Final Progress Report

Proposal No. IBD-0045R
Principal Investigator:  Gerhard Rogler, M.D., Ph.D.
Applicant Organization:  University of Regensburg (Germany)
Project Title:  Role of CARD15/NOD2 for oxidative burst reaction and NF-kappaB activation in intestinal macrophages
Period of Award:  October 1, 2003 - February 28, 2006

1.  Summary of project aims

Initially the proposed project had the following aims:

1.1 Study of oxidative burst reaction in NOD2/CARD15 mutated CD patients ex vivo in comparison to patients without NOD2/CARD15mutations.

(In previous experiments we had found a complete lack of oxidative burst reaction in intestinal macrophages from normal mucosa and a high induction in Crohn’s disease We had assumed that if NOD2/CARD15 mutations play a role in tissue destruction and oxidative burst activation higher values in mutated patients would occur.)

1.2. Determination of NF-κB activation in vivo in intestinal macrophages in correlation to NOD2/CARD15 mutations.

(As it is still unclear whether mutations of NOD2/CARD15 in humans are followed by a loss or gain of functions mutation these experiments would help to understand the consequence of those mutations for the mucosal immune system in IBD patients.)

 
1.3 Investigation of the functions of NOD2/CARD15 forms on oxidative burst activity and NF-κB activation in vitro

(Wildtype as well as Leu1007fsinsC NOD2/CARD15 were to be introduced into primary IMACs by microinjection.)

2.  Accomplishments towards meeting those aims

Aim 1

29 control patients with NOD2/CARD15 wildtype, 6 control patients with NOD2/CARD15 variants (3 SNP8 heterozygous, 2 SNP 12 heterozygous, 1 SNP 8 heterozygous), 27 CD patients with NOD2/CARD15 wildtype and 14 patients with heterozygous NOD2/CARD15 variants (7 SNP13 heterozygous, 4 SNP 8 heterozygous, 3 SNP 12 hetrozygous) have been analysed. The results are given in Figure 1.



Figure 1: Oxidative burst activity of macrophages isolated from intestinal mucosa of controls and Crohn’s disease patients. Differences for NOD2 WT and mut groups did nor reach a level of significance.

Differences for NOD2 WT and mutated groups did nor reach a level of significance. The difference between controls and CD patients that had been reported by our group before could be confirmed (Figure 2)

 
 
Figure 2: Oxidative burst activity of macrophages isolated from intestinal mucosa of controls and Crohn’s disease patients. A significant difference between controls and CD patients was observed.

It may be concluded that NOD2 mutations do not significantly influence oxidative burst activity of intestinal macrophages. This indicates that our first hypothesis was wrong.

Aim 2

24 control patients with NOD2/CARD15 wildtype, 5 control patients with NOD2/CARD15 variants, 21 CD patients with NOD2/CARD15 wildtype and 12 patients with heterozygous NOD2/CARD15 variants have been analysed.
 

In contrast to our analysis on oxidative burst we found a significant association of NF-kappaB activation an NOD2 status as visible from figure 3:
 

 
Figure 3: NF-kappaB activation in intestinal mucosa of controls and Crohn’s disease patients. A significant difference between WT and NOD2 mutated patients was observed.

These results were confirmed by Western Blot analysis.





Aim 3

We planed to microinject a NF-B-GFP reporter plasmid and wildtype NOD2/CARD15/ truncated NOD2/CARD15into primary macrophages from normal and inflamed mucosa (Part B of the grant proposal). Doing these experiments we faced a number of problems. The major problem was, that the primary intestinal macrophages do hardly re-attach to culture plastic surfaces. The only surface which was optimal for re-attachment (Primaria® plates, Falcon) could not be used, because the special treatment of the surface of these plates makes it impossible to use them under the microinjection microscope.


Another emerging problem was that intestinal macrophages are more sensitive to the microinjection procedure that monocytes, macrophage cell lines or in vitro differentiated macrophages. They simply “popped” after microinjection. We had to improve our technical skills and had to find a different room for the microinjection.
 
3) List of significant results

In addition to the results mentioned above we performed a number of experiments on the role of NOD2/CARD15 mutations for graft versus host disease. As these studies were very successful we focussed more on the studies mentioned below and reduced efforts to successful microinject NOD2 constructs into isolated macrophages.
 
NOD2/CARD15 variants and their role for graft versus host disease (GvHD)
 
DNA samples in 2 cohorts of 169 consecutive recipient (-R-) /donor (-D-) pairs from Regensburg and 102 R/D pairs from Newcastle and Vienna were typed for the presence of SNP 8,12 and 13 mutations using a sensitive Taqman PCR and results were compared with clinical outcome. In the first set of patients, the incidence of severe GvHD (and associated gastrointestinal GvHD) rose from 18% in 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). The association of NOD2/CARD15 mutations proved to be an independent risk factor for TRM in multivariate analysis. In the second cohort of patients, severe GvHD rose from 12% in wildtype D and R to 39% in pairs with either D or R mutations, and TRM doubled from 18% to 38% (p 0.05). Again, prognosis was poorest in 5 pairs with mutations of both, D and R with severe GvHD occurring in 4/5 patients and a TRM of 60% (p 0.01).
 
This observations obtained in 2 independent cohorts of patients indicate a major role of NOD2/CARD15 mutations in GvHD (especially GI-GvHD) following allogenic stem cell transplantation: A deficient antibacterial response in both, IEC/Paneth cells of the recipient’s mucosa and donor monocytes might result in increased bacterial translocation and subsequent mucosal inflammation. Assuming a comparable pathophysiology in GvHD and CD, these data suggest the hypothesis that the primary pathophysiology in a subgroup of CD patients is a IEC- and monocyte/macrophage defect and that alterations in T-cell function are secondary.

These results have been published in Blood and outlined as a Viewpoint in Nature Clinical Practise Gastroenterology.
 
In order to assess the role of NOD2/CARD15 variants on the long term outcome of allogeneic stem cell transplantation in a genetically homogenous group, we extended the above mentioned study and genotyped DNA from additional 225 recipients and their HLA-identical sibling donors from 4 European centres for NOD2/CARD15 SNPs. Results of genotyping were compared with clinical outcome: The strong association of NOD2/CARD15 variants with transplant related mortality (TRM) was confirmed in univariate and multivariate analysis: TRM increased from 20% in recipient/donor pairs without any NOD2/CARD15 variants to 47in the presence of 1 variant in either donor or recipient and further up to 74% in the presence of 2 or more variants (p < 0.002). NOD2/CARD15 SNPs were not associated with relapse rate but had a strong impact on overall survival. In an analysis of centre effects, the type of gastrointestinal decontamination was the only factor interfering with the prognostic significance of NOD2/CARD15 SNPs. Our data further support an interaction between gastrointestinal defense mechanisms, activation of the innate immune system and specific transplant related complications.
 
In the cohort of matched unrelated donors further analysis revealed that if donors had a SNP13 variant (n=23) there was a higher risk for GvHD and NRM. GvHD grade III/IV of these patients rose from 22 to 48% (p 0.007) and 1 yr NRM from 27% to 59% (p 0.007). Multivariate cox regression analysis for 1 yr NRM revealed a strongly increased risk of mortality in the presence of donor SNP13 variants (HR 3.7, 95% CI 1.77 –7.79, p 0.001) whereas other risk factors such as age, stage at the time of SCT and more important, mismatch at an allel- or even antigen-level did not reach significance. 1 yr NRM almost doubled in this high-group, but did not reach significance.
 
These data suggest a different but still relevant role of NOD2/CARD15 variants in matched unrelated donor transplants as compared to HLA identical sibling SCT. As NOD2/CARD15 SNP13 variants result in a stronger suppression of NOD2/CARD15 dependent function than other SNPs altered monocyte/macrophage functions in unrelated donors may contribute to these specific effects. Our data furthermore suggest for the first time that matching for a non-HLA gene could be more relevant than differentiated HLA-matching in URD-SCT and strongly support a role of intestinal bacterial translocation and a crucial role of macrophages which is in support of our intial hypotheses.

NOD2/CARD15 variants and their role for lung complications after stem cell transplantation

After we had shown that NOD2/CARD15 variants (SNPs) play a major role for the outcome after SCT we investigated whether it could also play a role for lung diseases. Bronchiolitis obliterans syndrome (BOS) is a life-threatening complication after allogeneic SCT with high mortality. Risk factors and pathophysiology have yet to be defined. NOD2/CARD15 is expressed not only in monocytes/macrophages and intestinal epithelial cells IEC but also in bronchial epithelial cells.
 
The association of NOD2/CARD15 SNPs (SNP 8, 12, 13) with the occurrence of BOS was investigated in 244 donor/recipient (D/R) pairs of patients receiving allogeneic SCT. Follow-up was performed for a mean of 1243 172 days (range: 327-2149). BOS was diagnosed by pathology or by functional airway obstruction (FEV1</= 80% of predicted).
 
12 patients developed BOS with a mean time point of diagnosis at 678 112 days after SCT (range: 186-1407). NOD2/CARD15 variants were observed at higher frequencies in D/R pairs developing BOS (donor: 60.0% vs. 12.9%, p=0.001; recipient: 50.0% vs. 15.0%; p=0.007). 50% of patients with BOS died between day +327 and day +1582 (mean: 844 195), whereas 6 patients are still alive (mean follow-up: 1642 170 days). Incidence of BOS rose from 1.7% (3/174) in D/R pairs without mutated SNPs to 10.0% (6/60) in pairs with mutated alleles in either donor or recipient (p=0.01) and to 30.0% (3/10) in pairs with mutated alleles in both donor and recipient (p=0.009). Survival did not differ between patients with BOS in relation to the presence/absence of NOD2/CARD15 mutations. Finally, we analyzed whether other transplant-related parameters contributed to the incidence of BOS. Conditioning regimen intensity, stem cell source, donor type (matched unrelated donor vs. HLA-identical sibling), recipient age, donor or recipient gender, and acute GVHD were not associated with BOS development, whereas chronic GVHD was confirmed as a risk factor for BOS (p=0.01).
 
Our data show that variants of the NOD2/CARD15 gene are not only associated with an increased risk to develop Crohn’s disease and GvHD after SCT but also with the development of lung disorders such as BOS. This indicates that either NOD2/CARD15 directly plays a role in the bronchial epithelial cells or bacterial translocation in the gut is relevant for lung disease.
A manuscript is in preparation.

NOD2/CARD15 variants and the role for mortality during sepsis
 
Subsequent to our finding of an association between NOD2/CARD15 variations and a significantly increased rate of transplant related mortality due to intestinal and pulmonary complications in stem cell transplantation we investigated whether NOD2/CARD15 variant could play a role in bacterial translocation during sepsis. To assess a possible contribution of variants in the NOD2/CARD15 gene to sepsis related mortality we investigated 132, prospectively characterised, consecutive patients with sepsis at three intensive care units at the University of Regensburg. PCR analysis for major SNPs 8,12, and 13 of the NOD2/CARD15 gene was performed and confirmed by sequencing of the coding regions. Sepsis related mortality (day 30) was increased in patients with NOD2/CARD15 coding variants (42% versus 31%) and was highest (57%) in 8 patients carrying the 1007insC variant (SNP13, p< 0.05). Multivariate analysis demonstrated the 1007insC genetic variant as an independent risk factor for sepsis related mortality. Therefore, our findings indicate a major role of NOD2/CARD15 variants and, therefore, impaired barrier function and bacterial translocation in the pathophysiology of early sepsis related death highlighting the important role of this protein for the function of the mucosal immune system.
 
Lay Summary

Mutations of a gene called NOD2/CARD15are found in 25-40% of patients with a specific type of chronic bowel inflammation, Crohn’s disease. While it is clear that mutations in this gene increase the risk to develop Crohn’s disease dramatically it is not clear how this happens. If NOD2/CARD15mutations are present Crohn’s disease occurs at earlier age and is more complicated by bowel obstructions. The reasons for this also are unknown.
 
NOD2/CARD15protein is mainly found in a certain cell type of the immune system called “macrophages”. Normal gut-macrophages are non-reactive cells, while in chronic bowel inflammation they show signs of activation. It has been unclear whether NOD2/CARD15 mutations are associated with reduced or increased activation of intestinal macrophages.
 
Our data indeed indicate an important role of those macrophages for intestinal inflammation not only in Crohn’s disease but also in other diseases such as lung infections and after stem cell transplantation. In patient samples we found increased activation of macrophages if NOD2/CARD15 mutations were detected answering the open question raised by mouse experiments.
 
Therefore our experiments and studies improved the understanding of the role of NOD2/CARD15 for the intestinal immune system.