Intravital Imaging of a Massive Lymphocyte Response in the Cortical Dura of Mice after Peripheral Infection by Trypanosomes

Abstract

Peripheral infection by Trypanosoma brucei, the protozoan responsible for sleeping sickness, activates lymphocytes, and, at later stages, causes meningoencephalitis. We have videoed the cortical meninges and superficial parenchyma of C56BL/6 reporter mice infected with T. b. brucei. By use of a two-photon microscope to image through the thinned skull, the integrity of the tissues was maintained. We observed a 47-fold increase in CD2+ T cells in the meninges by 12 days post infection (dpi). CD11c+ dendritic cells also increased, and extravascular trypanosomes, made visible either by expression of a fluorescent protein, or by intravenous injection of furamidine, appeared. The likelihood that invasion will spread from the meninges to the parenchyma will depend strongly on whether the trypanosomes are below the arachnoid membrane, or above it, in the dura. Making use of optical signals from the skull bone, blood vessels and dural cells, we conclude that up to 40 dpi, the extravascular trypanosomes were essentially confined to the dura, as were the great majority of the T cells. Inhibition of T cell activation by intraperitoneal injection of abatacept reduced the numbers of meningeal T cells at 12 dpi and their mean speed fell from 11.64 ± 0.34 μm/min (mean ± SEM) to 5.2 ± 1.2 μm/min (p = 0.007). The T cells occasionally made contact lasting tens of minutes with dendritic cells, indicative of antigen presentation. The population and motility of the trypanosomes tended to decline after about 30 dpi. We suggest that the lymphocyte infiltration of the meninges may later contribute to encephalitis, but have no evidence that the dural trypanosomes invade the parenchyma.

Author Summary

African trypanosomes are motile parasites that cause sleeping sickness. They multiply first in the blood then cause death mainly by effects on the brain: immune system cells, including T cells and dendritic cells, play major roles in this. Thinking we might see the attack on the brain, we infected mice with trypanosomes and used a two-photon microscope, which allowed us to image the superficial brain and the delicate tissue between the skull and the brain called the meninges without making a hole in the skull. The mice (which were anesthetized) had been genetically modified so that T cells and dendritic cells were fluorescent, as were the trypanosomes. We did not notice much happening in the brain itself, but in the meninges, in a compartment called the dura, huge numbers of T cells and dendritic cells appeared. Trypanosomes also moved from the blood into this compartment. Since T cells, dendritic cells and trypanosomes had not been videoed in the meninges before, we began by observing them carefully: their numbers, their movements and their interactions. The accumulation of lymphocytes is a sign of meningitis, a feature of infection by a wide range of pathogens and our results suggest interesting future work.