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of June 18, 2017.
Cutting Edge: Crohn's Disease-Associated
Nod2 Mutation Limits Production of
Proinflammatory Cytokines To Protect the
Host from Enterococcus faecalis-Induced
Lethality
Yun-Gi Kim, Michael H. Shaw, Neil Warner, Jong-Hwan
Park, Felicia Chen, Yasunori Ogura and Gabriel Núñez
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Copyright © 2011 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2011; 187:2849-2852; Prepublished online 17
August 2011;
doi: 10.4049/jimmunol.1001854
http://www.jimmunol.org/content/187/6/2849
Cutting Edge: Crohn’s Disease-Associated Nod2 Mutation
Limits Production of Proinflammatory Cytokines To
Protect the Host from Enterococcus faecalis-Induced
Lethality
Yun-Gi Kim, Michael H. Shaw, Neil Warner, Jong-Hwan Park,1 Felicia Chen,
Yasunori Ogura,2 and Gabriel Núñez
ceptibility to Crohn’s disease (CD) (4, 5). Three Nod2 variants, G908R, R702W, and a frameshift deletion mutation at
L1007 (L1007fsinsC), have been linked to CD development
(4, 5). Although the precise mechanisms by which Nod2
promotes disease remain unclear, CD-associated human
Nod2 variants exhibit reduced capacity to activate NF-kB
following MDP stimulation (2), suggesting that the loss of
Nod2 activation promotes CD.
Population studies have revealed that the main CDassociated Nod2 variants are present in ∼20% of the
healthy white individuals (6). Furthermore, there are marked
differences in the allelic frequencies of the Nod2 variants
among white populations and geographical regions in Europe
(7). Because of these observations, it has been suggested that
Nod2 variants linked to CD are under selection pressure
possibly through bacterial infections (8). In this article, we
show that Nod2 contributes to innate immune responses and
regulates survival following infection with Enterococcus faecalis, an enteric commensal that is a common cause of systemic
infection and lethality in humans (9). Using a newly developed mouse model in which the common CD-associated
frameshift mutation was introduced into the Nod2 locus, we
show that mice homozygous for the Nod2 mutation are impaired in MDP recognition and exhibit reduced susceptibility
to systemic infection with E. faecalis.
N
Materials and Methods
ucleotide-binding oligomerization domain 2 (Nod2),
a member of the Nod-like receptor family, is activated by muramyl dipeptide (MDP), a conserved
structure from bacterial peptidoglycan (1, 2). Upon MDP
recognition, Nod2 induces the activation of the transcription
factor NF-kB and MAPKs, leading to production of proinflammatory and antimicrobial molecules (1–3). The importance of Nod2 is underscored by the observation that several
genetic variants of Nod2 are associated with increased sus-
Department of Pathology and Comprehensive Cancer Center, University of Michigan
Medical School, Ann Arbor, MI 48109
1
Current address: Department of Biochemistry, College of Medicine, Konyang University, Daejeon, South Korea.
2
Current address: Division of Bacterial Pathogenesis, Graduate School of Medicine,
University of the Ryukyus, Nishihara-cho, Okinawa, Japan.
Received for publication June 7, 2010. Accepted for publication July 17, 2011.
This work was supported by National Institutes of Health Grant R01 DK61707. Y.-G.K.
was supported by training funds from the University of Michigan Comprehensive Cancer
Center, and M.H.S. was supported by Fellowship T32-HL007517 from the National
Institutes of Health.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001854
Mice
C57BL/6 mice were raised in our animal facility from breeders originally
purchased from The Jackson Laboratory (Bar Harbor, ME). Nod22/2 mice on
a C57BL/6 background have been reported (10). The construction of Nod2
Leu980fs-knock-in mice was described (11). Homozygous Nod2 Leu980fsknock-in (Nod2m/m) mice and heterozygous Nod2+/m littermates on a mixed
129-C57BL/6 background were backcrossed six times to C57BL/6 mice before being used for in vivo experiments. Control wild-type mice were derived
from crosses between heterozygous Nod2+/m mice backcrossed six times to
Address correspondence and reprint requests to Dr. Gabriel Núñez, Department of
Pathology and Comprehensive Cancer Center, University of Michigan Medical School,
1500 E. Medical Center Drive, Ann Arbor, MI 48109. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: BMDM, bone marrow-derived macrophage; CD,
Crohn’s disease; LTA, lipoteichoic acid; MDP, muramyl dipeptide; Nod2, nucleotidebinding oligomerization domain 2.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Nucleotide-binding oligomerization domain 2 (Nod2)
mutations including L1007fsinsC are associated with
the development of Crohn’s disease (CD). These CDassociated Nod2 mutations are common in healthy
white populations, suggesting that they may confer
some protective function, but experimental evidence
is lacking. Using a mouse strain that expresses
Nod22939iCstop, the equivalent of the L1007fsinsC mutation, we found that macrophages homozygous for
Nod22939iCstop are impaired in the recognition of muramyl dipeptide and Enterococcus faecalis, a commensal
bacterium that is a common cause of sepsis-associated
lethality in humans. Notably, Nod2 deficiency and
homozygocity for Nod22939iCstop were associated with
reduced production of TNF-a and IL-6 and lethality
after systemic infection with E. faecalis despite normal
bacteria loads. Consistently, inhibition of TNF-a signaling protected wild-type mice from E. faecalis-induced
lethality. These results suggest that the same Nod2 mutation can increase the susceptibility to CD, but also
protect the host from systemic infection by a common
enteric bacterium. The Journal of Immunology, 2011,
187: 2849–2852.
2850
CUTTING EDGE: CD Nod2 MUTATION AND E. FAECALIS-INDUCED LETHALITY
C57BL/6 mice. The animal studies were conducted under approved protocols
by the University of Michigan Committee on Use and Care of Animals.
Macrophage stimulation and infection
Bone marrow-derived macrophages (BMDMs) were prepared as previously
described (12). Lipoteichoic acid (LTA) was from InvivoGen and MDP was
from Bachem. E. faecalis strain OG1RF (ATCC 47077) was from the
American Type Culture Collection. Macrophages were infected with bacteria
for 60 min at 37˚C and washed twice with PBS and IMDM containing 50
mg/ml gentamycin to limit the growth of extracellular bacteria.
HEK293T bioassay for Nod1 and Nod2 stimulation
Nod1 and Nod2 stimulation was determined as previously reported (2).
Measurement of cytokines
Mouse cytokines were measured using ELISA kits from R&D Systems.
Immunoblotting
Nod2 knock-in cDNA sequencing
Total RNA extracted from BMDMs was reverse transcribed and the resulting cDNA was applied as a template for PCR with primers spanning Nod2
2939insC mutation (forward, 59-TTGAGTGTGCTCTTCGCTGT-39; reverse, 59-ACCAACCATCACGACTCCTC-39). Amplified products were
purified by QIAEXII (Qiagen) and their ends blunted with T4 DNA polymerase (Promega) and ligated into pCR-Blunt II-Topo (Invitrogen) for sequence analysis.
Mouse stimulation and infection
Adult mice (6–12 wk old) were administered MDP (300 mg/mouse) by i.p.
injection and serum IL-6 levels were determined 3 h after injection. To induce
endotoxin shock, mice were prestimulated with MDP (300 mg/mouse) i.p.
and challenged with ultrapure LPS (250 mg/mouse) from Escherichia coli
O55:B5 (Sigma-Aldrich) i.p. 24 h later. For infection, mice were given E.
faecalis (1 3 108 CFU/animal) i.p. and bacterial loads in liver and spleen were
determined by plating. TNF-a blocking IgG was a gift of T. Moore (University of Michigan).
Statistical analysis
Statistically significant differences between the two groups were determined by
the two-tailed t test with unequal variance (Aspin–Welch t test). Bacterial
counts of infected mice were analyzed by a Mann–Whitney U test. The
survival rate of infected mice was analyzed using the log-rank test. Differences
were considered significant when p values were , 0.05.
E. faecalis, macrophages were infected with the bacterium at
different bacterial/macrophage ratios and cytokine responses
were measured. Infection of wild-type macrophages with E.
faecalis induced the production of TNF-a and IL-6 and this
response was reduced in macrophages lacking Nod2 (Fig. 1A,
Supplemental Fig. 1B). Consistently, phosphorylation of JNK
and Ik-Ba induced by E. faecalis infection was delayed and/or
reduced in Nod22/2 macrophages, whereas little or no difference was detected in p38 and ERK phosphorylation (Fig.
1B, Supplemental Fig. 1C). These results indicate that Nod2
contributes to the cytokine response triggered by E. faecalis in
macrophages.
Nod2 deficiency protects mice from systemic infection with E. faecalis
To assess whether Nod2 regulates the susceptibility to systemic
infection, wild-type and Nod22/2 mice were injected i.p. and
survival was monitored over time. Notably, ∼80% of wildtype mice succumbed to infection as compared with only
∼20% of the Nod22/2 mice (Fig. 2A). The increased survival
of Nod22/2 mice was associated with reduced levels of IL-6
and TNF-a in the blood when assessed 3 h postinfection (Fig.
2B). The reduced cytokine production was not explained by
differential bacterial clearance because we found similar bacterial burden in the spleen and liver of wild-type and Nod22/2
Results and Discussion
Nod2 contributes to the recognition of E. faecalis
The innate immune sensors that are involved in the recognition
of E. faecalis, a common cause of infection and lethality in
humans, are unknown. To assess a role for Nod2 in the
recognition of E. faecalis, we first determined whether products from the bacterium can induce the activation of Nod2
using an NF-kB reporter assay. The culture supernatant from
E. faecalis induced robust Nod2-dependent NF-kB activation
whereas extracts from the bacterial cell pellet induced little or
no stimulation (Supplemental Fig. 1A). These results suggest
that the bulk of the Nod2 stimulatory activity is released by
the bacterium during growth. Consistent with the lack of the
critical g-D-glutamyl-meso-diaminopimelic acid dipeptide
motif in the peptidoglycan of E. faecalis, neither the bacterial
pellet nor the culture supernatant induced Nod1-dependent
NF-kB activation (Supplemental Fig. 1A). To determine
whether Nod2 plays a role in the immune response to live
FIGURE 2. Nod2 deficiency protects mice from systemic infection with E.
faecalis. A, Mouse survival over time after i.p. infection with 109 E. faecalis in
Nod2+/+ (n = 37) and Nod22/2 mice (n = 37). B, Serum IL-6 and TNF-a
levels 3 h postinfection with 109 E. faecalis in Nod2+/+ and Nod22/2 mice. C,
Nod2+/+ (n = 5–6) and Nod22/2 mice (n = 6) were infected i.p. with 108
E. faecalis. Bacterial loads were determined in the spleen of mice 1, 3, and 5 d
postinfection by plating. D, Mouse survival over time after i.p. infection with
109 E. faecalis in the presence of TNF-a blocking Ab or control IgG in wildtype mice (n = 10).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Immunoblotting for mouse IkB-a, phospho–IkB-a, p38 and phospho-p38,
phospho-ERK, and phospho-JNK (Cell Signaling Technology, Beverly, MA)
was performed as described (10). To detect Nod2, protein extracts from
mouse small intestines were immunoprecipitated using a rabbit polyclonal
anti-Nod2 Ab (13), followed by immunoblotting with anti-Nod2 mAb (13).
Binding was revealed by ECL (Pierce).
FIGURE 1. Nod2 contributes to the recognition of E. faecalis. A, BMDMs
from Nod2+/+ and Nod22/2 mice were infected with E. faecalis at the indicated bacterial/macrophage ratio (B/M). Cell-free supernatants were analyzed for production of TNF-a 24 h postinfection by ELISA. *p , 0.05,
**p , 0.01, ***p , 0.001. B, BMDMs from Nod2+/+ and Nod22/2 mice
were infected with E. faecalis at a B/M of 10 for the indicated times and
assessed for MAPK and NF-kB activation using phospho-specific Abs. Results
are given as means 6 SD of triplicate cultures of cells from a single mouse and
are representative of at least three independent experiments.
The Journal of Immunology
2851
mice (Fig. 2C, Supplemental Fig. 2A). These results indicate
that Nod2 regulates susceptibility to systemic infection with
E. faecalis and this is associated with reduced production of
TNF-a and IL-6 without affecting bacterial clearance.
TNF-a is critical for induction of lethality in mice infected with
E. faecalis
Mice homozygous for Nod22939iCstop mutation exhibit impaired
recognition of MDP
Monocytes from individuals homozygous for the common
CD-associated L1007fsinsC Nod2 mutation exhibit impaired
cytokine response after stimulation with MDP (14). However, mouse macrophages homozygous for the equivalent
L1007fsinsC Nod2 mutation displayed increased NF-kB activation in response to MDP, which suggested that this genetic variant acts as a gain-of-function mutation in the mouse
(16). To clarify these puzzling observations, we used a new
strain of mice in which a Nod2 mutation homologous to the
human L1007fsinsC was introduced into the Nod2 locus by
homologous recombination (11). Sequencing of cDNA prepared from macrophages of homozygous Nod22939iCstop mice
revealed the expected cytosine insertion followed by the TAG
translation termination codon resulting in a truncated Nod2
protein lacking the last 33 aa residues (Supplemental Fig. 3A,
3B). Immunoblotting analysis of Nod2 immunoprecipitates
showed comparable levels of endogenous Nod2 in the intestinal tissue of wild-type and homozygous Nod2m/m mice
(Supplemental Fig. 3C). To assess the function of the
Nod22939iCstop mutation, we stimulated macrophages from
wild-type, Nod22/2, and Nod2m/m mice with MDP and
monitored MAPK and NF-kB activation. Stimulation of
wild-type macrophages with MDP induced phosphorylation
of MAPKs and IkBa, which was greatly reduced or abrogated
in Nod2-deficient and homozygous Nod22939iCstop macrophages (Fig. 3A). Consistently, costimulation of macrophages
from wild-type mice with MDP and LTA increased the
production of TNF-a and IL-6 when compared with the
response observed with LTA alone, which was impaired in
Nod22/2 and Nod2m/m macrophages (Fig. 3B, Supplemental
Fig. 3D). Furthermore, i.p. administration of MDP induced
the secretion of IL-6 in the serum of wild-type mice, but not
in Nod2m/m mice (Fig. 3C). To further assess the function of
the Nod22939iCstop mutation in vivo, wild-type and Nod2m/m
mice were injected i.p. with 300 mg MDP and 24 h later with
250 mg LPS, a protocol that promotes lethality in mice (3).
Nod2m/m mice were protected from lethality induced by ad-
FIGURE 3. Mice homozygous for Nod22939iCstop mutation exhibit impaired recognition of MDP. A, BMDMs from Nod2+/+, Nod22/2, and
Nod2m/m mice were stimulated with 10 mg/ml MDP for the indicated times.
Cell extracts were immunoblotted with Abs that detect total and phosphorylated (activated) forms of the indicated proteins. Results are representative of
at least three separate experiments. B, BMDMs were stimulated with 10 mg/
ml LTA in the presence or absence of 10 mg/ml MDP. Cell-free supernatants
were analyzed for production of TNF-a by ELISA. Results are representative
of at least three separate experiments. **p , 0.01, between cultures with and
without MDP. C, Nod2+/+ and Nod2m/m mice (n = 10/group) were administered MDP by i.p. injection. Blood samples were collected 3 h after
MDP administration and IL-6 levels were determined by ELISA. **p , 0.01,
between Nod2+/+ and Nod2m/m mice. D, Survival of Nod2+/+ (n = 14) and
Nod2m/m mice (n = 9) primed with MDP (300 mg) and challenged with LPS
(250 mg). The survival of each mouse genotype was monitored over time.
Results are given as means 6 SD.
ministration of LPS and MDP when compared with wildtype mice (Fig. 3D). These results indicate that the mouse
Nod22939iCstop mutation is insensitive to MDP stimulation
in vitro and in vivo.
FIGURE 4. Nod22939iCstop homozygocity is associated with reduced response to E. faecalis in vitro and in vivo. A, BMDMs from Nod2+/+, Nod22/2,
and Nod2m/m mice were infected with E. faecalis at indicated bacterial/
macrophage ratio (B/M). Cell-free supernatants were analyzed for production
of TNF-a by ELISA. *p , 0.05, **p , 0.01, and ***p , 0.001. B, Mouse
survival over time postinfection of wild-type, Nod2+/m, and Nod2m/m mice
with 109 E. faecalis. C, Serum IL-6 and TNF-a levels 3 h postinfection of
indicated mice with 109 E. faecalis. Results are given as means 6 SD.
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Given that increased survival of Nod22/2 mice after E. faecalis
infection was associated with reduced amounts of TNF-a, we
asked whether inhibition of TNF-a protects mice from infection induced lethality. Infection of mice i.p. induced 100%
mortality in mice treated with control IgG, whereas only 20%
of the mice injected with anti–TNF-a blocking Ab succumbed to infection (Fig. 2A). Treatment with anti–TNF-a
Ab did not inhibit the production of TNF-a or IL-6 induced
by E. faecalis infection (Supplemental Fig. 2B). In contrast,
administration of anti–IL-6 blocking Ab did not affect survival of mice infected with E. faecalis (Supplemental Fig. 2C,
2D). The results indicate that TNF-a, but not IL-6, is important for lethality associated with E. faecalis infection in our
mouse model.
2852
CUTTING EDGE: CD Nod2 MUTATION AND E. FAECALIS-INDUCED LETHALITY
Impaired cytokine responses induced by E. faecalis in macrophages
homozygous for Nod22939iCstop mutation
We next examined the role of the Nod22939iCstop mutation in
the regulation of proinflammatory cytokine responses postinfection with E. faecalis. Infection of wild-type macrophages
with E. faecalis induced secretion of IL-6 and TNF-a and
these responses were significantly impaired in macrophages
homozygous for the Nod22939iCstop mutation (Fig. 4A). As
a comparison, Nod22/2 and homozygous Nod22939iCstop
macrophages produced comparable amounts of IL-6 and
TNF-a in response to bacterial infection that was lower than
in wild-type macrophages (Fig. 4A, Supplemental Fig. 4).
These results indicate that the Nod22939iCstop mutation is
associated with defective recognition of E. faecalis in macrophages.
Reduced susceptibility to systemic infection with E. faecalis in mice
homozygous for Nod22939iCstop mutation
Acknowledgments
We are grateful to Richard Flavell for Nod22/2 mice, Joel Whitfield from the
Cellular Immunology Core Facility of the University of Michigan Cancer
Center for ELISA assays, Sharon Koonse for animal husbandry, and Mizuho
Hasegawa for advice.
Disclosures
The authors have no financial conflicts of interest.
References
1. Girardin, S. E., I. G. Boneca, J. Viala, M. Chamaillard, A. Labigne, G. Thomas,
D. J. Philpott, and P. J. Sansonetti. 2003. Nod2 is a general sensor of peptidoglycan
through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278: 8869–8872.
2. Inohara, N., Y. Ogura, A. Fontalba, O. Gutierrez, F. Pons, J. Crespo, K. Fukase,
S. Inamura, S. Kusumoto, M. Hashimoto, et al. 2003. Host recognition of bacterial muramyl dipeptide mediated through NOD2: implications for Crohn’s
disease. J. Biol. Chem. 278: 5509–5512.
3. Kobayashi, K. S., M. Chamaillard, Y. Ogura, O. Henegariu, N. Inohara, G. Nuñez,
and R. A. Flavell. 2005. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 307: 731–734.
4. Hugot, J. P., M. Chamaillard, H. Zouali, S. Lesage, J. P. Cézard, J. Belaiche,
S. Almer, C. Tysk, C. A. O’Morain, M. Gassull, et al. 2001. Association of
NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature
411: 599–603.
5. Ogura, Y., D. K. Bonen, N. Inohara, D. L. Nicolae, F. F. Chen, R. Ramos,
H. Britton, T. Moran, R. Karaliuskas, R. H. Duerr, et al. 2001. A frameshift
mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 411:
603–606.
6. Hugot, J. P., I. Zaccaria, J. Cavanaugh, H. Yang, S. Vermeire, M. Lappalainen,
S. Schreiber, V. Annese, D. P. Jewell, E. V. Fowler, et al; for the IBD International Genetics Consortium. 2007. Prevalence of CARD15/NOD2 mutations
in Caucasian healthy people. Am. J. Gastroenterol. 102: 1259–1267.
7. Cavanaugh, J. 2006. NOD2: ethnic and geographic differences. World J. Gastroenterol. 12: 3673–3677.
8. Gasche, C., M. Nemeth, P. Grundtner, C. Willheim-Polli, P. Ferenci, and
R. Schwarzenbacher. 2008. Evolution of Crohn’s disease-associated Nod2
mutations. Immunogenetics 60: 115–120.
9. Goossens, H. 1999. The epidemiology of vancomycin-resistant enterococci. Curr.
Opin. Infect. Dis. 12: 537–541.
10. Kim, Y. G., J. H. Park, M. H. Shaw, L. Franchi, N. Inohara, and G. Núñez. 2008.
The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host
defense after exposure to Toll-like receptor ligands. Immunity 28: 246–257.
11. Travassos, L. H., L. A. Carneiro, M. Ramjeet, S. Hussey, Y. G. Kim, J. G.
Magalhães, L. Yuan, F. Soares, E. Chea, L. Le Bourhis, et al. 2010. Nod1 and Nod2
direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of
bacterial entry. Nat. Immunol. 11: 55–62.
12. Celada, A., P. W. Gray, E. Rinderknecht, and R. D. Schreiber. 1984. Evidence for
a g-interferon receptor that regulates macrophage tumoricidal activity. J. Exp. Med.
160: 55–74.
13. Ogura, Y., S. Lala, W. Xin, E. Smith, T. A. Dowds, F. F. Chen, E. Zimmermann,
M. Tretiakova, J. H. Cho, J. Hart, et al. 2003. Expression of NOD2 in Paneth cells:
a possible link to Crohn’s ileitis. Gut 52: 1591–1597.
14. van Heel, D. A., S. Ghosh, M. Butler, K. A. Hunt, A. M. Lundberg, T. Ahmad,
D. P. McGovern, C. Onnie, K. Negoro, S. Goldthorpe, et al. 2005. Muramyl
dipeptide and Toll-like receptor sensitivity in NOD2-associated Crohn’s disease.
Lancet 365: 1794–1796.
15. Maeda, S., L. C. Hsu, H. Liu, L. A. Bankston, M. Iimura, M. F. Kagnoff,
L. Eckmann, and M. Karin. 2005. Nod2 mutation in Crohn’s disease potentiates
NF-kB activity and IL-1b processing. Science 307: 734–738.
16. Shaw, M. H., N. Kamada, N. Warner, Y. G. Kim, and G. Nuñez. 2011. The everexpanding function of NOD2: autophagy, viral recognition, and T cell activation.
Trends Immunol. 32: 73–79.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
We next examined the susceptibility of wild-type and Nod2m/m
mice to i.p. administration of E. faecalis. In these experiments,
we crossed heterozygous Nod2+/m mice with homozygous
Nod2m/m mice to generate littermate mice heterozygous and
homozygous for theNod22939iCstop mutation. We then
challenged wild-type as well as heterozygous Nod2+/m and
homozygous Nod2m/m littermates i.p. with E. faecalis and
assessed cytokine production and mouse survival over time.
Nod2m/m mice exhibited increased survival compared with
wild-type mice (67 versus 25%; p = 0.05) after i.p. challenge
with E. faecalis (Fig. 4B). In contrast, heterozygous Nod2+/m
and wild-type mice exhibited comparable survival after bacterial challenge (Fig. 4B). The increased survival of Nod2m/m
mice was associated with reduced serum levels of IL-6 and
TNF-a (Fig. 4C). These results indicate that homozygocity
for the Nod22939iCstop mutation promotes survival after systemic infection with E. faecalis.
Using a newly developed model of the L1007fsinsC Nod2
mutation, we show that the resulting Nod2 truncated protein is
impaired in MDP recognition and thus behaves as a loss-offunction mutation similar to that found in human cells (2,
14). There is no a clear explanation to account for the differences in results between our model and the model published by Maeda et al. (15). In contrast to our animal model,
a TAG termination codon was not introduced after the cytosine insertion at position 2939 in the model reported by
Maeda et al. (15), which is predicted to result in a frameshift
mutation leading to the addition of 42 aa not present in the
CD-associated or normal Nod2 protein. Thus, it is possible
that differences in the resulting Nod2 mutant protein between
the two models could explain, at least in part, the discrepancy
in results. We show in this study that mice homozygous for
the CD-associated Nod22939iCstop mutation displayed impaired production of proinflammatory cytokines and improved survival in response to systemic infection with E.
faecalis. These results suggest that the CD-associated
Nod22939iCstop mutation exhibits a dual function. Namely,
it promotes the susceptibility to CD, but it also protects the
host by limiting the production of harmful cytokines induced
by systemic infection with E. faecalis. Because MDP syner-
gizes with TLR ligands in the production of proinflammatory
cytokines, it is likely that the role for Nod22939iCstop mutation
in limiting the production of harmful cytokines and lethality
in response to bacterial infection is not limited to E. faecalis.
However, most studies to date have shown increased susceptibility of Nod22/2 mice to infections with microbial
pathogens (16). Thus, the contribution of Nod2 mutations in
evolutionarily selecting for individuals who are L1007fsinsC
remains unclear.