My second journal reading!

1. How can microbial interactions with
the blood–brain barrier
modulate astroglial and neuronal function?
Grab DJ, Chakravorty SJ, van der Heyde H, Stins MF
Cellular Microbiology (2011) 13(10), 1470–1478
Journal Reading
dr. Ersifa Fatimah
dr. Paulus Sugianto, SpS(K)

2. Summary
The emerging of NVU concept
• Homeostatic signalling :
Neuronal – Glial – vascular Endothelial
 maintaining normal brain function
• Observed in various neurological diseases: infection,
degenerative dis., seizure, stroke, etc
BBB endothelial activation: Role & impact is unclear
• Beneficial vs Detrimental ?
BBB as relay station in modulate astroneuronal
functioning (scenarios & factors)
• Penetrate vs Sequestered in vasculature (Cerebral Malaria)
2

3. The BBB endothelium & the NVU
BBB: ‘just’ a lining for vessels?
• Highly selective barrier between blood – CNS
• To allow blood to flow & nutrients to pass into underlying tissue
• Tight junction characteristic (compare: liver – fenestrae – plasma
exchange)
Endothelium in different tissues is very heterogeneous in
expression of surface rec. & response to stimuli
• Interaction with underlying tissues: Extracellular matrix, astrocytes,
pericytes
• Brain-trophic BBB-derived factors can promote differentiation of
neuroprecursor cells.
3

4. Endothelial-activating components (incl. microbial
involvement) initiate certain brain diseases
• Gut-derived LPS (HIV-1, alcohol abuse, depression) 
activate brain endothelium  astroglial activation signals /
modulate neurological functioning  sickness-related
behavioral changes
• Modulation of microglial function by peripheral inflammation
also depends on the prior history & immune status
The role of BBB-EC activation in the development of
neurological diseases is still an understudied area of
research
4

5. In vitro BBB endothelial models to study its
involvement in neurological diseases
In vitro model
• Single- / multi-cell
Culture of human brain EC
• Primary / Immortalized
Non-human / Non-Brain EC
Elicitation BBB-like characteristic
• Cultured on brain spesific extracellular matrix
• In combination multi-cell co-cultures
• Physical factors (vascular sheer stress, RBC interaction, TEER)  complex models
(available)
5

6. Microbes, BBB & neurological dysfunction;
to cross or not to cross?
High plasma concentration of neurotropic microbes
BBB crossing mechanism: transcellular, paracellular,
Trojan Horse
Neurons, microglia, astrocytes can be infected /
activated by microbial toxins
Innate immunity: microbial ligands + TLR  signal
transduction via NFKB
Release of mediators by activated astroglia  alter
neurological function, affect BBB function & integrity
6

7. Not all pathogens that cause
neurological disease breach the vascular
barrier and enter the CNS
• In low numbers, circulating microbes & toxins might not
penetrate into CNS but can still activate BBB-EC to release
signals
• Mild stimulation  increase expression of cell adhesion
molecules, release cytokine/ chemokine
• Stronger stimulation  opening intercellular junction &
increases barrier permeability  exposing brain to neurotoxic
plasma substances, or antibody to ion channel  affecting
neurological function
• How brain EC activation contributes in neuropathogenesis?
7

8. 8

9. Plasmodium falciparum
• Parasite lives in an infected RBC (PRBC)
remodels the PRBC surface with parasite-
encoded proteins (PfEMP-1)  bind to
endothelial receptor (ICAM-1) 
sequestration of PRBCs in vascular lumen
???  neurological symptoms
9

10. PRBC-mediated activation of the host BBB-EC  increase expression of luminal
ICAM-1 & polarized release of cytokines to both luminal side & basal side.
Traditionally cytokines are thought to be released in luminal side in order to
alarm the immune system. However, circulating cytokines can also shuttle
across the BBB into CNS.
Add the conditioned BBB medium to astrocyte & neuronal culture 
concentration dependent activation of astrocytes & neurons disruption of
axonal transport & retraction of neuronal extensions. (~ axonal damage in
autopsies of px with CM)
10

11. Toxoplasma gondii
BALB/c & C57BL/6
Retinal EC & BUVEC
murine models
• ↑ induction of MHC I • ↑ transcripts for E-,
& II Ag P-selectin, VCAM-1,
• ↑ Cell adhesion ICAM-1 within 1-4 h
molecules • ↑ inflammatory
expression mediators: RANTES,
• ↑ ALCAM expression IL-8, GM-CSF, COX2,
• ↑ BBB permeability iNOS, MCP-1,
Fractaline, GRO-1, T. gondii crosses BBB 
IP10 persist at least microglial infection &
up to 72 h post astrocytes activation 
infection activation & loss BBB integrity
11

12. Trypanosoma brucei
Upregulate brain endothelial
Release IL-6, CXCL-8, CCL-2,
expression of ICAM-1, VCAM-
TNF-α
1, E-selectin
Trypanosoma transmigration
requires cystein proteases
Brain endothelial potentially
Gαq-mediated Ca & Protease-
contribute to ↑ MMP2 in CSF
Activated Receptor-2
(in vitro study)
signalling (in vitro & gene-
profiling studies)
12

13. Borrelia burgdorferi
B. burgdorferi (/+ co-infection Anaplasma
phygocytophilum-infected neutrophils)  activation
of BBB-EC
↑ expression of host cell adhession molecules,
plasminogen activator & its receptor
↑ release of cytokines, chemokines, metalloprotease
13

14. HIV
• Loss of junctional molecules  BBB breakdown
(biopsy & post mortem samples in vivo)
• High viral titres + secreted viral protein 
activate BBB-EC  expression cell adhesion
molecule, release cytokines (brain side)  affect
neurological functioning
• Indirect pathogen-induced peripheral stimulus
can also alter neurological function, doesn’t
readily enter the CNS, but accumulates in BBB-EC
but still astroglial activation is observed. (ie: LPS)
14

15. Mechanism for BBB-related
neurological Modulation
Reduced release of neuroprotective secretions vs
Increased release of cytokine/chemokine, MMP
BBB-derived cytokines  glial cell migration, angiogenesis,
tumorigenesis, wound healing, modulate astrocyte-
neuronal interaction  neurological dysfunction
Which particular cytokines are involved? How they will
affect neuronal functioning? Their targets brain cell? 
Unclear
15

16. Astrocyte secrete
In vitro BBB-CM models
CXCL1,2,3
• ↑ CCL20 transcript & • CXCR2 present in • Alteration of the
release neuron  regulate chemokine/ receptor
• ↑ CCR6 in cell APMA type glutamate ratio in the brain may
resembling neuroglia receptor function  be a mechanism to
(oligodendrocyte)  ↑ receptor modulate neuronal
neuronal signal cooperativety & function, but when
modulation/ postsynaptic the BBB is
transmission transmission overactivated, this
amplitude may lead to neuronal
• CXCL1  modulate dysfunction
Purkinje neuron
activity & ↑ ERK
phosphorylation in
cortical neuron
16

17. Reactive Nitrogen
MMP & NO NO
Species
• Released from BBB • Alters connexin  • Trigger lipid
EC  modulate reduction of inter- peroxidation 
matrix – BBB EC astrocytic gap- affect brain
interaction  ~ junction parenchyma &
barrier integrity communications vasculature 
• synaptic function &  could also affect neurocognitive
long term astrocyte–BBB-EC sequelae (ie:
potentiation ~ or astrocyte– sepsis)
cleavage NR1 neurons
subunit of NMDA communications
receptor & ICAM-5
17

18. Conclusions
An alteration in the ‘resting’ status of the
BBB-Ec by neurotropic microbes can lead to
modulation of astroglial/neuronal function
An activated BBB could function as a
communication-relay station between
peripheral signals to the brain can
contribute to symptoms ranging from
mild symptom to serious neurological
dysfunction
18

19. 19

20. • It is not clear at what point the altered release of these
factors would tip the balance from playing a beneficial
role in astroneuronal homeostasis and repair to a
detrimental one leading to neurological dysfunction
• Many questions still remain and further clarification of
interactions between the BBB-Ec and underlying CNS
components are needed  lead to novel therapeutic
targets for treating neurological dysfunction in a wide
variety of disorders & be beneficial in the repair of
astroneuronal function
20

21. Critical Appraisal
1. Focus on a specific question
2. Clearly defines the questions being addressed
3. Methods of conducting search for relevant primary studies are described
4. Primary studies are critically appraised, preferably in relation to explicit
methodologic criteria
5. When results of primary studies are being presented, research design and
population studied are described
6. Quantitative data from primary studies are summarized, preferably with CI or P
values
7. Author obviously biased (-)
8. No references or a scanty list of references (-)
9. Summary statements regarding important issues are merely followed by one
or more references (or no references) without further description of the
studies or their results (-)
10. Magnitude of effect is discussed
21

22. Thank You
22

23. How can microbial interactions with
the blood–brain barrier
modulate astroglial and neuronal function?
--Review of the Concepts
Ersifa Fatimah, dr
PPDS Neurologi
Universitas Airlangga – RSUD dr Soetomo
Surabaya, 2012

24. THE NEUROVASCULAR UNIT /
BLOOD-BRAIN BARRIER

29. Tight Junction
• Specific structures present between cells such as
brain microvascular cells that ‘seal’ the
monolayer and prevent passage of
macromolecules between the luminal and
abluminal sides of the cells.
• It has a crucial role in maintaining the integrity of
the BBB.
• It is composed of molecules such as vascular
endothelial (VE)-cadherin, zona occludens 1,
junction adhesion molecules 1 to 3, or occludin.

30. BBB-EC
• Microvascular Ecs, one cell is found per cross-section of a
vessel (in capillaries).
• Not fenestrated, highly functional adherens, tight junctions
[can be identified via the expression of zona occludens (ZO)-1,
occludin, claudin, and junctional adhesion molecules (JAM-1,2,3) 
high transendothelial electrical resistance that helps reduce
paracellular flux of ions and small charged molecules in vivo]
• Brain ECs display higher numbers of mitochondria, have a
low level of pinocytosis, and present higher numbers of
microvilli at their surface.
• Brain ECs have a specialised basement membrane that is
essential in the maintenance of the BBB.

31. • Astrocytes participate in the upregulation of several
transport systems : GLUT-1, the L- and A-amino acid carrier
systems , P-glycoprotein.
• Participate in the functioning of the metabolic BBB,
whereby molecules that enter brain ECs due to their
lipophilic nature or because of their affinity for some
transport system can be converted by metabolic processes
into chemical compounds that cannot cross their
abluminal membrane. [Ex: L-dopa carried into the EC cytoplasm by
LAT-1 transporter is enzymatically converted into dopamine and
dihydroxyphenylacetic acid that are sequestered there]

32. Nomenclature & Principal Functions of Glial Cells

33. Astrocytes in BBB
• The most numerous cell types in the brain
• Astrocytes are central to the health of the CNS.
• Modulate synaptic transmission & ionic composition of
the brain, control metabolic processes & microvascular
behaviour, produce neurotrophins.
– In recent years, it has become clear that their activities are
drastically modified by ischaemia/ hypoxia & by cytokines,
both of which are involved in several neuroinflammatory
diseases.
• Provide physical support for neurons, control the
extracellular milieu, act as a bioenergetic regulator, and
influence vascular properties, for example, BBB integrity
and blood flow.

34. • A glial syncytium with gap junctions  role in
maintaining homeostasis, response amplification, and
rapid cell–cell signalling via calcium waves.
– Astrocytes play an essential role in CNS homeostasis via the
numerous cooperative metabolic processes they establish
with neurons, including neurotransmitter recycling, supply of
energy metabolites, and mediation of neurovascular
coupling.
– Many of these astrocytic functions are mediated via gap
junction communication. The combination of hexameric
connexin Cx proteins determines the types of ions and
small molecules that are able to pass from the cytoplasm
of one cell into the next and therefore have a direct
influence over astrocyte syncytium communication.
– Distinct astrocyte subpopulations establish different connexin
expression patterns in the CNS depending on physiological
requirements of the tissue.

35. • Modulation of the CNS microenvironment including
extracellular K+ homeostasis and pH.
• Astrocytes remove excess glutamate, the major excitatory
neurotransmitter in the brain. When released in excess,
glutamate is neurotoxic and can trigger neuronal cell
death. Astrocytes remove excess glutamate from the
extracellular space.
• Astrocytes supply glutamine to maintain glutamatergic
neurotransmission–astroglial glutamate transport is
essential for neuronal glutamatergic transmission by
operating the glutamate–glutamine shuttle.
• Regulation of blood flow – subserving ‘neurovascular
coupling’.
• Regulation of water movement via aquaporin AQP-4 and
AQP-9 on vascular endfeet.

36. • Astrocytes both produce and respond to
immunomodulators, for example, through cytokine (ciliary
neurotrophic factor, CNTF; leukaemia inhibitory factor, LIF)
and chemokine (chemokine receptor, CCR; C-X-C
chemokine receptor, CXCR) receptors and signalling.
• Formation of astrocytic scar – consisting of astrogliosis –
which can have both beneficial and deleterious
consequences on the brain parenchyma

37. PATOPHYSIOLOGY OF
CNS INFECTION

38. Mechanisms by which pathogens cross the brain endothelial monolayer.
The b2 adrenergic receptor (b2R) and the chemokine receptor, CCR5, are given as examples; several other
receptors are known to serve as tools for central nervous system (CNS) invasion, as thoroughly reviewed in.
Abbreviations: MF, monocyte–macrophage; MPs, microparticles. From Trends in Parasitology, 2012

39. Figure 1. Schematic diagram of the leukocyte adhesion and transmigration
cascade.
Given an inflammatory stimulus, leukocytesinitially loosely adhere on the vascular ECs, rolling along
the blood vessel wall via transient selectin-mediated interactions (1). During the activation stage, both the
EC and leukocyte begin to upregulate expression and/or activity of adhesion receptors on the cellsurfaces
(2), and this is required for initiating the firm adhesion stage (3). Finally, leukocytes exit the bloodstream,
crossing theendothelium by the process known as transmigration or diapedesis (4). The mechanisms and
pathways by which transmigration occurs are poorly understood ….

40. Modes of leukocyte TEM: paracellular
versus transcellular.
• Leukocytes can transmigrate across the endothelium via two
independent routes. Use of the "paracellular" route requires
transient junctional disruption as leukocytes migrate between
adjacentECs. Paracellular TEM may involve a series of PECAM-
enriched EC surface-connected membrane compartments that are
located adjacent to the junctional region. Conversely, "transcellular"
TEM involves leukocyte passage directly through an individual EC,
bypassing the need to disassemble EC junctions. Local fusion
of caveolae or vesiculo-vacuolar organelles may be a potential
mechanism of transcellular pore formation. Recent data suggest
that transcellular TEM is strongly dependent on the formation of
"invasive podosomes" that are extended by the leukocyte, while EC
apical cup structures may aid in both types of TEM.

41. Figure 2. Molecular components of EC and EC-leukocyte interactions.
ECs contain both adherens junctions and tight junctions. Transmembrane proteins located along the
paracellular cleft of two adjoining ECs interact, and thus provide the physical barrier to the
transmigrating leukocyte. Cytoplasmic junctional proteins provide a link between the transmembrane
proteins and the cellcytoskeleton. Leukocytes interact via integrins to EC adhesion molecules present on the
apical surface. In endothelial cells, adherens junctions and tight junctions can be interspersed along the
entire length of the lateral membrane.

42. Figure 3. Low molecular weight GTPases, via downstream effectors, are key participants
of EC signaling pathways involved in leukocyte TEM.
(A) Signals transduced by EC adhesion molecules downstream of leukocyte engagement regulate activity of
lowmolecular weight GTPases. GEFs activate the GTPase, enabling it to interact with downstream effectors. In
turn, GAPs aid thehydrolysis of GTP to GDP, inactivating the GTPase and inhibiting downstream signaling events.
(B) Effector signaling downstream from GTPase activation can control cell adhesion, cytoskeleton remodeling,
and membrane dynamics. In turn, this influences barrier function, membrane fusion events, and the formation
of cytoskeleton-enriched structures such as apical cups that are involved inleukocyte TEM.

43. Figure 4. Schematic diagram of signaling events initiated downstream of ICAM-
1 engagement.
Leukocyte binding to ICAM-1triggers diverse signaling pathways within the EC (highlighted in
red). Phosphorylation of target proteins (Section 4.1.3.), particularly the VE-cadherin complex (Section 4.1.4.),
production of ROS (Section 4.1.5.), activation of Rho family GTPases (Section 4.1.1.), and calcium
signaling (Section 4.1.2.) are centrally involved. These pathways all contribute to the junctional disruption
and/or actin remodeling that is permissive for leukocyte TEM to occur.

44. Figure 5. Schematic diagram of signaling pathways initiated downstream of VCAM-1
engagement
Leukocyte adhesion to VCAM-1 mainly signals via Rac1-mediated ROS generation. ROS inhibition of
phosphatases and activation of redox-sensitive kinases serve to increase phosphorylation of junctional proteins,
and together with production of MMPs, leads to junctional disruption. The Rac effector PAK has also been
implicated in actin remodeling via MLC-generated tension and contractility.