RESUMO
HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling.
Assuntos
HIV-1/metabolismo , Leishmania/crescimento & desenvolvimento , RNA de Cadeia Dupla/metabolismo , eIF-2 Quinase/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , Ativação Enzimática , Humanos , Interleucina-10/metabolismo , Espaço Intracelular/parasitologia , Leishmania/metabolismo , Leishmaniose/metabolismo , Leishmaniose/parasitologia , Leishmaniose/patologia , Macrófagos/enzimologia , Macrófagos/parasitologia , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
It is widely accepted that tumor necrosis factor alpha (TNF-α) plays a critical role in the development of tissue and nerve damage in leprosy and during the reactional episodes of acute inflammation. Thalidomide (N-α-phthalimidoglutarimide), a drug used to treat leprosy reaction, modulates immune response, inhibits inflammation and NF-κB activity. Here we investigated whether thalidomide inhibits NF-κB activation induced by Mycobacterium leprae, p38 and ERK1/2 MAPK activation. EMSA and supershift assays were performed to investigate NF-κB activation in response to M. leprae and its modulation following in vitro treatment with thalidomide. Luciferase assay was assayed in transfected THP-1 cells to determine NF-κB transcriptional activity. Flow cytometry and immunofluorescence were used to investigate p65 accumulation in the nucleus. Immunoblotting was used to investigate p38 and ERK1/2 phosphorylation. Following activation of PBMC and monocytes with M. leprae, the formation and nuclear localization of NF-κB complexes composed mainly of p65/p50 and p50/p50 dimers was observed. Induction of NF-κB activation and DNA binding activity was inhibited by thalidomide. The drug also reduced M. leprae-induced TNF-α production and inhibited p38 and ERK1/2 activation. Definition of the activation mechanisms in cells stimulated with M. leprae can lead to the development of new therapy applications to modulate NF-κB activation and to control the inflammatory manifestations due to enhanced TNF-α response as observed in leprosy and in leprosy reactions.
Assuntos
Hansenostáticos/farmacologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/patogenicidade , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Talidomida/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/microbiologia , DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Hanseníase/metabolismo , Hanseníase/microbiologia , Hanseníase/patologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We investigated the type I interferon (IFN-1)/PKR axis in the outcome of the Leishmania (Leishmania) amazonensis infection, along with the underlying mechanisms that trigger and sustain this signaling pathway. Reporter assays of cell extracts from RAW-264.7 macrophages infected with L. (L.) amazonensis or HEK-293T cells cotransfected with TLR2 and PKR promoter constructions were employed. Primary macrophages of TLR2-knockout (KO) or IFNR-KO mice were infected, and the levels of PKR, IFN-1, and superoxide dismutase 1 (SOD1) transcript levels were investigated and compared. Immunohistochemical analysis of human biopsy lesions was evaluated for IFN-1 and PKR-positive cells. Leishmania infection increased the expression of PKR and IFN-ß on induction of PKR-promoter activity. The observed effects required the engagement of TLR2. TLR2-KO macrophages expressed low IFN-ß and PKR levels postinfection with a reduced parasite load. We also revealed the requirement of PKR signaling for Leishmania-induced IFN-1 expression, responsible for sustaining PKR expression and enhancing infection. Moreover, during infection, SOD1 transcripts increased and were also enhanced when IFN-1 was added to the cultures. Remarkably, SOD1 expression was abrogated in infected, dominant-negative PKR-expressing cells. Finally, lesions of patients with anergic diffuse cutaneous leishmaniasis exhibited higher levels of PKR/IFN-1-expressing cells compared to those with single cutaneous leishmaniasis. In summary, we demonstrated the mechanisms and relevance of the IFN-1/PKR axis in the Leishmania infection.