Tissue barriers of the human placenta to infection with Toxoplasma gondii

Abstract

Toxoplasma gondii is a ubiquitous, obligate intracellular parasite capable of crossing the placenta to cause spontaneous abortion, preterm labor, or significant disease in the surviving neonate. Exploration of the cellular and histological components of the placental barrier is in its infancy, and both how and where T. gondii breaches it are unknown. The human placenta presents two anatomical interfaces between maternal cells and fetal cells (trophoblasts): (i) the villous region where maternal blood bathes syncytialized trophoblasts for nutrient exchange and (ii) the maternal decidua, where mononuclear, extravillous trophoblasts anchor the villous region to the uterus. Using first-trimester human placental explants, we demonstrate that the latter site is significantly more vulnerable to infection, despite presenting a vastly smaller surface. This is consistent with past findings concerning two vertically transmitted viruses and one bacterium. We further explore whether three genetically distinct T. gondii types (I, II, and III) are capable of preferential placental infection and survival in this model. We find no difference in these strains' ability to infect placental explants; however, slightly slower growth is evident in type II (Prugniaud [Pru]) parasites relative to other cell types, although this did not quite achieve statistical significance.

Fig 1

Comparison of in vivo placental structure to placenta explant model. (A) Structure and orientation of fetus and placenta in uterus at ∼6 weeks of gestation. Fetal structures are represented in shades of blue and purple while maternal structures are in shades of red. Maternal structures: MY, myometrium; SA, spiral arteries; DD, decidua (uterine lining during pregnancy); IVS, intervillous space filled with maternal blood. Fetal structures: VT, villous tree; CP, chorionic plate; UC, umbilical cord; AF, amniotic fluid. (B) Close-up view of the maternal-fetal interface as indicated by the inset in panel A. Maternal blood surrounds the villous tree composed of anchoring villi (AV) and floating villi (FV), which are covered by a syncytiotrophoblast (SYN) that is underlaid by subsyncytial cytotrophoblasts (sCTB) and a basement membrane. The sCTB layer grows increasingly discontinuous in later trimesters. Gas and nutrient exchange with the maternal blood occurs across the syncytiotrophoblast to supply fetal capillaries (not shown) in the villous stroma (STR). At the uterine wall, extravillous cytotrophoblasts (EVT) anchor the villous tree in the decidua. Some invade the decidua and move away from the tip to remodel maternal spiral arteries (not shown), with altered gene expression patterns as they move. (C) Six-week placental explant anchored in Matrigel. Bar, 1 mm. (D) Cartoon representation of the relevant structures shown in panel C. B. Mem, basement membrane.

Fig 2

T. gondii infects the placenta at extravillous cytotrophoblasts (EVT). (A) Two-dimensional (2D) projection of confocally scanned whole-mount placental explant infected for 24 h with type II T. gondii. Red, F-actin (phalloidin); green, GFP-expressing T. gondii; blue, DNA (DAPI); bar, 100 μm. Curve shows the integrated GFP signal across the image's axes, with peaks in the EVT region of anchoring villi (AV) while infectious foci in floating villi (FV) are rare. (B and E) Infected explants were sectioned at 24 h p.i. and stained with anti-hCG (syncytial marker; red) and DAPI (blue). Micrographs shown here feature type II T. gondii; bars, 100 μm. (B and C) Representative T. gondii locations at 24 h: EVT, sCTB, SYN. (C) Parasites in sCTB (arrows) were at times associated with breaks in syncytiotrophoblast (bSYN). (D) Locations of all three parasite types in two sections from each of three placentas show the majority are found in EVT at 24 h p.i. Bars, standard errors of the means (SEM). (E) Enzymatic removal of the SYN results in increased parasite infections along exposed sCTB on villus arms.

Fig 3

Comparison of T. gondii strains' invasion of placental explants. (A) Representative whole-mount placental explant infected for 5 h with type III T. gondii and stained with anti-SAG1 (red-stained parasites) without permeabilization. All living or recently dead parasites express GFP and are therefore green; extracellular parasites also stained red. This “inside-out” stain reveals parasites predominantly localized to EVT, SYN, or STR. White, DAPI. Bar, 100 μm. (B) Intracellular parasites were counted in AV and/or FV and normalized to the infectious dose as determined by PFU. Each dot represents the average of three fields per one placenta. * versus † denotes statistically similar groups (P < 0.04). Bars, SEM. (C) Average counts of extracellular attached parasites in each region (AV or FV) across all strains indicate a slight but not statistically significant pathogen preference for AV. (D) Counts of intracellular parasites as a fraction of total parasites found in AV and FV regions across all strains show invasion is significantly more likely (P > 0.0005) in AV. (C and D) Three AV or FV fields in each of three placentas per each of three strains. Bars, standard deviations (SD).

Fig 4

Parasite replication in trophoblasts within placental explants relative to replication in HFF. (A) Representative examples of parasitic vacuole sizes after 24-h or 48-h infection in HFF. Green, GFP-expressing type I T. gondii; white, DNA; red (outside), anti-SAG1 (stained parasites), added without permeabilization (none visible here). Bar, 10 μm. (B) Number of parasites per vacuole was scored in placental histological sections (top) or HFF cells (bottom) at 24 and 48 h p.i. Each bar represents the average of three placentas or HFF samples. The P value (between graphs) denotes the result of t tests comparing placenta and HFF percentages of single-parasite vacuoles across the three samples. (C) The log₂ transformation of parasites/vacuole yields the average number of replications per parasite in HFF and placenta samples from 24 to 48 h for each parasite type (calculated from data in panel B). Of note, panel C gives logarithmically greater weight to larger vacuoles in comparison to panel B: 1-parasite vacuoles have not replicated and thus received scores of zero, while 4-parasite vacuoles were weighted twice as heavily as 2-parasite vacuoles and 8-parasite vacuoles received three times the 2-parasite weight. Some cell lysis occurred by 48 h for type I (see Materials and Methods). Bars, SEM.

Fig 5

Dissemination of T. gondii throughout placenta. (A) AV and FV in histological sections were scored for presence/absence of T. gondii at 24, 48, or 72 h p.i. AV were scored as either colonized at the EVT-rich tip or at the tip and extending toward the fetus from the tip (Tip + Mid). FV colonization was uncommon, especially by 72 h. Bars, SEM. (B) High-magnification examination of T. gondii (here, type II) dissemination in histological sections stained with anti-hCG (syncytial marker; red) or anti-human E-cadherin (red-stained sCTB boundaries), showing that the basement membrane presents a barrier to infection of STR, where fetal vessels are found. Green, GFP-expressing T. gondii; blue, DAPI. Bar, 100 μm. (C) Whole-mount placental explants were imaged on a confocal microscope and villus cross sections reconstructed to further explore the integrity of the basement membrane barrier. In this representative 72-h-p.i. image, multiparasite vacuoles can be found ringing portions of the sCTB layer but not in STR or SYN. Dashed line, basement membrane; dotted line, probable SYN/sCTB boundary. Red, F-actin (phalloidin), which strongly stains cytotrophoblasts but weakly stains SYN; green, GFP-expressing T. gondii; blue, DAPI; yellow, autofluorescent granules found in SYN. Bar, 20 μm.

Fig 6

Localization of subsyncytial cytotrophoblast (sCTB) infections. (A) Multiparasite vacuole locations were scored across all three T. gondii types in whole-mount placental explants over 72 h (see Materials and Methods). Most are in EVT, while sCTB vacuoles can be found either at SYN breaks or in the apparent absence of such breaks. (B to D) Representative Z-series reconstructions via confocal imaging illustrate each of these three possibilities. Red, F-actin (phalloidin), which strongly stains cytotrophoblasts but weakly stains SYN; green, GFP-expressing T. gondii; blue, DAPI; yellow, autofluorescent granules found in SYN. Dashed line, SYN/CTB boundary; dotted line, basement membrane. Bar, 20 μm. (B) Infections of EVT in an anchoring villus at the SYN boundary, T. gondii type I at 24 h p.i. (C) Infected sCTB underlying apparently intact SYN, as in this image of T. gondii type I at 24 h p.i. (D) Vacuole in floating villus exhibiting damage in SYN, as evidenced by a discontinuous SYN layer with sCTB protruding from it. This representative image was taken 24 h p.i. with T. gondii type III. This may be the normal process of SYN renewal; however, nearby areas of discontinuity (*) suggest damage.