the apoptotic pathways in which NOD2 might be implicated will be performed.  The spectrum of activated caspases will be assessed by affinity labeling of active caspases and Western blots and the release of cytochrome c and processing of Bcl-2 proteins will be monitored by immunoblots.  To confirm the potential activated apoptotic pathway, (intrinsic or extrinsic) cells will be transfected with plasmids coding for caspase inhibitors.  Because a defective T-cell apoptosis, resulting in autoreactive T-cell clones, is of paramount importance in CD, the role of NOD2 and the influence of CD-associated NOD2-variants on T-cell apoptosis will be studied in a further experimental step. For this, the influence of activated primary monocytes with different NOD2 genotypes on susceptibility of T-cells to undergo apoptosis will be investigated using T-cell lines and primary isolated human lymphocytes.  In further experiments, monocyte-independent variations in T-cell apoptosis from individuals bearing different NOD2 genotypes will be studied using primary isolated peripheral blood lymphocytes and lamina propria lymphocytes.  To see if the results obtained in isolated cells can be extrapolated to the intestinal mucosa, the last experimental step, which might be the fundament of future work, will be the assessment of apoptosis hallmarks in immunocompetent cells in the intestinal mucosa.  For this, biopsy specimens from individuals with different NOD2 genotypes will be used.

We hypothesize that impaired apoptosis of immunocompetent cells caused by mutations in NOD2 could lead to the inappropriate initiation and resolution of inflammatory processes in response to bacterial stimuli resulting in perturbations of host homeostasis and the state of chronic inflammation, which is the hallmark of Crohn's disease.