Mutations in Toll predispose to Aspergillus fumigatus infection Lemaitre et al. Cell 1996; 86: 973-83 Innate Immune Receptors for Protozoan Parasites 1)Identify.

Mutations in Toll predispose to Aspergillus fumigatus infection

Lemaitre et al. Cell 1996; 86: 973-83

Innate Immune Receptors for Protozoan Parasites

1) Identify relevant innate immune receptors;2) Define parasite targets for innate immune receptors;

3) Define their role on host:parasite interaction, and disease outcome;

4) Elaborate prophylactic/therapeutic interventions employing protozoan derived “PAMPs” .

Beutler, Ann. Rev. Immunol., 2006

The innate immune system senses the

invasion of pathogenic

microorganisms through the Toll-like

receptors (TLRs), which recognize

specific molecular patterns that are

present in microbial components.

Beutler, Ann. Rev. Immunol., 2006

Plasma Membrane from Trypanosoma cruzi

GIPL

GPI

Mucin

Polypeptide core containing

O-linked oligosaccharides

Protozoan Associated Molecular Patterns

Complete blockade of TNF- and nitric oxide synthesis by macrophages from TLR2 KO, TLR6 KO and CD14 KO

exposed to tGPI-mucins but not to live trypomastigotes

0

2

4

6

8

10

TN

F-a

lph

a n

g/m

l

medium LPS poly IC tGPIm T.cruzi

C57Bl/6 +serumC57Bl/6 w/o serum CD14-/- w/o serum

0

1

2

3

4

5

6

7

8

medium tGPIm T.cruzi LPS

TN

F-a

lph

a n

g/m

l

C57Bl/6

TLR2-/-

TLR6-/-

TLR4 mediates NF-B-dependent cellular activation and pro-inflammatory activity upon exposure to purified

ceramide containing GIPL.

Functional expression of TLR4 confers proinflammatory responsiveness to Trypanosoma cruzi GIPLs and higher resistance to infection Oliveira A.C. et. al., J. Immunol. 173 (2004)

GIPL

surface glycolipid

0

20

40

60

80

100

0 6 12 18 24 30 36 42

% s

urv

iva

lDays post-infection

7 8 9 10 11 12

Pa

ras

ite

nu

mb

er/

5u

l o

f b

loo

d

0

2000

4000

6000

8000

10000 C57BL/6

TLR2/TLR4-/-

TLR2-/-

TLR4-/-

CD14-/-

Lack of TLR2, TLR4 and CD14 does not affect host resistance to acute infection with

Trypanosoma cruzi

TcMUCII

C-Class CpG

B-Class CpG

A-Class CpG

T. cruzi, T. brucei and L. major orthologs

Immunostimulatory CpG motifs are concentrated on TcMUC and VIPER genes from T. cruzi genome

TcMUCIMucin-like protein

DGF-1MASP

Trans-sialidase

Hypothetical proteinL1Tc

VIPER

20k 40k 60k 80k 100k 120k 140k 160k 180k 200k 220k

Stimulatory oligonucleotides derived from T. cruzi genome

TN

F-

ng/

ml

0

2

4

6

8

10

121

,5

0,3

0,0

6

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

B22

0

Med

ium

7909

Neg

ativ

e

B61

6

B34

4

B29

7

B18

91

B14

00

B59

9

B33

8

B27

8

B19

0

LPS

Macrophages

[M]0

2

4

6

8

10

12

14

16

18

[M]

Dendritic Cells

IL-1

2(p

40)

ng/

ml

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

1,5

0

,30

,06

B22

0

Med

ium

7909

Neg

ativ

e

B61

6

B34

4

B29

7

B18

91

B14

00

B59

9

B33

8

B27

8

B19

0

LPS

C57BL/6

TLR4-/-

TLR9-/-

Co-localization of TLR9, T. cruzi and LAMP-1 in DCs primed with ODN containing CpG motifs

Me

diu

m

NF

kB

re

po

rte

r a

cti

vit

y

(RL

U,

tho

us

an

ds

)

0

20

40

60

80

100

120

140 HEK TLR7

HEK TLR9

Me

diu

m

TC

DN

AU

ntre

ate

d

TC

DN

AU

ntre

ate

d

TC

DN

AN

uc

lea

se

TC

DN

AM

eth

ylas

e

DOTAP

Genomic DNA

days post-infection

% s

urv

ival

0

20

40

60

80

0 6 12 18 24 30 36 420

2000

4000

6000

8000

10000

6 7 8 9 10 11 12 13

Par

asit

e n

um

ber

/5u

l of

blo

od

Cyt

oki

ne

pg

/ml

0

400

800

1200

1600

IFN- TNF IL-10

DTR+DT+DCWT

DTR+DT+DCTLR9-/-

100

DTR+DTDTR+DT+DCwtDTR+DT+DCTLR9-/-

TLR9 expression on dendritic cells is essential for optimal IFN- production and host resistance acute

infection with Trypanosoma cruzi

3d mice with combined deficiency on TLR3/TLR7/TLR9 are highly susceptible to infection with

Trypanosoma cruzi

UNC93B1 and MyD88 are important elements for the optimal CD8 responses during early stage of infection with

Trypanosoma cruzi

Resposta protetora desafio com CL ou Y

Clone CL-14 de Trypanosoma cruzi

Origem: Cepa infectante CL

Menos invasivo

Parasitemia e Mortalidade: negativos

Base Molecular: deficiência expressão da gp82

CL-14

Tripomastigota de CL-14 expressando NY-ESO-1 APC Linfócito TCD8+

MHC IAmastigotes

Epimastigote Trypomastigote metacyclic

Amastigote Trypomastigote

Merge NY-ESO-1

Dapi Dic

Merge NY-ESO-1

Dapi Dic

Merge NY-ESO-1

Dapi Dic

Merge NY-ESO-1

Dapi Dic

NY-ESO-1 Amastin

CL-1

4 w

ild ty

pe

CL-1

4 N

Y-ES

O-1

His

(+)

CL-1

4 N

Y-ES

O-1

His

(-)

CL-1

4 N

Y-ES

O-1

GP6

3

CL-1

4 w

ild ty

pe

CL-1

4 N

Y-ES

O-1

His

(+)

CL-1

4 N

Y-ES

O-1

His

(-)

CL-1

4 N

Y-ES

O-1

GP6

3

Expression of the tumor antigen NY-ESO-1 by the highly attenuated CL-14 strain of T. cruzi

CL-14 NY-ESO-1

CL-1

4 N

Y-ES

O-1

His

(-)

CL-1

4 W

T

CL-1

4 N

Y-ES

O-1

His

(+)

CL-1

4 N

Y-ES

O-1

GP6

3

rNY-

ESO

-1

CD8 NY-ESO-1CD4 NY-ESO-2TSKB18RPMI

CL-14 strain of T. cruzi expressing NY-ESO-1 induces strong humoral and cellular specific immune responses

and protects mice against tumor development

T. cruzi derived CpG, but not GIPLs, pontentiates humoral and cellular immune responses specific for

NY-ESO-1 and protects mice against tumor development

CL

-14

NY

-ES

O-1

Conclusions:

1)Trypanosoma cruzi derived GPI anchors and DNA (unmethylated CpG motifs) act as TLR agonists.

2) Nucleotide sensing TLRs appear to be critical ones for eliciting protective immune responses during T. cruzi infection.

3) Optimal CD4+ T as well as CD8+ T cell responses elicited by T. cruzi are dependent on both MyD88 and UNC93B1 activities.

4) CL-14 strain induces a strong T-cell mediated immune response and protection against a melanoma cell line (B16) expressing the tumor antigen NY-ESO-1.

Acknowledgements:IMPAR - CPqRR-FIOCRUZ/UFMGBruno GalvãoCaroline F. JunqueiraCatherine Ropert Flávia RodriguesHelton Santiago Marco A. S. Campos Eneida P. Valente

.

University of Massachusetts - USABraulia CaetanoDouglas T. GolenbockEicke LatzMariane MeloPeggy ParrocheCheri Sirois

Osaka University – Japan Scripps Res. Inst. –USAShizuo Akira Bruce Beutler

National Institutes of Health - USAAndre BaficaRomina GoldszmidAlan Sher

Inst. Ciencias Biomedicas UFRJJose Oswaldo PreviatoLucia Mendonça-PreviatoMaria Bellio

Inst. Ciencias Biologicas – UFMGDaniella BartholomeuJacqueline Alvarez-LeiteSantuza M. R. TeixeiraEgler Chiari

Ludwig Institute for Cancer Research - USAAndrew SimpsonJonhatan SkipperLoyd OldGerd Ritter

UNIFESPMaurício M. Rodrigues