2 billion infections each year

3 million deaths

Liver

Mosquito

sporozoite

Erythrocyte

merozoite

gametocyte

Life Cycle of PlasmodiumP. falciparumP. vivaxP. malariaeP. ovale

Ring

Trophozoite

Merozoites

Schizont

Fever

Hypoglycemia

Respiratory problems

Miscarriage

Anemia

Cerebral Malaria

(major causes of death in

Plasmodium falciparum infections)

Symptoms of malaria

Malaria in humans

. Repeated infections required for clinical immunity

. Chronic infection

. Antibody responses can be short-lived

. Immunity is lost in a short time without exposureto infection

. Major complications are associated with regulation/dysregulation of inflammatory responses

Different rodent models of malaria

Lethal

Non-lethal

Plasmodium chabaudi chabaudi (AS)

1. Non-lethal in most mouse strains

2. Sequestration of trophozoites and schizonts

3. Antigenic variation, multigene families (cir)

4. Severe complications: anemia, hypoglycemiacerebral complications

Mechanisms of immunity in mice• CD4+ T cells and B cells important for

clearance and immunity to re-infection

CD4-/-

μMTNo B cells

Days of infection

% RBCInfected

Log scale

Wild type

~30%

Days of infection

Antibody

% R

BC

inf e

cte d

Immune response to mouse model of malariaPlasmodium chabaudi

5 10 15 20 25 30Nucleated cell number

1x 109

5 x 108

Th1 Th2IFNγ > Helper Helper > IFNγ

Innate responses

3m

• High levels of TNF are associated with severe anaemia and cerebral malaria.

• Low IL-10 responses, or ratios of plasma IL-10 to TNFαof <1 in severe anaemia and cerebral malaria.

• Anti-inflammatory cytokine, IL-10, protects against CM in rodent model.

An imbalance in anti-inflammatory and pro-inflammatoryresponses rather than absolute levels of one cytokine,may be important in pathogenesis of severe malaria.

Can any of the pathology of malaria be attributed to the host response?

0

10

20

30

40

% re

duct

ion

IL-10-/- IL-10+/+

*

b

Increased parasite sequestration

More disease and higher IFNγ/TNF in IL-10KO mice

0

25

50

75

100

IFN

γμg

/ml

IL-10KOIL-10KO

0

0.2

0.4

0.6

0.8

WT

WTTNFα

% s

tarti

ng w

eigh

t Body weight

Hem

atoc

rit(%

)12Days

Anemia

WT

IL-10

Oedema

Infected IL-10 WT

Cerebral oedema and haemorrhages in infected IL-10 KO mice

haemorrhage

Infected IL-10 KO WT

Treatment with anti TNFα Abs reduces brain haemorrhages and cerebral oedema in P. chabaudi infected IL-10 KO mice

haemorrhages

Days post infection

0 10 20 30

+ anti TNFα

% m

ice

with

hae

mor

rhag

e

+ control Ab

0

10

20

30

40

anti-TNF Control Abug E

vans

blu

e/ w

et w

eigh

t of b

rain oedema

50%

Severe symptoms of malaria in P.chabaudi infections(and other rodent malarias) are partly a result of

host inflammatory responses such as TNFα.

IL-10 can regulates this response

? source of IL-10? What other cytokines are involved? How much of a role does parasite variation play

Days of infection

Antibody

% R

BC

inf e

cte d

Immune response to mouse model of malariaPlasmodium chabaudi

5 10 15 20 25 30Nucleated cell number

1x 109

5 x 108

Th1 Th2IFNγ > Helper Helper > IFNγ

Innate responses

3m

CD4 T cells specific for a peptide of Merozoite Surface Protein 1 (MSP1) are used to study presentation and T cell by

dendritic cells

B5 Tg:CD4 Tg cells Vα2/Vβ8 TCRBALB/cMSP138 (aa 1151-1171)MHC class II I-Ed.

33

19

38

30

83Transgenic CD4 T cells

Ring

Trophozoite

Merozoites

Schizont

Presentation of MSP1 peptide to Tg CD4 T cells in vivo:

maximum cell division andmaximum numbers of IFNγ− and IL-10- producing cellsbetween days 2 and 5

Activation and division of Tg cells in vivo is low between 5 and 8d

DC exhaustion? Suppression?

Cannot determine which splenic APC activate Tg T cells

Do different splenic DC induce different responses??

CD8

CD

11c

CD8- CD8+

CD8+ and CD8- conventional DC in spleen

CD8+ and CD8- DC from naive mice can induce MSP1 TCR Tg CD4 T cells to proliferate and produce cytokines

8-DC

8+DC

or + Schizonts + Tg CD4 T cell

CytokineIn supernatant

6d αCD3/28

CD8+ DC

CD8- DC101 102 103 104 1050

1000020000300004000050000600007000080000

APC number

prol

ifera

tion

(cpm

) Proliferation

0

1234567

25

50

75

100

125

0

10

20

30

40

50

0

5

10

15

20

25

IFNγ IL-10 IL-2IL-4

ng/m

lproliferation

Increase in CD11c+ DC during infection

d0 d5 d8 d14 d20

Thy 1.2CD11c IgD

0 7 12 17050

100150200250

Days of infection

DC

num

ber (

x10-

6 )

Increase in CD8-CD11c DC in spleens of infected miceand apoptosis of CD8+ DC

Days of infection

20

40

60

80

Num

ber o

f CD

11c

DC

pe

r spl

een

(x10

-6)

CD8+

CD8-

100

75

50

25

0Num

ber o

f ap

opto

tic c

ells

per s

plee

n (x

10-5

)

ApoptosisCell number

0 5 6 9 20 0 5 6 9 20

Can both CD8+ and CD8- CD11c+ DC present Ag and activate Tg T cells in vivo?

P. chabaudi

CD8

CD

11c

Sort CD8+ and CD8- DC from spleenat different times of infection

Incubate with Tg CD4 T cells 6d

(proliferation)

Restimulate with α CD3 and and αCD28 2d

IFNγ, IL-4, Il-10, IL-2 ELISA

Normal BALB/c

Day 7Day 0

At day 7 of infection only CD8- DC induce T cell proliferation.

10-2 10-1 10 0 10 1 10 2 10 30

10000

20000

30000

40000

50000

60000

10 -2 10 -1 10 0 10 1 10 2 10 30

10000

20000

30000

40000

50000

60000

CD8+ DC

CD8- DC

+peptide

+peptide

Number of DC

3 H in

corp

orat

ion

(cpm

)

IFNγ

IL-4

IL-10

IL-2

0

25

50

75

02468

0

5

10

15

0

10203040

Cytokine production by naive Tg cells stimulated byCD8 + or CD8- DC taken from infected mice

Cytokine( ng or pg) per 104 cells

CD8-

CD8+

Cell recovery:40 x104

Cell recovery:3 x104

MHC Class II expression on splenic myeloid DC during P. chabaudi infection

CD8+ DC

CD8- DC

0 5 6 9 200

1000

2000

3000

4000

5000

6000

Days post infection

MFI

0 5 6 9 20500

1000

1500

0 5 6 9 20100

200

300

400

500

0 5 6 9 2050

100

150

200

250

0 5 6 9 200

5

10

15

20

CD86 CD40M

ean

fluor

esce

nce

inte

nsity

CD8+

CD8-

Upregulation of CD86 and CD40 on CD11c+ DC during infection

Days of Infection

Splenic CD11c + dendritic cells but not pDC from uninfected micetake up P. chabaudi-infected RBC, present MSP-1 peptides, Induce specific Tg CD4 T cells to proliferate, and to produce IL-2, IL-4, IL-10 and IFNγ

Only splenic CD11c+CD8- DC( day 7 of infection) induce T cell proliferation and significant levels of IL-4, and IL-10 in MSP1-specific Tg CD4 T cells.

Our data suggest that the switch in CD4 T cells responsesobserved in Plasmodium chabaudi infected mice may bethe result of presentation by different DC which have been modified by the infection.

Conclusions

Mouse models show many characteristics of human malaria

Host inflammatory response plays a role in pathology of malaria

Infected RBC activate DC through PAMP/PRR (eg TLR4, 9?)

Splenic DC regulate the CD4 T cell response, and therefore caninfluence pathology and immunity

Questions:

Characteristics of this large population of CD11c+CD8- cells that are present in the infected spleen?

are they generated in the spleen or do they migrate?do they have anti parasite activity?

Do DCs at different times of infection activate Tregsor induce T cell anergy?

Memory?

Division of Parasitology

Anne-Marit SponaasCecile VoisineEmma CadmanTracey LambVicky MillinsRobin StephensLatifu Sanni

Divisions of Molecular Immunology and Immunoregulation

Anne O’GarraAndre BoostraGitta StockingerDimitris Kioussis