BRIEF RESEARCH REPORT
published: 08 December 2020
doi: 10.3389/fcell.2020.613733

Neutrophils Return to Bloodstream
Through the Brain Blood Vessel After
Crosstalk With Microglia During
LPS-Induced Neuroinflammation
Yu Rim Kim 1,2† , Young Min Kim 3† , Jaeho Lee 1,2 , Joohyun Park 1,2 , Jong Eun Lee 1,2 andYoung-Min Hyun 1,2*
1

Edited by:Zhichao Fan,UCONN Health, United States
Reviewed by:Rongrong Liu,
Northwestern University,
United States
Bo Liu,University of California, Berkeley,
United States
Souvarish Sarkar,
Brigham and Women’s Hospital andHarvard Medical School,
United States*Correspondence:
Young-Min Hyun
ymhyun@yuhs.ac† These authors have contributed

equally to this work
Specialty section:
This article was submitted toCell Adhesion and Migration,a section of the journal
Frontiers in Cell and Developmental
Biology
Received: 03 October 2020
Accepted: 20 November 2020
Published: 08 December 2020
Citation:
Kim YR, Kim YM, Lee J, Park J,
Lee JE and Hyun Y-M (2020)
Neutrophils Return to Bloodstream
Through the Brain Blood Vessel After
Crosstalk With Microglia During
LPS-Induced Neuroinflammation.
Front. Cell Dev. Biol. 8:613733.
doi: 10.3389/fcell.2020.613733

Department of Anatomy, Yonsei University College of Medicine, Seoul, South Korea, 2 BK21 PLUS Project for Medical
Science, Yonsei University College of Medicine, Seoul, South Korea, 3 Department of Medicine, Yonsei University Collegeof Medicine, Seoul, South Korea

The circulatory neutrophil and brain tissue-resident microglia are two important immune
cells involved in neuroinflammation. Since neutrophils that infiltrate through the brain
vascular vessel may affect the immune function of microglia in the brain, close
investigation of the interaction between these cells is important in understanding
neuroinflammatory phenomena and immunological aftermaths that follow. This study
aimed to observe how morphology and function of both neutrophils and microglia areconverted in the inflamed brain. To directly investigate cellular responses of neutrophils
and microglia, LysMGFP/+ and CX3 CR1GFP/+ mice were used for the observation ofneutrophils and microglia, respectively. In addition, low-dose lipopolysaccharide (LPS)
was utilized to induce acute inflammation in the central nervous system (CNS) of
mice. Real-time observation on mice brain undergoing neuroinflammation via twophoton intravital microscopy revealed various changes in neutrophils and microglia;
namely, neutrophil infiltration and movement within the brain tissue increased, while
microglia displayed morphological changes suggesting an activated state. Furthermore,neutrophils seemed to not only actively interact with microglial processes but also
exhibit reverse transendothelial migration (rTEM) back to the bloodstream. Thus, it
may be postulated that, through crosstalk with neutrophils, macrophages are primed
to initiate a neuroinflammatory immune response; also, during pathogenic events inthe brain, neutrophils that engage in rTEM may deliver proinflammatory signals toperipheral organs outside the brain. Taken together, these results both show thatneuroinflammation results in significant alterations in neutrophils and microglia and lay
the pavement for further studies on the molecular mechanisms behind such changes.
Keywords: neuroinflammation, neutrophil, microglia, reverse transendothelial migration, two-photon intravital
imaging

Abbreviations: BBB, Blood-brain barrier; CNS, Central nervous system; GFP, Green fluorescent protein; LPS,
Lipopolysaccharide; rTEM, reverse Transendothelial migration; SPF, Specific pathogen-free; TEM, Transendothelial
migration; WGA, Wheat-germ agglutinin.

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INTRODUCTION

MATERIALS AND METHODS

Neuroinflammation is generally defined as the response of brain
cells to infection and other sources of cell death, involving
infiltration of circulating immune cells to the brain. Such
infiltration of immune cells occur due to microglial and glial
cell activation and blood-brain barrier (BBB) dysfunction during
the pathogenesis of various illnesses, such as Alzheimer’s disease,
Parkinson’s disease, and Amyotrophic lateral sclerosis (Shastri
et al., 2013; Mammana et al., 2018).
Neutrophils are commonly known as the earliest responders
to acute inflammation, aiding the initiation and continuationof immune reactions throughout the human body (Nathan,2006). Neutrophils are highly versatile cells with various immune
functions, such as inflammation mediation, microbial capture via
granular proteins, and repair of sterile wounds (Kruger et al.,2015). In particular, during neuroinflammation, neutrophils
participate in the general immune response by signaling todiverse cell types, including endothelial cells, mesenchymal stem
cells, lymphocytes, and microglia (Ahn et al., 2020). Recent
reports have emphasized the variety of roles neutrophils can playin neuroinflammation, where brain resident cells participate in acoordinated fashion (Liu et al., 2018).
Microglia, the resident macrophages of the central nervous
system (CNS), are distinguished from other glial cells, suchas astrocytes and oligodendrocytes by their gene expression,
morphology, and function (Ransohoff and Perry, 2009;
Kettenmann et al., 2011; Zhao et al., 2019). In contrast to otherglial cells, microglia function as the primary reacting cells forregulating neuroinflammatory response by phagocytizing andremoving myelin inhibitors, debris and dead cells in the CNS (Liet al., 2005; Zhao et al., 2019). Microglia also take part in innate
and adaptive immunity by regulating immune tolerance (Saijo
and Glass, 2011). Microglia are functionally and morphologicallydivided into three forms: the ramified, activated and ameboid
morphologies. Ramified microglia, with a small cell body andlong branches, have no functional capability of phagocytosis
and antigen presentation but maintain an immunologicallystable environment. When ramified microglia are stimulated
by neurodegeneration, endotoxin, interferon, or endothelial
activation, activation pathways cause them to transform into
activated microglia. Activated microglia exhibit thicker andmore retracted branches and possess the ability exhibit antigen
presentation and phagocytosis. Additionally, activated microglia,
when changed to the ameboid shape, display free movement
during phagocytosis but do not engage in antigen presentationand inflammation (Cai et al., 2014). In addition, excessive or
long-term activation of microglia induces neuronal death and anincrease in pro-inflammatory cytokines.
In this study, we aimed to observe the effects oflipopolysaccharide (LPS)-induced neuroinflammation on
neutrophils and microglia within brain tissue of live mice. To
this end, we attempted to obtain visual evidence of the effects
of neuroinflammation on neutrophils and microglia using
two-photon intravital imaging, which may then serve as a basis
for further research on the molecular and mechanistic bases ofsuch modifications.

MiceFrontiers in Cell and Developmental Biology | www.frontiersin.org

LysMGFP/+ (Faust et al., 2000) and CX3 CR1GFP/+ (Jung et al.,2000) mice, in which the lysozyme and the CX3 CR1 geneare replaced by green fluorescent protein (GFP), respectively,
were obtained and used for the visualization of neutrophil andmicroglia. All mice were kept in a specific pathogen-free (SPF)
room, a light cycle from 7:00 AM to 7:00 PM at 23 ± 2◦ C,
and 55 ± 10% humidity. All procedures were conducted inaccordance with the guidelines of the Institutional Animal Careand Use Committee of Yonsei University College of Medicine,South Korea (IACUC, 2019-0097).

Identification of Mouse Genotype
Genotyping for each strain (LysMGFP/+ and CX3 CR1GFP/+mice) was performed using a Genomic DNA Prep Kit (BioFACT,South Korea). A toe of 7–10 day-old mice was severed, and thenDNA extraction from the acquired toe was conducted using the
Genomic DNA Prep Kit. Template DNA (50 ng/µl), primers and2×Taq PCR master mix2 10 µl (BioFACT, South Korea) weremixed in a PCR tube, in which distilled water was added up to
20 µl reaction volume.

Cranial Window Surgery
The cranial window was implanted on the calvaria for intravital
brain imaging as previously described (Baik et al., 2014). Mice
were deeply anesthetized using intraperitoneal injection of zoletil(Virbac, France) at a dose of 30 mg/kg and rompun (Bayer,
Germany) at a dose of 10 mg/kg. Body temperature in each mouse
was maintained at 37 ± 0.5◦ C using heating pads during cranial
window surgery (Supplementary Figure 1). Mice were fixed ina stereotaxic instrument (Live Cell Instrument, South Korea)
during all procedures. A cranial window of 5 mm in diameter was
made in the right hemisphere. The head skin and the periosteum
on the calvaria were removed from between the eyes to the caudal
region of the ears. Between the lambda and bregma regions on
the right hemisphere, a circular opening was carved with a micro
drill, frequently washed with cool phosphate-buffered saline(PBS), and sealed with a round coverslip (diameter = 5 mm) using
tissue glue on the skull using the optical microscope. A metalframe was glued and fixed using dental cement (B.J.M laboratory,
Israel) on the borders of the cranial window and skull area forfilling imaging area with distilled water. The metal frame wasassembled with a stereotactic head fixation device attached tothe heating plate.

Two-Photon Intravital Microscopy
Mice were anesthetized using intraperitoneal injection of Zoletil
at a dose of 30 mg/kg and rompun at a dose of 10 mg/kg
during imaging. The imaging stage was composed of a XY microstage and a stereotactic head fixation device connected to a DC
temperature controller (Supplementary Figure 1A). Both twophoton microscopies with W Plan-Apochromat 20×/1.0 water
immersion lens from Carl-Zeiss, Germany (LSM7MP) and from
IVIM Technology, South Korea (IVM-M) were used for imaging

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data generation. LysMGFP/+ mice were intravenously injectedwith 70-kDa Texas red dextran (2.5 mg/kg, Sigma-Aldrich,Germany) for visualizing blood vessels. CX3 CR1GFP/+ mice wereintravenously injected with CF405M-conjugated Wheat germ
agglutinin (WGA) (2.5 mg/kg, Biotium, CA, United States) for
visualizing blood vessels and PE-conjugated anti-Ly6G antibody(0.1 mg/kg, BioLegend, CA, United States) for observing
neutrophils. For two-photon excitation, each mouse brain was
excited with light of 800 nm and 880 nm wavelength for imaging
of green, red, and blue. All images were acquired at a resolution of
512 × 512 pixels using steps of size 1 µm to a depth of 40–50 µm
for 1 min (Park et al., 2018; Lee et al., 2019).

RESULTSLPS-Induced Systemic Inflammation
Triggers Intravascular Adhesion and
Infiltration of Neutrophils Through Brain
Blood Vessels
To investigate the effect of inflammatory status via LPS injection,
the weight of mice injected with LPS was compared to thatof the control group, as weight loss is a hallmark of systemic
inflammation. The LPS group showed an 11.75% loss inbody weight compared to the control group, confirming that
inflammation had indeed been induced in the LPS-injected
mice (Figure 1A). Previous studies have demonstrated thatneutrophils are recruited in the brain during LPS-induced
systemic inflammation to fulfill their roles in the immune
response (Zhou et al., 2009; He et al., 2016). Our results
confirmed this result, as neutrophil extravasation to the brain
parenchyma was observed more frequently in response to LPS
injection. In addition, an increased number of neutrophils
were observed, which resulted from intravascular adhesion
and infiltration (Figures 1B,C and Supplementary Video 1).
Consistent with such findings, transendothelial migration (TEM)of neutrophils was also facilitated in the LPS group, where
neutrophils actively emerged out to the brain parenchyma(Figure 1D and Supplementary Video 2). Altogether, these data
demonstrate that LPS injection and the subsequent inflammatory
consequences that follow yield a considerable increase inneutrophil influx to the brain parenchyma.

LPS-Induced Neuroinflammatory Mouse
Model
Previous studies established that LPS-induced inflammationin the periphery can prompt immune responses in thecentral nervous system (Ebersoldt et al., 2007; Zhao et al.,2019). To investigate the migratory patterns of neutrophil andmicroglia during neuroinflammation, mice were treated withdaily intraperitoneal injections of lipopolysaccharide (1.0 mg/kg,
Sigma-Aldrich, Germany) for 2 consecutive days. Control mice
were intraperitoneally injected with daily PBS injections for 2
consecutive days. Intravital imaging was performed at 6 h after
LPS injections for 2 consecutive days.

Imaging Data Analysis
Volocity (PerkinElmer, MA, United States), Imaris (Bitplane,
Switzerland), and Fiji/Image J (NIH, United States) were used for3D and 4D imaging data analysis.

Neutrophils Exhibit Active Motility in the
Brain Parenchyma During
Neuroinflammation
Along with an increment in cell number, neutrophils displayed
an increase in motility, as exhibited in various motion-related
criteria. The motility of neutrophils was determined and assessedusing various factors, including track length, track velocity,
displacement, and meandering index. The track length andtrack velocity of migrating neutrophils in the brain parenchyma
were significantly higher in the LPS group compared to thecontrol group, indicating more active locomotion in response
to LPS injection. Furthermore, these results revealed that
infiltrated neutrophils showed constant migration within a
20 µm radius for 30 min, suggesting significant motility ofneutrophils during neuroinflammatory response; in addition, alower meandering index compared to the control group may
signify more directionality in neutrophil movement in LPSinjected mice (Figures 1E–I). Thus, these results indicate thatduring neuroinflammation, the motility of neutrophils in thebrain parenchyma is notably increased, suggesting a change inthe molecular and biochemical profile of the neutrophils.

Chemokine Microarray
Following LPS stimulation, brains of mice were lysed by adding
protease inhibitor cocktail (Roche, Germany) containing PROPREP (Intron biotechnology, South Korea) and 1% Triton X-100.
Cytokine and chemokine levels were detected using Proteome
Profiler Mouse Cytokine Array Panel A (R&D systems, MN,
United States) according to the manufacturer’s instruction. The
Array kit detected C5/C5a, G-CSF, M-CSF, GM-CSF, sICAM-1,
IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL7, IL-10, IL-13, IL-12p70, IL-16, IL-17, IL-23, IL-27, CXCL1,CXCL2, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCL1,
CCL2, CCL3, CCL4, CCL5, CCL11, CCL12, CCL17, TIMP1, TNF-α, and TREM-1. The blots were analyzed using thequick spots tool in HLImage++ (Western Vision Software,
UT, United States).

Statistical Analysis
All experiments were repeated at least three times. Statisticalanalyses were expressed as mean ± standard error of themean (S.E.M). Statistical analysis was performed using Prism(GraphPad software, CA, United States). For comparison of twogroups, unpaired two-sided Student t-tests were applied. p-values
less than 0.05 were considered statistically significant.
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Inflammation in the Brain Triggers
Numerical Reduction and Morphological
Shortening of Microglia
As the predominant innate immune cell population that is
resident to the brain, microglia play a role in the process of3

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FIGURE 1 | Quantitative analysis of neutrophil infiltration from the blood vessel to brain parenchyma. (A) Representative graph of weight loss in the LPS-treatedgroup compared to the control group. The % change in body weight was calculated as (body weight before LPS treatment/body weight after LPS treatment) × 100.
Data indicate mean ± SEM using Student’s t-test (****p < 0.0001, n = 7 mice per group). (B) LysMGFP/+ mice were used to visualize neutrophils (green) via TPIM.
Texas red dextran was i.v. injected to stain blood vessels (red). Representative images are shown from the brain of mice injected with PBS (control) or LPS,
respectively. Scale bar, 100 µm (see Supplementary Video 1). (C) Measurement of infiltrated neutrophils in the control and LPS groups. Data indicate mean ± SEM
using Student’s t-test (*p < 0.05, n = 3 mice per group of three independent experiments). (D) A series of representative time-lapse images show neutrophil
infiltration into inflamed brain parenchyma of LysMGFP/+ mice. Scale bar, 50 µm (see Supplementary Video 2). (E) Overlay of the representative migration tracks ofneutrophils in brain parenchyma for 30 min. x-, y-axis (length), -100 to 100 µm. n = 30 cells per group, PBS or LPS-treated mice for three independent experiments.
Migration was quantitatively assessed with tracking analyses: (F) displacement (µm), (G) meandering index (displacement/length), (H) velocity (µm/min), and (I)length (µm) in two different conditions for 30 min. Data indicate mean ± SEM using Student’s t-test (****p < 0.0001, n = 30 cells per group of three independent
experiments).

neuroinflammation. Therefore, it was important that the effectsof LPS injection and the ensuing inflammatory aftermaths on
microglia were investigated. Specifically, it had previously been

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suggested that neutrophils may interact with microglia; as our
aforementioned results indicate various changes in neutrophils
during neuroinflammation, it is crucial to unravel any microglial

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Neutrophil-Microglia Crosstalk in
Inflamed Brain Parenchyma Leads toEngulfment of Neutrophils by Microglia
During the neuroinflammatory response, neutrophils have beenthought to actively interact with cells in the vicinity, such as
astrocytes, microglia, and adaptive immune cells (He et al.,2016; Girbl et al., 2018). Imaging data from the present study
supports this idea, as contact between infiltrated neutrophils andbrain tissue-resident microglia was observed (Figures 3A,B and
Supplementary Video 5). After neutrophils made contact withmicroglial processes, microglia seemed to engulf the neutrophils,
indicating the possibility of molecular crosstalk between thetwo cell groups. On the other hand, during neutrophilmicroglial contact, nearby microglial processes were observed
to stretch toward the point of contact (Figures 3C,D and
Supplementary Video 6). Such phenomena further reinforces theidea that contact between neutrophil and microglia accompanies
intercellular crosstalk, affecting not only the cells making
contact but also the surrounding environment. Overall, while
the underlying mechanisms are yet to be revealed, theseresults strongly suggest that inflammatory states not only
prompt crosstalk and contact between neutrophils and microglia,
but also attract other microglia toward sites of neutrophilmicroglia contact.

FIGURE 2 | Numerical reduction and morphological shortening of microglia in
LPS-treated mice. CX3 CR1GFP/+ mice were used to visualize microglia(green) in brain parenchyma using TPIM. CF405M-conjugated lectin was i.v.
injected to stain blood vessels (blue). PE-conjugated anti-Ly6G antibody wasi.v. injected to label neutrophils (red). (A) Representative images from brains ofmice injected with PBS (control) and LPS, respectively (see Supplementary
Video 3). Scale bar, 100 µm. (B) Measurement of microglia numbers. Data
indicate mean ± SEM using Student’s t-test (*p < 0.05, n = 3 mice per groupof three independent experiments). (C) Representative image showing
difference in microglial morphologies between control and LPS groups. Scalebar, 30 µm (see Supplementary Video 4). (D) Quantification of microglia
soma size. Data indicate mean ± SEM using Student’s t-test (****p < 0.0001,
more than 70 cells were analyzed per group from three independent
experiments).

Reverse Transendothelial Migration of
Neutrophils Is Observed in Inflamed
Brain Parenchyma
An interesting event regarding neutrophils that have beenreported in previous literature is reverse transendothelial
migration (rTEM), where neutrophils that had extravasated outof the blood vessel re-enters the bloodstream (Colom et al.,2015; Wu et al., 2016; Burn and Alvarez, 2017). While rTEM
had been observed in various parts of the body, imaging resultsfrom the present study are the first to suggest that the process
also takes place in brain blood vessels (Figures 4A,B and
Supplementary Videos 7, 8). Neutrophils engaging in rTEM
first approach the blood vessel, and after a period of movement
along the perivascular region, re-enters and eventually gets
swept away by the bloodstream. Albeit a rare phenomenon,
rTEM of neutrophils that had been exposed to inflammatoryenvironments may lead to significant repercussions, as it ispossible that the molecular profile of re-entering neutrophils isaltered; nevertheless, the present results suggest that rTEM is areadily observable phenomenon during neuroinflammation.
To investigate the molecular aftereffects of LPS injection that
may have stimulated neutrophil rTEM, brain chemokine arrays
were conducted to detect changes in chemokine expression levels
following LPS injection (Figure 4C). As a result, levels of thechemokines CXCL1, CXCL10, CXCL13, CCL2, and CCL5 were
found to be increased, a consequence which may affect thefunction and motility of microglia and neutrophils. As such, it
may be postulated that increased levels of the aforementioned
chemokines had stimulated neutrophil migration and rTEM as
presented above.

changes during similar situations in order to pinpoint any
biomolecular interactions between the two innate immune
cell populations (Sevenich, 2018). Imaging data in the brain
showed that the number of microglia in LPS-injected mice
had decreased compared to the control group, a phenomenon
that may have been caused by a variety of apoptosis-inducing
agents (Figures 2A,B and Supplementary Video 3; Steff et al.,
2001; Fortin et al., 2005). Also, while microglia in the control
group predominantly displayed the morphology of ramified
microglia, those in the LPS group showed the morphologyof activated microglia, with short and thick processes andenlarged cell bodies (Figure 2C and Supplementary Video 4).
To focus on a more detailed analysis, such as the microglia
cell soma, we quantified the change in microglia soma size
between control and LPS group. Compared to control mice,the average of soma size increased (56.85 vs. 68.32 µm2 )(Figure 2D). The result demonstrated the LPS exposure leads toactivated microglia that features bigger soma size than ramified
microglia called “resting” cells (Kozlowski and Weimer, 2012;
Davis et al., 2017). Taken together, these results demonstratethat LPS-induced neuroinflammation may activate microglia,
leading to morphological alterations that suggest an activatedstate (Liu and Hong, 2003).

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FIGURE 3 | Crosstalk between microglia and neutrophils in inflamed brain parenchyma. CX3 CR1GFP/+ mice were used to visualize microglia (green) in brain
parenchyma using TPIM. CF405M-conjugated lectin was i.v. injected to stain blood vessels (blue). PE-conjugated anti-Ly6G antibody was i.v. injected to label
neutrophils (red). (A) A series of representative time-lapse images showing contact between microglia and neutrophils (white circle). Scale bar, 40 µm (seeSupplementary Video 5). (B) Magnification of a region of interest, indicated by a white circle in (A), in 3D. Scale bar per 1 unit. Scale bar, 12.7 µm. (C) A series ofrepresentative time-lapse images showing elongation of microglial processes toward site neutrophil-microglial contact (white square). Scale bar, 40 µm (seeSupplementary Video 6). (D) Direction of microglial process elongation from 0 to 50 min (yellow-dotted arrow).

diseases. In particular, it has been suggested that neutrophils
accumulate in regions rich in amyloid-β deposits via the integrin
LFA-1; in addition, CXCL12 and CCL2 levels in the CNS wereshown to be principal factors resulting in neutrophil infiltration(Zenaro et al., 2015; Echeverria et al., 2016; Jiang et al., 2016).
In line with results from previous literature, expression levelsof certain chemokines were increased after LPS injection inthis study; in specific, among the aforementioned chemokines,
CXCL1 and CCL2 demonstrated a surge in expression, alongwith CXCL13, CXCL10, and CCL5. CXCL1 and CCL2 are wellknown for their roles in stimulating neutrophil migration, while
CXCL10, CCL2, and CCL5 have been reported to be releasedby neutrophils in inflammatory situations, mostly in order torecruit other innate or adaptive immune cells (Kobayashi, 2008;
Sawant et al., 2016; Capucetti et al., 2020). Therefore, chemokinesthat are chemotactic for neutrophils, such as CXCL1 or CCL2
may be the principal factor driving the various migratory
phenomena observed in this paper: neutrophil extravasation
and rTEM. In addition, it may be postulated that LPS-induced
neuroinflammation had caused phenotypical modifications inneutrophils, prompting them to release chemokines that yieldfurther steps down the inflammatory cascade, including further
recruitment of neutrophils or chemoattraction of adaptive

DISCUSSION
The CNS had been known to be an “immune privileged site,”most likely due to the presence of the highly impenetrable
BBB; yet recent studies have demonstrated flexibility in the
BBB in response to inflammation-related needs and stimuli(Kanashiro et al., 2020). Indeed, in neuroinflammatory situations,
neutrophils and CNS-resident microglia are suggested toparticipate in the inflammatory response, as supported by theimaging results of this study. With such visual evidence at
hand, investigation of the molecular bases behind the observed
phenomena is of significant importance, especially in clinicalsettings. In previous literature, the molecules responsible forthe recruitment of neutrophils to neuroinflammatory sites havebeen given substantial attention. For instance, in the case of
autoimmune diseases, such as multiple sclerosis or experimental
autoimmune encephalomyelitis, neutrophils are known to be
attracted by the release of CXCL1, CXCL2, and CXCL5, which
are in turn stimulated by different molecules, such as IL-17
or IFN-γ (Christy et al., 2013; Simmons et al., 2014). The
behavior of neutrophils in neurodegenerative diseases including
Alzheimer’s has also been a target of scrutiny, as neutrophil
infiltration is known to contribute to the exacerbation of such

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FIGURE 4 | Reverse-transendothelial migration of neutrophils in inflamed brain parenchyma. LysMGFP/+ mice were used to visualize neutrophils (green) during
neuroinflammation. Texas red dextran was i.v. injected to stain blood vessels (red). (A) A series of representative time-lapse images showing neutrophil crawlingalong the perivascular region in inflamed brain parenchyma. Tracking of neutrophil (white line). Scale bar, 50 µm (see Supplementary Video 7). (B) A series ofrepresentative time-lapse images showing reverse-transendothelial migration of neutrophils from brain parenchyma into blood vessels. Tracking of neutrophil (whiteline). Scale bar, 20 µm. (C) Brain chemokine array results demonstrating altered expression levels of chemokines following LPS injection. Data indicate mean ± SEM
using Student’s t-test (*p < 0.005, ***p < 0.001). Three independent experiments were performed.

immune cells. On the other hand, CXCL13 plays a role inadaptive immune cell organization, being especially chemotactic
for B cells; yet, it is not known to be expressed in neutrophils(Havenar-Daughton et al., 2016).
One of the most interesting phenomena that deserve attention
in the present study is rTEM, the reverse migration of neutrophils
from the brain parenchyma back to the bloodstream. Although
its exact purpose and mechanism are yet to be clearly defined,
rTEM has been observed in numerous previous studies, andaccumulation of relevant data is continuing to provide new
insight into the topic (Burn and Alvarez, 2017). For instance,
several studies have attempted to pinpoint the molecular signals

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that seem to induce or influence rTEM; in one study, coldinducible RNA-binding protein (CIRP) has been suggested
to stimulate neutrophil rTEM in septic conditions via an
increase in neutrophil elastase and a decrease in junctional
adhesion molecule-C (JAM-C) (Jin et al., 2019). In addition,
the chemoattractant leukotriene B4 (LTB4) has been proposed
to be a potential factor which causes proteolytic cleavage of
JAM-C via neutrophil elastase, further reinforcing the possibleroles of neutrophil elastase and JAM-C in rTEM (Colomet al., 2015). Along with JAM-C, netrin-1, a protein highly
expressed in endothelial cells and is known to disrupt leukocytetransendothelial migration, has also been proposed as a factor

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observations were made in neuroinflammatory situations via
different inducers (systemic inflammation via LPS injection vs.
ischemic stroke) demonstrates that neutrophil-microglia contact
might be a general phenomenon in neuroinflammation, raisingquestions regarding the molecular mechanisms or cell signaling
underlying such interactions. Despite the fact that studies onsuch topics are scarce, previous research has suggested thatmolecules, such as RGD peptides of GlcNAC may hinder
microglia-neutrophil interactions in vitro, possibly laying thepavement for further mechanistic studies (Neumann et al., 2008).
Furthermore, previous research on neutrophil rTEM indicatethat the majority of neutrophils that engage in rTEM hadprior interaction with macrophages, strongly suggesting a roleof macrophages promoting neutrophil rTEM not only in thebrain but also in other organs in general (Burn and Alvarez,2017). In particular, the redox-SRC family kinase (SFK) signaling
pathway was shown to be relevant to the rTEM-inducing
capabilities of macrophages, with p22phox and Yes-related
kinases being key players (Tauzin et al., 2014). While contact
with macrophages was not necessary for neutrophil rTEM,
the number of neutrophils that undergo rTEM significantly
diminished in a setting that lacked macrophages; such previous
data, combined with the results of the present study, presents theneed to delve further into the impact of neutrophil-macrophageinteractions on neutrophil rTEM. Meanwhile, combining thepresent results with observations from previous literature, we
may even speculate that the neutrophils that had interacted withmicroglia in the brain parenchyma had underwent molecularmodifications that predispose them to engage in rTEM; inthis sense, neutrophil-microglia crosstalk may be crucial in the
resolution of inflammation in the brain.
One key discrepancy between previous studies on neutrophil
rTEM and the present study is the rate in which rTEM wasobserved; although it had been proposed that up to 80% of theneutrophils that accumulated in inflammatory sites may engage
in rTEM, the phenomenon was not seen as frequently in the brain(Mathias et al., 2006; Yoo and Huttenlocher, 2011). This may
be possibly due to the complexity and relative impenetrability of
the BBB, which often results in various complications regarding
leukocyte migration (Ransohoff et al., 2003). In this context,
further research is necessary to investigate why rTEM is observedin a much lower frequency in the brain. At the same time,
the occurrence of rTEM, however rare as it may be, alsopresents a need to decipher the brain-specific mechanisms behindrTEM through the BBB. The aforementioned netrin-1 may be areasonable starting point, as the molecule is known to be involved
in BBB function (Podjaski et al., 2015).
In the present study, we have demonstrated thatneuroinflammation induced by LPS injection yields increased
recruitment and mobility of neutrophils in the brain parenchyma,
while activating microglia, as observed from their morphologicalchanges. In addition, neutrophils that had infiltrated brain
tissue were seen to engage in neutrophil-microglia interaction,
where microglia engulfed the neutrophils in contact. Such aphenomenon also attracted nearby microglia, inducing dendritic
movement toward the site of contact. Furthermore, infiltrated
neutrophils also exhibited rTEM, returning to the bloodstreamthat may hinder rTEM (Ly et al., 2005; Podjaski et al., 2015). In
particular, it has been shown that netrin-1 activation is dependenton hypoxia inducible factor 1 alpha (Hif-1α), which in turn seems
to promote continuation of inflammation and prevent neutrophil
clearance from perivascular regions (Rosenberger et al., 2009;
Elks et al., 2011). Furthermore, Tanshinone IIA, a compound
originated from an Asian medicinal herb, has been shown topromote inflammation resolution via the induction of neutrophil
apoptosis and rTEM (Robertson et al., 2014). Yet, as Tanshinones
have also been suggested to play an inhibitory role on NFkB,
AP-1, and STAT1 activation and thus possess anti-inflammatory
functions, additional research on the matter is necessary toconfirm the role of Tanshinones in the context of rTEM andneutrophil clearance (Xu et al., 2008; Tang et al., 2011).
Yet while the molecular mechanisms underlying rTEM is
crucial, another important issue is perhaps the consequencesof neutrophil rTEM, especially the effects of the neutrophils
returning to the bloodstream on other organs. Post-rTEM
neutrophils were suggested to play diverse roles, including
inhibition of T cell proliferation, neutrophil clearance fromtissues, enhancement of reactive oxygen species (ROS) andneutrophil extracellular trap (NET) formation, and exacerbationof systemic inflammation (Buckley et al., 2006; Mathias et al.,2006; Woodfin et al., 2011; Yoo and Huttenlocher, 2011;
Brinkmann and Zychlinsky, 2012; Pillay et al., 2012; Cheng and
Palaniyar, 2013). Notably, the molecular profile of neutrophilsthat had underwent rTEM exhibited high levels of CD11b, CD54,
and ICAM-1, while showing low levels of CD62L, CXCR1, andCXCR2 markers (Walcheck et al., 1996; Buckley et al., 2006;
Woodfin et al., 2011). The distinguishable molecular expression
pattern in neutrophils that had returned to the bloodstream
presents various inquiries, including what effects such patterns
may have on the function of neutrophils after rTEM. Anotherimportant question that arises would be how those neutrophils
acquired such a state; one likely hypothesis is that the neutrophils
experienced molecular modification via active interaction withother cells. For example, it had been postulated that the highexpression of ICAM-1 on the surface of post-rTEM neutrophils
may have resulted from a mechanism that resembles trogocytosis,
by which neutrophils acquired ICAM-1 high membranes fromendothelial cells (Joly and Hudrisier, 2003; Burn and Alvarez,2017).
In this context, it is natural to speculate whether neutrophilsthat had been chemically stimulated or modified by interactions
with microglia engage in rTEM, thereby spreading inflammatory
signals from the brain to other peripheral organs of the body.
Although the accumulation of neutrophils in inflammatory
conditions is a well-established concept, the interaction between
neutrophils and microglia during neuroinflammation has yet
to be extensively studied. Our study presents compelling
evidence of neutrophil-microglial contact, which also led tofurther mobilization of nearby microglia toward the site ofcontact. Previously, the same phenomenon had been observed
via two-photon imaging in a stroke model, where neutrophils
infiltrated the brain parenchyma following cortical ischemia andmicroglia engulfed the neutrophils, in line with the results ofthe present study (Neumann et al., 2018). The fact that identical

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Neutrophil rTEM in the Brain

FIGURE 5 | Neutrophils and microglia exhibit various behavioral and phenotypical responses to LPS-induced neuroinflammation. A graphic illustration describing the
responses of neutrophils and microglia to LPS-induced neuroinflammation, as identified via two-photon microscopy. Upon LPS injection and the following
neuroinflammatory situations, neutrophils first respond by extravasating into the brain parenchyma. Meanwhile, microglia are activated and thus undergomorphological changes, such as enlargement of cell bodies and shortening of processes. Such activated microglia interact with infiltrated neutrophils, possibly
causing molecular modifications on the neutrophils; thereafter, neutrophils engage in reverse transendothelial migration, returning to the bloodstream.

mice, an AD mouse model, using two-photon microscopy;
Zenaro et al., later confirmed similar results in 3×Tg-AD mice,
another murine model of AD. As such, further studies that
corroborate the results of the present study using disease-specificmodels of neuroinflammation may help investigating the clinicalimplications of neutrophil infiltration, neutrophil-microglia
interaction, and neutrophil rTEM in neuroinflammation. Taking
such future research suggestions into consideration, this paper
presents strong visual data on the behavior of neutrophils in
response to neuroinflammatory situations and may establish thegroundwork for future research on the molecular mechanisms
underlying innate immune cell responses to neuroinflammationand systemic inflammation alike.

after entering the brain parenchyma (Figure 5). Despite suchcompelling evidence, this paper possesses several limitations.
First and foremost, it would benefit greatly from further researchthat is able to observe rTEM at a higher frequency, as oneof the biggest gaps between data in this paper and data on
rTEM studies was that between the rate of rTEM observation.
Another limitation is that in this study, we were not able toquantify meaningful ratios and proportions of cells exhibiting
desired behavior. For instance, the ratio of neutrophils thatengaged in extravasation to those that circulated within the
blood vessel, the ratio of neutrophils that infiltrated to thebrain parenchyma to those that interacted with microglia,
and the ratio of neutrophils that interacted with microglia tothose that exhibited rTEM are all of much importance butwere unattainable due to technological issues. Also, as mostof the results of this paper are of an observational nature,
future studies utilizing in vivo or in vitro methods may assistin underpinning the biomolecular mechanisms behind theobservations of this paper, specifically in relation to neutrophilmicroglial interactions. Finally, while the current study utilized
LPS injection to induce neuroinflammation, the variety ofneuroinflammatory models existing today will be of tremendous
usefulness in verifying the present results, in perhaps a moreclinical setting. For example, in our previous data, we had
demonstrated neutrophil infiltration into the brain parenchyma
and accumulation around amyloid beta plaques in 5XFAD

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

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional
Animal Care and Use Committee of Yonsei University College ofMedicine, South Korea.

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Neutrophil rTEM in the Brain

AUTHOR CONTRIBUTIONS

glue, d. cover glass (diameter = 5 µm), e. metal frame, f. pen, g. dental cement,h. resin tip, i. forceps, j. a pair of scissors, k. forceps, l. micro forceps, m. micro
drill. (C) Surgical procedure of cranial window surgery for intravital brain imaging. i.
Fix the mouse in a stereotactic heating plate, ii. Remove head skin and theperiosteum, iii. Coordinate predestinate location carved on right hemisphere, iv.
Moisturize a circular opening with a drop of isotonic saline solution and seal acircular opening with a 5 µm coverslip, v. Glue and fix the metal frame on theborders of the cranial window and skull area using dental cement, vi. Place themouse on a head fixation device for intravital imaging.

YRK performed the experiments, wrote the manuscript, andanalyzed the data. YMK wrote the manuscript and analyzed
the data. JL and JP performed the experiments. JEL helped
design the study. Y-MH directed the study design and wrote themanuscript. All authors contributed to the article and approved
the submitted version.

Supplementary Video 1 | TPIM of brain parenchyma in LysMGFP/+ mice injectedwith PBS (control) and LPS, respectively. Green, neutrophil (GFP); Red, bloodvessels (Texas Red dextran). Scale bar, 100 µm.

FUNDING

Supplementary Video 2 | TPIM of neutrophil infiltration during
neuroinflammation. Green, neutrophil (GFP); Red, blood vessels (Texas Red
dextran); White, track line of neutrophil. Scale bar, 50 µm.

This research was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korea
government (MSIT) (2016M3C7A1905098 to JEL and
2020R1A4A1019009 to Y-MH).

Supplementary Video 3 | TPIM of brain parenchyma in CX3 CR1GFP/+ mice
injected with PBS (control) and LPS, respectively. Green, microglia (GFP); Red,
neutrophils (Ab Ly6G); Blue, blood vessels (Lectin). Scale bar, 100 µm.

ACKNOWLEDGMENTS

Supplementary Video 4 | TPIM of morphological changes in microglia in brain
parenchyma. Green, microglia (GFP); Red, neutrophils (Ab Ly6G); Blue, bloodvessels (Lectin). Scale bar, 30 µm.

We thank Medical Illustration & Design, part of the Medical
Research Support Services of Yonsei University College ofMedicine, for all artistic support related to this work.

Supplementary Video 5 | TPIM showing contact between microglial processes
and neutrophils in inflamed brain parenchyma. Green, microglia (GFP); Red,
neutrophils (Ab Ly6G); Blue, blood vessels (Lectin). Scale bar, 40 µm.
Supplementary Video 6 | TPIM showing elongation of microglial processes
toward point of microglia-neutrophil contact (white arrow). Green, microglia (GFP);Red, neutrophils (Ab Ly6G); Blue, blood vessels (Lectin).

SUPPLEMENTARY MATERIALThe Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fcell.2020.
613733/full#supplementary-material

Supplementary Video 7 | TPIM showing neutrophil crawling along theperivascular region in inflamed brain parenchyma. Green, neutrophil (GFP); Red,
blood vessels (Texas Red dextran); White, tracking of neutrophil. Scale bar,
50 µm.

Supplementary Figure 1 | Cranial window surgery setup for brain intravital
imaging. (A) Brain chambers with heating plates for maintaining body temperature
at 37 ± 0.5◦ C. a. Metal and rubber heating plates, b. DC temperature controller,c. Stereotactic instrument for cranial window surgery, d. Head fixation device for
imaging. (B) Subsidiary tools for surgery a. cotton swab, b. air blower, c. tissue

Supplementary Video 8 | TPIM showing neutrophil reverse-transendothelial
migration from brain parenchyma into blood vessel. Green, neutrophil (GFP); Red,
blood vessels (Texas Red dextran); White, tracking of neutrophil; Blue, thereplacement of reverse-migrated neutrophil. Scale bar, 20 µm.

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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.
Copyright © 2020 Kim, Kim, Lee, Park, Lee and Hyun. This is an open-access article
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