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Intravital imaging of a massive lymphocyte response in the cortical dura of mice after peripheral infection by trypanosomes.

Coles JA, Myburgh E, Ritchie R, Hamilton A, Rodgers J, Mottram JC, Barrett MP, Brewer JM - PLoS Negl Trop Dis (2015)

Bottom Line: 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 population and motility of the trypanosomes tended to decline after about 30 dpi.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom; Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

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.

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T cell movement in the dura.A. Tracks of extravascular CD2+ T cells over 25 min in an uninfected mouse (S10 Video). A Z-stack 30 μm deep was acquired. The blood marker was dextran-fluorescein. B. Tracks of T cells at 40 dpi in a Z-stack 75 μm thick (S12 Video). Time along tracks is color coded over the 22 min duration of the acquisition, spheres indicate detected cells at the time point indicated (green, about 9 min). C. The total number of T cells was divided by the number of stationary T cells (with mean speed < 1 μm/min), and plotted on a log scale as a function of dpi. For the logarithm of this ratio vs dpi, r2 = 0.57 and the slope differs from zero with p = 0.0005. Each point is for one imaging area in one mouse. D. A side view of the tracks in B showing that movement was mainly close to horizontal; a few cells moved downwards or upwards at deeper levels. E. The mean track speed of each T cell was averaged for each mouse and average values were calculated for groups corresponding to short (0–5 dpi), medium (11–25 dpi) and longer periods of infection (26–30 dpi). Error bars are SEMs, Ns are 4, 6, 4 mice. F. (Displacement/track duration) of T cells for durations 300–800 sec in three uninfected mice (black) and three infected mice (red). Horizontal lines show medians.
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pntd.0003714.g006: T cell movement in the dura.A. Tracks of extravascular CD2+ T cells over 25 min in an uninfected mouse (S10 Video). A Z-stack 30 μm deep was acquired. The blood marker was dextran-fluorescein. B. Tracks of T cells at 40 dpi in a Z-stack 75 μm thick (S12 Video). Time along tracks is color coded over the 22 min duration of the acquisition, spheres indicate detected cells at the time point indicated (green, about 9 min). C. The total number of T cells was divided by the number of stationary T cells (with mean speed < 1 μm/min), and plotted on a log scale as a function of dpi. For the logarithm of this ratio vs dpi, r2 = 0.57 and the slope differs from zero with p = 0.0005. Each point is for one imaging area in one mouse. D. A side view of the tracks in B showing that movement was mainly close to horizontal; a few cells moved downwards or upwards at deeper levels. E. The mean track speed of each T cell was averaged for each mouse and average values were calculated for groups corresponding to short (0–5 dpi), medium (11–25 dpi) and longer periods of infection (26–30 dpi). Error bars are SEMs, Ns are 4, 6, 4 mice. F. (Displacement/track duration) of T cells for durations 300–800 sec in three uninfected mice (black) and three infected mice (red). Horizontal lines show medians.

Mentions: The activation state of T cells and the nature of their environment are reflected in the speed and pattern of their movement [19, 21, 34, 49–51]. To look for changes in T cell behavior in the cortical meninges during the progression of trypanosomiasis, we made videos, acquiring Z-stacks at 15–30s intervals over periods of up to 45 min. Fig 6A shows typical tracks in an uninfected mouse. Three T cells were almost stationary, one moved rapidly 28 μm along a blood vessel then careered off (S10 Video). In videos from infected mice, a greater proportion of the cells were moving (with speed >1 μm/min; Fig 6B and 6C and S11 Video). Particularly in infected mice, motile cells showed no evident preference for the vicinity of blood vessels.


Intravital imaging of a massive lymphocyte response in the cortical dura of mice after peripheral infection by trypanosomes.

Coles JA, Myburgh E, Ritchie R, Hamilton A, Rodgers J, Mottram JC, Barrett MP, Brewer JM - PLoS Negl Trop Dis (2015)

T cell movement in the dura.A. Tracks of extravascular CD2+ T cells over 25 min in an uninfected mouse (S10 Video). A Z-stack 30 μm deep was acquired. The blood marker was dextran-fluorescein. B. Tracks of T cells at 40 dpi in a Z-stack 75 μm thick (S12 Video). Time along tracks is color coded over the 22 min duration of the acquisition, spheres indicate detected cells at the time point indicated (green, about 9 min). C. The total number of T cells was divided by the number of stationary T cells (with mean speed < 1 μm/min), and plotted on a log scale as a function of dpi. For the logarithm of this ratio vs dpi, r2 = 0.57 and the slope differs from zero with p = 0.0005. Each point is for one imaging area in one mouse. D. A side view of the tracks in B showing that movement was mainly close to horizontal; a few cells moved downwards or upwards at deeper levels. E. The mean track speed of each T cell was averaged for each mouse and average values were calculated for groups corresponding to short (0–5 dpi), medium (11–25 dpi) and longer periods of infection (26–30 dpi). Error bars are SEMs, Ns are 4, 6, 4 mice. F. (Displacement/track duration) of T cells for durations 300–800 sec in three uninfected mice (black) and three infected mice (red). Horizontal lines show medians.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400075&req=5

pntd.0003714.g006: T cell movement in the dura.A. Tracks of extravascular CD2+ T cells over 25 min in an uninfected mouse (S10 Video). A Z-stack 30 μm deep was acquired. The blood marker was dextran-fluorescein. B. Tracks of T cells at 40 dpi in a Z-stack 75 μm thick (S12 Video). Time along tracks is color coded over the 22 min duration of the acquisition, spheres indicate detected cells at the time point indicated (green, about 9 min). C. The total number of T cells was divided by the number of stationary T cells (with mean speed < 1 μm/min), and plotted on a log scale as a function of dpi. For the logarithm of this ratio vs dpi, r2 = 0.57 and the slope differs from zero with p = 0.0005. Each point is for one imaging area in one mouse. D. A side view of the tracks in B showing that movement was mainly close to horizontal; a few cells moved downwards or upwards at deeper levels. E. The mean track speed of each T cell was averaged for each mouse and average values were calculated for groups corresponding to short (0–5 dpi), medium (11–25 dpi) and longer periods of infection (26–30 dpi). Error bars are SEMs, Ns are 4, 6, 4 mice. F. (Displacement/track duration) of T cells for durations 300–800 sec in three uninfected mice (black) and three infected mice (red). Horizontal lines show medians.
Mentions: The activation state of T cells and the nature of their environment are reflected in the speed and pattern of their movement [19, 21, 34, 49–51]. To look for changes in T cell behavior in the cortical meninges during the progression of trypanosomiasis, we made videos, acquiring Z-stacks at 15–30s intervals over periods of up to 45 min. Fig 6A shows typical tracks in an uninfected mouse. Three T cells were almost stationary, one moved rapidly 28 μm along a blood vessel then careered off (S10 Video). In videos from infected mice, a greater proportion of the cells were moving (with speed >1 μm/min; Fig 6B and 6C and S11 Video). Particularly in infected mice, motile cells showed no evident preference for the vicinity of blood vessels.

Bottom Line: 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 population and motility of the trypanosomes tended to decline after about 30 dpi.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom; Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus