ABSTRACT
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.
Subject(s)
Interferon Type I/metabolism , Leishmania mexicana , Leishmaniasis, Cutaneous/enzymology , Leishmaniasis, Cutaneous/immunology , Toll-Like Receptor 2/metabolism , eIF-2 Kinase/metabolism , Animals , Glycosphingolipids/immunology , Host-Parasite Interactions , Humans , Leishmania mexicana/immunology , Leishmania mexicana/pathogenicity , Leishmaniasis, Cutaneous/genetics , Leishmaniasis, Diffuse Cutaneous/enzymology , Leishmaniasis, Diffuse Cutaneous/genetics , Leishmaniasis, Diffuse Cutaneous/immunology , Macrophages, Peritoneal/enzymology , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/parasitology , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Promoter Regions, Genetic , Signal Transduction , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , Toll-Like Receptor 2/deficiency , Toll-Like Receptor 2/genetics , Transfection , eIF-2 Kinase/geneticsABSTRACT
The evolution of Leishmania infection depends on the balance between microbicidal and suppressor macrophage functions. Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a classic antiviral protein, is able to regulate a number of signaling pathways and macrophage functions. We investigated the possible role of PKR in the modulation of Leishmania infection. Our data demonstrated that Leishmania amazonensis infection led to PKR activation and increased PKR levels. Consistently, in macrophages from PKR knockout 129Sv/Ev mice and RAW-264.7 cells stably expressing a dominant-negative (DN) construct of PKR (DN-PKR), L. amazonensis infection was strongly reduced. The treatment of infected macrophages with the synthetic double-stranded RNA poly(I:C), a potent PKR inductor, increased L. amazonensis intracellular proliferation. This effect was reversed by 2-aminopurine (2-AP), a pharmacological inhibitor of PKR, as well as by the expression of DN-PKR. NO release induced by dsRNA treatment was inhibited by L. amazonensis through NF-kappaB modulation. PKR activation induced by dsRNA also resulted in IL-10 production, whose neutralization with specific antibody completely abrogated L. amazonensis proliferation. Our data demonstrated a new role of PKR in protozoan parasitic infection through IL-10 modulation.
