ORIGINAL RESEARCH
published: 24 November 2020
doi: 10.3389/fmolb.2020.596366

Delivery of Periodontopathogenic
Extracellular Vesicles to Brain
Monocytes and Microglial IL-6
Promotion by RNA Cargo
Jae Yeong Ha 1 , Song-Yi Choi 1 , Ji Hye Lee 2 , Su-Hyung Hong 1 and Heon-Jin Lee 1,3*
1Department of Microbiology and Immunology, School of Dentistry, Kyungpook National University, Daegu, South Korea,
Department of Oral Pathology, Dental and Life Science Institute, School of Dentistry, Pusan National University, Yangsan,South Korea, 3 Brain Science and Engineering Institute, Kyungpook National University, Daegu, South Korea
2

Edited by:
Olga N. Ozoline,
Institute of Cell Biophysics (RAS),
Russia
Reviewed by:Simon Swift,
The University of Auckland,
New Zealand
Yosuke Tashiro,
Shizuoka University, Japan*Correspondence:Heon-Jin Lee
heonlee@knu.ac.kr
Specialty section:
This article was submitted toProtein and RNA Networks,
a section of the journal
Frontiers in Molecular Biosciences
Received: 19 August 2020
Accepted: 02 November 2020
Published: 24 November 2020
Citation:
Ha JY, Choi S-Y, Lee JH,
Hong S-H and Lee H-J (2020)
Delivery of Periodontopathogenic
Extracellular Vesicles to Brain
Monocytes and Microglial IL-6
Promotion by RNA Cargo.
Front. Mol. Biosci. 7:596366.
doi: 10.3389/fmolb.2020.596366

Gram-negative bacterial extracellular vesicles (EVs), also known as outer membrane
vesicles (OMVs), are secreted from bacterial cells and have attracted research attention
due to their role in cell-to-cell communication. During OMV secretion, a variety ofcargo such as extracellular RNA (exRNA) is loaded into the OMV. The involvement of
exRNAs from a range of bacteria has been identified in several diseases, however, theirmechanism of action has not been elucidated. We have recently demonstrated that
OMVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans
can cross the blood–brain barrier (BBB) and that its exRNA cargo could promote
the secretion of proinflammatory cytokines in the brain. However, it was unclear
whether the brain immune cells could actually take up bacterial OMVs, which
originate outside of the brain, in an appropriate immune response. In the present
study, using monocyte-specific live CX3CR1-GFP mice, we visualized OMV-colocalized
meningeal macrophages and microglial cells into which bacterial OMVs had been loaded
and intravenously injected through tail veins. Our results suggested that meningeal
macrophages uptake BBB-crossed OMVs earlier than do cortex microglia. BV2 cells(a murine microglia cell line) and exRNAs were also visualized after OMV treatment andtheir proinflammatory cytokine levels were observed. Interleukin (IL)-6 and NF-κB of BV2
cells were activated by A. actinomycetemcomitans exRNAs but not by OMV DNA cargo.
Altogether, these findings indicate that OMVs can successfully deliver exRNAs into brain
monocyte/microglial cells and cause neuroinflammation, implicating a novel pathogenic
mechanism in neuroinflammatory diseases.
Keywords: periodontitis, small RNA, extracellular vesicle, Aggregatibacter actinomycetemcomitans, outermembrane vesicle

INTRODUCTION
Bacterial extracellular vesicles (EVs), also known as outer membrane vesicles (OMVs) in gramnegative bacteria, are around 10–300 nm sized particles released during all growth stages(Bonnington and Kuehn, 2014). Extracellular RNAs (exRNAs) are RNAs that are incorporated inEVs, which can transport them to other cells. Moreover, as bacterial exRNAs are well protected

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exRNAs. We anticipate that our results will shed light on thenovel mechanisms underlying immune signals to the brain in
response to systemic pathogenic infection.

by EV membranes from the harsh milieu outside of cells, they
have been suggested to function as intercellular communication
molecules (Choi et al., 2017a; Das et al., 2019). Not only RNAs but
also various types of cargo such as proteins, DNA, and lipids aretransported by EVs, however, RNAs have gained more researchattention due to their sustained biological activities in recipient
cells (Lee, 2019). In addition, a recent study suggested thepotential involvement of bacterial exRNAs in systemic diseases
and their use as biomarkers (Lee, 2020). The majority of exRNAs
in OMVs are small RNAs in the size range of 15 and 40
nucleotides (nts) (Ghosal et al., 2015), similar in size to eukaryotic
microRNAs (miRNAs) or miRNA-sized small RNAs (msRNAs)in bacteria, implying their regulatory role in host cells (Lee andHong, 2012; Kang et al., 2013; Blenkiron et al., 2016; Koeppen
et al., 2016). Although their precise mechanism of action has
not been clearly elucidated, microbial exRNAs are known tohave certain roles in host gene regulation, immune response,and diseases (see the review Lee, 2020). For these reasons, thedegree of penetration of bacterial EVs to tissues and organs
from circulation in the blood from their originated bacterial
residency remains an important question to uncover the functionof bacterial exRNAs and their relationship with systemic diseases.
A previous short report asserted that intracisternallyinoculated Haemophilus influenzae type b OMVs could increase
the permeability of BBB in rat, which was tested by using
radioisotope-labeled albumin (Wispelwey et al., 1989), but it was
not clear whether OMVs themselves could actually cross the
BBB until our recent demonstration of the evidence (Han et al.,2019). We reported that OMVs from the periodontopathogen
Aggregatibacter actinomycetemcomitans (Aa) could cross the
BBB after intracardiac injection, being detectable in the cortex
of mice by using brain clearance technique. Furthermore, we
observed that exRNAs contained in the Aa OMVs activate
TNF-α in the mouse brain cortex (Han et al., 2019). However,it was not clear what type of brain cells were responsible forpromoting the production of TNF-α by Aa exRNAs even though
the exRNAs of Aa increased the expression of TNF-α in theactivated human-macrophage-like cells.
Mononuclear phagocytes, i.e., monocytes and macrophages,
are key players in protective immunity and homeostasis(Ginhoux and Jung, 2014). Microglial cells are resident
monocytes/macrophages in the central nervous system (CNS)
whose primary role as immune defense against infection is criticalin modulating neuroinflammation as well as the development ofseveral neurodegenerative disorders such as Alzheimer’s disease(AD) (Block et al., 2007; Ginhoux et al., 2013; Li and Barres,2018).
In the present study, to explore the neuroinflammatory
effect of pathogenic OMVs, we characterize how intravenously
injected Aa OMVs can be taken up by monocyte-derived cells
using intravital imaging analysis of monocyte marker-expressing
CX3CR1-GFP knock-in mice. Tail-vein-injected OMVs and
exRNA cargo were delivered into meningeal macrophages andcortex microglial cells in a time-sequential manner. Moreover,analysis of the effect of Aa exRNAs on the murine microglial
BV2 cell line showed that a proinflammatory cytokine other than
TNF-α, Interleukin (IL)-6, is promoted in microglia cells by Aa

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MATERIALS AND METHODS
CultureA. actinomycetemcomitans (Aa, ATCC 33384) was inoculated onbrain heart infusion (BHI; Difco, Sparks, MD, United States) agar
plates in an anaerobic incubator. After 24 h, the colonies were
picked and cultured in BHI media for 48 h. Picked colonies werealso checked for ribosomal DNA to avoid any contamination.
The anaerobic incubator was maintained at 37◦ C in 5% CO2 , 5%
H2 , and 90% N2 . Aa were grown anaerobically in BHI until thedesired optical density (OD) was reached (around 0.7 of OD600 ).
Supernatants were collected for OMV purification andbacterial pellets were collected for RNA isolation andpurity evaluation.
The BV2 cells, mouse macrophage-like microglial brain
cells, were maintained on 6-well plates with 5× 105 cells perwell in DMEM medium supplemented with 10% FBS and
100 IU/ml penicillin G. The complete medium was changed
three times a week.

Animals
The G-protein-coupled receptor CX3CR1 is expressed in humanmonocytes, and CX3CR1-GFP transgenic mice are widely usedin studies on microglial cells (Jung et al., 2000). The CX3CR1GFP mice (B6.Cg-Ptprca Cx3cr1tm1Litt/LittJ, The JacksonLaboratory, Bar Harbor, ME, United States) were housed inaccordance with the standard guidelines for the care anduse of laboratory animals, the animal protocols having been
approved by the Institutional Animal Care and Use Committee(IACUC) of the Korean Advanced Institute of Science andTechnology (KAIST, Daejeon, South Korea) for the company
IVIM Technology (Daejeon, South Korea). All mice underwent
surgery under anesthesia, and all efforts were made to minimize
their suffering. Mice were individually housed in ventilated,temperature- and humidity-controlled cages (22.5◦ C, 52.5%)
under a 12:12 h light:dark cycle and provided with standard dietand water ad libitum.

OMV Sample Preparation
Aa OMV samples were prepared as previously described (Hanet al., 2019). Briefly, OMVs were obtained from the filteredand concentrated supernatant using ExoBacteria OMV Isolation
Kits (SBI, Mountain View, CA, United States) according to
the manufacturer’s protocol. The isolated OMVs were treatedwith 1 µl of RNase A (1 U/µl, Thermo Fisher Scientific,
Wilmington, DE, United States) and DNase I (2 U/µl, Thermo
Fisher Scientific) to make a 1.5 ml volume of the OMVs, which
was then incubated at 37◦ C for 25 min. This was done to remove
any residual nucleic acids in the OMV preparation. The OMVs
were purified again using the ExoBacteria OMV Isolation Kits to
get rid of unnecessary remained enzymes. Transmission electron

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micrographs (TEM) of Aa OMVs (pre-purification and purified)were compared in Supplementary Figure S1.
For physical lysis, the OMVs were frozen and thawed
five times followed by sonication using a sonicator (100 W
and 20 KHz; KSC-100 portable ultrasonicator, Korea Process
Technology, Seoul, South Korea) for 30 s and then placed on ice
for 30 s. Subsequently, 1 µl of RNase A and/or DNase I was added
to 1 ml of the lysed OMVs and incubated at 37◦ C for 25 min. The
purified OMVs were checked for sterility by spreading 10 µl of
the purified OMVs on BHI agar plates for 3–4 days and stored at−80◦ C until use.
For staining, the OMVs (resuspended in 100 µl in PBS,
without calcium and magnesium; GE Healthcare Bio-Sciences,
Pittsburgh, PA, United States) were incubated with 2 µM SYTORNASelect Green Fluorescent Cell Stain Kit (Thermo Fisher
Scientific) and/or 10 µM red fluorescent Lipophilic Tracer DiD(1,1’-dioctadecyl-3, 3, 39, 39-tetramethylindodicarbocyanine, 4chlorobenzenesulfonate salt; Thermo Fisher Scientific) for 1 h
at 37◦ C. The samples were washed once with PBS followed byultracentrifugation at 150,000 × g for 1 h at 4◦ C.
Both intact OMVs and lysed OMVs were analyzed forlipopolysaccharide (LPS) concentrations, for which the Pierce
LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher
Scientific) was used according to the manufacturer’s instructions.

Japan; magnifications: × 25; numerical aperture: 1.1) was used
to visualize the brain cellular uptake of OMVs. During theintravital imaging, the body temperature of the mouse wasmaintained at 37◦ C using a homeothermic controller. Mice wereanesthetized using intramuscular injections of a cocktail mixtureof zoletil (30 mg/kg) and xylazine (10 mg/kg). The imaging
analysis was conducted as previously described (Ahn et al.,2017). All experiments were performed in duplicate using two
independent OMV sample preparations and at least two mice foreach experiments.
All the intravital imaging experiments conducted using mice
were performed by IVIM Technology according to institutionaland national guidelines.

Confocal Microscopic Imaging
The OMVs and RNAs inside were stained as described earlier
in the OMV sample preparation section of “Materials andMethods.” Stained OMVs were added to BV2 cells cultured onchamber slides and incubated for 24 h at 37◦ C, then washedfour times with 500 µl of PBS, followed by incubation withDAPI (Vector Laboratories, Burlingame, CA, United States).
The stained OMVs with the added cells were visualized on alaser scanning confocal microscope (LSM Zeiss 800; Carl Zeiss
Microscopy, Jena, Germany) equipped with an objective lens(magnifications: × 40 with water immersion; numerical aperture:
1.2; resolution: 0. 28 µ m).

OMV Analysis
The OMVs underwent nanoparticle tracking analysis using
the NanoSight system (NanoSight NS300; Malvern Panalytical,
Malvern, United Kingdom) according to the manufacturer’s
protocols. Samples were diluted 100 times in a total volumeof 1 ml of PBS. PBS was introduced into the chamber using
a 1 ml syringe and assessed whether it was free from particles
and whether the chamber was clean (i.e., no light scattering).
Particle size was measured based on Brownian movement andparticle concentration was quantified in each sample. Protein
quantification of OMVs and OMV lysates were done using
Bradford reagent (Bio-Rad, Hercules, CA, United States) and the
NanoSight system as previously described (Han et al., 2019).

Total RNA Extraction
Total RNA was extracted using both TRIzol (Invitrogen,
Carlsbad, CA, United States) and the miRNeasy Mini Kit(QIAGEN, Valencia, CA, United States) according to the
manufacturers’ protocols, with some modifications. Each sample
was resuspended in 300 µl TRIzol reagent (Invitrogen).
Resuspension was homogenized for 30 s using a Kontes
Pellet Pestle Cordless Motor (Sigma-Aldrich, St. Louis, MO,
United States) and placed on ice for 30 s, the steps being
repeated 5 times for full homogenization. Then, an additional
700 µl TRIzol was added to the samples and incubated atroom temperature for 5 min. 1-bromo-3-chloropropane (200µl, Sigma-Aldrich) was then added to each sample, followed
by incubation for 3 min. Next, the samples were centrifuged at
13,499 × g for 15 min at 4◦ C using a centrifuge (Smart R17
plus, Hanil Science, Incheon, South Korea). The supernatant(approximately 750 µl) of each sample was mixed with 750 µlof 100% isopropanol. The mixed samples were incubated at 20◦ C for 2 h. The columns of miRNeasy Mini Kit (QIAGEN)
were additionally used to purify the small RNA-enriched RNA
and clean RNA according to the manufacturer’s protocol.

Intravital Imaging
Three weeks before intravital imaging, cranial window surgery of
CX3CR1-GFP mice was accomplished according to a previously
published protocol (Holtmaat et al., 2009). Briefly, the skin andskull were removed followed by drilling to remove the cranialbone. The exposed brain was covered by glass, and dental cement
was applied on the top of the skull and part of the cover glass,sealing off the exterior. All the exposed skull and wound edges
were covered with dental cement. The strategy used for the
intravital image acquisition of Aa OMV-injected mice is depicted
in Figure 1A. Mice were housed for 3 weeks for stabilization
before conducting intravital imaging.
After performing mouse tail vein (intravenous, i.v.) injectionsof DiD-stained OMVs (100 µl; approximately 3.0 × 108
particles) and anti-CD31 monoclonal antibody (60 µl; 553708,
BD Bioscience) conjugated with Alexa Fluor 555 (A- 20009,
Invitrogen), a laser scanning intravital confocal microscope(IVM-C, IVIM Technology) with an objective lens (Nikon,

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qRT-PCR
Total RNA (1 µg) was reverse-transcribed using OmniScript(QIAGEN). The qRT-PCR was performed with diluted
cDNA using primer sets for TNF-α, IL-1β, IL-6, and βactin (Supplementary Table S1). The PCR was performed in
96-well plates using the 7,500 Real-Time PCR System (Applied
Biosystems, Foster City, CA, United States). The expression

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FIGURE 1 | Delivery of Aa OMVs into microglial cells. (A) Schematic diagram of intravital imaging process performed in this study. (B) Nanoparticle tracking analysis(NTA) of Aa OMVs used in this study. OMVs from Aa were analyzed using the NanoSight system to visualize the size distribution (X-axis) and particle numbers(Y -axis). (C) Intravital image was captured 24 h after Aa OMV i.v. injection. OMVs were colocalized with GFP-positive microglial cells. Scale bar: 50 µm. Additional
Figures can be found in Supplementary Figure S3.

of each gene was determined from three replicates in a single
qRT-PCR experiment.

membrane using Pierce Power Blotter (Thermo Fisher Scientific)
at 110 V (80 mA) for 80 min. The membrane was incubated
in 5% skim milk/0.1% TBS-Tween 20 at room temperature for
1 h, followed by incubation with a 1:1,000 final diluted NFκB p65 (#8242, Cell Signaling Technology) and phospho-p65
antibody (Ser536, #3033, Cell Signaling Technology) and thenan anti-rabbit secondary antibody conjugated with horseradish
peroxidase (#7074, Cell Signaling Technology). The control for
loading was assessed using a β-actin antibody (at a dilution of
1:1,000, SC-47778, Santa Cruz Biotechnology Inc., Santa Cruz,
CA, United States).

Immunoassays of Cytokines
BV2 cells were incubated with purified OMVs for 24 h,after which the filtered supernatants using 0.22 µm porefilter (Sigma-Aldrich) were analyzed for four cytokines (IL1β, IL-6, TNF-α, and IFN-γ) using a quantitative multiplex
ELISA (Mouse Cytokine Panel 2 (4-plex); QUANSYS
Biosciences, Logan, UT, United States) according to the
manufacturer’s instruction. Data were analyzed using the
Q-View Software (Quansys Biosciences). All experiments wereconducted in triplicate.

Statistical Analysis
All data are presented as mean ±SD. Differencesamong sample group values were analyzed using a oneway ANOVA and post hoc analyses were performed
using the Tukey Test. All analyses were conductedusing Origin 8.0 software (OriginLab, Northampton,

Western Blotting
For the western blotting of NF-κB p65 and phospho-p65, 20 µg ofwhole cell lysates was used for SDS polyacrylamide gel-running,
followed by transfer to a polyvinylidene difluoride (PVDF)

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MA, United States), and p< 0.05 were considered to bestatistically significant.

region of colocalized monocytes/macrophages (the dotted
area in the magnified images) inside and outside of themeningeal blood vessel was also observed at 4–8 h, asseen in the magnified images in the region marked withyellow squares (Figure 2C). Colocalized monocytes (marked
in yellow in the merged images) in the dotted area areclearly observed in both the exterior and lumen of themeningeal blood vessel.
To observe whether the injected OMVs can reach microglial
cells, intravital images of cortex were captured at 24 and 48 h
as more time was required for observation. Three random field
images (Figure 3A) revealed that the OMVs (red) taken up by
GFP-positive microglial cells (Figure 3A, arrowheads). Moreover,in the magnified stack images acquired during confocal imaging
shown in Figure 3A, the OMV-colocalized microglial cells
are clearly visualized in the exterior region of the vessel at
48 h (Figure 3B, arrowheads of S1 and S2 random fields).
Although we could not observe whether the OMVs had crossed
the BBB and colocalized with cortex microglial cells at 8 hof OMV injection, it was assumed that OMVs that werei.v. injected into the tail crossed the BBB and reached thecortex sometime after 8 h (Figure 3C). These data demonstratethat OMVs can pass through the BBB and internalize intomeningeal macrophages, thus allowing cargo into microglial
cells of the brain.

RESULTS
Intravital Imaging Reveals That
Tail-Vein-Injected Aa OMVs Can Enter
Mouse Brain Monocytes
Unlike eukaryotic EV (exosomes), which have been shownto deliver cargo to the brain after crossing BBB (Matsumoto
et al., 2017; Yuan et al., 2017), little is known about theability of peripheral bacterial EVs to reach the brain; werecently suggested that Aa exRNAs are the primary causative
agents promoting the secretion of proinflammatory cytokines
in macrophages and the mouse brain (Han et al., 2019).
We therefore intended to confirm, using intravital imaging
technique, whether brain monocytes or microglial cells cantake up bacterial OMVs and their cargo when injected
through the tail vein.
To investigate whether Aa OMVs can be taken up by
mouse microglial cells, we first investigated the Aa OMVs
injected (tail i.v. injection) into monocyte-specific CX3CR1GFP mice using the intravital imaging system (Figure 1A).
The number and size distribution of Aa OMVs used in thisstudy were analyzed using a nanoparticle tracking analysis
system. The majority of OMVs were in the size range of
100–280 nm (Figure 1B). As the NTA system could notprecisely detect particle sizes < 60 nm (Bachurski et al.,2019), some smaller OMVs of Aa might have been missed.
Therefore, the actual particle numbers might be greater thanthe measurement results we present here. Our previous study
found that Aa OMVs apparently crossed the mouse BBB
after 24 h of intracardiac injection (Han et al., 2019). To
reduce the risk of cardiac damage, we switched to i.v. tailinjections to administer stained Aa OMVs. Membrane-stained(red) OMVs were colocalized with GFP+ microglial cells 24 hafter the injection of Aa OMVs into the cortex (Figure 1C andSupplementary Figure S3).
To ensure detection of localized of OMVs, brain bloodvessels were also fluorescently labeled by i.v. injection ofanti-CD31 antibody 1 h before imaging. At an imaging
depth of around 80 µm (from surface of the brain), wheremouse meninges are located, the GFP-positive meningeal
monocytes/macrophages were first identified using intravital
imaging analysis. As shown in Figure 2A, the majority of
OMVs in the meningeal blood vessel lumen are observed
at 4 h, suggesting that tail-injected OMVs can disseminate
into several organs through blood vessels. OMVs were taken
up by GFP-positive “patrolling” monocytes/macrophages in
the lumen of vessel (yellow arrow), whereas some OMVs
began to cross the blood vessel wall and colocalize withmeningeal macrophages at 8 h (white arrowhead in Figure 2A).
The magnified and stack images of monocytes/macrophages
shown in Figure 2A at 8 h clearly demonstrate the OMVs
and GFP-positive cell colocalization (Figure 2B). Another

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Aa OMVs Are Delivered Into the BV2Microglial Cells
We previously reported that Aa OMVs can penetrate macrophagecells along with their RNA cargo (Han et al., 2019). Due
to differences between macrophages and microglial cells, werepeated the confocal microscopy analysis of OMVs with the
RNA cargo. As the SYTO RNA-Select reagent is membranepermeable, we could detect RNA colocalization with the OMV
membrane inside BV2 cells (Figure 4). The stained OMVs
along with the RNA cargo were incubated with BV2 cells
for 24 h, followed by counterstaining with DAPI to visualize
the nuclei. Observation indicated OMVs and RNA cargo caninternalize microglial cells, consistent with previous studies
conducted using other cell types (Ho et al., 2015; Blenkiron
et al., 2016; Koeppen et al., 2016; Choi et al., 2017a; Han et al.,2019).

Aa exRNAs in OMVs Activate IL-6 in BV2Microglial Cells Through NF-κB
Activation
To explore the effect of Aa OMVs and the RNAs in OMVs
on BV2 cells, we treated BV2 cells with Aa OMVs and
OMV lysate for 16 h (the LPS levels being approximately 50
ng/ml in both OMV and OMV lysates). We infected around
4.5 × 108 particles of Aa OMVs to the cells (5 × 105 cells),
the higher number of OMVs resulting in cell death under
our experimental condition (cell viability results are shown in
Supplementary Figure S2).
We performed 4-flex cytokine (IL-1β, IL-6, TNF-α, and IFNγ) arrays from the BV2 cells treated with Aa OMVs and OMV

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FIGURE 2 | Visualization of tail-vein-injected OMVs and meningeal macrophages of live CX3CR1-GFP mice. (A,B) OMVs taken up by monocytes were compared
between 4 and 8 h after OMV injection. Confocal microscopic acquisition results show the OMV distribution before and 4 and 8 h after OMV i.v. injection of mice.
(A) The white dotted line delineates the lumen of blood vessels. White arrowheads indicate Aa OMV-colocalized meningeal macrophages, and yellow arrows indicate
OMV-colocalized monocytes/macrophages in the lumen of vessel. Scale bar: 50 µm. (B) Representative image stacks (5 µm intervals) acquired during confocal
imaging of mice with GFP-positive microglial cells for OMVs injected (8 h) shown in (A). The white dotted area refers to the colocalization of OMVs (red) and
CX3CR1-GFP-positive monocytes (green). Scale bar: 50 µm; magnified scale: 20 µm. (C) Colocalized monocytes/macrophages (dotted areas in the magnified
images) in the exterior and lumen of the meningeal blood vessel were compared. Magnified images (scale bar: 20 µm) of colocalized monocytes (shown in yellow inthe merged images) in the dotted area are clearly seen in both the exterior and lumen of the meningeal blood vessel.

lysate to investigate the effect of Aa OMVs and exRNAs on
microglial cells. The levels of IL-1β and IFN-γ were too low to
detect, whereas IL-6 and TNF-α levels were detectable. However,unlike macrophage-like U937 cells (Han et al., 2019), TNF-α
was not detectable in the BV2 cells treated OMVs and OMVlysates (Supplementary Table S2). The level of IL-6 protein
measured from accumulated culture media of BV2 cells was
upregulated by intact OMVs but not in OMV lysates treatedwith nucleases (DNase and RNase). This result indicated thatthe nucleic acid cargo of Aa OMVs is responsible for theactivation of IL-6. Moreover, OMV lysates were treated with
DNase or RNase to examine whether DNA or RNA is further
responsible for the elevated IL-6 secretion. The result indicatedthat RNA rather than DNA cargo is the pivotal factor forIL-6 promotion because the OMV lysate treated with only
RNase demonstrated less IL-6 secretion than that treated with
only DNase (Figure 5A). Similar results were obtained at the

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RNA transcript level of IL-6, which was measured by qRTPCR (Figure 5B).
The human IL-6 gene promoter possesses a bindingsite for NF-κB, which activates the expression of IL-6
[known to be induced by various stimuli such as LPS,
TNF-α, and Poly (I:C) (Tanaka et al., 2014; Kumar,2019)]. Our previous investigation demonstrated thatOMV RNA of Aa promoted the activation of NF-κB
via TLR8 in macrophage U937 cells (Han et al., 2019).
Therefore, to explore the effect of Aa exRNAs on NF-κB
activation, we performed western blotting using antiphospho-p65 antibody to detect phosphorylated p65,
which is the active subunit of NF-κB. We found thatremoval of RNA cargo of OMVs by treatment with
RNase reduced phosphorylation activity, whereas total p65
expression showed no apparent differences (Figure 5C).
The overall findings indicated that Aa exRNAs promote

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FIGURE 3 | Visualization of tail-vein-injected OMVs and microglial cells in the cortex of live CX3CR1-GFP mice. (A) OMVs taken up by microglial cells were imaged at
24 and 48 h after OMV injection. White arrowheads indicate OMVs taken up by microglial cells. Fluorescence-dye-conjugated anti-CD31 antibody was injected 1 h
before imaging. Three different regions were captured. Scale bar: 50 µm. (B) Representative image stacks (5 µm intervals) acquired during confocal imaging ofexperimental mice with GFP-positive microglial cells for OMVs injected (48 h). The arrowheads refer to the colocalization of BBB-crossed OMVs (red) and
CX3CR1-positive microglial cells (green) localized in the exterior of vessels. Scale bar: 50 µm; magnified scale: 20 µm. (C) Confocal microscopic acquisition results
showing the OMV distribution before OMV injection (–1 h) and 8, 24, and 48 h after OMV i.v. injection of mice. The white dotted rectangle in the left panel was
magnified. Right panel: magnified images of OMVs taken up by microglial cells (dotted circles). Scale bar: 100 µm; magnified scale: 20 µm.

been investigated with regard to AD (Wang et al., 2015).
However, there is limited information regarding the effect ofpathogenic exRNAs in microglial cells on proinflammatory
cytokine secretion.
The meninges consist of three layers, the dura mater, thearachnoid mater, and the pia mater. The arachnoid materis a membrane barrier that separates the dura mater fromthe remaining portion of the brain and has well-regulated
junctions, similar to the BBB (Rua and McGavern, 2018).
Studies conducted using CX3CR1-GFP transgenic micereported a high density of meningeal macrophages in the
dura mater and pia mater (Chinnery et al., 2010). Alongwith other CNS macrophages (perivascular and choroid
plexus), meningeal macrophages are non-parenchymal immune
modulators at brain boundaries (Goldmann et al., 2016; Ruaand McGavern, 2018). Therefore, their immune response
against infection could have pivotal consequences for CNS
homeostasis and diseases. However, these macrophages
have received little research attention with respect to oral
pathogens and commensal bacteria in the context of brain
pathogenesis due to the tight defenses of the CNS. Recently,
considerable effort has been devoted to understanding tiny

the production of IL-6 in microglial BV2 cells throughNF-κ B activation.

DISCUSSION
Among the numerous bacteria involved in periodontitis, Aa
is a small gram-negative bacterium previously well-known for
causing localized aggressive periodontitis (Henderson et al.,2010). Aa is also implicated in various systemic diseases suchas non-alcoholic fatty liver disease (Komazaki et al., 2017),
diabetes (Demmer et al., 2015), and rheumatoid arthritis(Konig et al., 2016). Furthermore, recent evidence suggeststhat Aa and other oral microorganisms are associated withneuroinflammatory diseases such as AD (Laugisch et al., 2018;
Dominy et al., 2019; Díaz-Zúñiga et al., 2019; Han et al., 2019;
Wu et al., 2019). However, whether bacteria themselves directlyor their derivatives are involved in diseases remains debatable(Lee, 2020).
Microglial cells are activated by various stimuli such as
LPS, IFN-γ, and β-amyloid and also release proinflammatory
cytokines such as IL-6, IL-1β, and TNF-α, which have

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FIGURE 4 | Delivery of Aa OMVs and RNA into BV2 cells. Aa OMVs were prestained with the lipid tracer dye DiD (red) and RNA-specific dye SYTO RNASelect(green). Stained OMVs (approximately 4.5 × 108 particles) were incubated with BV2 cells on a chamber slide for 24 h at 37◦ C. DAPI was also counterstained withDAPI (blue) to visualize the nuclei. (A) Confocal microscopy analysis of Aa OMVs revealed colocalized OMVs and the RNA cargo inside (overlay). Bar = 20 µm. (B,C)
3D rendering of confocal fluorescence images. Bar = 10 µm.

particles originated from bacteria (i.e., bacterial EVs) and theirroles as messengers in human diseases given their ability totravel freely from cell to cell in the body (Choi et al., 2017b;
Lee, 2019).
In the present study, we demonstrated for the first time,using intravital imaging analysis, the transport of OMVs fromperipheral introduction to brain microglial cells through themeninges. The delivery of bacterial EVs into the brain after
crossing the BBB suggests that infection at any place in the
body can cause a fatal immune response in the brain. Since
the number of human commensal bacteria has been estimatedto be the same as the total number of human cells (Sender
et al., 2016), bacterial EVs would be continually produced
and the effect of chronic brain stimulation by bacterial EVs
from commensal bacteria may be attributed bacterial OMVs
crossing the BBB.

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OMVs are taken up by host cells through various methods,
and it is known that Aa OMVs are internalized into cellsby membrane fusion or clathrin-mediated endocytosis(Rompikuntal et al., 2012; Thay et al., 2014). Our results
clearly demonstrated that Aa OMVs and RNA cargo (i.e.,
exRNA) can enter into microglial cells (Figure 4).
It is known that LPS isolated from Aa has the ability tostimulate the production of TNF-α, IL-1β, and IL-6 in humanwhole blood (Schytte Blix et al., 1999) and microglial cells (DíazZúñiga et al., 2019), however, little is known about the effect ofother pathogenic factors of Aa on microglial cells. Because intact
LPS of OMVs might have less effect than purified or released
forms of OMVs, internalized OMVs and their cargo may activate
various immune signals as cytosolic LPS activates caspase-11 andexRNAs enhance the TLR8 pathway (Vanaja et al., 2016; Hanet al., 2019).

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FIGURE 5 | ExRNAs of Aa activate IL-6 in BV2 cells through the NF-κB signaling pathway. (A) Secreted IL-6 protein levels were upregulated at 16 h after treatment
with intact OMVs. IL-6 secretion by OMV lysate was decreased by RNase-only treated OMV lysates in BV2 cells at 16 h. (B) qRT-PCR analysis revealed that thetranscript levels of IL-6 activation by Aa OMV lysates were decreased by RNase-only pretreatment at 16 h. (C) NF-κB activation (phosopho-p65, upper panel) was
significantly decreased by RNase-only-treated OMV lysates compared with DNase-treated OMV lysates at 16 h. Total NF-κB p65 (middle panel) and actin (bottom
panel) levels were assessed for controls. BV2 cells were seeded onto 6-well plates (5 × 105 cells/well) and treated with Aa OMVs (approximately 4.5 × 108
particles/well in 2 ml of media) and OMV lysates (the same amount of proteins as in 4.5 × 108 OMV particles). Data are presented as mean ± SD from three
independent experiments. The letters (a–c) indicate significant differences at p < 0.05.

it elevates the activity of proinflammatory signaling pathways andis associated with a large number of immune diseases (Luo andZheng, 2016). Therefore, the increased IL-6 production in BV2
microglial cells by Aa exRNAs might be a novel causative agent ofneuroinflammatory diseases.
Furthermore, our previous data showing that increased
production of TNF-α by Aa OMVs in the mouse brain (Hanet al., 2019) might be modulated by other cell types such as

Interestingly, murine BV2 microglial cells reacted in a manner
unlike human U937 macrophage cells upon treatment with Aa
OMVs and exRNAs. Because we used a relatively low number(4.5 × 108 particles) of OMVs compared to that in a previous
study (4.5 × 1010 particles) to infect macrophage cells while
avoiding BV2 cell damage, the lower dose of OMVs may besimilar to the actual immune response that occurs in vivo. Amongthe various proinflammatory cytokines, IL-6 is the main player as

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astrocytes suggests that activated microglial cells induce thesecretion of TNF-α by astrocytes (Liddelow et al., 2017). As TNFα stimulates the induction of IL-6 production as observed in brain
pericytes and astrocytes and pericyte-originated IL-6 activated
BV2 microglial cells (Matsumoto et al., 2018), various types ofbrain cells interact with each other via cytokines they produce in
response to exogenous bacterial EVs and their cargo.
Taken together, our findings reveal a previously unrecognizedmechanism of RNA transport in bacterial EV-based induction ofbrain immune response to peripheral bacterial infections. Further
studies are necessary to investigate the range of bacterial EVs and
exRNAs that are constantly produced and affect entire organs. It
is also necessary to further explore strategies for preventing thechronic stress caused by bacterial EVs and exRNAs in relevant
immune diseases.

Advanced Institute of Science and Technology (KAIST, Daejeon,South Korea).

AUTHOR CONTRIBUTIONS
JH, S-YC, and JL contributed to the data collection and analysis.
S-HH contributed to the data analysis and critically revised themanuscript. H-JL drafted the manuscript and contributed to thedata collection and analysis as well as conception and design.
All authors gave their final approval and agreed to be held
accountable for all aspects of the work.

FUNDING
This research was supported by the Basic Science Research
Program through the National Research Foundation of
Korea (NRF), funded by the Korean Government (MSIT
2017R1A5A2015391 and 2018R1D1A3B07043539).

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

SUPPLEMENTARY MATERIALETHICS STATEMENTThe Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fmolb.
2020.596366/full#supplementary-material

The animal study was reviewed and approved by the Institutional
Animal Care and Use Committee (IACUC) of the Korean

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Conflict of Interest: The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interest.
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