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

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

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

T. cruzi infection: Consequences of apoptosis Efferocytosis

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

Blocking apoptosis in T. cruzi infection Caspase inhibition Efferocytosis

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),

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),

Treatment with zVAD in vivo increases macrophage activation

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

Blocking apoptosis in T. cruzi infection Anti-FasL Efferocytosis

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,

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

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

Modifying immune regulation through opsonization of apoptotic cells Antibodies Efferocytosis

Differential reaction of chagasic IgG with apoptotic lymphocytes % of Max Annexin V + CHA CT % CHA % IgG (H+L) A OD (Arbitrary Units) Migration Distance (Arbitrary Units) CT B C Position 403 Peak values (AU) Position 901 Position 1055Position 1022 Cha

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

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

Control of parasite replication requires engagement of FcγRs

Previous immunization with apoptotic cells reduces parasitemia in vivo Dm28c dpi

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