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