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

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.

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.