RESUMO
Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-γ in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis.
Assuntos
Moléculas de Adesão Celular/imunologia , Células Dendríticas/imunologia , Imunidade Inata , Macrófagos/imunologia , Proteínas de Protozoários/imunologia , Ácidos Siálicos/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Animais , Interleucina-12/imunologia , Camundongos , Camundongos Knockout , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Toxoplasmose Animal/genéticaRESUMO
Several studies have shown that conformational changes of ß(2)-glycoprotein I (ß(2)GPI) when bound to negatively charged components expose cryptic epitopes and subsequent binding of anti-ß(2)GPI from patients with antiphospholipid syndrome (APS). However, the role of the carbohydrate chains of ß(2)GPI in this anti-ß(2)GPI reactivity is poorly understood. We therefore studied the reactivity and inhibition of anti-ß(2)GPI antibodies from APS patients with native, partially glycosylated ß(2)GPI (pdß(2)GPI; without sialic acid) and completely deglycosylated ß(2)GPI (cdß(2)GPI). To determine the potential biologic importance of these glycoforms and their interaction with anti-ß(2)GPI in vitro, stimulation assays were performed with the U937 cell line. Circular dichroism (CD) and fluorescence analysis of the three ß(2)GPI forms were also studied. We found an increased reactivity of anti-ß(2)GPI against pdß(2)GPI and cdß(2)GPI compared to native ß(2)GPI. Both deglycosylated ß(2)GPI isoforms showed higher inhibition of the anti-ß(2)GPI reactivity than the native protein in soluble-phase. Likewise, the antibody/ß(2)GPI/glycoform complexes increased the synthesis of IL-6, IFNγ and TNFα and the expression of HLA-DR, CD14 and CD11c in U937 cells. CD and fluorescence studies of the glycoforms yielded considerable changes in the fluorescence signals. Our work suggests that the partial or complete removal of the carbohydrate chains uncover cryptic epitopes present in ß(2)GPI. The differentiation and increased synthesis of pro-inflammatory cytokines by U937 cells in vitro may have pathogenetic implications.
Assuntos
Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/imunologia , beta 2-Glicoproteína I/química , beta 2-Glicoproteína I/imunologia , Adulto , Anticorpos Anticardiolipina/sangue , Síndrome Antifosfolipídica/etiologia , Estudos de Casos e Controles , Diferenciação Celular , Citocinas/biossíntese , Epitopos/química , Epitopos/imunologia , Feminino , Glicosilação , Humanos , Imunoglobulina G/sangue , Masculino , Monócitos/imunologia , Monócitos/patologia , Ácidos Siálicos/química , Ácidos Siálicos/imunologia , Células U937 , Adulto JovemRESUMO
Hybridomas secreting monoclonal antibodies to Nippostrongylus brasiliensis antigens were generated by hybridization of IR983F myeloma cells with spleen cells from Lou/M/Wol rats infected with living third-stage larvae. Antibodies specific either for larval or worm antigens were identified by enzyme-linked immunosorbent assays with Nippostrongylus brasiliensis fragments, homogenates and secretions as antigens. The results demonstrate that all antibodies which recognized larval antigens (38 antibodies) also reacted with worm surfaces. Ten antibodies were specific only for worm antigens. Ten antibodies reacted with worm homogenate, three antibodies recognized components of worm secretion and 17 antibodies combined with acetylcholinesterase. The epitope specificity was investigated by the capacity of various glycosides, aminoacids, N-acetylneuraminic acid and phosphorylcholine to inhibit the binding to worm fragments. The analysis revealed that alpha-methylglucoside, alpha-methylmannoside, N-acetylglucosamine, N-acetylgalactosamine, fucose and the amino acids leucine, phenylalanine, tyrosine, serine, tryptophan did not combine with the antigen-binding sites of the antibodies. Proline, arginine and histidine, however, displayed inhibitory effects. With N-acetylneuraminic acid as inhibitor three groups of antibodies could be discriminated. At a concentration of 10-20 mM, phosphorylcholine was a potent inhibitor for all antibodies.