Defining the parasitological and immunological basis of cerebral pathology during murine experimental cerebral malaria

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

Cerebral malaria (CM) is a severe complication of some human malaria infections. CM is believed to be caused by the sticking (sequestration) of malaria-infected red blood cells and/or specific types of immune cells (white blood cells) in the small blood vessels of the brain. These blood vessels become clogged, blood flow is reduced and the lack of oxygen and other nutrients causes the patient to go into a coma or to have fits.

In order to prevent or treat cases of CM, we need a better understanding of the cellular and molecular processes that lead to sequestration of red and white blood cells in the brain. It is impossible to carry out these studies in human malaria patients, but a clinically similar syndrome (experimental CM; ECM) develops in mice infected with particular strains of rodent malaria parasites. Even so, until now, it has been impossible to visualise the key events leading up to ECM in an intact mouse brain.

I propose to use novel microscopic imaging techniques to visualise the migration, localisation and consequences of parasite and immune cell sequestration in the brains of infected mice and to identify the key molecules and cells involved in development of ECM.

Technical Summary

Cerebral pathology (CM) is a life-threatening consequence of human Plasmodium falciparum infection and a comparable syndrome (experimental CM; ECM) is seen in susceptible strains of mice infected with P. berghei ANKA. Cerebral accumulation of both parasitised erythrocytes (iRBC) and leucocytes has been implicated in the initiation of CM and ECM, but the sequence of events and the relative importance of these two cell types remains unclear. The advent of new tools for high resolution tracking of individual live cells gives us an opportunity, for the first time, to definitively address these shortfalls in our knowledge. I will use dynamic multi-photon imaging of living brains and thick brain sections of malaria-infected mice, combined with transgenic fluorescently-tagged parasites and various fluorescently-tagged leucocyte populations, to examine the intracerebral migration and localisation of iRBC and leucocytes, enabling the elucidation of the cellular and molecular events that direct these pathological events. Intracerebral parasitic accumulation patterns and leucocyte migration and function will be compared during P. berghei ANKA (ECM causing) infection of C57BL/6 (ECM susceptible) and BALB/c (ECM resistant) mice and with P. berghei NK65 (non-ECM promoting parasite strain) infection of C57BL/6 mice to define the basis of cerebral pathology.

By integrating imaging techniques with standard immunological approaches (e.g. cell depletion, flow cytometry and real-time PCR) I will determine the phenotype and function of brain infiltrating leucocytes; determining whether specific leucocyte populations (e.g. CD4+ and CD8+ T cells, monocytes) migrate to, and exert effector functions in, specific areas of the brain; and whether this correlates with sites of iRBC sequestration and/or with sites of brain pathology (haemorrhage, oedema, endothelial damage). I will examine whether these responses (homing, sequestration and cytokine production) change during secondary P. berghei ANKA infections (utilising a drug-cure infection model), to investigate the generation of immunological memory to infection and how this alters susceptibility or resistance to ECM.

Finally, I will determine whether the leucocyte populations that localise in the brain and contribute to development of ECM differ from the leucocytes that are required to control parasite replication and eliminate the infection. For example, pathogenic lymphocytes may differ from protective populations in their effector function (production of cytokines, lytic molecules, superoxides etc), tissue homing (chemokine receptor expression) or regulation (regulatory receptor expression). Putative biomarkers of protective or pathogenic leucocyte populations will be validated using relevant transgenic or gene knock-out mice.

Publications

10 25 50

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Gwyer Findlay E (2013) IL-27 receptor signaling regulates CD4+ T cell chemotactic responses during infection. in Journal of immunology (Baltimore, Md. : 1950)

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Riley EM (2010) Neuropathogenesis of human and murine malaria. in Trends in parasitology

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Strangward P (2018) Targeting the IL33-NLRP3 axis improves therapy for experimental cerebral malaria. in Proceedings of the National Academy of Sciences of the United States of America

 
Description MRC Centenary Award
Amount £80,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2012 
End 08/2013
 
Description New Investigator Project Grant
Amount £485,704 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2011 
End 09/2014
 
Description Responsive mode research grant
Amount £670,000 (GBP)
Funding ID MR/R010099/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2021
 
Description research grant
Amount £450,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2014 
End 04/2017
 
Title P. berghei ANKA TagRFP 
Description P. berghei ANKA parasites were transfected with the far red optimized reporter TagRFP, which was inserted under the EF-1alpha promoter. These parasites are optimized for 2 photon imaging. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2011 
Provided To Others? Yes  
Impact The far red (excitation >1000nm) reporter parasites has enabled us to image and locate parasite deeper within tissues. The red shift of the parasites allows multiple colour imaging to visualise multiple processes/events simultaneously. 
 
Title P. berghei ANKA mKateS158 
Description Transgenic P. berghei ANKA parasites where the fluorescent far red optimised reporter mKateS158 was inserted into the parasite genome under the EF-1alpha promoter 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2011 
Provided To Others? Yes  
Impact The far red (excitation >1000nm) reporter parasites has enabled us to image and locate parasite deeper within tissues. The red shift of the parasites allows multiple colour imaging to visualise multiple processes/events simultaneously. 
 
Title P. berghei NK65-OVA plasmid 
Description P. berghei NK65 parasite genetically engineered to express an episomal plasmid (maintained under drug selection pressure) that expresses OVA. Useful for following surrogate parasite specific T cell immune responses 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2012 
Provided To Others? Yes  
Impact Manuscript in preparation. 
 
Title P. berghei mCherry 
Description Transgenic P. berghei ANKA parasites were created where the fluorescent reporter mCherry was inserted into the parasite genome under the Ef-1alpha promoter 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2011 
Provided To Others? Yes  
Impact Enabled the visualization of parasites within the brain of infected mice by two-photon microscopy 
 
Title Cerebral malaria drug cure whole brain RNA-seq database 
Description Whole brain RNA-seq datasets from mice pre and post-drug treatment of cerebral malaria, identifying the transcriptomic changes enforced in the brain following drug treamtent and assessing the temporal recovery of the brain following treatment of the condition. The database can be used to identify new adjunct treatments for the condition 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact None yet but validation of predicted targets for adjunct treatments is ongoing 
 
Title Exposure-induced resistance microarray database 
Description Whole genome mouse microrray datasets examining differential gene expression in brains of 4 groups of mice experiencing: 1. Primary infection with Plasmodium berghei ANKA. Mice have late-stage experimental cerebral malaria (day 7 of infection); 2. 4th infection (repeated exposure-induced resistance model). Mice are resistant to experimental cerebral malaria (day 7 of 4th infection); 3. uninfected but age matched; 4. Mice that have experienced 3 rounds of infection-drug cure but are currently uninfected (30 days after clearance of parasites after 3rd infection). 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact We have used these datasets to examine the molecular basis of parasite-exposure induced resistance to experimental cerebral malaria. The datasets will be deposited and shared with investigators when the work is published 
 
Description Generation of transgenic Plasmodium berghei parasites 
Organisation University of Edinburgh
Department Institute of Immunology and Infection Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated targeting vectors for insertion in Plasmodium berghei ANKA parasites
Collaborator Contribution Inserted targeting vectors into parasites
Impact Transgenic P. berghei ANKA parasites optimised for 2 photon imaging: P. berghei ANKA-mCherry P. berghei ANKA-TagRFP P. berghei ANKA-mKate Collaborators provide molecular parasitology expertise to develop the parasites for use in immunological and parasitological studies.
Start Year 2009
 
Description Generation of transgenic parasites 
Organisation Leiden University Medical Center
Country Netherlands 
Sector Academic/University 
PI Contribution Instigated the collaboration to make the transgenic malaria parasites. Tested and validated the developed parasites.
Collaborator Contribution Generated he trasngenic OVA-expressing Plasmodium berghei NK65 parasites by reverse purifying existing plasmids from transgenic parasites. Expanded upon the original collaboration and generated oher transgenic parasite lines where the OVA is inserted under diffeent promoters.
Impact Manuscripts are still in preparation. 3 lines of OVA expressing Plasmodium berghei ANKA and 3 lines of OVA expressing Plasmodium berghei NK65
Start Year 2011
 
Description Integrating research in murine and human cerebral malaria 
Organisation Liverpool School of Tropical Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We bring the expertise in murine experimental cerebral malaria (ECM) and the detailed investigation of ECM patogenesis, pathology and resolution.
Collaborator Contribution The partners bring expertise in human cerebral malaria and tools and methods for studying the pathogenesis of human cerebral malaria. They also allow access to human post-mortem brain samples to study.
Impact A research paper: http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006267 and a MRC project grant: MR/R010099/1
Start Year 2016
 
Description Modelling of T cell behaviour in the brain 
Organisation University of Manchester
Department School of Computer Science
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated the data to be modelled
Collaborator Contribution Applied mathematical modelling to the complex data sets to allow us to better understand T cell tafficking and behaviour in the brain of malaria infected mice
Impact Collaboration only recently established
Start Year 2012