RESUMO
Lipids from microorganisms are ligands of Toll like receptors (TLRs) and modulate the innate immune response. Herein, we analyze in vitro the effect of total lipid extracts from Trypanosoma cruzi amastigotes of RA and K98 strains (with polar biological behavior) on the induction of the inflammatory response and the involvement of TLRs in this process. We demonstrated that total lipid extracts from both strains induced lipid body formation, cyclooxygenase-2 expression and TNF-α and nitric oxide release in macrophages, as well as NF-κB activation and IL-8 release in HEK cells specifically through a TLR2/6 dependent pathway. We also evaluated the inflammatory response induced by total lipid extracts obtained from lysed parasites that were overnight incubated to allow the action of parasite hydrolytic enzymes, such as Phospholipase A1, over endogenous phospholipids. After incubation, these total lipid extracts showed a significantly reduced pro-inflammatory response, which could be attributed to the changes in the content of known bioactive lipid molecules like lysophospholipids and fatty acids, here reported. Moreover, analyses of total fatty acids in each lipid extract were performed by gas chromatography-mass spectrometry. Our results indicate a relevant role of T. cruzi lipids in the induction of a pro-inflammatory response through the TLR2/6 pathway that could contribute to the modulation of the immune response and host survival.
Assuntos
Lipídeos/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 6 Toll-Like/imunologia , Receptores Toll-Like/imunologia , Trypanosoma cruzi/metabolismo , Animais , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Ácidos Graxos/imunologia , Células HEK293 , Humanos , Imunidade Inata , Interleucina-8/metabolismo , Gotículas Lipídicas , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Fosfolipases A1/genética , Fosfolipases A1/metabolismo , Proteínas Recombinantes , Trypanosoma cruzi/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Triatoma infestans is the most relevant vector of Chagas disease in the southern cone of South America. Since its genome has not yet been studied, sequencing of Expressed Sequence Tags (ESTs) is one of the most powerful tools for efficiently identifying large numbers of expressed genes in this insect vector. RESULTS: In this work, we generated 826 ESTs, resulting in an increase of 47% in the number of ESTs available for T. infestans. These ESTs were assembled in 471 unique sequences, 151 of which represent 136 new genes for the Reduviidae family. CONCLUSIONS: Among the putative new genes for the Reduviidae family, we identified and described an interesting subset of genes involved in development and reproduction, which constitute potential targets for insecticide development.
Assuntos
Vetores de Doenças , Etiquetas de Sequências Expressas , Estudos de Associação Genética/métodos , Triatoma/genética , Animais , Argentina , DNA Complementar/química , DNA Complementar/genética , Perfilação da Expressão Gênica/métodos , Análise de Sequência de DNARESUMO
Intestinal extracts of Triatoma infestans induce cell differentiation of Trypanosoma cruzi epimastigotes into the infective metacyclic form. Part of this effect can be explained by the presence of haemoglobin fragments, which stimulate trypanosomal adenylate cyclase. In this work we examined the metacyclogenic activity of lipids present in this intestinal extract. We found that lipid extracts of the intestinal extract have significant stimulatory effects that reside with the free-fatty-acid fraction, especially oleic acid. These compounds stimulate de novo diacylglycerol formation and protein kinase C activity in the parasite. Moreover, metacyclogenesis is stimulated by phorbol esters and cell-permeant diacylglycerol, while protein kinase C down-regulation or incubation with inhibitors of this kinase abrogates this effect. These results indicate that free fatty acids are a novel signal, inducing metacyclogenesis, acting through a pathway involving diacylglycerol biosynthesis and protein kinase C activation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Ácidos Graxos não Esterificados/farmacologia , Sulfonamidas , Trypanosoma cruzi/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Bucladesina/farmacologia , Divisão Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Diglicerídeos/biossíntese , Diglicerídeos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ácidos Graxos não Esterificados/farmacocinética , Intestinos/química , Isoquinolinas/farmacologia , Ácido Oleico/farmacocinética , Ácido Oleico/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Triatoma/química , Trypanosoma cruzi/citologia , Trypanosoma cruzi/metabolismoRESUMO
A peptide from hindguts of the Triatoma infestans, the hematophagous Chagas' insect vector, activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes (proliferative and non-infectious forms) to metacyclic trypomastigotes (non-proliferative and infectious forms). The peptide was purified from hindguts of insects fed two days before with chicken blood. After purification, the peptide showed upon SDS-PAGE a single band of about 10 kDa. The sequence for 20 residues of the amino terminus of this peptide was: H2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln-Gln-Ala-Trp-Glu-Lys-Ala- Ala-Ser-His. This sequence corresponds to the amino terminus of chicken alpha D-globin. A synthetic peptide with the sequence of the 40 amino acids corresponding to the amino terminus of alpha D-globin, also stimulated T. cruzi adenylyl cyclase activity and promoted metacyclogenesis