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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.

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

1 Mutations in Toll predispose to Aspergillus fumigatus infection Lemaitre et al. Cell 1996; 86: 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.

2 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

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5 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 TNF-alpha ng/ml mediumLPSpoly ICtGPImT.cruzi C57Bl/6 +serum C57Bl/6 w/o serum CD14-/- w/o serum mediumtGPImT.cruziLPS TNF-alpha ng/ml C57Bl/6 TLR2-/- TLR6-/-

6 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

7 % survival Days post-infection Parasite number/5ul of blood C57BL/6 TLR2/TLR4-/- TLR2-/- TLR4-/- CD14-/- Lack of TLR2, TLR4 and CD14 does not affect host resistance to acute infection with Trypanosoma cruzi

8 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 TcMUCI Mucin-like protein DGF-1 MASP Trans-sialidase Hypothetical protein L1Tc VIPER 20k40k 60k 80k100k120k 140k 160k 180k200k220k

9 Stimulatory oligonucleotides derived from T. cruzi genome TNF- ng/ml ,5 0,3 0,06 1,5 0,3 0,06 1,5 0,3 0,06 1,5 0,3 0,06 1,5 0,3 0,06 1,5 0,3 0,06 1,5 0,3 0,06 1,5 0,3 0,06 1,5 0,3 0,06 1,5 0,3 0,06 1,5 0,3 0,06 1,5 0,3 0,06 B220 Medium 7909 Negative B616B344B297 B1891 B1400 B599B338B278 B190 LPS Macrophages C57BL/6 TLR4-/- TLR9-/-

10 Co-localization of TLR9, T. cruzi and LAMP-1 in DCs primed with ODN containing CpG motifs Medium NFkB reporter activity (RLU, thousands) HEK TLR7 HEK TLR9 Medium TC DNA Untreated TC DNA Untreated TC DNA Nuclease TC DNA Methylase DOTAP Genomic DNA

11 TLR9 expression on dendritic cells is essential for optimal IFN- production and host resistance acute infection with Trypanosoma cruzi

12 3d mice with combined deficiency on TLR3/TLR7/TLR9 are highly susceptible to infection with Trypanosoma cruzi

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

14 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 I Amastigotes

15 Epimastigote Trypomastigote metacyclic Amastigote Trypomastigote MergeNY-ESO-1 DapiDic MergeNY-ESO-1 DapiDic MergeNY-ESO-1 DapiDic MergeNY-ESO-1 DapiDic NY-ESO-1 Amastin CL-14 wild type CL-14 NY-ESO-1 His(+) CL-14 NY-ESO-1 His(-) CL-14 NY-ESO-1 GP63 CL-14 wild type CL-14 NY-ESO-1 His(+) CL-14 NY-ESO-1 His(-) CL-14 NY-ESO-1 GP63 Expression of the tumor antigen NY-ESO-1 by the highly attenuated CL-14 strain of T. cruzi

16 CL-14 NY-ESO-1 CL-14 NY-ESO-1 His (-) CL-14 WT CL-14 NY-ESO-1 His (+)CL-14 NY-ESO-1 GP63rNY-ESO-1 CD8 NY-ESO-1 CD4 NY-ESO-2 TSKB18 RPMI CL-14 strain of T. cruzi expressing NY-ESO-1 induces strong humoral and cellular specific immune responses and protects mice against tumor development

17 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-ESO-1

18 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.

19 Acknowledgements: IMPAR - CPqRR-FIOCRUZ/UFMG Bruno Galvão Caroline F. Junqueira Catherine Ropert Flávia Rodrigues Helton Santiago Marco A. S. Campos Eneida P. Valente. University of Massachusetts - USA Braulia Caetano Douglas T. Golenbock Eicke Latz Mariane Melo Peggy Parroche Cheri Sirois Osaka University – Japan Scripps Res. Inst. –USA Shizuo Akira Bruce Beutler National Institutes of Health - USA Andre Bafica Romina Goldszmid Alan Sher Inst. Ciencias Biomedicas UFRJ Jose Oswaldo Previato Lucia Mendonça-Previato Maria Bellio Inst. Ciencias Biologicas – UFMG Daniella Bartholomeu Jacqueline Alvarez-Leite Santuza M. R. Teixeira Egler Chiari Ludwig Institute for Cancer Research - USA Andrew Simpson Jonhatan Skipper Loyd Old Gerd Ritter UNIFESP Maurício M. Rodrigues


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