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J. Exp. Med. Vol. 206 No. 10 2101-2110
BRIEF DEFINITIVE REPORT
Nucleotide-binding oligomerization domain 2
(NOD2) belongs to the family of intracellular
NOD-like receptors, which are present in var-
ious cell types (Bertin et al., 1999; Inohara et al.,
2001). NOD2 has a C-terminal leucine-rich
repeat region for sensing microbial products, a
central nucleotide-binding domain (NACHT),
and an N-terminal CARD (caspase activation
and recruitment domain). NOD2 detects mur-
amyl dipeptide (MDP), a molecule which is
produced during the synthesis and degradation
of peptidoglycan, a cell wall component of most
bacteria (Inohara and Nunez, 2003). Stimula-
tion of NOD2 induces recruitment of Rip2
(also called RICK) at the N-terminal CARD
domain. Activation of Rip2 leads to subsequent
steps that ultimately result in activation and
nuclear translocation of NF-B and transcrip-
tion of its target genes.
NOD2 has been studied in human Crohn’s
disease and in mouse models for inflammatory
bowel disease. Single nucleotide polymorphisms
(SNPs) near, or within, the NOD2 leucine-
rich repeat region (G908R, L1007insC, and
R702W) constitute genetic risk factors for the
development of Crohn’s disease (Hugot et al.,
2001; Ogura et al., 2001). Mutations in the
NACHT region of NOD2 are linked to other
inflammatory diseases such as Blau syndrome
(Miceli-Richard et al., 2001) and early onset
sarcoidosis (Kanazawa et al., 2005). Several
groups have investigated the role of NOD2
in experimental models for intestinal inflamma-
tion, which has led to the development of three
hypotheses regarding the function of NOD2.
Kobayashi et al. (2005) found greater suscepti-
bility to enteral infection with Listeria monocyto-
genes and reduced expression of defensins,
antibacterial peptides produced by intestinal
Paneth cells, in NOD2/ mice. Watanabe et al.
Marcel R.M. van den Brink:
Abbreviations used: allo-BMT,
allogenic BM transplantation;
allo-HSCT, allogeneic hemato-
poietic stem cell transplantation;
AML, acute myeloid leukemia;
GVHD, graft-versus-host dis-
ease; MDP, muramyl dipeptide;
MLN, mesenteric LN; MLR,
mixed leukocyte reaction;
oligomerization domain 2; SNP,
single nucleotide polymorphism;
TCD-BM, T cell–depleted BM;
TLR, toll-like receptor.
K. Brandl’s present address is Dept. of Genetics, the Scripps
Research Institute, La Jolla, CA 92037.
NOD2 regulates hematopoietic cell function
during graft-versus-host disease
Olaf Penack,1,3 Odette M. Smith,1 Amy Cunningham-Bussel,4 Xin Liu,4
Uttam Rao,1 Nury Yim,1 Il-Kang Na,1,3 Amanda M. Holland,1,4
Arnab Ghosh,1 Sydney X. Lu,1 Robert R. Jenq,1,2 Chen Liu,5
George F. Murphy,6 Katharina Brandl,1 and Marcel R.M. van den Brink1,2
1Department of Immunology and 2Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10065
3Department of Hematology and Oncology, Charité, Campus Benjamin Franklin, Berlin 12200, Germany
4Department of Immunology and Microbial Pathogenesis, Weill Cornell Medical College, New York, NY 10065
5Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville,
6Program in Dermatopathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
Nucleotide-binding oligomerization domain 2 (NOD2) polymorphisms are independent risk
factors for Crohn’s disease and graft-versus-host disease (GVHD). In Crohn’s disease, the
proinflammatory state resulting from NOD2 mutations have been associated with a loss of
antibacterial function of enterocytes such as paneth cells. NOD2 has not been studied in
experimental allogeneic bone marrow transplantation (allo-BMT). Using chimeric recipients
with NOD2/ hematopoietic cells, we demonstrate that NOD2 deficiency in host hemato-
poietic cells exacerbates GVHD. We found that proliferation and activation of donor T cells
was enhanced in NOD-deficient allo-BMT recipients, suggesting that NOD2 plays a role in
the regulation of host antigen-presenting cells (APCs). Next, we used bone marrow chime-
ras in an experimental colitis model and observed again that NOD2 deficiency in the hema-
topoietic cells results in increased intestinal inflammation. We conclude that NOD2
regulates the development of GVHD through its inhibitory effect on host APC function.
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The Journal of Experimental Medicine
NOD2 REGULATES GVHD | Penack et al.
mine the effect of NOD2 deficiency in allo-BMT donors on
the development of GVHD, we first used an MHC-disparate
allo-BMT model, B6 (H-2b)→BALB/c (H-2d). Lethally ir-
radiated recipients received T cell–depleted BM (TCD-BM),
and GVHD was induced by the addition of donor splenic
T cells to the allograft. We used NOD2/ donors and WT
donors as the source for TCD-BM or T cells. We did not
detect significant differences in lethal GVHD between the
four different groups receiving WT BM + WT T cells, WT
BM + NOD2/ T cells, NOD2/ BM + WT T cells, or
NOD2/ BM + NOD2/ T cells (Fig. 1 E, left). To confirm
that our observations were not strain dependent or model
dependent, we performed similar experiments in an MHC-
matched model with minor histocompatibility disparities,
B6 (H-2b)→LP (H-2b) (Fig. 1 E, right). We conclude that
NOD2 deficiency of the allo-BMT donor (either donor
T cells or BM) has no significant impact on the development
of GVHD and does not regulate alloactivation of donor
T cells. Our data suggest that NOD2 has no cell-intrinsic role
in the regulation of T cell activity during GVHD despite the
increasing evidence that innate immune receptors, such as
TLRs, can modulate T cell function and activation during
inflammation (Dabbagh and Lewis, 2003; Caron et al., 2005;
MacLeod and Wetzler, 2007).
NOD2/ allo-BMT recipients have increased systemic
and target organ GVHD
To investigate the role of NOD2 in regulating GVHD in
allo-BMT recipients, we first used the MHC-disparate
B10BR (H-2k)→B6 (H-2b) model. We found significantly
more lethal GVHD in B6 NOD2/ allo-BMT recipients as
compared with B6 WT allo-BMT recipients (Fig. 2 A, left).
We confirmed these findings in the MHC-matched LP
(H-2b)→B6 (H-2b) model (Fig. 2 A, right). To analyze target
organ GVHD, we performed histopathological analyses and
found significantly more GVHD in the terminal ileum, co-
lon, and liver, as well as a trend toward more skin GVHD in
NOD2/ versus WT allo-BMT recipients (Fig. 2 B). The
numbers of tissue-infiltrating alloreactive CD3+ T cells in
GVHD target organs were increased in NOD2/ allo-BMT
recipients (Fig. 2, C and D). To investigate thymic GVHD,
we determined the number of thymocytes and their subsets
at day 21 after allo-BMT. We found a significantly reduced
number of CD4+/CD8+ double-positive thymocytes in
NOD2/ allo-BMT recipients (Fig. 2 E), which is the char-
acteristic feature of thymic GVHD (Hollander et al., 1994).
We conclude that NOD2 deficiency in allo-BMT recipients
exacerbates systemic and organ GVHD. Our results are in
agreement with clinical data connecting NOD2 SNPs in
allo-BMT recipients to increased incidence and severity of
GVHD (Holler et al., 2004, 2006). A limitation of our study is
that in humans the association between NOD2 and GVHD
was observed in individuals with heterozygous SNPs, whereas
our studies used homozygous NOD2-deficient allo-BMT
recipients to investigate the effect of NOD2 deficiency during
GVHD. This is similar to studies in mice and men regarding
(2004, 2006) demonstrated increased sensitivity to experi-
mental colitis in NOD2/ mice, which was driven by
T helper type 1 cells and increased IL-12 release. Maeda et al.
(2005) created a knockout/knockin transgenic NOD2 mouse
using the mouse equivalent to a human mutation (L1007insC)
and found abundant IL-1 production in response to MDP.
Collectively, these experimental data do not allow for one
unified conclusion regarding the mechanism of NOD2-
mediated regulation of inflammatory diseases.
Recently, SNPs in the NOD2 gene locus have been
linked to graft-versus-host disease (GVHD), which is the
major complication after allogeneic hematopoietic stem cell
transplantation (allo-HSCT). GVHD is a systemic inflamma-
tory disease caused by alloreactive donor T cells and charac-
terized by tissue damage in gut, liver, and skin. Holler et al.
(2004, 2006) found an association between SNPs (alleles 8,
12, and 13) of the NOD2 gene in the donor or host and a
higher incidence of GVHD, as well as increased transplant-
related mortality. Interestingly, these findings were only par-
tially confirmed by Elmaagacli et al. (2006), who also found
an increased incidence of GVHD when NOD2 SNPs were
present in both the donor and recipient but reduced inci-
dence of GVHD when the NOD2 SNPs were present in the
donor only. In addition, Mayor et al. (2007) connected NOD2
SNPs with an increased leukemia relapse rate after allo-
HSCT, which could not be confirmed by other investigators
(Holler et al., 2008). Collectively, it appears that NOD2 SNPs
of allo-HSCT recipients influence posttransplant immunity;
however, the underlying mechanism is unclear. In the pres-
ent study, we aim to clarify the underlying role of NOD2 in
mouse models for allogenic BM transplantation (allo-BMT),
colitis, and tumors.
RESULTS AND DISCUSSION
NOD2 deficiency in donor BM cells and/or donor T cells
does not affect the development of GVHD
To address contradictory clinical studies showing that NOD2
SNPs of the allo-HSCT donor can either exacerbate GVHD
(Holler et al., 2004, 2006) or ameliorate GVHD (Elmaagacli
et al., 2006), we investigated the effect of NOD2 deficiency
of allo-BMT donors in our GVHD models. We first assessed
whether NOD2 deficiency affects allogeneic donor T cells.
We transferred CFSE-labeled B6 WT or B6 NOD2/
T cells to lethally irradiated BALB/c recipients and found simi-
lar T cell proliferation (Fig. 1 A), T cell activation (Fig. 1 B),
and expression of the gut-homing molecule LPAM (Fig. 1 C).
Next, we performed mixed leukocyte reactions (MLRs) us-
ing CD5+-selected B6 WT or B6 NOD2/ splenocytes as
effectors and irradiated BALB/c splenocytes as stimulators.
We found no significant differences in the proliferation rate
between WT T cells and NOD2/ T cells (Fig. 1 D). In
addition, we found no significant differences in the prolifera-
tion rate between WT T cells and NOD2/ T cells when
we added different toll-like receptor (TLR) and NOD2 li-
gands (MDP, LPS, Pam3Cys, Flagellin, and PGN) alone or in
combination with the MLRs (unpublished data). To deter-
JEM VOL. 206, September 28, 2009
BRIEF DEFINITIVE REPORT
labeled allogeneic B10BR T cells into B6 WT or B6
NOD2/ allo-BMT recipients. We found that the in vivo
proliferation of alloreactive donor T cells was significantly
increased in NOD2/ allo-BMT recipients (Fig. 2 F). Next,
we performed allo-BMTs in the B10BR→B6 model and
harvested organs at day 14 after allo-BMT. We found that
the number of donor T cells was significantly increased in
spleen and mesenteric LNs (MLNs) in NOD2/ allo-BMT
recipients (Fig. 2 G, left). However, the percentages of CD4+
T cells, CD8 + T cells, and CD44+/CD62L effector T cells
were not significantly different (Fig. S1). We next deter-
mined the alloactivation status of donor T cells by their CD25
expression, which is a reliable marker for alloactivation in our
the role of NOD2 in Crohn’s disease (Hugot et al., 2001;
Watanabe et al., 2004, 2006, 2008; Kobayashi et al., 2005).
NOD2 polymorphisms resulting in impaired NOD2 func-
tion were associated with increased susceptibility to Crohn’s
disease. Studies in NOD2-deficient mice demonstrated an
increased susceptibility to experimental colitis, whereas mice
with a NOD2 transgene resulting in increased NOD2 func-
tion were resistant to experimental colitis.
The activation and proliferation of alloreactive donor T cells
is increased in NOD2/ allo-BMT recipients
To study the mechanisms underlying increased GVHD in
NOD2/ allo-BMT recipients, we first transferred CFSE-
Figure 1. NOD2 deficiency in donor BM cells and/or donor T cells does not affect the development of GVHD. (A–C) Lethally irradiated (8.5 Gy)
BALB/c allo-BMT recipients received 5 × 106 CFSE-labeled B6 WT or B6 NOD2/CD5+ T cells. Recipient spleens were harvested 96 h after transfer of
T cells; n = 8/group; combined data from two independent experiments are shown. (A) Identical CFSE histogram overlays of WT and NOD2/ donor T cells
(left), as well as similar total number of CFSEhigh and CFSElow donor T cells. (B) Similar up-regulation of the alloactivation marker CD25 on WT B6 donor
T cells versus NOD2/ donor T cells. (C) Similar expression of the gut-homing marker LPAM on WT B6 donor T cells versus NOD2/ donor T cells.
(D) MLR with WT versus NOD2/CD5+ T cells and irradiated BALB/c stimulators. Combined data of three independent experiments are shown; n = 12/group.
(E) Lethally irradiated BALB/c (8.5 Gy) or LP (11Gy) allo-BMT recipients were transplanted with 5 × 106 B6 WT TCD-BM versus B6 NOD2/ TCD-BM and
106 B6 WT T cells versus B6 NOD2/ T cells. Combined data from four independent experiments are shown; n = 15–45/group. Error bars indicate SEM.
NOD2 REGULATES GVHD | Penack et al.
Figure 2. NOD2/ allo-BMT recipients have increased systemic and target organ GVHD because of enhanced activation and proliferation
of alloreactive donor T cells. (A) Lethally irradiated (11 Gy) B6 WT versus B6 NOD2/ allo-BMT recipients received 5 × 106 B10BR TCD-BM or 5 × 106 LP
TCD-BM with or without 106 B10BR T cells or 2 × 106 LP T cells. Combined data from four independent experiments are shown; n = 45/group. (B-E) Le-
thally irradiated (11 Gy) B6 WT versus B6 NOD2/ allo-BMT recipients received 5 × 106 B10BR TCD-BM + 106 B10BR T cells. Organs were harvested at day
21 after allo-BMT. Combined data from two independent experiments are shown; n = 10/group. (B) Histopathologic GVHD scores in target organs show-
ing increased GVHD in NOD2/ allo-BMT recipients. (C) CD3+ histochemistry images showing increased infiltration of T cells (brown) in liver tissue of
NOD2/ allo-BMT recipients. Bars, 50 µm. (D) Quantification of T cell infiltration in GVHD target organs by CD3+ histochemistry. (E) Increased thymic
GVHD in NOD2/ allo-BMT recipients demonstrated by reduced numbers of CD4+/CD8+ double-positive thymocytes. Shown are flow cytometric plots
and the absolute numbers of DP (CD4+/CD8+), DN (CD4/CD8), CD4+, and CD8+ thymocytes. (F) Lethally irradiated (11 Gy) B6 WT versus B6 NOD2/
allo-BMT recipients received 5 × 106 CFSE-labeled B10BR CD5+ T cells. Recipient spleens were harvested 96 h after transfer of T cells; n = 8/group; com-
bined data from two independent experiments are shown. CFSE histogram overlays of donor T cells in WT allo-BMT recipients versus NOD2/ allo-BMT
recipients (left); ratio of proliferating donor T cells (right, top) and absolute numbers of donor T cells (right, bottom) showing increased donor T cell pro-
liferation in NOD2/ allo-BMT recipients. (G and H) Lethally irradiated (11 Gy) B6 WT versus B6 NOD2/ allo-BMT recipients were transplanted with
JEM VOL. 206, September 28, 2009
BRIEF DEFINITIVE REPORT
determined the alloactivation status of donor T cells by their
CD25 expression and found that donor T cells were signifi-
cantly more activated in NOD2/→WT chimeric allo-BMT
recipients as compared with WT→NOD2/ chimeric allo-
BMT recipients (Fig. 3 D, middle). Additionally, we found a
significantly reduced percentage of FoxP3+/CD4+ regulatory
T cells in the spleen (percentage of FoxP3+ in total CD4+
splenocytes) in NOD2/→WT chimeric allo-BMT recipi-
ents (Fig. 3 D, top right). The expression of LPAM on donor
T cells in MLNs was similar in both chimeric allo-BMT re-
cipients (Fig. 3 D, bottom right). We conclude that NOD2
deficiency in the hematopoietic system exacerbates GVHD,
whereas NOD2 deficiency in the nonhematopoietic system
has no significant impact on the development of GVHD in
NOD2/ allo-BMT recipients.
NOD2/ DCs have a higher activation status and increased
ability to induce T cell proliferation during GVHD
Based upon our finding that donor T cell proliferation is in-
creased in allo-BMT recipients with NOD2 deficiency in the
hematopoietic system, we hypothesized that NOD2/ DCs
might have a higher activation status and increased ability to
induce T cell proliferation during GVHD. We therefore per-
formed allo-BMTs in the B10BR→B6 model and harvested
spleens at day 7 after allo-BMT. We found that the absolute
numbers of host DC subsets in the spleen were not significantly
different before and after allo-BMT in WT versus NOD2/
allo-BMT recipients (Fig. 4 A). We then quantified the ex-
pression of the activation markers and costimulatory mole-
cules CD40, CD80, and CD86 on host (CD11c+H2kb+) DCs
before allo-BMT and at day 7 after allo-BMT. We found a
significantly increased up-regulation of activation markers on
host NOD2/ DCs in contrast to WT DCs during GVHD
(Fig. 4, B and C). The increase in the expression of MHC
class II molecules, which was already high at baseline, did not
reach statistical significance (unpublished data). To study DC
function, we selected splenic DCs from WT and NOD2/
allo-BMT recipients with GVHD (day 7) and used them as
stimulators in MLRs. We found that B6 NOD2/ DCs had a
significantly increased ability to induce proliferation of alloge-
neic B10BR T cells as compared with B6 WT DCs (Fig. 4 D).
These data supports the hypothesis that NOD2 can negatively
regulate the activity and function of host DCs, resulting in
increased alloactivation and proliferation of donor T cells in
NOD2/ allo-BMT recipients. Our results provide a pos-
sible cellular mechanism for recent clinical studies suggest-
ing a regulatory role of NOD2 on the incidence of GVHD
and transplant-related mortality in allo-HSCT recipients with
SNPs in the gene locus encoding NOD2 (Holler et al., 2004,
2006; Elmaagacli et al., 2006).
GVHD models (Alpdogan et al., 2003), and found that donor
T cells were significantly more activated in NOD2/ allo-
BMT recipients as compared with WT allo-BMT recipients
(Fig. 2 G, middle). When we analyzed FoxP3+/CD4+ regula-
tory T cells in the spleen, we found a significantly reduced
percentage of FoxP3+/CD4+ cells (of total CD4+ splenocytes)
in NOD2/ allo-BMT recipients (Fig. 2 G, top right). The
percentages of IL-17+CD4+, IFN-+CD4+, and Tbet+CD4+
T cells were not different (Fig. S1). There was no difference
between the expression of the gut-homing molecule LPAM
(47 integrin) on donor T cells in MLNs in NOD2/ and
WT allo-BMT recipients (Fig. 2 G, bottom right). We de-
tected no differences in donor cell types, such as B cells, NK
cells, granulocytes, monocytes, and DCs in NOD2/ allo-
BMT recipients (Fig. 2 H). We conclude that increased
GVHD in NOD2/ allo-BMT recipients is the result of en-
hanced alloactivation and proliferation of donor T cells.
NOD2 deficiency in the hematopoietic system
of the allo-BMT recipient exacerbates GVHD
Given that NOD2 is expressed in both hematopoietic and
nonhematopoietic cells types, including antigen-presenting
cells and intestinal epithelial cells, respectively, it is not clear
what relative contributions of these cells types is to the devel-
opment of GVHD. To address this, we created BM chimeras
in which the NOD2 deficiency was only manifest in one of
these systems. BM chimeras were created by performing
syngeneic BMTs after lethal irradiation, using B6 WT and
B6 NOD2/ as donors or as recipients (WT→NOD2/ or
NOD2/→WT). Using B6 Ly5.1+ mice as donors, we found
the donor chimerism of hematopoietic cell subsets to be
>95% in GVHD target organs, as well as in secondary lym-
phoid organs at day 90 after syngeneic BMT (Fig. S2). We then
used the BM chimera, WT mice, and NOD2/ mice as
allo-BMT recipients in the B10BR→B6 GVHD model. We
found that NOD2/ and NOD2/→WT chimeric allo-
BMT recipients had a greater GVHD-induced mortality rate
as compared with WT and WT→NOD2/ chimeric allo-
BMT recipients (Fig. 3 A, left). We confirmed our findings
in the minor mismatched LP→B6 model (Fig. 3 A, right).
To analyze target organ GVHD, we performed histopatho-
logical analyses and found significantly more GVHD in the
terminal ileum, colon, liver, and skin in NOD2/→WT
chimeric allo-BMT recipients versus WT→NOD2/ chi-
meric allo-BMT recipients (Fig. 3 B). The numbers of tissue-
infiltrating CD3+ T cells in GVHD target organs were
increased in NOD2/→WT chimeric allo-BMT recipients
(Fig. 3 C). We found that the number of donor T cells was
significantly increased in the spleen and MLNs in NOD2/→
WT chimeric allo-BMT recipients (Fig. 3 D, left). We next
5 × 106 B10BR TCD-BM + 106 B10BR T cells. Organs were harvested at day 14 after allo-BMT. Combined data from two independent experiments are shown;
n = 10/group. (G) Absolute numbers and activation status (CD25+) of donor CD3+ T cells in spleen and MLNs. Percentage of FoxP3+ cells in CD4+ splenocytes
(top right) and expression of the gut homing marker LPAM in MLNs (bottom right) are shown. (H) Absolute numbers of donor B cells, NK cells, monocytes,
DCs, and granulocytes in spleen. Error bars indicate SEM.
NOD2 REGULATES GVHD | Penack et al.
mechanism for the enhanced ability of NOD2/ DCs to in-
duce T cell proliferation, we performed allo-BMTs in the
B10BR→B6 model and measured cytokines/chemokines in
serum with a multiplex system at day 7 after allo-BMT. We did
not detect significant differences in serum levels of IFN-,
IL-1, IL-1, IL-2, IL-6, IL-7, IL-9, IL-10, IL-12 p40,
In previous studies using NOD2/ mice in inflamma-
tory bowel disease models, it was suggested that increased
TLR signaling led to a massively increased production of cy-
tokines such as IL-12. (Watanabe et al., 2004, 2006, 2008;
Strober et al., 2006; Yang et al., 2007) To determine whether
increased cytokine/chemokine production is the underlying
Figure 3. NOD2 deficiency of the hematopoietic system of the allo-BMT recipient aggravates GVHD. (A–D) Chimeric mice with NOD2 defi-
ciency either in the hematopoietic system or in the nonhematopoietic system were created by syngeneic BMT (B6 WT→B6 NOD2/ or B6 NOD2/→B6
WT). After 90 d, lethally irradiated (11 Gy) B6 WT versus B6 NOD2/ versus chimeric allo-BMT recipients were transplanted with 5 × 106 B10BR TCD-BM
or 5 × 106 LP TCD-BM + 2 × 106 B10BR T cells or 3 × 106 LP T cells. (A) Survival curves. Combined data from two independent experiments are shown;
n = 16/group. (B-E) Lethally irradiated (11 Gy) B6 WT→B6 NOD2/ or B6 NOD2/→B6 WT chimeric allo-BMT recipients were transplanted with 5 × 106
B10BR TCD-BM + 106 B10BR T cells. Organs were harvested at day 14 after allo-BMT. Combined data from two independent experiments are shown;
n = 8/group. (B) Histopathologic GVHD scores in GVHD target organs. (C) Quantification of T cell infiltration in GVHD target organs by CD3+ histochemistry.
(D) Absolute number and activation status (CD25+) of donor CD3+ T cells in spleen and MLNs, percentage of FoxP3+ regulatory T cells (top right), and ex-
pression of the gut-homing marker LPAM in MLNs (bottom right). Error bars indicate SEM.
JEM VOL. 206, September 28, 2009
BRIEF DEFINITIVE REPORT
in cytokine production in specified cell populations. How-
ever, our results are in agreement with recent data, which
showed that APCs from humans with NOD2 SNPs do not
have increased cytokine production (van Heel et al., 2005).
Consequently, there might be alternative molecular mech-
anisms that can enhance APC function and activity in NOD2/
allo-BMT recipients during inflammatory diseases such as
colitis and GVHD.
NOD2 deficiency of the hematopoietic system exacerbates
Next, we analyzed whether results from our GVHD experi-
ments also apply to other inflammatory diseases. Because the
pathophysiology of GVHD and inflammatory bowel disease
has many similarities, we performed experiments using the
TNBS (2,4,6-trinitrobenzenesulfonic acid) colitis model. TNBS
colitis is a transmural inflammation often used as a model for
inflammatory bowel disease (Scheiffele and Fuss, 2002). Ad-
ministration of TNBS leads to modification of intestinal pro-
teins that function as haptens. We rectally challenged B6 WT
or B6 NOD2/ mice with 5 mg TNBS in 50% ethanol. We
found a significantly higher weight loss in NOD2/ mice
than in WT mice during colitis (Fig. 5 A). We next compared
the NOD2/→WT chimeric to the WT→NOD2/ chi-
meric mice (90 d after syngeneic BMT) in the TNBS coli-
tis model. We found significantly more weight loss in
NOD2/→WT chimeric mice versus the WT→NOD2/
chimeric mice (Fig. 5 B). We harvested colons at day 3 after
induction of colitis, performed histopathological analysis, and
found significantly higher colitis scores in NOD2/→WT
chimeric mice as compared with the WT→NOD2/ chimeric
mice (Fig. 5, C and D). Our data demonstrate that NOD2
deficiency in the hematopoietic system, but not in the non-
hematopoietic system, exacerbates both experimental GVHD
and experimental TNBS colitis. These results suggest that loss
of intestinal epithelial cell function causing impaired antibac-
terial resistance, which has been proposed as a mechanism for
increased inflammation in NOD2/ mice (Kobayashi et al.,
2005), is not responsible for the increased GVHD and colitis.
Our results are in agreement with previous findings (Watanabe
et al., 2004, 2006, 2008; Strober et al., 2006; Yang et al.,
2007) that found increased susceptibility to different types of
experimental colitis in NOD2/ mice, which was the result
of the enhanced ability of NOD2/ APCs to trigger inflam-
matory T cell responses.
NOD2/ mice have intact resistance against
In response to mixed clinical results on a possible role of
NOD2 in antitumor immunity (Mayor et al., 2007; Holler et al.,
2008), we performed tumor challenges with hematologic and
solid tumors. We studied the role of NOD2 in antitumor re-
sponses in two tumor models: acute myeloid leukemia (AML;
C1498) and melanoma (B16). When we challenged B6 WT
or B6 NOD2/ mice intravenously with C1498 or B16, we
found no significant differences in survival (unpublished
IL-12 p70, IL-15, IL-17, IP-10, LIX, MCP-1, MIG, MIP-1,
MIP-1, Rantes, and TNF in NOD2/ allo-BMT re-
cipients versus WT allo-BMT recipients (Fig. S3). Although
serum levels were not different we cannot exclude differences
Figure 4. NOD2/ DCs have a higher activation status and in-
creased ability to induce T cell proliferation during GVHD. Lethally
irradiated (11 Gy) B6 WT versus B6 NOD2/ allo-BMT recipients were
transplanted with 5 × 106 B10BR TCD-BM + 2 × 106 B10BR T cells. Organs
were harvested at day 7 after allo-BMT. Donor and host-derived DCs were
distinguished by MHC class I disparity in flow cytometry (H2k vs. H2b).
Combined data from two independent experiments are shown; n = 8/
group. (A) Quantification of host DC subsets by flow cytometry. (B) Quan-
tification of activation marker expression of host DCs by flow cytometry
before allo-BMT and at day 7 after allo-BMT. (C) Histogram overlays of
activation marker expression of host DCs during GVHD. (D) MLR using
105 splenic DCs (CD11+ selection) from B6 WT allo-BMT recipients versus
B6 NOD2/ allo-BMT recipients as stimulators and 105 B10BR T cells (CD5+
selection). Shown is the thymidine H3 uptake (cpm) between 48 and 72 h
of MLR. Error bars indicate SEM.
NOD2 REGULATES GVHD | Penack et al.
Flow cytometry and MLR. Most antibodies were obtained from BD.
DCs were purified by positive selection with CD11c (N418) MicroBeads
(Miltenyi Biotec) from digested spleens. Purity was between 90 and 95%.
Splenic T cells were enriched by CD5+ selection and cocultured. After 48 h,
thymidine (1 µCi [0.037 MBq] [3H]thymidine/well) was added and, after 72 h,
thymidine uptake was measured (TopCount; Packard).
Proliferation of CFSE-labeled T cells in vivo. T cells were selected us-
ing CD5 MicroBeads (Miltenyi Biotec), stained with 5 µM CFSE (Invitro-
gen), and transplanted into lethally irradiated allogeneic recipients.
Measurement of cytokines and chemokines. Serum levels were determined
using the Milliplex Mouse Cytokine/Chemokine Immunoassay (Millipore).
data). Bioluminescence imaging demonstrated that early
tumor growth was slightly reduced in B6 NOD2/ mice in
both models (Fig. S4). We conclude that NOD2/ has no
major role in antitumor immunity against C1498 AML and
MATERIALS AND METHODS
Mice and allo-BMT. Animal protocols were approved by the Memorial Sloan-
Kettering Cancer Center Institutional Animal Care and Use Committee.
NOD2/ mice (Kobayashi et al., 2005) were backcrossed to a B6 background
(n = 11). BMT experiments and bioluminescent imaging was performed as de-
scribed previously (Terwey et al., 2005; Zakrzewski et al., 2006, 2008).
Figure 5. NOD2 deficiency in the hematopoietic system regulates experimental colitis. (A) TNBS colitis (5 mg TNBS in 50% ethanol) was induced
in B6 WT or B6 NOD2/ mice. Higher weight loss of NOD2/ mice during colitis. Combined data from three independent experiments are shown;
n = 12/group. (B–D) First, chimeric mice with NOD2 deficiency either in the hematopoietic system or in the nonhematopoietic system were created by synge-
neic BMT (B6 WT→B6 NOD2/ or B6 NOD2/→B6 WT). After 90 d, TNBS colitis (5 mg TNBS in 50% ethanol) was induced. Colons were harvested at day
3 after induction of colitis. Combined data from two independent experiments are shown; n = 8/group. (B) Higher weight loss of NOD2/→WT chimera
versus WT→NOD2/ chimera during colitis. (C) Hematoxylin and eosin–stained images of the colon at day 3 after induction of colitis. Bar, 200 µm.
(D) Histological scoring showing increased colitis in the hematopoietic NOD2-deficient chimera. Error bars indicate SEM.
JEM VOL. 206, September 28, 2009
BRIEF DEFINITIVE REPORT
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Induction and assessment of TNBS colitis. Induction of TNBS colitis
was performed as published previously (Scheiffele and Fuss, 2002). Severity
of colitis was assessed using a semiquantitative scoring system (Scheiffele and
Statistics. Groupwise comparisons were done with the Mann-Whitney
test, one-way ANOVA test, or the Wilcoxon matched pairs test where ap-
propriate. Survival data were analyzed with the Mantel-Cox log-rank test.
Online supplemental material. Fig. S1 shows that donor T cell subsets are
not different in NOD2/ versus WT allo-BMT recipients. In Fig. S2, the do-
nor chimerism at day 90 is given. Fig. S3 shows serum levels of cytokines and
chemokines during GVHD. Fig. S4 demonstrates that NOD2/ mice have
intact resistance against experimental tumors. Online supplemental material
is available at http://www.jem.org/cgi/content/full/jem.20090623/DC1.
We would like to thank Renier Brentjens, Richard Flavell, Ivan J. Fuss, Atsushi Kitani,
Katia Manova, Yan Nikhamin, Gabriel Nunez, Eric Pamer, Carles Ubeda-Morant, Joan
Zhiqiong Yang, and James Young.
This research was supported by National Institutes of Health grants RO1-
HL069929 (to M. van den Brink), RO1-CA107096 (to M. van den Brink), RO1-
AI080455 (to M. van den Brink), R01 HL084815 (to G.F. Murphy), and PO1-CA33049
(to M. van den Brink). Support was also received from the Ryan Gibson Foundation,
the Elsa U. Pardee Foundation, the Byrne Fund, the Emerald Foundation, the
Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center
funded by Mr. William H. Goodwin and Mrs. Alice Goodwin, the Commonwealth
Foundation for Cancer Research, the Bobby Zucker Memorial Fund (to M. van den
Brink), the Deutsche Forschungsgemeinschaft (to O. Penack), the Deutsche
Krebshilfe (to I. Na and A. Ghosh), and the Alexander von Humboldt foundation
(to K. Brandl).
The authors have no conflicting financial interests.
Submitted: 18 March 2009
Accepted: 11 August 2009
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