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Differential Expression of NLRP3 among
Chantal Mattmann and Jürg Tschopp
Schroder, Isabel Ferrero, Philippe Menu, Aubry Tardivel,
Greta Guarda, Manuel Zenger, Amir S. Yazdi, Kate
2011; 186:2529-2534; Prepublished online 21
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The Journal of Immunology
Differential Expression of NLRP3 among Hematopoietic Cells
Greta Guarda,*,1Manuel Zenger,*,†,1Amir S. Yazdi,* Kate Schroder,*,‡
Isabel Ferrero,xPhilippe Menu,* Aubry Tardivel,* Chantal Mattmann,*
and Ju ¨rg Tschopp*
Although the importance of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in health and
disease is well appreciated, a precise characterization of NLRP3 expression is yet undetermined. To this purpose, we generated
a knock-in mouse in which the Nlrp3 coding sequence was substituted for the GFP (enhanced GFP [egfp]) gene. In this way, the
expression of eGFP is driven by the endogenous regulatory elements of the Nlrp3 gene. In this study, we show that eGFP
expression indeed mirrors that of NLRP3. Interestingly, splenic neutrophils, macrophages, and, in particular, monocytes and
conventional dendritic cells showed robust eGFP fluorescence, whereas lymphoid subsets, eosinophils, and plasmacytoid
dendritic cells showed negligible eGFP levels. NLRP3 expression was highly inducible in macrophages, both by MyD88-
and Trif-dependent pathways. In vivo, when mice were challenged with diverse inflammatory stimuli, differences in both
the number of eGFP-expressing cells and fluorescence intensity were observed in the draining lymph node. Thus, NLRP3
levels at the site of adaptive response initiation are controlled by recruitment of NLRP3-expressing cells and by NLRP3
induction. The Journal of Immunology, 2011, 186: 2529–2534.
pattern-recognition receptors consists of the intracellular NOD-
like receptors (NLRs) that survey the cytoplasm for the presence
of invaders or damage. Distinct NLRs are key components for the
assembly of inflammasomes, multiprotein platforms serving the
maturation of inflammatory mediators (1).
Awell-studied example of an inflammasome is the NLR family,
pyrin domain containing 3 (NLRP3, also known as NALP3 or
cryopyrin) inflammasome. Upon activation, NLRP3 oligomerizes
and, through the adaptor protein apoptosis-associated speck-like
protein containing a caspase recruitment domain, recruits caspase-1
to the complex (1, 2). Once caspases are brought into close
proximity within the inflammasome platform, autocatalytic
processing leads to their activation, which in turn mediates the
cleavage of the proinflammatory cytokines IL-1b and IL-18 into
their bioactive forms. The signal required for NLRP3 inflamma-
nnate immune cells recognize pathogens or danger using a
limited number of germ-line–encoded receptors called
pattern-recognition receptors. One important family of
some formation can be of a heterogeneous nature, including par-
ticulate stimuli, pathogens, pore-forming toxins, or extracellular
IL-1b is an important proinflammatory cytokine that induces
the expression of other cytokines and chemokines, crucial for the
onset of the immune response, to resolve the infection and pro-
mote the healing process. For this reason, IL-1b is implicated in
the control of several pathogens (3–7). However, when its pro-
duction is not properly controlled, this cytokine can cause or worsen
several inflammatory disorders, such as cryopyrin-associated pe-
riodic syndromes, which are caused by activating mutations in
the Nlrp3 gene (8).
Published data documenting the expression profile of NLRP3
are not exhaustive and are partially conflicting (9–12). It is widely
accepted that NLRP3 expression can be induced in monocytes and
macrophages upon exposure to inflammatory stimuli (9, 10, 13,
14). There are isolated reports of NLRP3 expression in gran-
ulocytes and in B and T lymphocytes (10, 11). Because some
reports showing NLRP3 expression in particular tissues or cell
types were not substantiated by later studies, the pattern of NLRP3
expression is not yet well defined. Furthermore, little is known on
the expression of this protein in vivo, under normal conditions or
upon stimulation. Thus, despite the importance of the NLRP3 in-
flammasome in the inflammatory response and in several dis-
eases, the complete expression profile of NLRP3 is yet undocu-
Previously, NLRP3-deficient mice were generated by in-frame
insertion of the enhanced GFP (egfp) gene at the Nlrp3 locus
(15). As such, mice are simultaneously knocked out for the Nlrp3
gene, and the transcription of the fluorescent reporter is controlled
by the endogenous Nlrp3 regulatory elements. These mice, to
which we refer to as eGFP knock-in (KI), represent a very useful
tool to characterize the expression of NLRP3, a protein otherwise
difficult to study given the scarcity of specific Abs and their un-
suitability for FACS analysis. In the present study, we examined
the expression of NLRP3 in vitro and in vivo, under steady-state
and inflammatory conditions, taking advantage of the NLRP3 re-
*Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland;
†Brain Mind Institute, E´cole Polytechnique Fe ´de ´rale de Lausanne, 1015 Lausanne,
Switzerland;‡Monash Institute of Medical Research, Monash University, Melbourne,
Victoria 3800, Australia; and
Branch, University of Lausanne, 1066 Epalinges, Switzerland
xLudwig Institute for Cancer Research, Lausanne
1G.G. and M.Z. contributed equally to this work.
Received for publication August 10, 2010. Accepted for publication December 14,
This work was supported in part by the Swiss National Science Foundation and by
the Institute for Arthritis Research.
Address correspondence and reprint requests to Dr. Ju ¨rg Tschopp, Department of
Biochemistry, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges,
Switzerland. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this article: BMDM, bone marrow-derived macrophage; cDC,
conventional dendritic cell; DC, dendritic cell; eGFP, enhanced GFP; Het, heterozy-
gous; HPRT, hypoxanthine phosphoribosyltransferase; KI, knock-in; LN, lymph
node; MDP, muramyl dipeptide; NLR, NOD-like receptor; NLRP3, NOD-like re-
ceptor family, pyrin domain containing 3; pDC, plasmacytoid dendritic cell; PGN,
peptidoglycan; poly(I:C), polyinosinic-polycytidylic acid; WT, wild-type.
by guest on June 13, 2013
Materials and Methods
Six- to 12-wk-old (unless otherwise indicated) C57BL/6, eGFP KI (15),
Trif2/2(16), and MyD882/2(17) mice were housed at the animal facility
of the University of Lausanne. All animal procedures were conducted in
compliance with Swiss federal legislation for animal experimentation.
Bone marrow-derived macrophages (BMDMs) were generated as pre-
viously described (18). Liver was harvested from PBS-perfused animals.
Blood was collected from the tail vein. Spleen cell suspensions were
prepared by grinding the organs through mesh filters. CD11b+splenocytes
were isolated by MACS (Miltenyi Biotec), by using a two-step labeling
procedure. First, splenocytes were incubated with allophycocyanin-labeled
anti-CD11b (M1/70) Ab (eBioscience). Next, anti-allophycocyanin micro-
beads were used to magnetically label and select CD11b+cells (Miltenyi
Biotec). For spleen dendritic cell (DC) analysis, cell suspensions were ob-
tained after collagenase digestion as described in detail elsewhere (19).
Murine tissue panel was purchased from Clontech. Total RNA extraction
from macrophages was done using an RNeasy kit (Qiagen) with on-column
DNase digestion according to the manufacturer’s instructions. RNA con-
centration was measured by a NanoDrop 1000 spectrophotometer (Thermo
Scientific). cDNA synthesis was performed using SuperScript II reverse
transcriptase on a Mastercycler gradient (Eppendorf). Gene expression was
quantified using a LightCycler 480 (Roche) with SYBR Green (Roche).
Expression was normalized relative to the control gene hypoxanthine phos-
phoribosyltransferase (Hprt) using the Roche LightCycler advanced rela-
tive quantification software. Primer sequences are available upon request.
In vitro stimulation experiments
The medium and the culture conditions used are described elsewhere (18).
BMDM stimulations were done using 20 ng/ml ultrapure LPS, 2 mg/ml
polyinosinic-polycytidylic acid (poly(I:C)), 15 mg/ml peptidoglycan (PGN;
all from Invivogen), 1 mg/ml mouse TNF (Alexis Biochemicals), 15 mg/ml
muramyl dipeptide (MDP; Bachem), or 2.5 mg/ml CpG 1826 (59-CCATG-
ACGTTCCTGACGTT-39) (Microsynth). Inflammasome activation was
for 150 min.
In vivo stimulations
Mice were injected s.c. in the footpad with PBS, 500 ng mouse TNF
(Alexis), 50 mg CpG 1826 (Microsynth), or 150 mg MDP (Bachem) in a
volume of 30 ml. Sixteen hours later, mice were sacrificed and popliteal
lymph node (LN) cells were analyzed by FACS.
Rabbit polyclonal Ab to b-actin was purchased from Abcam, and the
monoclonal anti-NLRP3 (NALP3) Ab was from AdipoGen (Cryo-2).
Abs and flow cytometry
Cells were preincubated with anti-mouse CD16/32 (2.4G2) culture super-
natant to block FcRs, then washed and surface-stained using combinations
(L3T4), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD19 (1D3), anti-
F4/80 (BM8), anti–PDCA-1 (eBio927), and anti-NK1.1 (PK136) were
purchased from eBioscience; and anti-B220 (RA3-6B2), anti–Ly-6C (AL-
21), and anti–Ly-6G (1A8) were purchased from BD Biosciences. Allo-
phycocyanin-Cy7–labeled streptavidin was purchased from eBioscience.
Allofthe Abs werelabeledwithanappropriatecombination offluorophore.
Propidium iodide (Sigma-Aldrich) was used to exclude dead cells from in
vitro cultures. Samples were analyzed on either FACSCalibur or FACS-
using FlowJo software (Tree Star, Ashland, OR).
For in vitro experiments, statistical analyses were calculated with an un-
paired Student t test (GraphPad Prism version 5.0; GraphPad Software).
For in vivo experiments, differences between stimulated groups and ref-
erence group were calculated using one-way ANOVAwith a Dunnett post-
test (GraphPad Prism version 5.0).
eGFP fluorescence correlates with NLRP3 expression
To reliably use eGFP fluorescence as a measure of NLRP3 ex-
pression in the eGFP KI cells (Supplemental Fig. 1), we first
confirmed that the level of the reporter mirrors that of NLRP3
protein expression. Because it is known that NLRP3 expression is
strongly induced upon LPS treatment (9), we stimulated BMDMs
from eGFP KI, heterozygous (Het), and wild-type (WT) mice with
LPS. Over time, the levels of Nlrp3 mRNA expression in WT and
Het BMDMs were coherent with those of egfp mRNA in KI and
Het BMDMs, showing a peak at 6 h and decreasing overnight
(Fig. 1A). Note, however, that the decrease in egfp mRNA had
a slightly delayed kinetics than the one of Nlrp3. A clear gene
dose effect was observed for Nlrp3 and egfp gene transcripts in
Het BMDMs compared with WT or KI BMDMs, respectively.
We next examined whether the expression of eGFP would be
consistent with NLRP3 also at the protein level. When eGFP
fluorescence was determined by FACS, we observed higher basal
pression at both mRNA and protein levels. WT,
eGFP Het, and KI BMDMs were treated 1.5, 3, and
6 h, and overnight with LPS or left untreated. A, egfp
and Nlrp3 mRNA expression, relative to Hprt, was
determined by quantitative PCR (qPCR). Means and
SDs of technical triplicates are depicted. B, eGFP
fluorescence as determined by FACS. Values shown
on the right side of the graph refer to the respective
geometric mean fluorescence intensities (MFIs). C,
NLRP3 protein levels (upper) compared with b-actin
loading control (lower) were assessed by Western
blot. One representative experiment of three is shown
eGFP is a reporter for NLRP3 ex-
2530 DIFFERENTIAL EXPRESSION OF NLRP3 AMONG HEMATOPOIETIC CELLS
by guest on June 13, 2013
levels of eGFP fluorescence in eGFP KI and Het BMDMs as
compared with the WT BMDM controls, with Het BMDMs dis-
playing an intermediate level between WT and KI BMDMs (Fig.
1B). The strongest increase in fluorescence was observed when
stimulating with LPS overnight. As expected, WT BMDMs
showed no significant change in fluorescence following LPS
treatment. We then determined NLRP3 protein expression by
Western blot. A basal level of NLRP3 protein could be observed in
unstimulated WT and Het cells, but not in eGFP KI control cells
(Fig. 1C). This was consistent with the basal eGFP fluorescence
detected in unstimulated Het and eGFP KI cells. The strongest
expression of NLRP3 was observed after a minimum of 6 h
stimulation. Het BMDMs showed again an intermediate level of
NLRP3 expression, thus confirming a clear gene dose effect of the
Nlrp3 and the egfp alleles. In agreement with observations at the
mRNA level, eGFP protein also showed slightly delayed kinetics
as compared with NLRP3.
Taken together, these results indicate that eGFP fluorescence can
be used as a reliable reporter for NLRP3 expression.
Basal NLRP3 levels in BMDMs suffice for inflammasome
Next, we sought to understand whether the induction of NLRP3
upon LPS treatment was dependent on MyD88 or Trif activation.
For this purpose, we stimulated WT, MyD882/2, or Trif2/2
BMDMs with LPS (Fig. 2A). Although at reduced potency, cells
deficient for either MyD88 or Trif were still able to upregulate
NLRP3, suggesting that both TLR 4 signaling pathways triggered
by LPS can induce NLRP3 expression. We therefore reasoned that
also the Trif-dependent TLR3 agonist poly(I:C) as the MyD88-
dependent TLR9 agonist CpG DNA would upregulate NLRP3
expression. Indeed, CpG and poly(I:C) both caused an increase in
NLRP3 levels (Fig. 2A). We also stimulated BMDMs with the
inflammatory cytokine TNF, with the NOD2 ligand MDP, and
with the TLR2 agonist PGN. All of these treatments increased the
levels of NLRP3 protein, as shown by Western blot, as mirrored
by a raise in the eGFP fluorescence values in KI cells (Fig. 2B,
2C). Taken together, proinflammatory stimuli of diverse nature
can induce NLRP3 expression in BMDMs, and in the case of TLR
agonists both MyD88- and Trif-dependent signaling pathways can
be alternatively used.
Because we observed detectable, albeit weak, NLRP3 expres-
sion prior to LPS stimulation, we hypothesized that the NLRP3
inflammasome would be competent for activation without prior
stimulation with one of the above-mentioned proinflammatory
molecules. To this purpose, we treated cells with two potent NLRP3
activators, nigericin and alum, in the presence or absence of 5 h
prior LPS priming. As shown in Fig. 2D, although the extent of
caspase-1 activation was strongly enhanced by prior priming with
LPS, caspase-1 cleavage was detectable also in LPS unprimed
cells. Not surprisingly, pro–IL-1b induction and mature IL-1b
secretion were observed exclusively when cells were LPS treated,
as pro–IL-1b synthesis is known to be dependent on the priming
NLRP3 is primarily expressed by myeloid cells such as
conventional DCs and monocytes
To determine NLRP3-expressing organs, Nlrp3 mRNA expression
was tested in various murine tissues. Nlrp3 was most strongly
transcribed in secondary lymphoid organs, namely the spleen and
the LNs, and in organs densely populated by immune cells, such
as the lung and the liver (Fig. 3A). A few reports also suggested
that IL-1 secretion by keratinocytes is NLRP3-dependent (20–22).
However, we could detect neither a significant shift in eGFP
fluorescence by the eGFP KI keratinocytes nor NLRP3 expression
in WT keratinocytes by Western blot (Supplemental Fig. 2). Taken
together, our data suggest that NLRP3 is strongly expressed in
immune cells. This prompted us to more closely examine eGFP
expression within cells of the hematopoietic system.
First, we examined expression of the NLRP3 reporter in spleno-
cytes; eGFP expression was almost exclusively found in cells
expressing the myeloid marker CD11b (Fig. 3B). This indicates
that within the spleen, the myeloid compartment predominates
over the lymphoid compartment for NLRP3 expression. Similar
results were obtained when bone marrow, blood, and liver were
analyzed (Supplemental Fig. 3). In s.c. LNs and thymus the eGFP
reporter was expressed by a small percentage of the few CD11b+
cells, suggesting that NLRP3 is poorly expressed in these lym-
phoid organs under resting conditions (Supplemental Fig. 3). To
detectable caspase-1 cleavage. A, BMDMs of WT,
MyD882/2, or Trif2/2origin were stimulated for 7 h
with LPS, CpG, or poly(I:C). NLRP3 expression was
analyzed by Western blot. B and C, WT and eGFP KI
BMDMs were treated overnight with CpG, TNF,
MDP, or PGN. NLRP3 expression was assessed by
Western blot (B), while eGFP mean fluorescence in-
tensity (MFI) was measured by FACS (C). Means and
SDs of three individual experimental points are
depicted in the graph. *p # 0.05, **p # 0.01, ***p #
0.001, unpaired Student t test, two-tailed. D, WT
BMDMs were either left unprimed or treated 5 h with
LPS. Then cells were stimulated for 2 h 30 min with
nigericin (nig.) or alum. Caspase-1 and IL-1b cleav-
age and secretion were assessed by Western blot on
cell supernatants (SN). The levels of pro–caspase-1,
pro–IL-1b, and NLRP3 were measured in cell ex-
tracts (XT). One representative experiment of at least
two is shown (A–D).
Basal NLRP3 levels mediate weak but
The Journal of Immunology2531
by guest on June 13, 2013
corroborate the eGFP reporter pattern with endogenous NLRP3
expression, we separated CD11b+from CD11b2cells of WT
spleen and assessed NLRP3 expression by Western blot (Fig. 3C).
This supported a tight correlation between eGFP fluorescence and
NLRP3 protein expression also ex vivo.
To more precisely define NLRP3 expression in cell types of
the hematopoietic system, we analyzed the eGFP fluorescence in
specific splenic subpopulations (Fig. 3D). T cells, B cells, and NK
cells of eGFP KI mice showed no or a minimal increase of eGFP
fluorescence compared with WT control mice, further corrobo-
rating that NLRP3 is barely expressed by lymphoid cells under
resting conditions. Plasmacytoid DCs (pDCs) also showed negli-
gible eGFP fluorescence. In contrast, splenic conventional DCs
(cDCs), Ly6Chighmonocytes, and macrophages all displayed a
very clear shift between WT and eGFP KI. Interestingly, in the
granulocyte compartment, eosinophils did virtually not express
eGFP whereas neutrophils did. These results thus indicate that
NLRP3 is expressed, among hematopoietic cells and under resting
conditions, by myeloid cells and most strongly by cDCs, mono-
cytes, and, to a lesser extent, by macrophages and neutrophils.
Inflammatory cells with increased NLRP3 expression are
recruited to inflamed LNs
Finally, to address the question of whether NLRP3 expression is
also increased by proinflammatory stimuli in vivo, we s.c. injected
MDP, TNF, CpG, or saline only in the footpad of eGFP KI and
WT mice. Popliteal LNs were harvested 16 h postinjection and
eGFP-expressing cells were examined. We identified by FACS the
fluorescent cells in the eGFP KI LNs as cDCs, monocytes, and
neutrophils (Fig. 4). Although MDP, TNF, and CpG all shared the
ability to increase LN cellularity (Supplemental Fig. 4A), eGFP-
expressing cDCs, monocytes, and neutrophils were most strongly
recruited to the LN draining the site of TNF and CpG injection.
Additionally, at a single cell level, monocytes and neutrophils
strongly augmented their eGFP emission in response to these two
stimuli, suggesting Nlrp3 promoter activation. In contrast, the
eGFP fluorescence in cDCs recovered from untreated or treated
mice was similar, suggesting that cDCs do not further upregulate
PCR in a panel of different murine tissues (mean and SD). B, Splenocytes were harvested from WT, eGFP Het, and KI mice, and eGFP mean fluorescence
intensity (MFI) was assessed by FACS in CD11blow, CD11bint, or CD11bhighpopulations. C, Splenocytes from WT animals were separated into CD11b2
and CD11b+fractions. NLRP3 expression in these was assessed by Western blot. As a control, total splenocytes were extracted from eGFP KI and WT
mice. D, Splenic T cells (CD3+), B cells (CD19+), NK cells (NK1.1+, CD32), cDCs (CD11chi, PDCA12, F4/80int/2, CD11bint), pDCs (CD11cint, PDCA1+,
F4/80int/2), monocytes (CD11bhi, Ly6Chi, Ly6G2, SSClow), macrophages (F4/80hi, FSCint–hi, CD11bint), neutrophils (CD11bhi, Ly6Ghi), and eosinophils
(CD11bhi, SSChi, Ly6Cint, Ly6Glow–int) from WT, eGFP Het, and KI mice were assessed for their eGFP fluorescence by flow cytometry. The numbers in
each histogram correspond to the MFI. One representative experiment of two (C) or three is shown (B, D).
NLRP3 is highly expressed by cDCs and monocytes. A, Nlrp3 mRNA expression relative to Hprt mRNA was determined by quantitative
WT and eGFP KI mice were s.c. injected in the footpad either with MDP,
TNF, CpG, saline only, or left untreated. Sixteen hours postinjection,
popliteal LNs were harvested. cDC, monocyte, and neutrophil recruitment
to the popliteal LNs and the eGFP mean fluorescence intensity (MFI) of
these populations were measured by FACS. Absolute cDC, monocyte, or
neutrophil cell numbers recruited to the draining LNs of eGFP KI are
shown on the left part of each panel, while their eGFP MFI is depicted on
the right part. Data represent means and SEM of three individual experi-
mental points. *p # 0.05, **p # 0.01, ***p # 0.001, one-way ANOVA
with Dunnett post-test.
Recruitment of eGFP-expressing cells to the inflamed LN.
2532DIFFERENTIAL EXPRESSION OF NLRP3 AMONG HEMATOPOIETIC CELLS
by guest on June 13, 2013
NLRP3 expression in an inflammatory environment. Importantly,
for all three cell types, the intensity of eGFP was higher compared
with the emission by WT cells, which remained constant upon the
different inflammatory conditions, indicating the specificity of the
fluorescence detected (Supplemental Fig. 4B).
In conclusion, these data indicate that augmented expression of
NLRP3 in draining LNs is achieved by two mechanisms; that is,
NLRP3 reporter-expressing cells are recruited to the LN, and also
individual cells upregulate expression of the NLRP3 reporter.
The NLRP3 inflammasome is a pivotal host platform for the
sensing of endogenous and exogenous danger and for the sub-
sequent orchestration of inflammatory responses. Despite this cen-
tral role, its expression is still poorly characterized. We thus gen-
erated a reporter mouse by substituting the coding sequence for
Nlrp3 with the egfp coding sequence. By using BMDMs, we first
demonstrated that eGFP regulation indeed mirrored that of NLRP3.
This was further confirmed in splenocytes, where both eGFP of KI
mice and NLRP3 of WT mice were coherently present almost
exclusively in myeloid CD11b+cells, and thus we validated the use
of our reporter mouse for interrogating the expression of NLRP3
in specific cellular subsets.
As already described, NLRP3 expression is highly inducible by
various TLRs (13). TLRs can signal via two pathways that depend
on two different adaptor proteins, MyD88 and Trif. Consistent
with what has been suggested by a previous report (13), our results
indicate that each pathway can individually upregulate NLRP3.
In line with that, TLR4, which triggers both pathways in parallel,
can even more strongly augment NLRP3 expression (9, 13,
14). Inducers of NLRP3 expression were, however, not limited to
TLRs. TNF and the NOD2 agonist MDP were equally effective,
suggesting that NLRP3 is inducible by any pathway that activates
the proinflammatory transcription factor NF-kB (9, 13, 14). In
agreement with published data, we detected basal levels of
NLRP3 in BMDMs also without LPS priming (13). However, in
contrast to what was observed for ATP (13), this NLRP3 basal
expression supported weak, but detectable, caspase-1 activation
upon exposure to nigericin or alum. This difference might be ex-
plained by the longer duration of alum and nigericin stimulation
compared with the usual very short stimulations used for ATP.
Longer time courses might allow positive feedback loops to take
place and amplify caspase-1 cleavage over time.
Nlrp3 mRNA is predominantly expressed in lymphoid organs
and organs highly populated by immune cells (10). Consistent
with previous publications and with the widespread use of bone
marrow-derived DCs for studying the NLRP3 inflammasome, we
found that splenic cDCs show very strong NLRP3 expression (3,
10, 14). In contrast to cDCs, pDCs presented negligible NLRP3
reporter levels. This compartmentalization of NLRP3 expression
among DCs might favor the local secretion of IL-1b by activated
tissue-resident cDCs, while avoiding more systemic production of
this cytokine by pDCs, which are mainly circulating cells. Splenic
monocytes also showed very high eGFP expression, consistent
with the common use of monocytes as a model for NLRP3-ex-
pressing cells (9, 15), whereas macrophages presented modest
reporter expression. This observation might suggest that mono-
cytes recently recruited to the site of inflammation could activate
the inflammasome more efficiently than do fully differentiated
macrophages, which may need an additional stimulation provided,
for example, by TLRs, to re-express high NLRP3 levels. Eosi-
nophils did not express NLRP3 reporter, while in agreement with
previous observations, neutrophils showed a clear expression (10,
11). Our results also indicate that NLRP3 reporter is barely ex-
pressed by splenic lymphoid B, T, and NK cells, an observation
that diverges with some previous publications (10, 11), but which
may be due to differences in expression profiles between mice and
humans or differences in T cell activation status.
Primary murine keritoncytes appear not to express NLRP3; we
could not detect NLRP3 by Western blot in WT or eGFP fluo-
rescence in KI cells. Importantly, previous literature showing basal
NLRP3 protein expression was based on studies with human
keratinocytes. It is therefore possible that expression of NLRP3
diverges in keratinocytes from humans and mice, or that NLRP3
expression requires to be induced in these cells.
Finally, we compared the in vivo effects of different inflam-
matory stimuli on NLRP3 expression in the draining LNs. Where-
as the dose of MDP used was a poor stimulator, TNF and CpG
were potent inducers of eGFP expression among cells of the pop-
liteal LNs. These two stimuli not only recruited higher numbers
of cDCs, monocytes, and neutrophils to the draining LN, but also
efficiently upregulated NLRP3 reporter expression in neutrophils
unchanged by challenge, suggesting that this cell type already
expresses NLRP3 at a maximal level. Given the importance of the
NLRP3 concentration in the efficacy of inflammasome activation
(13), it seems likely that NLRP3 levels are tightly regulated to
avoid spontaneous activation leading to chronic inflammation.
Mounting evidence supports a crucial role for the NLRP3 in-
flammasome in infectious disease control and in the priming of
T cell responses, as exemplified by the discovery that the vaccine
adjuvant alum activates the inflammasome and the function of the
NLRP3 inflammasome in generating antitumoral immunity (3–7,
23). Thus, a detailed knowledge of the regulation of NLRP3 ex-
pression is crucial to better understand immune system function
and regulation. We anticipate that the NLRP3-eGFP KI mouse we
have characterized in this study will enable such future studies.
We thank Rosa Castillo and Danny Labes for technical support.
The authors have no financial conflicts of interest.
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