REVIEWS

Organ-specific protection mediated

by cooperation between vascular
and epithelial barriers
Ilaria Spadoni1, Giulia Fornasa1 and Maria Rescigno1,2
Abstract | Immune privilege is a complex process that protects organs from immune-mediated
attack and damage. It is accomplished by a series of cellular barriers that both control immune
cell entry and promote the development of tolerogenic immune cells. In this Review, we describe
the vascular endothelial and epithelial barriers in organs that are commonly considered to be
immune privileged, such as the brain and the eye. We compare these classical barriers with
barriers in the intestine, which share features with barriers of immune-privileged organs, such as
the capacity to induce tolerance and to protect from external insults. We suggest that when
intestinal barriers break down, disruption of other barriers at distant sites can ensue, and this may
underlie the development of various neurological, metabolic and intestinal disorders.

Inflammatory bowel diseases
(IBDs). Chronic disorders of the
intestine characterized by
severe inflammation and
mucosal tissue destruction.
There are two main forms:
Crohn’s disease, which is a
granulomatous segmental
inflammation affecting any part
of the intestine, and ulcerative
colitis, which is a mucosal
inflammation involving the
rectum and extending for a
variable distance along the
colon.
1
Department of Experimental
Oncology, European
Institute of Oncology,
20139 Milan, Italy.
2
Dipartimento di Scienze
della Salute, Università degli
Studi di Milano,
20122 Milan, Italy.
Correspondence to M.R.
maria.rescigno@ieo.it
doi:10.1038/nri.2017.100
Published online 4 Sep 2017
The original concept of ‘immune privilege’ was coined to
explain the failure of certain tissues to reject implanted
grafts1. Many tissues and organs are considered to be
immune privileged — they include the brain, retina,
testis and feto–maternal interface. However, this concept
of immune privilege has recently been extended to include
sites of immune tolerance induction, such as the intestine,
which is tolerant to food antigens and the trillions of com-
mensal microorganisms2–6, and tumours7. This extended
definition of immune privilege to include the intestine
does not refer to an exclusion of immune cells but rather
to the capacity to avoid the induction of systemic immu-
nity against dietary antigens and the microbiota. In this
respect, immune privilege seems to be a much more
complex process than previously appreciated and can
involve different mechanisms depending on the organ.
It is established by at least two mechanisms: a passive
physical restriction of leukocyte access across vas­cular
barriers, such as the blood–brain barrier (BBB)8,9, and
an active mechanism that involves immune-­regulatory
processes, such as those at the blood–cerebrospinal
fluid (CSF) interface and the intestinal epithelial barrier
(IEB), that ensure the skewing of immune cells towards a
regulatory phenotype.
In this Review, we broaden the model of immune
gates proposed by Shechter et al.10 and highlight the
role of the intestine in establishing immune privilege,
both in the intestine and in other tissues. We analyse
the function of barrier systems, which consist of vas-
cular barriers that restrict the trafficking of immune
cells and of epithelial barriers that are more permissive
to antigens and leukocytes10. In many organs, such as
the intestine and brain, these barriers work together
to form a complex protective layer. This is necessary
because the intestine has to preserve the microbiota
but, at the same time, prevent it from entering the
systemic circulation11. In the central nervous system
(CNS), endothelial and epithelial barriers allow the
immunosurveillance of the CNS without the risk of
perturbing the homeostasis of the brain parenchyma12.
These barriers in the intestine and brain have similar-
ities and differences in their anatomy, properties and
development, and we review recent efforts that have
revealed a functional connection between vascular
barriers at different sites of the body, especially the
intestine and brain. The use of experimental mouse
models, together with genomics, metagenomics and
metabolomics, has shed light on the mechanisms that
underlie barrier development and maintenance and
has highlighted the role of the intestinal microbiota
in shaping the properties of the intestinal and brain
barriers. Indeed, intestinal microorganisms can influ-
ence angiogenesis13,14, modulate intestinal function15,
shape BBB properties and influence behaviour 16–19. The
microbiota is also involved in the establishment of an
efficient vascular barrier in other organs, such as the
testis20 and eye21. Thus, it is evident that tissue barriers
are dynamic during the steady state; however, much of
the knowledge about barrier modifications comes from
disease conditions, such as neurological disorders,
meta­b olic disorders and inflammatory bowel diseases
(IBDs). We first describe the nature of the different

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Meninges
Vascularized tissue membranes
that envelop superficial central
nervous system areas and
enclose the parenchyma.
The meninges are composed of
three layers: the dura mater
(outermost, beneath the skull),
the arachnoid mater and the
pia mater (the innermost layer,
which is proximal to the
parenchyma).
Astrocyte
A type of glial cell that is found
in the vertebrate brain and is
named for its characteristic
star-like shape. Astrocytes
provide both mechanical and
metabolic support for neurons,
thereby regulating the
environment in which neurons
function.
Experimental autoimmune
encephalomyelitis
(EAE). An experimental model
for the human disease multiple
sclerosis. Autoimmune disease
is induced in experimental
animals by immunization with
myelin or peptides derived
from myelin. The animals
develop a paralytic disease
with inflammation and
demyelination in the brain and
spinal cord.
Pericytes
Cells that are embedded in the
vascular basement membrane
of microvessels. They make
close contact with endothelial
cells, and this interaction is
essential for the maintenance
of vessel function, as well as for
the regulation of angiogenesis
and vascular remodelling.
barriers and then examine how several pathologies are
linked to the disruption of these barriers, with a focus
on how barriers in different organs can influence each
other in pathological settings.
The brain barriers
Anatomy of the BBB. The CNS is protected by differ-
ent layers. The outermost barrier, beneath the inner
surface of the skull, is formed by the meninges, namely,
the dura mater, arachnoid mater and pia mater, which
surround the brain and the CSF. This layer is permissive
to immune cell trafficking (FIG. 1a). Underneath the pia
mater, the glia limitans forms a barrier that restricts the
trafficking of T cells. This barrier consists of astrocyte
endfeet and the parenchymal basement membrane that
covers blood vessels and the brain parenchymal surface
(FIG. 1a). Deeper within the brain, two further barri-
ers are present: the BBB, which involves parenchymal
capill­aries, and the blood–CSF barrier (BCSFB), which
is localized at the choroid plexus in the brain ventricles.
These two barriers have different cellular compositions
and permeability characteristics; the BCSFB establishes
a much tighter barrier to soluble tracers and higher elec-
trical resistance than the BBB. They are also character-
ized by differences in permissiveness to immune cells,
conferring diverse degrees of immune privilege to these
regions of the brain22; tissue grafts transplanted into
cerebral ventricles are rejected, but they are not rejected
if transplanted into parenchymal tissue23,24.
The BBB is present across the capillaries and
post-capillary venules of the CNS25, the core element of
which is the blood microvasculature that is composed
of specialized endothelial cells. The endothelial cells of
the BBB are characterized by low rates of pinocytosis, a
lack of fenestrations and continuous intercellular junc-
tional complexes formed by tight junction and adherens
junction proteins that limit the paracellular leakage of
molecules. The passage of molecules that are necessary
for the CNS and for the clearance of toxic agents is medi-
ated by several specific transporters25. However, the infil-
tration of immune cells from the blood into the healthy
brain is limited by the endothelial cells of the BBB owing
to their low expression of leukocyte adhesion molecules
and the production of mediators, such as sonic hedgehog
(SHH), that limit immune cell adhesion, migration and
pro-inflammatory cytokine production26. This special-
ized function of the parenchymal endothelium resides in
the post-capillary venules, the vascular segment in which
the endothelial and parenchymal basement membranes
create a perivascular space that can accommodate rare
antigen-presenting cells (APCs)27–30. Interestingly, CNS-
resident APCs have been shown to be able to sample CNS
antigens and activate CD4+ and CD8+ T cells. The infiltra-
tion of activated T cells into the CNS is restricted during
the steady state31; however, they can cross the BBB in the
absence of neuroinflammation and independent of their
antigen specificity. Activated T cells can enter the CNS
through subpial venules or subarachnoid veins, or they
can reach the lepto­meningeal spaces through the choroid
plexus. If T cells do not recognize their cognate antigen
presented by APCs, they do not cross the glia limitans
and will undergo apoptosis. By contrast, upon antigen
recognition, activated T cells become encephalitogenic
and start to produce pro-inflammatory cytokines that
alter BBB properties and trigger the upregulation of the
expression of chemokines that recruit myeloid cells.
Myeloid cells are responsible for the cleavage of
dys­troglycan at the membrane of astrocyte endfeet, and
this process allows the subsequent infiltration of immune
cells across the glia limitans into the CNS parenchyma and
promotes the development of experimental autoimmune
enceph­alomyelitis (EAE)12, a mouse model of multiple
sclerosis (MS).
Formation of the BBB. Early studies of quail–chick
transplantation demonstrated that the barrier
characteristics of endothelial cells are not intrinsic and
are induced by the CNS environment 32,33. Indeed, newly
formed sprouting vessels express junctional proteins and
nutrient transporters, show high levels of transcytosis
and express leukocyte adhesion molecules 25. The
characteristics of the BBB are induced and maintained by
the interaction of brain endothelial cells with pericytes,
astrocytes and components of the extracellular matrix,
which together are referred to as the neurovascular unit
(NVU) (FIG. 1b).
Formation of the BBB starts when endothelial pro-
genitor cells extend from the perineural vascular plexus
into the embryonic neuroectoderm through a process
that is guided by neural progenitor cells that secrete par-
acrine factors34 such as vascular endothelial growth fac-
tor (VEGF)35 and WNT ligands. Embryonic deletion of
the genes encoding components of the WNT–β‑catenin
signalling pathway is lethal, as endothelial cells fail to
extend into the CNS parenchyma and instead form
large haemorrhagic aggregates that lack expression of
BBB-specific genes34,36. However, postnatal in­­activation
of the β‑catenin pathway in endothelial cells leads to
the reduced expression of claudin 3, a component of
tight junctions, and increased expression of plasma-
lemma vesicle-associated protein (PLVAP), a marker
of vesi­cular transport, and these changes correlate with
vascular leakage and reduced BBB maturation37. Thus,
the canonical WNT–β‑catenin signalling pathway is
fundamental for brain angiogenesis and for BBB matur­
ation. In addition to the β‑catenin pathway, adhesion G
protein-coupled receptor A2 (ADGRA2; also known as
GPR124) is expressed by developing brain endothelial
cells and is necessary for normal angiogenesis and for
barrier development 38–40.
After capillary formation, the second stage
of BBB development is mediated by the interaction of
endo­thelial cells with pericytes and astrocytes, which
promote the ‘sealing’ of the BBB. Mice deficient
in platelet-­derived growth factor receptor‑β (Pdgfrb)
or platelet-derived growth factor subunit B (Pdgfb)
completely lack CNS pericytes, exhibit endothelial cell
hyperplasia and show increased vessel diameter and vas-
cular permeability, which results in micro­haemorrhages
and perinatal lethality 41,42. Moreover, the absence of peri-
cytes during brain development has been associated with
increased expression of markers of a more permeable

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a Meninges and BLMB

Skull bone

Dura mater

Arachnoid mater
Subarachnoid space b BBB: exclusive barrier
BLMB
Neuron
Pia mater
Astrocyte
Glia limitans APOE
Microglia

Tight junction Brain SHH

parenchyma

ANG1

RA

Pericyte

Basement
membrane

Meninges

Tight
junction
Endothelium

Post-capillary Parenchymal c BCSFB: regulatory barrier

Ventricle capillary
Brain parenchyma

CSF Cuboidal Tight Ependymal cells

epithelium junction

Basal membrane

Fenestrated capillary
Choroid plexus stroma

Figure 1 | Cellular components of brain barriers. a | The meninges
(comprising the dura mater, arachnoid mater and pia mater) form the
outermost barrier that protects the brain parenchyma. The meningeal
microvessels in the subarachnoid space, which is located between the
arachnoid mater and pia mater, are composed of a non-fenestrated
endothelium that contains tight junctions but is not ensheathed by
astroglial endfeet. The pia mater, which covers the surface of the central
nervous system (CNS), surrounds these blood vessels to form the blood–
leptomeningeal barrier (BLMB), which is permeable to solutes and
immune cells. b | The blood–brain barrier (BBB) is necessary for brain
homeostasis and neuron preservation, as it controls the transport of
solutes and fluids and the extravasation of immune cells into the CNS
parenchyma. The BBB consists of capillary endothelial cells that are
sealed by complex junctions mainly formed NaturebyReviews
claudin| 5, astrocyte
Immunology
endfeet that encircle the abluminal side of the vasculature and pericytes.
Together, these cellular components of the BBB form the functional unit
called the neurovascular unit (NVU). Neurons and microglia are
considered to be part of the NVU, as they can influence barrier function.
Sonic hedgehog (SHH), angiopoietin 1 (ANG1), apolipoprotein E (APOE)
and retinoic acid (RA) are produced by astrocytes and promote BBB
integrity. c | The cuboidal epithelial cells of the choroid plexus form the
blood–cerebrospinal fluid (CSF) barrier (BCSFB). These epithelial cells are
connected by tight junctions and have a polarized distribution of
numerous transporters. The endothelium of the choroid plexus does not
form a barrier but is permeable to allow the delivery of water from the
blood to the epithelium, which is necessary for CSF production.

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Fenestrated capillaries
A non-continuous vascular bed
characterized by the presence
of pore-like subcellular
structures, or fenestrae, that
are responsible for transcellular
exchange of molecules.
Fenestrated endothelia are
located in the intestine,
pancreas, endocrine glands,
glomeruli of the kidney and
liver sinusoids.
Müller cells
The major type of glial cell that
is found in the retina. These
cells contribute to retinal
homeostasis and function and
regulate the tightness of the
blood–retinal barrier.
Diabetic retinopathy
A condition affecting people
with diabetes that causes
progressive damage to the
retina, the light-sensitive tissue
at the back of the eye.
vascular barrier, such as PLVAP and leukocyte adhe-
sion molecules. By contrast, features of a ‘tight’ barrier,
such as expression of junctional proteins and transport-
ers, are not influenced by a lack of pericytes42,43. Pericyte
coverage is also important for the regulation of BBB
integrity in adults; CNS pericyte depletion during age-
ing leads to a reduction in brain microcirculation and
an increase in neuro­inflammatory responses, which
precipitate changes in neuronal structure and behav-
iour 42–44. An additional role of pericytes in the NVU
is the establishment of astrocyte–­vessel contact and
astrocyte polarization during the postnatal period42,43.
Pericytes induce the polarization of astrocyte endfeet
that enclose neuronal processes and the ablu­minal side
of blood vessels. Moreover, many of the factors secreted
by astrocytes have been identified as promoters of BBB
integrity during postnatal development and in adult-
hood. One of these is SHH, which induces the expression
of junctional proteins, reduces vascular permeability and
promotes immune quiescence by downregulating the
expression of pro-inflammatory cytokines and reduc-
ing leukocyte adhesion and extravasation26. In addition,
astrocytes enhance barrier properties through the secre-
tion of angiopoietin 1 (REF. 45), apolipoprotein E46–48 and
retinoic acid49 (FIG. 1b).
Components of the BCSFB. Unlike the BBB, which is a
true vascular barrier, the BCSFB in the choroid plexus
is an epithelium-based barrier 10 that reflects its ability
to secrete CSF (reviewed in REFS 50–52). The ventri­
cular choroid plexus is lined by a polarized monolayer of
cuboidal epithelial cells that are joined by junctional com-
plexes, which are mainly composed of claudins 1, 2, 3, 9, 11
and 19 (REFS 53–55). The ventricular epithelium surrounds
a network of fenestrated capillaries that are not ensheathed
by astrocyte endfeet 56 (FIG. 1c), unlike the endothelium
in the parenchyma. This permeable endothelium is
necessary for the delivery of water from the blood to the
epithelium for CSF production56 and also for immuno­
surveillance of the CNS10. Immunosurveillance is achieved
by an afferent and efferent connection with cervical and
lumbar lymph nodes, to which parenchymal antigens are
drained (reviewed in REF. 12).
Thus, the CNS is protected by two barriers, namely,
the endothelial BBB and the epithelial BCSFB, that
together provide both passive and active protection.
Similar strategies of protection can also be found in other
organs, such as the eye and intestine, as described below.
The blood–retinal barriers
Immune privilege of the anterior chamber of the eye
is achieved by distinct, overlapping mechanisms of
protection, both passive and active57. The first ele-
ments of protection are a physical separation between
antigens in the eye and the immune system owing
to the blood–­retinal barrier (BRB) and a paucity of
efferent lymphatic vessels; these elements ensure
that the anterior chamber drains into the blood and
not the lymph. The anterior chamber is connected
directly to the blood circulation via Schlemm’s canal,
which is a lymphatic-like vessel58. Accordingly, under
homeostatic conditions, the BRB prevents the entry
of pathogens, allergens and any kind of protein into
the eye. The second element of protection is provided
by a strong anti-inflammatory environment and a
skewed immune response to antigens that prevents
inflammatory reactions.
Anatomy of the BRB. Similar to the barriers of the
CNS, the BRB comprises two physical barriers with
distinct locations, cell compositions and immune-­
skewing properties. The inner BRB, which is located
in the inner layers of the neural retina near the vit­
reous body, is a true endothelial barrier. It is formed by
a specialized non-fenestrated endothelium surrounded
by astrocytes, Müller cells and pericytes, which together
form the retinal NVU (reviewed in REF. 59) (FIG. 2a).
Astrocytes60,61, Müller cells62,63 and pericytes60,64,65 are
thought to contribute to the proper functioning of
the inner BRB. Indeed, in non-primate mammals, the
development of the retinal vasculature starts after birth
when it is immediately ensheathed by pericytes64 and
astrocyte endfeet 61. However, it is still unclear how the
retinal endothelium acquires barrier properties. It has
been shown that although the major cellular compo-
nents of the BRB are in close contact immediately after
birth, the endothelium acquires barrier characteristics
only after 10 days64,65; whether this is a result of micro-
biota colonization of the intestine remains to be estab-
lished. Studies of transgenic animal models in which
pericytes, astrocytes or Müller cells have been depleted
indicate that all these cell types are key regulators of
the induction of BRB properties. For example, mice
that lack pericyte coverage of retinal endothelial cells
show decreased expression of claudin 5 and increased
expression of PLVAP compared with control mice,
which suggests increased paracellular and trans­cellular
transport 64,65. Further evidence for the importance of
pericytes in the maintenance of a functional BRB is
provided by studies on diabetic retinopathy, in which
a reduction in pericyte numbers caused increased
permeability of retinal vessels to injected tracers and
ensuing inflammation65. Indeed, pericyte depletion
induced the expression of pro-inflammatory genes,
such as Tnf, Icam1, Il6 and Ccl2, by endothelial cells,
inducing perivascular infiltration of macrophages
responsible for retinal vessel damage. By contrast,
macrophage depletion in the mouse model of diabetic
retinopathy suppressed retinal oedema and promoted
the recovery of vessel organization65.
In addition to the inner endothelial-based barrier,
the BRB has an outer epithelial layer that is permissive
to transcellular transport. It is composed of the retinal
pigment epithelium, which separates the outer neural
retina from the fenestrated choriocapillaries (FIG. 2b). The
retinal pigment epithelium has an important role in reg-
ulating the homeostasis of the retina, as it is involved
in the transport of glucose, lactose, retinol (which are
necessary for visual function), fatty acids and major
com­ponents of photoreceptors and in the removal of
metabolic waste and water from the subretinal space
(reviewed in REFS 66,67).

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Aqueous
humour

b Outer BRB: regulatory barrier

a Inner BRB: true barrier

Müller cell Rods and cones Sub-retinal

space

Tight
junction
Pericyte Retinal
Basement pigment
membrane epithelial
cell
CD95L PDL1 Galectin 1 TGFβ IL-10
Bruch’s
membrane
Fenestrated
capillary
Tight
Endothelial junction Choroid plexus
cell
Sclera

Figure 2 | The blood–retinal barrier. The neural retina is equipped with barrier systems that regulate ion, water and
Nature Reviews
molecule flux out of the retina. The blood–retinal barrier (BRB) can be separated into two components: | Immunology
the inner BRB
formed by tight junctions that seal neighbouring capillary endothelial cells (part a) and the outer BRB, which has tight
junctions that restrict paracellular trafficking between retinal pigment epithelial cells (part b). Both layers are fundamental
for maintaining the immune-privileged status of the eye and for regulating retinal homeostasis and visual function.
PDL1, programmed cell death 1 ligand 1; TGFβ, transforming growth factor‑β.

Aqueous humour
The clear immunosuppressive
and anti-inflammatory fluid
that fills the anterior chamber
of the eye.
Anterior
chamber-associated
immune deviation
(ACAID). A form of eye-derived
tolerance in which T helper 1
(TH1)- and TH2‑mediated
immunity is suppressed but
non-inflammatory adaptive
immune effectors are present.
Immune regulation by the BRB. In addition to being
a physical barrier to ensure homeostasis of the eye,
the BRB creates an immunosuppressive milieu that
inhibits the activation of immune cells as they cross
the barrier. Hence, similar to the BBB and BCSFB,
the inner and outer BRB function together to pro-
mote immune privilege. The outer BRB also expresses
immunoregulatory molecules that inhibit lympho-
cyte activation, such as transforming growth factor‑β
(TGFβ), cytotoxic T lymphocyte antigen 4 (CTLA4),
thrombospondin 1, programmed cell death 1 ligand
1 (PDL1) and galectin 1, and molecules that promote
apoptosis, such as CD95 ligand (also known as FAS
ligand) and TNF-related apoptosis-inducing ligand
(TRAIL; also known as TNFSF10)10,68,69. The retinal
pigment epithelium of the BRB secretes immuno-
modulatory mediators into the aqueous humour to
create an environment that dampens immune and
inflammatory responses within the eye. The inhibi-
tory factors include macrophage migration inhibitory
factor (MIF), IL‑10, retinoic acid, immuno­suppressive
neuropeptides, such as α‑melanocyte-stimulating
hormone and vasoactive intestinal peptide, and
complement regulatory proteins70–72.
The immune-privileged status of the eye is also
achieved by a systemic component known as anterior
chamber-associated immune deviation (ACAID), which
mediates the downregulation of cellular and humoral
immune responses to antigens that are encountered in
the anterior chamber of the eye73. Specifically, an anti-
gen experimentally injected into the anterior chamber
or released after injury induces a skewed response that
includes the generation of antigen-specific CD4+ and
CD8+ regulatory T cells and non-complement-binding

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Lamina propria
The layer of mucosal tissue
directly under the mucosal
epithelial cell surface in which
effector cells for mucosal
immunity reside.
Goblet cells
Mucus-producing cells found in
the epithelial-cell lining of the
intestine and lungs.
Paneth cells
Specialized enterocytes that
are present at the base of the
crypts in the intestinal
epithelium and that produce
antimicrobial proteins and
peptides, including
phospholipase A2 and
defensins.
antibodies at the expense of potentially destruc-
tive T helper 1 (TH1) cell, TH2 cell and delayed-type
hypersensitivity inflammatory reactions74.
The intestinal barriers
Structural barriers. The gastrointestinal tract is a com-
plex ecosystem that contains the microbiota, which has
evolved together with the host species and established a
mutualistic relationship. The microbiota is required by
the host for harvesting energy from food by de­­grading
complex molecules and for forming a first barrier of
protection against potential pathogens, whereas the
host provides nutrients and a niche to the microbiota75.
Despite this mutualistic relationship, the presence of
the microbiota poses immense health challenges to
the host. Accordingly, several strategies have evolved
in the intestine to protect the host from the threat of
infection and, at the same time, allow the host to toler-
ate the microbiota. These strategies comprise a struc-
tural compartmentalization that minimizes the access
of bacteria into the underlying lamina propria and their
systemic dissemination and a specialized immune bar-
rier that ensures intestinal bacteria and food antigens
are tolerated (reviewed in REF. 76). Defects in these bar-
riers are responsible for microbial infection, IBDs and
food allergies.
The physical exclusion of the intestinal microbiota
from the systemic circulation is achieved by two cellular
barriers, which include some of the same characteristics
that are present in the BBB and BCSFB of the brain and
in the inner and outer BRB of the eye. Just beneath the
intestinal mucus, the thickness of which differs depend-
ing on the location in the intestine77, the first cellular
barrier is provided by a monolayer of enterocytes that
cover the entire mucosa. Similar to the choroid plexus
epithelium, the IEB is a semipermeable barrier that
limits the passage of molecules and microorganisms
through the paracellular route owing to the presence of
tight junctions and adherens junctions between entero-
cytes. Specialized enterocytes, such as goblet cells, which
produce mucus, and Paneth cells, which secrete anti-
microbial peptides, provide additional support for the
maintenance of intestinal barrier function (reviewed in
REFS 75,78) (FIG. 3a).
The second intestinal barrier — the gut–vascular bar-
rier (GVB) — is composed of the intestinal endo­thelium,
which actively blocks haematogenous bacterial dis­
semination5. Intestinal endothelial cells of the GVB share
many key characteristics with other vascular barriers,
such as specialized intercellular junctional complexes
that reduce the paracellular trafficking of molecules and
the expression of different classes of transporters (such as
ATP-binding cassette transporters and sugar transport-
ers)5,79. Similar to the structure of the BBB, endothelial
cells in the intestine are closely associated with pericytes
and enteric glial cells (the intestinal counterpart of brain
astrocytes), which together form the gut–vascular unit 5
(FIG. 3b). The role of these cells in the development and
maintenance of the GVB in healthy and diseased states
has not yet been investigated. However, some studies
suggest that the enteric glial cells have an important role
in preserving intestinal epithelial and endothelial bar-
rier integrity. Transgenic mice that lack enteric glial cells
die from intestinal disease owing to increased epithelial
permeability and microvascular disturbances that result
in the spread of bacteria into the blood circulation80,81.
Intestinal glial cells could support barrier function by
releasing soluble factors, such as S-nitrosoglutathione,
which is known to restore epithelial barrier integrity in
mice that lack enteric glial cells82. Interestingly, it has
been shown that the formation and maintenance of the
network of enteric glial cells in the lamina propria are
guided by the microbiota83. The microbiota is also in­­
dispensable for angiogenesis in the intestine13; therefore,
it is possible that it could influence the GVB directly and
indirectly through enteric glial cells, although this needs
to be formally demonstrated.
Maintenance of GVB integrity, as for BBB integ-
rity 34,36, is regulated by activation of the canonical
WNT–β‑catenin signalling pathway 5. Indeed, mice
in which β‑catenin is constitutively activated in endo­
thelial cells are more resistant to Salmonella infection;
they show reduced systemic bacterial dissemination, a
lack of PLVAP expression, which is a marker of vascular
permeability, and no increase in GVB permeability 5.
Immune regulation at the IEB. Oral tolerance is a state
of mucosal and systemic immune unresponsiveness to
orally ingested food antigens4. This process requires the
uptake of antigens mediated by CX3CR1+ mononuclear
phagocytic cells, the transfer of these proteins to migra-
tory CD103+ dendritic cells (DCs) via the gap junction
protein connexin 43 (also known as gap junction‑α1
protein) (REF. 84) and the migration of CD103+ DCs
to the draining lymph nodes85,86, in which they promote
the differentiation of regulatory T cells that have intesti-
nal-homing properties87. Alternatively, soluble antigens
can be directly delivered from the intestinal lumen to
tolerogenic CD103+ DCs by goblet cells88.
Epithelial cells are also involved in the process of oral
tolerance induction by releasing TGFβ, retinoic acid and
thymic stromal lymphopoietin, which are required for
the generation of DCs with a tolerogenic phenotype89,90.
Food antigens also have access to the intestinal vascula-
ture through which they spread systemically 91. Unlike
soluble antigens, commensal microorganisms must be
excluded from the blood circulation to avoid the devel-
opment of systemic immune activation. In the steady
state, intestinal microorganisms can be found in mes-
enteric lymph nodes (but not in the liver 92) that act as a
firewall to avoid systemic dissemination via the lymph
through the thoracic duct 93.
Regulatory or protective barriers?
On the basis of their structure, cellular components
and anatomical location (parenchyma or periphery),
the barriers described above can be grouped into three
main categories: first, protective barriers consisting of
tightly regulated endothelial layers, such as the BBB and
the inner BRB; second, more permissive endothelial
layers, such as the GVB; and third, immunomodulatory
selective gateways consisting of epithelial cells working

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b GVB
alone, such as the BCSFB, or working together with an
endothelial barrier, such as the IEB with the GVB or the
Enteric
glial cell outer BRB with the inner BRB.
In the steady state, endothelial barriers are considered
to be protective immunological barriers, as they physi-
cally block leukocyte migration. The BBB endothelium
suppresses parenchymal invasion by leukocytes through
Pericyte
Basement the production of mediators such as IL‑25, which
membrane induces the upregulation of junctional components and
a IEB the downregulation of pro-encephalitogenic cytokines94.
Fenestrated In addition, parenchymal vessels do not express the
Lumen endothelium
ad­­hesion molecule P‑selectin, thereby reducing leuko-
Tight cyte recruitment 95, or express CXC-chemokine ligand
junction 12 on the abluminal side, thereby retaining mono-
Intestinal
nuclear cells in the perivascular space96. By contrast,
microbiota immunosurveillance occurs in the choroid plexus,
where the BCSFB allows the migration of CD4+ effec-
tor memory T cells specific for CNS antigens97. In addi-
tion, and similarly to other epithelial barriers such as
the IEB, the BCSFB can skew immune cells towards
a regulatory phenotype owing to the production of
Mucus TGFβ, vaso­active intestinal peptide98, prostaglandin D2
layer (REF. 99) and IL‑1 receptor antagonist (IL‑1RA)100. Cells
in the choroid plexus, as in the intestine87,101,102, express
Goblet the enzyme indoleamine 2,3‑dioxygenase (IDO), which
cell is responsible for tryptophan catabolism, and retinoic
acid metabolic enzymes, which are involved in the inhi-
Epithelial
bition of T cell activity and the induction of regulatory
cell T cells at the barrier interface10. In the eye, similar prop-
Capillary
Tight junction network erties of immune cell exclusion apply to the inner endo­
thelial BRB, and the ability to skew immune responses is a
CX3CR1+ DC
feature of the outer BRB10.
GSNO For all the barriers described above, the loss of func-
tionality and leukocyte invasion into the correspond-
CD103+ DC
? ing parenchyma can lead to pathological conditions10.
However, it is worth noting that immune cell migra-
Basement
membrane RA Enteric tion into an immune-privileged site is not harmful in all
glia Arteriole cases. Indeed, mice lacking adaptive immune cells show
Treg cell Venule Nerve marked impairments of hippocampal neuro­genesis,
TGFβ TSLP memory and spatial learning abilities103. The T cells
responsible for hippocampal plasticity recognize CNS-
derived autoantigens103, indicating that the BBB may
Lymphatic
vessel be less restrictive than originally thought. Lymphocyte
Lamina propria
patrolling of the CNS is crucial for controlling viral
infections, as illustrated by the failure of the clinical
Figure 3 | The intestinal barriers. In the intestine, epithelial-
Natureand endothelial-based
Reviews | Immunology trial testing the α4β1 integrin (VLA4)‑specific antibody
barriers can be identified. a | The intestinal epithelial barrier (IEB) is the first layer of
defence that prevents the uncontrolled translocation of commensal bacteria from natalizumab in patients with MS104. Natalizumab has
the intestinal lumen into the lamina propria. Goblet cells, which produce the mucus robust beneficial effects on MS disease activity by pre-
layer, and Paneth cells (not depicted), which secrete antimicrobial peptides, give venting extravasation through the BBB of lymphocytes
additional support to the maintenance of mucosal barrier function. A rim of tight expressing α4β1 integrin or α4β7 integrin. However,
junctions seals the epithelial barrier and controls paracellular trafficking. In the by also blocking the recruitment of virus-specific
lamina propria, the mucosal immune system participates in the induction of oral cytolytic cells, treatment with natalizumab has been
tolerance. b | Blood capillaries are found just below the intestinal epithelium. They associated with an increased risk of JC virus infec-
consist of endothelial cells that express junctional proteins to form the gut–vascular tion and the development of progressive multifocal
barrier (GVB). Closely associated with the endothelial cells are enteric glial cells and leukoencephalopathy 104.
pericytes, which together form the gut–vascular unit. Enteric glia cells are involved
Given these examples, it is important to high-
in preserving the integrity of the epithelial and endothelial barriers through the
release of S‑nitrosoglutathione (GSNO). In the healthy state, the GVB controls the light that barrier classification as epithelial or endo­
type of antigens that translocate into the systemic circulation and prohibits the thelial in some cases could be too general, as barrier
entry of the intestinal microbiota. DC, dendritic cell; RA, retinoic acid; TGFβ, systems could work differently depending on their
transforming growth factor‑β; Treg cell, regulatory T cell; TSLP, thymic stromal function and location. For example, size exclusion by
lymphopoietin. the brain and by intestinal vascular barriers is different.

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Pyramidal neurons The BBB has a size exclusion threshold of 500 Da, pyramidal neurons107. The same disorders can also affect
Type of nerve cells with a whereas the GVB is permeable to molecules as large the BRB in pigs108. Increased permeability of the BRB is
pyramidal-shaped cell body as 4 kDa. This probably reflects the need of the GVB to associated with a leak of plasma components into the
(soma) and two allow the passage of nutrients and antigens for tolerance retina, leading to IgG deposition in the ganglion cell
distinct dendrite trees (the
longer apical and the shorter
induction. In addition, the GVB can afford to be less layer 108. Together, these reports indicate that vascular
basal trees). They are present stringent than the BBB because the first intestinal barrier barriers in different regions of the body are linked and
in most mammalian forebrain is provided by the epithelial layer that directly faces the that they may be disrupted under the same pathological
structures (cerebral cortex, intestinal lumen. conditions, suggesting common mechanisms of
hippocampus and amygdala),
Another degree of complexity is created by the influ- maintenance and protection.
and they are associated with
advanced cognitive functions.
ence of the environment on the barriers — for example, What drives the initial breakdown of vascular barri-
the interplay between the microbiota and the intestinal ers remains to be established, but the observation that
barriers (FIG. 4). Germ-free mice, which lack a normal the IEB is affected in several disorders raises the possi-
intestinal microbiota, have an impaired BBB both in bility that a disturbed epithelial barrier (‘leaky gut’) may
fetal and adult phases owing to a dysregulation of junc- be responsible for vascular barrier breakdown. This is
tional occludin and claudin 5 expression16. However, particularly evident in the intestine, where we demon-
this defect in BBB tight junctions can be repaired strated that pathogens that are capable of disrupting the
when mice are re‑colonized with the microbiota from IEB, such as Salmonella enterica subsp. enterica serovar
mice raised in specific pathogen-free conditions or Typhimurium, can also disrupt the GVB. However, the
are monocolonized with bacteria that produce short- capacity of S. Typhimurium to dismantle the GVB and
chain fatty acids (SCFAs)16. The exact mechanisms by disseminate systemically is an active mechanism that is
which the microbiota affects BBB maturity and function used by the bacteria itself and that depends on its patho­
remain unknown. One possibility is that the intestinal genicity island 2 (REF. 5). Thus, IEB disruption alone is
microbiota influences the IEB and/or the GVB, favour- not sufficient for the microbiota to enter the systemic
ing the translocation of bacteria-derived metabolites105 circulation through the GVB.
into the blood and thus influencing more barriers at A series of reports has shown that patients with
once. Additionally, the microbiota might modulate both autism spectrum disorders (ASDs) experience gastro­
intestinal barrier permeability and brain development intestinal discomfort, which was initially thought
through neurotransmitters (such as serotonin)106. to have a neurobehavioural aetiology rather than a
gastro­e nteric aetiology 109–111. However, subsequent
Barriers and diseases studies have shown that gastrointestinal symptoms in
Neurological disorders.Chronic metabolic disorders individuals with an ASD are associated with mild lev-
associated with nephropathy or retinopathy, such as els of mucosal inflammation112,113. Assessment of intes-
diabetes mellitus or hypercholesterolaemia, can result tinal permeability identified that a high percentage
in an increase in BBB permeability in the cerebral of patients with ASDs (36.7%) and their first-degree
cortex and a progression towards Alzheimer disease, relatives (21.2%) presented with increased permea-
concomitant with deposition of amyloid-β peptide in bility compared with healthy children (4.8%), which
supports the hypothesis that a disturbed IEB and/or
GVB is a distinctive feature of patients with ASDs114.
Gut microbiota
These reports suggest that modifications of the IEB
are linked to dis­orders affecting sites that are far from
Barrier homeostasis the intestine. In this context, both animal and human
studies support a connection between increased intes-
tinal permeability and neurological disorders, includ-
ing ASDs, schizophrenia, Parkinson disease and
GVB and IEB BBB BRB Alzheimer disease. In particular, a post-mortem study
• Tight junction expression • Tight junction expression • Neovascularization has shown that the expression of several genes associ-
modulation modulation • Immune cell recruitment ated with tight junctions is dysregulated in the brains
• Formation of the enteric • Barrier permeability and activation of patients with ASDs and schizophrenia. In particular,
glia network regulation • Establishment of tolerance
• Angiogenesis and vascular • Microglia activation levels of mRNAs encoding claudin 5 and claudin 12 are
remodelling • Modulation of behaviour upregulated in the brain cortex of patients with ASDs
• Modulation of barrier
permeability compared with those of patients with schizophrenia
• Establishment of oral and and healthy controls, whereas claudin 3 expression is
mucosal tolerance upregulated both in patients with schizophrenia and in
those with ASDs115. Expression of the matrix metallo­
Figure 4 | Impact of the microbiota on physiological barrier functions.
Nature ReviewsThe intestinal
| Immunology proteinases MMP2 and MMP9, as well as of trans­
microbiota can affect the physiology and barrier functions in the intestine and in distant
locator protein (TSPO), is also upregulated in patients
organs. In the intestine, it reduces intestinal permeability, induces the establishment of
the gut–vascular unit necessary for correct functioning of the gut–vascular barrier (GVB) with ASDs, whereas tissue-type plasminogen activa-
and enhances mucosal immunity and oral tolerance. At distant sites, such as the tor (tPA) and allograft inflammatory factor 1 (AIF1;
brain and eye, it is involved in modulating vascular barrier permeability and immune also known as IBA1) are downregulated in patients
cell recruitment and activation. BBB, blood–brain barrier; BRB, blood–retinal barrier; with schizophrenia. Together, these data suggest that
IEB, intestinal epithelial barrier. neuro­inflammation (indicated by increased MMP9

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Dysbiosis
A condition in which the
balance of the bacterial
communities that constitute
the intestinal microbiota is
altered. Dysbiosis may be a
predisposition factor for
several diseases.
Lewy pathology
A pathology characterized by
the presence of abnormal
α‑synuclein aggregates (bodies)
in the residual neurons of the
substantia nigra and in the
enteric nervous system. Lewy
bodies are the histological
hallmark of Parkinson disease.
Microglia
Phagocytic cells of myeloid
origin that are involved in the
innate immune response in the
central nervous system.
Microglia are thought to be the
major brain-resident
macrophages.
Autophagy
A specialized process involving
the degradative delivery of a
portion of the cytoplasm or of
damaged organelles to the
lysosome. Internalized
pathogens can also be
eliminated by this pathway.
Genotype
BBB
• Anxiety and depression
• Autism spectrum disorders
• Parkinson disease,
Alzheimer disease and
schizophrenia
Figure 5 | Effect of dysbiosis and barrier failure on disease onset. Many genetic
and environmental factors affect the composition of the Nature
Dysbiosis has been linked to barrier impairments and the development of diseases,
including anxiety and neurological disorders, metabolic disorders and autoimmune
diseases. Several studies have reported an association between these diseases and
increased intestinal permeability that, in turn, may lead to dysregulation of the
blood–brain barrier (BBB) or blood–retinal barrier (BRB). GVB, gut–vascular barrier;
IEB, intestinal epithelial barrier.
and TSPO expression) in patients with ASDs results in
compensatory mechanisms that aim to increase barrier
integrity (indicated by increased claudin 5 and claudin
12 expression at the mRNA level)116. However, protein
levels of claudin 12 do not increase in patients with
ASDs, suggesting a failure to increase barrier tight junc-
tions. The expression of tight junction proteins is also
dysregulated in the intestine of patients with ASDs com-
pared with healthy controls, which is consistent with the
leaky gut hypothesis115. Similar data have been obtained
from a mouse model of obesity, in which it was shown
that anxiety-like behaviour was linked to an impair-
ment of intestinal barriers, increased expression of
neuro­inflammatory markers (such as MMP9, AIF1 and
Toll-like receptors 4 and 2) in the prefrontal cortex and
reduced expression of BBB junctional proteins (such as
claudin 5 and tight junction protein ZO1)117. Together,
these results indicate that ASD is often accompanied
by BBB and IEB defects, linking these two barriers in a
gut–brain axis. Given that most of these studies assessed
the expression of tight junction proteins in the intestine
without separating epithelial cells from endothelial cells,
it is not clear whether patients with ASDs have a defect
in the GVB as well as in the IEB.
What could be the implications of an impaired IEB
or BBB in the development of these disorders? Defects
in the IEB that may be due to microbiota dysbiosis may
also lead to alterations in the GVB and subsequent
abnormal translocation of nutrients, microbial prod-
ucts or even microorganisms into the systemic circula-
tion (FIG. 5). These intestine-derived factors may affect
the BBB. Alternatively, because the different vascular
Stress Diet Antibiotics Inflammation
Gut microbiota
Dysbiosis
Barrier function impairment
GVB and IEB BRB
• Metabolic disorders • Type 1 diabetes or
• Autoimmune disorders hypercholesterolaemia-
(coeliac disease) associated retinopathy
• Inflammatory bowel diseases • Autoimmune uveitis
• Food allergies
Reviews
intestinal | Immunology
microbiota.
barriers share common pathways for their formation
and maintenance, genetic defects or environmental
triggers affecting one of the pathways involved in bar-
rier formation may concomitantly affect several barri-
ers in the body. This may explain why diabetes-related
nephropathies and retinopathies are often associated
with BBB disruptions107. A relationship between the
intestinal microbiota, IEB permeability and ASDs has
been shown in a mouse model of maternal immune
activation-induced experimental autism118. Changes
in the microbiota due to inflammation during preg-
nancy were suggested to cause intestinal barrier defects
in the offspring that are responsible for ASD develop-
ment. Oral administration of the commensal bacteria
Bacteroides fragilis could correct IEB permeability and
microbiota alterations, leading to reduced symptoms
of ASDs118. Moreover, studies in the context of EAE
have demonstrated that the intestinal microbiota can
influence neurological inflammation119.
Colonic inflammation has been reported in patients
with other neurological disorders such as Parkinson dis-
ease, independent of the presence of an enteric Lewy pathol-
ogy120. Interestingly, patients with Parkinson disease show
α‑synuclein aggregation in neurons of the gastro­intestinal
tract, suggesting that the disease pathology arises in the
intestine; this pathology does not seem to be a conse-
quence of inflammation, as patients with IBD did not dis-
play similar aggregation patterns121. Further evidence for
the progression of Parkinson disease pathology from the
intestine to the brain is provided by a recent report show-
ing active retrograde transport of aggregated α‑synuclein
via the vagal nerve (which links to the intestine) to the
dorsal motor neuron122. This begs the question of how
and why α‑synuclein aggregates in the intestine and
whether α‑synuclein aggregation is linked to increased
per­m eability of the IEB. Studies of α‑synuclein-­
overexpressing (ASO) mice, which present a model
of Parkinson disease, confirm a role for the intestinal
microbiota in the disease123. ASO mice raised in germ-
free conditions show reduced signs of motor neuron
impairment123. It is likely that this effect of the micro­biota
occurs during a postnatal phase, as germ-free ASO mice
that are recolonized with a normal micro­biota display
features of Parkinson disease, and antibiotic treatment
of adults reduces symptoms of Parkinson disease123. Most
of the Parkinson disease-inducing effects of the micro­
biota can be recapitulated by microbiota-­derived SCFAs.
SCFAs have been shown to be important for the matura-
tion of brain mononuclear phagocytes, known as micro-
glia124. SCFAs may therefore cross the BBB and induce
the maturation of microglia, which are then responsi-
ble for mediating neuroinflammation and α‑synuclein
aggregation. The microbiota or its meta­bolites may
also affect autophagy, thereby impeding the clearance of
α‑synuclein aggregates123. A similar acceleration of the
disease was observed when the microbiota from patients
with Parkinson disease was transferred into germ-free
mice, suggesting that dysbiosis leads to disease progres-
sion, presumably through the release of SCFAs123. An
important question that arises is whether this dysbiosis
also promotes increased permeability of the IEB and/or

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GVB (maybe through altered expression of as‑yet-un-
known receptors) and translocation of SCFAs and other
microbiota-derived metabolites into the bloodstream.
It is unlikely that this effect on the barrier is due to SCFAs,
as they have been shown in vitro to increase epithelial
integrity rather than increase permeability 125.
An additional mechanism to account for diseases
mediated by defective vascular barriers may relate
to nutrient transport. A recent report has shown that
impaired amino acid transport at the BBB may partic-
ipate in ASD development 126. Mice lacking the solute
carrier transporter Slc7a5, a neutral amino acid trans-
porter, specifically in endothelial cells have an imbalance
of amino acid transport into and out of the brain and
an accumulation of histidine in the brain that may be
involved in causing ASDs126. Histidine may affect the
metabolism of brain immune cells. Loss-of-function
mutations in this amino acid transporter have been
described in some families with ASDs126. This mutation,
when present, occurs in every vasculature in the body;
therefore, a role for amino acid transport may also be
relevant at other endothelial barriers, in particular in
the intestine, for the translocation of amino acids into
the blood.
Metabolic and autoimmune disorders. An increase in
intestinal permeability has been observed in metabolic
and autoimmune disorders and seems to precede overt
disease. In type 1 diabetes (T1D), for example, patients
with preclinical evidence of the disease also show
increased intestinal permeability, which suggests that
diabetes occurs in association with an entero­pathy 127,128.
The same is true in spontaneous rodent models of dia-
betes129. In addition, there is a clear correlation between
coeliac disease and T1D130. It has been postulated that
in the event of higher permeability of the IEB, dietary
antigens may cross the epithelium, become immuno-
genic and cause autoimmunity 131. Another possible
consequence of increased intestinal permeability is
increased translocation of bacterial products or constit-
uents that may be inflammatory, thereby inducing sys-
temic inflammation. However, what drives the increase
in intestinal permeability is not clear. Dysbiosis has
been associated with T1D and is character­ized by an
under-representation of taxa that produce SCFAs132.
Special diets that release SCFAs (either acetate or
butyrate) have been shown to provide partial pro-
tection against T1D, and full protection is conferred
by diets containing both acetate and butyrate132. The
mechanism of action of butyrate involves the induction
of regulatory T cells, whereas that of acetate involves
a reduction of autoreactive T cells133. Although the
combination of acetate- and butyrate-containing diets
promotes intestinal integrity 133, protection against T1D
is most likely a result of the barrier-­protecting effect
exerted by acetate134.
An indication that T1D may be associated with vas-
cular impairment comes from the compromised integ-
rity of the BRB in patients with T1D135, which leads to
increased frequencies of retinopathies, particularly in
individuals with pregnancy-induced hypertension136.
It would be interesting to determine whether vascular
barriers are affected by dietary intervention to reduce
diabetic complications.
Metabolic disorders are also linked to other dis-
eases. Major depressive disorder has comorbidities
with type 2 diabetes mellitus, irritable bowel syndrome
and obesity 137. All these diseases have been associated
with microbiota dysbiosis, reduced physical activity,
poor diet and a leaky gut (FIG. 5). Microbiota dysbiosis
may lead to the release of metabolites that may affect
IEB and GVB permeability, on the one hand, and the
enteric nervous system, on the other, with a direct effect
on the CNS138. In both cases, the effects propagate to
the whole organism and may lead to comorbidities. One
interesting hypothesis is that the regulatory function of
the IEB is lost during dysbiosis and that this induces a
dysregulation of the GVB. Finally, the microbiota or its
metabolic products may access systemic sites, thereby
contributing to low-grade systemic inflammation and
leading to several disorders or comorbidities, depending
on the genetic predisposition of the affected individual.
Intestinal disorders. IBDs are associated with micro-
bial dysbiosis139, altered bile acid transformation140 and
submucosal bacterial translocation141. Nearly 30% of
patients with IBDs have extra-intestinal manifestations
of the disease, including arthropathies, cutaneous and
ocular manifestations and liver and biliary dis­orders142.
Given that increased epithelial permeability is asso-
ciated with metabolic and/or neurodegenerative dis­
orders, it is not clear why extra-intestinal manifestations
do not occur in all patients with IBDs. It is likely that,
in most of the cases, the GVB functions normally and
can therefore completely or partly prevent the trans­
location of microorganisms or their metabolic products
to systemic organs despite a disrupted IEB. The opposite
may be true in patients with coeliac disease, who show
increased serum levels of liver transaminases even when
on a gluten-free diet 5. This suggests that liver damage
occurs owing to an impaired GVB even in the absence
of damage to the IEB.
Conclusions and perspectives
In this Review, we describe how endothelial and epi­thelial
barriers often cooperate to establish immune privilege.
Endothelial barriers can be more or less stringent depend-
ing on the organ that they protect. At body sites that are
classically considered to be immune privileged, barriers
tend to exclude immune cells from aberrantly entering
the organ (such as the BBB in the brain and the BRB in
the eye). Less stringent barriers, such as the GVB, are not
meant to protect the organ from leuko­cyte migration but
rather to avoid the dissemination of unwanted molecules
to the whole organism. In other words, the GVB protects
the whole body rather than the intestine. Protection of
the intestine is guaranteed by the mucus layer and the
epithelial barrier. The IEB has another important func-
tion in promoting the establishment of tolerance, thereby
acting as an immuno­regulatory gate. Hence, the whole
body can be considered to be a series of protective barri-
ers: the IEB, which protects the body from the microbiota

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but also tolerates it; the GVB, which protects the body
from unwanted molecules that cross the IEB; and the
other vascular barriers of the body, which protect classical
immune-privileged organs. These organs have additional
regulatory gates that promote immune tolerance and
avoid inflammatory responses by leukocytes and thereby
ensure organ homeostasis and proper development.
In several diseases, the IEB and possibly the GVB are
compromised, allowing leakage of unwanted molecules
into the circulation and perhaps causing damage to the
other barriers. Knowledge of how barriers are formed
and disrupted may lead to the identification of new
thera­peutic targets to re-establish the epithelial barrier,
the endothelial barrier or both. For example, patients
with coeliac disease who, even after following a gluten-­
free diet, experience liver damage (as indicated by
high blood transaminase levels) may benefit from the
administration of drugs aimed at restoring the GVB.
Similarly, patients with ASDs or ‘high-risk’ individ­
uals with increased intestinal permeability may benefit
from treatments aimed at restoring the IEB and GVB
to both treat and prevent their disorders. In addition,
future research aimed at understanding the mechanisms
controlling intestinal barriers may help to determine the
mechanisms of barrier formation at other sites and vice
versa. Attention should be focused particularly on the
microbiota and its postbiotics and on the WNT and
SHH signalling pathways.

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Acknowledgements
This work is supported by the European Council of Research
(Consolidator grant HomeoGUT, 615735), the Italian
Association for Cancer Research (AIRC) and the Italian
Ministry of Health (Ricerca finalizzata). G.F. is supported by
the Italian Ministry of Health (grant GR‑2013‑02359806)
and by Fondazione Cariplo (grant 2016–0472).
Author contributions
I.S. and M.R. wrote the article and reviewed and edited the
manuscript before submission. G.F. contributed to research-
ing data for the article and reviewed and edited the manu-
script before submission.
Competing interests statement
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

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