Introduction to neuroimmunology

(repetita juvant)
Roberto Furlan, San Raffaele Scientific Institute

Giano bifronte. Testa fittile da Vulci, II sec. a. C.

(Roma, Museo Nazionale Etrusco di Villa Giulia).
Rabat, September 27th 2023
“Immune privilege is a characteristic of specialized tissues
and sites in the body. Immune-privileged sites allow foreign
grafts to survive for extended, often indefinite intervals,
and immune-privileged tissues survive for extended, often
indefinite intervals at conventional sites.”
Body sites and tissues that are immune privileged
Sites Tissues
Eye: cornea, anterior chamber Eye: cornea, lens, pigment
vitreous cavity and subretinal
space epithelium and retina
Brain: ventricles and striatum Brain and spinal cord
Pregnant uterus Placenta
Ovary Ovary
Testis Testis
Adrenal cortex Liver
Hair follicles
Hamster cheek pouch Hamster cheek pouch
Certain tumours Certain tumours

Modified from: Nature Rev.Immunol. 2003; 3:879

van Dooremaal JC. Die Entwicklung der in
fremden Grund versetzten lebenden Gewebe.
Albrecht Von Graefes Archiv von Klinische und
Experimentelle Ophthalmologie 19:358-373,
1873.

Medawar, P. Immunity to homologous grafted

skin. III. The fate of skin homografts
transplanted to the brain, to subcutaneous
tissue, and to the anterior chamber of the eye.
British Journal of Experimental Pathology. 129,
58–69, 1948.
 (auto)immune reaction in the CNS

Auto-Antigen loco-regional lymph node

Dendritic lymphatics
cell
costimulus
inflammation!
Naïve T
Induction site lymphocyte

drugs

oligo
Activated
M A N T lymphocytes

blood stream
Target organ
 Basis of immune-privilege

• Anatomy (lymphatic vessels, blood

vessels)
• MHC expression
• Clearance of infiltrating cells
• Regulation
 Synopsis
Immune-privilege in the CNS:
• Anatomy
• MHC expression
• Cell clearance
• Regulation
• Immunological properties of
neural cells
• Cytokines in the CNS
 The brain as immune
privileged site
• The CNS is not drained by a lymphatic
vasculature.
• The CNS parenchyma is separated from the the
blood stream by the BBB and brain endothelium
expresses low levels of adhesion molecules.
• MHC expression is absent or very low in CNS
resident cells (restricted to perivascular and
meningeal macrophages).
• Clearance of CNS-infiltrating lymphocytes
during an immune-mediated pathological
process is very efficient.
MS HSV-e

MS MS
 The Immune-privileged
Status of the Brain is
not Absolute
• CNS is not drained by a fully developed
lymphatic vasculature:
• There are no classical lymphatic vessels in the brain
but there are other routes to channel CNS interstitial
fluid into lymph and blood circulation (Bradbury et al.,
1980; Widner et al., 1988; Cserr et al., 1992)
 CSF/Brain-Lymph Node Routes
• Brain-lymph nodes (cerebral interstitial fluid):
via cerebral perivascular spaces plus
passage from subarachnoid space of Brain CSF
olfactory lobes into submucous spaces of
nose and thus to lymph (Bradbury et al.,
1981).
• CSF-lymph nodes (CSF circuit): via
prolongations of subarachnoid space around
Regional
the olfactory nerves leading into the Blood
Lymph Nodes
interstitial spaces of the nasal submucosa
(Bradbury and Westrop, 1983).
• Antigen-specific responses occur in the deep
cervical lymph nodes after intracerebral
antigen deposition (Widner et al., 1988).
• Antigens deposited in the lateral ventricles Spleen
drain preferentially to the blood with a high
response in the spleen (Widner et al., 1988).
 Indian Ink: Interstitial Fluid Bulk
Flow along the Perivascular route

Injection

MCA
Perivascular Bulk Flow
Routes for Interstitial Fluid Tracer

Artery

Capillary

Tracer

(Roxana Nnadi 2002)

 Different Functions for the Perivenular:Perivenous spaces
and the Pericapillary:Periarterial spaces
Vein
Artery

Entry of Lymphocytes
into CNS
Apoptosis

Drainage of
Interstitial
Fluid Capillary Venule

Courtesy of Prof. Roy Weller

 The Immune-privileged
Status of the Brain is
not Absolute
• CNS parenchima is separated from blood
circulation by an unusually tight
endothelium forming the BBB:
• The endothelial BBB is tight but not tight enough to
let pass at least some immune cells (Hickey, 1991);
• Activated T cells can readily transverse the BBB
irrespective of their antigen specificty (Wekerle et al.,
1991; Martino et al, 1994).
The blood-brain barrier:
- does not allow the passage of
macromolecules such as antibodies
The brain endothelium:
- expresses low levels of adhesion
molecules making trafficking of
celle vs. the CNS difficult
 Different Functions for the Perivenular:Perivenous spaces
and the Pericapillary:Periarterial spaces
Vein
Artery

Entry of Lymphocytes
into CNS
Apoptosis

Drainage of
Interstitial
Fluid Capillary Venule

Courtesy of Prof. Roy Weller

Eur. J. Immunol. 2004. 34: 2955–2963
Eur. J. Immunol. 2004. 34: 2955–2963
1955
Continuity of CSF with
Brain
Woollam & Millen 1955

1995
No Continuity of CSF
with Brain:
CSF and ISF drainage
pathways are separate
Gray’s Anatomy 1995
 J Anat. 2015 Nov;
227(5):702-3

First described by Paolo Mascagni in «Vasorum lymphaticorum corporis

humani historia et ichnographia» (1787).

Lmyphatics of the dura mater follow blood vessels exiting … through the “foro
spinoso” … of the skull. They join those coming from pterigoideal muscles and
go to the (lymphatic) glands in proximity of the internal jugular vein.
 first step in neuroinflammation (postcapillary venules): the passage of T cells
and macrophages across the vascular wall is not necessarily related to pathology

astrocyte endfeet isolate

the vascular
compartment (vessels
and perivascular spaces)
from the brain
parenchyma

Ransohoff RM,
Engelhardt B. Nat Rev
Immunol 2012;12:623

3 compartments with 7 layers separate the blood (red) from the neuropil
endothelium (grey ring), media (light blue), Virchow–Robin space (orange), glia limitans (green),
inner and outer vascular basement membranes (1 and 2, mid-blue), and the basement
membrane on top of the glia limitans (3, light blue)
A, astrocyte; B, blood; E, endothelium; GL, glia limitans; M, media; MФ, macrophage; P, pericyte; PC, perivascular cell; T, T cell; VRS,
Virchow–Robin space
First step in neuroinflammation
 second step in neuroinflammation: it depends on ‘vessel-associated’ antigen-
presenting cells (JM), and ectoenzymes (MMP-2, MMP-9) that cleave components
of the third basement membrane
(perivascular restimulation of T cells is required for the induction of such ectoenzymes by T
cells or macrophages); it is possibly related to pathology

A, astrocyte; B, blood; E, endothelium; GL, glia limitans; JM, juxtavascular microglia; M, media; MФ, macrophage; P, pericyte; PC,
perivascular cell; T, T cell; VRS, Virchow–Robin space.
The Immune-privileged
Status of the Brain is
not Absolute
• Expression of MHC antigens is
very low in the CNS
• Brain cells do not normally
express MHC products which
can, however, be induced by
strong proinflammatory stimuli
(Wekerle et al., 1986: Lampson,
1987).
 MHC Expression in
the Brain
• MHC-antigen expression:
• Normal conditions: no MHC expression
• Mild inflammation: microglia (Class I and II)
• Severe inflammation: neuroectodermal cells
such as astrocytes and ependymal cells (Vass
and Lassmann, 1990)

Block of electrical activity in neurons

induces MHC-expression
(Neumann et al, 1995 and 1996)
 Electrically
silent neurons
treated with
IFN-g express
MHC class I

Neumann et al.,
J Exp Med. 1997 185:305
 Immune-privilege in the CNS: clearance
R5
of infiltrating inflammatory cells
R5

R1
R5
R1 R1

File: 28/5/04.017 #5File: 28/5/04.010 #1 File: 28/5/04.011 #2

File: 28/5/04.018 #6 File: 28/5/04.012 #3
#4
Quad % Gated % Quad
Total % Gated % Total Quad % Gated %
Quad
Total % Gated % Total Quad % Gated % Total
% Total
UL 14.59 UL
8.10 5.26 0.08 UL 14.87 2.26
8.68 UL 19.43 14.13 UL 4.37 1.32
UR UR
61.52 34.16 40.53 0.63 UR 34.61 5.27
27.02 UR 55.38 40.28 UR 6.91 2.09
LL 4.81 LL
2.67 23.95 0.37 LL 24.85 3.78
2.76 LL 4.57 3.33 LL 37.15 11.22
LR LR
19.08 10.59 30.26 0.47 LR 25.67 3.91
5.84 LR 20.62 15.00 LR 51.57 15.58
 Signaling to microglia
Chemokine receptors
CCR2, CCR3, CCR5, CXCR4, CX3CR1

Death receptors/ligands
Fas (CD95)/FasL (CD95L)

Complement receptors
CR1, CR3, CR4, C1qRp

Fc receptors
FCg, RI, RII, RIII

Other receptors Microglia: morphology

EP2, PPAR-g These cells are small in size with a few
highly branched processes. They are
Cytokine receptors believed to be derived from
IFNgR, TNFRI, TNFRII, IL-1RI, IL-12R, IL- monocytes which enter the CNS
15R, IL-18R, IL-4R, IL-10R, IL-13R, TGFbR during development. Microglia in the
injured or diseased CNS express
Prostaglandin receptors properties characteristic of an
CD40, OX2R/CD200R antigen-presenting cell and participate
in CNS immune responses.
Pattern recognition receptors
CR3 (lectine site), mannose R, CD14/TLR4
 Microglia-derived inflammatory and
immunoregulatory mediators

Pro-inflammatory cytokines
IL-1, IL-6, IL-12, IL-15, IL-18

Chemokines
IL-8, IP10, MIP-1a, MIP-1b,
MCP-1, Rantes, MDC, MIP3-b

Cytotoxic molecules
iNOS, nitric and oxigen radicals

Microglia are the primary phagocytes of

Prostanoids
PDG2, PGE2, Tromboxane B2
Anti-inflammatory cytokines
IL-10, TGFb, IL-1ra
the brain and take part in inflammatory
reactions. Here a microglial cell (M)
exhibits dense phagocytosed particles in
its cytoplasm (arrow). Its morphology is
different from those of astrocytes (A) and
oligodendrocytes (O) in the field.
 Astrocytic-derived inflammatory and
immunoregulatory mediators
Pro-inflammatory cytokines
IL-1, IL-6 (a), TNFa The cell body of an
astrocyte spans 10-20
Growth factors µm and its processes
radiate out for another
NGF, FGF-II, EGF, LIF, NT-3, GMF, 20-30 µm, forming the
CNTF, PDGF, FGF-R, IFGF (a), stellate shape of the
astrocyte
EGF-R (a)

Cytotoxic molecules
iNOS (a), GDN (a), oxidoreductive
enzymes (a), cathepsin D and G (a)

Recognition molecules
PSA-NCAM, NCAM (a), APP (a), G-CAM
(a), integrin, N-cadherin, laminin, tenascin
(a), proteoglycans, H-CAM (a), VCAM-I,

Astrocytes in rat hippocampus (blu) in

Anti-inflammatory cytokines close contact with synapses
TGFb (a)
Immunological features of neurons

MHC-antigen expression in neurons:

•Normal conditions and mild inflammation: no MHC expression
•Severe inflammation inducing block of electrical activity: MHC-
expression
 Cytokines in EAE/MS
Nature of Association Disease
modulated by
Cytokines Active Remission/ Antibody or
disease/ suppression inhibitors
relapse
IL-1 + - Ye s
IL-2 + - NR
IL-3 + - NR
IL-4 - +
IL-6 + - NR
IL-10 - +
IL-12 + - Ye s
IL-13 - +
IL-18 + - Ye s
IFNg + + Ye s
TNFa + - Ye s
LTa + - Ye s
TGFb - +
IFNa ? +

Owens et al., 2001

 Cytokines in EAE/MS
Cytokine gene Modification Effect on EAE
Caspase-1 Gene disruption
IL-3
Overexpression (GFAP)
IL-4 Overexpression (lymphocytes)
IL-4 Gene disruption
IL-6 Overexpression (GFAP)
IL-6 Gene disruption
IL-10 Overexpression (MHC II)
IL-10 Gene disruption
IL-12 Overexpression (T cells)
IL-12 Gene disruption
IFNa Overexpression (GFAP)
IFNg Overexpression (GFAP, MBP)
IFNg Gene disruption
TNFa Overexpression (glial
progenitors, GFAP, NF, MBP)
TNFa Gene disruption
LTa Gene disruption
TGFb Overexpression (GFAP)
Modified (a lot) from Owens et al., 2001
SYNAPTIC PLASTICITY IN A MS MODEL
(MOG35-55-INDUCED EXPERIMENTAL AUTOIMMUNE
ENCEPHALOMYELITIS, EAE)

registering
stimulating electrode
electrode
 Pharmacological isolation of GABA- and
glutamate-mediated synaptic transmssion
GABA Glutamate
in 30 µM APV in 10 µM bicuculline
+ 10 µM CNQX

X
X
Presynaptic alterations:
EAE increases the frequency of glutamatergic
spontaneous excitatory postsynaptic currents
(sEPSCs)
 Expression and phosphorylation of
glutamate AMPA receptors are increased
in EAE
Arc/Arg3.1
HC

EAE 20dpi
TNF-alpha mimics the synaptic
defects of EAE mice
TNF-alpha mediates the effects of EAE and of
microglia on AMPA-mediated transmission
 Conclusions
• The immune-privileged status of the CNS (and the
other immune-privileged sites) is not absolute.
• Immune reactions occur in the CNS after
inflammatory and/or degenerative stimuli.
• In response to pathogenic stimuli, CNS-resident
cells closely interact with immune-cells coming
from the peripheral circulation or resident in the
regional lymph nodes.
• Auto-immunity in the CNS may occur as a
consequence of the failure of mantainance
immune-privilege