Intravital placenta imaging reveals microcirculatory dynamics impact on sequestration and phagocytosis of Plasmodium-infected erythrocytes

Malaria in pregnancy is exquisitely aggressive, causing a range of adverse maternal and fetal outcomes prominently linked to Plasmodium-infected erythrocyte cytoadherence to fetal trophoblast. To elucidate the physiopathology of infected erythrocytes (IE) sequestration in the placenta we devised an experimental system for intravital placental examination of P. berghei-infected mice. BALB/c females were mated to C57Bl/6 CFP+ male mice and infected with GFP+ P. berghei IE, and at gestational day 18, placentas were exposed for time-lapse imaging acquisition under two-photon microscopy. Real-time images and quantitative measurements revealed that trophoblast conformational changes transiently restrain blood flow in the mouse placental labyrinth. The complex dynamics of placental microcirculation promotes IE accumulation in maternal blood spaces with low blood flow and allows the establishment of stable IE-trophoblast contacts. Further, we show that the fate of sequestered IE includes engulfment by both macrophagic and trophoblastic fetal-derived cells. These findings reinforce the current paradigm that IE interact with the trophoblast and provide definitive evidence on two novel pathogenesis mechanisms: (1) trophoblast layer controls placental microcirculation promoting IE sequestration; and (2) fetal-derived placental cells engulf sequestered IE

Inhale, exhale: Why particulate matter exposure in animal models are so acute? Data and facts behind the history

We present a dataset obtained by extracting information from an extensive literature search of toxicological experiments using mice and rat animal models to study the effects of exposure to airborne particulate matter (PM). Our dataset covers results reported from 75 research articles considering paper published in 2017 and seminal papers from previous years. The compiled data and normalization were processed with an equation based on a PM dosimetry model. This equation allows the comparison of different toxicological experiments using instillation and inhalation as PM exposure protocols with respect to inhalation rates, concentrations and PM exposure doses of the toxicological experiments performed by different protocols using instillation and inhalation PM as exposure methods. This data complements the discussions and interpretations presented in the research article "Inhale, exhale: why particulate matter exposure in animal models are so acute?" Curbani et al., 2019Instituto Capixaba de Ciências e Administração | Ref. 007/201

Early IL-10 production is essential for syngeneic graft acceptance

We performed a comparative study and evaluated cellular infiltrates and anti-inflammatory cytokine production at different time-points after syngeneic or allogeneic skin transplantation. We observed an early IL-10 production in syngeneic grafts compared with allografts. This observation prompted us to investigate the role of IL-10 in isograft acceptance. For this, we used IL-10 KO and WT mice to perform syngeneic transplantation, where IL-10 was absent in the graft or in the recipient. The majority of syngeneic grafts derived from IL-10 KO donors did not engraft or was only partially accepted, whereas IL-10 KO mice transplanted with skin from WT donors accepted the graft. We evaluated IL-10 producers in the transplanted skin and observed that epithelial cells were the major source. Taken together, our data show that production of IL-10 by donor cells, but not by the recipient, is determinant for graft acceptance and strongly suggest that production of this cytokine by keratinocytes immediately upon transplantation is necessary for isograft survival. J. Leukoc. Biol. 92: 259-264; 2012.FAPESPFAPESPCNPqCNPqFCTFC

Control of Uterine Microenvironment by Foxp3+ Cells Facilitates Embryo Implantation

Implantation of the fertilized egg into the maternal uterus depends on the fine balance between inflammatory and anti-inflammatory processes. Whilst regulatory T cells (Tregs) are reportedly involved in protection of allogeneic fetuses against rejection by the maternal immune system, their role for pregnancy to establish, e.g., blastocyst implantation, is not clear. By using 2-photon imaging we show that Foxp3(+) cells accumulated in the mouse uterus during the receptive phase of the estrus cycle. Seminal fluid further fostered Treg expansion. Depletion of Tregs in two Foxp3.DTR-based models prior to pairing drastically impaired implantation and resulted in infiltration of activated T effector cells as well as in uterine inflammation and fibrosis in both allogeneic and syngeneic mating combinations. Genetic deletion of the homing receptor CCR7 interfered with accumulation of Tregs in the uterus and implantation indicating that homing of Tregs to the uterus was mediated by CCR7. Our results demonstrate that Tregs play a critical role in embryo implantation by preventing the development of a hostile uterine microenvironment.DFG grants: (ZE526/4-2, SFB854TP7), Wilhelm Sander Stiftung Germany grant: (2009.022.1), Helmholtz Alliance for Immunotherapy, FCT, Medical Faculty Otto-von-Guericke University PhD grant

In Vivo Approaches Reveal a Key Role for DCs in CD4+ T Cell Activation and Parasite Clearance during the Acute Phase of Experimental Blood-Stage Malaria

Dendritic cells (DCs) are phagocytes that are highly specialized for antigen presentation. Heterogeneous populations of macrophages and DCs form a phagocyte network inside the red pulp (RP) of the spleen, which is a major site for the control of blood-borne infections such as malaria. However, the dynamics of splenic DCs during Plasmodium infections are poorly understood, limiting our knowledge regarding their protective role in malaria. Here, we used in vivo experimental approaches that enabled us to deplete or visualize DCs in order to clarify these issues. To elucidate the roles of DCs and marginal zone macrophages in the protection against blood-stage malaria, we infected DTx (diphtheria toxin)-treated C57BL/6.CD11c-DTR mice, as well as C57BL/6 mice treated with low doses of clodronate liposomes (ClLip), with Plasmodium chabaudi AS (Pc) parasites. The first evidence suggesting that DCs could contribute directly to parasite clearance was an early effect of the DTx treatment, but not of the ClLip treatment, in parasitemia control. DCs were also required for CD4+ T cell responses during infection. The phagocytosis of infected red blood cells (iRBCs) by splenic DCs was analyzed by confocal intravital microscopy, as well as by flow cytometry and immunofluorescence, at three distinct phases of Pc malaria: at the first encounter, at pre-crisis concomitant with parasitemia growth and at crisis when the parasitemia decline coincides with spleen closure. In vivo and ex vivo imaging of the spleen revealed that DCs actively phagocytize iRBCs and interact with CD4+ T cells both in T cell-rich areas and in the RP. Subcapsular RP DCs were highly efficient in the recognition and capture of iRBCs during pre-crisis, while complete DC maturation was only achieved during crisis. These findings indicate that, beyond their classical role in antigen presentation, DCs also contribute to the direct elimination of iRBCs during acute Plasmodium infection.São Paulo Research Foundation grants: (2011/24038-1 [MRDL], 2009/08559-1 [HBdS], CAPES/IGC 04/ 2012 [MRDL, CET])

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

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

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

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