Activation of innate immune pathways by Epstein-Barr virus DNA using Drosophila melanogaster as a model system

Abstract

Introduction: The mammalian Epstein-Barr virus (EBV) is associated with several malignancies and autoimmune diseases. Following EBV infection and establishment of latency, recurrences frequently occur resulting in potential viral DNA shedding, which may then trigger the activation of immune pathways. A previous study conducted at the Department of Experimental Pathology, Immunology and Microbiology indicated that levels of IL-17, a pro-inflammatory cytokine associated with several autoimmune diseases, is increased in response to EBV DNA injection in mice. Whether other pro-inflammatory pathways are induced in EBV DNA pathobiology remains to be investigated. The molecular complexity of mammalian immune systems makes it challenging to study differential activities of specific immune pathways in response to a particular challenge. In this study we used Drosophila melanogaster to identify innate immune pathways that are activated in response to EBV DNA. Methods: To assess the activation of the three major Drosophila immune pathways Toll, Immune deficiency (IMD), and Janus kinase-signal transducers and activators of transcription (JAK-STAT) in response to EBV DNA, wild type adult flies were injected with 70, 140 or 280 EBV DNA copies. As a non-viral DNA control, wild type flies were injected with 0.01, 0.02 or 0.05 pg of Staphyloccocus epidermidis DNA. In addition, flies that overexpress Toll, IMD and JAK-STAT pathway components were used as positive controls. Transcriptional levels of downstream targets of each pathway were measured by real time reverse transcriptase PCR. We also investigated the effect of EBV DNA on activating the cellular arm of the fly innate immunity. Wild type flies that were treated as described above were bled and hemocytes were counted using a Neubauer chamber. Results: Upon injection of 70 copies of EBV DNA into flies, the transcriptional level of diptericin, indicative of the IMD pathway, was increased by 115 folds on day 1 but not day 3 post-injection compared to flies injected with ste