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Article
The NLRP12 Sensor Negatively Regulates
Autoinflammatory Disease by Modulating
Interleukin-4 Production in T Cells
Highlights
d
d
Deficiency in NLRP12 promotes the generation of
hyperinflammatory T cell responses
Induction of EAE in Nlrp12 / mice results in atypical
neuroinflammatory disease
Authors
John R. Lukens, Prajwal Gurung, ...,
Hongbo Chi,
Thirumala-Devi Kanneganti
Correspondence
d
NLRP12 is an intrinsic regulator of T cells
[email protected]
d
Dysregulated IL-4 production causes T-cell-induced
inflammation in Nlrp12 / mice
In Brief
Lukens et al., 2015, Immunity 42, 654–664
April 21, 2015 ª2015 Elsevier Inc.
http://dx.doi.org/10.1016/j.immuni.2015.03.006
Kanneganti et al. identify the NOD-like
receptor protein, NLRP12, as a novel
negative regulator of T cell activation.
Their work defines a T-cell-autonomous
role for NLRP12 in the suppression of
inflammatory cytokine production and
suggests that dysregulated NLRP12mediated signaling in T cells is sufficient
to drive inflammatory disease.
Immunity
Article
The NLRP12 Sensor Negatively Regulates
Autoinflammatory Disease by Modulating
Interleukin-4 Production in T Cells
John R. Lukens,1,2 Prajwal Gurung,1 Patrick J. Shaw,1 Maggie J. Barr,1 Md. Hasan Zaki,3 Scott A. Brown,1 Peter Vogel,4
Hongbo Chi,1 and Thirumala-Devi Kanneganti1,*
1Department
of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
for Brain Immunology and Glia (BIG), Department of Neuroscience, University of Virginia, Charlottesville, VA 22908, USA
3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
4Animal Resources Center and the Veterinary Pathology Core, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.immuni.2015.03.006
2Center
SUMMARY
Missense mutations in the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain
containing family of gene 12 (Nlrp12) are associated
with periodic fever syndromes and atopic dermatitis
in humans. Here, we have demonstrated a crucial
role for NLRP12 in negatively regulating pathogenic
T cell responses. Nlrp12 / mice responded to antigen immunization with hyperinflammatory T cell responses. Furthermore, transfer of CD4+CD45RBhi
Nlrp12 / T cells into immunodeficient mice led to
more severe colitis and atopic dermatitis. NLRP12
deficiency did not, however, cause exacerbated
ascending paralysis during experimental autoimmune encephalomyelitis (EAE); instead, Nlrp12 /
mice developed atypical neuroinflammatory symptoms that were characterized by ataxia and loss of
balance. Enhanced T-cell-mediated interleukin-4
(IL-4) production promotes the development of atypical EAE disease in Nlrp12 / mice. These results
define an unexpected role for NLRP12 as an intrinsic
negative regulator of T-cell-mediated immunity and
identify altered NF-kB regulation and IL-4 production
as key mediators of NLRP12-associated disease.
INTRODUCTION
Nucleotide-binding oligomerization domain (NOD)-like receptors
(NLRs) compose a family of intracellular sensor molecules that
are involved in the regulation of inflammatory signaling in
response to infection and cellular stress (Kanneganti, 2010; Kanneganti et al., 2007; Lukens et al., 2012). Recent studies have
revealed pivotal roles for NLR-mediated inflammation in a spectrum of diverse human autoimmune and inflammatory disorders
(Kanneganti and Dixit, 2012; Lamkanfi et al., 2011; Lukens et al.,
2011). The vast majority of NLRs to date have been defined as
activators of inflammatory signaling in innate immune cells. For
instance, the prototypical NLR members NOD1 and NOD2
654 Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc.
initiate proinflammatory NF-kB and mitogen activated protein kinase (MAPK) signaling in response to their direct recognition of
bacterial peptidoglycan fragments (Strober et al., 2006). Multiple
NLRs have also been described to promote the activation and
secretion of interleukin-1b (IL-1b) and IL-18 by coordinating the
assembly of the inflammasome complex (Shaw et al., 2011; Strowig et al., 2012). Negative regulation of inflammatory signaling
has also recently been ascribed to NLRP6, NLRP12, and
NLRX1 (Anand et al., 2012; Chen et al., 2011; Elinav et al.,
2011; Lei et al., 2012; Normand et al., 2011; Xia et al., 2011;
Zaki et al., 2011). However, the cellular and molecular mechanisms that direct the suppression of inflammation by this new
class of inhibitory NLRs remain to be formally elucidated.
NLRP12 is a recently identified member of this class of inhibitory NLR proteins. Missense mutations in Nlrp12 lead to periodic
fever syndromes and atopic dermatitis in humans (Borghini et al.,
2011; Jéru et al., 2008, 2011; Macaluso et al., 2007), although little is known about how defective NLRP12-mediated signaling
contributes to these autoinflammatory disorders. In vitro studies
with human and mouse macrophages suggest that NLRP12 is a
negative regulator of toll-like receptor (TLR)-induced cytokine
production (Lich et al., 2007; Williams et al., 2005; Zaki et al.,
2011, 2014). Moreover, NLRP12-mediated suppression of proinflammatory signaling was recently shown to play a central role in
the attenuation of colon inflammation and tumorigenesis in mice
(Allen et al., 2012; Zaki et al., 2011). These studies establish
NLRP12 as a critical regulator of inflammatory responses in
innate immune cells. However, the putative role and importance
of NLRP12 in regulating T cell responses during inflammatory
disease progression has not been characterized. T cells are centrally involved in the pathogenesis of numerous autoinflammatory diseases, to which they participate in both tissue destruction
and the sustained recruitment of inflammatory cells through their
release of effector cytokines and chemokines (Goverman, 2009).
In this study, we focused on investigating the pathophysiological role of NLRP12 in shaping autoinflammatory T cell responses. Deficiency in NLRP12 promoted the generation of
hyperinflammatory T cells in response to antigen immunization.
Nlrp12 / mice did not, however, exhibit exacerbated demyelinating disease in the T-cell-driven experimental autoimmune
encephalomyelitis (EAE) mouse model. Rather, the absence of
NLRP12 provoked an alternative form of neuroinflammation
A
C
Figure 1. NLRP12 Deficiency Promotes the
Generation of Hyperinflammatory T Cell Responses
WT and Nlrp12 / mice were immunized with MOG
peptide in CFA adjuvant and mice were harvested
on day 27.
(A) Splenocytes were restimulated directly ex vivo
and the intracellular production of IFN-g, IL-17, and
TNF-a by CD4+ T cells was determined. Pooled data
are presented in the right panel.
(B) Splenocytes were stimulated for 48 hr with MOG
peptide and cytokine production was measured by
ELISA.
(C) Expression of activation markers by splenic
CD4+ T cells.
Data are representative of at least four independent
experiments with three to six mice per group. All
graphs depict mean ± SEM. *p < 0.05, **p < 0.01,
***p < 0.001; Student’s t test. See also Figure S1.
B
that was characterized by atypical EAE symptoms that included
loss of balance and ataxia. This development of atypical demyelinating neuroinflammation in Nlrp12 / mice was found to be
dependent on dysregulated IL-4 production and we further identify NLRP12 as an intrinsic negative regulator of T cells.
RESULTS
NLRP12 Negatively Regulates In Vivo T Cell Responses
NLRs have emerged as central regulators of inflammatory
signaling, and multiple NLRs have been identified as critically
involved in the regulation of proinflammatory cytokine production by antigen-presenting cells. In contrast, the ability of NLRs
to modulate T cell responses and how NLR-dependent control
of T cells influences autoinflammatory disease progression remains poorly defined. Thus, to elucidate the role of NLRP12 in
shaping T cell responses, wild-type (WT) and NLRP12-deficient
mice were immunized with MOG peptide in CFA adjuvant. T cells
isolated from the spleens of Nlrp12 / mice at day 27 after immunization displayed a hyperinflammatory phenotype and produced higher amounts of interferon-g (IFN-g), IL-17, and tumor
necrosis factor-a (TNF-a) after restimulation (Figure 1A). Likewise, Nlrp12 / splenocytes were also found to secrete
increased amounts of cytokines after stimulation with MOG peptide (Figure 1B). Surface marker expression analysis revealed
that Nlrp12 / T cells expressed higher amounts of CD44 and
CD69, and markedly downregulated CD62L relative to WT cells
(Figure 1C), which suggests that T cells are in a hyperactivated
state in NLRP12-deficient mice. To further characterize the abil-
ity of NLRP12 to modulate T cell responses
in a separate model, we evaluated T cell
activation status and effector cytokine production after DSS-induced inflammation.
In agreement with what was observed
in the antigen immunization model,
Nlrp12 / T cells isolated from dextran sodium sulfate (DSS)-challenged mice also
exhibited a more activated phenotype
(CD44hiCD62Llo) and produced greater
amounts of inflammatory cytokines (Figure S1). Collectively,
these results establish NLRP12 as a negative regulator of T cell
activation in models of antigen immunization and DSS-induced
colitis.
NLRP12-Deficient Mice Develop Atypical EAE Disease
To delineate the role of NLRP12 in the regulation of T-cell-driven
autoimmune disease progression, we evaluated demyelinating
neuroinflammatory disease in the experimental autoimmune
encephalomyelitis (EAE) model. Given our initial observation
that Nlrp12 / T cells were hyperinflammatory after antigen immunization (Figure 1), we hypothesized that NLRP12-deficient
mice would develop exacerbated neuroinflammatory disease.
In contrast to our expectations, classical clinical disease scores
that are based on ascending paralysis were consistently lower in
Nlrp12 / mice (Figure 2A). Instead, Nlrp12 / mice presented
with atypical symptoms of neuroinflammatory disease that
included loss of balance and ataxia (Figure 2B and Movies S1
and S2). Histological analysis revealed that atypical neuroinflammatory disease in Nlrp12 / mice was associated with enhanced
mononuclear cuffing in the brain (Figures 2C and 2D). Likewise,
protection against paralyzing demyelination in NLRP12-deficient
mice was further confirmed by Luxol fast blue staining (LFB) of
spinal cord sections (Figure S2). Consistent with previous
studies that linked atypical EAE disease with extensive infiltration of neutrophils into the central nervous system (CNS)
(Kroenke et al., 2010; Stromnes et al., 2008), neutrophils also
represented an increased fraction of the infiltrating myeloid cell
population in Nlrp12 / mice (Figures 2E and 2F). Furthermore,
Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc. 655
Mice with classical or
aytpical EAE disease (%)
2
1
P<0.001
7
E
F
Nlrp12-/-
WT
105
105
21.8
104
46.2
104
103
Gr-1
Gr 1
D
Nlrp12-/-
WT
103
102
13.5
0
0
10
2
10
3
10
4
10
102
17.8
0
5
0
10
2
10
3
10
4
10
5
CD11b
_______
**
1.5
1.0
0.5
0.0
WT
Nlrp12-/-
50
25
20
_______
***
15
10
5
0
WT
Nlrp12-/-
2.0
_______
*
1.5
1.0
0.5
0.0
4
WT
Nlrp12-/-
_______
**
3
2
1
0
40
30
20
10
0
the proportions of neutrophils to other myeloid cells were
increased in the periphery of diseased Nlrp12 / mice (Figure 2G). In addition, non-classical neuroinflammatory disease
in Nlrp12 / mice was associated with increased frequencies
of Foxp3-expressing regulatory T (Treg) cells in both the periphery (Figure 2H) and the CNS (Figure 2I). Although multiple NLRs
have been identified to play important roles in the orchestration
of inflammasome-mediated cytokine production (Vladimer et al.,
2012), atypical EAE disease in Nlrp12 / mice was not associated with altered generation of IL-1b or IL-18 (Figure S3).
Dysregulated IL-4 Production Promotes Atypical
Neuroinflammatory Disease in Nlrp12–/– Mice
To investigate the immunological factors that contribute to the
development of non-classical neuroinflammatory disease symptoms in Nlrp12 / mice, we next explored whether differences in
clinical disease parameters were associated with differential
regulation of T-cell-mediated cytokine production in the CNS.
Deficiency in NLRP12 was not, however, found to affect the production of IFN-g, IL-17, or TNF-a by CD4+ T cells in the CNS (Fig656 Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc.
Nlrp12-/-
WT
I
H
2.0
Foxp3+ CD4+ T cells (%)
Ratio neutrophils:myeloid cells
G
Classical
0
12
17
22
27
Days post immunization
C
Atypical
Foxp3+ CD4+ T cells (%)
0
100
Mononuclear cuffing score
3
Figure 2. Nlrp12–/– Mice Develop Atypical
EAE Disease
B
WT (n=29)
Nlrp12-/- (n=32)
Ratio neutrophils:myeloid cells
Clinical score (classical)
A
WT
Nlrp12-/-
WT and Nlrp12 / mice were immunized with
MOG+CFA and pertussis toxin to induce EAE.
(A) Classical clinical scores that are based on
ascending paralysis.
(B) Percentages of mice that developed classical
(ascending paralysis) or atypical (ataxia and
impaired balance) EAE disease symptoms.
(C) H&E staining of brain lesions. The arrows
highlight areas of extensive mononuclear cuffing
(original magnification 3 20).
(D) Mononuclear cuffing histology scores from WT
(n = 7) and Nlrp12 / (n = 5) brain sections.
(E) Frequencies of CNS-infiltrating cells that are
neutrophils (CD11b+Gr-1+) or other myeloid cells
(CD11b+Gr-1 ) on day 28.
(F) Ratios of neutrophils to other myeloid cells in
the CNS.
(G) Ratios of neutrophils to other myeloid cells in
the spleen.
(H and I) Frequencies of CD4+ T cells that express
Foxp3 in the spleen (H) and CNS (I). Each point
represents an individual mouse.
Data are representative of four independent experiments with three to six mice per group.
Statistical significance in (A) was determined by
two-way ANOVA. Statistical significance in (D) and
(F)–(I) was determined by Student’s t test. All
graphs depict mean ± SEM. *p < 0.05, **p < 0.01,
***p < 0.001. See also Figure S2 and Movies S1
and S2.
_______
*
ure 3A). Our knowledge of the cellular and
molecular mediators that potentiate atypical EAE currently remains limited, but a
few studies have pointed to important
roles for IL-4 in the induction of atypical
neuroinflammation (Delgoffe et al., 2011;
Nlrp12-/WT
Wensky et al., 2001). Therefore, we next
assessed whether dysregulated IL-4 production promoted atypical neuroinflammatory disease in
Nlrp12 / mice. We observed greater production of IL-4 by
both CNS and splenic CD4+ T cells in Nlrp12 / mice (Figures
3A and 3B). Furthermore, restimulation of NLRP12-deficient
splenocytes with myelin-specific antigen provoked markedly
enhanced secretion of IL-4 (Figure 3C).
To confirm that dysregulated IL-4 production is indeed involved
in dampening ascending paralysis while conversely spurring the
development of atypical EAE disease symptoms in Nlrp12 /
mice, we treated mice with anti-IL-4 neutralizing antibody to
disrupt IL-4-mediated signaling. In agreement with published reports (Bettelli et al., 1998; Falcone et al., 1998; Furlan et al., 1998;
Shaw et al., 1997), blockade of IL-4 during EAE caused exacerbated classical disease symptoms in WT mice (Figure 3D).
Furthermore, ablation of IL-4 during EAE caused NLRP12-deficient mice to develop classical symptoms of demyelinating neuroinflammatory disease that included severe ascending paralysis
and morbidity (Figure 3D). Collectively these results suggest a
causal role for hyperinflammatory IL-4 production in driving atypical EAE disease progression in Nlrp12 / mice.
Figure 3. Dysregulated IL-4 Production Promotes Atypical Neuroinflammatory Disease
in Nlrp12–/– Mice
A
B
C
D
NLRP12 Is Dispensable for Thymic Development
To elucidate the level at which NLRP12 influences T cell activation, we first evaluated the influence of NLRP12 deficiency on
T cell development. NLRP12 deficiency was not found to promote major perturbations in positive T cell selection under homeostatic conditions, and standard thymic development flow
cytometry gating strategies revealed normal distribution of thymocytes in Nlrp12 / mice (Figures S4A and S4B). Moreover,
genetic deletion of Nlrp12 failed to alter the accumulation of
CD4 CD8 , CD4+CD8+, CD4+CD8 , or CD4 CD8+ thymocytes
(Figure S4C). Analysis of the double-negative (DN) population via
CD44 and CD25 discrimination also ruled out a homeostatic role
for NLRP12 in negative selection (Figures S4D and S4E). These
results indicate that NLRP12 is dispensable for normal thymic
development and suggest a role for NLRP12 in the regulation
of mature T cell responses.
NLRP12 Shapes Peripheral T Cell Development in
Naive Mice
To formally address whether the absence of NLRP12 causes
alterations in peripheral T cell development and activation under homeostatic conditions, we next evaluated T cell numbers,
cytokine production, and Treg cell frequencies in naive mice.
We detected enhanced numbers of peripheral CD4+ and
CD8+ T cells in Nlrp12 / mice (Figure S4F). NLRP12-deficient
T cells also displayed a more inflammatory phenotype and
produced greater amounts of IFN-g and IL-17 after brief restimulation (Figure S4G). Consistent with a role for NLRP12 in
shaping T cell development in the periphery, we also observed
greater frequencies of Foxp3-expressing Treg cells in naive
Nlrp12 / mice (Figure S4H). These results indicate that
NLRP12 is involved in the regulation of mature T cell development and activation. However, NLRP12 appeared to exert a
more prominent effect on T cells responses after overt stimula-
WT and Nlrp12 / mice were immunized with
MOG+CFA and pertussis toxin to induce EAE and
mice were harvested either on day 27 (A and B) or
day 29 (C).
(A) Frequencies of CD4+ T cells that express IFN-g,
IL-17, TNF-a, or IL-4 in the CNS.
(B) Frequencies of IL-4-producing CD4+ T cells in
the spleen.
(C) Splenocytes were stimulated with MOG peptide
for 48 hr and IL-4 production was measured by
ELISA.
(A–C) Data are representative of three independent
experiments with three to six mice per group. Statistical significance was determined by Student’s
t test.
(D) Classical ascending paralysis clinical scores for
WT and Nlrp12 / EAE mice that were treated with
either vehicle control or 500 mg of anti-IL-4 blocking
antibody on days 1, 0, 2, 4, 6, 8, and 13. Data are
representative of two independent experiments
with 5–14 mice per group. Statistical significance
was determined by two-way ANOVA.
All graphs depict mean ± SEM. *p < 0.05, **p < 0.01,
***p < 0.001. See also Figure S3.
tion, as was observed in the antigen immunization model
(Figure 1).
NLRP12 Is an Intrinsic Negative Regulator of T Cell
Activation
We were next interested in determining whether NLRP12 modulates T cell activation through an extrinsic role in antigen-presenting cells (APCs) or through an intrinsic function in T cells.
As a first approach we evaluated the expression of Nlrp12
mRNA transcripts in various immune cell populations in an effort
to help identify the cell types that NLRP12 could potentially function in to shape T cell responses. We found that Nlrp12 mRNA
was most highly expressed in polymorphonuclear leukocytes
(PMNs), CD4+ T cells, and CD8+ T cells (Figure 4A). Nlrp12
mRNA expression was also detected in resting dendritic cells
and macrophages, but its expression in these antigen-presenting cells was markedly less in comparison to T cells and
PMNs. To ascertain whether the observed expression of
Nlrp12 in T cells influences functional responses, in vitro
T cell thymidine incorporation assays were conducted. Purified
Nlrp12 / T cells displayed more extensive thymidine incorporation than WT T cells in response to anti-CD3 stimulation (Figure 4B), which implicates NLRP12 in the attenuation of T cell
expansion.
It is possible that defects in Treg-cell-mediated suppressive
functions are responsible for the hyperactive T cell responses
that are observed in Nlrp12 / mice. Therefore, to formally test
this possibility, in vitro Treg cell suppression assays were conducted. In agreement with our anti-CD3 thymidine incorporation
results, we also observed enhanced proliferation by CFSE dye
dilution of Nlrp12 / T cells in the absence of Treg cells. Furthermore, the suppressive capacity exhibited by Treg cells that
lacked NLRP12 was equal to that of WT Treg cells in classical
in vitro suppression assays (Figure 4C), and Nlrp12 / Treg cells
Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc. 657
150
CPM (x1000)
30
20
10
s
N
__
** __
*
50
0
0.1 0.25 0.5
1
CD3 (µg/ml)
PM
lls
ce
ce
__
* __
*
2
C
D
8+
T
4+
T
c
C
D
iti
dr
en
D
lls
lls
ce
ag
ph
WT
Nlrp12-/-
100
0
0
ro
ac
M
C
Figure 4. NLRP12 Negatively Regulates
T Cell Responses
B
40
es
Relative Nlrp12 expression
A
Ratio of Treg cells relative to CD4+ T cells
CFSE
control
0
1:1
1:4
1:8
WT CD4+ T cells
WT Treg cells
(A) Relative expression of Nlrp12 in purified
macrophages (Ly6G CD11b+), dendritic cells
(CD11c+MHCIIhi), CD4+ T cells (Thy1.2+CD4+), CD8+
T cells (Thy1.2+CD8+), and polymorphonuclear
leukocytes (PMNs) (Ly6G+CD11b+).
(B) Purified naive T cells isolated from WT and
Nlrp12 / mice were stimulated with plate bound
anti-CD3 for 48 hr and the incorporation of
thymidine was measured during the final 8 hr.
(C) Purified conventional CD4+ T cells (CD4+
CD62Lhi) and Treg cells (CD4+CD25+) were isolated from WT and Nlrp12 / mice by flow cytometry sorting. Conventional CD4+ T cells were
labeled with CFSE and stimulated with anti-CD3
and irradiated splenocytes. Treg cell suppression
assays were then conducted by mixing conventional CD4+ T cells and Treg cells in various
combinations and at different ratios.
All graphs depict mean ± SEM. *p < 0.05, **p <
0.01; Student’s t test. See also Figures S4 and S5.
Count
WT CD4+ T cells
Nlrp12-/- Treg cells
CFSE
did not display any impairment in IL-10 production (Figure S4I).
Conversely, NLRP12-deficient T cells were less responsive to
the suppression exerted by increasing concentrations of WT
Treg cells, and conventional Nlrp12 / T cells were more likely
to undergo division even when WT Treg cells were present at
high numbers (Figure 4C). These findings indicate that the
altered hyperinflammatory T cell responses that were observed
in Nlrp12 / mice are most likely not the result of dysfunctional
Nlrp12 / Treg cell responses. Rather, they indicate that dysregulated Nlrp12 / effector T cells are responsible for the
observed T-cell-mediated effects in NLRP12-deficient mice.
NLRP12 Is an Intrinsic Negative Regulator of In Vivo
T Cell Responses
To confirm an intrinsic role for NLRP12 in the regulation of T cell
responses in vivo, 1:1 competitive adoptive T cell transfer assays
were carried out. For these experiments, congenically mismatched WT and NLRP12-deficient T cells were adoptively
transferred into Rag1 / recipient mice. After 2 weeks of homeostatic expansion, CD45.2+ Nlrp12 / T cells markedly outcompeted CD45.1+ WT T cells in both the lymph nodes (LNs) and
spleens of recipient mice (Figure S5A). Nlrp12 / T cells also expressed greater amounts of proinflammatory cytokines on a
per-cell basis (Figure S5B). To ascertain whether NLRP12 also
658 Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc.
intrinsically controls T cell responses
under pathophysiological conditions, we
generated 1:1 chimera mice with
Nlrp12-/- CD4+ T cells
CD45.2+ Nlrp12 / and CD45.1+ WT
WT Treg cells
bone marrow cells. After reconstitution,
mice were immunized with MOG peptide
plus CFA adjuvant and T cell responses
were monitored in the spleen at day 21.
Nlrp12-/- CD4+ T cells
Nlrp12-/- Treg cells
Intriguingly, NLRP12-deficient T cells outcompeted WT T cells in the periphery after stimulation (Figure S5C). Furthermore,
T cells produced greater
Nlrp12 /
amounts of IFN-g and IL-17 on a percell basis in the 1:1 chimeric mice (Figure S5C). These results
establish an unexpected, intrinsic role for NLRP12 in the negative regulation of T cell responses under pathophysiological
conditions.
NLRP12 Can Regulate Inflammatory Disease through
T-Cell-Intrinsic Functions
Defective NLRP12 expression was shown to promote exacerbated inflammatory disease in models of colon damage and
tumorigenesis (Zaki et al., 2011). However, the cellular mechanisms responsible for enhanced inflammation in Nlrp12 /
mice have not been formally elucidated. To ascertain whether
Nlrp12 / T cells directly promote hyperinflammation and disease pathology, we transferred CD4+CD45RBhi T cells from
WT or Nlrp12 / mice into Rag1 / mice and tracked the development of colitis. In comparison to the injection of WT T cells,
adoptive transfer of Nlrp12 / T cells provoked more severe
weight loss (Figure 5A) and intestinal inflammation (Figures 5B
and 5C). The development of aggravated disease in Nlrp12 /
T cell transfer mice was associated with enhanced recruitment
of inflammatory cells and cell death in the afflicted mucosa (Figures 5D and S6). Adoptive transfer of Nlrp12 / CD4+CD45RBhi
T cells into Rag1 / mice also provoked the development of
atopic dermatitis in recipient mice (Figure 5E), and this was
A
WT
B
Figure 5. Nlrp12–/– T Cells Are Pathogenic
C
15
110
100
* * * *
WT (n=5)
Nlrp12-/- (n=5)
90
D
Nlrp12-/-
*
0
5
9
Macrophage
Neutrophils
_____
**
10
0
1 2 3 4 5 6 7 8
Weeks post T cell transfer
T cell
Clinical Score
Weight Loss (%)
120
WT
Nlrp12-/-
TUNEL
WT
Purified CD4+CD45RBhi WT or Nlrp12 / T cells
were adoptively transferred into Rag1 / mice.
(A) Percent weight change over time.
(B) Representative large intestine histology at
9 weeks after adoptive transfer of CD4+CD45RBhi
T cells (original magnification 3 20).
(C) Combined colon histology scores.
(D) Representative immunohistochemistry staining
of T cells (anti-CD3), macrophages (anti-F4/80),
neutrophils (anti-Gr-1), and cell death (TUNEL) in
inflamed colon tissue (original magnification 3 20).
(E) Representative H&E ear sections (original
magnification 3 20).
(F) Combined ear histology scores. Each point
represents an individual mouse and the line represents the mean ± SEM.
Data are representative of four independent experiments with three to seven mice per genotype.
All graphs depict mean ± SEM. *p < 0.05, **p <
0.01; Student’s t test. See also Figure S6.
Nlrp12-/-
WT
Nlrp12-/-
F
Ear histology score
E
15
____
*
10
5
0
WT
Nlrp12-/-
associated with more severe skin pathology (Figure 5F). Cutaneous inflammation in this model is typically associated with
dysregulated T helper 2 (Th2) cell-associated inflammation
(Davenport et al., 2002; Leon et al., 2006; Schön et al., 1997),
suggesting that altered IL-4 production might also be contributing to disease pathology.
We next determined the mechanisms by which Nlrp12 /
T cells induce intestinal inflammation and atopic dermatitis. We
found that Nlrp12 / T cells accumulated in greater numbers
(Figure 6A) and exhibited a more activated phenotype (Figure 6B)
in the T cell transfer colitis model. To investigate the contributions of altered inflammatory cytokine production in exacerbated
disease progression, we evaluated cytokine production after
overnight (16 hr) anti-CD3 stimulation. T cell receptor (TCR)mediated activation of cells isolated from Nlrp12 / CD4+
CD45RBhi T cell transfer mice resulted in more robust secretion
of proinflammatory cytokines including IL-6, TNF-a, and GMCSF (Figure 6C, left). Furthermore, Nlrp12 / T cells produced
potent amounts of Th2-cell-associated
cytokines including IL-4, IL-5, and IL-13
(Figure 6C, right), which helps to explain
the development of atopic dermatitis in
these mice. Consistent with the more pronounced production of Th2-cell-derived
cytokines in Nlrp12 / T cell transfer
mice, the expression of YM1 and MBP,
which are markers that have been associated with Th2-cell-mediated inflammation, were also markedly increased in the
CD4+CD45RBhi
colons of Nlrp12 /
T cell transfer mice (Figure S7; Ponomarev
et al., 2007). These results identify
NLRP12 as a negative regulator of
T-cell-induced inflammatory disease and
suggest that unchecked T-cell-mediated
cytokine production centrally contributes
to disease pathology.
Because our findings in the CD4+CD45RBhi T cell transfer
model of colitis indicated that NLRP12 can influence autoinflammatory disease pathogenesis through specific actions in T cells,
we were next interested in elucidating whether NLRP12-dependent regulation of T cells responses contributed to the altered
EAE disease phenotype that was observed in Nlrp12 / mice.
To this end we evaluated the development of demyelinating neuroinflammatory disease in the passive transfer model of EAE disease. Consistent with our previous data showing abrogated
classical EAE disease in germline NLRP12-deficient mice (Figure 2), the transfer of enriched myelin-specific Nlrp12 /
T cells into Rag1 / recipient mice resulted in less severe
paralyzing neuroinflammatory disease (Figure 6D). As an independent approach, WT or Nlrp12 / thymocytes were also
adoptively transferred into T-cell-deficient mice (Tcra / mice)
and EAE was induced. In agreement with our previous results,
Tcra / mice that were reconstituted with Nlrp12 / thymocytes
were also protected from severe paralyzing EAE disease
Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc. 659
A
C
B
D
Figure 6. Dysregulated T Cell Activation and
Cytokine Production Contribute to the Pathogenicity of Nlrp12–/– T Cells
Purified CD4+CD45RBhi WT or Nlrp12 / T cells
were adoptively transferred into Rag1 / mice.
(A) Total numbers (mean ± SEM) of CD4+ T cells in
the mesenteric LNs (MLNs) and spleen at 9 weeks
after adoptive transfer.
(B) Expression of CD69 by adoptively transferred
CD4+ T cells in the MLNs and spleen.
(C) WT (n = 5) and Nlrp12 / (n = 5) splenocytes
were stimulated overnight with anti-CD3 and
cytokine production was measured by ELISA.
(D) Passive transfer EAE was induced in Rag1 /
recipient mice by transferring enriched myelinspecific CD4+ T cells that were generated and
expanded from WT and Nlrp12 / mice. Classical
signs of EAE disease (ascending paralysis) were
assessed over time.
(E) T-cell-deficient (Tcra / ) mice were reconstituted with WT or Nlrp12 / thymocytes. Mice
were then immunized with MOG plus CFA to
induce EAE, and the development of classical
signs of clinical disease (ascending paralysis) were
assessed over time.
All graphs depict mean ± SEM. *p < 0.05, **p <
0.01; Student’s t test. See also Figure S7.
E
(Figure 6E). These studies in T cell transfer models of disease
thus implicate critical T-cell-intrinsic roles for NLRP12 in
the regulation of autoinflammation and autoimmune disease
pathogenesis.
NLRP12 Negatively Regulates NF-kB Signaling in T Cells
Previous studies suggest that NLRP12 dampens TLR-induced
NF-kB activation in macrophages and dendritic cells (Lich
et al., 2007; Williams et al., 2005; Zaki et al., 2011), but the specific molecular pathways that are controlled by NLRP12 in
T cells remain unknown. Many of the cytokines that were found
to be enhanced in Nlrp12 / T cell transfer colitis mice are
known to be regulated by NF-kB-dependent signaling (Figure 6C), so we were interested in determining whether
NLRP12 limits T-cell-induced inflammatory disease through its
regulation of NF-kB activation. Indeed, transfer of Nlrp12 /
CD4+CD45RBhi T cells resulted in enhanced phosphorylation
of IkBa (Figures 7A and 7B). Moreover, we also detected greater
NF-kB2 processing (conversion of p100 to p52) in Nlrp12 /
CD4+CD45RBhi T cell transfer colon lysates, which indicates
660 Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc.
enhanced non-canonical NF-kB signaling
in the absence of NLRP12 (Figures 7A
and 7B). To elucidate whether NLRP12
is involved in the global dampening of
proinflammatory signaling or whether it
is specifically involved in the NF-kB
pathway, we also evaluated the regulation of other important signaling pathways that are known to drive disease
pathology in the T cell transfer colitis
model. Exacerbated inflammatory disease in CD4+CD45RBhi Nlrp12 / T cell
transfer mice was not, however, associated with altered ERK,
p38, or STAT3 signaling (Figures 7A and 7B). Importantly, we
also observed enhanced canonical and non-canonical NF-kB
signaling in the brains of NLRP12-deficient mice during EAE
(Figures 7C and 7D).
To determine whether NLRP12-mediated regulation of NF-kB
activation in T cells potentially contributes to the altered inflammatory signaling that was observed in these two models of autoimmunity, we evaluated signaling in purified T cells. We found
that stimulation of Nlrp12 / T cells with anti-CD3 plus antiCD28 led to enhanced phosphorylation of IkBa and greater
amounts of processing of p100 to p52 (Figure 7E). Furthermore,
pharmacological inhibition of NF-kB activation with SC514 treatment rescued the hyperproliferation exhibited by Nlrp12 /
T cells stimulated with anti-CD3 plus anti-CD28 (Figure 7F).
Inhibition of NF-kB signaling with SC514 was also found to return
IL-4 secretion by Nlrp12 / T cells to wild-type amounts (Figure 7G). Collectively, these findings identify NLRP12 as a negative regulator of both canonical and non-canonical NF-kB
signaling in T cells.
A
C
E
Figure 7. NLRP12 Negatively Regulates
Both Canonical and Non-canonical NF-kB
Signaling
B
D
F
G
DISCUSSION
Mutations in Nlrp12 have been described to cause autoinflammatory disorders in humans (Borghini et al., 2011; Jéru et al.,
2008, 2011; Macaluso et al., 2007), but the cellular and molecular
mechanisms involved in NLRP12-mediated regulation of
immune responses remain poorly defined. In this study,
we focused on elucidating the roles of NLRP12 in shaping inflammatory T cell responses and T-cell-mediated disease. We
discovered that the induction of experimental autoimmune
encephalomyelitis (EAE) in NLRP12-deficient mice provoked
the generation of hyperinflammatory myelin-specific T cell responses. Nlrp12 / T cells did not, however, cause exacerbated
demyelinating disease in the T-cell-driven EAE model. In
contrast, Nlrp12 / mice developed atypical neuroinflammatory
disease symptoms that included loss of balance and ataxia.
Moreover, the development of non-classical EAE disease in
mice that lack NLRP12 was characterized by altered brain
(A and B) Purified CD4+CD45RBhi WT or Nlrp12 /
T cells were adoptively transferred into Rag1 /
mice. Colons were collected from WT and
Nlrp12 / CD4+CD45RBhi transfer mice at 9 weeks
after adoptive transfer.
(A) Immunoblot analysis of colon lysates. Each lane
represents an individual mouse.
(B) Densitometry quantification of band intensity.
Data are representative of four independent experiments with three to seven mice per genotype.
(C and D) WT and Nlrp12 / mice were immunized
with MOG+CFA and pertussis toxin to induce EAE
and mice were harvested on day 29. Immunoblot
analysis of brain lysates.
(D) Densitometry quantification of band intensity.
Data are representative of three independent experiments with three to five mice per genotype.
(E–G) Purified WT and Nlrp12 / T cells were
stimulated with anti-CD3 (1 mg/ml) plus anti-CD28
(1 mg/ml) in the presence or absence of the NF-kB
inhibitor SC514 (3 mg/ml).
(E) Lysates were probed for total and phosphorylated IkBa, ERK p100/p52, and GAPDH.
(F) Counts per minute (CPM) of T cells at 72 hr after
stimulation with anti-CD3 and anti-CD28.
(G) Secretion of IL-4 at 72 hr after stimulation with
anti-CD3 and anti-CD28.
All graphs depict mean ± SEM. *p < 0.05, **p <
0.01; Student’s t test.
pathology and a myeloid cell CNS infiltrate that was dominated by neutrophils.
We demonstrated that NLRP12 is an
intrinsic negative regulator of T cells and
that NLRP12 deficiency in T cells was
sufficient to induce colitis and atopic
dermatitis in the CD4+CD45RBhi T cell
transfer model. Mechanistically, dysregulated NF-kB activation and enhanced IL-4
production were found to contribute to
T-cell-induced inflammation in Nlrp12 / mice. Our findings
define a previously unknown role for NLRs in the negative regulation of T cell activation and unravel a T-cell-intrinsic function for
NLRP12 in the suppression of inflammatory cytokine production.
Furthermore, our discovery that NLRP12 deficiency leads to
unchecked IL-4 production helps to explain how missense mutations in Nlrp12 contribute to the development of atopic dermatitis in humans.
Utilization of the EAE model system has helped to define the
inflammatory factors that contribute to demyelination and paralysis in multiple sclerosis (MS) pathogenesis. However,
considerably less is known about the conditions and pathways
that induce many of the other disease symptoms that are common in MS patients, including ataxia, impaired balance, and
loss of sensation. Our findings presented here suggest that
investigation of EAE in Nlrp12 / mice could provide a muchneeded model system to formally characterize the immune factors that trigger non-paralyzing neuroinflammatory disease
Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc. 661
symptoms in MS patients. Importantly, excessive production of
both classical inflammatory factors (IFN-g, IL-6, GM-CSF) and
Th2-cell-associated cytokines (IL-4 and IL-5) have been
observed in patients with severe MS (Hedegaard et al., 2008;
Hohnoki et al., 1998; Link et al., 1994). However, the physiological role of Th2 cell cytokines in MS disease pathogenesis has
remained elusive. In the EAE mouse model, IL-4 has been
described to provide protection against classical ascending
paralysis (Bettelli et al., 1998; Falcone et al., 1998; Furlan
et al., 1998; Shaw et al., 1997). In agreement with these findings, we observed that enhanced IL-4 production limited the
development of paralysis in NLRP12-deficient mice. Unchecked IL-4 production during EAE has also been shown to
induce atypical neuroinflammatory disease symptoms (ataxia
and issues with balance), which is consistent with the IL-4associated atypical neuroinflammatory disease phenotype
that was observed in Nlrp12 / mice (Delgoffe et al., 2011;
Wensky et al., 2001).
Th2-cell-associated cytokines also play pathogenic roles
in intestinal inflammatory disease. In particular, Th2 cell cytokines are highly expressed in combination with Th1-cell-associated cytokines in ulcerative colitis (UC) patients (Fuss et al.,
1996), and Th2 cell cytokines are believed to be the major
drivers of pathology in UC (Cho, 2008). Likewise, Th2 cell cytokines potentiate UC-like intestinal disease in the oxazolone
colitis mouse model (Boirivant et al., 1998; Heller et al., 2002;
Mizoguchi et al., 1999). In these studies, it was shown that
Th2 cell cytokines cause inflammation to the intestinal
epithelial layer and that neutralization of IL-4 with blocking antibody treatment rescues oxazolone-mediated colitis. Similarly,
hyperinflammatory IL-4 production has also been described
to cause exacerbated disease in the CD4+CD45RBhi T cell
transfer colitis model (Fort et al., 2001). In this setting, Th2
cell cytokines promote the upregulation of major hisotocmpatibility complex (MHC) class II expression in the lamina propria
of the colon and also stimulate the expression of exorbitant
amounts of both Th1- and Th2-cell-associated cytokines in
the intestinal mucosa. Our findings demonstrate prominent
roles for NLRP12 in the attenuation of colitogenic T cell responses and identify NLRP12 as an attractive candidate for
the treatment of Th2-cell-associated intestinal inflammatory
disease.
In summary, we identify NLRP12 as a negative regulator of
inflammatory T cell responses and T-cell-mediated disease.
NLRP12 intrinsically regulates T cell activation, and dysregulated
NLRP12-mediated signaling in T cells is sufficient to induce inflammatory disease. However, deficiency in NLRP12 did not provoke more severe demyelinating disease and paralysis in the
EAE model. In contrast, Nlrp12 / mice developed atypical neuroinflammatory disease that was characterized by worsened
brain pathology and excessive production of both traditional
inflammatory cytokines (IL-6, GM-CSF, TNF-a) and IL-4.
Furthermore, dysregulated T-cell-mediated NF-kB signaling
and downstream IL-4 were found to contribute to inflammatory
responses in Nlrp12 / mice. These findings define additional
roles for NLRP12 as an intrinsic attenuator of T cell responses
and IL-4-associated inflammation. Moreover, they suggest that
NLRP12-targeted therapies might provide a novel treatment
strategy in T-cell-mediated disease.
662 Immunity 42, 654–664, April 21, 2015 ª2015 Elsevier Inc.
EXPERIMENTAL PROCEDURES
Mice
Nlrp12 / mice have been previously described (Zaki et al., 2011). Tcra /
mice were kindly provided by Dr. Maureen McGargill (St. Jude Children’s
Research Hospital) and Rag1 / mice were received from Dr. Terrence L. Geiger (St. Jude Children’s Research Hospital). Mice at 6–12 weeks of age were
used. All mice were kept in specific-pathogen-free conditions within the
Animal Resource Center at St. Jude Children’s Research Hospital. Animal protocols were approved by the Institutional Animal Care and Use Committee of
St. Jude Children’s Research Hospital.
Flow Cytometry Protocols and Antibodies
For the flow cytometry cellular analysis, the following monoclonal antibodies
were used: CD4 (L3T4), IFN-g (XMG1.2), IL-17A (eBio17B7), IL-4 (11B11),
MHCII (M5/114.15.2), CD11b (M1/70), CD19 (6D5), CD44 (IM7), Ly-6G (1A8),
CD25 (3C7), B220 (RA3-6B2), and Gr-1 (RB6-8C5) from eBioscience and
TCR-b (H57-597), CD8 (53-6.7), Foxp3 (FJK-16 s), CD62L (MEL-14), CD69
(H1.2F3), TNF-a (MP6-XT22), CD11c (N418), CD45.1 (A20), and CD45.2
(104) from Biolegend.
Nlrp12 Expression
Macrophages (Ly6G CD11b+), dendritic cells (CD11c+MHCIIhi), CD4+ T cells
(Thy1.2+CD4+), CD8+ T cells (Thy1.2+CD8+), and polymorphonuclear leukocytes (PMNs) (Ly6G+CD11b+) were purified by flow cytometry sorting and total
RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s instructions. 1 mg of RNA was reverse transcribed to cDNA with random RNAspecific primers using the high-capacity cDNA reverse transcription kit
(Applied Biosystems). Transcript amounts of Nlrp12 and Gapdh were analyzed
with SYBR-Green (Applied Biosystems) according to the manufacturer’s
recommendations.
Thymidine Incorporation Assay
Thy1.2+ T cells were isolated from WT and Nlrp12 / mice via positive magnetic bead enrichment (Miltenyi Biotec). Naive T cells (CD44loCD62Lhi) were
then purified via FACS sorting. Purified naive T cells were stimulated with
increasing concentrations of plate bound anti-CD3 in triplicate wells for
48 hr. During the last 8 hr of stimulation, T cells were pulsed with [3H]thymidine
and the amount of incorporated [3H]thymidine was measured as counts per
minute (cpm).
Mixed Bone Marrow Chimeras
Bone marrow from WT (CD45.1+) and Nlrp12 / (CD45.2+) mice was flushed
from the femurs, filtered through a 40 mm filter, and mixed at a 1:1 ratio. Mixed
bone marrow cells (total 5–10 3 106 cells) were transferred by tail vein injection
into lethally irradiated (1,000 rad) Rag1 / mice. 1:1 bone marrow chimera
mice were allowed to reconstitute for 2 months before immunization with
MOG peptide in CFA. Congenic CD45 markers were utilized to verify
chimerism.
CD4+CD45RBhi T Cell Transfer Colitis Model
Splenocytes and peripheral LNs were harvested from WT and Nlrp12 / mice
and bulk T cells were positively enriched with anti-Thy1.2 magnetic beads
(Miltenyi Biotec). CD4+CD45RBhi T cells were purified from the bulk T cell population by FACs sorting. Rag1 / recipient mice received 5 3 105 WT or
Nlrp12 / CD4+CD45RBhi T cells by intraperitoneal injection and mice were
weighed once every week to measure percent weight change.
EAE Model
Mice were immunized subcutaneously with 100 mg MOG35–55 peptide
(MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA (Difco Laboratories) with
500 mg Mycobacterium tuberculosis on day 0. Mice also received 200 ng of
pertussis toxin (List Biological Laboratories) by intraperitoneal injection on
days 0 and 2. Classical disease severity was assessed daily by assigning clinical scores according to the following ascending paralysis scale: 0, no disease;
1, tail paralysis; 2, weakness of hind limbs; 3, paralysis of hind limbs; 4, paralysis of hind limbs and severe hunched posture; 5, moribund or death. The incidence of atypical disease was recorded based on the development of balance
issues and ataxia. To evaluate the role of IL-4 in Nlrp12-mediated atypical neuroinflammatory disease, EAE was induced in WT and Nlrp12 / mice as
described above. Mice received either vehicle control or 500 mg anti-IL-4
blocking antibody (11B11) by intraperitoneal dosage on days 1, 0, 2, 4, 6,
8, and 13, and classical clinical scores were assigned based on ascending paralysis development.
Additional Supplemental Experimental Procedures, including histopathology and immunohistochemistry, competitive T cell adoptive transfer, chronic
DSS model, Treg cell suppression assay, passive and thymocyte transfer
EAE transfer models, and CFSE T cell proliferation assay, are available online.
Chen, G.Y., Liu, M., Wang, F., Bertin, J., and Núñez, G. (2011). A functional role
for Nlrp6 in intestinal inflammation and tumorigenesis. J. Immunol. 186, 7187–
7194.
Statistics
All results are presented as mean ± SEM. Two-way ANOVA analysis was utilized to evaluate differences in clinical EAE scores over time. All other p values
were calculated by two-tailed unpaired Student’s t tests. p values < 0.05
were considered significant. p values are denoted by *p < 0.05, **p < 0.01,
***p < 0.001.
Delgoffe, G.M., Pollizzi, K.N., Waickman, A.T., Heikamp, E., Meyers, D.J.,
Horton, M.R., Xiao, B., Worley, P.F., and Powell, J.D. (2011). The kinase
mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2. Nat. Immunol. 12, 295–303.
Cho, J.H. (2008). The genetics and immunopathogenesis of inflammatory
bowel disease. Nat. Rev. Immunol. 8, 458–466.
Davenport, C.M., McAdams, H.A., Kou, J., Mascioli, K., Eichman, C., Healy, L.,
Peterson, J., Murphy, S., Coppola, D., and Truneh, A. (2002). Inhibition of proinflammatory cytokine generation by CTLA4-Ig in the skin and colon of mice
adoptively transplanted with CD45RBhi CD4+ T cells correlates with suppression of psoriasis and colitis. Int. Immunopharmacol. 2, 653–672.
SUPPLEMENTAL INFORMATION
Elinav, E., Strowig, T., Kau, A.L., Henao-Mejia, J., Thaiss, C.A., Booth, C.J.,
Peaper, D.R., Bertin, J., Eisenbarth, S.C., Gordon, J.I., and Flavell, R.A.
(2011). NLRP6 inflammasome regulates colonic microbial ecology and risk
for colitis. Cell 145, 745–757.
Supplemental Information includes seven figures, Supplemental Experimental
Procedures, and two movies and can be found with this article online at http://
dx.doi.org/10.1016/j.immuni.2015.03.006.
Falcone, M., Rajan, A.J., Bloom, B.R., and Brosnan, C.F. (1998). A critical role
for IL-4 in regulating disease severity in experimental allergic encephalomyelitis as demonstrated in IL-4-deficient C57BL/6 mice and BALB/c mice.
J. Immunol. 160, 4822–4830.
AUTHOR CONTRIBUTIONS
J.R.L., P.G., and T.-D.K. designed the study. J.R.L., P.G., P.J.S., M.H.Z., and
M.J.B. performed experiments. P.V. performed and analyzed the histopathology data. S.A.B. made IL-4 neutralizing antibodies. H.C. provided essential reagents and scientific insights. J.R.L., P.G., and T.-D.K. analyzed data and
wrote the manuscript.
ACKNOWLEDGMENTS
P.G. is a postdoctoral fellow supported by Paul Barrett Endowed Fellowship
from St. Jude Children’s Research Hospital. This work was supported by the
National Institute of Arthritis and Musculoskeletal and Skin Diseases, part of
the NIH, under Award Number AR056296 (T.-D.K.); the National Cancer Institute, part of the NIH, under Award Number CA163507 (T.-D.K.); the National
Institute of Allergy and Infectious Diseases, part of the NIH, under Award Number AI101935 (T.-D.K.); and ALSAC.
Received: September 29, 2014
Revised: January 9, 2015
Accepted: March 18, 2015
Published: April 14, 2015
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