Investigating the mechanisms of brain innate immune activation following bacterial infection in Drosophila

Abstract

AbstractBackgroundCentral Nervous System (CNS) infections caused by bacteria and resulting immune responses could underlie neurodegenerative diseases such as Alzheimer’s disease (AD). Neuroinflammation is a well‐known component of AD furthering disease pathology and progression. However, the mechanisms by which infectious agents contribute to neuroinflammatory reactions and neurodegeneration are not fully understood. The model organism Drosophila melanogaster shares conserved mechanisms of innate immune activation and neural development with mammals, and thus represents an excellent system for the investigation of pathogen‐associated neurodegeneration. Previous work in flies has shown that bacterial brain injection results in age‐dependent neurodegeneration and impaired locomotor activity. Furthermore, these phenotypes depend on NF‐κB in both glia and neurons, suggesting that long‐term inflammatory processes take place in both cell types. However, a detailed picture of innate immune pathway activation in the Drosophila brain following bacterial infection is lacking. Here, we investigate the activation in the brain of the two Drosophila NF‐ κB pathways, Toll and Immune deficiency (IMD), in response to infection with Gram‐positive and Gram‐negative bacteria, respectively.MethodFly brains are infected by pricking heads with a thin, metal needle, dipped into concentrated pellets of overnight cultured bacterial solutions. Activation of NF‐κB pathways in brain cell types is assayed using immunohistochemistry and confocal microscopy of flies carrying fluorescent NF‐κB reporters. The dependence of canonical NF‐κB pathway components is assayed using quantitative reverse‐transcription PCR (RT‐qPCR) using RNA isolated from dissected brains of control‐injected or bacterially injected flies.ResultOur preliminary results show that brain bacterial injection leads to the activation of GFP reporters for both Toll and IMD pathways in multiple glial subtypes in the fly brain 24h following infection. We have optimized a protocol for brain‐specific gene expression of NF‐κB targets and are currently examining the response to bacterial challenge of multiple mutants for components of the Toll and IMD pathways.ConclusionWe have established a Drosophila model of bacterial brain infection that allows studying the long‐term consequences of pathogen‐mediated neuroinflammatory reactions associated with neurodegeneration. Our system could be used to identify the processes of immediate protection as well as the long‐term effects of brain infection.