RESUMO
Macrophage migration inhibitory factor (MIF) is a proinflammatory molecule in mammals that, unusually for a cytokine, exhibits tautomerase and oxidoreductase enzymatic activities. Homologues of this well conserved protein are found within diverse phyla including a number of parasitic organisms. Herein, we produced recombinant histidine-tagged Toxoplasma gondii MIF (TgMIF), a 12-kDa protein that lacks oxidoreductase activity but exhibits tautomerase activity with a specific activity of 19.3 µmol/min/mg that cannot be inhibited by the human MIF inhibitor ISO-1. The crystal structure of the TgMIF homotrimer has been determined to 1.82 Å, and although it has close structural homology with mammalian MIFs, it has critical differences in the tautomerase active site that account for the different inhibitor sensitivity. We also demonstrate that TgMIF can elicit IL-8 production from human peripheral blood mononuclear cells while also activating ERK MAPK pathways in murine bone marrow-derived macrophages. TgMIF may therefore play an immunomodulatory role during T. gondii infection in mammals.
Assuntos
Fatores Inibidores da Migração de Macrófagos , Macrófagos , Proteínas de Protozoários , Toxoplasma , Toxoplasmose , Animais , Cristalografia por Raios X , Humanos , Interleucina-8/química , Interleucina-8/genética , Interleucina-8/imunologia , Interleucina-8/metabolismo , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Toxoplasma/química , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasma/metabolismo , Toxoplasmose/genética , Toxoplasmose/imunologia , Toxoplasmose/metabolismoRESUMO
The ability of CD8(+) T cells to act as cytolytic effectors and produce interferon-γ (IFN-γ) was demonstrated to mediate resistance to Toxoplasma gondii in murine models because of the recognition of peptides restricted by murine major histocompatibility complex (MHC) class I molecules. However, no T gondii-specific HLA-B07-restricted peptides were proven protective against T gondii. Recently, 2 T gondii-specific HLA-B*0702-restricted T cell epitopes, GRA7(20-28) (LPQFATAAT) and GRA3(27-35) (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702 and elicited IFN-γ from peripheral blood mononuclear cells of seropositive HLA-B*07 persons. Herein, these peptides were evaluated to determine whether they could elicit IFN-γ in splenocytes of HLA-B*0702 transgenic mice when administered with adjuvants and protect against subsequent challenge. Peptide-specific IFN-γ-producing T cells were identified by enzyme-linked immunosorbent spot and proliferation assays utilizing splenic T lymphocytes from human lymphocyte antigen (HLA) transgenic mice. When HLA-B*0702 mice were immunized with one of the identified epitopes, GRA7(20-28) in conjunction with a universal CD4(+) T cell epitope (PADRE) and adjuvants (CD4(+) T cell adjuvant, GLA-SE, and TLR2 stimulatory Pam(2)Cys for CD8(+) T cells), this immunization induced CD8(+) T cells to produce IFN-γ and protected mice against high parasite burden when challenged with T gondii. This work demonstrates the feasibility of bioinformatics followed by an empiric approach based on HLA binding to test this biologic activity for identifying protective HLA-B*0702-restricted T gondii peptides and adjuvants that elicit protective immune responses in HLA-B*0702 mice.