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Pericytes regulate vascular immune homeostasis in the CNS

20 May 2019-bioRxiv (Cold Spring Harbor Laboratory)-pp 644120

TL;DR: It is shown that pericytes indirectly restrict immune cell transmigration into the CNS under homeostatic conditions and during autoimmune-driven neuroinflammation by inducing immune quiescence of brain endothelial cells.
Abstract: Brain endothelium possesses several organ-specific features collectively known as the blood-brain barrier (BBB). In addition, trafficking of immune cells in the healthy central nervous system (CNS) is tightly regulated by CNS vasculature. In CNS autoimmune diseases such as multiple sclerosis (MS), these homeostatic mechanisms are overcome by autoreactive lymphocyte entry into the CNS causing inflammatory demyelinating immunopathology. Previous studies have shown that pericytes regulate the development of organ-specific characteristics of brain vasculature such as the BBB and astrocytic end-feet. Whether pericytes are involved in the control of leukocyte trafficking remains elusive. Using adult, pericyte-deficient mice ( Pdgfb ret/ret ), we show here that brain vasculature devoid of pericytes shows increased expression of VCAM-1 and ICAM-1, which is accompanied by increased leukocyte infiltration of dendritic cells, monocytes and T cells into the brain, but not spinal cord parenchyma. Regional differences enabling leukocyte trafficking into the brain as opposed to the spinal cord inversely correlate with the pericyte coverage of blood vessels. Upon induction of experimental autoimmune encephalitomyelitis (EAE), pericyte-deficient mice succumb to severe neurological impairment. Treatment with first line MS therapy - fingolimod significantly reverses EAE, indicating that the observed phenotype is due to the massive influx of immune cells into the brain. Furthermore, pericyte-deficiency in mice that express myelin oligodendrocyte glycoprotein peptide (MOG 35-55 ) specific T cell receptor ( Pdgfb ret/ret ; 2D2 Tg ) leads to the development of spontaneous neurological symptoms paralleled by massive influx of leukocytes into the brain, suggesting altered brain vascular immune quiescence as a prime cause of exaggerated neuroinflammation. Thus, we show that pericytes indirectly restrict immune cell transmigration into the CNS under homeostatic conditions and during autoimmune-driven neuroinflammation by inducing immune quiescence of brain endothelial cells.

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Pericytes regulate vascular
immune homeostasis in the CNS
Journal Article
Author(s):
Török, Orsolya; Schreiner, Bettina; Schaffenrath, Johanna; Tsai, Hsing-Chuan; Maheshwari, Upasana; Stifter, Sebastian A.; Welsh,
Christina; Amorim, Ana; Sridhar, Sucheta; Utz, Sebastian G.; Mildenberger, Wiebke; Nassiri, Sina; Delorenzi, Mauro; Aguzzi,
Adriano; Han, May H.; Greter, Melanie; Becher, Burkhard; Keller, Annika
Publication date:
2021-03-09
Permanent link:
https://doi.org/10.3929/ethz-b-000475206
Rights / license:
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Originally published in:
Proceedings of the National Academy of Sciences of the United States of America 118(10), https://doi.org/10.1073/
pnas.2016587118
This page was generated automatically upon download from the ETH Zurich Research Collection.
For more information, please consult the Terms of use.

Pericytes regulate vascular immune homeostasis in
the CNS
Orsolya Török
a,b
, Bettina Schreiner
c,d
, Johanna Schaffenrath
a,b
, Hsing-Chuan Tsai
e
, Upasana Maheshwari
a,b
,
Sebastian A. Stifter
c
, Christina Welsh
c
, Ana Amorim
c
, Sucheta Sridhar
a,b
, Sebastian G. Utz
c
,
Wiebke Mildenberger
c
, Sina Nassiri
f
, Mauro Delorenzi
f
, Adriano Aguzzi
g
, May H. Han
e
, Melanie Greter
c
,
Burkhard Becher
c
, and Annika Keller
a,b,1
a
Department of Neurosurgery, Clinical Neuroscience Center, University Hospital Zürich, Zürich Univer sity, 8091 Zürich, Switzerland;
b
Neuroscience
Center Zürich, University of Zürich and ETH Zürich, 8057 Zürich, Switzerland;
c
Institute of Experimental Immunology, University of Zürich, 8057 Zürich,
Switzerland;
d
Department of Neurology, University Hospital Zurich, 8091 Zürich, Switzerland;
e
Department of Neurology and Neurological Sciences,
Stanford University, Stanford, CA 94305;
f
Bioinformatics Core Facility, Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland; and
g
Institute of
Neuropathology, University Hospital Zürich, 8091 Zürich, Switzerland
Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved January 26, 2021 (rec eived for review Augus t 16, 2020)
Pericytes regulate the development of organ-specific characteristics
of the brain vasculature such as the bloodbrain barrier (BBB) and
astrocytic end-feet. Whether pericytes are involved in the control of
leukocyte trafficking in the adult central nervous system (CNS), a
process tightly regulated by CNS vasculature, remains elusive. Using
adult pericyte-deficient mice (Pdgfb
ret/ret
), we show that pericytes
limit leukocyte infiltration into the CNS during homeostasis and au-
toimmune neuroinflammation. The permissiveness of the vasculature
toward leukocyte trafficki ng in Pdgfb
ret/ret
mice inversel y corre lates
with vessel pericyte coverage. Upon induction of experimental auto-
immune encephalomyelitis (EAE), pericyte-deficient mice die of severe
atypical EAE, which can be reversed with fingolimod, indicating that
the mortality is due to the massive influx of immune cells into the
brain. Additionally, administration of anti-VCAM-1 and antiICAM-1
antibodies reduces leukocyte infiltration and diminishes the severity
of atypical EAE symptoms of Pdgfb
ret/ret
mice, indicating that the
proinflammatory endothelium due to absence of pericytes facilitates
exaggerated neuroinflammation. Furthermore, we sho w that the pres-
ence of myelin peptide-specific peripheral T cells in Pdgfb
ret/ret
;2D2
tg
mice leads to the development of spontaneous neurological symptoms
paralleled by the massive influx of leukocytes into the brain. These
findings indicate that intrinsic changes within brain vasculature can
promote the development of a neuroinflammatory disorder.
pericyte
|
bloodbrain barrier
|
leukocyte trafficking
|
autoimmune neuroinflammation
|
MOG
3555
specific T cell receptor
C
entral nervous system (CNS) vasculature possesses specific
features collectively referred to as the bloodbrain barrier
(BBB), which localizes to endothelial cells. The BBB ensures the
delivery of essential nutrients while preventing the entry of
xenobiotics into the brain. In addition, brain endothelial cells re-
strict the invasion of leukocytes into the brain parenchyma, thus
contributing to the immune privilege of the CNS. BBB function is
induced by neural tissue and established by all cell types consti-
tuting the neurovascular unit (NVU). Pericytes and mural cells
residing on the abluminal side of capillaries and postcapillary ve-
nules regulate several features of the BBB (1, 2). Studies on Pdgfb
and Pdgfrb mouse mutants, which exhibit variable pericyte loss,
have demonstrated that pericytes negatively regulate endothelial
transcytosis, which, if not suppressed, leads to increased BBB per-
meability to plasma proteins (1, 2). In addition, pericyte-deficient
vessels show abnormal astrocyte end-feet polarization (1). Thus,
pericytes regulate several characteristics of the brain vasculature
during development and in the adult organism (1, 2). Whether the
nonpermissive properties of the brain vasculature to leukocyte
trafficking in the adult organism are regulated by pericytes has not
been addressed. Interestingly, increasing evidence points to the role
of pericytes in leukocyte extravasation in peripheral organs such as
theskinandthestriatedmuscleandintumors(35).
Increased vascular permeability to plasma proteins and im-
mune cells accompanies neurological disorders such as multiple
sclerosis (MS), stroke, and Alzheimers disease (reviewed in refs.
68). In MS, a chronic inflammatory and degenerative neuro-
logical disorder (9), autoreactive lymphocytes infiltrate the CNS
parenchyma, leading to focal inflammatory infiltrates, demye-
lination, axonal damage, and neurodegeneration. These infil-
trating immune cells could induce vascular dysfunction, including
permeability to plasma proteins such as fibrinogen (1012). On
the contrary, disruption of the BBB has been shown to precede
the infiltration of inflammatory cells and the formation of de-
myelinating lesions in MS patients (13). Therefore, it is impor-
tant to understand roles of different cell types and alterations in
cellcell communication at the NVU, which may facilitate entry
of autoimmune T cells as well as the anatomical localization of
lesions. However, knowledge about how pericytes contribute to
the development of the disease is still limited.
This prompted us to investigate whether pericytes, which regu-
late several aspects of the BBB phenotype of endothelial cells, also
regulate immune cell trafficking into the CNS during homeostasis
and neuroinflammation. We show that pericytes play a crucial role
Significance
The CNS vasculature tightly regulates the passage of circulating
molecules and leukocytes into the CNS. In the neuroinflammatory
disease multiple sclerosis (MS), these regulatory mechanisms fail,
and autoreactive T cells invade the CNS via blood vessels, leading
to neurological deficits depending on where the lesions are lo-
cated. The region-specific mechanisms directing the development
of such lesions are not well understood. In this study, we inves-
tigated whether pericytes regulate CNS endothelial cell permis-
siveness toward leukocyte trafficking into the brain parenchyma.
By using a pericyte-deficient mouse model, we show that intrinsic
changes in the brain vasculature due to absence of pericytes fa-
cilitate the neuroinflamm atory cascade and can influence the lo-
calization of the neuroinflammatory lesions.
Author contributions: O.T., B.S., M.H.H., M.G., B.B., and A.K. designed research; O.T., B.S.,
J.S., H.-C.T., U.M., S.A.S., C.W., A. Amorim, S.S., S.G.U., W.M., and A.K. performed research;
M.G. and B.B. contributed new reagents/analytic tools; O.T., B.S., J.S., U.M., S.A.S., C.W.,
A. Amorim, S.S., S.G.U., S.N., M.D., A. Aguzzi, M.H.H., M.G., and A.K. analyzed data; and
O.T. and A.K. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
This open access article is distributed under Creative Commons Attribution-NonCommercial-
NoDerivatives License 4.0 (CC BY-NC-ND).
1
To whom correspondence may be addressed. Email: annika.keller@usz.ch.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/
doi:10.1073/pnas.2016587118/-/DCSupplemental.
Published March 2, 2021.
PNAS 2021 Vol. 118 No. 10 e2016587118 https://doi.org/10.1073/pnas.2016587118
|
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in the regulation of BBB features related to the restricted leu ko c yt e
trafficking into the CNS parenchyma, both under physiological and
pathophysiological conditions. We show that the permissiveness to
leukocyte trafficking into the CNS inversely correlates with the vessel
pericyte coverage, suggesting that vascular inflammation of the CNS
due to alterations in the cellular composition of the NVU can direct
the spatial distribution of neuroinflammation.
Results
Increased Expression of Leukocyte Adhesion Molecules in the Brain
Vasculature in Pericyte-Deficient Adult Mice.
To address the ques-
tion of whether pericytes regulate immune cell trafficking into the
CNS, we used a pericyte-deficient mouse line, Pdgfb
ret/ret
,which
shows 90% reduction in brain pericyte numbers and 75% re-
duction in pericyte vessel coverage in adult animals (1). Earlier
studies have shown that pericyte deficiency in embryos leads to
increased mRNA levels of leukocyte adhesion molecules (LAMs)
on endothelial cells (2). We analyzed published microarray data of
the adult Pdgfb
ret/ret
brain microvasculature (1) and detected a
deregulation of several LAMs, including vascular cell adhesion
molecule 1 (VCAM-1; log2 = 0.43, P = 0.007), in the adult brain
microvasculature abated in pericyte numbers (SI Appendix, Fig.
S1A). To corroborate these findings, we investigated whether
strongly reduced pericyte coverage in adult mice leads to changes
of LAMs at the protein level. We focused on the expression of
VCAM-1 and intercellular adhesion molecule 1 (ICAM-1), which
play a major role in the cascade of immune cell transmigration
into tissues (14). We detected a zonated endothelial expression of
both LAMs in control mice (Fig. 1 A and B), similar to a published
study (15). In brains of Pdgfb
ret/ret
mice, the zonated expression
pattern was lost and paralleled by a conspicuously stronger staining
of VCAM-1 and ICAM-1 (Fig. 1 A and B), which colocalized with
the endothelial marker podocalyxin (Fig. 1 C and D). Quantifica-
tion of VCAM-1 and ICAM-1 vessel surface coverage in the ce-
rebral cortex and in the striatum showed a significant increase of
VCAM-1 and ICAM-1 expression in Pdgfb
ret/ret
mice compared
with controls (Fig. 1E). Thus, pericyte deficiency results in an in-
creased expression of LAMs on the brain vasculature in adult mice.
Leukocyte Extravasation into the Brain in Pericyte-Deficient Adult
Mice.
We next asked whether increased expression of LAMs on
the brain endothelium of pericyte-deficient mice is accompanied
ControlPdgfb
ret/ret
VCAM-1 VCAM-1+CD13VCAM-1+Collagen-IV
A
E
B ICAM-1 ICAM-1+Collagen-IV ICAM-1+CD13
ControlPdgfb
ret/ret
C
ICAM-1
Podocalyxin
D Merge
Podocalyxin
VCAM-1 Merge
Fig. 1. Increased expression of LAMs on pericyte-deficient brain vasculature. (AD) Immunofluorescent stainings showing the expression of VCAM-1 (A)and
ICAM-1 (B ) on brain vessels in the striatum of naïve control and pericyte-deficient mice ( Pdgfb
ret/ret
). Vascular basement membrane was visualized with
collagen IV (in green) and pericytes with CD13 (in cyan) immunostaining. High-magnification images of the vasculature of Pdgfb
ret/ret
mice showing coloc-
alization of VCAM-1 ( C) and ICAM-1 (D) staining (in magenta) with the endothel ial marker podocalyxin (in green). (E ) Quantification of vascular surface
coverage of VCAM-1 and ICAM-1 in the cortex and striatum in control and pericyte-deficient mice (Pdgfb
ret/ret
; n = 3 mice per genotype). Unpaired t test was
used to determine statistical significance. Data are presented as the mean ± SD. (Scale bars: A and B, 100 μm; C and D,50μm.)
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PNAS Török et al.
https://doi.org/10.1073/pnas.2016587118 Pericytes regulate vascular immune homeostasis in the CNS
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by leukocyte infiltration into the brain parenchyma. We first
analyzed the presence of CD45
hi
leukocytes in different anatomical
regions of the brain by immunofluorescent staining and confocal
imaging. Adult Pdgfb
ret/ret
mice showed numerous CD45
hi
leukocyte
infiltrates in the brain parenchyma (Fig. 2A). High-magnification
images revealed that CD45
hi
cells were found in the vessel lumen
and in the brain parenchyma and clustered around blood vessels in
the brains of Pdgfb
ret/ret
mice,whereas,incontrolmice,CD45
hi
cells
were detected, but they resided in the lumen of blood vessels (Fig. 2
B and D). CD45
hi
leukocyte infiltrates can be distinguished from
CD45
lo
microglia based on their round morphology and the lack of
expression of the microglia marker P2Y12 (SI Appendix,Fig.S2A).
Microglia surrounding CD45
hi
infiltrates in corpus callosum in
Pdgfb
ret/ret
mice show a slightly altered morphology of shorter and
thicker dendrites (SI Appendix,Fig.S2A), and occasional local al-
teration in microglia morphology was also detected in the cerebral
cortex (SI Appendix,Fig.S2B). Pdgfb
ret/ret
miceshowaslightlyin-
creased number of microglia in the brain but not in the spinal cord
(SI Appendix,Fig.S2C and D). Although all cerebral regions in
Pdgfb
ret/ret
mice showed altered vascular expression of LAMs and
immune cell infiltrates, quantification of extravasated leukocytes in
different brain regions showed that corpus callosum and striatum
contained more transmigrated CD45
hi
cells compared with cortex
(Fig. 2C). Notably, immunohistochemical analysis of CD45
hi
cells in
the spinal cord of Pdgfb
ret/ret
mice showed the absence of immune cell
infiltrates in the spinal cord parenchyma (SI Appendix,Fig.S1B).
Having established that the brain parenchyma of Pdgfb
ret/ret
mice
contains CD45
hi
cells, we used flow cytometry to identify immune
cell populations. To characterize immune cell populations, leuko-
cytes were isolated from the CNS and analyzed by flow cytometry.
Flow cytometric analysis confirmed the immunohistochemistry
findings and showed increased frequencies of CD45
hi
cells in the
CNS of pericyte-deficient mice. The majority of cells in the brains of
Pdgfb
ret/ret
mice were CD45
hi
CD11b
+
myeloid cells (mainly com-
posed of Ly6C
hi
monocytes and CD11c
+
MHC-II
high
dendritic cells
[DCs]) and T cells (Fig. 2E). Quantification of the absolute cell
numbers of manually gated immune cell subsets in the brain showed a
significant increase in the number of CD45
hi
CD11b
+
myeloid cells,
Ly6C
hi
MHC-II
+
monocyte-derived cells (MdCs), Ly6C
hi
monocytes
(Ly6G
Ly6C
hi
MHC-II
myeloid cells), DCs (3.5% of the
CD45
hi
CD11b
+
population), and CD4
+
and CD8
+
T cell populations
compared with controls (Fig. 2F). Similarly to immunohistochemical
analysis, no difference was detected in CD45
hi
cells in the spinal cord
between control and Pdgfb
ret/ret
mice using flow cytometry analysis
(SI Appendix,Fig.S1C and D).
We next investigated the temporal aspect of leukocyte infiltra-
tion. ICAM-1 expression was detected by the developing vascula-
ture in postnatal brains (postnatal day [P] 6, P16, P25) in control
and Pdgfb
ret/ret
mice (SI Appendix,Fig.S3). We did not detect
CD45
hi
infiltrates in brain parenchyma at investigated postnatal
stages (P6, P16, P25) in Pdgfb
ret/ret
mice; however, frequent CD45
hi
cells were detected inside the vessel lumen in 16- and 25-d-
old Pdgfb
ret/ret
mice (SI Appendix,Fig.S4). Thus, extravasation of
leukocytes into brain parenchyma in Pdgfb
ret/ret
mice takes place after
P25, a time point when the angiogenesis has gradually diminished
and vasculature begins to stabilize (16).
Taken together, our data show that, in the absence of pericytes,
the adult brain vasculature becomes permissive for leukocyte entry
and that the infiltrated leukocyte population consists mostly of
Ly6C
hi
monocytes, MdCs, DCs, and T cells.
Characterization of Leukocyte Populations in Peripheral Organs in
Pericyte-Deficient Adult Mice.
We next analyzed leukocyte pop-
ulations in blood as well as in primary and secondary lymphoid
organs to ensure that the increased number of leukocytes in the
brains of pericyte-deficient animals is not caused by peripheral al-
terations. The total cell number in thymus, spleen, axillary and
inguinal lymph nodes, and blood was determined using an auto-
mated cell counter with isolated cells stained for further flow
cytometry analysis. The cell number in the thymus, spleen, lymph
node, and blood was comparable between Pdgfb
ret/ret
and control
mice (SI Appendix,Fig.S5A). Subsequent analysis of leukocyte
populations did not show a skewing between Pdgfb
ret/ret
and control
mice in blood, lymph nodes, and spleen (SI Appendix,Fig.S5BD).
There was no difference in the total leukocyte or neutrophil count
in blood (SI Appendix,Fig.S5B), indicating the absence of systemic
inflammation in Pdgfb
ret/ret
mice. Histological examination of lym-
phoid organs did not show any differences in the spatial organiza-
tion of T and B cells (SI Appendix,Fig.S5EG) between Pdgfb
ret/ret
and control mice.
We also investigated whether organs other than the brain pre-
sented with leukocyte infiltration or a spontaneous inflammation in
Pdgfb
ret/ret
mice. Flow cytometry analysis of immune cell populations
was performed in the lung, liver, and small intestine of control and
Pdgfb
ret/ret
mice. No difference in any of the analyzed immune cell
populations was observed between control and Pdgfb
ret/ret
mice
(SI Appendix,Fig.S6).
Thus, the increased number of infiltrated leukocyte subsets in
the brain of adult pericyte-deficient mice is not due to increased
numbers in the blood or peripheral organs.
Spatial Differences in Pericyte Coverage in the CNS of Pdgfb
ret/ret
Mice. Previous studies have shown a negative correlation between
pericyte coverage and BBB permeability in the brain (1, 2, 1719).
We therefore asked whether selective leukocyte infiltration into
different CNS regions in Pdgfb
ret/ret
mice can be explained by dif-
ferences in capillary pericyte coverage in the brain and spinal cord.
Therefore, we determined pericyte coverage in several other brain
regions in addition to already reported areas (e.g., cortex and deep
brain regions) (1, 17). Pericyte coverage in Pdgfb
ret/ret
mice was
significantly reduced also in the cerebellum and brainstem (42%
and 54%, respectively; SI Appendix,Fig.S7A and B). In agree-
ment with reduced pericyte coverage, we observed a significantly
increased VCAM-1 and ICAM-1 coverage in the brainstem and
cerebellum of Pdgfb
ret/ret
mice (SI Appendix,Fig.S7CF) and nu-
merous CD45
hi
cell infiltrates (SI Appendix,Fig.S7GJ). Quan-
tification of vessel surface pericyte coverage in the spinal cord
showed that Pdgfb
ret/ret
mice also have a significantly reduced cap-
illary pericyte coverage compared with control animals in this CNS
region (Fig. 3 A and B). However, the observed reduction (26%)
of pericyte coverage in the spinal cord vasculature of Pdgfb
ret/ret
mice is notably less than the previously reported reduction of
pericyte coverage in the cortex or deep brain regions (75%)
(1, 17) or in the brainstem and cerebellum (54% and 42%, re-
spectively; SI Appendix,Fig.S7A and B). In agreement with rel-
atively complete pericyte coverage, the pattern and morphology of
spinal cord vasculature of Pdgfb
ret/ret
mice appeared similar to
control mice (Fig. 3A). The difference in vessel pericyte coverage
between spinal cord and different brain regions in Pdgfb
ret/ret
mice
indicates that spinal cord pericytes are less dependent on the
platelet-derived growth factor (PDGF) BPDGF receptor β sig-
naling axis for vascular recruitment.
Finally, we investigated whether higher capillary pericyte coverage
in the spinal cord of Pdgfb
ret/ret
mice parallels normalized expression
of VCAM-1 and ICAM-1. Indeed, the expression of VCAM-1 and
ICAM-1 on the spinal cord vasculature showed a similar zonal ex-
pression pattern in control and Pdgfb
ret/ret
mice and only a slight in-
crease in VCAM-1 and ICAM-1 coverage (34%; Fig. 3 C F).
Based on these data, we conclude that regional differences in
the degree of capillary pericyte coverage in the CNS determine
the extent to which the brain vasculature is permissive to leu-
kocyte entry into the CNS under homeostasis.
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Control Pdgfb
ret/ret
Collagen-IV + CD45
A
C
ControlPdgfb
ret/ret
Podocalyxin + CD45 CD45B Podocalyxin + CD45
E Control Pdgfb
ret/ret
F
D
+
**
CD45 Ly6G
CD
11b
CD
1
1b
MHC-II
Ly6C
CD3
CD19
CD4
CD8
CD
11b
CD11b
CD45 Ly6G
Ly6C
MHC-II
CD19
CD3
CD8
CD4
Gated on live singlets
Gated on live singlets
Brain
D
Fig. 2. Increased extravasation of leukocytes in the brain parenchyma of naïve pericyte-deficient brain. (A) Overview images of the periventricular areas in
the brains of control and pericyte-deficient mice (Pdgfb
ret/ret
). Arrowheads indica te leukocyte infiltrates (CD45, in red) in the parenchyma of Pdgfb
ret/ret
mice.
Blood vessels are visualized by collagen IV staining (in green). (B) High-magnification images showing a parenchymal infiltrate of CD45
hi
leukocytes (in red,
asterisk) in the corpus callosum of Pdgfb
ret/ret
mice. In control mice, few CD45
hi
leukocytes (arrowheads) are found only in the lumen of blood vessels
(podocalyxin, in green). (C) Quantification of CD45
hi
leukocytes in different anatomical regions in the brains of control and Pdgfb
ret/ret
mice (n = 3). (D)
Quantification of extravasated CD45
hi
leukocytes in different anatomical regions in the brains of control and Pdgfb
ret/ret
mice (n = 3). One-way ANOVA
followed by Tukeys post hoc test was used to determine the statistical significance. (E) Representative flow-cytometry pseudocolor plots showing the manual
gating of microglia and other immune cell populations in the brain of naïve control and Pdgfb
ret/ret
mice. (F) Quantification of the absolute cell numbers of
detected immune cell populations using flow cytometry in the brain (n = 78 mice per genotype). Pooled data from two independen t experiments. Statistical
significance was determined using unpaired t test (DCs, CD11c
+
, MHC-II
high
, and CD4
+
and CD8
+
T cells) or MannWhitney U test (CD45
hi
cells, CD45
hi
CD11b
+
cells, MdCs, Ly6C
hi
monocytes, neutrophils, and B cells). Data are presented as the mean ± SD. (Scale bars: A, 250 μm; B, 100 μm; Insets,20μm.)
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https://doi.org/10.1073/pnas.2016587118 Pericytes regulate vascular immune homeostasis in the CNS
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Citations
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Liqun He1, Maarja Andaloussi Mäe1, Lars Muhl2, Ying Sun1  +25 moreInstitutions (6)
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TL;DR: It is shown that endothelial cells do not express angiotensin-converting enzyme-2 (ACE2), the SARS-CoV-2 receptor, and pericytes and microvascular smooth muscle cells express ACE2 in an organotypic manner, suggesting thatpericytes limit endothelial pro-thrombotic responses.
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Journal ArticleDOI
Juliane Gust1, Juliane Gust2, Rafael Ponce, W. Conrad Liles1  +3 moreInstitutions (4)
Abstract: Chimeric antigen receptor (CAR) T cells provide new therapeutic options for patients with relapsed/refractory hematologic malignancies. However, neurotoxicity is a frequent, and potentially fatal, complication. The spectrum of manifestations ranges from delirium and language dysfunction to seizures, coma, and fatal cerebral edema. This novel syndrome has been designated immune effector cell-associated neurotoxicity syndrome (ICANS). In this review, we draw an arc from our current understanding of how systemic and potentially local cytokine release act on the CNS, toward possible preventive and therapeutic approaches. We systematically review reported correlations of secreted inflammatory mediators in the serum/plasma and cerebrospinal fluid with the risk of ICANS in patients receiving CAR T cell therapy. Possible pathophysiologic impacts on the CNS are covered in detail for the most promising candidate cytokines, including IL-1, IL-6, IL-15, and GM-CSF. To provide insight into possible final common pathways of CNS inflammation, we place ICANS into the context of other systemic inflammatory conditions that are associated with neurologic dysfunction, including sepsis-associated encephalopathy, cerebral malaria, thrombotic microangiopathy, CNS infections, and hepatic encephalopathy. We then review in detail what is known about systemic cytokine interaction with components of the neurovascular unit, including endothelial cells, pericytes, and astrocytes, and how microglia and neurons respond to systemic inflammatory challenges. Current therapeutic approaches, including corticosteroids and blockade of IL-1 and IL-6 signaling, are reviewed in the context of what is known about the role of cytokines in ICANS. Throughout, we point out gaps in knowledge and possible new approaches for the investigation of the mechanism, prevention, and treatment of ICANS.

17 citations


Journal ArticleDOI
Maarja Andaloussi Mäe1, Liqun He2, Liqun He1, Sofia Nordling3  +22 moreInstitutions (8)
Abstract: Rationale: Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain d...

16 citations


Journal ArticleDOI
TL;DR: Current understanding on the characterization of pericytes, their roles in maintaining the integrity of the blood–brain barrier, and their contributions to neuroinflammation and neurorepair are discussed.
Abstract: Pericytes are contractile cells that extend along the vasculature to mediate key homeostatic functions of endothelial barriers within the body. In the central nervous system (CNS), pericytes are important contributors to the structure and function of the neurovascular unit, which includes endothelial cells, astrocytes and neurons. The understanding of pericytes has been marred by an inability to accurately distinguish pericytes from other stromal cells with similar expression of identifying markers. Evidence is now growing in favor of pericytes being actively involved in both CNS homeostasis and pathology of neurological diseases, including multiple sclerosis, spinal cord injury, and Alzheimer's disease among others. In this review, we discuss the current understanding on the characterization of pericytes, their roles in maintaining the integrity of the blood-brain barrier, and their contributions to neuroinflammation and neurorepair. Owing to its plethora of surface receptors, pericytes respond to inflammatory mediators such as CCL2 (monocyte chemoattractant protein-1) and tumor necrosis factor-α, in turn secreting CCL2, nitric oxide, and several cytokines. Pericytes can therefore act as promoters of both the innate and adaptive arms of the immune system. Much like professional phagocytes, pericytes also have the ability to clear up cellular debris and macromolecular plaques. Moreover, pericytes promote the activities of CNS glia, including in maturation of oligodendrocyte lineage cells for myelination. Conversely, pericytes can impair regenerative processes by contributing to scar formation. A better characterization of CNS pericytes and their functions would bode well for therapeutics aimed at alleviating their undesirable properties and enhancing their benefits.

7 citations


Cites background from "Pericytes regulate vascular immune ..."

  • ...A recent study has shown that pericyte knockout (in Pdgfβret/ret mice) resulted in increased leukocyte adhesion molecules and enhanced leukocyte infiltration into the EAE cerebrum (but not the spinal cord), suggesting that pericytes may have differential functions in migration of leukocyte subsets, and in a niche-dependent manner (Torok et al., 2019)....

    [...]



References
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Journal Article
01 Jan 2014-MSOR connections
TL;DR: Copyright (©) 1999–2012 R Foundation for Statistical Computing; permission is granted to make and distribute verbatim copies of this manual provided the copyright notice and permission notice are preserved on all copies.
Abstract: Copyright (©) 1999–2012 R Foundation for Statistical Computing. Permission is granted to make and distribute verbatim copies of this manual provided the copyright notice and this permission notice are preserved on all copies. Permission is granted to copy and distribute modified versions of this manual under the conditions for verbatim copying, provided that the entire resulting derived work is distributed under the terms of a permission notice identical to this one. Permission is granted to copy and distribute translations of this manual into another language, under the above conditions for modified versions, except that this permission notice may be stated in a translation approved by the R Core Team.

229,202 citations


Journal ArticleDOI
Matthew E. Ritchie1, Belinda Phipson2, Di Wu3, Yifang Hu1  +4 moreInstitutions (5)
TL;DR: The philosophy and design of the limma package is reviewed, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.
Abstract: limma is an R/Bioconductor software package that provides an integrated solution for analysing data from gene expression experiments. It contains rich features for handling complex experimental designs and for information borrowing to overcome the problem of small sample sizes. Over the past decade, limma has been a popular choice for gene discovery through differential expression analyses of microarray and high-throughput PCR data. The package contains particularly strong facilities for reading, normalizing and exploring such data. Recently, the capabilities of limma have been significantly expanded in two important directions. First, the package can now perform both differential expression and differential splicing analyses of RNA sequencing (RNA-seq) data. All the downstream analysis tools previously restricted to microarray data are now available for RNA-seq as well. These capabilities allow users to analyse both RNA-seq and microarray data with very similar pipelines. Second, the package is now able to go past the traditional gene-wise expression analyses in a variety of ways, analysing expression profiles in terms of co-regulated sets of genes or in terms of higher-order expression signatures. This provides enhanced possibilities for biological interpretation of gene expression differences. This article reviews the philosophy and design of the limma package, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.

13,819 citations


Journal ArticleDOI
Annika Armulik1, Guillem Genové1, Maarja Mäe1, Maya H. Nisancioglu1  +11 moreInstitutions (4)
25 Nov 2010-Nature
TL;DR: A novel and critical role for pericytes is indicated in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the blood–brain barrier.
Abstract: The blood–brain barrier is a gatekeeper between the central nervous system and the rest of the body, and is made up of vascular endothelial cells. Previous work upheld the notion that the barrier was formed postnatally as a result of signalling from non-neuronal cells called astrocytes to endothelial cells. Now, two independent studies demonstrate that the barrier is in fact formed during embryogenesis, with the critical factor being the interaction between blood-vessel-surrounding cells called pericytes and epithelial cells. A better understanding of the tight relationship between pericytes, neuroendothelial cells and astrocytes in blood–brain barrier function will contribute to our understanding of the breakdown of the barrier during central nervous system injury and disease. The blood–brain barrier (BBB) is made up of vascular endothelial cells and was thought to have formed postnatally from astrocytes. Two independent studies demonstrate that this barrier forms during embryogenesis, with pericyte/endothelial cell interactions being critical to regulate the BBB during development. A better understanding of the relationship among pericytes, neuroendothelial cells and astrocytes in BBB function will contribute to our understanding of BBB breakdown during central nervous system injury and disease. The blood–brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS)1. The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport2. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes3, but the relative contribution of these different components to the BBB remains largely unknown1,3. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB.

1,782 citations


"Pericytes regulate vascular immune ..." refers methods in this paper

  • ...(e) 714 Immunohistochemical staining of T cells (CD3, in red) on coronal sections of the spinal cords 715 showing two regions (1, 2) after active induction of EAE in control (on day 16 716 postimmunization) and Pdgfb mice (on day 11 postimmunization)....

    [...]


Journal ArticleDOI
12 Apr 2002-Science
TL;DR: It is shown that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolites of the immunosuppressive agent FTY720.
Abstract: Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S1P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S1P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S1P receptors may result in therapeutically useful immunosuppression.

1,562 citations


Journal ArticleDOI
25 Nov 2010-Nature
TL;DR: Pericytes regulate functional aspects of the blood–brain barrier, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration.
Abstract: Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood-brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. Owing to the close spatial relationship between astrocytes and endothelial cells, it has been hypothesized that astrocytes induce this critical barrier postnatally, but the timing of BBB formation has been controversial. Here we demonstrate that the barrier is formed during embryogenesis as endothelial cells invade the CNS and pericytes are recruited to the nascent vessels, over a week before astrocyte generation. Analysing mice with null and hypomorphic alleles of Pdgfrb, which have defects in pericyte generation, we demonstrate that pericytes are necessary for the formation of the BBB, and that absolute pericyte coverage determines relative vascular permeability. We demonstrate that pericytes regulate functional aspects of the BBB, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells. Pericytes do not induce BBB-specific gene expression in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration. These data indicate that pericyte-endothelial cell interactions are critical to regulate the BBB during development, and disruption of these interactions may lead to BBB dysfunction and neuroinflammation during CNS injury and disease.

1,372 citations


"Pericytes regulate vascular immune ..." refers methods in this paper

  • ...(e) 714 Immunohistochemical staining of T cells (CD3, in red) on coronal sections of the spinal cords 715 showing two regions (1, 2) after active induction of EAE in control (on day 16 716 postimmunization) and Pdgfb mice (on day 11 postimmunization)....

    [...]


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