Dynamic Imaging in Viral Encephalitis Defines Unique Roles for Chemoattractants.

ABSTRACT
Background-
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.

IMPORTANCE
Targeted 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 BBB

OBJECTIVES
- 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.

BACKGROUND
Herpes simplex virus (HSV) encephalitis is a devastating disease of brain inflammation characterised by blood-brain barrier (BBB) breakdown and leucocyte migration, particularly early neutrophils, forming perivascular 'cuffs'. No immune therapy is established therefore 10-30% die and most survivors have neurological morbidity. Targeted therapy which mitigates deleterious leucocyte migration and BBB breakdown is desperately required. Leucocytes migration is determined by chemokines, but increasingly atypical chemoattractant proteins/receptors are recognised. However, their role in neutrophil migration and behaviour in the brain, such as the production of neutrophil-extracellular traps (NETs), are not known.
Together the Co-PIs have the individual techniques to image this in real time by Multiphoton Intravital Microscopy (MP-IVM).

AIM
Determine the relative contribution of typical/atypical chemoattractants on neutrophil migration into the brain and the behaviour driving BBB breakdown.

SPECIFIC AIMS
1. Establish specific neutrophil migration behaviours which are associated with BBB breakdown in a murine model of HSV encephalitis.
2. Determine the contribution of typical/atypical chemoattractant receptor/ligand interactions driving this behaviour.
3. Interrogate the differential production of NETs and associated processes in BBB breakdown

METHODS
1. In an intrathecal murine model of HSV inoculation, neutrophil migration and BBB breakdown will be imaged by MP-IVM in LysM-Green Fluorescent mice. Typical/atypical chemoattractant expression will be determined by RT-Polymerase Chain Reaction and confirmed by Enzyme-Linked Immunosorbant Assay.
2. Candidate chemoattractants in knock-out or antibody-treated mice will be interrogated by MP-IVM in relation to neutrophil migration and BBB breakdown
3. NETs will be imaged by MP-IVM and single-cell RNAseq of transmigrated neutrophils will explore associated processes in the UK Co-PI's BBB model

FOSTERING LASTING COLLABORATION
The Michael and Miyabe laboratories investigate host immune processes, particularly those driving leucocyte migration, in viral infection and autoimmune disease respectively. MP-IVM is a powerful tool with which they both work and through which they are able to interrogate this leucocyte migration in real-time in vivo. This approach has broad potential to study host immune-mediated disease, either due to viral infection or in association with autoimmune disease. There is significant potential overlap between these processes during inflammation of the central nervous system (CNS).

Following the platforms and publications arising from the proposed work in this application they will seek future collaborative funding to conduct studies of the inflammatory processes underlying leucocyte migration in CNS vasculitis in response to viral infection with Varicella zoster virus and autoimmunity in a CNS lupus model.

Together these platforms and the UK-Japan collaboration will be a significant step towards these early career researchers becoming leaders in the field of leucocyte migration in brain inflammation.

SCIENTIFIC IMPACT
The burgeoning field of "Atypical" chemoattractant receptor/ligand interactions, particularly with regards to neutrophil behaviour, is one in which the Japanese Co-PI has played an important role, resulting in high-impact publications, such as with regards to a novel role for C5a receptor in arthritis. However, these processes have not been explored in viral encephalitis and we currently have little understanding of their roles at the BBB.

The UK Co-PI has determined the critical chemoattractants and associated cytokines which correlate with BBB breakdown in clinical samples and developed a murine model and BBB model of viral encephalitis through which these findings can be replicated and interrogated.

Together, using these techniques and platforms in a clinically-relevant model of brain infection provides the opportunity to identify those typical/atypical chemoattractants which determine the neutrophil migration and behaviours which initiate and perpetuate BBB permeability. This has the potential for substantive advances of our understanding of leucocyte migration processes at this critical barrier between the bloodstream and the brain.

MEDICAL IMPACT
Despite antiviral therapy many patients with HSV encephalitis will die and the majority of survivors are left with significant neurological morbidity. Although corticosteroids are sometimes given in extremis, their broad immunosuppressive effects risk licencing uncontrolled viral replication. Targeted adjunctive therapy is desperately needed which can reduce cerebral oedema and BBB breakdown without impairing viral replication. The critical step in this process is determining the key chemoattractant receptor/ligand interactions which drive this so that existing or novel targeted therapies can be investigated.

SOCIAL IMPACT
Whilst individual cases of HSV encephalitis are rare, the societal impact is huge; in part as those affected are usually otherwise healthy people. As treatment is limited to antiviral therapy, based on clinical trials in the 1980's, most survivors have significant neurological disability and many are unable to return to work and care for their family; consequently many require long-term support. Typical medical-negligence cases exceed £3-4M due to the significant loss of quality of life for those adversely affected.

EXPLOITATION POTENTIAL.
Viral infection is the leading cause of encephalitis and is associated with marked mortality/morbidity. Identifying the key chemoattractants which drive neutrophil migration and BBB breakdown will make it possible to investigate targeted therapy which ameliorate this process; thereby establishing either a novel therapy or new therapeutic indication for an existing treatment, which has implications for viral encephalitis more broadly.