ORIGINAL RESEARCH
published: 27 August 2021
doi: 10.3389/fimmu.2021.650176

Prevention of Diabetes-AssociatedCognitive Dysfunction Through Oral
Administration of Lipopolysaccharide
Derived From Pantoea agglomerans
Haruka Mizobuchi 1*, Kazushi Yamamoto 1, Masashi Yamashita 1, Yoko Nakata 2,
Hiroyuki Inagawa 1,2,3, Chie Kohchi 1,2 and Gen-Ichiro Soma 1,2,3
1 Control of Innate Immunity, Collaborative Innovation Partnership, Kagawa, Japan, 2 Research and Development Department
Macrophi Inc., Kagawa, Japan, 3 Research Institute for Healthy Living, Niigata University of Pharmacy and Applied Life
Sciences, Niigata, Japan

Edited by:Michael D. Burton,
The University of Texas at Dallas,
United States
Reviewed by:
Gyorgy Fejer,
University of Plymouth,
United Kingdom
Zhongcheng Xin,
Second Hospital of Tianjin Medical
University, China*Correspondence:
Haruka Mizobuchi
mizobuchi@shizenmeneki.org
orcid.org/0000-0002-6667-4820
Specialty section:
This article was submitted to
Molecular Innate Immunity,
a section of the journal
Frontiers in Immunology
Received: 06 January 2021
Accepted: 13 August 2021
Published: 27 August 2021
Citation:
Mizobuchi H, Yamamoto K,
Yamashita M, Nakata Y, Inagawa H,
Kohchi C and Soma G-I (2021)
Prevention of Diabetes-AssociatedCognitive Dysfunction Through Oral
Administration of Lipopolysaccharide
Derived From Pantoea agglomerans.
Front. Immunol. 12:650176.
doi: 10.3389/fimmu.2021.650176

Diabetes-related cognitive dysfunction (DRCD) is a serious complication induced bydiabetes. However, there are currently no speciﬁc remedies for DRCD. Here, we showthat streptozotocin-induced DRCD can be prevented without causing side effects throughoral administration of lipopolysaccharide (LPS) derived from Pantoea agglomerans. Oraladministration of LPS (OAL) prevented the cerebral cortex atrophy and tau
phosphorylation induced by DRCD. Moreover, we observed that neuroprotective
transformation of microglia (brain tissue-resident macrophages) is important for
preventing DRCD through OAL. These ﬁndings are contrary to the general recognitionof LPS as an inﬂammatory agent when injected systemically. Furthermore, our results
strongly suggest that OAL promotes membrane-bound colony stimulating factor 1 (CSF1)
expression on peripheral leukocytes, which activates the CSF1 receptor on microglia,
leading to their transformation to the neuroprotective phenotype. Taken together, thepresent study indicates that controlling innate immune modulation through the simple andsafe strategy of OAL can be an innovative prophylaxis for intractable neurological diseases
such as DRCD. In a sense, for modern people living in an LPS-depleted environment, OAL
is like a time machine that returns microglia to the good old LPS-abundant era.
Keywords: microglia, lipopolysaccharide, oral administration, neuroprotection, cognitive dysfunction, dementia

INTRODUCTION
Diabetes affects 463 million people worldwide, and one of its more serious complications is diabetesrelated cognitive dysfunction (DRCD) (1). However, there is still no radical preventive or curative
treatment for DRCD. Accordingly, its prevention is a crucial to sustained global health.
To address this problem, we focused on oral administration of lipopolysaccharide (LPS) derived
from Pantoea agglomerans. Our previous study showed that oral administration of LPS derived

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from P. agglomerans (OAL) prevents high fat diet-induced
cognitive dysfunction (2). In addition, P. agglomerans isreportedly used as a food preservative (3, 4), and the safety of
OAL is objectively guaranteed based on Organization for
Economic Co-operation and Development standards (5–7).
Based on these ﬁndings, we hypothesized that OAL could
prevent DRCD.
Here, we demonstrate the effect of OAL on preventing DRCD
using an intracerebroventricular injection model of the diabetes
inducer, streptozotocin (STZ), which has already been
established as a DRCD model (8–10). Furthermore, in order toelucidate the DRCD prevention mechanism utilized by OAL, wedepleted microglia, which are brain tissue macrophages, andperformed genetic analysis of isolated microglia. The results
revealed that transformation to neuroprotective microglia isrequired for DRCD prevention by OAL. Importantly, it isstrongly suggested that signal activation of colony stimulating
factor 1 receptor (CSF1R) by the membrane-bound form CSF1 isinvolved in this transformation to neuroprotective microglia.

and frozen at -80°C for cytokine analysis. Brains were collected forhistopathological analysis and isolation of microglia.

LPS Treatment
Puriﬁed LPS derived from P. agglomerans (Macrophi Inc.,
Kagawa, Japan) was used in this study. LPS derived from P.
agglomerans was puriﬁed to over 99% according to the methods
described previously (11). LPS derived from P. agglomerans hasvery low nucleic acid and protein contamination [protein
contamination was 0.5%, nucleic acid contamination was lessthan 0.35% (w/w)] and activates macrophages in very small
amounts (1.6 ng/ml) (11). It was conﬁrmed that the LPS content
of the D12450B diet was lower than that of the common mousediet MF (Oriental Yeast Co., Ltd, Tokyo, Japan) or CE-2 (CLEA
Japan Inc., Tokyo, Japan) (Supplementary Table 1). For oral
LPS administration, LPS was dissolved in drinking water (sterile
distilled water) and applied at 1 mg/kg body weight (BW)/day.
The dose of LPS was determined to be the sufﬁcient dose
required to achieve preventive effects as estimated fromprevious studies (2, 12). The drinking water was changed
weekly and the concentration of LPS was adjusted according to
the average body weight and amount of water consumption. We
previously conﬁrmed that the LPS in drinking water was not
signiﬁcantly degraded in a week (12).
For the DRCD model, LPS was orally administrated to mice 1
week before STZ-injection until the end of the experiment (n = 17–
19). For OAL without intracerebroventricular STZ injection, naïve
mice were orally administered LPS (1 mg/kg/day, for 1 week) (n =5). For intraperitoneal administration of LPS, naïve mice wereintraperitoneally injected with LPS (4 mg/kg BW, single-dose) (n =5), and samples were collected 4 h after LPS injection.

MATERIALS AND METHODS
Animals and Tissue Sample Preparation
Six-week-old male C57BL/6 mice (20–22 g) were purchased from
SLC, Inc., Shizuoka, Japan, and were acclimated for 1 week. All
mice (3–5 mice per cage) were maintained under speciﬁc
pathogen-free conditions in a temperature- and humiditycontrolled room under a 12-h light/dark cycle with unrestricted
access to food and water. Mouse diet D12450B was purchasedfrom Research Diets, Inc., New Brunswick, NJ, USA. At the end of
the experiments, mice were anaesthetized under 4% isoﬂuranevapor using the simple inhalation anesthesia device NARCOBIT-E(Natsume Seisakusho Co., Ltd., Tokyo, Japan) and euthanized bycardiac puncture whole blood collection. The animal experiments
were reviewed and approved by the Animal Care and Use
Committee of the Control of Innate Immunity CIP (ApprovalNo. 18-04, 18-12, 18-13, and 20-01).
The experiment was carried out according to the Law for the
Humane Treatment and Management of Animals Standards
Relating to the Care and Management of Laboratory Animals
and Relief of Pain (Ministry of the Environment, Japan), theFundamental Guidelines for Proper Conduct of Animal
Experiments and Related Activities in Academic ResearchInstitutions (Ministry of Education, Culture, Sports, Scienceand Technology, Japan), and the Guidelines for Proper
Conduct of Animal Experiments (the Science Council ofJapan). Animal health and well-being was also assessed inaccordance with the guidelines described above.
At the end of the experiment, whole blood was collected bycardiac puncture under anesthesia with 4% isoﬂurane vapor.
Plasma/serum were collected after centrifugation for 10 min at
500 ×g for cytokine analysis and limulus amebocyte lysate (LAL)
assay. Peripheral white blood cells were collected for RNA analysis.
Bone marrows (BMs) were collected for isolation of CD11b+ cells,
RNA analysis, and primary culture. Hippocampuses were collected

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Intracerebroventricular STZ Injection
STZ was purchased from Sigma-Aldrich, St Louis, MO, USA. Under
anesthesia with 4% isoﬂurane vapor, mice were ﬁxed using the brain
stereotaxic apparatus SR-5M-HT (Narishige, Tokyo, Japan) andadministrated a single injection of STZ (6.6 mg/kg, dissolved insaline 5 µl) into the right lateral ventricle with the microinjector,
IMS-20, and micromanipulator SMM-100 (Narishige). The
stereotaxic coordinates were + 0.3 mm anterior, + 1.0 mm lateral(right) and + 2.5 mm ventral from bregma. After the skin suture,
antibiotic ointment (20 mg/g Chloramphenicol, 5 mg/g
Fradiomycin, 100,000 U/g Nystatin, Daiichi Sankyo HealthcareCo., Ltd, Tokyo, Japan) was dabbed on the wound. Control shammice were administrated 5 µl saline into the right lateral ventricle.
After the surgery, mice were monitored daily for pain/discomfort
and infections in accordance with the guidelines describedabove. We checked for proper placement of the needle bydelivering 7 ml of 5% Trypan Blue Dye (Nacalai, Kyoto, Japan)(Supplementary Figure 1).

Microglia Depletion
The CSF1R inhibitor PLX3397 (Pexidartinib) was purchasedfrom Chemgood, Glen Allen, VA, USA. PLX3397 wasformulated based on the D12450B diet (Research Diets) at aconcentration of 400 mg/kg chow. The components of the
PLX3397-containing diet are shown in Supplementary2

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Table 2. Mice were orally administered PLX3397 and LPS 1 week
before STZ-injection until the end of the experiment.

for 10–20 min. After washing in 0.5% acetic acid, the tissues wereincubated in 0.1% gold chloride for 5 min. Then, the tissues were
ﬁxed in 1% sodium thiosulfate for 5 min and counterstained with
0.1% nuclear fast red for 2 min.
For immunostaining of Iba1, the tissues were incubated with
anti-Iba1 antibody at room temperature (RT) for 15 min (Wako,
Osaka, Japan) after blocking. Then, the tissues were incubatedwith HRP-conjugated anti-rabbit IgG antibody at 4°C for 1 h(Leica Biosystems, Buffalo Grove, IL, USA). Enzymatic color
development was performed using DAB (Leica Biosystems).
To evaluate cerebral atrophy, the thickness of the cerebral
cortex was measured using imaging software NIS-Element(Nikon, Tokyo, Japan) in the Klüver-Barrera stained brains.
For quantitative analysis of tau inclusion, argyrophilic grains
were counted in Gallyas-Braak stained brains.

Morris Water Maze (MWM) Test
To assess spatial learning and memory, the MWM test was carriedout 3 weeks after STZ-injection as previously described (2), withminor modiﬁcations. Brieﬂy, the experimental apparatus consistedof a circular tank 100 cm in diameter and 40 cm in height ﬁlled withwater to 30 cm, maintained at 23 ± 1°C and rendered opaque withwhite ink. The area of the pool was conceptually divided into four
equal quadrants, and cards with different shapes (circle, square,
triangle or cross) were placed on the wall of each quadrant. A
removable circular platform 10 cm in diameter was submerged 1 cm
below the water surface and placed approximately in the midpointof one quadrant, deﬁned as the target quadrant. Each mouseconsecutively received a pre-training session (1 day), training
session (4 days), and probe session (1 day).
One day before the ﬁrst training session, each mouse received apre-training session to make it aware of the escape platform. The
mouse was put on the platform for 20 s, given a 30 s free swim, andthen assisted in swimming back to the platform. The next day, theﬁrst training session was conducted to assess spatial learning ability.
From the next day, training session continued for four consecutive
days. One each trial, the mouse was released into the water at a
randomly assigned starting position, facing the pool’s wall. The
mouse was given 60 s to ﬁnd the platform and was allowed to stayon it for 20 s after reaching the escape platform. The spatial learning
ability of each mouse was identiﬁed as the time elapsed between
releasing and locating the platform, deﬁned as escape latency. If themouse failed to ﬁnd the platform within 60 s, it was gently guided tothe platform and kept there for 20 s, and the escape latency wasrecorded as 60 s. Each mouse underwent four trials per day, and theaverage value of the escape latency was calculated. One day after
the last training session, the probe test was performed to assess thespatial reference memory ability of the mice. The platform wasremoved from the pool, and each mouse was placed in the pool at astarting position located opposite the target quadrant and allowed toswim freely for 60 s. The time spent swimming in the target
quadrant was recorded. A video camera was hung above thecenter of the pool to record the swimming paths of the mice. The
swimming trajectory was visualized and the swimming distance/velocity was measured using AnimalTracker software (13).

Isolation of Primary Microglia From
Adult Mouse
Primary microglial cells were isolated from adult mouse brain by
enzymatic digestion as described previously (14), with minor
modiﬁcations. Brieﬂy, the brain was removed and kept in ice
cold PBS. The brain tissue was chopped ﬁnely with a ﬁne sharp
razor. The whole brain homogenate was then incubated in DMEM
containing 1.2 units/ml dispase II, 1 mg/ml papain (SigmaAldrich), 100 units/ml penicillin and 100 mg/ml streptomycin(Thermo Fisher Scientiﬁc, Waltham, MA, USA), 20 units/ml
RNase inhibitor (Promega, Madison, WI, USA) and 10 units/ml
DNase I (Takara Bio, Shiga, Japan) for 30 min at 37°C. The
digestion was terminated by the addition of PBS containing 10%
fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), and thecells were then centrifuged for 5 min at 300 ×g at RT. The pellets
were resuspended in PBS containing 0.5% bovine serum albumin(BSA; Sigma-Aldrich) and 2 mM EDTA (Wako), then centrifuged
for 5 min at 300 ×g at RT. The cell suspension was passed througha 70-mm cell strainer (Corning, Durham, NC, USA) and myelin
was removed using the debris removal solution (Miltenyi Biotec,
Bergisch Gladbach, Germany). After myelin removal, the cells
were incubated with anti-CD11b antibodies conjugated tomagnetic beads (20 ml/brain; Miltenyi Biotec) in PBS containing
0.5% BSA and 2 mM EDTA for 15 min at 4°C. After washing, theCD11b+ cells were separated using autoMACS® proseparator(Miltenyi Biotec) and used as microglia. As a control, CD11b+
cells were isolated from BM in a similar manner.

Histochemical Analysis
Brains were ﬁxed with 4% paraformaldehyde and embedded inparafﬁn. The cut surface was uniﬁed at the same position of the
hippocampal dentate gyrus. Klüver-Barrera staining, GallyasBraak staining, and immunostaining of Iba1 were performed
with the standard procedure. Brieﬂy, Klüver-Barrera staining was
performed as follows. The tissues were stained with 0.1% Luxol
fast blue at 56°C overnight and differentiated in 0.05% lithium
carbonate solution for 30 s. Then, the tissues were counterstainedwith 0.1% cresyl violet acetate for 10 min. For Gallyas-Braakstaining, the tissues were placed in 5% periodic acid for 5 min
and incubated in silver iodide solution for 1 min. After washingin 0.5% acetic acid, the tissues were placed in developer solution

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Flow-Cytometric Analysis of Iba1
Isolated CD11b+ cells from brain or BM were ﬁxed andpermeabilized using ﬁxation/permeabilization solution (BDBiosciences, San Jose, CA, USA) for 20 min at 4°C. Afterwashing, the cells were incubated with anti-Iba1 antibody(Wako) for 30 min at 4°C followed by incubation with Alexa
Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientiﬁc)for 30 min at 4°C. The median of ﬂuorescent intensity (MFI) was
measured in total 5,000-count cells using a Beckman Coulter
Gallios ﬂow cytometer and Kaluza for Gallios software version
1.3 (Beckman Coulter, Indianapolis, IN, USA).

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reverse transcription using ReverTra Ace qPCR RT Master Mix(TOYOBO, Osaka, Japan), according to the manufacturer’s
instructions. Real-time PCR assay was carried out using 2 ml ofcDNA as the template and 10 ml of Power SYBR Green PCR MasterMix (Thermo Fisher Scientiﬁc) on the Stratagene Mx 3005P QPCR
System (Agilent Technologies, Santa Clara, CA, USA).
The primers are listed in Supplementary Table 3. m-CSF1
and s-CSF1 were distinguished by speciﬁc primers (19). The data
was analyzed based on 2−DDCt method and normalized by
GADPH expression using the MxPro software version 4.10
(Agilent Technologies). The thermal cycling conditions for the
PCR were 95°C for 10 min for polymerase activation, followed by
45 cycles of 95°C for 15 s for denaturation, and 60°C for 1 minfor extension.

Determination of Ab1-42 and Cytokines(ELISA)The levels of Ab1-42 in isolated microglia were determinedusing a commercial ELISA kit (Wako). Protein was extracted
from isolated microglia in ice-cold 70% (v/v) formic acid
(Wako, Tokyo, Japan) followed by centrifugation at 20,000 ×g
for 1 h at 4°C. The supernatant was neutralized with a 20-folddilution in 1M Tris buffer, and used for Ab1-42 ELISA. The
concentration of Ab1-42 was normalized against the number ofisolated microglia.
The levels of IL-6 and TNF-a in plasma were determinedusing a commercial ELISA kit (BioLegend, San Diego, CA, USA).
The levels of CSF1 in plasma and brain were determined using acommercial ELISA kit (R&D systems, Minneapolis, MN, USA).
The snap-frozen brain tissues were homogenized in ice-cold PBS
containing 1% protease inhibitor cocktail (GE Healthcare UK
Ltd., Buckinghamshire, UK) and 1% phosphatase inhibitor
cocktail (Nacalai), followed by sonication for 5 min andcentrifugation at 12,000 ×g for 10 min at 4°C. The supernatant
was used for CSF1 ELISA. The total protein concentration ineach sample was determined by a BCA assay kit (Thermo Fisher
Scientiﬁc) and the concentration of target protein in tissues werereported as pictograms of cytokine relative to the protein
content. Absorbance was measured using the iMark microplate
reader (BIO RAD, Hercules, CA, USA), and data was analyzed
using the Microplate Manager 6 software (BIO RAD).

LAL Assay
The total abundance of LPS in serum was analyzed as described
previously (2) using a kinetic turbidimetric assay with the
Limulus Amebocyte Lysate (LAL) assay kit and a Toxinometer
ET-6000 computer operated kinetic incubating tube reader(Wako). The serum was diluted 1:10 in pyrogen-free water(Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) andpreheated to 70°C for 10 min prior to analysis. Data was analyzed
using the Toximaster QC7 software.

Cell Culture
Bone marrows were collected from femurs by ﬂushing with PBS.
After RBC lysis, BMC (1 × 107 cells/ml) were seeded in 96-well tissue
culture plates (n = 5) and cultured in Dulbecco’s modiﬁed Eagle’s
medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine
serum (Sigma-Aldrich, St Louis, MO, USA), 100 U/ml penicillin and100 mg/ml streptomycin (Life Technologies, Carlsbad, CA, USA), at
37°C in 5% CO2. To evaluate CSF1 production by LPS, BMC weretreated with or without LPS (1–104 pg/ml, puriﬁed LPS derived from
Pantoea agglomerans, Macrophi Inc., Kagawa, Japan). RNA andculture supernatants were collected at 4 h and 24 h after the last LPS
treatment, respectively.
The murine microglial cell line C8-B4 microglia, which wasestablished from brain cerebella, was purchased from theAmerican Type Culture Collection, Manassas, VA, USA. C8B4 microglia (2 × 105 cells/ml) were seeded in 12-well tissue
culture plates (n = 3, in triplicate) and cultured in Dulbecco’s
modiﬁed Eagle’s medium (Wako) supplemented with 10% fetal
bovine serum (Sigma-Aldrich), 100 U/ml penicillin and 100 mg/
ml streptomycin (Life Technologies), at 37°C in 5% CO2. C8-B4
microglia were treated with or without 80 ng/ml CSF1
(Promega). After 16 h of incubation, the culture supernatant
was collected and RNA was extracted from the C8-B4 microglia.
For the phagocytosis assay, C8-B4 microglia were incubatedwith HiLyte Fluor 488-labeled Ab1-42 (1 mg/ml; AnaSpec,
Fremont, CA, USA) or ﬂuorescent latex beads (Fluoresbrite
YG Microspheres 2.0 mm; Polysciences, Warrington, PA, USA)at a cell/bead ratio of 1:5 for 3 h after 16 h of treatment with
CSF1. After washing with PBS, C8-B4 microglia were detached
by 0.25% trypsin treatment (Life Technologies), and the MFI ofphagocytosed beads in the cells were measured using a Beckman

Microarray Analysis
Microarray analysis was performed at the Cell Innovator,
Fukuoka, Japan. Brieﬂy, the RNA samples of microglia were
quantiﬁed with Agilent 2200 TapeStation (Agilent Technologies,
Santa Clara, CA, USA). Each 50 ng of total RNA was labeled usinga Low-Input QuickAmp Labeling Kit (Agilent Technologies). The
RNA was hybridized with SurePrint G3 Mouse GE microarray 8 ×
60K v2 (Agilent Technologies) using pooled sample (n = 4), andthe signals were detected using a DNA microarray scanner(Agilent Technologies). Data was quantiﬁed by Feature
Extraction software (Agilent Technologies). Normalization was
performed by a quantile algorithm with statistical processing
software R [R Core Team (2019). R: A Language andEnvironment for Statistical Computing. R Foundation for
Statistical Computing, Vienna, Austria].
The ratio (non-log-scaled fold change) and Z scores (15) were
calculated from the normalized signal intensities of each probe
for comparison between control and experimental samples.
Then, the criteria for regulated genes were deﬁned as follows:for upregulated genes, Z score ≧ 2.0 and ratio ≧ 1.5-fold; fordownregulated genes, Z score ≦ 2.0 and ratio ≦ 0.66 (16). The
heat map was created by using the distance from the median of
the log2 converted signal value with MultiExperiment Viewer(MeV) (17, 18).

Quantitative RT-PCR
RNA was extracted using the RNeasy micro kit (QIAGEN,
Hilden, Germany) and cDNA was synthesized using the

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Coulter Gallios ﬂow cytometer with Kaluza software(Beckman Coulter).
To evaluate the neuroprotective effect of MCS, a murine
neuroblast cell line Neuro-2a, which was established frombrain neuroblastoma, was cultured with CSF1-treated MCS.
Neuro-2a cells were provided from the Japanese Collection of
Research Bioresources Cell Bank, Osaka, Japan. Neuro-2a cells
were seeded in 24-well tissue culture plates (4 × 105 cells/ml) andpre-cultured in Eagle’s minimal essential medium with nonessential amino acids (Wako) supplemented with 10% fetal
bovine serum (Sigma-Aldrich), 100 U/ml penicillin and 100
mg/ml streptomycin (Life Technologies), at 37°C in 5% CO2
for 24 h (n = 3). Then, the medium was replaced by CSF1-treated
MCS. After culturing with CSF-1 treated MCS for 24 h, 400 µM
STZ (Sigma-Aldrich) was added to the culture medium, andNeuro-2a cells were cultured for another 24 h. Finally, the cellnumber was counted after detachment by treatment with 0.25%
trypsin for 2 min, and the survival rate was calculated by staining
with Trypan Blue Dye (Nacalai).

analyzed by one-way ANOVA followed by Tukey’s multiple
comparisons test or two-way ANOVA followed by Sidak
multiple comparisons test. Student’s t test was used to compare
differences from two independent groups. A p-value < 0.05 wasconsidered signiﬁcantly different. Representative experiments
were conducted at least twice, independently.

RESULTS
Prevention of DRCD by OAL
To demonstrate DRCD prevention through OAL, spatial learning
memory was evaluated using the Morris water maze (MWM)
procedure with the STZ-induced DRCD model (Figure 1A). In
training session, STZ-injected mice showed reduced learning
ability as their escape latency was signiﬁcantly longer thansaline-injected controls and LPS-treated mice. By contrast, theloss of learning ability was prevented by OAL. The escape latencyof LPS-treated mice was comparable to saline-injected controls(Figure 1B). In probe test, the time spent in the target quadrant of
STZ-injected mice was signiﬁcantly shorter than saline-injected
controls, indicating the declined memory performance. On thecontrary, LPS-treated mice did not decline memory ability as they
exhibited signiﬁcantly longer time spent in the target quadrant
than STZ-injected mice (Figure 1C). As shown in Figure 1C, it

Statistical Analysis
Statistical analysis was performed using the GraphPad Prism 6.0
software package (GraphPad Software Inc., San Diego, CA).
Results were presented as the mean ± standard error of themean (SE). The differences between the groups of mice were

A

B

C

D

F

E

G

FIGURE 1 | Microglia are indispensable for preventing diabetes-related cognitive dysfunction (DRCD) through oral administration of LPS (OAL). (A) The experimental
schedule of DRCD and OAL. (B, C) Prevention of deterioration of the learning ability and memory performance induced by OAL (n = 17–19). (D, E) PLX3397
reversed the prevention of DRCD by OAL (n = 5–7). (F) Prevention of cerebral cortex atrophy by OAL (n = 5–8). Bars, 200 mm. (G) Prevention of accumulation ofargyrophilic grains by OAL (n = 5–8). Bars, 10 mm. Means ± SE are presented. *,†p or a,bp < 0.05 for one-way ANOVA with Tukey’s multiple comparisons or twoway ANOVA with Sidak multiple comparisons. DW, distilled water; i.c.v., intracerebroventricular; p.o., per os.

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was evident from the swimming trajectory that LPS-treated mice
memorized the target location. Furthermore, the athletic ability
such as velocity and total swimming distance did not signiﬁcantly
vary between the groups (Supplementary Figure 2). Therefore,
these results indicate that OAL prevents DRCD.

not peripheral monocytes, highly express Iba1 (microglia
marker), indicating that the contamination of peripheral
monocytes was extremely low (Figure 3A). In addition, thenumber of microglia was not signiﬁcantly different between
STZ-injected and LPS-treated mice (Figure 3B).
As shown in Figure 3C, Ab contained in isolated microglia
was declined in STZ-injected mice, but was not declined in LPStreated mice as well as in saline-injected controls. whereas thediabetes-induced suppression of Ab phagocytosis by microglia
was prevented by OAL. The result indicates that OAL promotes
Ab phagocytosis of microglia.
Second, comprehensive gene analysis by microarray was used
to extract the notable differentially expressed genes from theoverall tendency of gene expression. Microarray analysis using
pooled sample provided 1,061 candidate genes upregulated in
OAL-microglia (Supplementary Material 1 and Supplementary
Figure 4A), showing that the gene expression patterns of OALmicroglia are distinct from healthy microglia and microglia in
DRCD-developed mice (DRCD-associated microglia).
Additionally, gene ontology term analysis revealed that pathways
related to immune function, neuronal survival, and tissue repair
were upregulated in OAL-microglia (Supplementary Figure 4B).
Quantitative RT-PCR was performed to distinguish
representative neuroprotective genes from those ﬂuctuating in
OAL-microglia. As shown in Figure 3D, the following
neuroprotective genes are signiﬁcantly upregulated in OALmicroglia compared to DRCD-associated microglia: CSF1R, IL10, IL-12B, prostaglandin-E2 EP4 receptor (encoded by Ptger4gene), c-Jun, and heat shock protein (HSP) family (encoded by
Hsp90aa1, Hspb1, Dnaja4, and Dnajb4 genes). In addition, gene
expression of IL-12B, EP4 receptor, and HSPb1 was promoted in
OAL-microglia compared to microglia of healthy mice.
However, OAL did not affect the gene expression of
inﬂammatory mediators in microglia. The gene expression of
inﬂammatory mediators, such as IL-1b, IL-6, tumor necrosis
factor (TNF)-a and nitric oxide synthase (NOS) 2, was not
signiﬁcantly different between OAL-microglia and DRCDassociated microglia (Figure 3E). These results indicate that
OAL-microglia, which are characterized by high expression ofneuroprotective genes, differ in gene expression from DRCDassociated microglia and microglia of healthy mice.

Microglia Are Indispensable for Preventing
DRCD Through OAL
To investigate whether microglia are necessary for DRCD prevention
through OAL, we depleted microglia using the PLX3397 (20, 21).
Microglia depletion by PLX3397 was conﬁrmed by reduced numberof cells expressing Iba1 (Supplementary Figure 3). It wasdemonstrated that microglia depletion reversed the preventive
effect of OAL on DRCD (Figures 1D, E). Furthermore, microglial
depletion did not reduce the spatial learning memory of salineinjected healthy controls
Next, to investigate the pathological mechanism of DRCD
prevention through OAL, a histological analysis of the brain was
performed. STZ injection induced cerebral cortex atrophy,
whereas OAL prevented diabetes-induced cerebral atrophy,
which was ablated by microglial depletion (Figure 1F). As
shown in Figure 1G, Gallyas-Braak staining revealed diabetesinduced accumulation of argyrophilic grains, which are mainlycomposed of hyperphosphorylated tau protein (22). Contrarily,
the argyrophilic grains were hardly observed in the brains ofsaline-injected controls and LPS-treated mice. The prevention ofargyrophilic grain accumulation by OAL was cancelled by
PLX3397 administration. These results indicate thatsuppression of cerebral atrophy and tau phosphorylation via
microglia are the preventive mechanisms of DRCD through OAL.

OAL-Microglia Are Distinct From
Inﬂammatory Microglia Induced by
Systemic Injection of LPS
The characteristics of microglia transformed by OAL (OALmicroglia) in naïve mice were compared to that of inﬂammatory
microglia induced by intraperitoneal injection with LPS (LPSi.p.). First, the systemic effects of LPS administration were
evaluated. As shown in Figures 2A, B, OAL did not induce
weight loss and systemic elevation of inﬂammatory cytokines,
unlike LPS i.p. (4 mg/kg). In addition, LPS was not detected in
the blood of mice that received OAL unlike LPS i.p. (Figure 2C).
Second, gene expression analysis of OAL-microglia in naïve
mice showed that OAL-microglia did not promote inﬂammatory
mediators (IL-1b, IL-6, TNF-a, and NOS2) nor suppress antiinﬂammatory mediator (PPARg), unlike microglia of mice thatreceived LPS i.p. (Figure 2D). These results indicate that OALmicroglia are distinct from inﬂammatory microglia transformed
by LPS i.p. These results indicate that the characteristics of OALmicroglia are fundamentally different from those of
inﬂammatory microglia induced by systemic LPS injection.

CSF1R Signaling Is Involved in theTransformation to Neuroprotective
Microglia Through OAL
Ligands for CSF1R include CSF1 and IL-34. CSF1 is also
expressed as two isoforms, the membrane-bound form (mCSF1) and secretory form (s-CSF1) (23). Gene expression
analysis of CSF1R ligands in peripheral leukocytes revealed that
only m-CSF1 expression was promoted by OAL (Figure 4A). LPS
stimulation promoted m-CSF1 gene expression in primarycultured bone marrow cells (Figure 4B). However, OAL did
not alter the expression of s-CSF1 and IL-34 in peripheralleukocytes (Figures 4A, C, D). These results suggested that
CSF1R activation by m-CSF1 of peripheral leukocytes inducestransformation to neuroprotective OAL-microglia.

Characterization of Neuroprotective
Microglia Transformed Through OAL
We next performed further characterization of OAL-microglia in
the DRCD model. First, it was conﬁrmed that isolated microglia,

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A

B

C

D

FIGURE 2 | OAL-microglia are distinct from inﬂammatory microglia transformed by intraperitoneal injection of LPS. (A) No weight loss by OAL in naïve mice without
STZ i.c.v. (n = 5). (B) No increase in serum inﬂammatory cytokines by OAL. (C) Undetectable serum LPS by OAL. (D) OAL-microglia showed distinct gene
expression patterns from inﬂammatory microglia transformed by LPS i.p. Means ± SE are presented. *p < 0.05 for Student’s t test, one-way ANOVA with Tukey’s
multiple comparisons, or two-way ANOVA with Sidak multiple comparisons. n.s., not signiﬁcant; n.d., not detected; i.p., intraperitoneal.

expression of IL-10 and HSP40 (encoded by Dnaja4gene) (Figure 5C).
To investigate whether CSF1R-stimulated microglia prevent
diabetes-induced neuropathy similar to OAL-microglia in vivo,
STZ was applied to neurons treated with CSF1R-stimulated
microglia culture supernatant (CSF1R-stimulated MCS). As
shown in Figure 5D, it was demonstrated that CSF1-treated
MCS suppressed STZ-induced neuronal cell death (Figure 5D).
indicating the neuroprotective effect of CSF1R-stimulated
microglia. These results demonstrate that neuroprotective
transformations like OAL-microglia in vivo can be reproducedwith CSF1R-stimulated microglia.

Thus, we examined whether neuroprotective transformations
similar to OAL-microglia in vivo could be reproduced throughCSF1R-stimulation of microglia in vitro. C8-B4 microglia, one of
the most widely used in vitro assay systems, is a cell line whose
homology with microglia in vivo has been sufﬁciently conﬁrmed
based on marker expression and immune responsiveness(Figure 5A and Supplementary Figure 5) (24–27). Therefore,
the cell line was used to characterize CSF1R-stimulated microglia
in vitro.
Consistent with OAL-microglia in vivo, CSF1R-stimulated
microglia showed promotion of phagocytosis (Figure 5B). In
addition, CSF1R-stimulated microglia also promoted gene

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A

B

C

D

E

FIGURE 3 | Neuroprotective microglia are transformed through OAL. (A) High expression of Iba1 in isolated microglia (n = 3). (B) Number of isolated microglia(n = 17–19). (C) Ab1-42 levels in isolated microglia (n = 4–6). (D, E) Upregulation of neuroprotective genes and unchanged inﬂammatory genes in isolated microglia
through OAL (n = 4–6). Means ± SE are presented. *p or a,bp < 0.05 for one-way ANOVA with Tukey’s multiple comparisons or two-way ANOVA with Sidak multiple
comparisons. n.s., not signiﬁcant. a,b,cp < 0.05 for two-way ANOVA with Sidak multiple comparisons. The different letters (the symbols of a, b, c) indicate statisticallysigniﬁcant difference between groups in multiple comparison test.

preventing effect of OAL on cognitive dysfunction is found at
higher LPS doses (1 mg > 0.3 mg/kg/day) (2). Based on the above
results, the LPS dose was set to 1 mg/kg/day in this study.
Here, we demonstrate the effect of OAL on preventing DRCD
as MMM test showed that OAL prevented the loss of bothlearning (Figure 1B) and memory ability (Figure 1C) induced bydiabetes. The preventing effect of OAL on high fat diet-induced
cognitive dysfunction has also been demonstrated in ourprevious study, in which mice were individually bred toaccurately control LPS intake (2). Because the preventing effectof OAL on cognitive dysfunction in this study is consistent withDISCUSSION
Our previous studies have revealed that OAL is effective inmaintaining homeostasis (2, 12). We previously showed that theimmunoprotective effect of OAL increases depending on the LPS
dose. Phagocytosis of peritoneal macrophages induced by OAL isenhanced with higher LPS dose (10~1000 mg/kg/day) (28). In highfat diet-fed apoE-deﬁcient mice, the preventive effect of OAL tosuppress weight gain and increasing blood glucose level is observed
at higher LPS doses (1 mg > 0.3 mg/kg/day) (12). Similarly, insenescence-accelerated prone 8 mice fed a high-fat diet, theFrontiers in Immunology | www.frontiersin.org

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A

B

C

D

FIGURE 4 | Promotion of m-CSF1 on peripheral blood leukocytes by OAL. (A) Upregulated m-Csf1 through OAL in peripheral blood leukocytes (n = 5).
(B) Upregulated m-Csf1 by LPS in primary bone marrow cells. (C, D) No increase in s-CSF1 levels through OAL in DRCD [(C), n = 13] and naïve mice [(D), n = 5].
Means ± SE are presented. *p < 0.05 for Student’s t test or one-way ANOVA with Tukey’s multiple comparisons. n.s., not signiﬁcant.

for the prevention of DRCD through OAL, although they do notaffect cognitive ability under physiological conditions.
Histological analysis showed that OAL prevented diabetesinduced cerebral atrophy and the accumulation of argyrophilic
grains (Figures 1F, G), which are mainly composed of
hyperphosphorylated tau protein and are frequently seen inassociation with neurodegeneration and cognitive decline (22).
Therefore, it is indicated that suppression of cerebral atrophy
and tau phosphorylation via microglia are the preventive
mechanisms of DRCD through OAL.
Thus, while microglia are necessary to prevent DRCD
through OAL, DRCD cannot be prevented without OAL, evenin the presence of microglia. It was shown that the characteristics
of OAL-microglia are distinct from those of DRCD-associatedmicroglia. Therefore, transformation to the neuroprotective
microglia induced by OAL is important for DRCD prevention.
Based on the above results, we next characterized the OALmicroglia. Generally, LPS is known to induce inﬂammation throughtransformation into inﬂammatory microglia when systemicallyinjected (29, 30). Therefore, the characteristics of OAL-microglia
in naïve mice were compared to that of inﬂammatory microglia.
As shown in Figure 2, unlike LPS i.p., OAL did not induceside effects such as weight loss and systemic inﬂammation(Figures 2A, B), or inﬂammatory microglia (Figure 2D). It
has been reported that immune training (immune tolerance)

the previous study, it is considered that there is almost no
variation in the amount of water consumed among individuals.
In addition, we previously reported that the phagocytic activityenhancing effect of peritoneal macrophages induced by OAL was
not observed in Toll-like receptor 4 (TLR4)-deﬁcient mice (28).
Therefore, the protective effects induced by OAL are considered
to be TLR4 dependent. Furthermore, considering that the LPS
derived from P. agglomerans used in this study has very low
nucleic acid and protein contamination and activates
macrophages in very small amounts (1.6 ng/ml) (11), it isreasonable to conclude that the effect obtained is due to LPS
derived from P. agglomerans.
Microglia play a central role in the innate immune system andthe maintenance of homeostasis in the central nervous system(CNS). Since our previous study suggested that OAL enhances
phagocytosis of amyloid beta (Ab) by microglia (2), we consideredthat microglia may also play an important role in the prevention of
DRCD through OAL. According to our hypothesis, microglia
depletion using PLX3397 revealed that microglia are necessary for
DRCD prevention through OAL (Figures 1D, E).
Furthermore, microglial depletion did not reduce the spatial
learning memory of saline-injected healthy controls, which wasconsistent with a previous study (20). Because PLX3397 does notaffect the number of other CNS cell types and circulating leukocyte
subsets (20, 21), these results indicate that microglia are indispensable

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A

B

C

D

FIGURE 5 | Neuroprotective microglia are transformed by CSF1R signaling. (A) Dendrite protrusion of CSF1R-stimulated microglia. Bars, 50 (upper) and 10 mm(lower). (B) Promoted phagocytosis of CSF1R-stimulated microglia. (C) Upregulation of neuroprotective genes in CSF1R-stimulated microglia. (D) Prevention ofdiabetes-induced neuronal death by CSF1R-stimulated MCS. Bar, 50 mm. Means ± SE are presented. *p or a,bp < 0.05 for Student’s t test or two-way ANOVA with
Sidak multiple comparisons. MCS, microglia culture supernatant. n.s., not signiﬁcant; n.d., not detected. a,b,cp < 0.05 for two-way ANOVA with Sidak multiple
comparisons. The different letters (the symbols of a, b, c) indicate statistically signiﬁcant difference between groups in multiple comparison test.

received OAL unlike LPS i.p. (Figure 2C), indicating that orally
administrated LPS does not directly impact microglia.
Furthermore, it was revealed that characteristics of OALmicroglia were distinct form DRCD-associated microglia(Figure 3). Because the number of microglia was not signiﬁcantly
different between STZ-injected and LPS-treated mice (Figure 3B),
we concluded that OAL induces qualitative rather than quantitativetransformation of microglia. Consistent with our previous studies

with repetitive low-dose LPS i.p. transforms microglia into a
neuroprotective phenotype. However, mild sickness, weight loss,
and systemic inﬂammation are often associated with LPS i.p.
(31). In contrast, our results challenge the stereotype of LPS as aninﬂammation inducer by showing that the characteristics of
OAL-microglia are fundamentally different from those of
inﬂammatory microglia induced by systemic LPS injection.
Additionally, LPS was not detected in the blood of mice that

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activate CSF1R signaling in microglia via the “just-in-timesystem” method.
Schwartz et al. reported that the cerebral choroid plexus
functions as a location for crosstalk between CNS cells and the
peripheral immune system (54). Under physiological conditions,peripheral immune cells regularly patrol to maintain CNS
homeostasis in the choroid plexus. In contrast, underpathological conditions, peripheral immune cells sense theabnormality in the choroid plexus and prevent neuropathy
through crosstalk with CNS cells by secreting neuroprotective
molecules. Therefore, the cerebral choroid plexus is likely to act
as a communication site for restrictive signaling between mCSF1 of peripheral leukocytes and CSF1R on microglia.
In agreement with the theory of Schwartz et al., OAL had little
effect on the expression of neuroprotective genes in the microglia
of naïve mice (Supplementary Figure 6). Therefore, it isconsidered that CSF1R on microglia is activated by m-CSF1 ofperipheral leukocyte only under pathological conditions such as
CNS diabetes, which induces transformation to neuroprotective
microglia to prevent DRCD. Assuming such an abnormality
detection system, m-CSF1 is more plausible than s-CSF1.
Finally, we investigated whether CSF1R stimulation could
reproduce the characteristics of OAL-microglia (Figure 5). It was
shown that CSF1R-stimulated microglia promoted phagocytosis(Figure 5B) and gene expression of IL-10 and HSP40 (encoded by
Dnaja4 gene) (Figure 5C). Since the expression of the EP4 receptor
and other HSPs is promoted downstream of IL-10 (55, 56), it can besaid that CSF1R-stimulated microglia almost reproduces thecharacteristics of OAL-microglia in vivo, such as high phagocytosis
and neuroprotective gene expression. Furthermore, since CSF1Rstimulated microglia prevented diabetes-induced neuronal death(Figure 5D), it is indicated that OAL-microglia in vivo can be
reproduced with CSF1R-stimulated microglia. Therefore, it isstrongly proposed that transformation to neuroprotective microglia
via CSF1R activation is involved in the mechanism of DRCD
prevention by OAL.
To prove our hypothesis that CSF1R signal mediates thetransformation to neuroprotective OAL-microglia in vivo, we
needed a system that functionally inhibits only the CSF1R signal
without affecting the survival of microglia. However, the problem
is that inhibition of CSF1R induces microglia death because the
CSF1-CSF1R signal controls not only the function of microglia
but also their survival and proliferation (20, 21). In fact, PLX3397
used for microglia depletion in this study is a CSF1R inhibitor.
Therefore, the next task is to develop alternative approaches totest our hypothesis. Our discovery that the CSF1R activation by
m-CSF1 is a key factor that mediates the transformation toneuroprotective OAL-microglia is a major step towards
elucidating the DRCD prevention mechanism through OAL.
In conclusion, the present study demonstrates that OAL
prevents DRCD by transforming microglia to a neuroprotective
phenotype. In addition, activation of CSF1R on microglia
mediated by m-CSF1 expressed on peripheral leukocytes
strongly suggests that the CSFR1-mCSF1 interaction facilitates
the mechanism of microglia transformation (Figure 6). Other
groups also have reported that physiological mucosal exposure towhich suggested that OAL-microglia contributed to the preventionof cognitive dysfunction by promoting the phagocytosis of Ab (2,28), the diabetes-induced suppression of Ab phagocytosis by
microglia was prevented by OAL (Figure 3C). This is considereda background mechanism for preventing tau phosphorylation by
OAL (Figure 1G) since Ab deposition triggers tau protein
phosphorylation leading to neuropathy (32).
Gene analysis revealed that some neuroprotective genes arehighly expressed in OAL-microglia compared to DRCDassociated microglia (Figure 3D). CSF1R regulates survival andproliferation of macrophages and its signal induces transformation
into neuroprotective microglia (33–36). IL-10 is an antiinﬂammatory cytokine and interestingly synergizes with IL-12B toregulate inﬂammation in tumor models (37). Activation of EP4
receptor suppresses brain inﬂammation (38–40), and also
synergizes with CSF1 signaling (41). c-Jun forms activator protein
1 (AP-1), a transcription factor, which is activated downstream of
CSF1 signal involving in transformation to anti-inﬂammatory
macrophage (34, 42). AP-1 is also involved in the induction of
IL-10, IL-12, HSP (43–46). Hsp90aa1, Hspb1, Dnaja4, and Dnajb4
genes encode HSP family, and exogenous HSP promotes Ab
phagocytosis of microglia and induces neuroprotection (47).
These results indicate that OAL-microglia are neuroprotective andqualitatively different from DRCD-associated microglia.
Considering our results above, we speculated that there must be a
mediator that connects OAL and the transformation to neuroprotective
microglia in the DRCD model. We focused on CSF1R as a potential
mediator as it was one of the highly expressed genes in OAL-microglia.
It has been reported that CSF1R signaling induces transformation toneuroprotective microglia via c-Jun (33–36, 42).
Furthermore, peripheral levels of CSF1, a CSF1R ligand,correlate with CSF1R levels in the CNS (48, 49), and peripheral
administration of CSF1 improves neurological disorders (50–52).
Therefore, we hypothesized that OAL increases expression of theperipheral CSF1R ligand, which activates the CSF1R signal toinduce the transformation to neuroprotective microglia.
It was revealed that gene expression of m-CSF1, not s-CSF1 or
IL-34, was promoted by OAL in peripheral leukocytes(Figures 4A, C, D), and that LPS stimulation promoted mCSF1 gene expression in primary cultured bone marrow cells(Figure 4B). Therefore, it is suggested that m-CSF1, whose
expression is promoted on peripheral leukocytes by OAL, is amediator that induces transformation to neuroprotective OALmicroglia via CSF1R. It is yet unsolved mechanisms by which
OAL promotes only m-CSF1, not s-CSF1. Concerning the point
above, since Csf1 gene is transcribed into m-CSF1 or s-CSF1
mRNA forms via alternative splicing (23), OAL is thought toinﬂuence the regulation of mRNA splicing of Csf1 gene.
Since both m-CSF1 and s-CSF1 bind to CSF1R and induce
transformation to homogeneous macrophages (53), the maindifference between the two is assumed to be the mechanism oftransmission. In other words, s-CSF1 acts systemically in an
endocrine manner, whereas m-CSF1 acts locally in a juxtacrine
or paracrine manner (23). Since CSF1R is speciﬁcally expressedin microglia in the brain, it is considered that m-CSF1, whose
expression is promoted on peripheral leukocytes by OAL, may

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FIGURE 6 | A model of transformation into neural microglia through OAL. OAL induces m-CSF1 expressing leukocytes, which patrol and maintain CNS
homeostasis under physiological conditions. Under diabetes-induced pathological conditions, microglia transform to a neuroprotective phenotype mediated by
m-CSF1 in a juxtacrine/paracrine manner, and prevent neuronal damage.

blood ﬂow (63). In addition, because LPS from P. agglomerans isabundant in the organic foods we consume on a daily basis (6, 66–68),
humans have a dietary experience of LPS from P. agglomerans.
Therefore, although more careful safety studies will be required in
the future, LPS from P. agglomerans is thought to be the suitable LPS
considering the application of OAL to human therapy.
Based on the hygiene hypothesis (69), human beings used tointake LPS naturally from the organic environment. Accordingly,
microglia used to be activated physiologically (6, 66, 67). However,the present age is described as the LPS-lost era, in which humanbeings live in an unnaturally clean and inorganic environment. In asense, OAL is like a time machine that returns microglia back to thegood old LPS-abundant era, as if DeLorean of “Back to the Future”.

LPS maintains homeostasis via tissue-resident macrophages (57,58), which supports our ﬁndings.
Microglia depletion has been recently proposed as a new
solution to neuropathy. Indeed, microglia shift to a proinﬂammatory phenotype in some neurological diseases, andtherefore microglia depletion leads to anti-inﬂammatory andneuroprotective effects (21, 59). However, since microglia play
diverse roles in CNS homeostasis, microglia depletion is an
impractical strategy leading to collapse of the brain immune
system and homeostasis (60, 61). However, it is an innovative
method of preventing DRCD by controlling microglialtransformation in a simple and safe way through OAL.
Previous study compared the biological activity of LPS from P.
agglomerans with that of LPS from Escherichia coli and that of LPS fromEnterobacterium (the rice symbiotic bacterium) at the cellular level,
showing that there is not much difference in the biological activity of thethree (62). However, LPS derived from P. agglomerans has been provensafe in animal studies when administered orally (no-observed adverseeffect level 4500 mg/kg/day) (5–7). Also in humans, it is conﬁrmed that
10 mg/kg/day OAL does not induce systemic inﬂammation bymeasuring biomarkers such as white blood cell count, red blood cellcount, aspartate aminotransferase, alanine aminotransferase,
creatinine, C-reactive protein, and immunoglobulin A in humanperipheral blood samples (63). Moreover, OAL at the dose improves
hyperglycemia, hyperlipidemia (64), reduced bone density (65), andFrontiers in Immunology | www.frontiersin.org

DATA AVAILABILITY STATEMENTThe original contributions presented in the study are included in
the article/Supplementary Material. Further inquiries can bedirected to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Careand Use Committee of the Control of Innate Immunity CIP.

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supported by a grant from the Cross-ministerial Strategic
Innovation Promotion Program (SIP-No. 14533073) of theCouncil for Science from Technology and Innovation (CSTI)
in the Cabinet Ofﬁce of the Japanese Government and the
National Agriculture and Food Research Organization(NARO). CSTI and NARO had no role in the study design,
data collection and analysis, decision to publish, or preparationof the manuscript.

AUTHOR CONTRIBUTIONS
HM, HI, and G-IS conceptualized the study and coordinated theexperiments. HM, KY, and MY performed the experiments. HM
performed data curation and formal analysis. YN and CK
provided resource of LPS. HI and G-IS acquired the funding
and administrated the project. HM wrote the manuscript
supervised by G-IS, HI, CK, and YN with contribution from all
authors. All authors contributed to the article and approved thesubmitted version.

SUPPLEMENTARY MATERIALFUNDING

The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/ﬁmmu.2021.
650176/full#supplementary-material

This study was funded by the Control of Innate Immunity
Collaborative Innovation Partnership. This study was also

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Mizobuchi et al.

Prevention of Dementia With LPS

Conﬂict of Interest: HM, KY, MY, HI, and G-IS are employed by the Control of
Innate Immunity, Collaborative Innovation Partnership. YN, HI, CK, and G-IS are
employed by Macrophi Inc. This does not affect our adherence to journal policies
on sharing data and materials.

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