Estudo da modula????o do metabolismo lip??dico de c??lulas dendr??ticas humanas na infec????o por Toxoplasma gondii

Abstract

A toxoplasmose ?? uma doen??a de alta preval??ncia no mundo, causada pelo Toxoplasma gondii, um parasita intracelular obrigat??rio. O T. gondii possui mecanismos de modula????o do metabolismo do seu hospedeiro que garantem sua sobreviv??ncia e a instala????o da infec????o cr??nica. Um dos tipos celulares mais permissivos ?? infec????o e multiplica????o do T. gondii s??o as c??lulas dendr??ticas (DC), paradoxalmente as c??lulas apresentadoras de ant??geno mais eficazes, com capacidade de deflagrar uma resposta imune protetora eficaz e duradoura. Neste trabalho, estudamos a modula????o do metabolismo lip??dico de c??lulas dendr??ticas humanas na infec????o por T. gondii. Mostramos que o parasita induziu a matura????o da c??lula e um padr??o misto de resposta inflamat??ria, com altas concentra????es das citocinas pr??-inflamat??rias IL-6 e TNF-??? e da citocina antiinflamat??ria IL-10. Ap??s 3 horas de infec????o, verificamos que T. gondii induziu a express??o g??nica de ciclooxigenase-2 (COX-2). De forma importante, observamos por qRT-PCR que a infec????o com T. gondii regulou positivamente a express??o do gene do receptor nuclear (RN) regulado por lip??dios PPAR???, mas n??o do LXR???. J?? a express??o do mRNA das mol??culas envolvidas no transporte e estoque de lip??dios, FABP4 e ADRP, alvos do PPAR???, e ABCA1, alvo do LXR???, foram aumentadas pela infec????o de 3 horas com o parasita. Utilizando duas t??cnicas distintas (BODIPY e colora????o com ??smio) avaliamos a biog??nese de corp??sculos lip??dicos (CL) ap??s infec????o com o T. gondii e constatamos que essas organelas n??o foram induzidas ap??s 3 horas de infec????o. Entretanto, ap??s 24 horas, 90% das DC apresentaram CL e o n??mero de CL por DC foi estatisticamente maior. Observamos a presen??a de CL em DC n??o infectadas pelo parasita, mostrando que a indu????o da biog??nese de CL ?? um fen??meno par??crino, n??o dependente da infec????o celular. Avaliamos tamb??m a import??ncia do PPAR??? na infec????o por T. gondii, atrav??s do tratamento das DC com seu agonista ou antagonista. Ap??s 3 horas, apenas os genes ADRP, FABP4 e ABCA1, alvos dos receptores nucleares, foram modulados. Por ??ltimo, investigamos a influ??ncia do T. gondii na express??o das mol??culas apresentadoras de ant??genos lip??dicos. Por citometria de fluxo, constatamos que n??o h?? altera????o na express??o de membrana dessas mol??culas. Contudo, por qRT-PCR, observamos que o T. gondii regula negativamente a express??o dos genes cd1d e cd1e. Em conclus??o, mostramos que T. gondii foi capaz de regular positivamente o metabolismo lip??dico das DC e negativamente as mol??culas apresentadoras de lip??dios CD1, sem a participa????o essencial de PPAR??? nesses processos.Toxoplasmosis is a worldwide high prevalence disease caused by Toxoplasma gondii, an obligate intracellular parasite. T. gondii developed mechanisms for modulating the metabolism of its host, ensuring their survival and the chronic infection. Dendritic cells (DC) are one of the most permissive cell types to T. gondii infection and replication and are, paradoxically, considered the most effective antigen presenting cells, triggering an effective and long-lasting protective immune response. In this work, we studied the modulation of lipid metabolism in human dendritic cells infected with T. gondii. We showed that the parasite induced cell maturation, and a mixed pattern of inflammatory response with high levels of proinflammatory cytokines IL-6 and TNF-???, and anti-inflammatory cytokine IL-10. After 3h of infection, we found that T. gondii induced increased gene expression of cyclooxygenase-2 (COX-2). Importantly, we observed by qRT-PCR that T. gondii infection upregulated the nuclear receptor (RN) PPAR??? gene expression, while the levels of LXR??? were unchanged. Furthermore, the mRNA expression of molecules involved in the transport and storage of lipids, FABP4 and ADRP, PPAR??????targets, and ABCA1, LXR??? target, were also increased at 3h of infection with the parasite. In parallel, using two different techniques, (BODIPY and osmium staining), we evaluated the biogenesis of lipid droplets (LD) after infection with T. gondii, and observed that these organelles were not induced after 3 hours of infection. However, after 24 hours, 90% of DC showed LD and the number of LD per DC was statistically enhanced. We observed the presence of LD in DC not infected with the parasite, showing that induction of biogenesis of LD is a paracrine phenomenon not dependent on cell infection. We also evaluated the role of PPAR??? in T. gondii infection by treating DC with its agonist or antagonist. After 3 hours of infection only the nuclear receptor target genes ADRP, FABP4 and ABCA1 were modulated. Finally, we investigated the influence of T. gondii in the expression of lipid antigens presenting molecules. By flow cytometry, we observed no variation in expression of these molecules in cell membrane. However, by qRT-PCR, we found that the T. gondii downregulated gene expression of cd1d and cd1e. In conclusion, we showed that T. gondii was able to upregulate the DC lipid metabolism, and downregulate CD1 lipid presentation. Apparently, the RN PPAR??? has no essential role in this process