NLRP10 is a NOD-like receptor essential to initiate adaptive immunity by dendritic cells.
ABSTRACT NLRs (nucleotide-binding domain leucine-rich-repeat-containing receptors; NOD-like receptors) are a class of pattern recognition receptor (PRR) that respond to host perturbation from either infectious agents or cellular stress. The function of most NLR family members has not been characterized and their role in instructing adaptive immune responses remains unclear. NLRP10 (also known as PYNOD, NALP10, PAN5 and NOD8) is the only NLR lacking the putative ligand-binding leucine-rich-repeat domain, and has been postulated to be a negative regulator of other NLR members, including NLRP3 (refs 4-6). We did not find evidence that NLRP10 functions through an inflammasome to regulate caspase-1 activity nor that it regulates other inflammasomes. Instead, Nlrp10(-/-) mice had a profound defect in helper T-cell-driven immune responses to a diverse array of adjuvants, including lipopolysaccharide, aluminium hydroxide and complete Freund's adjuvant. Adaptive immunity was impaired in the absence of NLRP10 because of a dendritic cell (DC) intrinsic defect in emigration from inflamed tissues, whereas upregulation of DC costimulatory molecules and chemotaxis to CCR7-dependent and -independent ligands remained intact. The loss of antigen transport to the draining lymph nodes by a subset of migratory DCs resulted in an almost absolute loss in naive CD4(+) T-cell priming, highlighting the critical link between diverse innate immune stimulation, NLRP10 activity and the immune function of mature DCs.
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ABSTRACT: The innate immune system is composed of a wide repertoire of conserved pattern recognition receptors (PRRs) able to trigger inflammation and host defense mechanisms in response to endogenous or exogenous pathogenic insults. Among these, Nucleotide-binding and Oligomerization Domain (NOD)-like Receptors (NLRs) are intracellular sentinels of cytosolic sanctity capable of orchestrating innate immunity and inflammatory responses following the perception of noxious signals within the cell. In this review, we elaborate on recent advances in the signaling mechanisms of NLRs, operating within inflammasomes or through alternative inflammatory pathways, and discuss the spectrum of their effector functions in innate immunity. We describe the progressive characterization of each NLR with associated controversies and cutting edge discoveries.Cytokine & Growth Factor Reviews 12/2014; · 8.83 Impact Factor
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ABSTRACT: Dendritic cells (DCs) are critical for regulating CD4 and CD8 T cell immunity, controlling Th1, Th2, and Th17 commitment, generating inducible Tregs, and mediating tolerance. It is believed that distinct DC subsets have evolved to control these different immune outcomes. However, how DC subsets mount different responses to inflammatory and/or tolerogenic signals in order to accomplish their divergent functions remains unclear. Lipopolysaccharide (LPS) provides an excellent model for investigating responses in closely related splenic DC subsets, as all subsets express the LPS receptor TLR4 and respond to LPS in vitro. However, previous studies of the LPS-induced DC transcriptome have been performed only on mixed DC populations. Moreover, comparisons of the in vivo response of two closely related DC subsets to LPS stimulation have not been reported in the literature to date. We compared the transcriptomes of murine splenic CD8 and CD11b DC subsets after in vivo LPS stimulation, using RNA-Seq and systems biology approaches. We identified subset-specific gene signatures, which included multiple functional immune mediators unique to each subset. To explain the observed subset-specific differences, we used a network analysis approach. While both DC subsets used a conserved set of transcription factors and major signalling pathways, the subsets showed differential regulation of sets of genes that 'fine-tune' the network Hubs expressed in common. We propose a model in which signalling through common pathway components is 'fine-tuned' by transcriptional control of subset-specific modulators, thus allowing for distinct functional outcomes in closely related DC subsets. We extend this analysis to comparable datasets from the literature and confirm that our model can account for cell subset-specific responses to LPS stimulation in multiple subpopulations in mouse and man.PLoS ONE 01/2014; 9(6):e100613. · 3.53 Impact Factor
Article: NLR proteins and parasitic disease.[Show abstract] [Hide abstract]
ABSTRACT: Parasitic diseases are a serious global health concern. Many of the most common and most severe parasitic diseases, including Chagas' disease, leishmaniasis, and schistosomiasis, are also classified as neglected tropical diseases and are comparatively less studied than infectious diseases prevalent in high income nations. The NLRs (nucleotide-binding domain leucine-rich-repeat-containing proteins) are cytosolic proteins known to be involved in pathogen detection and host response. The role of NLRs in the host response to parasitic infection is just beginning to be understood. The NLR proteins NOD1 and NOD2 have been shown to contribute to immune responses during Trypanosoma cruzi infection, Toxoplasma gondii infection, and murine cerebral malaria. The NLRP3 inflammasome is activated by T. cruzi and Leishmania amazonensis but also induces pathology during infection with schistosomes or malaria. Both the NLRP1 and NLRP3 inflammasomes respond to T. gondii infection. The NLRs may play crucial roles in human immune responses during parasitic infection, usually acting as innate immune sensors and driving the inflammatory response against invading parasites. However, this inflammatory response can either kill the invading parasite or be responsible for destructive pathology. Therefore, understanding the role of the NLR proteins will be critical to understanding the host defense against parasites as well as the fine balance between homeostasis and parasitic disease.Immunologic research. 07/2014;
NLRP10 is a NOD-like receptor essential to initiate
adaptive immunity by dendritic cells
Stephanie C. Eisenbarth1*, Adam Williams2*, Oscar R. Colegio3, Hailong Meng4, Till Strowig2, Anthony Rongvaux2,
Jorge Henao-Mejia2, Christoph A. Thaiss2, Sophie Joly5, David G. Gonzalez1, Lan Xu1,3, Lauren A. Zenewicz2, Ann M. Haberman1,
Eran Elinav2, Steven H. Kleinstein4,6, Fayyaz S. Sutterwala5,8& Richard A. Flavell2,7
NLRs (nucleotide-binding domain leucine-rich-repeat-containing
receptors; NOD-like receptors) are a class of pattern recognition
tious agents or cellular stress1,2. The function of most NLR family
members has not been characterized and their role in instructing
adaptive immune responses remains unclear2,3. NLRP10 (also
known as PYNOD, NALP10, PAN5 and NOD8) is the only NLR
lacking the putative ligand-binding leucine-rich-repeat domain,
and has been postulated to be a negative regulator of other NLR
members, including NLRP3 (refs 4–6). We did not find evidence
that NLRP10 functions through an inflammasome to regulate
caspase-1 activity nor that it regulates other inflammasomes.
Instead, Nlrp102/2mice had a profound defect in helper T-cell-
drivenimmune responsestoadiverse arrayofadjuvants,including
lipopolysaccharide, aluminium hydroxide and complete Freund’s
adjuvant. Adaptive immunity was impaired in the absence of
NLRP10 because of a dendritic cell (DC) intrinsic defect in
emigration from inflamed tissues, whereas upregulation of DC
costimulatory molecules and chemotaxis to CCR7-dependent and
to the draining lymph nodes by a subset of migratory DCs resulted
inanalmostabsolutelossinnaive CD41T-cell priming,highlight-
ing the critical link between diverse innate immune stimulation,
NLRP10 activity and the immune function of mature DCs.
To elucidate the in vivo biological role of NLRP10, we generated
seemed healthy without evidence of autoimmunity or tumour forma-
tion, and had a normal composition and activation profile of immune
cells including T and B lymphocytes in the periphery, bone marrow
and thymus (data not shown). Peritoneal macrophages or bone-
marrow-derived dendritic cells (BMDCs) from Nlrp102/2mice
stimulated with Toll-like receptor (TLR) agonists or NLRP3 inflam-
masome activators secreted normal levels of IL-1b, TNF-a and IL-6
(Supplementary Fig. 2a–c), indicating that loss of NLRP10 does not
affect caspase-1 or NLRP3 inflammasome function. To test in vivo
whether NLRP10 acts as a negative regulator of NLRs, we tested the
to ovalbumin (OVA) and aluminium hydroxide (alum) in a T-helper
type 2 (TH2)-driven asthma model7,8or complete Freund’s adjuvant
(CFA; mycobacteria-based) with myelin oligodendrocyte glycoprotein
(MOG) peptide in the IL-17-producing T-helper cell (TH17)-driven
model of experimental autoimmune encephalomyelitis (EAE)9.
Surprisingly, Nlrp102/2mice had a profound defect in adaptive
immunity in both models. TH2 responses in the lung, lymph nodes
(LN) and systemic antibody production were significantly reduced in
a marked reduction in MOG-specific IL-17 and IFN-c production
from the spleen, LNs and spinal cord (Fig. 1d–f and Supplementary
(LPS) as an adjuvant in an intranasal TH1/neutrophil airway inflam-
mation model10was also defective in Nlrp102/2mice (Fig. 1g).
Together, these findings suggest that Nlrp102/2mice have a global
defect in adaptive immunity upon immunization with multiple
adjuvants. Bone marrow chimaeric mice in which NLRP10 deficiency
was limited to the haematopoietic compartment failed to respond to
marrow-derived cells was sufficient to recapitulate the phenotype
To test if the Nlrp102/2mice have a defect in T-cell-driven adap-
tive immune processes, we compared the immunization response to
the hapten trinitrophenyl (TNP) linked to either keyhole limpet
haemocyanin (KLH) or Ficoll. In this model, anti-TNP antibodies
are generated by activated B cells in either a T-cell-dependent
(KLH) or T-cell-independent (Ficoll) manner11,12. Anti-TNP IgG1
antibodies were severely diminished with TNP-KLH (Fig. 2d), but
there was no defect in T-cell-independent IgG3 (Fig. 2e) antibody
production to TNP-Ficoll. Therefore, Nlrp102/2B cells are not
intrinsically impaired but T-cell activation, either secondary to a
T-cell-intrinsic or T-cell-extrinsic defect, is severely impaired in
Nlrp102/2mice. Nlrp102/2T cells can be primed and differentiated
into cytokine-producing helper T-cell subsets (Supplementary Fig. 4)
in vitro. Furthermore, adoptively transferred T-cell receptor (TCR)
Therefore, we concluded that Nlrp102/2mice fail to initiate adaptive
immune responses, possibly because of a T-cell-extrinsic defect in
To evaluate T-cell priming in vivo, we adoptively transferred
TCR transgenic OT-II T cells into wild-type and Nlrp102/2mice.
T cells divided in wild-type, but not Nlrp102/2, hosts (Fig. 2f), indi-
in the absence of NLRP10. As dendritic cells are the primary antigen-
presenting cell (APC) controlling the activation fate of naive T cells
following immunization, we tested whether DC maturation was
defective in Nlrp102/2mice13. Nlrp102/2BMDCs in vitro and splenic
DCs in vivo upregulated all requisite stimulatory molecules necessary
for effective T-cell priming, including major histocompatibility com-
plex (MHC) class II and B7 family member CD86 following LPS
exposure (Supplementary Fig. 6a, d and data not shown). Similarly,
06520, USA.5Inflammation Program, Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242, USA.6Interdepartmental Program in Computational Biology and Bioinformatics, Yale
Center, Iowa City, Iowa 52241, USA.
*These authors contributed equally to this work.
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vitro (Supplementary Fig. 6b) and in vivo (Supplementary Fig. 6e).
Antigen-pulsed Nlrp102/2BMDCs were also capable of activating
naive CFSE-labelled wild-type OT-II T cells in vitro (Supplementary
Fig. 6c). Therefore DC maturation following innate stimulation was
intact; yet, Nlrp102/2BMDCs loaded with protein antigen and adop-
tively transferred into mice harbouring CFSE-labelled naive OT-II T
cells were unable to activate these T cells (Fig. 3a), indicating that the
T-cell-priming defect in Nlrp102/2mice was due to a loss of DC–T-
cell interactions. To test this, we used the traditional model of FITC
(fluorescein isothiocyanate) skin painting to track migration of DCs
from the skin to the LN14. FITC-painted Nlrp102/2mice contained
fewFITC1DCsin the draining LNat 18h,similarto mice lacking the
both wild-type and Nlrp102/2mice (data not shown), indicating that
Nlrp102/2DCs were viable and capable of capturing antigen, but
failed to reach the LN. Nlrp102/2mice also demonstrated a profound
absence of antigen-containing DCs in the draining LN following sub-
cutaneous injection of a fluorescently labelled antigen (0.5–5.0mg
OVA-AF647) at 18h (Fig. 3c) or any time point evaluated out to
12days following immunization (data not shown). This defect in
antigen-containing DCs in the LN could be partially overcome in
Nlrp102/2mice upon exposure to high antigen doses (50mg OVA)
(Fig. 3c), as previously observed in CCR7-deficient mice14. This is
weak T-cell responses14,17. Evaluation of mediastinal LNs after inhala-
indicated that Nlrp102/2DCs were unable to transport antigen to the
draining LN, yet bead-containing DCs were present in the lung
(Supplementary Fig. 8). Therefore Nlrp102/2mice have a profound
defect in DC-dependent transport of antigen to lymph nodes.
Lymphoid-tissue-resident CD8a1and plasmacytoid DCs were not
different between wild-type and Nlrp102/2mice (Supplementary
the LN via the bloodstream. In contrast, migratory DCs which take
antigen from inflamed tissue to the LN18and express high levels of
MHC class II, but intermediate levels of CD11c, were affected by the
loss of NLRP10. Specifically, within this group we found that the
CD11b1CD2072CD1032DC subset, which is primarily responsible
for priming helper T cells, is nearly absent in the LN of immunized
Nlrp102/2mice (Supplementary Fig. 7), again similar to the defect
observed in CCR7-deficient mice14,19–21. Consistent with the finding
that antigen-containing mature DCs were present normally in the ear
or lung of immunized Nlrp102/2mice (Supplementary Fig. 8), we
found no DC subset deficiency in any non-lymphoid tissue from
knockout mice (Supplementary Fig. 9), indicating that the develop-
ment and migration of CD11b1DCs to peripheral tissues is intact in
tion to the LN is cell intrinsic or extrinsic, we co-injected labelled
activated wild-type and Nlrp102/2BMDCs into wild-type or
Nlrp102/2host mice. Although wild-type DCs were found in the
LN 18h after subcutaneous injection, very few Nlrp102/2DCs were
1015202530 3540 45
OVA lgG1 (ng ml−1 x 103)
IL-13 (pg ml−1)
Cell number (x105)
Cell number (x105)
IL-17 (pg ml−1)
Total lgE (ng ml−1)
Mean clinical score
Figure 1 | Nlrp102/2mice have a global defect in adaptive immune
13 (b) and serum antibodies (c) from wild-type (WT) and Nlrp102/2mice
sensitized intraperitoneally with OVA/alum and challenged intranasally with
culture IL-17 concentration (f) from wild-type and Nlrp102/2mice following
sensitization with MOG/CFA and pertussis toxin (n58–10 mice from one of
way ANOVA in d. g, Bronchoalveolar lavage from wild-type and Nlrp102/2
mice sensitized and challenged intranasally with OVA/LPS (n53–5 mice per
group). *P,0.0001; **P,0.001; ***P,0.023. All error bars show s.e.m.
Per cent of maximum
TNP lgG3 (OD450 nm)
TNP lgG1 (OD450 nm)
d e f
W>W W>K K>W K>K Naive
W>K K>W K>K Naive
Cell number (x105)
IL-13 (pg ml−1)
OVA lgG1 (ng ml−1)
Figure 2 | Nlrp102/2mice cannot mount T-cell-
dependent adaptive immune responses.
marrow chimaeras (donor.recipient; W, wild
type; K, Nlrp102/2) immunized and challenged as
in Fig. 1a–c (n53–7 mice per group from one of
three independent experiments). d, Serum TNP-
specific IgG1 in TNP-KLH-immunized wild-type
or Nlrp102/2mice. P,0.0022 by one-way
TNP-Ficoll-immunized mice (n53–5 mice per
group from one of two independent experiments,
KO, Nlrp102/2). f, CFSE dilution of labelled wild-
type OVA-specific OT-II T cells in the draining
after OVA/LPS immunization. One of three mice
per group is shown. *P,0.033, **P,0.0001. All
error bars show s.e.m.
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present (Fig. 3d), regardless of the presence or absence of NLRP10 in
the host (Supplementary Fig. 10), confirming that Nlrp102/2DCs
were incapable of reaching the LNs due to a cell-intrinsic defect. We
conclude that NLRP10 is essential for DC-mediated transport of
antigen to the LN from multiple peripheral sites following maturation
by a wide range of innate stimuli.
Given the similarity of our findings to those described for CCR7-
deficient mice, we postulated that Nlrp102/2DCs might have a defect
in CCR7 expression. However, Ccr7 messenger RNA (Supplementary
Fig. 11a) and CCR7 surface expression (Fig. 4a) in LPS-treated
Nlrp102/2BMDCs was equivalent to wild-type BMDCs. Consistent
a gradient of CCL19 and CCL21 as well as a CCR7-independent
chemokine, CXCL12 (SDF-1), and the signalling sphingolipid
S1P14,22,23in trans-well assays (Fig. 4b and Supplementary Fig. 11b,
c), indicating that CCR7 sensing and signalling was intact and that
general DC kinesis was not affected in Nlrp102/2mice. Despite the
apparently normal homing properties of Nlrp102/2DCs in vitro,
splenic DCs failed to relocalize to the T-cell zone in the spleen follow-
requires numerous integrated steps, including emigration from the
initial site of residence, the ability to home towards a chemokine
gradient, transmigration through the lymphatic endothelial layer
and the physical machinery to do so15,24–26. Yet, we found no defect
in the ability of Nlrp102/2DCs to traverse an endothelial monolayer,
we postulated that NLRP10 regulates the emigration of DCs from
inflamed tissue. To visualize DC movement in vivo, we used two-
photon laser scanning microscopy to follow co-injected fluorescently
the tissue using Imaris software revealed that wild-type DCs actively
moved away from the injection site and surveyed the surrounding
tissue, whereas the majority of Nlrp102/2DCs remained near the site
of injection despite their active extension of lamellipodia (Fig. 4c).
their ability to exit inflamed tissues, suggesting a defect in a molecular
of particular adhesion molecules including b1, b2 or b3 integrin
chains, DC-SIGN nor altered in vitro adhesion to ICAM-1, ICAM-2,
fibronectin or collagen I (data not shown). However, such surface
expression or isolated in vitro adhesion assays will not reveal defects
in chemokine receptor–integrin activation pathways. Therefore, to
identify novel molecules potentially involved in NLRP10-dependent
DC function, we used an unbiased gene array approach on Nlrp102/2
were differentially expressed (q value ,0.05 and absolute fold-change
.1.2) in Nlrp102/2DCs compared to wild-type DCs (Fig. 4d).
Restricting the analysis to genes that were more than two-fold up/
down regulated at baseline and after LPS stimulation revealed only
three differentially expressed genes: Il4ra, Mmp13 and Gdpd3 (Sup-
plementary Fig. 13). The first two genes identified were either not
differentially expressed in Nlrp102/2DCs at the protein level or do
We were most intrigued by the aberrant regulation of a molecule
Per cent of maximum
OVA (–) DCsOVA (+) DCs
0.0 μg0.5 μg5.0 μg50.0 μg
0.04 0.10 4.47
0.66 1.33 0.10 15.40
Figure 3 | Nlrp102/2dendritic cells do not take antigen to the draining
lymph node. a, Dilution of lymph node CFSE1OT-II T cells adoptively
transferred into wild-type hosts 3days after injection with wild-type or
Nlrp102/2OVA-peptide-pulsed (solid line) or -unpulsed (shaded histogram)
BMDCs. One of three mice per group is shown. b, FITC1CD11c1MHCIIhi
DCs in thedraining LN of wild-type, Ccr72/2or Nlrp102/2mice painted with
1% FITC. One representative experiment out of four. c, CD11b1CD11c1DCs
in the LN of wild-type or Nlrp102/2mice injected with indicated doses of
Nlrp102/2BMDCs were labelled with CFSE or CellTrace Violet and co-
injected into CD45.1 mice with LPS. Inguinal lymph nodes were analysed for
CD45.21CD11c1MHCII1BMDCs. One of two mice per group from one
experiment of seven.
LPS: − − + +
8 10 12 14
Upper well: − −
Lower well: − CCL19 CXCL12
PT − PT
Figure 4 | Nlrp102/2dendritic cells cannot emigrate from inflamed tissue
but remain responsive to chemokines. a, CCR7 surface expression on wild-
type, Ccr72/2or Nlrp102/2BMDCs stimulated with LPS. One representative
with pertussis toxin (PT) before use. Representative of three experiments. All
error bars show s.e.m. c, Cell-track displacement rate of BMDCs labelled with
wild type) or CFSE (Nlrp102/2) co-injected into the ears of wild-type hosts and
imaged 4h later by intravital two-photon laser scanning microscopy;
*P,0.0012. d, Heat map of log2-transformed gene expression values in
Nlrp102/2and wild-type BMDCs treated with and without LPS.
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with no known mammalian function—glycerophosphodiester phos-
phodiesterase domain containing 3 (GDPD3) (Supplementary Fig.
13c)—because its domain structure suggests that it has multiple trans-
membrane regions and structural homology to glycerophosphodiester
phosphodiesterase 1 (GDE1). Gdpd3 was more than 80-fold upregu-
lated in Nlrp102/2BMDCs, compared to wild-type DCs by real-time
PCR (Supplementary Fig. 13c). GDPD family members catalyse the
hydrolysis of glycerophosphoinositol, but in addition have functions
in cell morphology, motility and G protein signalling downstream of
phospholipid metabolism and the motility of mature DCs. Subsequent
work characterizing GDPD3 will potentially provide important clues
on how NLRP10 regulates a fundamental cellular process in DCs
during inflammation. Althoughno other NLR or TLR reported to date
affects adaptive immunity globally in this way, there is a growing body
of literature to indicate that multiple NLRs and related signalling
molecules are involved in controlling different aspects of DC func-
tion29,30. The finding that inhibition of a single NLR, NLRP10, can
paralyse mature DCs could have a profound impact on the approach
to treating misguided adaptive immune responses driving allergy and
alum or intranasal OVA/LPS. Mice were then challenged intranasally with OVA.
EAE was elicited using MOG peptide, CFA, heat-inactivated Mycobacterium tuber-
culosis and Bordetella pertussis toxin. For TNP immunizations, TNP-KLH or TNP-
flowcytometry at6h.Forantigen tracing,AlexaFluor 647-OVA and LPS wereco-
transfer labelled BMDCs were co-injected into the flank of wild-type mice, and
OVA-peptide-loaded BMDCs were injected subcutaneously, and inguinal LNs
collected 3days later to evaluate T-cell proliferation. For intravital microscopy,
labelled BMDCs were co-injected intradermally and imaged using an upright
two-photon laser scanning microscope, and quantified using Imaris software. For
gene expression analysis an Affymetrix Mouse Gene 1.0 ST Array was used.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 13 September 2011; accepted 2 March 2012.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We would like to thank R. Medzhitov and M. Albert for discussion
S.C.E. was supported by T32HL007974, K08AI085038 and Yale CTSA (UL1
(DRG 108-09), the Yale CTSA (UL1 RR024139 and 5KL2RR024138), the Yale SPORE
in Skin Cancer(1 P50 CA121974) andthe Dermatology Foundation. E.E. is supported
by Cancer Research Institute, the American Physicians for Medicine in Israel
Foundation, and the United States-Israel binational Foundation grant. A.M.H., D.G.G.
and in vivo imaging were supported by Yale Rheumatologic Disease Research Core
Center P30AR053495. F.S.S. was supported by R01AI087630 and an Edward
an Investigator of the Howard Hughes Medical Institute.
Author Contributions S.C.E. and A.W. wrote the manuscript, designed, performed and
mice, S.J. performed in vitro inflammasome activation, O.R.C. and J.H.-M. assisted with
trans-well assays and performed real-time PCR, L.A.Z. assisted with EAE experiments,
T.S. assisted withTNPimmunizations,A.R. assisted with intravenousLPS experiments,
E.E. provided technical assistance with DC isolations, C.A.T. performed
immunofluorescence experiments, H.M. and S.H.K. performed array analysis, D.G. and
A.M.H. performed intravital microscopy and quantification. R.A.F. assisted in
experimental design and interpretation. S.C.E. and R.A.F. directed the project.
Author Information The microarray data discussed in this publication have been
accession number GSE36009. Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to S.C.E. (email@example.com) or R.A.F.
2 6 A P R I L 2 0 1 2 | V O L 4 8 4 | N A T U R E | 5 1 3
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Materials. All reagents were purchased from Sigma except Imject alum (Pierce)
and incomplete Freund’s adjuvant (IFA) (Difco) unless indicated otherwise.
Antibody pairs for ELISA were purchased from R&D Systems (IL-1b), BD
Pharmingen (IL-5, IL-6, IL-17 and IFN-c) or from eBioscience (TNF-a). OVA-
specific IgG1 was measured by ELISA as previously described10and secondary
antibodies were purchased from BD Pharmingen. TNP-specific IgG3 and IgG1
was performed as above with TNP-OVA used for coating instead of OVA and
horseradish-peroxidase-conjugated secondary antibodies (Bethyl).
gene, containing the translation-initiation codon ATG, were replaced by a neo
cassette flanked by two loxP sites (Supplementary Fig. 1a). The Nlrp10-targeting
vector was electroporated into C57BL/6 embryonic stem (ES) cells (Bruce4).
Homologous recombinant ES cells were identified by Southern blot analysis and
were microinjected into BALB/c blastocysts. Chimaeric offspring were back-
crossed to C57BL/6 mice, and germline transmission was confirmed by PCR of
tail genomic DNA. Screening of Nlrp102/2mice with the primers 59-TAG
AGTGGATACCCAGCACACACG-39 and 59-CATCTCGTAAGTGGAACTTC
AGCG-39 amplifies a 700-base pairs product from the wild-type allele; primers
59-TAGAGTGGATACCCAGCACACACG-39 and 59-AACGAGATCAGCAGC
CTCTGTTC-39 amplify a 594-bp product from the targeted allele. RT–PCR ana-
confirmed the absence of Nlrp10 mRNA in Nlrp102/2mice. Primers used for
RT–PCR analysis were as follows: Nlrp10, 59-GGAGCTTGTAGACTACCTCA-
39, 59-AAAGTCTCCACATCGACAGG-39; Hprt, 59-GTTGGATACAGGCCA
used as all wild-type controls. All naive mice were C57BL/6 unless otherwise
indicated in the figure. RAG1-deficient (B6.129S7-Rag1tm1Mom/J) and OT-II
(B6.Cg-Tg(TcraTcrb)425Cbn/J) and CCR7-deficient mice were purchased from
backgroundfromNCI.Allprotocols used inthis studywere approved bythe Yale
Institutional Animal Care and Use Committee.
intraperitoneally on day 0 with50mg of ovalbumin (Grade V; Sigma) adsorbed on
of Imject alum. Mice were challenged intranasally with 25mg of ovalbumin in PBS
on days 21, 22 and 23 and killed for analysis on day 25. For sensitization by
inhalation, 100mg Ova with an additional 0.05mg LPS in 50ml PBS was given
intranasally on days 0, 1 and 2 as previously described10. Mice were challenged
for analysis. Naive mice (wild type) were not sensitized intraperitoneally but
received an intranasal OVA challenge.
Bronchoalveolarlavage analysis.Micewere primedandchallengedasindicated.
by cannulation of the trachea and lavage of the airway lumen with 3ml PBS. Red
and cytospin slides were prepared by haematoxylin and eosin staining with Diff-
Quick (Dade Behring).
Ex vivo lymphocyte restimulation. Draining lymph nodes (inguinal or
mediastinal) were removed and single-cells suspensions were generated.
cells (see below) in the presence or absence of 200mgml21of OVA for 48h.
Syngeneic T-cell-depleted splenocytes were used as antigen-presenting cells and
were prepared by complement lysis using antibodies to CD4 (GK1.5), CD8
(TIB105) and Thy1 (Y19), followed by treatment with mitomycin C.
Experimental autoimmune encephalomyelitis model. EAE was elicited
and scored as previously described33. Briefly, on day 0 mice received bilateral
subcutaneousflank injectionof50mg of MOG(MEVGWYRSPFSRVVHLYRNGK)
Mycobacterium tuberculosis (Difco Labs). A dose of 200ng of Bordetella pertussis
toxin (PT; LIST Biological Labs) was injected intraperitoneally on days 0 and 2.
Micewere monitoreddaily andscoredasfollows: 1,flaccidtail; 2,partialunilateral
complete bilateralhindlimb paralysis; and5,moribund. After disease resolution(a
majority of the paralysis resolved), micewere challenged with 50mg MOG peptide
collected, pooled and 53106cells were stimulated with 10mgml21MOG peptide
48h later, supernatant was collected for cytokine analysis by ELISA. Naive mice
(wild type) did not receive MOG peptide.
TNP model. 300mg TNP-KLH (29:1) and 100mg TNP-Ficoll (90:1) (both from
14. Mice were killed on day 30 and anti-TNP antibodies were analysed by ELISA
Intravenous LPS model. Mice were given 25mg of LPS by retro-orbital injection.
Mice were bled at 90min and 6h and spleens were removed at 6h. Spleens were
treated with collagenase D (Roche) at 37uC for 45min, red blood cells were lysed
and samples were stained and analysed by flow cytometry. Blood was allowed to
clot at room temperature, serum isolated and analysed by ELISA.
AF647, Molecular Probes) were injected with 10mg LPS subcutaneously in the
flank bilaterally of wild-type and Nlrp102/2mice. 18h post injection inguinal
lymph nodes were removed, digested with collagenase D for 1h at 37uC and
stained for flow cytometry analysis.
DC transfer experiments. BMDCs were labelled with either 2mM CFSE or 2mM
CellTrace Violet at 37uC for 5min. 33106labelled BMDCs were mixed in equal
numbers and injected into each flank of CD45.1 C57BL/6 mice. After 1day,
draining inguinal lymph nodes were removed, digested with collagenase D for
1h at 37uC before antibody staining and analysis by flow cytometry. For in vivo
OVA peptide (ISQAVHAAHAEINEAGR) or nothing, extensively washed and
13106were injected subcutaneously in the flank. Inguinal LNs were harvested
3days later to evaluate T-cell proliferation.
Bone marrow chimaeras. Bone marrow was flushed from femurs, red cells were
engraftment mice were maintained on antibiotics and 2weeks after transplant,
chimaerism was assessed using congenic CD45 markers. All mice used in the
experiments demonstrated at least 92% haematopoietic engraftment.
T-cell proliferation studies. From the spleen and LNs of OT-II transgenic mice
on a RAG1-deficient background, CD41cells were prepared by positive selection
using CD4 Miltenyi beads (L3T4) as per the manufacturer’s instructions. CD4-
andthen washedonce in FBS andtwicewith PBS.Forin vivo studies 33106cells
were transferred into mice by retro-orbital injection. After 1day, mice were
challenged by subcutaneous injection of 0.25mg OVA protein with either 100ml
CFA or 5mg LPS in each flank and 3days later, inguinal LNs were removed and
analysed by flow cytometry. Forin vitro studies 13105OT-II T cells were stimu-
lated with 13104wild-type or Nlrp102/2BMDCs loaded with 100mg OVA or
BSA in a 96-well plate for 3days.
In vitro T-cell activation and skewing. For in vitro T-cell skewing, polyclonal
wild-type or Nlrp102/2T cells were stimulated with plate-bound anti-CD3
(10mgml21) and anti-CD28 (2mgml21). T-cell skewing was accomplished for
TH1 cultures with IL-12 (3.5ngml21), IL-2 (0.1ngml21) and anti-IL-4 (10mg
ml21); for TH2 cultures with IL-4 (20ngml21), IL-2 (0.1ngml21) and anti-
TGF-b (0.5ngml21) and anti-IFN-c (10mgml21; XMG1.2) and anti-IL-4
(10mgml21;11B11). After 5days, cells were collected and restimulated with
plate-bound anti-CD3 (10mgml21) for 8h and supernatant was analysed by
Flow cytometry protocols and antibodies. CD11b (M1/70), CD45.1 (A20),
Valpha2 TCR (B20.1), CD4 (RM4-5), CD19 (6D5), CD3e (145-2C11), CD86
(GL1) flow cytometry antibodies were from BD. CD11c (N418), CD45.2 (104),
B220 (RA3-6B2) were from Biolegend. IAIE(M5/114.15.2) was from eBioscience.
Intracellular cytokine staining was done using the BD Cytofix/Cytoperm kit and
according to manufacturer’s protocol. IL-17A (TC11-18H10) and IFN-c
ester (CFSE) was from Invitrogen. CCR7 staining was performed on BMDCs after
from BD Pharmingen at 1:100 at 37uC for 40min.
In vitro DC and macrophage stimulation. The generation of thioglycollate-
elicited peritoneal and bone-marrow-derived macrophages and bone-marrow-
derived dendritic cells has been described previously31,34. For Supplementary
Fig. 2a, cells were primed by stimulating with 50ngml21LPS from Escherichia
coli serotype 0111:B4 (InvivoGen) for 16–18h before stimulation ATP or alum.
For ATP-stimulated cells, the medium was changed at 20min. and all stimulants
were replaced. All other TLR ligands were used at the concentration indicated in
the figure legend. Type A CpG (InvivoGen), heat-killed M. tuberculosis (Difco),
PolyI:C (Amersham) and Imiquimod (R837; InvivoGen). To assess antigen pro-
or 4uC. Cells were then washed, stained with antibodies and analysed by flow
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Relative gene expression analysis. RNA from cells was isolated using TRIzol
(Invitrogen) and RNA was subjected to reverse transcriptase with Superscript II
(Invitrogen) with oligo(dT) primer in accordance with the manufacturer’s pro-
tocol. cDNA was quantified using commercially available primer/probe sets
(Applied Biosystems) by real-time PCR and analysed with the DCt(change in
amplification reactions during each PCR quantification. Results are presented as
levels relative to Hprt. The following primer/probes were used: Ccr7
Mm1301785_m1, Gdpd3 Mm00470322_m1, Il4ra Mm00439634_m1, Mmp13
Mm00439491_m1, Hprt Mm00446968_m1.
was administered intravenously by retro-orbital injection. After 4h spleens were
harvested and treated with collagenase D and DNase I at 37uC for 45min, red
blood cells were lysed and samples were stained and analysed by flow cytometry.
Intranasal latex bead delivery. Yellow-green fluorescent 0.5-mm latex particles
(Polysciences) were diluted 1/25 in PBS and 50ml was administered intranasally
with 1mg LPS35. 18h later draining mediastinal lymph nodes were harvested and
digested with collagenase D before staining and analysis by flow cytometry. In
parallel, lung was minced into small pieces, incubated with a cocktail of
150Uml21collagenase type I (Worthington Biochemical Corp) and 20mgml21
DNase I in media supplemented with 10% FBS for 30min at 37uC. After passing
through a 70-mm mesh, the single-cell suspension was stained and analysed by
FITC painting. The ventral portion of each ear was painted with 20ml of 1%
fluorescein isothiocyanate (FITC) in carrier solution (1:1 v/v acetone:dibutyl
phthalate)14,23. After 18h draining lymph nodes were collected, digested with
collagenase D, stained and analysed by flow cytometry. In parallel, ears were split
into dorsal and ventral halves and floated dermal side down on dispase
(2mgml21, Roche) for 30min at 37uC. Ears were then minced and incubated
in the presence of collagenase D (5mgml21), DNase I (0.1mgml21) and
hyaluronidase (2mgml21MP Biomedicals) for 30min at 37uC. After passing
through a 70-mm mesh the resulting single-cell suspension was stained and ana-
lysed by flow cytometry.
Trans-well assay. BMDCs stimulated with LPS (1mgml21) overnight were
with 0.1% fatty acid-free BSA (Sigma). Chemokines were suspended in RPMI
supplemented with 0.1% fatty acid free BSA at 100ngml21. 600ml of each was
added into a 24-well non-tissue-culture-treated plate containing a 6.5mm
Transwell insert with a 5.0-mm pore size (Corning), and allowed to equilibrate
for 30–45min in tissue culture incubator before the addition of 100ml of cells at a
concentration of 13107ml to the upper chamber. After 3h migrated cells were
harvested from the lower chamber and counted using a haemocytometer. For
toxin (100ngml21). For controls BMDCs were in media containing respective
chemokines before adding to the upper chamber.
Intravenous LPS and spleen immunofluorescence staining. Wild-type and
and fixed with 4% PFA solution for 4h at 4uC and then treated with increasing
prepared using a cryostat (Leica) at a working temperature of 219uC. Frozen
sections were blocked in 5% fetal bovine serum for 30min at room temperature.
Slides were incubated at 4uC with primary antibodies to CD3 (clone17A2, BD
Pharmingen) and CD11c (biotinylated, clone HL3, BD Pharmingen) followed by
incubation with AlexaFluor 647-labelled chicken anti-rat IgG (Molecular Probes)
and phycoerythrin-labelled streptavidin Biolegend) for 2h at room temperature.
Slides were then dried and mounted using ProLong Antifade mounting medium
(Invitrogen). Images were acquired on a PerkinElmer Ultraview Spinning disk
confocal microscope and images were processed using Volocity software
Intravital microscopy. BMDCs stimulated with LPS (1mgml21) overnight were
harvested, washed twice in ice-cold PBS before staining with either CFSE or
CMTMR (Molecular Probes). After washing twice with fetal calf serum and twice
with PBS, 0.53105–13105cells in 10ml of PBS and 2.5mg LPS were injected
intradermallyinto eachear (withorwithout anequalmixture ofunlabelled DCs).
Imaging of dendritic cell motility in ear skin of mice was performed using an
upright two-photon laser scanning microscope. For image acquisition, an
Olympus BX61WI fluorescence microscope with a 320, 0.95 numerical aperture
(NA) water immersion Olympus objective and dedicated single-beam LaVision
TriM laser scanning microscope (LaVision Biotec) was controlled by Imspector
software. The microscope was outfitted with a Chameleon Vision II Ti:Sapphire
laser (Coherent) with pulse pre-compensation. Emission wavelengths of 390–
480nm (blue, for second harmonic generation emissions), 500–550nm (green,
three photomultiplier tubes (Hamamatsu). Mice were anaesthetized with an
intraperitoneal injection of ketamine (100mgkg21) and xylazine (10mgkg21)
mouse was placed on a custom-designed stereotaxic restraint platform with ear
plane of deep anaesthesia was maintained using a mixture of isofluorane gas and
photon laser was tuned to a wavelength of 850nm. Volocity software
(Improvision) was used to create QuickTime formatted movies of image
sequences. All movies are displayed as two-dimensional maximum intensity pro-
jections of the time-resolved image stacks. The displacement rate of cells in the
algorithm in Imaris software (Bitplane/Perkin Elmer). All cell tracks were indi-
vidually examined to confirm that they reported the behaviour of a single cell.
Only viable cells with track origins at least 10mm from an injection site were
included in quantitative analysis.
Statistical analysis. We performed statistical analysis using a one-way ANOVA
with a Bonferroni multiple comparison post test unless otherwise indicated. We
considered P,0.05 to be statistically significant. Error bars represent s.e.m. of
to generate these error bars.
Affymetrix array. DNA microarray analysis was performed on two independent
BMDCs treated overnight with LPS (1mgml21). RNA was isolated with a Qiagen
RNeasy MiniKit and was hybridized to Mouse Gene 1.0 ST Array (Affymetrix) at
the Yale Keck Microarray Facility. The microarray analysis was carried out with
package. Differential gene expression was defined by two criteria: (1) an absolute
fold-change$1.2 of knockout samples relative to wild-type samples and (2) a
statistically significant change in expression as determined by LIMMA with a
Benjamani–Hochberg false discovery rate cutoff q,0.05. The microarray data
discussed in this publication have been deposited in NCBI’s Gene Expression
Omnibus36and are accessible through GEO Series accession number GSE36009
Western blot analysis. Electrophoresis of proteins was performed with the
NuPAGE system (Invitrogen) in accordance with the manufacturer’s protocol.
In brief, BMDCs were suspended in lysis buffer (Cell Signaling) containing a
protease inhibitor cocktail (Roche). Lysates from an equal number of BMDCs
were separated on a NuPAGE gel and transferred to a PVDF (poly(vinylidene
difluoride)) membrane by electroblotting. To detect MMP13, rabbit polyclonal
anti-MMP13 antibody (ab39012) from Abcam was used.
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