Toll-like receptor 4 stimulation with the detoxified
ligand monophosphoryl lipid A improves Alzheimer’s
Jean-Philippe Michauda, Maxime Halléb, Antoine Lamprona, Peter Thériaulta, Paul Préfontainea, Mohammed Filalia,
Pascale Tribout-Joverb, Anne-Marie Lanteigneb, Rachel Jodoinb, Christopher Cluffc, Vincent Brichardd, Rémi Palmantierd,
Anthony Pilorgetb, Daniel Larocqueb,1, and Serge Rivesta,1
aNeuroscience Laboratory, Department of Molecular Medicine, Centre Hospitalier Universitaire de Québec Research Center, Laval University, Québec City, QC,
Canada G1V 4G2;bGlaxoSmithKline Vaccines, Laval, QC, Canada H7V 3S8;cGlaxoSmithKline Vaccines, Hamilton, MT 59840; anddGlaxoSmithKline Vaccines,
B-1330 Rixensart, Belgium
Edited by Shizuo Akira, Osaka University, Osaka, Japan, and approved December 19, 2012 (received for review September 1, 2012)
Alzheimer’s disease (AD) is the most common cause of dementia
worldwide. The pathogenesis of this neurodegenerative disease,
currently without curative treatment, is associated with the accu-
mulation of amyloid β (Aβ) in brain parenchyma and cerebral vas-
culature. AD patients are unable to clear this toxic peptide, leading
to Aβ accumulation in their brains and, presumably, the pathology
associated with this devastating disease. Compounds that stimulate
the immune system to clear Aβ may therefore have great therapeu-
tic potential in AD patients. Monophosphoryl lipid A (MPL) is an
LPS-derived Toll-like receptor 4 agonist that exhibits unique immu-
nomodulatory properties at doses that are nonpyrogenic. We show
here that repeated systemic injections of MPL, but not LPS, signif-
icantly improved AD-related pathology in APPswe/PS1 mice. MPL
treatment led to a significant reduction in Aβ load in the brain of
these mice, as well as enhanced cognitive function. MPL induced
a potent phagocytic response by microglia while triggering a mod-
erate inflammatory reaction. Our data suggest that the Toll-like re-
ceptor 4 agonist MPL may be a treatment for AD.
innate immunity|microglial cells|monocytes|phagocytosis|
characterized by the accumulation of amyloid beta (Aβ) and
neurofibrillary tangles in the brain parenchyma (1). Inflammation,
which occurs in parallel with the progression of the disease, is
is an area of active investigation. Whereas chronic activation of
microglial cells by Aβ can trigger the exaggerated release of cyto-
kines and neurotoxic mediators that could be detrimental to neu-
rons, microglia can also clear Aβ via increased phagocytosis and
proteolytic degradation, which may be neuroprotective (2).
Toll-like receptors (TLRs) on the surface of microglial cells have
been shown to bind Aβ, which triggers downstream intracellular
signaling cascades (3, 4). Microglia deficient in TLR2, TLR4, or
the coreceptor CD14 are not activated by Aβ and do not exhibit
a phagocytic response (5). Transgenic AD mice lacking TLR4 have
stimulation of microglial cells with TLR2-, TLR4-, or TLR9-specific
agonists accelerates Aβ clearance both in vitro and in vivo (3, 6, 7).
moiety derived from Salmonella minnesota R595 LPS (8). This
TLR4 ligand is at least 100-fold less pyrogenic than LPS yet main-
tains many of the immunomodulatory properties of LPS (9). Im-
portantly, MPL is safe in humans and has been administered to
millions of patients as a component of several vaccine formulations
such as the Cervarix vaccine (10). We investigated herein the
chronicuseofthenonpyrogenicTLR4 agonist MPLand compared
it with a strong TLR4 ligand (LPS) in a mouse model of AD.
Although the therapeutic potential of innate immune activa-
tion for AD is being evaluated in preclinical models, this concept
lzheimer’s disease (AD) is a neurodegenerative pathology
has not been tested in humans. We propose that the age-related
defects in immune cell function (11) commonly found in aging
diseases such as AD (12) can be reconciled with a prophagocytic
phenotype, yet mildly proinflammatory, which may lead to an
improved clearance of Aβ.
Our data demonstrate that chronic, systemic administration of
MPL ameliorates AD-like pathology by decreasing the cerebral
Aβ load through the stimulation of the phagocytic capacity of
innate immune cells.
MPL Drives a Distinct TLR4 Stimulation from LPS. MPL is derived
R595 by three main chemical modifications: (i) elimination of the
core oligosaccharide, (ii) hydrolysis of the 1-phosphate from the
the3-position ofthedisaccharide (Fig.1 AandB).Theabsence of
the 1-phosphate on the MPL molecule was suggested to weaken
(13). This presumably induces a structural change in the TLR4
receptor complex that alters the recruitment of the adaptor pro-
teins to the intracellular domain (13). Such a structural change
may account for the distinct signaling properties of MPL, which
predominantly activates the TLR4-TRAM (TRIF-related adap-
tor molecule)-TRIF (TIR-domain-containing adaptor protein
inducing IFN-β) pathway over the more proinflammatory TLR4-
MAL (MyD88-adaptor-like protein)-MyD88 (myeloid differen-
tiation primary-response protein 88) signaling pathway (14). This
differential use of intracellular adaptor proteins may be key to
explaining the distinct effects observed after exposure of cells to
MPL or LPS.
To characterize the ligand–receptor interaction of MPL with
the TLR4 receptor complex we used the HEK293 cell line
transfected with TLR4, MD2, and CD14 genes, as well as an
NF-κB and AP-1 reporter system. At the highest concentration
of MPL tested (20 μg/mL), the activation of NF-κB and AP-1 was
at a level comparable to a 200-fold lower concentration of LPS
(0.1 μg/mL) (Fig. 1C). A neutralizing antibody directed against
TLR4 inhibited the response to MPL. Incubating TLR2-trans-
fected HEK293 cells with up to 2.5 μg/mL of MPL did not induce
any activation of NF-κB and AP-1 (Fig. 1D), indicating that the
Author contributions: J.-P.M., A.L., A.P., D.L., and S.R. designed research; J.-P.M., M.H., A.L.,
P.T., P.P., M.F., P.T.-J., A.-M.L., and R.J. performed research; J.-P.M., M.H., A.L., P.P., and D.L.
analyzed data; and J.-P.M., M.H., A.L., C.C., V.B., R.P., A.P., D.L., and S.R. wrote the paper.
Conflict of interest statement: M.H., P.T.-J., A.-M.L., R.J., C.C., V.B., R.P., A.P., and D.L. are
employees of GlaxoSmithKline Vaccines. This research was supported in part by
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 29, 2013
| vol. 110
| no. 5
activation was mediated only by TLR4 (because not more than 1
μg/mL MPL was used to treat cells in the following experiments).
In accordance with the results observed in HEK293 cells, the
degradation kinetics of the NF-κB inhibitor, IκBα, seemed both
delayed and of a lower magnitude in MPL-treated microglia,
even though the difference between MPL and LPS did not reach
statistical significance (Fig. 1E).
In addition to NF-κB activation, MAPK cascades transduce
signals from TLR activation, with effects on gene transcription
and posttranscriptional modifications of cytoplasmic targets (15).
We assessed the phosphorylation kinetics of three MAPKs (ERK,
JNK, and p38) after stimulation of microglia with LPS and MPL.
LPS induced strong phosphorylation of both ERK and JNK in
microglia at 30 and 60 min poststimulation, whereas MPL had
little effect (Fig. 1 F and G). LPS and MPL induced a similar level
of p38 phosphorylation in microglial cells (Fig. 1H). This finding
confirms a previous report (16) that both of these TLR4 ligands
activate the p38 pathway to the same degree.
MPL Induces a Low Inflammatory Response by Microglia in Vitro.
Microglial cells are highly dynamic cells that can undergo rapid
actin cytoskeleton remodeling to migrate or phagocytose foreign
material. In vitro microglia activated by LPS exhibit a profound al-
teration in cell morphology, changing from a nonactivated amoe-
boid shape (Fig. 2A) to an elongated and multipolar morphology
(Fig. 2B). MPL also stimulated cytoskeletal remodeling (Fig. 2C),
but to a lower extent than LPS. Similar differences (MPL < LPS)
were observed for cell migration (Fig. 2E and Fig. S1). To evaluate
whether these morphological/motility changes correlated with in-
nate immune activation, we compared the expression of cellular
markers of microglia activation after treatment with either MPL
or LPS. In contrast to LPS, MPL treatment did not induce the ex-
pression of cyclooxygenase-2 (COX-2) (Fig. 2F) or formation of
nitrite (Fig. 2H), two classic markers of LPS-induced microglial
activation. Whereas MPL did not trigger the production of cyto-
kine-inducible NOS (iNOS) in microglia (Fig. 2I), treatment with
either MPL or LPS led to similar decreases in Arginase 1 (Arg1)
protein expression (Fig. 2J), a sensitive marker of alternative
these two enzymes are generally inversely regulated (17). This
reflects the multifaceted effects of the distinct TLR4 stimulation
by MPL. Microglia treated with MPL induced both TNF-α mRNA
(Fig.2K) andprotein(Fig.2G) expression,althoughatlowerlevels
compared with LPS. Both MPL and LPS induced similar levels of
CCL2 mRNA expression (Fig. 2L). MPL and LPS induced equiv-
alent levels of TLR2 gene expression after 2 h of incubation (Fig.
S2). These results demonstrate that MPL induces a low proin-
flammatory and transitory innate immune response in microglia
while still inducing characteristic properties such as migration and
MPL Induces a Strong Phagocytic Response. Because MPL stimu-
whether it could also modulate the phagocytic potential of these
cells. Microglial cells treated with MPL showed a significant in-
B). We then investigated the ability of MPL-treated cells to in-
ternalize Aβ peptides. MPL-stimulated microglia exhibited a sig-
nificant increase in the internalization of Aβ1–42oligomers (Fig.
3C). Phagocytized Aβ oligomers colocalized with Lamp-2 in MPL-
treated microglia, suggesting they were transported to lysosomes
following phagocytosis (Fig. 3D and Fig. S3).
Monocytes are cells that would likely be targeted by peripheral
monocytes (Fig. 3E). The phagocytic response observed in MPL-
treated microglia correlates with actin remodeling (Fig. 2 D and E)
such as Aβ. On the surface of microglia, SR-As are important for
adherence and phagocytosis of Aβ (18) but do not stimulate the
production of reactive oxygen species (19). These results demon-
strate that MPL induces a strong phagocytic response.
MPL Induces a Low Inflammatory Response While Triggering a Strong
Monocytopoiesis in Mice. To determine whether the low in-
in vivo, we measured several cytokines and chemokines in the sera
of either MPL or LPS. MPL elevated the levels of most cytokines
and chemokines that were measured, but the levels were
acylmonophoryl lipid A (MPL) chemically extractedfromSalmonella minnesota
end glucosamine, and (iii) removal of the acyl chain from the 3-position of the
disaccharide. (C) TLR4-specific activation of NF-κB and AP-1 in HEK293 cells
expressing TLR4, MD-2, and CD14 following stimulation with MPL and LPS for 5
h (n = 3). In contrast, (D) no TLR2 activation is observed in HEK293-TLR2 cells
Pam3CSK4 was used as a positive control (n = 3). Using the same concentration
of ligands (1 μg/mL), (E) degradation of IκBα in BV-2 microglia is delayed and
milder following MPL treatment compared with LPS. BV-2 cells incubated with
MPL exhibit significantly lower (F) ERK1/2 and (G) JNK1/2 phosphorylation
compared with LPS. However, (H) p38 phosphorylation in MPL-stimulated BV-2
cells was not statistically different from that in LPS-treated cells (n = 4–6 for E–
H). The lanes were run on the same gel but were noncontiguous. Data are
expressed as the means ± SEM; **P < 0.01 vs. PBS.
Distinct mechanisms mediating LPS- and MPL-induced TLR4 signaling.
| www.pnas.org/cgi/doi/10.1073/pnas.1215165110Michaud et al.
substantially lowerthan what wasobserved inLPS-treated animals
(Fig. 4). LPS induced considerably more TNF-α and IL-6 than
no longer detectable 6 h after MPL injection. MPL did not stim-
ulate the release of IL-1β at the two time points examined (Fig.
4D). The levels of the chemokines CCL3, IP-10, CXCL-1, and
CCL2 were all increased in MPL-treated mice but to lower con-
centrations than in the LPS group (Fig. 4 F–I).
We also examined the inflammatory response in the brains of
these mice by analyzing TLR2 and TNF-α mRNA expression, two
sensitive markers of microglia activation (20). LPS induced robust
TLR2 and TNF-α expression in circumventricular organs (CVOs),
choroid plexus, and throughout the brain parenchyma, whereas
Interestingly, whereas i.p. injection of either MPL or LPS stimu-
lated similar expansion of blood monocytes (Fig. 5), MPL induced
a milder proinflammatory cytokine response compared with LPS.
MPL Treatment Improves AD-Related Pathology of APPswe/PS1 Mice.
To investigate whether treatment with MPL might affect AD-re-
by i.p. injection to APPswe/PS1 mice for 12 consecutive weeks be-
ginning when the mice were 3 mo old. Cognitive function and Aβ
deposition were assessed for each mouse 2 and 3 wk after the final
injection, respectively. Using a T water maze behavioral test to
assess hippocampus-based spatial learning and memory, experi-
ments were conducted to determine whether clearance of Aβ cor-
with the PBS-treated control group, the MPL-treated APPswe/PS1
mice showed significant improvement in cognitive functions
(Fig. 6I). LPS treatment, however, did not lead to significant
improvement in the cognitive performances of APPswe/PS1 mice.
Administration of MPL caused a significant reduction in the
number and size of Aβ deposits, as well as the quantity of soluble
PBS control group; Fig. 6 A–G). Compared with controls, the size
and number of Aβ plaques were considerably greater in LPS-
treated animals, whereas the level of soluble Aβ monomers was
microglia were stimulated for 24 h with 1 μg/mL of LPS or MPL. F-actin
(green) was stained with phalloidin to expose different cell morphologies:
(A) nonactivated amoeboid shape, (B) LPS-activated elongated and multi-
polar morphology, and (C) an MPL-induced intermediate phenotype. (Scale
bar, 100 μm.) (D) Total protrusion length was measured in a minimum of 70
cells per treatment group. (E) The migration of BV-2 cells was assessed using
the scratch assay test (n = 3). (F) No induction of COX-2 was observed fol-
lowing MPL stimulation in contrast to LPS (n = 3–4). (G) A strong TNF-α se-
cretion was seen in the media of LPS-incubated cells, whereas low levels
were detected for MPL-treated cells (n = 6). (H and I) MPL did not generate
nitrites or iNOS expression in comparison with the robust induction by LPS
(n = 3–6). (J) Arginase 1 levels were similarly reduced following LPS or MPL
stimulation (n = 3). (K) Two hours after cell stimulation, the transcriptional
activation of TNF-α was lower in MPL-treated cells than in those treated with
LPS. (L and M) Both MPL and LPS induced comparable levels of CCL2 and
TLR2 mRNA at 2 h poststimulation (n = 3). The lanes were run on the same
gel but were noncontiguous. Data are expressed as the means ± SEM. *P <
0.05, **P < 0.01, ***P < 0.001 (vs. PBS);&&P < 0.01,&&&P < 0.001 (vs. LPS).
MPL induces a low inflammatory response in microglia. BV-2
microglia were stimulated with 1 μg/mL of LPS or MPL for 18 h. (A) Both MPL
and LPS stimulate the phagocytosis of fluorescent Escherichia coli beads by
microglia (n = 3). (B) The intracellular localization of these beads (green) was
validated by confocal microscopy. (Scale bar, 10 μm.) (C) Mean fluorescence
intensity of internalized fluorescent Aβ oligomers was evaluated by flow
Aβ oligomers (red) were colocalizing with the lysosomal Lamp-2 proteins
(green). (Scale bar, 10 μm.) (E) Distinct wild-type mice received a single LPS (20
μg) or MPL (50 μg) i.p. injection, and 24 h later 5 μg of fluorescent Aβ was
injected via tail vein. Monocytes were isolated 2 h later and significantly more
receptor A (SR-A) mRNA was induced in microglia after 24 h of incubation
with LPSorMPL.Dataareexpressedas themeans± SEM.*P < 0.05,**P< 0.01,
***P < 0.001 (vs. PBS);&P < 0.05,&&P < 0.01,&&&P < 0.001 (vs. LPS).
MPL stimulates phagocytosis in microglia and monocytes. BV-2
Michaud et al.PNAS
| January 29, 2013
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after i.p. injections of MPL was determined to investigate whether
MPL treatment led to lower Aβ levels by promoting phagocytosis
percentages of Aβ-positive cells than those of the control group
MPL treatment can stimulate the clearance of Aβ and improve
cognitive function of APPswe/PS1 mice. To understand whether an
be responsible for these beneficial effects, we generated APPswe/
PS1 chimeric mice in which bone marrow cells were GFP+/−. The
number of these cells, mostly found in perivascular spaces and the
choroidplexus,wasnot increasedfollowing12weeklyinjections of
MPL in APPswe/PS1 mice (Fig. S5).
Repeated Weekly Injections of MPL in APPswe/PS1 Mice Generate a
Transient Innate Immune Response and Do Not Create an Immune
Tolerance.We examined the pathways previously explored (following
an acute injection of MPL) to assess the effects of the chronic
treatment regimen used in APPswe/PS1 mice. Both TLR2 and
TNF-α gene expression in the CVOs vanished 3 wk after the last
injection of MPL (Fig. S6). In chronically LPS-challenged mice,
we could not observe TNF-α mRNA in the brain, and we detected
a very weak or no TLR2 signal. In the periphery, monocytes were
at normal levels 6 d after the last injection (Fig. S7B). Blood
TNF-α, IL-6, IL-10, CCL2, IFN-γ, and IL-12p70 were all at base-
line concentrations 3 wk after the last injection and not statistically
different among treatment groups (Fig. S8). To evaluate whether
repeated weekly injections of MPL induced immune tolerance in
APPswe/PS1 mice, we measured its ability to trigger TNF-α and
CCL2 release (Fig. S9) as well as expansion of monocytes (Fig. S7A)
in blood after 12 weekly injections. Essentially, MPL had effects
similar to a single acute injection (Figs. 4 and 5). This is in line
with evidence showing that endotoxin tolerance is not maintained
more than 5 d (21). These data demonstrate that repeated weekly
MPL injections do not produce a sustained state of immune acti-
vation and that the ligand remains able to activate TLR4 similarly to
a single acute injection.
The data reported here show that the TLR4 ligand MPL induces
phagocytosis of Aβ by mononuclear phagocytes yet elicits an in-
flammatory response that is considerably lower than generated
by LPS. Chronic systemic administration of MPL significantly
improved AD-related pathology in treated APPswe/PS1 mice,
evidenced by reduced levels of Aβ in the brain and improved
many studies highlighting the efficacy of finely tuned innate im-
mune activation fortreatment of AD in transgenic mice havebeen
published (2, 22). For instance, induction of the proinflammatory
cytokines IL-1β, TNF-α, IL-6, and IFN-γ correlated with reduced
Aβ deposition in AD mouse models (23–26). Recently, TNF-α
receptor ablation was shown to enhance AD pathology in trans-
genic mice, likely because of impaired Aβ phagocytosis (27). Ad-
ditionally, activation of microglia has been reported to induce
lysosome acidification and increased degradation of Aβ (28). In
this regard, MPL seems to induce a suitable degree of innate im-
mune stimulation that reduces the accumulation of Aβ and
improves spatial memory in APPswe/PS1 mice.
Stimulation of microglia and monocytes with MPL induced in-
creased phagocytosis of Aβ by these cells. Whereas it is widely
accepted that p38, ERK, JNK, and NF-κB all induce TLR-medi-
ated cytokine production, p38 has been shown to up-regulate
scavenger receptor expression and induction of phagocytic activity
(29, 30). Previous studies have also shown that the Aβ phagocytic
activity involving cell surface receptors such as SR-A is altered in
AD and aged mice (31, 32). Although MPL does not induce ERK
injection in mice. (A–I) Cytokine and chemokine profile in sera of wild-type
mice 2 and 6 h after an i.p. injection of LPS (20 μg) or MPL(50 μg). Results are
shown in relative units (RU), pg/mL or ng/mL (n = 4–5). The bars represent
mice injected i.p. with PBS, MPL (50 μg), or LPS (3 μg). Twenty-four hours
after the injection, LPS provoked a strong TLR2 induction in the circum-
ventricular organs, choroid plexus, and brain parenchyma, whereas MPL
triggered weak expression, mainly in circumventricular organs. (Scale bar, 500
μm.) *P < 0.05.
Mild activation of the immune system in response to systemic MPL
blood monocytes in mice. Wild-type
mice received a single i.p. injection of
PBS, MPL (50 μg), or LPS (20 μg) and
their blood was analyzed 24 h later by
flow cytometry. Cells were considered
to be monocytes by their expression of
CD11b and the lack of lineage (Lin−)
markers: CD3−, NK1.1−, Ly6G−, and
B220−(n = 5). Data are expressed as
the percentage of total CD45+leuko-
cytes excluding debris. The bars represent mean ± SD; **P < 0.01, ***P < 0.001 (vs. PBS).
MPL triggers the expansion of
| www.pnas.org/cgi/doi/10.1073/pnas.1215165110 Michaud et al.
or JNK and stimulates NF-κB to a lesser extent than LPS, it does
activate p38 strongly and drives the expression of SR-A in micro-
glial cells. These findings suggest that MPL selectively stimulates
p38 and promotes Aβ uptake while avoiding the extensive pro-
by other signaling pathways.
There are several possibilities to explain how peripheral ad-
ministration of MPL reduces Aβ levels in the brain and restricts
cognitive deficits in APPswe/PS1 mice. First, MPL-activated, highly
Aβ phagocytic myeloid cells in theperiphery could actas a sink for
Aβ in the blood. This sink effect could contribute to reduce the
level of Aβ in the brain via equilibrium-driven redistribution of Aβ
to the periphery, as observed in Aβ-specific immunotherapy (33).
This is in line with the increase in Aβ phagocytosis by blood mon-
ocytes stimulated with MPL. However, the increased proportion of
CD45+cells containing Aβ found in the brain of MPL-treated mice
suggests that peripheral clearance is not the exclusive mechanism
involved in eliminating Aβ. In this regard, it is noteworthy that
CD45+microglial cells have been shown to promote the clearance
of oligomeric Aβ (34). A compatible mode of action could be the
stimulation of microglia or macrophages located in the CVOs,
which lay outside the blood–brain barrier (BBB). Once activated,
these cells can mediate progressive release of cytokines across the
BBB and into the brain parenchyma (20). Finally, systemic admin-
from bone marrow, which move into the brain perivascular spaces
and clear Aβ via phagocytosis (35, 36).
Although LPS was found to induce Aβ phagocytosis better than
MPL in microglia and monocytes, chronic systemic LPS admin-
istration in APPswe/PS1 mice exacerbated the Aβ plaque load.
This result is consistent with published studies showing that acute
injection of LPS activates microglia and increases Aβ phagocy-
tosis (6, 37), whereas chronic exposure to LPS leads to higher
produced by chronic LPS stimulation is thought to be the main
cause for this phenomenon (38–40). Indeed, multiple lines of
evidence link inflammatory mediators to increased amyloido-
genesis by different mechanisms, including enhanced tran-
scription (41) and translation (42) of the APP gene and JNK-
dependent stimulation of gamma-secretase enzymatic activity
(43). The findings that MPL induces increased Aβ phagocytosis,
but only modest inflammation, could explain the highly efficient
reduction of Aβ levels in vivo by this TLR4 ligand compared
Mawuenyega et al. (44) recently demonstrated that AD is
characterized by an impairment in the clearance of Aβ, rather
than increased production. Notably, the reduction of factors
associated with hematopoiesis and phagocytosis has been asso-
ciated with development of AD (12). Moreover, it has been
shown that myeloid cells from patients with AD and mild cog-
nitive impairment phagocytose Aβ less efficiently than cells from
normal controls (31). Therapeutics promoting the elimination
of Aβ by scavenger cells such as monocytes, macrophages, or
microglia could be valuable tools in the fight against AD. We
demonstrate here that TLR4 stimulation with the detoxified li-
gand MPL significantly improved AD-related pathology. Al-
though the safety of the MPL treatment regimen used here has
not been confirmed in humans, this compound has been ad-
ministeredtohundreds of thousands of humans as anadjuvant in
different vaccines (9) and is currently used as a component of
a marketed human vaccine (Cervarix). Based on our findings, we
propose that MPL holds great promise as a safe and effective
treatment for this neurodegenerative disease.
Materials and Methods
Materials and methods are described in detail in SI Materials and Methods.
Ligand Preparation. The MPL immunostimulant was produced at Glax-
oSmithKline from the LPS of S. minnesota R595 following alkaline pro-
cedures, which have been described previously (45, 46).
NF-κB/AP-1 Activation Assay. Transfected HEK 293 cells with the expression
vectors encoding human TLR4, MD-2, and CD14 were further stably trans-
fected with the NF-κB reporter vector pNifty-2 secreted alkaline phospha-
tase. Cells were stimulated for 5 h in FCS-free medium containing different
concentrations of MPL and LPS or PBS.
Quantification of Cytokine and Chemokine Levels in Mouse Sera. Blood (sera)
cytokine and chemokine levels were measured in C57BL/6 mice using ELISA or
a Luminex mouse cytokine-chemokine kit after the i.p. injection of either LPS
(20 μg) or MPL (50 μg) at 2 or 6 h postinjection.
TLR2 in Situ Hybridization. In situ hybridization histochemical localization of
TLR2 on brain slices was performed using35S-labeled cRNA probes according
to a protocol described previously (47).
FACS Analysis on Mouse Whole Blood and Aβ1–42Uptake by Monocytes. C57BL/6
mice received an i.p. injection of PBS, MPL (50 μg), or LPS (20 μg). Twenty-four
hours after treatment, mice received 5 μg of fluorescent HiLyte Fluor 488-
labeled Aβ1–42via a tail vein injection and peripheral blood was harvested 2 h
later. Leukocytes were stained and acquired on a flow cytometer.
Transgenic MouseLinesandTreatment.Allprotocols were conducted according
to the Canadian Council on Animal Care guidelines, as administered by the
Laval University Animal Welfare Committee. MPL (50 μg), LPS (3 μg), or PBS
was administered once a week by i.p. injection in 3-mo-old APPswe/PS1 mice
for 12 consecutive weeks.
mice. MPL (50 μg), LPS (3 μg), or PBS was administered once a week by i.p.
injection in 3-mo-old APPswe/PS1 mice for 12 consecutive weeks. (A–C) Rep-
resentative Aβ immunoreactivity in cortex and hippocampus is shown in brain
sections of APPswe/PS1 mice injected with PBS, MPL, or LPS. (Scale bar, 500
μm.) (D and E) Compared with the PBS control group, the number and the
area of Aβ plaques are significantly reduced in the cortex of MPL-treated mice
(n = 9). (F) Extracellular Aβ monomers in mouse brains were quantified by
Western blot analysis. (G) MPL-treated mice had significantly fewer mono-
mers compared with controls, whereas the LPS group remained essentially
unchanged (n = 8). (H) Analysis of CD45+brain cells in 10-mo-old APPswe/PS1
after five consecutive daily i.p. injections of MPL (25 μg). Flow cytometry
analysis of CD45+brain cells that were immunoreactive for intracellular Aβ
(using monoclonal anti-Aβ 6E10 antibody) revealed a significant increase in
MPL-injected mice compared with the PBS control group (n = 4–5). (I) The
hippocampus-based spatial learning and memory of APPswe/PS1 mice was
evaluated in the T water maze behavioral test. APPswe/PS1 mice treated with
MPL had a significant improvement of their cognitive functions as shown by
their lower number of trials to reach the criterion in the reversal phase of the
test (n = 9–19). Each point represents a single mouse and the horizontal bars
are the mean for each group. *P < 0.05, **P < 0.01 (vs. PBS).
MPL treatment reduces Aβ levels and cognitive deficits in APPswe/PS1
Michaud et al.PNAS
| January 29, 2013
| vol. 110
| no. 5
Aβ Plaque Immunofluorescence and Stereological Analysis. To stain Aβ plaques, Download full-text
free-floating sections were immunolabeled with the monoclonal anti-Aβ
(6E10). The number of plaques and the area occupied by all Aβ-labeled pla-
ques were determined by stereological analysis as described previously (35).
Western Blot Analysis of Brain Samples. Monomeric Aβ and β-actin were
detected by immunoblotting with the monoclonal antibodies anti-Aβ (6E10)
and anti-β-actin (13E5).
Isolation of Brain Leukocytes and FACS Analysis. Brain leukocytes were iso-
lated by Percoll gradient, stained with anti–CD45-PE and anti-Aβ (6E10), and
analyzed by flow cytometry.
Left–Right Discrimination Learning. Mice were placed at the stem of a water-
filled T maze and choose to swim either left or right until they found the
submerged platform and escaped to it. The reversal learning phase was then
conducted 48 h later. During this phase, the same protocol was repeated
except that the mice were trained to find the new location of the escape
platform on the side opposite that on which they had learned during the
Additional Information. The experiments conducted with BV-2 cells are de-
scribed in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Martine Lessard, Marie-Pier Girouard, and
Marie-Michèle Plante for technical help. GlaxoSmithKline Vaccines and the
Canadian Institutes in Health Research (CIHR) supported this research. J.-P.M.
is supported by a doctoral Studentship from the CIHR, M.H. by a postdoctoral
fellowship from the Natural Sciences and Engineering Research Council, and
P.T. by a master scholarship from Bourse de Recherche en Milieu Pratique
Innovation (Fonds Québécois de la Recherche sur la Nature et les Technolo-
gies–Conseil de Recherches en Sciences Naturelles et en Génie). S.R. holds
a Canadian Research Chair in Neuroimmunology.
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| www.pnas.org/cgi/doi/10.1073/pnas.1215165110Michaud et al.