3rd Annual BMRP Investigator Meeting - Abstract

Role of NOD2/CARD15 for Oxidative Burst Reaction and NF-κB Activation in Intestinal Macrophages

Gerhard Rogler^a, Hans Herfarth, Julia Brenmoehl, Juergen Schoelmerich and Ernst Holler

Department of Internal Medicine I and Division of Hematology/Oncology, University of Regensburg (Regensburg, Germany)

Background: NOD2/CARD15 has been described as an intracytoplasmatic sensor for bacterial muramyl dipeptide (MDP).  Recently, a significant association of mutations in single nucleotide polymorphisms (SNPs) in the NOD2/CARD15 gene resulting in a diminished antibacterial defense has been reported as a major risk factor in Crohn’s disease (CD).  Due to the similarities of inflammatory changes in CD and graft-versus-host disease (GvHD) after bone marrow transplantation, we speculated that NOD2/CARD15 SNPs might play a role in the pathogenesis of GvHD.  Furthermore, we tested whether NOD2/CARD15 cause an increased activation or a loss of function/activation in intestinal macrophages (IMACs) by monitoring their oxidative burst reaction and NF-κB activation.

Methods: DNA samples from 350 bone marrow recipients and donors were typed for the presence of SNP 8, 12 and 13 mutations using a sensitive Taqman PCR.  Results were compared with clinical outcome.  Oxidative burst reaction and NF-κB activation were investigated by flow cytometry and immunohistochemistry in IMACs, which were isolated via anti-CD33 immunomagnetic beads.

Results: The incidence of severe GvHD (and associated gastrointestinal GvHD) rose from 18% in stem cell donor (D)/recipient (R) pairs without any mutations to 37% in pairs with either D or R mutations with a subsequent increase of treatment related mortality (TRM) from 33 to 60% (HR 2.3, p 0.002).  The effect of NOD2/CARD15 mutations was even more pronounced in the small subgroup of 11 D/R pairs where both D as well as R revealed mutated SNPs: severe GvHD rose from 22 to 55% (p 0.01) and TRM from 38% to 100% (p 0.001, HR 3.3).  Oxidative burst activity was 1.4 +/- 0.7 in WT controls, 2.0 +/- 1.4 in NOD2 mutated controls, 2.7 +/- 1.1 in WT CD patients and 3.8 +/- 2.8 in NOD2-mutated CD patients, indicating that NOD2/CARD15 mutations are followed by an increased cell activation of IMACs even in controls and more pronounced in CD patients.  The same trend was found for NF-κB activation.

Conclusion: Our observations indicate a major role of NOD2/CARD15 mutations in GI-GvHD following allogenic stem cell transplantation as an example for chronic mucosal inflammation.  A deficient antibacterial response in both IEC of the recipient’s mucosa and donor macrophages might result in increased bacterial translocation and subsequent mucosal inflammation.  Assuming a comparable pathophysiology in GvHD and CD, these data support the hypothesis that the primary pathophysiology in a subgroup of CD patients is a IMACs/IEC defect and that alterations in T-cell function are secondary.  In addition, our results indicate that an increased activation of IMACs is found in the mucosa of patients with CD bearing NOD2/CARD15 mutations, which is in conflict with the hypothesis of a simple loss of function mutation, but favors the hypothesis of a failure in suppression of TLR2 signalling as recently concluded from in vitro data.

^aPrincipal Investigator