Dynamic Imaging in Viral Encephalitis Defines Unique Roles for Chemoattractants.

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ABSTRACTBackground-Viral encephalitis, most commonly due to Herpes simplex virus (HSV), is a devastating disease of brain inflammation characterised by break down in the integrity of the blood-brain barrier (BBB) and the migration of immune cells, particularly early neutrophils, into the brain where they form perivascular 'cuffs' around blood vessels. No immune therapy is established therefore many patients die and most survivors have brain injury. Therapy which causes broad immune suppression risks uncontrolled viral replication. Therefore, targeted therapy which mitigates deleterious immune cell migration and BBB breakdown is desperately required. The migration of immune cells is determined by attractant proteins, termed chemokines, but increasingly atypical attractant proteins are recognised. However, this has not be investigated in encephalitis and their role in neutrophil behaviour in the brain, such as the production of neutrophil-extracellular traps (NETs), and the impact on BBB permeability are not known.Collaboration-Whilst both UK and Japanese PIs were completing their Post-Doctoral training in the Center for Immunology (MGH/Harvard) they noted significant added value in the investigatory skills required to address their overlapping disciplines. The UK PI established a multiphoton intravital microscopy (MP-IVM) model of viral encephalitis as a powerful tool to visualize BBB breakdown and immune cell migration in real-time.The Japanese PI has extensive experience of MP-IVM in non-CNS inflammation identifying novel roles for atypical attractant proteins and has established direct visualisation attractant proteins and also neutrophil behaviour.Specific aims-We will determine the relative contribution of typical/atypical attractants proteins on neutrophil migration into the brain and the post-migration behaviour driving BBB breakdown in HSV encephalitis.Hypotheses-Following HSV infection, co-ordination of specific typical/atypical attractant proteins cause neutrophils to migrate into the brain and drive BBB breakdown through NET production.Methodology and approach-- Use MP-IVM to determine neutrophil migratory behaviour in relation to BBB breakdown and the expression of typical/atypical attractants proteins and their receptors. - Interrogate the impact of the abrogation of these proteins on neutrophil behaviour and BBB breakdown.- Use MP-IVM to visualise the post-migration production of NETs and associated processes and use single cell approaches to explore wider up-regulation.IMPORTANCETargeted immune therapy which mitigates neutrophil migration and ameliorates BBB breakdown without allowing uncontrolled viral replication is desperately needed for viral encephalitis.KEY AIMS AND OBJECTIVES AIMS- Establish the specific neutrophil migration behaviour which correlates with BBB breakdown in a murine model of HSV encephalitis.- Determine the relative contribution of typical/atypical attractant protein-receptor interactions which drive this behaviour.- Interrogate the differential production of NETs and associated processes in the break down in integrity of the BBBOBJECTIVES- Share existing experience and skills between UK and Japanese PIs, including cranial windowing surgery for MP-IVM of the brain and also real-time attractant protein and neutrophil behaviour visualisation; building a strong partnership for future collaborative research.- Exchange Early Career Researchers for exposure of these techniques in UK and Japanese laboratory settings to begin to build capacity which lays the ground work for larger grants to establish leading laboratories in the field of immune cell-migration biology.- Use the complementary platforms of MP-IVM (Japan) and BBB modelling (UK) to demonstrate the additive roles of the two approaches which has broad potential as a basis for exploring immune cell migration in response to infection and autoimmunity.