Mécanismes effecteurs dans la malaria cérébrale: cellules de l’hôte et leurs microparticules. Conférence de la 8ème édition du Cours international « Atelier Paludisme » - COMBES Valéry

1. Mécanismes effecteurs dans la malaria
cerebrale: cellules de l’hôte et leurs
microparticules
Valéry Combes, PhD

2. Cerebral malaria
• Intravascular pathology with neuronal
damage without parasitised red blood cells
(PRBC) ever entering into the brain
parenchyma

3. Cerebral malaria
• In sub-Saharan Africa: children under 5
• In South-East Asia: young adult
• Cerebral capillaries and venules distended by
infected erythrocytes: sequestration
• Cerebral oedema and micro-hemorrhages are
features of endothelial alteration in CM
• No transmigration, maturation of monocytes
within vessels

4. Courtesy of Prof M Molyneux

5. •Without treatment: 100% fatal
•With treatment 70 to 80 %
survival
•Among survivors: ~20% long-
term sequelae (motor and
cognitive impairment)
Courtesy of Prof M Molyneux

6. Differentiating CM from other causes
of death
• Sequestration occurs in every P. falciparum infection
(including asymptomatic)
• Mere presence of parasites is not sufficient to
attribute illness or death to malaria
clinically defined CM
autopsy
Sequestration only No sequestration
(15%) (29%)
Sequestration
+ microvascular pathology
(56%) Taylor et al, Nat Med. 10: 143-145, 2004

7. Importance of retinopathy diagnostic
• So far, retinal examination is the only clinical
sign (non invasive) that distinguishes malarial
from non malarial coma
Retinal whitening
Macular
Peripheral
Vessel changes
Whitening (including orange vessels and
tramlining)
Capillary whitening
Retinal hemorrhages, predominantly
white-centered
Papilledema
Cotton wool spots Beare et al Am J Trop Med 2006

8. Hypotheses / Pathogenesis
• “mechanical theory” – vascular obstruction by
parasitised erythrocytes
– but … low correlation between parasitaemia and
mortality
• “immunopathology theory” – immune cells and
effector molecules
– but … anti-TNF therapies are not effective
• combination of the above ?
Berendt et al., Parasitol. Today 10: 412, 1994
Clark et al., Parasitol. Today 10: 410, 1994
Grau et al., Parasitol. Today 10: 408, 1994
Van der Heyde et al., Trends Parasitol 22: 503, 2006

9. The neurovascular lesion

10. Hunt & Grau
24: 491-499, 2003
Schofield & Grau
5: 722-735, 2005
Bleeding,
Hypoxia?,
Parenchymal +
Axonal & glial
damage
mechanisms?

11. Human CM: brain haemorrhage

12. Importance of interactions between
endothelium and host cells
• Endothelium expresses cytoadherence receptor for infected
erythrocytes (IE): ICAM-1, PECAM-1, CD36, VCAM-1, P-
selectin, ...
• Increased binding of T lymphocytes, monocytes, neutrophils,
NK, NK T cells and dendritic cells
• IE and host cell sequestration in deep vascular beds (brain,
lungs, kidneys) activate endothelium and lead to endothelial
lesion
• Endothelium: both produces and is modified mostly by pro-
inflammatory molecules
• Cytokines play a key role in the worsening of the endothelial
activation

13. INPUT OUTPUT
infected erythrocytes
endothelial
platelets cytokines cytokines MP
sCAMs
MP
CAM upreg.
sTM
monocytes
Brain endothelium BBB
adapted from Combes, Lou and Grau:
alteration
Endothelium in cerebral malaria. Aird
publications, 2007 parenchymal changes

14. Modelling cerebral malaria

15. Primate ANIMAL MODELS Mouse
Parasite Host CM Study Parasite Host CM Study
Squirrel CBA/J Yes • Host cell
P. falciparum Yes sequestration
monkey • Cytoadherence BALB/c No
• Sequestration P. berghei ANKA • Cytokines
DBA/2 Non fatal
• Rosettes • Chemokines
Gene KO Yes/No • CAM
• Brain vascular
P. coatneyi complications • T cell
Rhesus • PRBC adhesion P. berghei K176 No responses
P. knowlesi Yes
monkey receptors
P. yoelii 17XL rare •PRBC
P. fragile sequestration
P. chabaudi •Rosettes
Combes, de Souza, Renia,
IN VITRO MODELS
Hunt & Grau, 2005
Parasite Cells Study
• Cytoadherence
HUVEC/ HBEC/ HMEC/ HLEC • Role of CAM
• Release of soluble molecules
P. falciparum
• Monolayer alterations
HBEC / PRBC / Platelets • Interactions between cells
• Apoptosis
P. berghei ANKA versus Retinal wholemount • BBB changes
P. berghei K173 (also in vivo for functions) • Role of brain parenchymal cells

16. The murine model of CM

17. The murine model of CM
neurological Score Observation
phase
hyperparasitaemia 0 No discernible clinical signs
days
0 7 14 21
severe 1 Hunched posture, slightly
anaemia ruffled fur
cerebral
malaria 2 Very ruffled fur, incipient
motor impairment
Blood collected 3 Very ruffled fur, severe
motor impairment such
Platelet-free plasma as ataxia, hemiplegia
and paraplegia,
Using flow cytometry:
convulsions, fitting
Kinetics – Annexin V (PS ligand)
4 Very little movement, cold
Phenotyping – cell specific antibodies
to the touch

18. T cells, cytokines and their receptors

19. Cytokine interplay leading to TNF
overproduction in CM
malaria lymphoid organs bl ood
antigens IL-3 RECRUITMENT
GM-CSF
Th1 IF N-γ MO TNF
AMPLIFICATION
Th2
GENETIC
SUSCEPTIBILITY
IL-4 endothelial
suppressed changes

20. Hunt & Grau, 2003
Effector cells PLoS ONE
monocyte Togbe et al, 2008
CD8+ T cell
mTNF α β
mLTα1β2 α β
mLTα2β1
sLTα3
β
LTβR
TNFR2
Effects?
ICAM-1
upregulation
Unknown
target
Endothelial cell cell

21. Cytokines / chemokines
• TNF, LTα, IFNγ: pro-inflammatory
• IL-10:
– anti-inflammatory
– protection against experimental CM
– low levels associated with severe malaria
• IL-8 and MIP1-α:
– responsible for prolonged anaemia
– involved in the recruitment of leucocytes (lymphocytes and
monocytes)
• CXCL9, CXCL10
• CCR5
• CXCR3 (receptor for CXCL9 and 10)

22. Overview of immunopathological mechanisms in murine
cerebral malaria
leukocyte effector
activation molecules
α β
LTα/β
γ
IFNγ
malaria γ
IFNγ neuronal
immune microglia biochemical
products activation dysfunction
response changes
γ
IFNγ Fas/FasL
increased blood-brain barrier astrocyte
permeability to protein damage
(cytokines, malaria antigens)
Courtesy of Prof N. Hunt

23. Early events: d 1-4 Late events: d 5-8
innate immune
response CCR5
γδ
CD8+
NK α
sLTα3
IFN-γ
IFN-γ
adaptive immune
response
CD4+ Mφ
chemokines
Perforin
Ag presentation IL-3
memTNF
GM-CSF
MCP-1
TNFR2
APC
Lysis? Activation?
↗ numbers
adapted from Coltel et al., Curr. Neurovasc. Res. 2004
BBB disruption

24. In vitro modelling of cerebral
malaria

25. 1 Modelling cerebral malaria in
Immunostaining
(patient brain) vitro
2
cell BRAIN
isolation endothelial
cell
3
PRBC
co-cultures
Mo
PRBC
PLT
WBC PLT
PLT
platelets EC

26. Human brain microvascular endothelial cell
lines
co-cultures with PRBC, leucocytes, platelets

27. Platelets have a role in PRBC sequestration
and endothelial damage
PRBC
P
platelet P
P
pRBC
PRBC
P
PRBC
brain endothelium endothelium endothelium
Concomitant presence of platelets and PRBC on brain EC:
– decreased trans-endothelial electrical resistance
– increased permeability to 70 kDa FITC-dextran
– induction of apoptosis
Blood brain barrier damage haemorrhage
Wassmer et al., J. Infect. Dis. 189: 180-9, 2004

28. Modulation of brain endothelial genes
1 2
PRBC platelets
QUESTIONS
- is 1 ≠ 2?
- is 1 + 2 > 1?
endothelium
RNA µarrays

29. Hierarchical clustering of brain
endothelial gene responses
-2 0 +2
NRB pRB Tim Platelet TN
C C e s F
Barbier, Faille et al in preparation

30. For a False discovery rate (FDR) of 5%:
• Identification of 107 TNF-induced brain
endothelial genes
• Identification of 32 platelet-induced brain
endothelial genes
• NO differential expression when comparing
experimental conditions with NRBC and those
with pRBC

31. µ arrays: conclusions
• Platelets and TNF, but not pRBC alone, induce
transcriptional changes in HBEC
• pRBC act in interaction with platelets and TNF
to induce transcriptional changes in HBEC
• analysis of gene functional annotation:
responses of HBEC to platelets were identified
in genes involved in inflammation and
apoptosis
• pathogenic role of platelets in CM

32. A role for platelet during the early
events of the infection?
- Von Willebrand Factor -

33. Platelet-decorated vWF strings will bind PRBC
FLOW DIRECTION (D)
(C)
(B)
(A)
Weibel-Palade bodies
Hollestelle, M.J. et al (2006). Br J Haematol 133, 562-569.
PRBC: DAPI
Bridges et al, Blood 2009
Platelet: calcein AM

34. Microparticles: a new player in
CM pathogenesis

35. MP production: membrane
vesiculation
Platelet Monocyte Red blood cell Brain endothelial cell
PMP MMP RMP EMP
Microparticles: not bystanders but true biological effectors
• submicron elements produced by membrane remodelling
• released by resting, activated and apoptotic cells
• express phophatidylserine (PS) and antigens from their cell of origin
• display procoagulant, proinflammatory and proadhesive properties
• propagate signals

36. Vesiculation mechanisms
Agonists PS
Translocase
Ca2+ Scramblase
+ve
MP shedding
-ve
PS
Floppase
(ABCA1) Surface Ags
Cytoskeleton
Disruption
ER Ca2+ ↑↑ Ca2+ Calpain Talin

37. Microparticles in human CM?
• TNF enhances MP release by endothelial cells (Combes J Clin Invest 99)
• TNF levels are high in patients with CM (Grau N Engl J Med 89)
First study: EMP in CM patients in Malawi
p < 0.0001
p = 0.01
150 p = 0.005
EMP / µl plasma
100
50
0
Malaria - + + +
CM - + - +
SMA - - + +
Combes et al, JAMA 291:2542 (2004)

38. Massive MP release in CM patients
- Cameroon -
Annexin V: total CD41: platelets CD105: endothelium
**
*** **
CD235a: red blood cells CD11b: monocytes / neutrophils CD3: lymphocytes
*** *** *
Pankoui-Mfonkeu et al, PLoSONE (2010)

39. Biological and clinical correlations
• In CM patients, negative correlations between
platelet MP and:
– Blantyre coma score: r = -0.437, P = 0.0122
– Platelet count: r = -0.397, P = 0.0244
• In SA patients, correlation between erythrocyte
MP and
– haemoglobin levels: r = 0.359, p = 0.0154
Pankoui-Mfonkeu et al, PLoSONE (2010)

40. Conclusions
• highest levels of total and cell-specific MP in
CM patients
• platelet MP:
– levels were the highest
– correlated with important clinical and biological
parameters such as coma score and platelets
counts
– returned to normal values at discharge
MP: a relevant marker
• in the follow up of patients with CM?
• as indicator of the efficiency of patient management

41. A role for platelet-MP in modulating
PRBC cytoadherence?

42. Platelet-MP transfer platelet antigens on
brain EC surface
PMP membrane PKH67
CD36 / GPIV
New surface phenotype
for brain EC
Faille et al, FASEB J (2009)

43. Platelet-MP enhance PRBC cytoadherence
to brain EC
PMP on PMP
None EC on RBC
1
1 2 2
Faille et al., FASEB J 2009
al.,

44. PMP decorate VWF strings and allow
binding of PRBC
Histamine
Brightfield stimulated
HUVEC
PMP:PKH26 VWF strings
PRBC: DAPI

45. Conclusion
• Platelet MP can mediate PRBC binding to
stimulated endothelium in static and flow
conditions
• Platelet MP are internalised by brain EC and
are likely to modify endothelial response
In vivo, platelet MP could be responsible for
some of the effects attributed to platelets

46. Role of MP in the pathogenesis
of CM
- murine model -

47. Microparticles as pathogenic elements
during CM?
• Full protection of ABCA1-/- Infected ABCA1-/- mice
mice against CM have fewer circulating MP
Cerebral p < 0.05
phase ABCA1-/- 1000
Nber of MP / µl plasma
100
75 750
Survival (%)
50
500
25
ABCA1+/+ 250
0
0 7 14 21 0
Time after PbA infection (days)
Plasmodium - + - +
WT ABCA -/-
Combes, Coltel et al, Am J Pathol 166:295 (2005)

48. Elevated levels of MP detected upon
onset of CM
CBA C57BL/6
150 150
* **
125 125
M P / µ l of pla sm a
M P /µ l of pla sm a
100 100
75 75
µ
50 50
25 25
0 0
+
Non-infected CM Non-infected CM +

49. CM “resistant” DBA/2 mice with CM
show elevated levels of circulating MP
*
200 *
175 n =4
MP/µ l of plasma
150
125
100
75
50 n =6 n =3
25
0
NI CM - CM +
day 8 & 10
Only mice developing CM show MP rise

50. Levels of MP are related to the
severity of the disease
600 ***
500 ***
MP/µ l of plasma 400 ***
300
200
100
60
50
µ
40
30
20
10
0
0 1 2 3 4
CM score
Severity

51. What is the in vivo localisation of microparticles?
→ adoptive transfer experiments
MP donor i.v. recipient
PKH-26
Localisation in vessels?
Tissue distribution?
Normal CBA Normal CBA
Kinetics?
Toxic effects ?
CM+ CBA CM+ CBA ... ... ...

52. In vivo adoptive transfer & detection of MP
- whole blood detection -
3000
2000
MP/µ l of whole blood
NI recipient NI donor SUP
1000
NI recipient NI donor MP
250
NI recipient PbA donor SUP
200 NI recipient PbA donor MP*
150
PbA recipient NI donor SUP
100 PbA recipient NI donor MP
50 PbA recipient PbA donor SUP
0 PbA recipient PbA donor MP
0 1 2 3 4 5 6 10 20 30 40 50 60
Time (min)

53. In vivo adoptive transfer & detection of MP
brain smear preparations
NI CM+
DAPI MP donor
Lectin - Endothelium
PKH26 – MP
NI
MP recipient
CM+

54. Conclusions
In human studies: MP as marker of disease
• MP from various origins are detected in high number in
patients with neurological complications
• Platelet MP are the most abundant and their levels
correlate with severity of disease
In the mouse model: tool to understand MP function
• Equivalent observations
• Possibility of interventional studies to evaluate the
pathogenic potential of MP and their “homing”

55. CONCLUSIONS: endothelial cells suffer
interactions
leucocytes trigger pathology.........
and
platelets
pRBC cytokines
PMP
EMP
endothelium
Pick up P. falciparum antigens
present P. falciparum antigens?
determinism of susceptibility to CM?

56. University of Sydney University of Douala
Australia Cameroon
Vascular Immunology Unit Joel Pankoui-Mfonkeu
Fatima El-Assaad Innocent Gouado
Beryl Wen
Sharissa Latham Université
Université de la
Gerard Chan diterrané
Méditerranée
Julie Wheway France
Anelia Dietmann ABC transporters
Alban Millonig Andrew Mitchell
Ronan Jambou Giovanna Chimini
Valery Combes
Georges E. Grau Parasitology
Thierry Fusaï
Liverpool School of Tropical
Medicine & Hygiene, UK
Alister Craig
Malawi-Liverpool-
Malawi-Liverpool-Wellcome Trust
Clinical Research Programme
Malawi
Samuel C. Wassmer
Malcolm Molyneux & Terrie Taylor