Abstract
BackgroundLutzomyia longipalpis is the main vector of visceral leishmaniasis in Latin America. Sandfly immune responses are poorly understood. In previous work we showed that these vector insects respond to bacterial infections by modulating a defensin gene expression and activate the Imd pathway in response to Leishmania infection. Aspects of innate immune pathways in insects (including mosquito vectors of human diseases) have been revealed by studying insect cell lines, and we have previously demonstrated antiviral responses in the L. longipalpis embryonic cell line LL5.MethodsThe expression patterns of antimicrobial peptides (AMPs) and transcription factors were evaluated after silencing the repressors of the Toll pathway (cactus) and Imd pathway (caspar). AMPs and transcription factor expression patterns were also evaluated after challenge with heat-killed bacteria, heat-killed yeast, or live Leishmania.ResultsThese studies showed that LL5 cells have active Toll and Imd pathways, since they displayed an increased expression of AMP genes following silencing of the repressors cactus and caspar, respectively. These pathways were also activated by challenges with bacteria, yeast and Leishmania infantum chagasi.ConclusionsWe demonstrated that L. longipalpis LL5 embryonic cells respond to immune stimuli and are therefore a good model to study the immunological pathways of this important vector of leishmaniasis.
Highlights
Lutzomyia longipalpis is the main vector of visceral leishmaniasis in Latin America
Leishmania infantum chagasi (MHOM/BR/1974/PP75) obtained from the Leishmania collection of Instituto Oswaldo Cruz was maintained in M199 medium, pH 7.0, supplemented with 10 % fetal bovine serum and collected at exponential growth phase, washed with phosphate-buffered saline (PBS), and resuspended in fresh PBS at 107 parasites/ml for direct use in challenge procedures of LL5 cells
Silencing of the toll pathway repressor cactus affects antimicrobial peptides (AMPs) genes expression The role of the Toll pathway on AMP production was evaluated by examining the effect of silencing the Toll repressor cactus with double-stranded RNA for the cactus gene
Summary
Lutzomyia longipalpis is the main vector of visceral leishmaniasis in Latin America. Sandfly immune responses are poorly understood. In previous work we showed that these vector insects respond to bacterial infections by modulating a defensin gene expression and activate the Imd pathway in response to Leishmania infection. Aspects of innate immune pathways in insects (including mosquito vectors of human diseases) have been revealed by studying insect cell lines, and we have previously demonstrated antiviral responses in the L. longipalpis embryonic cell line LL5. Previous reports have demonstrated the value of insect cell lines to study complex immune responses. Cell lines of the insect vectors Aedes albopictus and Aedes aegypti have been shown to express various immune effector molecules, including antimicrobial peptides (AMPs) [2]. There are two established Lutzomyia longipalpis cell lines derived from embryonic tissues, LL5 [5] and Lulo [6]. Other Leishmania species were able to adhere to Lulo cells at different rates [8]
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