J Alzheimers Dis. 2013 (May 21); 35 (4): 697–713
Erik Hjorth, Mingqin Zhu, Veronica Cortes Toro, Inger Vedin, Jan Palmblad
Division of Neurodegeneration,
Department of Neurobiology,
Care Sciences & Society,
The use of supplements with omega-3 fatty acids (FAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is widespread due to proposed beneficial effects on the nervous and cardiovascular systems. Many effects of omega-3 FAs are believed to be caused by down-regulation and resolution of inflammation. Alzheimer's disease (AD) is associated with inflammation mediated by microglia and astrocytes, and omega-3 FAs have been proposed as potential treatments for AD. The focus of the present study is on the effects of DHA and EPA on microglial phagocytosis of the AD pathogen amyloid-β, on secreted and cellular markers of immune activity, and on production of brain-derived neurotrophic factor (BDNF). Human CHME3 microglial cells were exposed to DHA or EPA, with or without the presence of Aβ42. Phagocytosis of Aβ42 was analyzed by flow cytometry in conjunction with immunocytochemistry using antibodies to cellular proteins. Secreted proteins were analyzed by ELISA. Both DHA and EPA were found to stimulate microglial phagocytosis of Aβ42. Phagocytosis of Aβ42 was performed by microglia with a predominance of M2 markers. EPA increased the levels of BDNF in the culture medium. The levels of TNF-alpha were decreased by DHA. Both DHA and EPA decreased the pro-inflammatory M1 markers CD40 and CD86, and DHA had a stimulatory effect on the anti-inflammatory M2 marker CD206. DHA and EPA can be beneficial in AD by enhancing removal of Aβ42, increasing neurotrophin production, decreasing pro-inflammatory cytokine production, and by inducing a shift in phenotype away from pro-inflammatory M1 activation.
Keywords: Amyloid, brain-derived neurotrophic factor, cytokine, DHA, EPA, interleukin, M1, M2, resolution
From the FULL TEXT Article:
Alzheimer’s disease (AD) is a neurodegenerative
and progressive disease characterized by the impairment
and death of neurons, increased levels of the
amyloid-β (Aβ) peptide , increased presence of
intracellular tangles composed of hyperphosphorylated
forms of the microtubule protein tau , and
chronic inflammation [3–5]. Inflammation is prominent
in AD as evidenced by activated microglia
and astrocytes , and increased levels of proinflammatory
cytokines in the brain [3, 5].
On a clinical level, AD is characterized by cognitive
deficits that initially affect learning and memory,
but later in the disease are manifested by a global
cognitive decline. The clinical symptoms mirror the
pathological changes in the brain, where the neuronal
loss and plaque/tangle pathology begin in memoryrelated
areas (entorhinal cortex, hippocampus), and
then spread to other parts of the cortex [7, 8].
It is believed that increased levels of Aβ represent
one of the culprits for the disease . Aβ is produced by
intramembranous cleavage of the Aβ protein precursor
(AβPP), yielding a peptide that is prone to aggregation,
thus forming oligomers, fibrils, and plaques .
Studies on AD brains and data from animal studies
show the presence of activated glial cells around the
plaques [5, 11, 12]. It is unclear whether inflammation
is an initial driving force in the pathogenesis of AD or
a consequence of the disease. Aβ has been shown to
induce inflammation [13–19], with release of cytokines
and other inflammatory factors. In turn, inflammatory
cytokines have been shown to increase the production
of Aβ [15, 20, 21], suggesting the presence of a vicious,
self-reinforcing circle between inflammation and Aβ
production and, thus, a role for inflammation in the
primary pathology of AD.
Clearance of debris by phagocytosis and secretion of
neurotrophic growth factors from activated astrocytes
and microglia are activities related to inflammation
that can protect and improve the function of neurons
[22–24]. However, in the case of chronic inflammatory
states, such as in AD, the detrimental and tissuedamaging
activities of inflammation are dominating
. Early evidence of a role for inflammation in AD
pathogenesis originates from epidemiological studies,
showing reduced prevalence of AD in patients under
treatment with non-steroidal anti-inflammatory drugs
(NSAIDs) (, see ).
Dietary supplementation with the ω3 fatty acids
(FAs) docosahexaenoic acid (DHA) and eicosapentaenoic
acid (EPA) is very common, and has been
ascribed several health benefits related to the nervous
system [28–31]. Furthermore, the levels of polyunsaturated
fatty acids (PUFAs, i.e., ω3) have been shown
to be reduced in postmortem brain tissue , and
in plasma samples , from AD patients. Several
studies have shown that DHA and EPA can exert antiinflammatory
activities, and their beneficial effects are
commonly ascribed to this property (see [34, 35]).
In vitro studies have shown a suppressing effect by
EPA on inflammatory cytokine production in human
macrophages , and DHA has been shown to downregulate
inflammatory proteins in vitro  and in
vivo. Dietary supplementation with DHA-rich ω3 FAs
resulted in increased plasma concentrations of DHA
(and EPA) in AD patients, and this was associated with
reduced release of interleukin (IL)-1β, IL-6, and granulocyte
colony-stimulating factor from peripheral blood
mononuclear cells ex vivo . DHA is the ω3 FA that
has received most attention for its beneficial effects on
factors related to AD [40–44]. Authors of this study
performed the first completed intervention study in
which AD patients were treated with ω3-supplements
(the OmegAD study) . A beneficial effect was not
found, except in a subgroup with mild symptoms, suggesting
the importance of early intervention. Similar
studies performed after this study have provided little
support for a curative effect of ω3 FAs on established
AD, but rather emphasizing the findings of a potential
benefit on early AD .
DHA and EPA are intimately associated with the
resolution phase of inflammation. In this final stage
of the immune response, cleaning, healing, and regeneration
of the tissue take place for the restoration of
homeostasis, concurrent with a down-regulation of
the inflammatory response. The increased phagocytic
activity in the resolution phase is not associated with
inflammatory activation (non-phlogistic phagocytosis)
(e.g., ). Resolution is induced by specialized proresolving
mediators (SPMs), that are derivatives of
FAs [34, 35, 48]. The SPMs include the resolvin E
series, derived from EPA , and the resolvin D
series, derived from DHA [44, 49]. Studies on mice
have shown that healing and regeneration of the tissue
is promoted by increased levels of growth factors,
emphasizing the role of growth factors in resolution
An effective treatment for AD is lacking. Reducing
the levels of Aβ and the inflammation in the AD
brain, while at the same time increasing the neurotrophic
signaling, is a reasonable approach for the
treatment of AD. The capacity to phagocytose Aβ by
microglia has been demonstrated in in vitro studies
. Interestingly, this phagocytic capacitywas shown
to be reduced in microglia incubated with human
cryostat sections containing amyloid plaques , suggesting
that the environment in AD brain tissue has
“anti-resolving” properties. Increased clearance of Aβ
can be accomplished by stimulating microglia into
increased phagocytosis in vitro .
Phagocytosis in the acute phase of inflammation may have neurotoxic
consequences, not desirable from a treatment point
of view. However, the prominent phagocytic activity
during the resolution phase of inflammation is a
promising therapeutic target. Therefore, we have performed
a series of experiments on human microglial
cells, in which the capacity of DHA and EPA to stimulate
phagocytosis of Aβ42 was the primary outcome.
The effects on cell-associated and secreted markers and
effectors of immune activation were also investigated.
The M1/M2 polarization axis is commonly applied to
the interpretation of the responses of macrophages and
similar cells, such as microglia. To establish the effect
on phenotype polarization, we investigated the M1 activation
marker CD40 [55, 56] and the co-stimulatory
factor CD86.We also analyzed theM2markers CD163
and CD206, as well as the deactivating receptor
CD200R. CD163 and CD206 also have a direct link
to phagocytosis by their ability to mediate this through
recognition of pathogens or debris [57, 58].
We analyzed the secretion of the important memoryrelated
neurotrophin brain-derived neurotrophic factor
(BDNF), a protein shown to be decreased in the brain
of AD-patients [59, 60], and of the inflammatory (M1)
cytokines IL-6 and tumor necrosis factor (TNF)-α,
shown to be increased in AD [3, 61]. The antiinflammatory
M2 cytokine IL-10 was also measured.
MATERIALS AND METHODS
DHA and EPA were purchased from Nucheckprep
(Elysian, USA). Aβ42 conjugated with HiLyteFluor-
488 or biotin was obtained from Anaspec (Fremont,
USA). Dimethylsulfoxide (DMSO), Triton-X100, and
bovine serum albumin (BSA) were purchased from
Sigma (Stockholm, Sweden). Normal donkey serum
and fluorescence mounting medium were purchased
from DakoPatts (Stockholm, Sweden). Donkey antirabbit
Northern Light-647 conjugated antibodies, and
ELISA-kits for IL-6 IL-10, (TNF)-α, and BDNF were
purchased from R&D systems (Abingdon, United
Kingdom). Cell culture medium, phosphate-buffered
saline (PBS), GlutaMaxII, fetal calf serum (FCS), and
PBS-based enzyme-free cell dissociation buffer, were
purchased from Invitrogen (Stockholm, Sweden). Cell
culture bottles and multi-well plates were purchased
from BD Biosciences (Stockholm, Sweden).
Human microglial cells (CHME3) were obtained
as a kind gift from Prof. M. Tardieu, Neurologie
p´ediatrique, Hˆopital Bicˆetre, Hˆopitaux de Paris, Paris,
France. The CHME3 cells were cultured in T75 or
T175 bottles in culture medium (DMEM/high glucose
w/o sodium pyruvate, supplemented with GlutaMaxII
and 10% heat-inactivated FCS). The cells were subcultured
at confluence using enzyme-free cell dissociation
buffer, afterwashing once with PBS without Mg2+ and
For flow cytometry and analysis of secretory products,
the CHME3 microglial cells were seeded in
6-well plates. The CHME3 cells are quick proliferators,
and care must be taken so the cultures do not
become over-confluent. In our experience, starting the
experiments at 60–70% confluency of the cells produce
cultures of 80–85% confluency after 24 h, thus
Aβ42 was dissolved in DMSO to a concentration
of 5 mg/ml, and stored in darkness at +4°C until use
in a final concentration of 1 µg/ml for all experiments
on cells. DHA and EPA were diluted in 95%
EtOH to a concentration of 200mM and stored in
a nitrogen atmosphere. Before the experiments, the
cells are washed with serum-free medium, and then
treated with Aβ42 or vehicle (DMSO) together with
DHA, EPA, or vehicle (95% EtOH), also in serum-free
medium. The final concentration of DMSO in the cultures
was 0.02%, and the final concentration of EtOH
Assessment of aggregational forms of Aβ42 by immunoblotting
Aβ42 peptidewas diluted with DMEM/high glucose
culture medium to the final concentration of 1 µg/ml
(used in the cell experiments) and 5 µg/ml, and seeded
into a 24-well plate. Medium without Aβ42 was used
as control. All the samples were incubated at +37°C
and 5% CO2 in a cell culture incubator for 6 and 24 h.
Freshly dissolved Aβ42 peptide was prepared before
start of the electrophoresis. In order to keep the state
of aggregation ofAβ42 at the time points studied, all the
samples were treated with the cross-linker glutaraldehyde
at a final concentration of 2.5%. Equal volume
of sample buffer (75 µl) was added to each sample
(75µl) and incubated 10 min before loading the gel.
We found that Aβ42 is prone to stick to the plastic
of the cell culture well. In order to detach this, each
well was flushed with 75 µl sample buffer by trituration,
and then mixed with an equal volume of the
medium previously removed. Proteins were separated
by electrophoresis, followed by overnight transfer to
0.22mm nitrocellulose membrane. After transfer, the
membranes were incubated with 6E10 primary antibody
diluted 1:700 (Nordic Biosite, Sweden). The
6E10 antibody is raised against the COOH-terminus
of Aβ peptide. The membranes were then incubated
with horse-radish peroxidase (HRP)-conjugated sheep
anti-mouse secondary antibody diluted 1:1000 (GE
Healthcare, Sweden), after which chemiluminescence
(ECLTM Prime, GE Healthcare) reagent was applied
and the membranes analyzed with aCCDcamera (Fujifilm
LAS-3000 luminescent image analyzer).
The amount of fibrillar Aβ42 in the culture medium
was analyzed using the Thioflavin-T (ThT) assay. Culture
medium incubated with 0, 1, and 5 µg/mlAβ42 for
6 and 24 h, as well as medium in which the Aβ42 was
freshly dissolved, were analyzed. In short, ThT was
prepared in a stock solution of 25 mM, diluted in PBS
to 50 µM, in which the medium to be analyzed was
prepared to a final concentration of 25 µM ThT, and
incubated at +37°C for 20 min, followed by analysis in
a Tecan plate reader (excitation wavelength: 440 nm;
emission wavelength: 480 nm). To control the validity
of the ThT assay, Aβ42 (0, 2.5, 25, and 250 µg/ml) was
incubated in PBS for 96 h and analyzed with the same
Quantification of Aβ42 phagocytosis and cellular markers by flow cytometry
To investigate the influence of DHA and EPA on
phagocytosis of Aβ42 the CHME3 microglial cells
were exposed to 1µg/ml of this peptide together with
DHA or EPA, or vehicle (EtOH). The cells were
harvested at 2, 6, and 24 h, and analyzed by flowcytometry
for phagocytosis of Aβ42 and the presence
of inflammatory and phenotype markers. The levels
of secreted products were analyzed by ELISA (see
After the treatments, the CHME3 microglial cells
were dissociated with PBS-based enzyme-free dissociation
buffer, and centrifuged at 1500× g for 10 min.
The cells were then resuspended and fixed in 300 µl
of 1% para-formaldehyde in PBS for 40 min at room
temperature. Subsequently, the cells were washed by
addition of 10 ml PBS followed by centrifugation at
1500× g for 10 min, removal of supernatant, resuspension
in 300 µl of PBS, and then stored at +4°C in
Analysis of phenotype markers by directly conjugated antibodies
After harvesting and fixation, the cells were
incubated with phytoerythrin (Biolegend)-conjugated
antibodies to CD40 (diluted 1:200), CD86 (diluted
1:100) and CD200R (diluted 1:100), respectively, and
with AlexaFluor647 (Biolegend)-conjugated antibodies
to CD163 (diluted 1:50) and anti-CD206 (diluted
1:100). After incubation with the antibodies at +4°C
for 40 min, the cells were washed by adding PBS and
centrifuged at 2000× g for 10 min, after which the
pelleted cells were resuspended and analyzed with
flow-cytometry using the corresponding isotype control
(Biolegend) to set the limit of background for each
Enzyme-linked immunosorbent assay (ELISA)
The levels of IL-6, IL-10, TNF-α
, and BDNF were
analyzed in the cell culture medium obtained from
experiments terminated at 24 h. After dissociation and
centrifugation as described above, the culture medium
was stored at –20°C until analysis with commercially
available ELISA-kits. To detect the low levels of IL-
10 and TNF-α in the medium, the assay protocol was
adjusted so that the samples were incubated overnight
in +4°C in addition to the 2 h at room temperature. In
the case of IL-10 and TNF-α ELISAs, QuantaRed fluorescent
substrate (Thermo Scientific) was used instead
of the colorimetric substrate in the standard protocol.
Analysis of optical density (OD), or fluorescence, was
performed in a TECAN Safire2 plate reader.
The presented data are the result from 10–14 individual
experiments. In each experiment, the data for
each treatment were normalized to the averaged data
of that particular parameter in that experiment. To
allow for parametric statistical analysis, the normalized
data were logarithmized (natural logarithm, e),
after which the data were analyzed for systematic variance
using ANOVA. When significant variance was
found, pair-wise comparison between groups using
Fisher’s post hoc test was performed. The comparison
between different variables was performed with the
non-parametric Wilcoxon matched-pairs test on nonnormalized
and non-logarithmized data. The graphs
show the normalized and logarithmized data. All statistical
analyses were performed in Statistica v10
Assessment of aggregational forms of Aβ42
Immunoblottingwas performed to investigate which
aggregation forms of Aβ42 that the microglial cells
were exposed to in the experiments (Fig. 1). The concentration
(1 µg/ml) used in the treatment experiments,
as well as a higher concentration (5 µg/ml), were analyzed.
No bands were visible in the control (medium
without addition ofAβ42) (data not shown). The results
show a mixture of lengths of Aβ42 oligomers. Bands
corresponding to the size of monomers and dimers
were seen at all incubation times, as well as in the
freshly dissolved Aβ42 preparation, of both concentrations
(Fig. 1A). Due to the weak luminescence
signal of the bands from incubation of 1µg/ml Aβ42,
image analysis was performed on bands obtained from
5µg/ml Aβ42 (Fig. 1B). Bands corresponding to the
size of tetramers (19.2 kD), hexamers (26.8 kD), and
other large oligomers (>70 kD) were observed. Factorial
ANOVA (Fig. 1B) showed that the intensity of
bands corresponding to monomers was stronger than
that of tetramers, hexamers, and large oligomers after
0, 6, and 24 h of incubation (p < 0.005–0.05), indicating
a predominance of Aβ42 monomers. No fibrillar
Aβ42 was detected by the ThT-assay at any time point
or concentration tested (data not shown).
Effects of ω3 FAs on phagocytosis of Aβ42
Exposure of the human CHME3 microglial cells to
1µg/ml of Aβ42 for 2, 6, and 24 h did not result in any
visible changes in morphology or other apparent signs
of activation. Neither did the treatment with DHA or
EPA change the morphology.
Incubation of the microglia for 2 h with DHA in
the concentrations of 0.1, 0.5, and 1 µM, significantly
increased phagocytosis (p < 0.005, p < 0.05, and
p < 0.05, respectively) as compared to control (treatment
with Aβ42 alone) (Fig. 2A). At 24 h, all
concentrations increased the number of microglia
showing phagocytosis of Aβ42 (p < 0.005–0.05).
Treatment with EPA in the concentrations of
0.01, 0.5, and 1 µM for 2 h increased the number
of Aβ42-positive cells above control (Aβ42 alone)
(p < 0.005–0.05). Results from incubation for 6 h did
not reach significant variance. Similar to DHA, the
EPA-stimulated phagocytosis of Aβ42 regained vigor
after 24 h of incubation, showing a significant variance.
The post hoc test showed that 0.01, 0.5, and 1 µMEPA
increased the number of Aβ42-positive cells with 24%
above control level (p < 0.005–0.05) (Fig. 2B). At 24 h,
significant differences between these concentrations
indicate a bell-shaped dose-response curve.
Effects of ω3 FAs on cellular markers related to M1-activation
To investigate the immunological phenotype of the
human microglia, markers for M1 (CD40 and CD86)
activation were used.
Effects of ω3 FAs on cellular markers related to M2-activation
The basal level (vehicle control) of CD40-positive
microglia was 57.8% at 2 h, 53% at 6 h, and 58.4%
at 24 h. DHA did not produce any changes in the
number of positive cells until at 24 h (Fig. 3A), when
a decrease was observed with 0.5 µM (p < 0.05) and
1µM (p < 0.01), as compared to control.
The level of CD40-positive microglia when incubated
with Aβ42 alone was 40% at 2 h, 26.2% at 6 h,
and 21.2% at 24 h. No significant effects could be seen
upon co-incubation of the cells with Aβ42 and DHA.
Incubation with EPA did not have statistically
significant effects on the number of CD40-positive
microglia, but in the experiments on EPAtogether with
Aβ42, a significant decrease in the number of CD40-
positive cells could be seen at 6 h with 0.5µM EPA
(p < 0.0005, Fig. 3B), as compared with control (Aβ42
The basal level (vehicle control) of CD86-positive
microglia was 8.5% at 2 h, 7.5% at 6 h, and 10.4%
at 24 h. At 24 h, treatment with DHA resulted in
a statistically significant decrease in the number of
CD86-positive cells as compared to control (p < 0.05)
(Fig. 4A, left panel).
The level of CD86-positive microglia when incubated
with Aβ42 alone was 7.8% at 2 h, 4.2% at
6 h, and 6.6% at 24 h. For both DHA and EPA, coincubation
of the microglia with Aβ42 induced more
prominent effects than when each FA was added alone.
Co-incubation of the cells with Aβ42 and DHA gave
a hint of a bell-shaped dose-response curve, which
was supported by a significant variance in the data
(p < 0.0005, Fig. 4A, right panel). At 6 h, the lowest
(0.05µM), and highest (1 µM) concentrations ofDHA
resulted in a decrease in the number of CD86-positive
microglia as compared to Aβ42 alone (p < 0.01 and
and p < 0.005, respectively). At 24 h, the reduction in
CD86-positive cells induced by 0.05 and 1 µM DHA
was still present (p < 0.05). The image of a bell-shaped
dose-response relationship was even stronger than at
6 h and supported by significant variance in the data
(p < 0.05).
The only effect of EPA alone on the number of
CD86-positive microglia was a reduction by 0.5 µM
at 24 h (p < 0.05, Fig. 4B, left panel). Co-incubation of
the cells with EPA and Aβ42 also (similar to DHA)
resulted in data for which a bell-shaped dose-response
relationship could be discerned. In contrast to DHA,
the bell-shaped curve seen for EPA appeared already
at 2 h (Fig. 4B, right panel). Thus, at 2 h the lowest
(0.005µM) and highest (1µM) concentration of EPA
reduced the number of CD86-positive cells (p < 0.05
and p < 0.005, respectively). At 6 and 24 h, the effect
of EPA on CD86 had dissipated.
The number of cells bearing the M2 markers CD163
and CD206 was also investigated after exposure of
the microglia to FAs with and without Aβ42. There
was a smaller number of microglia with M2 markers
as compared to the M1 marker CD40 at basal
The basal level (vehicle) of CD163-positive
microglia was 9%at 2 h, 12% at 6 h, and 10.8% at 24 h.
The level of CD163-positive microglia when incubated
withAβ42 alonewas 8.6% at 2 h,15%at 6 h, and 13.4%
at 24 h. Treatment with DHA or EPA at any concentration
tested did not induce any significant change in
the number of CD163-positive cells. Thiswas true both
for cultures treated with FAs alone, and in combination
with Aβ42 (data not shown).
The basal level (vehicle control) of CD206-positive
microglia was 14.3% at 2 h, 9.5% at 6 h, and 10.9%
at 24 h. At 6 h, incubation with 0.5 and 1 µM DHA
reduced the number of CD206-positive cells as compared
to control (p < 0.05 and p < 0.005, respectively)
(Fig. 5). This result was reversed at 24 h, when 0.1 and
0.5µMDHA increased the number of CD206-positive
microglia (p < 0.005 and p < 0.05, respectively). The
highest dose of DHA resulted in a significant decrease
in the number of CD206-positive cells, as well as with
0.1 and 0.5 µM DHA, indicating a bell-shaped doseresponse
curve at 24 h.
The level of CD206-positive microglia when incubated
with Aβ42 alone was 12.9% at 2 h, 7.7% at 6 h,
and 9.8% at 24 h. Treatment with DHA together with
Aβ42 did not produce any significant changes in the
number of CD206-positive microglia at any concentration
or time point tested (data not shown).
Treatment with EPA alone or in combination with
Aβ42 did not produce any significant change in the
number of CD206-positive microglia at any concentration
or time point tested (data not shown).
Effects of ω3 FAs on the immunosuppressive receptor CD200R
The basal level (vehicle control) of CD200Rpositive
microglia was 8.9% at 2 h, 10.2% at 6 h, and
10% at 24 h. The level of CD200R-positive microglia
when incubated withAβ42 alonewas 5.9% at 2 h, 4.5%
at 6 h, and 8% at 24 h.
Treatment with DHA alone or together with Aβ42
did not produce any significant change in the number
of CD200R-positive microglia at any concentration or
time point tested (data not shown).
Upon incubation of the microglia with EPA alone,
there was no significant change in the number of
CD200R-positive microglia at any concentration or
time point tested (data not shown). Incubation of
the cells with both 1µM EPA and Aβ42 for 6 h
resulted in a significant reduction in the number
of CD200R-positive microglia as compared to Aβ42
alone (p < 0.005) (Fig. 6).
Microglial phenotype associated with phagocytosis
In order to establish the phenotype of microglia
performing phagocytosis of Aβ42, the proportion of
microglia showing immunoreactivity to each cellular
marker was analyses in the phagocytic and nonphagocytic
cell population, respectively (see Supplementary
data; available here: http://dx.doi.org/
10.3233/JAD-130131). Microglia belonging to the
phagocytic group were found to express the M2
markers CD163 and CD206 to a larger extent, and
to be less prone to express the M1 marker CD40,
by comparison of the number of cells labeled with
a specific marker using the Wilcoxon matched-pairs
test (see Supplementary Table 1). No significant
treatment effect could be observed on the distribution
of phagocytic microglia into M1 or M2 phenotypes.
Effects of ω3 FAs on the secretion of BDNF, IL-6,
IL-10, and TNF-α
The secretory products BDNF, IL-6, IL-10, and
TNF-α were measured in media collected after 24 h
incubation with DHA and EPA, alone or together with
The mean basal secretion of BDNF from the
CHME3microgliawas 463 pg/ml.Aβ42 at 1µg/ml did
not affect this secretion. Treatment with DHA, alone or
together withAβ42, did not alter the secretion of BDNF
(data not shown). After exposure of the microglia with
0.005 and 0.01µM EPA alone (Fig. 7A, left panel),
there was an increase in BDNF levels in the medium
(p < 0.05 and p < 0.01, respectively). This effect was
not seen in the presence of Aβ42 (Fig. 7A, right panel).
The mean basal level of IL-6 in the microglial culture
medium was 985 pg/ml. Aβ42 at 1µg/ml did not
affect this secretion, and neither did DHA (data not
shown). A small increase in the levels of IL-6 was seen
after incubation with 0.005 and 0.01µM EPA alone
(p < 0.05, Fig. 7B, left panel). This effect was not seen
in the presence ofAβ42 (p < 0.005–0.05) (Fig. 7B, right
The mean basal level of TNF-α in the microglial culture
medium was 27.8 pg/ml. Aβ42 at 1µg/ml did not
affect this secretion. There was no effect of DHA alone
(Fig. 7C, left panel), but when co-incubated withAβ42,
all concentrations of DHA produced a reduction in the
TNF-α levels in the medium, as compared to Aβ42
alone (Fig. 7C, right panel). The strongest effect was
seen with 0.1µM DHA (p < 0.005). Also, the levels
of TNF-α after incubation with DHA and Aβ42 were
significantly lower as compared to the levels after treatment
with DHA alone (p < 0.005–0.05). There was no
effect of EPA alone, or together with Aβ42, on the
secretion of TNF-α (data not shown).
The mean basal level of IL-10 in the microglial culture
medium was 5.4 pg/ml. Aβ42 at 1µg/ml did not
affect this secretion, and neither did DHA or EPA,
alone or together with Aβ42 (data not shown).
In the present study, microglia of the human cell line
CHME3 were exposed to the ω3 FAs DHA and EPA
in a context of AD. The data showed an increase in
phagocytic removal of Aβ42, and increased production
of growth factors, together with a decrease in inflammatory
factors. These findings support the notion of
beneficial effects of the FAs.
The microglia were incubated with 1µg/ml Aβ42
for time periods up to 24 h, during which no morphological
changes indicating activation could be seen. In
the experimental conditions used in this study, lownumber
oligomeric forms of Aβ42 were present, but
no fibrillar forms could be detected. The activation
elicited by microglia exposed to Aβ is a function of the
level and form of the Aβ, together with the expression
pattern of microglial proteins that recognize Aβ. In a
previous study on rat microglia, we found that the form
of Aβ was determinant of both magnitude and type of
cytokines secreted , emphasizing the importance of
the form of Aβ. Studies on human THP-1 monocytes
and murine microglia showed that Aβ-fibrils induce
inflammatory activation in a CD36-dependent manner
. The fact that fibrils were absent under the conditions
used in the present studies supports the view that
the microglia were not activated.
Both DHA and EPA had significantly stimulatory
effects on microglial phagocytosis of Aβ42 and it
appeared that this stimulation was biphasic, with an
immediate stimulation, followed by a period of relative
inertness, and then (at 24 h), stimulation again.
Considering the plethora of cellular mechanisms that
are influenced by DHA and EPA, it may be assumed
that one set of mechanisms, already in place at the start
of the experiments,was responsible for the stimulatory
effect at 2 h. The effects on phagocytosis observed at
24 h could hypothetically have been induced by events,
downstream of DHA and EPA, that require time to put
the molecular machinery mediating enhanced phagocytosis
in place. DHA and EPA are precursors for
the SPMs resolvin, maresin, and neuroprotectin [49,
63], which have been shown to stimulate phagocytosis
and down-regulate inflammation. SPMs may require
some time to build up to relevant concentrations,
which could be one explanation for the later effects
on phagocytosis. Microglial functions influenced by
DHAand EPAinclude ionic conductance , clustering
of pathogen-associated molecular pattern receptors
in lipid rafts , and activation of signaling molecules
such as peroxisome proliferator-activated receptor
(PPAR)- , all representing possible alternative
explanations for the effects observed in this study. Activation
of PPAR- has been shown to mediate both an
anti-inflammatory effect and the stimulation of phagocytosis
A study on mice fed with DHA showed that activated
microglia are converted to a quiescent phenotype,
and confirms an anti-inflammatory effect of DHA on
microglia . However, to our knowledge, the present
study is the first to address the effects of ω3 FAs on
microglial phagocytosis of Aβ42 and on their M1/M2
phenotype. Several studies have shown stimulatory
effects of ω3 FAs on other types of phagocytic cells
of human or animal origin, such as caprine monocytes
 and murine macrophages . Also, Halvorsen
and colleagues demonstrated that EPA increased adhesion
of bacteria to human monocytes, indicating a
positive effect on phagocytosis . Studies on neutrophils
and monocytes obtained from human subjects
given fish oil containing DHA and EPA daily for 2
months  showed an increase in phagocytic activity.
In contrast, 4 weeks treatment of mice with EPA or
DHAdid not affect the phagocytosis of ex vivo cultured
cells as compared to placebo (olive oil) .
Studies on the effects of ω3 FAs on phagocytosis
have, so far, been performed mainly on bacteria or
apoptotic leukocytes. However, a recently published
study on human macrophages ex vivo  showed a
positive influence on the phagocytosis of Aβ in the
presence of the DHA-derived resolvin D1. Interestingly,
this SPM was shown to prevent the increase
in transcription of inflammatory genes observed upon
incubation with Aβ .
In parallel with the studies on phagocytosis ofAβ42,
the effects of DHA and EPA on microglial phenotype
polarization were analyzed. Microglia expressing
the M1 phenotype markers CD40 and CD86 were
significantly decreased by treatment with DHA and
EPA. Interestingly, this effect was enhanced upon coincubation
with Aβ42. This leads us to hypothesize
that Aβ42 at the concentration used was not sufficient
to evoke a detectable activation, but induced changes
which made the microglia more responsive to DHA
The microglial cells labeled with the M1 marker
CD40 outnumbered the proportion of cells expressing
the M2 markers CD163 and CD206 by approximately
10:1. The effects of DHA and EPA on CD163 were
negligible. There was no effect of EPA on microglial
expression of CD206, whereas DHA showed a biphasic
effect, with a significant decrease in the number of
CD206-positive cells at 6 h, followed by an increase
at 24 h. Again, this result is suggestive of a polymodal
action of the ω3 FAs. Since CD206 (macrophage mannose
receptor 1) is associated with cellular ingestion,
it may be speculated that the biphasic effects are associated
with the effects on phagocytosis observed at the
same time points, i.e., no effect at 6 h, but a stimulatory
effect at 24 h. No study has yet investigated phagocytosis or ingestion of Aβ mediated by CD206. However,
mannose-binding lectin was shown to bind Aβ ,
suggesting that Aβ can be recognized by CD206. Titos
et al. demonstrated an increase in M2 markers, including
CD206, upon incubation of mouse macrophages
with DHA . Interestingly, similar results were
observed with the DHA-derived resolvin D1 .
Altogether, analysis of the microglial phenotype
showed that microglia performing phagocytosis of
Aβ42 had a lower expression of M1 markers, while the
expression of M2 markers was higher. This indicates
that under the conditions used microglia performing
phagocytosis of Aβ42 carry the M2 phenotype and
that M2-polarized microglia are responsible for removing
extracellular Aβ. It may be speculated that the
inflammation present in the AD-brain, characterized
by increased levels of proinflammatory factors, contributes
to the overabundance of Aβ in the AD-brain
by hindering attainment of theM2phenotype which, as
indicated by our data, may be more efficient in removing
Aβ. Support for this theory is provided by findings
of increased expression of M1 markers such as CD40
, and major histocompatibility complex Class II
(MHC-II) [77, 78], in theADbrain. Additional support
comes from in vitro studies showing M2 polarization
and stimulated phagocytosis of Aβ in rat microglia
that were stimulated with IL-4 , and from a study
in which Th2 cytokines were found to reduceAβ accumulation
and improve cognition in a transgenic mouse
model resembling AD .
The membrane receptor CD200R is known to be
expressed on microglia and macrophages  and
to mediate a de-activating effect upon binding its
ligand CD200 (OX-2), which is expressed on neurons
and astrocytes [82, 83]. Dysfunction of the
CD200-CD200R pathway has been shown to increase
microglial activation and neurodegeneration in a rat
model of Parkinson’s disease . Importantly, the
expression of CD200 and CD200R has been shown to
be decreased in brains fromADpatients .We found
that only a small proportion of the CHME3 microglia
expressed CD200R. The effects of DHA and EPA on
CD200R were not significant, with the exception of
1µMEPA, inducing a decrease after 6 h of incubation.
A decrease in CD200R is not desirable from a perspective
of treatment of AD, in view of its association
with a detrimental neuroinflammation. However, further
studies will be required to examine the relevance
of this down-regulation in the context of other, more
beneficial, effects of EPA.
In studies on the effects of the ω3 FAs on microglial
secretion, we found a significant increase in IL-6 by
0.005 and 0.01 µM EPA, an effect that disappeared
with higher concentrations. The marked increase in
IL-6 secretion by the lowest concentration of EPA was
nullified upon co-incubation with Aβ42. Most studies
suggest an inhibiting effect of EPA (and DHA)
on IL-6 production . However, in those studies
an activating stimulus was used to induce a robust
IL-6 secretion, which was not the case in response
to 1µg/ml Aβ42 used in the present study (also
reported previously ). For example, the increase
in IL-6 production by mouse adipocytes induced by
bacterial lipopolysaccharide (LPS), an archetypical
pro-inflammatory activator,was inhibited by EPA.
The toll-like receptor (TLR)-4, a receptor for LPS,
needs to be assembled in lipid rafts of the cellular membrane
to transduce its pro-inflammatory signal .
One of the actions of DHA and EPA is the inhibition of
the clustering of immune-related proteins in lipid rafts
, which may explain the anti-inflammatory effect
in conditions where TLR-4 ligands are present. In support
of our findings on IL-6, a study on rat lymphocyte
function ex vivo showed that fish oil, which contains
DHA and EPA, significantly increased the levels of
The present study is the first to address the influence
of DHA and EPA on microglial production of
BDNF in vitro and to show a stimulatory effect of EPA
on BDNF production. Earlier studies have described
the positive influence of DHA on BDNF expression in
vitro in induced pluripotent stem cells , and in the
CNS [90–92]. Similarly to IL-6, the lowest concentrations
of EPA resulted in an increase in BDNF levels
that disappeared at higher concentrations. The effect
on BDNF was abolished when EPA was co-incubated
with Aβ42, lending support for the result on depression
ofBDNFproduction byAβ42 observed previously
. It may be speculated that activation of the CRE
motifs present on both the BDNF and IL6 genes are
responsible for the stimulation of production of these
two mediators, which could explain the synchrony with
regard to effective concentrations of EPA.
It could also
explain the annulment of the stimulatory effect upon
co-incubation withAβ42. It has been shown thatCREB
is inhibited by Aβ42 , which can account for the
lack of effect of EPA on BDNF and IL-6 upon coincubation.
However, there are no data in the literature
on the effects of EPA on CREB activation. Considering
that DHA was shown to activate CREB in primate
hippocampal neurons , it is conceivable that EPA
may have a similar action. A positive correlation was
found between the level of BDNF and the percentage
of phagocytic microglia (Supplementary Table 2),
supporting the notion of a concerted restoration of the
tissue by phagocytosis and growth factors, in line with
the theory on inflammatory resolution.
Incubation of the microglia with DHA resulted
in a strong inhibition of the secretion of the proinflammatory
cytokine TNF-α, whereas the effect of
EPA was less prominent. However, these effects of
DHAand EPAwere only observed upon co-incubation
with Aβ42. Several studies show an inhibiting effect of
these FAs on TNF-α production . Similar to what
has been discussed above for IL-6, a strong inducer
of TNF-α production, such as LPS, was used in those
studies, and the low concentration of Aβ42 was not
sufficient to affect the production of TNF-α, but may
have induced changes that made the microglia more
responsive to DHA and EPA.
The level of the anti-inflammatory M2 cytokine
IL-10 was unaltered upon incubation with DHA or
EPA, except for an increase upon incubation with
the lowest concentration of EPA together with Aβ42.
This is different from responses by peripheral blood
mononuclear cells  and macrophages  showing
increased IL-10 secretion upon treatment with DHA.
The study is the first to investigate the effects of
DHA and EPA on human microglia. The stimulatory
effects of DHA and EPA on microglial phagocytosis
of Aβ42, together with the down-regulatory effects on
pro-inflammatory markers, and stimulatory effects on
the neuroprotective factor BDNF, indicate that these
ω3 FAs activate a pro-resolving program in human
microglia. However, our results also suggest multiple
pathways of action of DHA and EPA with different
time frames of activities. This creates a complex network
of responses that may have different emphasis in
different conditions, a hypothesis which has implications
for the efficiency of a potential treatment based
on ω3 FAs depending on the stage of disease. We also
show that low concentrations of Aβ42, which do not
activate microglia, can still influence responses to other
types of stimulation. The present results support the
idea that ω3 FAs, and the pathways with which they
are associated, are promising treatment targets for AD
and other neurodegenerative disorders.
Grants supporting this work from the Swedish
Research Council (072194), Swedish Brain Power,
The Knut and Alice Wallenberg Foundation, Karolinska
Institutet Research funds, The Swedish Brain
Foundation, Stiftelsen f¨or Gamla Tj¨anarinnor, The
Swedish Alzheimer Foundation, Gun och Bertil
Stohnes stiftelse, Petrus och Augusta Hedlunds Stiftelse,
and Stockholm County Council, are gratefully
acknowledged. The authors thank Professor Charles N.
Serhan (Department of Anesthesiology, Perioperative
and Pain Medicine, Harvard Medical School, Boston,
USA) for helpful discussions.
Authors’ disclosures available online (http://www.jalz.
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