Trophoblast topology dynamics transiently remodels maternal blood spaces.

<p>(<b>A</b>) Intravital sequential images showing gradual occlusion (dotted circle) and opening (solid line and arrow) of blood space areas (black regions); in blue placental tissue (see Video S3); (<b>B</b>) Sequential images of a 4.3 min intravital acquisition showing intermittent blood flow as represented by IE stop-go movement: full line arrows point to IE before moving while the dashed arrow indicates movement (see Video S4). (<b>C</b>) Tracking of individual IE (indicated in b) show distance travelled by infected cells in the movement phase (M) alternating with stationary phases (S). (<b>D</b>) Diagram illustrating blood flow disturbance by transient trophoblast topological changes that occlude maternal blood space (MBS) and interrupt blood flow (1 to 3); dashed rectangle indicates the imaging plane incidence and shows only events captured on that plane by the 2-photon microscopy (4–6); the third dimension is inferred by compiling sequential images. Possible involvement of “Coan-Burton” bridges is illustrated (C-BB). Red dots: erythrocytes; dashed semi-circle delimits MBS undergoing occlusion; FC: fetal capillary; in blue, placental tissue. Scale bars 20 µm. Illustration of placental tissue fraction (<b>D</b>) was adapted from scanning electron micrograph from Coan <i>et al. </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003154#ppat.1003154-Watson1" target="_blank">[18]</a>.</p

Stationary behavior of parasitized erythrocytes in the placenta.

<p>Intravital imaging was performed on G18, five days after infection with <i>P. berghei</i>-ANKA GFP+ IE. (<b>A</b>) Sequential images of an infected cell (arrow) that moves towards the trophoblast and progressively acquire stationary behavior (2 last images) (see Video S7); (<b>B</b>) Two images within 150 seconds interval showing stationary IE (arrow) in a “niche” while other IE travel according to blood flow (see Video S8); (<b>C</b>) Images show infected cell (arrow) in transient contact with fetal-derived tissue structure for approx. 80 s (see Video S9); (<b>D,E,F</b>) Velocity plots of IE depicted in <b>A,B</b> and <b>C</b>, respectively; arrows in the legends refer to IE indicated in the respective images (arrow). Infected cells were tracked in images from the videos to ascertain individual cell velocity. Blue: placental tissue; green: IE Scale bar: 20 µm.</p

Labyrinth structure in non-infected (A–C) and infected placenta (D,E) at G18.

<p>Infection was performed with <i>P. berghei-</i>ANKA on G13. (<b>A</b>) Sagittal section of the placenta highlighting the labyrinth (La); (<b>B,C</b>) Magnified area of the labyrinth of non-infected placenta showing maternal blood space (MBS), fetal capillary (FC) identified by the presence of endothelial cells (EC), cytotrophoblast (C) and syncytiotrophoblast (S) which together form the interhaemal membrane (IM) as depicted in (<b>C</b>); (<b>D</b>) Thickening of trophoblast layer (delimited area) between maternal and fetal circulations in infected placenta (arrow point to IE); (<b>E</b>) Infected erythrocytes (arrows) are restricted to maternal compartment. MBS: maternal blood space; FC: fetal capillary. Scale bars: 1 mm (<b>A</b>); 50 µm (<b>B,E</b>); 25 µm (<b>C,D</b>).</p

Blood flow heterogeneity in non-infected placenta.

<p>Placental imaging was performed on G18 in non-infected pregnant BALB/c mouse as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003154#ppat-1003154-g002" target="_blank">Figure 2</a>. (A) Three different time points of a 5 min intravital acquistion (600×) showing regions of low (full line arrows), intermediate (dashed line arrows) or high blood flow (open arrowhead); blood fluid was labeled with Dextran-Rhodamine (red) and blood cells are in black (see Video S5); (B) Areas of high (full line), intermediate (dashed line) and low flow (dotted line) were delimited within the maternal blood space for quantification of mean pixel values in each frame using Fiji imageJ and data was plotted in (C). *p<0.05; **p<0.01; ***p<0.0001 (one-way ANOVA with Tukey's multiple comparison test). Scale bar: 50 µm.</p

Blood flow determines movement of infected erythrocytes in the placental labyrinth.

<p>Placental imaging was performed on G18 in infected pregnant BALB/c mouse. (A) Sequential time-points of a 1.6 min intravital acquisition (Video S6) showing IE that are stationary (full line arrows) in areas of low blood flow (dashed line) or moving (dashed arrows) in high flow areas (full line); (B) Maximum intensity projection along the z axis of sequential frames collected during 37.8 s (55.8 s to 93.6 s; illustrated in A); arrows point to stationary IE in low flow area (as depicted in A); in high blood flow areas (full line) various IE represents parasite movement across time. Z-project was performed using Fiji imageJ. Red: Dextran-Rhodamine labeled blood fluid; green: IE; blue: CFP+ trophoblast; FC: fetal capillary. Scale bar: 30 µm.</p

Fate of stationary parasitized erythrocytes in the placenta includes migration across blood spaces and phagocytosis.

<p>(<b>A,B,C</b>) Intravital imaging was performed on G18, five days after infection with <i>P. berghei</i>-ANKA GFP⁺ IE. (<b>A</b>) Sequential images show stationary IE (arrows) that traverse the “Coan-Burton bridge” (C-BB) that divide maternal blood space (MBS) remaining stationary in that site (see Video S11); (<b>B</b>) Velocity plot of IE depicted in <b>A</b>; arrows in the legends refer to IE indicated in the image. Dashed arrow indicates direction of flow; velocity of IE was calculated in the area highlighted in the diagram. (<b>C</b>) Sequential images show fetal-derived phagocyte (arrow) migration towards an IE (arrowhead) and engulfment of the infected cell (see Video S12). (<b>D,E,F</b>) Immunohistochemistry of <i>P. berghei</i>-ANKA GFP⁺ infected placental sections (G18) stained with biotinilated anti-Mac-1 antibody developed with streptavidin-HRP Cy3 conjugate. (<b>D</b>) Mac-1⁺ fetal–derived cells oriented towards MBS; images in dashed squares are magnified (upper and lower right). (<b>E</b>) Infected erythrocytes within Mac-1⁺ fetal-derived cells (upper left and upper right inset) and Mac-1⁻ fetal cells (upper right, lower right and lower left), magnified in insets (arrows point to GFP⁺ parasite-derived material). (<b>F</b>) Parasite-derived material within syncytiotrophoblast layer. Blue: CFP⁺ fetal-derived placental tissue; green: IE, red: Mac-1. Scale bar: 20 µm (A and C) and 10 µm (D, E F).</p

IE accumulation in the labyrinth is favored in regions of low blood flow.

<p>Placental imaging was performed on G18 in pregnant BALB/c mouse after infection with <i>P. berghei-</i>ANKA at G13. (<b>A</b>) Binary images with maternal blood spaces in white and trophoblast in black; maternal blood regions were delimited (red) before (<b>B</b>) and after (<b>C</b>) occlusion by a “Coan-Burton bridge” (arrow) (see Video S2) interrupting flow (white area). (<b>D</b>) Number of IE/region at each 5 time-points was recorded over a 300 s period. Tracking of individual IE was performed during the entire acquisition period in RI (<b>E</b>) and RIII (<b>G</b>) and during the first 75 s in RII (<b>F</b>). (<b>H</b>) Average velocity of individual (top) or grouped (bottom) IE in the indicated regions (one-way ANOVA with Tukey's post test; **<i>p</i><0.01; ***<i>p</i><0.001).</p

P2X7 receptor drives Th1 cell differentiation and controls the follicular helper T cell population to protect against <i>Plasmodium chabaudi</i> malaria

<div><p>A complete understanding of the mechanisms underlying the acquisition of protective immunity is crucial to improve vaccine strategies to eradicate malaria. However, it is still unclear whether recognition of damage signals influences the immune response to <i>Plasmodium</i> infection. Adenosine triphosphate (ATP) accumulates in infected erythrocytes and is released into the extracellular milieu through ion channels in the erythrocyte membrane or upon erythrocyte rupture. The P2X7 receptor senses extracellular ATP and induces CD4 T cell activation and death. Here we show that P2X7 receptor promotes T helper 1 (Th1) cell differentiation to the detriment of follicular T helper (Tfh) cells during blood-stage <i>Plasmodium chabaudi</i> malaria. The P2X7 receptor was activated in CD4 T cells following the rupture of infected erythrocytes and these cells became highly responsive to ATP during acute infection. Moreover, mice lacking the P2X7 receptor had increased susceptibility to infection, which correlated with impaired Th1 cell differentiation. Accordingly, IL-2 and IFNγ secretion, as well as T-bet expression, critically depended on P2X7 signaling in CD4 T cells. Additionally, P2X7 receptor controlled the splenic Tfh cell population in infected mice by promoting apoptotic-like cell death. Finally, the P2X7 receptor was required to generate a balanced Th1/Tfh cell population with an improved ability to transfer parasite protection to CD4-deficient mice. This study provides a new insight into malaria immunology by showing the importance of P2X7 receptor in controlling the fine-tuning between Th1 and Tfh cell differentiation during <i>P</i>. <i>chabaudi</i> infection and thus in disease outcome.</p></div

Splenic B6 or <i>P2rx7</i>^-/- CD4 cell co-transfer and protection against <i>Pc</i> infection.

<p>(A-D) Naïve CD4⁺ cells from B6 (CD45.1) and <i>P2rx7</i>^-/- (CD45.2) female mice were co-transferred into <i>Cd4</i>^-/- female mice that were then infected with 1 × 10⁶ <i>Pc</i>-iRBCs. Splenic CD4⁺ cells were analyzed at 30 days p.i. Non-infected mice were used as controls (day 0). The data were expressed as means ± SD (<i>n</i> = 5) of one representative experiment out of three. Significant differences were for the (*) indicated groups with <i>p</i> < 0.05, using the Mann Whitney U test (NS, not significant). (A) A schematic illustration of the experimental protocol is shown. (B) Dot plot shows CD45.1⁺CD4⁺ and CD45.1^-CD4⁺ cells at 30 days p.i. CD45.1⁺CD4⁺ and CD45.1^-CD4⁺ cell numbers per spleen are shown in the column bar graph. (C) Contour plots show PD1 and CD39 expression in CD4⁺ cells. PD1^hiCD39^loCD4⁺ and PD1^loCD39^hiCD4⁺ cell percentages are shown in the column bar graphs. (D) Histograms show T-bet and Bcl6 expression in PD1^hiCD39^loCD45.1⁺CD4⁺, PD1^loCD39^hiCD45.1⁺CD4⁺, PD1^hiCD39^loCD45.1^-CD4⁺ and PD1^loCD39^hiCD45.1^-CD4⁺cells. FMO controls are shown in the histograms. The MFIs of T-bet and Bcl6 expression are shown in the column bar graphs. (E-F) CD4⁺ cells from B6 and <i>P2rx7</i>^-/- female mice at 20 days p.i. were transferred into <i>Cd4</i>^-/- female mice that were infected with 1 × 10⁵ <i>Pc</i>-iRBCs. <i>Cd4</i>^-/- mice transferred with naïve B6 cells were used as controls. The data were expressed as means ± SD (<i>n</i> = 4–6) of one representative experiment out of three. Significant differences were observed for the (*) mice transferred with B6 cells at 0 and 20 days p.i. and (**) mice transferred with B6 and <i>P2rx7</i>^-/- cells at 20 days p.i. with <i>p</i> < 0.05, using the Mann Whitney U test. (E) A schematic illustration of the experimental protocol is shown. (F) Parasitemia curves are shown.</p

Splenic Tfh cell responses in infected B6 and <i>P2rx7</i>^-/- mice.

<p>(A-G) B6 and <i>P2rx7</i>^-/- female mice were analyzed at 7,14, 20, 30, 50, 100 and 130 days p.i. with 1 × 10⁶ <i>Pc</i>-iRBCs. Naïve mice were used as controls (day 0). The data were expressed as means ± SD (<i>n</i> = 3–5) of one representative experiment out of three. Significant differences were observed for the (*) B6 and <i>P2rx7</i>^-/- groups with <i>p</i> < 0.05, using the Mann Whitney U test (NS, not significant). (A) Hematoxilyn-eosin stained sections show splenic follicular hyperplasia in <i>P2rx7</i>^-/- mice (40x magnification; bar scales correspond to 200 μm). (^#) The mean areas of lymphoid follicles are shown. (B) Confocal immunofluorescence images (100x magnification; bar scales correspond to 400 μm) of splenic sections are shown. Tissue slices were stained for CD19 (green), CD4 (red) and GL7 (blue). (C) CD19⁺ and Fas⁺GL7⁺CD19⁺ cell numbers per spleen were determined by flow cytometry. (D) Contour plots show PD1, ICOS and Bcl6 <i>versus</i> CXCR5 expression in CD4⁺ cells. PD1⁺CXCR5⁺CD4⁺, ICOS⁺CXCR5⁺CD4⁺ and Bcl6⁺CXCR5⁺CD4⁺ cell numbers per spleen are shown in the column bar graphs. (E) PD1⁺CXCR5⁺CD4⁺ cell numbers per spleen were determined by flow cytometry. (F) IL-21 concentrations were determined by ELISA in the supernatants of splenocytes stimulated or not with iRBCs (splenocyte/3 iRBCs). (G) Anti-parasite IgM and IgG2c serum concentrations were determined by ELISA.</p