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Cutting Edge
Cutting Edge
Cutting Edge: Alum Adjuvant Stimulates Inflammatory
Dendritic Cells through Activation of the NALP3
Inflammasome
Mirjam Kool,* Virginie Pe´trilli,
†
Thibaut De Smedt,
‡
Aline Rolaz,
‡
Hamida Hammad,*
§
Menno van Nimwegen,* Ingrid M. Bergen,* Rosa Castillo,
†
Bart N. Lambrecht,
1,2
*
§
and
Ju¨rg Tschopp
1,2†
Adjuvants are vaccine additives that stimulate the im-
mune system without having any specific antigenic ef-
fect of itself. In this study we show that alum adjuvant
induces the release of IL-1

from macrophages and den-
dritic cells and that this is abrogated in cells lacking var-
ious NALP3 inflammasome components. The NALP3
inflammasome is also required in vivo for the innate im-
mune response to OVA in alum. The early production
of IL-1

and the influx of inflammatory cells into the
peritoneal cavity is strongly reduced in NALP3-defi-
cient mice. The activation of adaptive cellular immunity
to OVA-alum is initiated by monocytic dendritic cell
precursors that induce the expansion of Ag-specific T
cells in a NALP3-dependent way. We propose that, in
addition to TLR stimulators, agonists of the NALP3 in-
flammasome should also be considered as vaccine
adjuvants. The Journal of Immunology, 2008, 181:
3755–3759.
Activationof dendritic cells (DCs)
3
is a crucial mecha-
nism by which vaccine adjuvants stimulate protec-
tive adaptive immunity to cancer or microbial in-
fection. Agonists to several TLRs are in clinical development
as adjuvants (1). The most promising TLR ligand is an un-
methylated CpG dinucleotide containing DNA that stimu-
lates TLR9 in a pathway requiring the adaptor MyD88, lead-
ing to the activation of DCs (2, 3). Nonetheless, there is a
debate as to whether TLR stimulation is essential for the Ab-
enhancing effect of vaccine adjuvants like alum. Although
initial studies showed that MyD88- deficient mice are defec-
tive in mounting Th1 and B cell responses (4), this notion
has been recently challenged (5). It has also become clear
that an alternative pathogen detection system exists, in ad-
dition to TLRs, that relies on a family of intracellular recep-
tors called nucleotide-binding oligomerization domain-like
receptors (NLRs) (6– 8). In addition to pathogen-associated
molecular patterns, some of these NLRs also sense the pres-
ence of endogenous danger signals. The most studied NLR
member is NALP3, which, together with Cardinal and apo-
ptosis-associated speck-like protein containing a caspase re-
cruitment domain (ASC), forms a caspase-1 activating com-
plex, the so-called inflammasome (9). This complex is
activated by multiple agonists including a bacterially derived
muramyl dipeptide or endogenous ATP and uric acid, lead-
ing to the processing and release of IL-1

(10, 11). We there-
fore considered the possibility that NLRs, in addition to
TLRs, are implicated in the effect of some adjuvants.
Materials and Methods
Mice
Wild-type (WT), ASC
⫺/⫺
, IPAF
⫺/⫺
(gifts of V. Dixit, Genetech; IPAF is IL-
1

-converting enzyme protease activating factor), MyD88
⫺/⫺
, and
NALP3
⫺/⫺
mice (12) (all mice in C57BL/6 background) were bred at the Uni-
versity of Lausanne, Lausanne, Switzerland and at the University Hospital, Gh-
ent, Belgium. Male and female mice were used between the ages of 8 and 14 wk.
All in vivo experiments were approved by the animal ethics committee of Ghent
University.
Isolation and activation of mouse primary cells
Peritoneal macrophages were isolated 3 days after an i.p. injection with 10%
thioglycollate and primed in vitro with LPS as previously described (13). Uric
acid, uricase, and cytochalasin D were bought from Sigma-Aldrich and crystals
were generated as previously described (12).
Ags and adjuvant
OVA was purchased from Worthington Biochemical. In Ag-tracking experi-
ments, OVA-Alexa Fluor 647 (Molecular Probes) was used. Imject Alum
(Pierce Biochemicals; hereafter simply called alum) is a mixture of aluminum
hydroxide and magnesium hydroxide and was mixed at a 1:20 ratio with a
*Department of Pulmonary Medicine, Erasmus University Medical Centre, Rotterdam,
The Netherlands;
†
Department of Biochemistry, University of Lausanne, Epalinges, Swit-
zerland;
‡
TopoTarget Switzerland, Lausanne, Switzerland; and
§
Laboratory of Immuno-
regulation, University Hospital Ghent, Ghent, Belgium
Received for publication May 22, 2008. Accepted for publication July 14, 2008.
The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1
B.N.L. and J.T. shared supervision over the work.
2
Address correspondence and reprint requests to Dr. Ju¨rg Tschopp, Department of Bio-
chemistry, Chemin des Boveresses 155, 1066 Epalinges, Switzerland. E-mail address:
jurg.tschopp@unil.ch or Dr. Bart Lambrecht, Laboratory of Immunoregulation, Univer-
sity Hospital Ghent, Ghent, Belgium. E-mail address: bart.lambrecht@ugent.be
3
Abbreviations used in this paper: DC, dendritic cell; ASC, apoptosis-associated speck-
like protein containing a caspase recruitment domain; IPAF, IL-1

-converting enzyme
protease activating factor; MLN, mediastinal lymph node; MSU, monosodium urate;
NLR, nucleotide-binding oligomerization domain-like receptor; ROS, reactive oxygen
species; WT, wild type.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
solution of OVA Ag in saline, followed by stirring for at least 1 h. For im-
munization, 500
l of alum (crystalline magnesium hydroxide and amor-
phous aluminum hydroxide) suspension (1 mg) containing 10
gofOVA
(OVA-alum) was injected i.p. or, alternatively, 10
g of OVA in 500
l
saline was injected.
Detection of cellular influx in vivo after OVA-alum injection
In WT and NALP3
⫺/⫺
mice, 2, 6, and 24 h after injection the peritoneal cavity
was washed with 3 ml of PBS containing EDTA (peritoneal lavage) and the
mediastinal lymph nodes were harvested and used for flow cytometry as previ-
ously described (14).
In experiments where the role of IL-1

was examined, mice were treated
with anti-IL-1

mAb (200
g/ms i.p.; BioLegend) 5 min before OVA-alum
injection and sacrificed 24 h later. Detection of Ag-specific T cell responses was
done as previously described (14).
Statistical analysis
For all experiments, the difference between groups was calculated using the
Mann-Whitney Utest for unpaired data (GraphPad Prism version 4.0). Dif-
ferences were considered significant when p⬍0.05.
Results
Alum adjuvant activates the NALP3 inflammasome
We first exposed peritoneal macrophages obtained from WT,
NALP3
⫺/⫺
, and ASC
⫺/⫺
mice to various amounts of alum (see
Materials and Methods for composition). Even at low doses, a
substantial amount of processed IL-1

was detected in the cel-
lular supernatant, which compared well with the quantities of
this cytokine observed with crystalline monosodium urate
(MSU), one of the most potent activators of the NALP3 inflam-
masome (12) (Fig. 1A). IL-1

was almost completely absent in
supernatants of macrophages from NALP3- and ASC-deficient
mice, indicating a crucial involvement of NALP3 in caspase-1-
mediated, pro-IL-1

processing.
Activation of caspase-1 occurs not only upon assembly of the
NALP3 but also upon that of the IPAF inflammasome (15).
However, a contribution of this complex in alum-triggered
caspase-1 activation is unlikely, because alum-induced IL-1

secretion from macrophages derived from IPAF-deficient mice
was still observed (data not shown). Also, alum adjuvant con-
tinued to activate caspase-1 in the absence of MyD88, exclud-
ing a major role of TLRs in alum-mediated inflammasome ac-
tivation (Ref. 16 and data not shown).
Mechanisms of NALP3 inflammasome activation by alum
and uric acid crystals are similar
We next investigated the mechanisms by which alum acti-
vates the NALP3 inflammasome. Recent studies have shown
that NALP3 inflammasome activation is dependent on K
⫹
efflux (17, 18) and the generation of reactive oxygen species
(ROS) (17). K
⫹
efflux can be blocked by the addition of high
concentrations of K
⫹
(130 mM) to the extracellular me-
dium. The blockade of K
⫹
efflux resulted in the inhibition of
alum-induced caspase-1 activation and IL-1

secretion (Fig.
1B). Activation of the inflammasome was also reduced when
the ROS inhibitor (2R,4R)-4-aminopyrrolidine-2, 4-dicar-
boxylate (APDC) was added (Fig. 1B).
We could not exclude the possibility that activation of the
inflammasome by alum occurred indirectly through one of
the two endogenous danger signals known to activate
NALP3, namely ATP and uric acid (12, 19). ATP, which is
thought to be released from necrotic cells, potently activates
the inflammasome via binding to the P2X7 receptor (19),
whereas uric acid can form crystals that activate the inflam-
masome by an as yet poorly understood mechanism. We
have recently reported that extracellular ATP triggers in-
flammation by activating DCs (20) and that in vivo injection
of alum into the peritoneal cavity leads to uric acid (MSU)
release (14). It was therefore possible that MSU or ATP or
both, were released from alum-treated cells, leading to the
subsequent activation of the NALP3 inflammasome. How-
ever, we obtained no evidence for this hypothesis. Uricase,
which catalyzes the conversion of uric acid to allantoin (14),
had only a minor effect on IL-1

processing in vitro (Fig.
1B). Alum also still activated the inflammasome in macro-
phages of P2X7-deficient mice (data not shown).
Despite the fact that both ATP and MSU activate the
NALP3 inflammasome, the initial pathways leading to inflam-
masome activation are not identical. MSU is a particulate mat-
ter and requires endocytosis (12). In fact, endocytosis seems to
be required for particles in general, including asbestos and silica,
to activate the inflammasome (13). It was therefore expected
that alum also required endocytosis. The effect of alum, but not
ATP (data not shown), was abrogated by the actin-destabilizing
drug cytochalasin D (Fig. 1B). Taken together, NALP3 inflam-
masome activation in vitro by alum requires endocytosis of the
particle, K
⫹
efflux, and generation of ROS, but not release of
ATP or uric acid.
Recruitment of inflammatory cells to the site of alum injection is
decreased in the absence of NALP3
Intraperitoneal injection of alum is frequently used in mice to
induce cellular Th2 immunity and humoral immunity to ad-
sorbed protein Ags (21). To assess the role of the NALP3 in-
flammasome in alum adjuvanticity in vivo, we examined the
FIGURE 1. Role of NALP3 and ASC in alum-mediated IL-1

production.
A, Peritoneal macrophages from WT, NALP3
⫺/⫺
, and ASC
⫺/⫺
mice were
primed overnight with ultrapure LPS and then stimulated with different
amount of Imject Alum (Pierce Biochemicals). MSU (150
g/ml) was used as
a control. Production of mature IL-1

was measured by Western blotting in
supernatants (SN) and cell extracts (XT). B, Alum-induced IL-

secretion is
dependent on phagocytosis, K
⫹
efflux, and ROS. Peritoneal macrophages from
WZ mice were primed as described in Aand treated with alum in the presence
and absence of high concentrations of extracellular K
⫹
(130 mM), the ROS
inhibitor (2R,4R)-4-aminopyrrolidine-2, 4-dicarboxylate (APDC) (100
M),
cytochalasin D (2
M), and uricase (0.1 U/ml).
3756 CUTTING EDGE: INFLAMMASOME AND ALUM ADJUVANT
innate inflammatory response caused by alum upon i.p. injec-
tion of OVA adsorbed to alum. Twenty-four hours after injec-
tion there was a massive recruitment of neutrophils (Ly6G
⫹
,
F4/80
⫺
, and CD11b
⫹
cells), eosinophils (F4/80
dim
, Ly6G
⫹
,
and CD11b
⫹
cells), and inflammatory mononuclear cells
(CD11b
⫹
, F4/80
⫹
, and Ly6C
⫹
cells) into the peritoneal cavity
that was not seen when OVA was injected without alum (Fig.
2A). This innate response was severely reduced in NALP3-de-
ficient animals but nevertheless still present. Compared with in-
jection of saline or OVA alone, injection of OVA-alum induced
a rapid increase in the peritoneal cavity of the innate cytokines
IL-6 and IL-1

(Fig. 2B). Strikingly, in NALP3
⫺/⫺
mice the
increase in IL-1

was completely reduced to the level seen in
mice receiving saline or OVA, whereas the level of IL-6 was re-
duced but not abolished (Fig. 2B).
Activation of DCs is IL-1

and NALP3 dependent
DCs are seen as nature’s adjuvant and have a unique potential to
induce adaptive immunity. We have recently reported that the
induction of T cell adaptive immunity in vivo following the in-
jection of alum depends on uptake and transport of Ag from the
peritoneal cavity to the mediastinal LN by both resident
CD11c
⫹
DCs and inflammatory CD11b
⫹
Ly6C
⫹
monocytes
that become CD11c
⫹
DCs upon migration to the draining me-
diastinal lymph node (MLN) (14). To address the contribution
of inflammasome activation to this process, we measured the
uptake of fluorescent OVA in resident Ly6C
⫺
CD11c
⫹
DCs
following the injection of OVA or OVA-alum. Adsorption of
FIGURE 2. Innate inflammatory response induced by alum is NALP3 de-
pendent. NALP3
⫺/⫺
or WT mice were injected i.p. with OVA or OVA-alum.
A, Twenty-four hours after injection, the peritoneal lavage was taken and the
number of neutrophils (CD11b
⫹
Ly6C
⫹
Ly6G
high
F4/80
⫺
), eosinophils
(CD11b
⫹
Ly6C
int
Ly6G
int
F4/80
int
), and monocytes (CD11b
⫹
Ly6C
high
Ly6G
⫺
F4/80
int
) (were “int” is “intermediate”) was determined by flow cytometry. B,
Two hours after OVA or OVA-alum injection the peritoneal lavage was taken
and cytokine levels were determined in the supernatant by ELISA. Data shown
are mean ⫾SEM; ⴱ,p⬍0.05; ⴱⴱ,p⬍0.01; and ⴱⴱⴱ,p⬍0.001; n⫽4–6
mice per group.
FIGURE 3. Activation of DCs by alum is NALP3 dependent. A,
NALP3
⫺/⫺
or WT mice were injected i.p. with OVA-Alexa Fluor 647 or OVA-
AF647-alum. Twenty-four hours after injection the number of OVA
⫹
resident
Ly6C
⫺
CD11c
⫹
DCs was determined in the peritoneal lavage by flow cytom-
etry. B, Twenty-four hours after injection the number of CD11c
⫹
OVA
⫹
monocytes in the MLN was measured. C, Mice were treated with anti-IL-1

mAb before OVA-alum injection. The influx of CD11c
⫹
OVA
⫹
monocytes
into the MLN was measured 24 h after injection. D, CD86 and MHC II ex-
pressions were measured on the OVA
⫹
and OVA
⫺
monocytes in the MLN
24 h after the injection of OVA-alum in WT or NALP3
⫺/⫺
mice. Data shown
are mean ⫾SEM; ⴱ,p⬍0.05; n⫽4–6 mice per group.
3757The Journal of Immunology
OVA-Alexa Fluor 647 in alum led to a strong increase in DCs
carrying antigenic cargo in WT mice but not in NALP3
⫺/⫺
mice (Fig. 3A). We also followed the appearance of OVA-laden
Ly6C
⫹
inflammatory monocytes in the MLN (Fig. 3B). In WT
mice there was a time-dependent increase in the number of in-
flammatory monocytes in the MLN at 24 h, and a majority of
these carried OVA. In contrast, in NALP3
⫺/⫺
mice this in-
crease was severely diminished. This deficiency was most likely
explained by a defective generation of bioactive IL-1

in
NALP3
⫺/⫺
mice, as the alum-induced increase in OVA-laden
CD11c
⫹
monocytes in the MLN was severely reduced when
IL-1

was neutralized in vivo, implying an important func-
tional role for this cytokine in promoting monocyte migra-
tion and differentiation (Fig. 3C). Provision of costimula-
tion is a necessary requisite for the induction of adaptive
immunity. Injection of OVA-alum induced the up-regula-
tion of the costimulatory molecule CD86 on inflammatory
OVA
⫹
CD11c
⫹
Ly6C
⫹
monocytes in the MLN of WT mice,
but to a much lesser extent in NALP3
⫺/⫺
mice (Fig. 3D). A
similar effect was seen for MHC class II (Fig. 3D).
The reduced migration of OVA-laden Ly6C
⫹
monocytic
cells with DC characteristics to the mediastinal nodes of
NALP3
⫺/⫺
mice prompted us to study the activation of naive
OVA-specific CD4
⫹
T cells. For this, mice first received a co-
hort of CFSE-labeled T cells obtained from OT-II TCR Tg
mice followed by injection of OVA or OVA-alum, and accu-
mulation of OVA-specific T cells was measured in the lymph
nodes. In WT mice OVA-alum caused a marked increase in the
number of Ag-reactive T cells in the mediastinal LN at days 4
and 10 postimmunization (Fig. 4A) compared with injection of
OVA alone. In NALP3
⫺/⫺
mice, the number of T cells induced
by OVA-alum was severely reduced but still higher than that
obtained by the injection of OVA alone. These changes in the
accumulation of T cells were mainly due to altered degrees of T
cell division, as measured using CFSE dilution (Fig. 4B).
We finally measured the effect of NALP3 deficiency on the
type of immunoglobulins induced by OVA-alum cells (Fig.
4C). Mice receiving OVA alone have generally low Ab titers to
OVA (data not shown). In WT mice, OVA-alum induced the
production of IgE, IgG1, and somewhat lower levels of IgG2c.
In NALP3
⫺/⫺
mice there was a significant decrease in OVA-
specific IgE Abs and an increase in OVA-specific IgG2c Abs.
Strikingly, IgG1 levels were unaffected.
Discussion
In 1926, the adjuvant effect of aluminum compounds was first
described by Glenny et al. (22). Despite this early discovery, to
this day little is known about the mode of action of alum adju-
vant. Adjuvants containing a pathogen-associated molecular
patterns act as ligands for the TLRs. For example, TLR9 was
shown to be essential for the adjuvant effect of CpG oligode-
oxynucleotides (3). However, incubation of DCs with alum ad-
juvant fails to activate the DCs as indicated by the increased
expression of MHC class II and costimulatory molecules (23);
yet, several authors described an increased production of IL-1

secretion (24, 25). Mice lacking TLR signaling components
still mount a robust Ab responses if alum is given as an adjuvant
(5). Thus, alum adjuvants, in contrast to bacteria-derived adju-
vants, do not activate TLRs.
Entirely in agreement with two recent studies by Eisenbarth
and Li and colleagues (16, 26), our data show that alum adju-
vant triggers activation of the NALP3 inflammasome. The po-
tential of alum to trigger the NALP3 inflammasome leads to
early activation of the innate cytokine IL-1

and an innate cel-
lular immune response at the site of injection. Activation of the
NALP3 inflammasome and the subsequent release of IL-1

leads to the recruitment of immature monocytes and DCs. Pro-
duction of IL-1

also leads to the activation of inflammatory
monocytes and their migration to the lymph nodes draining the
peritoneum. This situation resembles the migration of skin
Langerhans cells to the lymph nodes, which also requires IL-1

for functional migration and maturation to occur (27). In
OVA-alum-immunized mice, OVA-laden DCs and inflamma-
tory monocytes take their cargo to the MLN and up-regulate
the expression of critical costimulatory molecules like CD86
with the subsequent expansion of Ag-specific T cells (14). All of
these steps are highly impaired in NALP3-deficient mice, indi-
cating that activation of the NALP3 inflammasome contributes
FIGURE 4. Induction of adaptive cellular immunity by alum is NALP3 de-
pendent. A, NALP3
⫺/⫺
or WT mice were injected with CFSE-labeled OT-2
OVA-TCR Tg cells 1 day before the i.p. injection of OVA or OVA-alum. Four
and 10 days after the injection the MLN was analyzed for T cell proliferation
with flow cytometry (n⫽4 mice; experiment was performed two times). B,
Representative CFSE profiles of adoptively transferred T cells on day 10 after
injection. Data are shown as mean ⫾SEM; ⴱ,p⬍0.05; n⫽5–11 mice per
group. C, On day 0, NALP3
⫺/⫺
or WT mice were injected i.p. with OVA-alum
and boosted i.p. on day 7 with OVA. At day 14, serum samples were taken and
OVA-specific IgE, IgG1, and IgG2c levels were determined by ELISA. Data are
shown as mean ⫾SEM; ⴱ,p⬍0.05; n⫽5–11 mice per group.
3758 CUTTING EDGE: INFLAMMASOME AND ALUM ADJUVANT
not only to the immediate inflammatory innate response at the
site of injection of alum but also to the generation of adaptive
cellular immunity.
Levels of serum Igs are also influenced in the NALP3-defi-
cient mice, as a decreased level of OVA-IgE and increased levels
of OVA-IgG2c are found. This pattern of Ig synthesis is most
likely the result of a shift toward IFN-
␥
-secreting Th1-like
helper cells necessary for Ig class switching to IgG2c and equally
able to suppress IgE synthesis. Interestingly, we found that
NALP3 does not influence IgG1 levels, whereas decreased levels
of this subclass were found in the Eisenbarth report (16). This
striking-difference may be due to a different experimental
setup, as in their study Ab generation was determined after an
intranasal challenge with OVA (16) whereas we boosted mice
by injecting OVA i.p. The data are most consistent with the
notion that NALP3-dependent IL-1

production by DCs
skews the adaptive cellular immune response toward a Th2 type
of response, as recently proposed (25).
Strikingly, alum adjuvant activates the NALP3 inflamma-
some directly in vitro, in agreement with two reports showing
IL-1

secretion from alum-treated DCs (24, 25). We have re-
cently found that alum also induces high-level production of
uric acid in vivo and that this increased level of uric acid was
required for the infiltration of inflammatory cells (14). Al-
though how this is done and which cells generate or release uric
acid upon alum administration are open questions, it suggests
that the increased level of uric acid leads to an amplification of
NALP3 inflammasome activation and, thus, IL-1

secretion.
Interestingly, uric acid was identified not only as one of the
most potent danger signals released from dying cells, but also as
an excellent adjuvant (28). Thus, we propose NALP3 as the
newest member of a list of innate receptors that could be ex-
ploited for the design of new adjuvants.
Acknowledgments
We thank V. Dixit (Genentech, San Francisco, CA) for the ASC
⫺/⫺
and
Ipaf
⫺/⫺
mice and S. Akira (Osaka University, Osaka, Japan) for the
MyD88
⫺/⫺
mice.
Disclosures
The authors have no financial conflict of interest.
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3759The Journal of Immunology
