Pericytes regulate vascular immune homeostasis in the CNS

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.

Summary

Introduction

• For more information, please consult the Terms of use.
• Pericyte | blood–brain barrier | leukocyte trafficking | autoimmune neuroinflammation | MOG35–55–specific T cell receptor Central nervous system (CNS) vasculature possesses specificfeatures collectively referred to as the blood–brain barrier (BBB), which localizes to endothelial cells.
• The authors show that pericytes play a crucial role Significance The CNS vasculature tightly regulates the passage of circulating molecules and leukocytes into the CNS.

Increased Expression of Leukocyte Adhesion Molecules in the Brain

• Having established that the brain parenchyma of Pdgfbret/ret mice contains CD45hi cells, the authors used flow cytometry to identify immune cell populations.
• Subsequent analysis of leukocyte populations did not show a skewing between Pdgfbret/ret and control mice in blood, lymph nodes, and spleen .
• The observed reduction (∼26%) of pericyte coverage in the spinal cord vasculature of Pdgfbret/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 .
• Finally, the authors investigated whether higher capillary pericyte coverage in the spinal cord of Pdgfbret/ret mice parallels normalized expression of VCAM-1 and ICAM-1.

Pericyte-Deficient Mice Present an Aggravated, Atypical Experimental

• The authors next investigated whether leukocyte-permissive vasculature modifies the course of autoimmune neuroinflammation.
• Previous studies have reported that ∼5% of 2D2 mice develop spontaneous EAE with classical symptoms (24).
• Of note, the ataxia scores of individual Pdgfbret/ret;2D2tg mice fluctuated over the monitored time period.
• Immune cell infiltrates in the brains of Pdgfbret/ret and Pdgfbret/ret;2D2tg mice consisted of Ly6Chi monocytes, MdCs, and CD4+ and CD8+ T cells (Fig. 7C).

Discussion

• Pericytes have been shown to regulate BBB integrity at the level of endothelial transcytosis (1, 2).
• In addition, increased vascular permeability to plasma proteins (e.g., fibrinogen) due to increased transcytosis in pericyte-deficient mice could contribute to leukocyte trafficking.
• Thus, the mechanism by which pericytes control leukocyte extravasation is likely multifaceted, and the increased leukocyte trafficking and aggravated neuroinflammation in pericyte-deficient mice is caused by a combination of changes in acellular (basement membrane, increased presence of plasma proteins in the brain parenchyma) and cellular components (endothelium, astrocytes) of the NVU .
• This relatively spared vasculature of spinal cord vessels with a higher pericyte coverage and relatively small upregulation of ICAM-1 and VCAM1 (∼3–4%) compared with brain vessels could explain the preferential location of neuroinflammation in the brain in Pdgfbret/ret and Pdgfbret/ret;2D2tg mice.
• BBB breakdown, one of the pathological hallmarks of MS (53, 54), is an early event in the formation of the inflammatory lesions and has been suggested to precede parenchymal inflammation (55).

Materials and Methods

• Transgenic mouse lines, reagents, immunohistochemistry and histochemistry, flow cytometry and data analysis, induction of EAE and scoring, FTY-720, anti–VCAM-1 and anti–ICAM-1 treatment, quantification of immunohistochemical and histochemical stainings, transmission electron microscopy, leukocyte isolation from tissues, Materials and Methods.
• Imaging was performed with equipment maintained by the Center for Microscopy and Image Analysis of the University of Zürich.
• The biology of the blood-brain barrier in health and disease.
• S. A. Lévesque et al., Myeloid cell transmigration across the CNS vasculature triggers IL-1β-driven neuroinflammation during autoimmune encephalomyelitis in mice.

Figures

Fig. 3. Pericyte coverage and expression of LAMs in the spinal cord vessels of Pdgfbret/ret mice. (A) Immunofluorescent staining of pericytes (CD13, in cyan) and vasculature (collagen IV, in red) in different anatomical regions of the CNS (spinal cord, striatum, cortex) in control and Pdgfbret/ret mice. The yellow dotted line outlines the cortical surface. (B) Quantification of vessel pericyte coverage in the spinal cord of control and Pdgfbret/ret mice (n = 3 mice per genotype). (C and D) VCAM-1 (C) and ICAM-1 (D) expression (in cyan) on the blood vessels (in red, collagen IV) in the spinal cord of control and Pdgfbret/ret mice. (E and F) Quantification of vascular surface coverage of VCAM-1 (E) and ICAM-1 (F) in the spinal cord of control and Pdgfbret/ret mice. (Scale bars: 100 μm.) Unpaired t test was used to determine statistical significance.

Fig. 7. Pericyte-deficient mice expressing myelin-specific TCR develop neurological symptoms accompanied by immune cell infiltration. (A) Scoring of cerebellar ataxia in Pdgfbret/ret × Pdgfbwt/ret;2D2tg crossing offspring. Dashed black line indicates the mean baseline score of control mice (ataxia score 1, occasional slip during the ledge test; n = 7–11 mice per genotype). (B) Immunofluorescent detection of CD45hi leukocyte infiltrates and microglia (in red) in the striatum in Pdgfbret/ret;2D2neg and Pdgfbret/ret;2D2tg mice. Blood vessels are in green (collagen IV). (C) Representative flow cytometry pseudocolor plots showing the manual gating of microglia and other immune cell populations in the brain. (C) Quantification of absolute cell numbers of immune cells (gated as shown in SI Appendix, Fig. S13A) in the brains of control mice and Pdgfbret/ret;2D2neg and Pdgfbret/ret;2D2tg animals. Pdgfbret/ret;2D2tg mice were euthanized at the peak of cerebellar ataxia symptoms (ataxia score of 6–9). Age of animals was 2–3 mo (C). Pooled data from four individual experiments. Data are presented as the mean ± SD. One-way ANOVA followed by Tukey’s post hoc test was used to determine the statistical significance. (Scale bars: 100 μm.)

Fig. 6. Combined treatment using anti–VCAM-1 and anti–ICAM-1 antibodies ameliorates symptoms of pericyte-deficient and control mice after the induction of EAE. (A) Scoring of clinical symptoms during the course of anti–VCAM-1 and anti–ICAM-1 mAb treatment after induction of active EAE of control mice. (B) Kaplan–Meier survival curves of isotype control and anti–VCAM-1 and anti–ICAM-1 monoclonal antibody (α-VCAM-1 and α-ICAM-1 mAb) treated Pdgfbret/ret mice after active induction of EAE (P = 0.05, log-rank test). (C) Scoring of clinical symptoms during the course of anti–VCAM-1 and anti–ICAM-1 mAb treatment after induction of active EAE in Pdgfbret/ret mice (C). (A–C) The EAE or ataxia score of each mouse is plotted. Arrowheads indicate when mice reached termination criteria and were euthanized for analysis. The experiment was terminated on day 20 (black dashed line; n = 4 mice treated with isotype control, n = 4 mice treated with anti–VCAM-1 and anti–ICAM-1 mAb). (D and E) Quantification of the absolute cell numbers of different immune cell populations (gating shown in SI Appendix, Fig. S12) in the brains (D) and spinal cords (E) of isotype control and anti–VCAM-1 and anti–ICAM-1 mAb-treated control and Pdgfbret/ret mice using flow cytometry. Controls, n = 4 mice per group; Pdgfbret/ret mice, n = 3 mice per group. Pooled data from two independent experiments. Data are presented as the mean ± SD. Two-way ANOVA followed by Šídák’s post hoc test was used to determine statistical significance between groups.

Fig. 1. Increased expression of LAMs on pericyte-deficient brain vasculature. (A–D) 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 (Pdgfbret/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 Pdgfbret/ret mice showing colocalization of VCAM-1 (C) and ICAM-1 (D) staining (in magenta) with the endothelial 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 (Pdgfbret/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, 50 μm.)

Fig. 4. Pericyte-deficient mice die with atypical EAE accompanied by increased leukocyte infiltration into the brain. (A) Kaplan–Meier survival curves after active induction of EAE. The experiment was terminated on day 15, indicated by black dashed line. Pooled data from two individual experiments (n = 11 mice per genotype). Survival curves showed statistical difference (P < 0.0001, log-rank test). (B) Scoring of neurological symptoms during the course of active EAE. The left y axis shows cerebellar ataxia scores of Pdgfbret/ret mice (in red), and the right y axis shows classical EAE scores of control mice (in black). Arrowheads indicate when individual mice were euthanized for flow-cytometry analysis. Materials and Methods includes detailed termination criteria. Each line represents symptoms of an individual mouse (n = 5 mice per genotype, showing two pooled experiments). (C) Kaplan–Meier survival curves after passive induction of EAE. The experiment was terminated on day 14, indicated by black dashed line (controls, n = 5; Pdgfbret/ret, n = 6). Survival curves showed a statistically significant difference (P = 0.0300, log-rank test). (D) Immunohistochemical staining of T cells (CD3, in red) of sagittal brain sections of the cerebellum and brainstem after active induction of EAE of control (on day 16 postimmunization) and Pdgfbret/ret mice (on day 11 postimmunization). (E) Immunohistochemical staining of T cells (CD3, in red) on coronal sections of the spinal cords showing two regions (1, 2) after active induction of EAE in control (on day 16 postimmunization) and Pdgfbret/ret mice (on day 11 postimmunization). Tissue sections were counterstained with hematoxylin (D and E). (F and G) Quantification of the absolute cell numbers of different leukocyte populations (gated as shown in SI Appendix, Fig. S10) in the brains (cerebrum, cerebellum, and brainstem; F) and spinal cords (G) of control (EAE score of 3–3.5) and Pdgfbret/ret (ataxia score, n = 9–10) mice using flow cytometry. Shown are pooled data from two individual experiments (n = 5 mice per genotype). Data are presented as the mean ± SD. Statistical significance was determined using unpaired t test (brain, CD45hi, MdCs, and CD4+ T and CD8+ T cells; spinal cord, CD4+ T and Ly6Chi monocytes) or Mann–Whitney U test (brain, Ly6Chi monocytes; spinal cord, CD45hi, CD8+ T cells, and MdCs). (Scale bars: D and E, 100 μm.)

Fig. 5. FTY-720 treatment rescues the lethality of pericyte-deficient mice after induction of EAE. (A) Kaplan–Meier survival curves of vehicle and FTY720–treated Pdgfbret/ret mice after active induction of EAE (P = 0.002, log-rank test). The experiment was terminated on day 25 (marked by a black dashed line; n = 5 mice treated with vehicle, n = 6 mice treated with FTY-720). (B) Scoring of clinical symptoms during the course of FTY-720 treatment after induction of active EAE of control and Pdgfbret/ret mice. The left y axis shows the cerebellar ataxia scores of Pdgfbret/ret mice (in red) and the right y axis the classical EAE scores of control mice (in black). FTY-720 administration (0.5 mg/kg) was started on day 4 postimmunization (black arrow), and the experiment was terminated on day 25 postimmunization. The ataxia score or EAE score of each mouse is plotted individually. Arrowheads indicate when Pdgfbret/ret mice reached termination criteria and were euthanized (n = 4–5 mice per group). (C and D) Quantification of the absolute cell numbers of the different immune cell populations (gating shown in SI Appendix, Fig. S11 F and G) in the brain (C) and spinal cords (D) of vehicle- and FTY-720–treated control and Pdgfbret/ret mice using flow cytometry. Controls, n = 3 vehicle-treated and n = 4 FTY-720–treated; Pdgfbret/ret mice, n = 3 vehicle-treated and n = 3 FTY-720–treated. Pooled data from two independent experiments. Data are presented as the mean ± SD. Two-way ANOVA followed by Šídák’s post hoc test was used to determine statistical significance between groups.

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 (Pdgfbret/ret). Arrowheads indicate leukocyte infiltrates (CD45, in red) in the parenchyma of Pdgfbret/ret mice. Blood vessels are visualized by collagen IV staining (in green). (B) High-magnification images showing a parenchymal infiltrate of CD45hi leukocytes (in red, asterisk) in the corpus callosum of Pdgfbret/ret mice. In control mice, few CD45hi leukocytes (arrowheads) are found only in the lumen of blood vessels (podocalyxin, in green). (C) Quantification of CD45hi leukocytes in different anatomical regions in the brains of control and Pdgfbret/ret mice (n = 3). (D) Quantification of extravasated CD45hi leukocytes in different anatomical regions in the brains of control and Pdgfbret/ret mice (n = 3). One-way ANOVA followed by Tukey’s 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 Pdgfbret/ret mice. (F) Quantification of the absolute cell numbers of detected immune cell populations using flow cytometry in the brain (n = 7–8 mice per genotype). Pooled data from two independent experiments. Statistical significance was determined using unpaired t test (DCs, CD11c+, MHC-IIhigh, and CD4+ and CD8+ T cells) or Mann–Whitney U test (CD45hi cells, CD45hiCD11b+ cells, MdCs, Ly6Chi monocytes, neutrophils, and B cells). Data are presented as the mean ± SD. (Scale bars: A, 250 μm; B, 100 μm; Insets, 20 μm.)

Full text

ETH Library
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 blood–brain 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 anti–ICAM-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
|
blood–brain barrier
|
leukocyte trafficking
|
autoimmune neuroinflammation
|
MOG
35–55
–specific T cell receptor
C
entral nervous system (CNS) vasculature possesses specific
features collectively referred to as the blood–brain 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(3–5).
Increased vascular permeability to plasma proteins and im-
mune cells accompanies neurological disorders such as multiple
sclerosis (MS), stroke, and Alzheimer’s disease (reviewed in refs.
6–8). 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 (10–12). 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
cell–cell 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
|
1of12
IMMUNOLOGY AND
INFLAMMATION
Downloaded at ETH-Bibliothek on March 24, 2021

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. (A–D) 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.)
2of12
|
PNAS Török et al.
https://doi.org/10.1073/pnas.2016587118 Pericytes regulate vascular immune homeostasis in the CNS
Downloaded at ETH-Bibliothek on March 24, 2021

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.S5B–D).
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.S5E–G) 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, 17–19).
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.S7C–F) and nu-
merous CD45
hi
cell infiltrates (SI Appendix,Fig.S7G–J). 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) B–PDGF 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 (∼3–4%; 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.
Török et al. PNAS
|
3of12
Pericytes regulate vascular immune homeostasis in the CNS https://doi.org/10.1073/pnas.2016587118
IMMUNOLOGY AND
INFLAMMATION
Downloaded at ETH-Bibliothek on March 24, 2021

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 Tukey’s 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 = 7–8 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 Mann–Whitney 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.)
4of12
|
PNAS Török et al.
https://doi.org/10.1073/pnas.2016587118 Pericytes regulate vascular immune homeostasis in the CNS
Downloaded at ETH-Bibliothek on March 24, 2021

Frequently asked questions

Q1. What are the future works in "Pericytes regulate vascular immune homeostasis in the cns" ?

In the future, it would be interesting to investigate early changes in the brain vasculature of MS patients and determine whether vascular alterations regulate the localization of MS lesions. 

Q2. What is the effect of FTY-720 on the brain endothelium?

Pericyte deficiency alters endothelial cell phenotype and leads to up-regulation of LAMs on the brain endothelium, which are important for leukocyte transmigration. 

Q3. What is the mechanism by which pericytes control leukocyte trafficking?

In addition, increased vascular permeability to plasma proteins (e.g., fibrinogen) due to increased transcytosis in pericyte-deficient mice could contribute to leukocyte trafficking. 

Q4. What is the role of vascular dysfunction in MS?

Since vascular dysfunction modulates leukocyte entry and neuroinflammation, vasoprotective therapies combined with preexisting treatments could lead to improved clinical outcomes in MS. 

Q5. What is the phenotype of Pdgfbret/ret mice?

After active induction of EAE, which replicates both the induction and effector phase of the disease, Pdgfbret/ret mice presented with a severe, early-onset (4–5 d postimmunization) atypical phenotype as well as reduced survival (Fig. 4 A and B and SI Appendix, Fig. S9A and Table S2). 

Q6. What is the effect of a humanized monoclonal antibody against 4-?

H-B iblio thek on Mar ch2 4,2 021natalizumab, a humanized monoclonal antibody against α4-integrin on leukocytes, has been proven to be an effective treatment in MS (25). 

Q7. What is the effect of FTY-720 on the death of Pdgfbret?

All IgG isotype control-treated Pdgfbret/retmice reached termination criteria similarly to previous experiments, whereas anti–VCAM-1 and anti–ICAM-1 treatment ameliorated the ataxia symptoms and rescued the mortality of Pdgfbret/ret mice (Fig. 6 B and C). 

Q8. What is the reason for the severe atypical phenotype in Pdgf?

Adoptive transfer (passive) EAE resulted in the same aggravated atypical EAE in Pdgfbret/ret mice as seen in active EAE (Fig. 4C), indicating that the severe phenotype is not due to a pathologically enhanced induction phase in pericyte-deficient mice. 

Q9. What causes the death of pericyte-deficient mice after induction of EAE?

the authors conclude that the mortality of pericyte-deficient mice after induction of EAE is caused by excessive entry of peripheral immune cells into the brain and neuroinflammation.