<i>In vivo</i> imaging of MPO-specific bioluminescence in mice infected with <i>B</i>. <i>pahangi</i> L3 larvae.

<p>BALB/c mice were injected with 50 L3 of <i>B</i>. <i>pahangi</i> into the RHS footpad (<i>B</i>.<i>p</i>) and with HBSS into the LHS footpad. On day 4 (<b>A</b>) and day 11 (<b>B</b>) post-infection mice were imaged using an IVIS spectrum 20 minutes after subcutaneous injection of 200 mg/kg luminol. Representative images of 3 mice per group are shown. The colour scale indicates bioluminescence radiance in photons/second/cm²/steradian. Graphs show the bioluminescence total flux (in photons/second) within the same footpad region of interest (ROI, red ovals in images). Each symbol shows the total flux for a single mouse, lines indicate the means (n = 7 mice) and error bars show SD (**<i>p</i> < 0.01 using a Mann-Whitney test).</p

<i>In vivo</i> imaging of MPO-specific bioluminescence in mice implanted with adult <i>B</i>. <i>pahangi</i>.

<p>BALB/c mice were infected intraperitoneally by transplantation with 10 female adult <i>B</i>. <i>pahangi (B</i>.<i>p)</i>. Sham-operated mice (Sham) received no worms and control mice (Ctl) received an intraperitoneal injection of HBSS but underwent no surgery. On day 17 post-infection mice were imaged using an IVIS spectrum 20 minutes after subcutaneous injection of 200 mg/kg luminol. Representative images of 2 mice per group are shown. The colour scale indicates bioluminescence radiance in photons/second/cm²/steradian. Graphs show the bioluminescence total flux (in photons/second) within the same abdominal region of interest. Each symbol shows the total flux for a single mouse, lines indicate the means (n = 4–6 mice) and error bars show SD (*<i>p</i> < 0.05 using a one-way ANOVA with Dunn’s post-test).</p

Histopathology of <i>B</i>. <i>pahangi</i> infected and uninfected limbs in a mouse model.

<p>BALB/c mice were injected with 50 L3 of <i>B</i>. <i>pahangi</i> into the RHS footpad and with HBSS into the LHS footpad and analysed on the days p.i. as indicated. <b>(A)</b> uninfected mouse, Day 21, HBSS-injected limb: there is no inflammation in the fascial plane <b>(B)</b> infected mouse, Day 7: a filarial nematode is present within the lumen of a dilated lymphatic vessel in the fascial plane. <b>(C)</b> infected mouse, Day 14 prominent lymphangiectasis and lymphangitis in the fascial plane. <b>(D)</b> infected mouse, Day 21: marked inflammatory infiltration obliterating the lumen of a lymphatic vessel. Inset: remnants of degenerate nematodes within the lumen surrounded by inflammatory cells. <b>(E)</b> infected mouse (same animal as section C), Day 14: the inflammatory infiltrate affecting the lymphatic vessel in the fascial plane is characterised by the presence of many eosinophils highlighted by the modified haematoxylin eosin method. <b>(F)</b> infected mouse, Day 21: nematodes surrounded by intense inflammatory infiltration comprising large numbers of eosinophils highlighted by the modified Congo Red protocol.</p

T cell movement in the dura.

<p><b>A</b>. Tracks of extravascular CD2⁺ T cells over 25 min in an uninfected mouse (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003714#pntd.0003714.s015" target="_blank">S10 Video</a>). A Z-stack 30 μm deep was acquired. The blood marker was dextran-fluorescein. <b>B</b>. Tracks of T cells at 40 dpi in a Z-stack 75 μm thick (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003714#pntd.0003714.s017" target="_blank">S12 Video</a>). 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). <b>C</b>. 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 <i>p</i> = 0.0005. Each point is for one imaging area in one mouse. <b>D</b>. A side view of the tracks in <b>B</b> showing that movement was mainly close to horizontal; a few cells moved downwards or upwards at deeper levels. <b>E.</b> 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. <b>F</b>. (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.</p

T cells contacted dendritic cells in the meninges.

<p><b>A-C</b>. A T cell that made contact with a dendritic cell for 5.4 min. During 25 min of imaging, the T cell indicated by the arrow contacted a dendritic cell (green, part of an agglomeration) at 18.3 min (<b>A</b>) remained in place (<b>B</b>), and left at 23.7 min (<b>C</b>). 25 dpi. <b>D,E</b>. A T cell (arrow, red) that remained in contact with a dendritic cell (yellow) throughout 20 min of imaging (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003714#pntd.0003714.s018" target="_blank">S13 Video</a>). 25 dpi. Both scale bars 20 μm. <b>F-I</b>. Illustrative plots of T cell speed changing with time. <b>J</b>. The mean skewness of the distribution of speed about the mean speed of at least 10 tracks per mouse was calculated and plotted against the number of T cells in the dura. Each symbol corresponds to one mouse. Purple symbols indicate three mice treated with abatacept.</p

Extravascular trypanosomes appear in the meninges.

<p><b>A.</b> GFP trypanosomes in the meninges at 32 dpi. Blue: collagen. Faint magenta: blood marker (Qdots). <b>B</b>. At the level of vertical penetrating vessels and parenchymal capillaries, intravascular trypanosomes were detected (arrow), but extravascular trypanosomes were not. 16 dpi. <b>C</b>. A trypanosome labeled by intravenous injection of furamidine. In this case, the nucleus gave red fluorescence (arrow) and the cytoplasm gave blue fluorescence. <b>D</b>. Trypanosomes (identifiable by their rapid movement as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003714#pntd.0003714.s010" target="_blank">S5 Video</a>) giving red fluorescence after labeling with i.v. furamidine. Furamidine also produced blue fluorescence from endogenous nuclei. Single plane, 25 dpi, CD-1 mouse. <b>E</b>. Numbers of trypanosomes in the meninges as a function of dpi (green triangles). Their appearance tends to be later than the increase in T cells (red lozenges). In 8 mice, T cell number had increased but no trypanosomes were observed in the meninges. <b>F.</b> The number of trypanosomes in the dura shows no dependence on paristemia. <b>G</b>. Numbers of trypanosomes in the dura 20–40 dpi in C57BL/6 mice and CD1 mice (from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003714#pntd.0003714.ref020" target="_blank">20</a>]).</p

Infection increases the number of dendritic cells in the meninges.

<p><b>A.</b> In uninfected mice, there was a small population of CD11c⁺ (EYFP) cells (yellow) in the meninges, mainly close to dural vessels (gray). <b>B</b>. The numbers increased by 12 dpi. SHG signal (blue) indicates the proximity of the skull; blood vessels are magenta. <b>C</b>. Approximate numbers of dendritic cells per unit area of meninges. Each symbol is the mean for one mouse. <b>D</b>. In a 3D reconstruction of a 139 μm Z-stack at 27 dpi, the dendritic cells are seen to be close under the skull (blue) and above or beside vessels (magenta). The grid spacing is 42.4 μm <b>E</b>. At 11 dpi in the same mouse as (<b>B</b>) a deeper XY plane shows only two CD11c⁺(YFP) cells, located on horizontal pial vessels embedded in the brain surface (arrows). Arrowheads point to vertical vessels below the pia mater.</p

Abatacept reduced the increase in meningeal T cells.

<p><b>A.</b> CD2⁺ (DsRed) cells in the meninges at 11 dpi, untreated control. Z-projection, 15 μm. Blue from SHG indicates the proximity of the skull. <b>B</b>. A companion mouse treated with abatacept. <b>C</b>. Numbers of meningeal T cells and dendritic cells, in uninfected controls, and untreated and treated mice at 11–12 dpi. Each symbol is the mean for several fields for one mouse. <i>p</i> values were calculated by Student's <i>t</i> test after log transformation. <b>D</b>. Mean speeds of T cells in three untreated and three treated mice. <b>E</b>. Parasitemia (blue) and numbers of meningeal trypanosomes (green) in infected mice at 11–12 dpi without or with abatacept treatment. Each symbol corresponds to one mouse.</p

T cells and CD11c+ cells show some interaction with the vascular endothelium.

<p><b>A.</b> A T cell (red, arrow) is almost stationary. <b>B.</b> The green blood marker image of the same frame confirms that the T cell was intravascular. <b>C</b>. 18 min later the T cell has moved only 2.5 μm. <b>D</b>. 20s after that it has moved out of the field. <b>D,E,F.</b> Frames at 2s intervals show a CD11c+ cell moving slowly (19.3 μm/s) in a blood vessel.</p

As infection progressed, trypanosomes in the dura made shorter displacements.

<p>Automated tracking suggests that at 11 dpi (<b>A</b>) extravascular trypanosomes make longer displacements compared to 32 dpi (<b>B</b>). Scale bar applies to both images. <b>C</b>. Maximum excursions measured on 12s videos. Each symbol is the mean of the maximum excursions for all trypanosomes imaged one image field. Pooled results from 3 mice 11–14 dpi and 2 mice 36 and 39 dpi. <b>D</b>. Another frame from the mouse of (<b>A</b>) showing trypanosomes and T cells in approximately the same plane, but apparently not interacting (see also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003714#pntd.0003714.s014" target="_blank">S9 Video</a>). All scale bars are 20 μm.</p