Universidade Federal do Rio de Janeiro Centro de Ciências da Saúde

1. Universidade Federal do Rio de Janeiro Centro de Ciências da Saúde Instituto de Biofísica Carlos Chagas Filho The importance of apoptosis for immune regulation in Chagas’ disease George A. DosReis

2. Fabrício Montalvão. IBCCF-UFRJ Landi V. Guillermo. IBCCF-UFRJ Geisy M. de Almeida. IBCCF-UFRJ Elizabeth M. Silva. IBCCF-UFRJ Marise P. Nunes. IOC-Fiocruz-RJ Juliana de Meis. IOC-Fiocruz-RJ Christina M. Takiya. Dept. Histologia-UFRJ Richard Siegel. NIAID-NIH, USA Celio Freire-de-Lima. IBCCF-UFRJ Marcela F. Lopes. IBCCF-UFRJ

3. Phagocytosis of apoptotic cells (efferocytosis) inactivates macrophages and exacerbates parasite replication through TGF-β, ODC and polyamine synthesis X Efferocytosis Lopes MF et al. Immunol. Today 21; 489-494, 2000 Freire-de-Lima CG et al. Nature 403; 199-203, 2000

4. T. cruzi infection: Consequences of apoptosis Efferocytosis

5. Apoptosis in T. cruzi infection Modify efferocytosis by: 1. Caspase inhibition 2. Anti-FasL treatment 3. Antibodies to apoptotic cells Efferocytosis

6. Blocking apoptosis in T. cruzi infection Caspase inhibition Efferocytosis

7. Caspase inhibition reduces lymphocyte apopotosis in vivo Silva, E. M. et al. Caspase inhibition reduces lymphocyte apoptosis and improves host immune responses to Trypanosoma cruzi infection. Eur. J. Immunol. 2007, 37 (3), 738-746.

8. Treatment with zVAD reduces parasitemia Silva, E. M. et al. Caspase inhibition reduces lymphocyte apoptosis and improves host immune responses to Trypanosoma cruzi infection. Eur. J. Immunol. 2007, 37 (3), 738-746.

9. Treatment with zVAD in vivo increases macrophage activation

10. The general caspase inhibitor zVAD reduces apoptosis of T lymphocytes in vitro and in vivo Treatment with zVAD increases immunity to T. cruzi infection

11. Blocking apoptosis in T. cruzi infection Anti-FasL Efferocytosis

12. Increased immunity to T. cruzi after treatment with anti-FasL in vivo Guillermo, L.V. et al. The Fas death pathway controls coordinated expansions of type 1 CD8 and type 2 CD4 T cells in Trypanosoma cruzi infection. J. Leukoc. Biol. 2007, 81, 942-951.

13. Anti-FasL in vivo accelerates and increases type 1 cytokine responses

14. Anti-FasL reduces apoptosis of CD4 and CD8 T cells Anti-FasL increases type 1 cytokine responses, increases macrophage NO production and reduces parasitemia Lymphocyte apoptosis is deleterious for T. cruzi Infection in vivo

15. Modifying immune regulation through opsonization of apoptotic cells Antibodies Efferocytosis

16. 100 80 60 40 20 0 A B OD (Arbitrary Units) Annexin V+ CT 50 40 30 % of Max CHACT - 26.1% CHA - 36.3% 20 10 0 400 600 800 1000 1 10 100 1000 10000 IgG (H+L) Migration Distance (Arbitrary Units) C Position 901 Position 403 Position 1022 Position 1055 Peak values (AU) Differential reaction of chagasic IgG with apoptotic lymphocytes Cha

17. Opsonization of apoptotic cells with chagasic IgG reduces the effect of efferocytosis on replication of T. cruzi IgG purified from T. cruzi infection (90 days) or age matched serum

18. Apoptotic cells opsonized with chagasic IgG increase TNF-α production Control of parasite replication is mediated by increased TNF-α production

19. Control of parasite replication requires engagement of FcγRs

20. Previous immunization with apoptotic cells reduces parasitemia in vivo Dm28c dpi

21. Conclusions Chagasic IgG opsonizes apoptotic cells and reduces the effect of efferocytosis on parasite replication The protective effect is mediated by increased TNF-α production The protective effect requires FcγRs Generation of autoantibodies against apoptotic cell antigens could play a protective role against parasite infection