Understanding cerebral inflammation in viral encephalitis - how does neuron-glial signalling drive blood-brain barrier permeability?

CONTEXT
Brain inflammation, termed 'encephalitis', is a devastating disease commonly caused by the cold-sore virus (herpes simplex virus-HSV). Inflammation occurs due to breakdown of the natural barrier with the bloodstream and the migration of immune-cells in. Despite antivirus medication the inflammation continues. Consequently, most patients die or have significant brain injury.

Sometimes patients are given steroids but their broad immune-suppression action risks increasing the virus, so targeted treatments are needed.

Immune-cells are attracted by inflammation-proteins; I identified several such proteins in patients with the greatest blood-brain barrier (BBB) breakdown and worst outcome, and developed a mouse-model to study this by visualising immune-cell movement in the brain.

To what extent BBB breakdown leads to immune cell migration or vice versa is not clear and it is not known how infected nerves communicate with other brain cells, and BBB cells.

Understanding this network of interactions is critical to develop targeted immune therapy which reduces brain inflammation and improves patient outcome, without increasing the amount of virus.

AIMS
1. Establish the inflammation proteins which most closely associated with BBB breakdown in HSV encephalitis and which are/are not steroid responsive

2. Interrogate how blocking these inflammation proteins alters BBB breakdown, immune cell migration and viral control

3. Determine how these immune proteins communicate between infected nerves, the other brain cells and the BBB cells

OBJECTIVES
1.From a unique cohort of HSV encephalitis patients who have received either steroids or placebo, I have collected blood and spinal fluid.
a.I will analyse these by both looking for specific inflammation proteins and also by assessing all proteins
b.I will compare them to the degree of BBB permeability from:
i.Amount of albumin protein which has leaked from the blood into the spinal fluid
ii.The volume of brain swelling on the brain scan (magnetic resonance imaging)

2.Take forward key inflammation proteins by determining their action, in a mouse model I established, using mice lacking the receptor for these inflammation proteins and/or block the inflammation protein using a neutralising antibody and determine:
a.Is morbidity decreased, using an established score?
b.Is BBB breakdown reduced, measuring the leakage of an intravenous dye into the brain?
c.Is viral control affected, using a plaque assay?
d.Is immune cell migration altered, using intravital microscopy to visualise immune cell movements in real time in the brain of an anaesthetised mouse?

3. In this model, I will analyse the generation of inflammation proteins and associated signals from each cell type in the brain and BBB over time to determine networks of communication up-stream and down-stream to key inflammation proteins.
a.I will then confirm these findings by examining the interaction between nerve cells, other brain cells, and BBB cells in a BBB model
i.In response to HSV infection of single cells and cultures of different cells which may interact
ii.I will interrogate whether blocking these communications alters BBB breakdown and immune cell migration

POTENTIAL APPLICATIONS AND BENEFITS
This is important so that I can develop targeted immune treatments to control inflammation, BBB breakdown, and leucocyte migration, without risking uncontrolled viral replication.

By establishing this for HSV it opens up treatment opportunities for a wide range of other brain infections.

Having built the capacity to generate hypotheses from clinical samples and with both a mouse model and a BBB model of brain infection, I will be excellently placed to determine the commonalities and differences between the immune response to HSV and other brain infections. Consequently, I will be able to drive forward targeted therapies and will be in a strong position to become a leader in the field.

CONTEXT
In the devastating brain infection herpes simplex virus (HSV) encephalitis, the inflammatory response, characterised by leucocyte migration, blood-brain barrier (BBB) breakdown and cerebral oedema, is associated with poor outcome, despite antivirals

Broad immunosuppression with corticosteroids may help or worsen outcome due to uncontrolled viral replication. I identified that BBB permeability and outcome correlate with several mediators in cerebrospinal-fluid (CSF), but corticosteroid impact is unknown

Targeted immunotherapy ameliorating BBB breakdown and detrimental leucocyte migration, without impairing viral control, is urgently needed. We must understand how HSV infection of neurons triggers inflammatory mediator cascades by glia and BBB breakdown

AIMS
1. Establish the mediators most closely associated with BBB permeabilisation in HSV encephalitis and which are/are not corticosteroid responsive

2. Interrogate effects of abrogating these mediators on BBB permeabilisation, leucocyte migration and viral control

3. Delineate mediator signalling networks between neurons, glia and BBB cells

METHODOLOGY
1. Analyse CSF mediators in a unique cohort of HSV encephalitis patients receiving corticosteroids/placebo (from RCT) by bead array/proteomic approaches relative to BBB permeability (CSF:serum albumin ratio and neuroimaging oedema quantification)

2. In my murine model, determine impacts of cognate receptor knock-outs and neutralising antibody-treatment, on phenotype, intravenous tracker leakage and
real-time leucocyte migration by intravital microscopy

3. In this model, use a single-cell RNA-sequencing time course and confirm findings in our BBB in vitro model, relative to leucocyte dynamics

OPPORTUNITIES
Understanding critical mediator pathways driving BBB breakdown and cerebral oedema in HSV encephalitis will direct targeted immunotherapy, has implications for other forms of encephalitis, and will help me become a field leader