of July 23, 2013.
This information is current as
of Alzheimer's Disease
Increases Plaque Burden in a Mouse Model
Cells Promotes Microglial Activation and
Production by Amyloid
Julie-Ann O'Reilly, Kingston H. G. Mills and Marina A.
Tara C. Browne, Keith McQuillan, Róisín M. McManus,
2013; 190:2241-2251; Prepublished online 30
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The Journal of Immunology
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The Journal of Immunology
IFN-g Production by Amyloid b–Specific Th1 Cells Promotes
Microglial Activation and Increases Plaque Burden in
a Mouse Model of Alzheimer’s Disease
Tara C. Browne,*,1Keith McQuillan,*,†,1Ro ´isı ´n M. McManus,*,†Julie-Ann O’Reilly,*
Kingston H. G. Mills,†,2and Marina A. Lynch*,2
Alzheimer’s disease (AD) is characterized by the presence of amyloid-b (Ab)–containing plaques, neurofibrillary tangles, and
neuronal loss in the brain. Inflammatory changes, typified by activated microglia, particularly adjacent to Ab plaques, are also
a characteristic of the disease, but it is unclear whether these contribute to the pathogenesis of AD or are a consequence of the
progressive neurodegenerative processes. Furthermore, the factors that drive the inflammation and neurodegeneration remain
poorly understood. CNS-infiltrating T cells play a pivotal role in the pathogenesis of multiple sclerosis, but their role in the
progression of AD is still unclear. In this study, we examined the role of Ab-specific T cells on Ab accumulation in transgenic mice
that overexpress amyloid precursor protein and presenilin 1 (APP/PS1). We found significant infiltration of T cells in the brains of
APP/PS1 mice, and a proportion of these cells secreted IFN-g or IL-17. Ab-specific CD4 T cells generated by immunization with
Ab and a TLR agonist and polarized in vitro to Th1-, Th2-, or IL-17–producing CD4+T cells, were adoptively transferred to APP/
PS1 mice at 6 to 7 mo of age. Assessment of animals 5 wk later revealed that Th1 cells, but not Th2 or IL-17–producing CD4+
T cells, increased microglial activation and Ab deposition, and that these changes were associated with impaired cognitive
function. The effects of Th1 cells were attenuated by treatment of the APP/PS1 mice with an anti–IFN-g Ab. Our study suggests
that release of IFN-g from infiltrating Th1 cells significantly accelerates markers of diseases in an animal model of AD.
Journal of Immunology, 2013, 190: 2241–2251.
patients treated with nonsteroidal anti-inflammatory drugs (1, 2);
these findings are supported by evidence of preventative effects of
these drugs in animal models of AD (3). Whereas the classical
characteristics of AD are the presence of amyloid-b (Ab) plaques
and neurofibrillary tangles, together with selective neuronal loss,
there is also evidence of innate immune activation in AD, with
activation of microglia, the primary resident immune cell of the
CNS. Activated microglia are found in the brain of AD patients
with mild to moderate dementia (4) and in a significant proportion
of cases with mild cognitive impairment (5). Microglia secrete
role for inflammation in the pathogenesis of Alzheimer’s
disease (AD) is suggested by epidemiological studies
that have reported a decreased incidence of AD in
inflammatory cytokines like IL-1b and TNF-a, which increase
activity and expression of secretases (6, 7), contributing to Ab
deposition and the early pathogenic changes in AD (8). Inflam-
matory cytokines released from activated microglia are known to
be potentially cytotoxic, but there is evidence indicating a positive
effect of an inflammatory environment on Ab clearance (9–11).
Microglia demonstrate significant plasticity and also adopt other
phenotypes that are associated with tissue repair (12). Further-
more, immune cells in the AD brain can have an alternative ac-
tivated state as well as the classical proinflammatory phenotype
(13). Cell-surface expression of MHC class II and costimulatory
molecules is enhanced on activated microglia (14, 15), enabling
them to act as APC. However, circulating cells, including T cells,
are infrequently observed in the normal CNS, although there is a
population of perivascular macrophages, distinct from microglia
(16–18), and these cells may play an important anti-inflammatory
function, perhaps mediated by a change in hypothalamic–pitui-
tary–adrenal axis function (19).
The blood–brain barrier plays a key role in protecting the brain,
restricting the entry of pathogens and macromolecules. An intact
blood–brain barrier is also important in restricting entry of cir-
culating cells, and increased blood–brain barrier permeability,
which is a characteristic of several neurodegenerative conditions
including multiple sclerosis, AD, and Parkinson’s disease (20–22),
is associated with infiltration of circulating immune cells. Studies
in multiple sclerosis and experimental allergic encephalomyelitis
(EAE), a mouse model of multiple sclerosis, have shown that
T cells, particularly IL-17–producing CD4+T cells (Th17) cells,
infiltrate the brain and spinal cord and are central to the patho-
genesis of the disease (23). The role of Th1 cells in CNS in-
flammation associated with EAE is more controversial, with some
studies suggesting that Th1 cells contribute to pathology and
*Trinity College Institute of Neuroscience, Trinity Biomedical Sciences Institute,
Trinity College Dublin, Dublin 2, Ireland; and†School of Biochemistry and Immu-
nology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2,
1T.C.B. and K.M. contributed equally to this work.
2K.H.G.M. and M.A.L. contributed equally to this work.
Received for publication April 4, 2012. Accepted for publication December 18, 2012.
This work was supported by grants from Science Foundation Ireland, the Health
Research Board of Ireland, and the Irish Research Council for Science, Engineering
Address correspondence and reprint requests to Prof. Kingston H.G. Mills, Immune
Regulation Research Group, School of Biochemistry and Immunology, Trinity Bio-
medical Sciences Institute, Trinity College Dublin, 152-160 Pearse Street, Dublin 2,
Ireland. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this article: Ab, amyloid-b; AD, Alzheimer’s disease; APP,
amyloid precursor protein; dH2O, distilled H2O; EAE, experimental autoimmune
encephalomyelitis; HBSS/FBS, HBSS containing 3% FBS; PS1, presenilin 1; RT,
room temperature; Th17 cells, IL-17–producing CD4+T cells; WT, wild-type.
at IRIS - IReL Consortia on July 23, 2013
others suggesting a protective role for IFN-g through inhibition of
Th17 cells. As well as their role in demyelination, the interaction
of T cells with microglia contributes to the inflammatory changes
observed in EAE (24).
T cells are also present in the brain of patients with AD (25–28),
and infiltration may result from increased expression of CXCR2
and MIP-1a on the T cells (29). Although T cells, in particular
Th2 or regulatory T cells, can have a protective role in the brain
(30, 31), the entry of activated effector T cells, particularly Th1 or
Th17 cells, into the brain in which inflammatory changes are on-
going, is likely to escalate the inflammatory cascade. Consistent
with this is the finding that Ab-induced release of inflammatory
cytokines from glia was exacerbated by Th1 and Th17 cells (32),
and this effect was attenuated by Th2 cells. Immunization with Ab
peptides, formulated with various adjuvants, is being evaluated
both in preclinical models and in the clinic as a potential therapy
for AD-based on Ab-mediated reduction of Ab plaque burden
(33). However, a proportion of AD patients who received a vac-
cine containing Ab peptide formulated with the adjuvant QS21
(AN1792) developed meningoencephalitis (34). It is possible that
the generation of certain subtypes of Ab-specific T cells may
contribute to inflammatory pathology in AD.
In this study, we used a transgenic mouse model of AD that
overexpresses amyloid precursor protein (APP) with the Swedish
mutation and exon-9–deleted presenilin 1 (PS1; APP/PS1 mice) to
determine whether Ab-specific T cell subsets can modulate Ab
burden and affect microglial activation. Ab-specific effector
T cells were generated by immunization with Ab and CpG, po-
larized in vitro to Th1, Th2, and Th17 cells, and adoptively
transferred to 6- to 7-mo-old APP/PS1 mice. We found that Ab-
specific Th1 cells increased Ab deposition and microglial ac-
tivation in APP/PS1 mice and negatively impacted on spatial
Mononuclear cells were prepared from the brain of APP/PS1 and WT
mice, and cells were surface stained with Abs specific for CD3, CD4, CD8,
intracellular IL-17, and IFN-g, and flow cytometric analysis was per-
formed. Mean frequency (A) and representative dot plots (B) of CD4+and
CD8+cells in brain of WT and APP/PS1 mice. Mean frequency (C) and
representative dot plots (D) of CD4+cells stained positively for IFN-g and
IL-17 in brain of APP/PS1 mice. (E) Mean frequency of CD8+in brain of
tissue prepared from WTand APP/PS1 mice. (F) Mean frequency of CD8+
cells stained positively for IFN-g and IL-17 in brain tissue prepared from
APP/PS1 mice. **p , 0.01, Student t test for independent means (n $ 4),
***p , 0.001, Student t test for independent means. Representative of
Th1 and Th17 infiltrate the brain of APP/PS1 mice.
T cells. Popliteal lymph nodes harvested from mice immunized with Ab
and CpG were cultured with Ab1–42in the presence of IL-12 to generate
Th1 cells, dexamethasone, IL-4, and anti–IFN-g to generate Th2 cells, or
IL-23 and anti–IFN-g to generate Th17 cells. IFN-g, IL-4, and IL-17
concentrations were determined by ELISA on supernatants removed 3 d
after stimulation with Ag and APC. Values are expressed as means 6 SEM
(n = 4); representative of four experiments.
Cytokine production by in vitro polarized Ab-specific
2242PATHOGENIC ROLE OF IFN-g IN MURINE ALZHEIMER’S MODEL
at IRIS - IReL Consortia on July 23, 2013
learning. Treatment of mice with anti–IFN-g Ab ameliorated these
changes, suggesting that release of IFN-g from infiltrating Th1
cells accelerates the pathology in these animals.
Materials and Methods
from The Jackson Laboratory and subsequently bred in a specific pathogen-
free unit in the Bioresources Unit in Trinity College Dublin. GFP micewere
a gift from Matthew Campbell, School of Genetics and Microbiology,
Trinity College Dublin. Mice used were transgenic animals on a C57/Bl6J
background expressing eGFP cDNA under the control of a chicken b-actin
promoter and CMV enhancer. All mice were maintained in controlled
conditions (temperature 22 to 23˚C, 12-h light-dark cycle, and food and
water ad libitum) under veterinary supervision, and experimentation was
carried out under a license granted by the Minister for Health and Children
(Ireland) and with the appropriate ethical approval.
Isolation and FACS analysis on mononuclear cell isolation
from CNS tissue
APP/PS1 mice and nontransgenic littermates were anesthetized with so-
dium pentobarbital (40 ml) and perfused intracardially with sterile ice-cold
PBS (20 ml). The brain was removed and placed in HBSS (2 ml) con-
taining 3% FBS (HBSS/FBS; Sigma-Aldrich). Tissue was dissociated
through a sterile 70-mm nylon mesh filter, washed with HBSS/FBS, and
centrifuged at 170 3 g for 10 min at room temperature (RT). The super-
natant was removed and the remaining pellet resuspended in HBSS/FBS
(2 ml) containing collagenase D (1 mg/ml; Roche) and DNAse I (10 mg/
ml; Sigma-Aldrich) and incubated for 1 h at 37˚C. Cells were washed in
HBSS/FBS and centrifuged at 1200 rpm for 5 min. Supernatants were
removed, and cells were resuspended in 1.088 g/ml Percoll (9 ml; Sigma-
Aldrich). This was underlaid with 1.122 g/ml Percoll (5 ml) and overlaid
with 1.072 g/ml Percoll (9 ml) followed by 1.030 g/ml Percoll (9 ml) and
finally PBS (9 ml). Percoll gradients were centrifuged at 1250 3 g for 45
min at 18˚C. Mononuclear cells were removed from between the 1.088/
1.072 and 1.072/1.030 g/ml interfaces, washed twice in HBSS/FBS, and
Mononuclear cells prepared from CNS tissue were prepared for intra-
cellular staining using a cell permeabilization kit (DakoCytomation). Cells
were centrifuged at 1200 rpm for 5 min before stimulation with X-Vivo
media (200 ml) containing PMA (10 ng/ml; Sigma-Aldrich), ionomycin
(1 mg/ml; Sigma-Aldrich), and brefeldin A (5 mg/ml; Sigma-Aldrich) for
5 h. Following stimulation, cells were centrifuged at 1200 rpm for 5 min
and resuspended. Low-affinity IgG receptors (FcgRIII) were blocked by
incubating cells in FACS buffer (50 ml/sample) containing CD16/CD32
FcgRIII (1:100) for 10 min at RT. Cells were incubated in 50 ml/sample
FACS buffer containing the appropriate FACS Abs for 15 min at RT and
fixed in IntraStain Reagent A (50 ml/sample; DakoCytomation) for 15 min
at RT. Cells were washed twice with FACS buffer and centrifuged at 1200
rpm for 5 min, permeabilized with IntraStain Reagent B (50 ml/sample;
DakoCytomation) plus intracellular Abs for 15 min at RT in the dark,
washed twice in FACS buffer, and centrifuged at 1200 rpm for 5 min.
Immunofluorescence analysis was performed on a DakoCytomation Cyan,
APP/PS1 mice that received Ab-specific Th1
cells. Ab-specific Th1, Th2, and Th17 cells,
generated as described in Fig. 2, were injected
i.v. into APP/PS1 mice at 6 to 7 mo of age.
Two weeks after injection, cognitive function
was analyzed in the Morris Water Maze test.
Training commenced after 1 d of habitation and
continued for 5 consecutive days on which the
mice underwent four 1-min trials with an in-
tertrial interval of 5 min. The day after the final
day of training, the platform was removed, and
mice were given a single 60-s probe trial. The
percentage of time each animal spent swimming
in the quadrant previously containing the plat-
form was measured. Path length was also
assessed. (A) The latency to reach the platform
of all groups. (B) Mean latency on day 5 of
training. (C) Sample paths for individual mice in
each treatment group. (D and E) The path length
taken to reach the platform. (F and G) In the
probe test, the percentage of the total time and
distance (i.e., path length) each animal spent
swimming in the quadrant was measured. *p ,
0.05, ANOVA; n $ 5; representative of two
experiments. Con, Control; Tg, transgenic.
Spatial learning is impaired in
The Journal of Immunology 2243
at IRIS - IReL Consortia on July 23, 2013
data acquired using Summit software (DakoCytomation), and the results
analyzed using FlowJo software (Tree Star).
Generation of Ab-specific T cell lines and in vivo transfer
WT mice were immunized in the footpad with Ab1–42(75 mg/mouse) and
CpG (25 mg/mouse) and boosted after 21 d. Mice were sacrificed 7 d later;
the spleens and popliteal lymph nodes were harvested and restimulated
with Ab1–42(25 mg/ml) in the presence of IL-12 (10 ng/ml) to generate
Th1 cells, dexamethasone (1 3 1028M), IL-4 (10 ng/ml), and anti–IFN-g
(5 mg/ml) to generate Th2 cells, or IL-23 (10 ng/ml) and anti–IFN-g (5 mg/
ml) to generate Th17 cells. After 4 d, IL-2 (5 ng/ml) was added to the Th1
and Th2 cell preparations, RPMI-1640 culture medium only was added to
the Th17 cell cultures, and incubation continued for a further 7 d. Cells
were washed and injected i.v. (15 3 106cells/mouse in 300 ml serum-free
medium) into 6- to 7-mo-old APP/PS1 mice. Control animals received in
300 ml serum-free medium alone. Behavior analysis was assessed 2 wk
after T cell transfer. Samples of supernatant were assessed by ELISA (see
below) for IFN-g, IL-4, IL-10, IL-17, and IL-5 production.
In a separate series of experiments, 6- to 7-mo-old APP/PS1 and WT
control mice were injected i.p. with anti–IFN-g Ab (600 mg) or a control
Ab (anti–b-galactosidase: 600 mg; R&D Systems) and after 24 h were
injected i.v. with Th1 cells (15 3 106cells/mouse) as described above.
Anti–IFN-g or anti–b-galactosidase Ab injections were repeated 3, 7, 10,
14, 17, 21, 24, 28, and 31 d after T cell transfer. Behavioral analysis was
assessed 21 d after T cell transfer.
Tracking of Ab-specific Th1 cells into the brain
Ab-specific Th1 were generated from GFP mice immunized with Ab1–42
and CpG, restimulated in vitro with Ab1–42and IL-12, and expanded with
IL-2 as described above. Cells were washed and injected i.v. (15 3 106
cells/mouse) into 6- to 7-mo-old APP/PS1 mice or WT mice. Mice were
sacrificed 14 d later and mononuclear cells prepared from CNS tissue.
Cells were stimulated with PMA and ionomycin and stained for surface
CD3, CD4, CD8, and intracellular IFN-g. Immunofluorescence analysis
was performed on a DakoCytomation Cyan as described above.
Gait was analyzed in WT and APP/PS1 mice using the footprint test to
assess stride length and hind and front limb base widths. Muscular strength
and coordination were assessed using the inverted screen and wire-hang
tests. Two days later, 2 wk after administration of Ab-specific T cells,
mice were tested for spatial memory in the Morris water maze. The pool
(1.2 m diameter; 0.6 m high; 0.24 m water depth; 0.15 m platform diameter
placed in the northwest quadrant of the pool; 0.13 m from the edge of the
pool) was sited in a well-lit room (22 to 23˚C), and distinct visual cues
were placed on the curtains that encircled the pool. Training commenced
after 1 d of habitation and continued for 5 consecutive days on which the
mice underwent four 1-min trials with an intertrial interval of 5 min. Each
trial ended when mice located the platform or after 60 s when mice that
failed to locate the platform were led to it; animals remained on the
platform for 20 s. The day after the final day of training, the platform was
removed, and mice were given a single 60-s probe trial. The percentage of
time each animal spent swimming in the quadrant previously containing
the platform was measured. Path length was also assessed.
Preparation of tissue
In the first study, in which the effect of transfer of Th1, Th2, and Th17 cells
was assessed, micewere killed 24 h after the last behavioral analysis. In the
second study, in which the effect of anti–IFN-g Ab was assessed, micewere
killed 34 d after the first injection of Ab. They were anesthetized with
sodium pentobarbital (40 ml; Euthatal; Merial Animal Health) and per-
fused intracardially with ice-cold PBS (20 ml). The brains were rapidly
removed, and one half of the brain was stored for later extraction and
analysis of Ab. The second half of the brain, which was used for immu-
nohistochemical analysis, was placed onto cork discs, coated with opti-
mum cooling temperature compound (Sakura Tissue-Tek), snap-frozen in
prechilled isopropanol, and stored at 280˚C. Before sectioning, the tissue
was allowed to equilibrate to 220˚C for 2 h. Sagittal sections (10-mm
thick) were prepared using a cryostat (Leica, Meyer, U.K.), mounted on
gelatin-coated (Flukaerland) glass slides, allowed to dry for 20 min, and
stored at 220˚C for later immunohistochemical analysis.
Detection of Ab
Snap-frozen cortical tissue was homogenized in five volumes (w/v) of
homogenizing buffer (SDS/NaCl in distilled H2O [dH2O] with proteases)
and centrifuged (15,000 rpm, 40 min, 4˚C). The supernatant samples were
removed to extract SDS-soluble Ab, and the pellets were kept for ex-
traction of insoluble Ab. Supernatants were equalized (3 mg/ml) with
homogenizing buffer using a BCA protein assay, and samples were neu-
tralized by the addition of 10% (w/v) 0.5 M Tris-HCl (pH 6.8). Samples
were stored at 220˚C for later detection of soluble Ab. Pellets were in-
cubated in guanidine buffer (50 ml; 5 M guanidine-HCl/50 mM Tris-HCl,
pH 8; Sigma-Aldrich) for 4 h on ice. Samples were centrifuged (15,000
rpm, 30 min, 4˚C), and the supernatant samples were equalized (0.3 mg/
ml) with guanidine buffer and stored at 220˚C for later detection of in-
soluble Ab using MSD 96-well multi-spot 4G8 Ab triple ultra-sensitive
assay kits according to the manufacturer’s instructions (Meso Scale Dis-
covery). Standards (Ab1–38, 0–3,000 pg/ml; Ab1–40, 0–10,000 pg/ml; Ab1–42,
0–3,000 pg/ml) and samples were added to the 96-well plates, incubated
cells migrate into the brain of APP/PS1 mice.
Ab-specific Th1 cells, generated from GFP
mice were injected i.v. into 6- to 7-mo-old WT
or APP/PS1 mice. Two weeks after injection,
mice were sacrificed, and mononuclear cells
were prepared from the brain. Cells were sur-
face-stained with Abs specific for CD3, CD4,
and CD8 and intracellularly stained for IFN-g,
and flow cytometric analysis was performed to
quantify GFP-expressing and IFN-g–secreting
T cells in the brain. (A) Results are mean ab-
solute number of the indicated cells in the
brain. (B) Sample FACS plots of GFP+T cells
(gated on CD3), IFN-g+CD8+, and IFN-g+
CD4+cells; number represent percentage pos-
itive. Data in (A) represent mean 6 SEM from
four animals per experimental group from one
experiment; data in (B) representative of four
mice. **p , 0.01, ***p , 0.001, Student t test
for independent means.
Transferred Ab-specific Th1
2244PATHOGENIC ROLE OF IFN-g IN MURINE ALZHEIMER’S MODEL
at IRIS - IReL Consortia on July 23, 2013
(2 h, RT), washed, and read in a Sector Imager plate reader (Meso Scale
Discovery) immediately after addition of the MSD read buffer. Ab con-
centrations were calculated with reference to the standard curves and
expressed as picograms per milliliter.
Cryostat sections were assessed for Ab plaque deposition by staining with
Congo red. Sections, equilibrated to RT, were fixed in ice-cold methanol
for 5 min, washed in PBS, and incubated at room temperature for 20 min in
an alkaline solution prepared by adding NaOH (2 ml; 1 M) to saturated
NaCl (200 ml; 80% ethanol in dH2O). Thereafter, sections were incubated
in filtered Congo red solution (0.2% Congo red dye in the same alkaline
solution) for 30 min, rinsed in dH2O, incubated in methyl green solution
(1% in dH2O) for 30 s, washed, and dehydrated by dipping in 80, 95, and
then 100% ethanol. Sections were dried, incubated in 100% xylene (3 3 5
min), mounted onto slides using dibutyl phthalate in xylene (RA Lamb),
and allowed to dry overnight.
To assess CD11b, sections were fixed in an acetone/ethanol mixture(1:1)
for 5–10 min, and endogenous peroxidase activity was blocked by incu-
bating in 0.3% H2O2in PBS for 5 min. Sections were washed, blocked in
10% rabbit serum (Vector Laboratories), incubated overnight at 25˚C in rat
anti-CD11b Ab (1:100 in 5% rabbit serum in PBS; clone 5C6; Serotec),
washed, and incubated for 2 h at RT in biotinylated rabbit anti-rat IgG
(1:200 in 5% rabbit serum in PBS; Vector Laboratories). Sections were
washed, incubated in Vectastain Elite ABC reagent (two drops of A/B in
5 ml PBS; Vector Laboratories) for 1 h at RT, washed, and developed using
the substrate 3,3 diaminobenzidine–enhanced liquid substrate system tet-
rahydrochloride (one drop solution B in 1 ml solution A) for ∼10 min until
the color developed and counterstained with 1% methyl green for 10 min.
Samples were dehydrated by dipping in graded ethanol (70, 80, 95, and
100%) and incubating in xylene (VWR International). Sections were
mounted with dibutyl phthalate in xylene, dried overnight, and stored at
RT. The sections were examined using an Olympus lx51 light microscope
(Olympus, Tokyo, Japan), and micrographs were taken using an Olympus
UCMAD3 (Olympus) at 340 magnification. Data were quantified using
the Immunoratio plugin (http:\\imtmicroscope.uta.fi/immunoratio/) avail-
able for the ImageJ software package (National Institutes of Health) (35).
Colocalization of Ab and CD11b was examined by confocal microscopy.
Frozen brain sections brought to RT, fixed in ice-cold methanol, washed,
permeabilized in 0.1% Trition (Sigma-Aldrich) in PHEM buffer, and
washed. Nonspecific binding was blocked by incubating sections in 10%
normal goat serum (2 h, RT), and sections were incubated overnight with
pan-Ab Ab (1:1000; Calbiochem) and rat anti-CD11b Ab (1:100, clone
5C6, AbD; Serotec) in 5% normal goat serum in PHEM buffer. Sections
were washed, incubated in secondary Ab ALEXA 488 (1:4000; Invitrogen)
and Alexa 546 (1:1000; Invitrogen; 90 min, RT), washed, mounted, and
analyzed using confocal microscopy (Axioplan 2; Zeiss).
Statistical analysis was performed using GraphPad Prism (GraphPad). Data
were analyzed using Student t test, two-way ANOVA, or one-way ANOVA
followed by Newman–Keuls post hoc test. Data are expressed as means
with SEM and deemed statistically significant when p , 0.05.
Th1 and Th17 cell are present in the periphery and infiltrate
the brains of APP/PSI mice
We used flow cytometry to assess the presence of T cells in the
brain of WTand APP/PS1 mice. We found that therewerevery few
CD3+CD4+cells in the brain of WT mice but a significantly
hanced Ab deposition in brains of APP/PS1 mice. APP/
PS1 mice mice were injected with Ab-specific Th1,
Th2, or Th17 cells as described in Fig. 3. (A) Cryostat
sections were stained with Congo red to assess Ab-
containing plaques in hippocampus and cortex; the
mean number of plaques was recorded (B). (C) The
concentrations of insoluble Ab1–42, Ab1–40, and Ab1–38
in the cortical tissue were quantified by ELISA. (D)
The concentrations of soluble Ab1–42and Ab1–40in
cortical tissue were quantified by ELISA. *p , 0.05,
**p , 0.01, ***p , 0.001, ANOVA, APP/PS1 versus
WT.+p , 0.05,++p , 0.01,+++p , 0.001, ANOVA
versus control untreated APP/PS1 mice (n = 5 to 6);
representative of two experiments. Con, Control.
Transfer of Ab-specific Th1 cells en-
The Journal of Immunology 2245
at IRIS - IReL Consortia on July 23, 2013
greater number in brain tissue prepared from APP/PS1 mice (***p ,
0.001; Student t test for independent means; Fig. 1A). Intracellular
staining revealed that a proportion of CD4+cells stained positively
for IFN-g and also for IL-17 (Fig. 1B–D). There was no genotype-
related difference in the number of CD3+CD8+cells in the brain
(Fig. 1E), although intracellular staining indicated that a greater
proportion of these cells stained positively for IFN-g compared with
IL-17 (**p , 0.01; Student t test for independent means; Fig. 1F).
Ab-specific Th1 cells impair cognitive function in APP/PS1
Having demonstrated the presence of Th1 and Th17 cells in the
brain of APP/PS1 mice, we set out to evaluate the effect of ad-
ministration of Ab-specific T cells on cognitive function, Ab
accumulation, and microglial activation in 6- to 7-mo-old APP/
PS1 mice in which early pathological changes have been reported
(36). To amplify Ab-specific T cells, WT mice were immunized
twice (0, 21 d) with Ab and CpG, an adjuvant known to promote
Th1 and Th17 responses. Short-term Ab-specific Th1, Th2, and
Th17 cell lines were generated by restimulation with Ag and APC
in the presence of polarizing mixture described in the Materials
and Methods section. This protocol resulted in the generation of
highly polarized populations of Th1, Th2, and Th17 cells; Th1
cells produced high levels of IFN-g and low IL-4 and IL-17, Th2
cells secreted high levels of IL-4 and low IL-17 and IFN-g, and
Th17 cells produced high levels of IL-17 and no IL-4 or IFN-g
(Fig. 2). After one round of Ag-stimulation, surviving T cells were
washed and injected i.v. (15 3 106cells/mouse) into 6- to 7-mo-
old APP/PS1 or WT mice. Mice were tested for spatial memory in
the Morris water maze 2 wk after administration of Ab-specific
T cells. The latency to reach the platform decreased over the
5-d training period but changes were similar in WT mice and
control-treated APP/PS1 mice or APP/PS1 mice that received
T cells (Fig. 3A), and no treatment effect was observed on day 5 of
training (Fig. 3B). The path length taken to reach the platform
decreased with training, except in APP/PS1 mice, which received
Th1 cells (Fig. 3D) as shown by the representative traces obtained
on day 5 (Fig. 3C). The mean path length on day 5 was signifi-
cantly increased in these mice compared with untreated APP/PS1
mice (*p , 0.05; ANOVA; Fig. 3E). In contrast, transfer of Th1
cells into WT mice had no significant effect on path length taken
to reach the platform or mean path length on day 5 (Supplemental
Fig. 1). The day after the final day of training, the platform was
removed, and mice underwent a single 60-s probe trial. The per-
centage of the total time and distance (i.e., path length) each an-
imal spent swimming in the quadrant that previously contained the
platform was significantly decreased in APP/PS1 mice that re-
ceived Th1 cells compared with untreated APP/PS1 mice (*p ,
0.05; ANOVA; n $ 5; Fig. 3F, 3G). Therefore, Th1 cell transfer
induces a deficit in spatial learning in APP/PS1 mice at an age at
which such deficits are not generally observed. Importantly, no
motor deficits were observed in these animals; stride length, hind
limb base width, and front limb base width were similar in all
groups of mice, and, on the hangwire task, there were no differ-
ences in the latency to fall between groups (data not shown).
These findings suggest that transfer of Th1, but not Th2 or Th17
cells, around the time of onset of Ab plaque formation impairs
cognitive function in APP/PS1mice.
We tracked the migration of transferred T cells into the CNS
using Ab-specific Th1 cells generated from GFP mice immunized
with Ab and CpG and polarized with IL-12. We found a higher
proportion of CD3+T cells in the brain of APP/PS1, compared
with WT, mice after transfer of Ab-specific Th1 cells (Fig. 4).
Furthermore, we detected GFP+cells in the brain 14 d following
transfer of Th1 cells, and this was significantly greater in APP/PS1
mice. Finally, we found that CD8+as well as CD4+cells infiltrated
cells increase CD11b immunoreactivity in hip-
pocampus and cortex of APP/PS1 mice. APP/
PS1 mice were injected with Ab-specific Th1,
Th2, or Th17 cells as described in Fig. 3. (A)
Microglial activation was assessed by CD11b
immunoreactivity. (B) Data are means 6 SEM.
*p , 0.05, ANOVA versus control (n = 3–5)
representative of two experiments. Con, Control.
Transfer of Ab-specific Th1
2246 PATHOGENIC ROLE OF IFN-g IN MURINE ALZHEIMER’S MODEL
at IRIS - IReL Consortia on July 23, 2013
the brain, and a significant number of these secreted IFN-g (Fig.
4). These findings suggested that at least a proportion of Ab-
specific Th1 cells migrate into the brain following systemic de-
livery, and this is more pronounced in APP/PS1 when compared
with WT mice. In addition, IFN-g–secreting CD8 T cells are de-
tected in higher numbers in the brains of APP/PS1 compared with
Ab-specific Th1 cells enhance Ab plaque burden and enhance
microglial activation in APP/PS1 mice
Ab deposition has been reported in the brain of APP/PS1 mice as
early as 6 mo of age (37). Cryostat sections prepared from the 6-
to 7-mo-old APP/PS1 mice used in this study confirm the presence
of Ab-containing plaques in cortex and hippocampus. Adoptive
transfer of Ab-specific Th1 cells markedly increased Ab load,
particularly in cortex, whereas transfer of Th2 or Th17 cells had
little effect (Fig. 5A). Mean plaque number was significantly
increased in sections prepared from mice that received Th1 cells
(*p , 0.05; ANOVA; Fig. 5B). Insoluble Ab1–38, Ab1–40, and
Ab1–42were all significantly increased in tissue prepared from
APP/PS1, compared with WT, mice (*p , 0.05; **p , 0.01;
ANOVA; Fig. 5C). Injection of Th1 cells induced a further
increase in the concentration of the three Ab species (+p , 0.05;++
p , 0.01, ANOVA, versus control APP/PS1 mice). Furthermore,
soluble Ab1–40and Ab1–42were also significantly increased in
tissue prepared from APP/PS1 following transfer of Th1 cells (Fig.
5D), although soluble Ab1–38was unchanged between treatment
groups (data not shown). Neither Th2 nor Th17 cells exerted any
significant effect on soluble or insoluble Ab.
havioral deficits. APP/PS1 mice were injected with Ab-specific Th1 cells
as described in Fig. 3, and mice were treated with anti–IFN-g Ab or
anti–b-galactosidase as a control Ab before and after injection of the cells.
Three weeks after injection, cognitive function was analyzed in the Morris
Water Maze test as described in Fig. 3. (A) The path length taken to reach
the platform. (B) Mean path length on day 5 of training. (C and D) In the
probe test, the time and path length in the quadrant that previously con-
tained the platform (expressed as a percentage of the total) was measured.
Data represent mean 6 SEM from four to five animals per experimental
group from two experiments. *p , 0.05, ANOVA, APP/PS1+Th1 cells
versus control APP/PS1 mice,+p , 0.05, ANOVA, APP/PS1 + Th1 cells
versus APP/PS1 plus Th1 cells plus anti–IFN-g Ab. Tg, Transgenic.
Anti–IFN-g Ab attenuated the effect of Th1 cells on be-
accumulation. APP/PS1 mice mice were injected with Ab-specific Th1
cells and treated with anti–IFN-g Ab or a control Ab as described in Fig. 6.
Mice were sacrificed 5 wk after cell transfer. Cryostat sections were
stained with Congo red to assess Ab-containing plaques in hippocampus
and cortex; the mean number of plaques was recorded (A), and the con-
centrations of insoluble Ab1–42(B), Ab1–40(C), and Ab1–38(D) were
quantified by ELISA in brain tissue prepared from APP/PS1 and WT mice.
Data represent mean 6 SEM from four to five animals per experimental
group from two experiments. **p , 0.01, ***p , 0.001, ANOVA; (n = 4–
6);+p , 0.05,++p , 0.01,+++p , 0.001, ANOVA; control APP/PS1 mice
versus APP/PS1 mice that received Th1 cells;xp , 0.05;xxxp , 0.001,
ANOVA, APP/PS1 + Th1 cells versus APP/PS1 + Th1 cells + anti–IFN-g
Ab. Con, Control.
Anti–IFN-g Ab attenuated the effect of Th1 cells on Ab
The Journal of Immunology 2247
at IRIS - IReL Consortia on July 23, 2013
Sections prepared from WT and APP/PS1 mice were assessed
for CD11b immunoreactivity as a measure of microglial acti-
vation. Immunoreactivity was negligible in sections of hippo-
campus and cortex prepared from WT mice (Fig. 6), whereas
CD11b staining was observed in both areas in some but not all
APP/PS1 mice. Quantification of the data indicated that CD11b
expression was markedly increased in APP/PS1 mice that re-
ceived Th1 cells, and the increase was significant in the case of
the cortex (*p , 0.05; ANOVA), where Th17 cells exerted a
Neutralization of IFN-g attenuates the effect of Th1 cells on
Having shown a specific effect of Th1 cells on spatial memory
and Ab accumulation, we assessed the role of the key Th1 cy-
tokine, IFN-g, by treating APP/PS1 mice with a neutralizing
anti–IFN-g Ab prior to, and following, Th1 cell transfer. There
was no significant effect of treatment on latency to reach the
platform (data not shown), confirming the data shown in Fig. 3.
However, we found that the path length taken to reach the
platform decreased with training in all groups except in APP/PS1
mice, which received Th1 cells (Fig. 7A), and analysis of the
mean data indicates that path length was significantly increased
in this group compared with APP/PS1 mice that did not receive
Th1 cells (*p , 0.05; ANOVA; Fig. 7B). Administration of anti–
IFN-g Ab significantly attenuated the Th1 cell–induced effect (+p ,
0.05, ANOVA, versus APP/PS1 mice that received Th1 cells). In
the probe test, treatment with Th1 cells decreased the percentage
of the total time and distance each animal spent swimming in the
quadrant that previously contained the platform (*p , 0.05;
ANOVA; Fig. 7C, 7D), confirming the findings presented in
Fig. 3. Treatment with anti–IFN-g significantly reversed the
effect of Th1 cells (+p , 0.05, ANOVA, versus APP/PS1 mice
that received Th1 cells).
Neutralization of IFN-g attenuates the effect of Th1 cells on Ab
Anti–IFN-g Ab attenuated the effects of Th1 cells on plaque
number and concentration of insoluble Ab1–38, Ab1–40, and Ab1–42
in tissue prepared from APP/PS1 mice (Fig. 8). These measures
were increased in tissue prepared from APP/PS1 mice compared
with WT mice (**p , 0.01, ***p , 0.001, ANOVA; Fig. 8), and
these were significantly increased by administration of Th1 cells
(+p , 0.05,++p , 0.01,+++p , 0.001, ANOVA, control APP/PS1
mice versus APP/PS1 mice that received Th1 cells). The increase
in Ab1–38, Ab1–40, and Ab1–42induced by Th1 cells was attenuated
when mice were treated with anti–IFN-g Ab (xp , 0.05,xxxp ,
0.001, ANOVA, APP/PS1 mice that received Th1 cells versus Ab-
treated APP/PS1 mice that received Th1 cells).
CD11b immunoreactivity was negligible in sections prepared
from hippocampus and cortex of WT mice (Fig. 9), whereas some
staining was observed in both areas in APP/PS1 mice. This was
greater in APP/PS1 mice that received Th1 cells, but this effect
was ameliorated to some degree in sections prepared from APP/
PS1 mice that received Th1 cells and anti–IFN-g Ab. Immuno-
reactivity was similar in sections prepared from control APP/PS1
mice and APP/PS1 mice, which received anti–IFN-g Ab. Analysis
of staining using confocal microscopy indicated that CD11b im-
munoreactivity (green staining; Fig. 10) was colocalized with Ab
deposition (red staining) in hippocampus and cortex. As shown in
Figs. 6 and 9, Ab accumulation was increased in sections prepared
from APP/PS1 mice, which received Th1 cells, and this effect was
attenuated by anti–IFN-g Ab treatment (Fig. 10). These findings
demonstrate that the impact of Th1 cells on Ab plaque burden and
microglial activation was mediated through IFN-g.
The significant finding of this study is that adoptive transfer of
Th1 cells increases Ab accumulation and microglial activation in
the effect of Th1 cells on CD11b immu-
noreactivity. APP/PS1 mice mice were
injected with Ab-specific Th1 cells and
treated with anti–IFN-g Ab or a control
Ab as described in Fig. 6. Mice were
sacrificed 5 wk after T cell transfer. (A) Mi-
croglial activation was assessed by CD11b
immunoreactivity in the cortex and hippo-
campus. (B) Data are means 6 SEM. Data
represent mean 6 SEM from four to seven
animals per experimental group from two
experiments. *p , 0.05, ANOVA, versus
control,+p , 0.05, ANOVA, versus Th1.
Anti–IFN-g Ab attenuated
2248PATHOGENIC ROLE OF IFN-g IN MURINE ALZHEIMER’S MODEL
at IRIS - IReL Consortia on July 23, 2013
the brain of 6- to 7-mo-old APP/PS1 mice and impairs perfor-
mance in a Morris water maze; these effects are attenuated by
treatment of mice with anti–IFN-g Ab.
It hasbeenrecognized forsometime thatTcells caninfiltratethe
brain (38, 39). T cell infiltration is significantly enhanced under
pathological conditions (for example, in multiple sclerosis and
EAE), and this is due, at least to some extent, to an increase in
blood–brain barrier permeability (23, 40). In this study, we report
that there is a significant increase in the number of CD3+CD4+
cells in the brain of APP/PS1 mice compared with WT mice and
that a proportion of these are Th1 and Th17 cells. Consistent with
this is the observation that significant numbers of peripheral T
cells are present in the postmortem brain of AD patients compared
with the relatively low numbers of cells in other degenerative
dementia cases and, importantly, that these cells are clustered in
areas of the brain in which pathology is more marked, such as the
hippocampus and limbic regions (25). However, the role of T cells
in the pathogenesis of AD is not clear, with circumstantial evi-
dence of both host protective and damaging roles for Ab-specific
T cells. Peripheral T cells specific for Ab1–40have been detected
in healthy individuals, but were absent in patients with AD (41),
possibly suggesting that Ab1–40-specific T cells may prevent the
development of Ab plaques. It has also been reported that Th1
cells directed against Ab1–42are present in young individuals but
decline with age and are lost in patients with AD, in whom IL-
10–producing regulatory T cells predominate (42).
Vaccine studies in mouse models have shown that immunization
with Ab42in CFA prevented the development of Ab plaques and
reduced the development of AD-like neuropathology (33, 43). The
protection was associated with Ab and could be mimicked by
passive transfer of Ab-specific Abs (44). There is also evidence
from a clinical trial that active immunization with Ab42, formu-
lated with the adjuvant QS21 (AN 1792), can reduce plaque
burden in AD patients (45), though a number of patients devel-
oped meningoencephalitis, and the trial was halted. Although the
cause of the meningoencephalitis is not clear, it has been sug-
gested that it could result from the induction of inflammatory
T cell responses (46). Interestingly, QS-21, the adjuvant used in
AD vaccine, has been shown to promote Th1 responses to coad-
ministered foreign Ag in mice (47). Our findings are consistent
with a pathogenic role for Th1 cells, at least in a mouse model
To evaluate the impact of different T cell subtypes on plaque
burden in the brain, we adoptively transferred Ab-specific Th1,
Th2, and Th17 cells into 6- to 7-mo-old APP/PS1 mice. Consistent
with previous findings (36), we found that there was some Ab
accumulation in the brain of the 6-mo-old APP/PS1 mice. This
was accompanied by increased concentrations of Ab1–42,Ab1–40,
and Ab1–38in cortical tissue. However, transfer of Th1 cells in-
creased deposition of Ab (determined by Congo red staining) and
markedly increased cortical Ab concentration. This suggests that
Ab-specific Th1 cells may play a role in the development of Ab
plaques in the brain. This was confirmed by treatment of mice
with a neutralizing anti–IFN-g Ab, which attenuated the effect of
Th1 cells on Ab accumulation. In contrast with the effect of Th1
cells, transfer of Th17 cells, which have been associated with
pathology in EAE and other autoimmune/inflammatory diseases,
and Th2 cells, which have a more anti-inflammatory function in
other diseases, did not enhance Ab accumulation in the brain.
These findings are consistent with our earlier report that Ab-
specific Th1 cells enhance proinflammatory cytokine production
and MHC class II and costimulatory molecule expression by Ab-
stimulated microglia, whereas Ab-specific Th2 cells suppress cy-
tokine production by glial cells (32).
Under resting conditions, microglia are maintained in a quies-
cent state in the brain because of the presence of neuroimmune
regulatory molecules that enable the interaction with other cells,
low concentrations of stimulatory factors such as IFN-g and other
inflammatory cytokines, and the presence of minimal numbers of
immune cells like T cells (13). However, microglial activation
occurs following any insult, and an activated state is a character-
istic of most, if not all, neurodegenerative diseases in which these
cells can assume the role of APC. Modest microglial activation
was observed in the hippocampus and cortex of 6- to 7-mo-old
APP/PS1 mice but transfer of Th1 cells markedly increased acti-
vation. This is consistent with our previous findings that showed
that Ab-specific Th1 cells increased microglial activation in vitro
(32). In parallel with its effect on Ab accumulation, treatment of
mice with anti–IFN-g Ab attenuated the effect of Th1 cells on
microglial activation. It is well established that IFN-g is among
the most potent activators of microglia (15, 48) and synergizes
with Ab to increase expression of cell-surface markers of activa-
tion and production of inflammatory cytokines (15, 49). Chakra-
barty et al. (50) reported that viral delivery of IFN-g gene
promotes microglial activation and clearance of Ab. We observed
that Th1 cells also promoted microglial activation but that this was
associated with an increase in Ab plaques. We do not have a de-
finitive explanation for the discrepancy in these studies other than
the differences in the experimental approaches: virally-delivered
IFN-g, which had effects, such as basal ganglia calcification, in
CD11b immunoreactivity. APP/PS1 mice were injected with Ab-specific
Th1 cells and treated with anti–IFN-g Ab or a control Ab as described in
Fig. 6. Mice were sacrificed 5 wk after T cell transfer. Microglial activation
and Ab deposition was assessed by confocal microscopy. Cells were
stained with DAPI (nuclei; blue), amyloid-b (red), and CD11b (green).
Original magnification 340 and enlarged panels 360. Data are repre-
sentative of five mice per experimental group from two experiments. Con,
Anti–IFN-g Ab attenuated the effect of Th1 cells on
The Journal of Immunology2249
at IRIS - IReL Consortia on July 23, 2013
WT as well as Tg mice, compared with i.v. injected Ab-specific
Th1 cells, in which the effects were largely confined to Tg mice.
One interpretation of the data, as suggested in this study, is that
anti–IFN-g prevents Th1 cell–induced activation of microglia, but
it is possible that the Ab treatment affects infiltration of cells,
perhaps by altering chemotaxis or exerting an effect on blood–
brain barrier permeability.
Our studies with Th1 cells expressing GFP demonstrated that at
least a proportion of the transferred Th1 cells did migrate from the
periphery into the CNS. Interestingly, IFN-g–secreting CD8 as
well as CD4 T cells were detected in the brain following i.v. in-
jection of Ab-specific Th1 cells. This is consistent with studies in
the EAE model that have demonstrated that Th1 cells preferen-
tially infiltrate the CNS and facilitate recruitment of other in-
flammatory T cells (51). Interestingly, the migration of T cells into
the brain and subsequent behavioral deficits was significantly
more pronounced following transfer of Ab-specific Th1 cells into
APP/PS1 when compared with WT mice. This may reflect the
higher Ab burden in the APP/PS1 mice and might suggest local
Ag stimulation of IFN-g–secreting T cells, which were at a sig-
nificantly higher frequency in the brains of APP/PS1 compared
with WT mice.
Previous studies from this laboratory have shown that Ab-
specific Th1 cells enhanced Ab-induced activation of microglia
(32). Furthermore, the increase in microglial activation in APP/
PS1 mice was accompanied by increased expression of inflam-
matory cytokines, including TNF-a and IL-1b (52). Interestingly,
TNF-a and IL-1b have been shown to increase activity and/or
expression of g- and b-secretases (6, 7), which leads to Ab de-
position. Although activated microglia may phagocytose and
remove Ab aggregates (50), IL-1b–expressing microglia are as-
sociated with Ab plaques and neurofibrillary tangles in the brain
of AD patients, where they correlate with progressive neuronal
damage (53). Furthermore, IL-1b can promote synthesis of APP in
endothelial cells (54). It has also been reported that IFN-g–in-
duced activation of microglia enhanced processing of APP and
suppressed Ab clearance (55). We found that Th1 cells, which
increase IL-1b expression by microglia (32), enhanced soluble
and insoluble Ab concentrations in the brains of APP/PSI mice.
However, it must be acknowledged that Ab potently activates
microglia in vitro and in vivo (56, 57), and therefore it is possible
that there may be a feedback loop, leading to persistent microglial
activation and Ab accumulation with the subsequent pathogenic
Although there was significant Ab accumulation in the brain of
6- to 7-mo-old APP/PS1 mice, we found no evidence of genotype-
related changes during the training phase in the Morris water maze
or during the probe test, contrasting with previous reports that
indicated a deficit in slightly older (8-mo-old) APP/PS1 mice (58,
59). It has been suggested that cognitive deficits correlate with
insoluble Ab in Tg2576 mice (60) and APP/PS1 mice (61), but
this view is not supported by the present findings. However, we
report that transfer of Th1 cells doubled the concentration of in-
soluble Ab in brain tissue, and this was associated with deterio-
ration in cognitive function in the probe test; this raises the
possibility that a threshold concentration of Ab must be reached
before an impact on spatial learning is exerted. In contrast to the
effect of Th1 cells on APP/PS1 mice, transfer of Th2 cells or Th17
cells exerted no effect in the spatial learning task or on either
plaque number or Ab accumulation. The present findings are at
variance with an earlier report that indicated that adoptive transfer
of a mixed T cell preparation improved performance of 10-mo-old
APP/PS1 mice in a radial arm maze task (62). Although no effect
on insoluble Ab or Ab plaque numbers was observed, the authors
suggested that microglia or monocytes were stimulated to clear
Ab because the distribution of Ab-immunoreactive cells in hip-
pocampus of mice that received T cells was similar to the distri-
bution of MHC class II–positive cells. More recent data from this
group suggested that the beneficial effects on behavior may be
Th2 cell–mediated because the effect was evident when T cells
had been incubated in vitro in the presence of IL-2 and IL-4 (31).
We have recently reported that the Ab-induced microglial acti-
vation in vitro is attenuated by Th2 cells (32), and the current
study found that Th2 cells, unlike Th1 cells, did not enhance
plaque burden in vivo.
Although beneficial effects of T cells in the brain have also been
observed (63), the evidence presented in this study indicates that
Th1 cells, but not Th2 or Th17 cells, contribute to Ab accumu-
lation and development of a functional deficit in APP/PS1 mice
during the early stages of development of pathology. In this
model, the effects appear to be mediated by IFN-g and are asso-
ciated with enhanced microglial activation, which may trigger
inflammatory changes that propagate a damaging cascade of
events and further development of pathology.
We thank Barry Moran (School of Biochemistry and Immunology, Trinity
College Dublin) for assistance with cell sorting and Mathew Campbell
(School of Genetics and Microbiology, Trinity College Dublin) for sup-
plying GFP mice.
K.H.G.M. is a cofounder and shareholder in Opsona Therapeutics and
TriMod Therapeutics Ltd., startup companies involved in the development
of immunotherapeutics. The other authors have no financial conflicts of in-
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