Final Progress Report
Proposal No. IBD-0052
Principal Investigator: Koichi Kobayashi, M.D., Ph.D.
Applicant Organization: Yale University (New Haven, Connecticut, U.S.A.)
Project Title: A role of innate immunity in the pathogenesis of inflammatory bowel disease
Period of Award: January 1, 2003 – February 28, 2005
A. Summary of project aims.
Several lines of evidence indicate that the activation of the immune system by bacterial flora may cause pathogenesis. For example, it has been observed that treatment with antibiotics may relieve the symptoms of inflammatory bowel disease (IBD) patients. Mouse models of IBD, such as IL-10 deficient mice, develop little pathology in the gut under germ-free conditions, although they develop severe pathology under conventional housing conditions. Our understanding of the pathogenesis of inflammatory disease involving the innate immune system is still in an early stage, however, the recent research strongly suggest that Toll-like receptors (TLR) and Nod proteins are important players. Thus, we proposed to study the effect of the innate immune system on mucosal immunity. We hypothesized that the increased response of the innate immunity aggravates IBD. Our proposal is to test this hypothesis by using mutant mice, which have either increased or decreased TLR response. Also, we proposed to study the association of the TLR system with Nod2, of which mutations are associated with Crohn’s disease.
B. Accomplishments toward meeting those aims.
The major accomplishments are as follows:
1. We found that Nod2 deficient cells respond to TLR stimulation normally.
2. We showed that Nod2 is critical to detect MDP in vitro and in vivo.
3. We found that Nod2 deficient mice are not susceptible to intravenous Listeria infection.
4. We found that Nod2 deficient macrophages respond normally to Listeria monocytogenes infection in vitro.
5. We observed a severe susceptibility of Nod2 deficient mice to Listeria infection via oral route.
C. A list of significant results.
1. Normal TLR response in Nod2 deficient cells.
We stimulated Nod2 deficient cells with various ligands for several TLRs and the production of cytokines (IL-6, IL-12, TNFα, IL-1 and IL-10) was analyzed by ELISA. We did not see any altered response upon TLR stimulation, including TLR2 stimulation in Nod2 deficient cells.
2. Critical role of Nod2 for the detection of MDP.
There was a synergistic effect for the production of inflammatory cytokines (IL-6, IL-12 and TNFα) in wild-type macrophages when stimulated with MDP and TLR ligands for TLR2, 3 or 4. In contrast, there was no such synergistic effect in Nod2 deficient cells, suggesting the critical role of Nod2 in the detection of MDP. We also challenged wild-type and Nod2 deficient mice with MDP and LPS in vivo. Wild-type mice showed severe susceptibility to endotoxin shock after MDP priming. However, Nod2 deficient mice were resistant even after MDP priming, again indicating that Nod2 is critical to recognize MDP in vivo.
3. Normal susceptibility of Nod2 deficient mice to intravenous Listeria infection.
We challenged Nod2 deficient mice with Listeria monocytogenes infection i.v. and bacterial load in various organs was counted. There was no difference between wild-type and Nod2 deficient mice for the susceptibility to the infection.
4. Normal response of Nod2-/- macrophages to Listeria monocytogenes infection in vitro.
We infected macrophages derived from Nod2 deficient mice. There was no altered response/susceptibility compared to wild-type macrophages.
5. Severe susceptibility of Nod2 deficient mice to Listeria infection via oral route.
When we challenged Nod2 deficient mice orally (by using gastric gavage), Nod2 deficient mice showed severe susceptibility, suggesting a pivotal role of Nod2 in intestinal immunity.
D. Lay summary of this report.
Our proposal was to show the association of the innate immune system with IBD. The gene encoding Nod2 protein is frequently mutated in Crohn’s disease patients, although the physiological function of Nod2 in the intestine has been elusive. We showed that Nod2 is critical to detect the bacterial cell wall component (MDP: muramyl dipeptide) both in vivo and in vitro. Furthermore, we showed that Nod2 protects the intestine from bacterial infection. Thus, our research has advanced the IBD field by revealing the physiological role of Nod2, which is of great importance for prevention/treatment of Crohn’s disease.