Placental syncytium forms a biophysical barrier against pathogen invasion

Abstract

Fetal syncytiotrophoblasts form a unique fused multinuclear surface that is bathed in maternal blood, and constitutes the main interface between fetus and mother. Syncytiotrophoblasts are exposed to pathogens circulating in maternal blood, and appear to have unique resistance mechanisms against microbial invasion. These are due in part to the lack of intercellular junctions and their receptors, the Achilles heel of polarized mononuclear epithelia. However, the syncytium is immune to receptor-independent invasion as well, suggesting additional general defense mechanisms against infection. The difficulty of maintaining and manipulating primary human syncytiotrophoblasts in culture makes it challenging to investigate the cellular and molecular basis of host defenses in this unique tissue. Here we present a novel system to study placental pathogenesis using murine trophoblast stem cells (mTSC) that can be differentiated into syncytiotrophoblasts and recapitulate human placental syncytium. Consistent with previous results in primary human organ cultures, murine syncytiotrophoblasts were found to be resistant to infection with Listeria monocytogenes via direct invasion and cell-to-cell spread. Atomic force microscopy of murine syncytiotrophoblasts demonstrated that these cells have a greater elastic modulus than mononuclear trophoblasts. Disruption of the unusually dense actin structure--a diffuse meshwork of microfilaments--with Cytochalasin D led to a decrease in its elastic modulus by 25%. This correlated with a small but significant increase in invasion of L. monocytogenes into murine and human syncytium. These results suggest that the syncytial actin cytoskeleton may form a general barrier against pathogen entry in humans and mice. Moreover, murine TSCs are a genetically tractable model system for the investigation of specific pathways in syncytial host defenses.

Figure 1. L. monocytogenes (LM) invades and replicates in murine trophoblast stem cells.

A. Colony forming units (CFU) per coverslip at 2, 5, 8, and 24 hours post-inoculation (p.i.) for the following L. monocytogenes (LM) strains were determined: wild type (wt), InlA-deficient (del-InlA), and murinized InlA (InlA^m). Average CFU per coverslip at 2 hours p.i. for each strain is based on nine independent experiments. Average CFU per coverslip at time points 5, 8, and 24 hours p.i. for each strain are based on three independent experiments. Bars represent standard error. Average CFU at 2 hours for InlA^m is 30-fold higher than for wild type and the p-value denotes statistical significance (p = 0.008 by Student's T-test). There is no difference in invasion for wild type versus del-InlA (p = 0.3 by Student's T-test). B. Immunofluorescence image of mTSC at 2 hours p.i.: LM is shown in green, nuclei in white, actin in red. Arrowheads point to foci of infection, Bar = 50 um.

Figure 2. Syncytiotrophoblasts (SYN) derived from murine trophoblast stem cells (TSC) resist direct invasion by L. monocytogenes (LM).

A. Schematic of differentiation process from TSC to SYN, which results in 65–77% of fused syncytium; the remaining cells in the dish are undifferentiated mononuclear trophoblasts (MNT). B. Panel i: Immunofluorescence images of 5-day differentiated multinuclear SYN (outlined and marked by blue star) demonstrates typical clustering of nuclei. Compare to surrounding MNT with stress-fibers, and clear cell boundaries. Bar = 50 um. Panels ii to v show close-up representative examples of actin structure of SYN: a diffuse meshwork of small actin filaments (iii and v show the actin channel of ii and iv respectively). Nuclei are shown in white, Bar = 10 um. C. SYN 2 hours p.i. with LM (green) showing resistant syncytial area (outlined and marked by blue star) neighboring infected mononuclear trophoblasts. Nuclei are shown in white; Bar = 50 um. D. Quantification of invasion of mononuclear trophoblasts (MNT) versus syncytium with two strains of LM – InlA^m-expressing and wt. Bacterial invasion at 2 hours p.i. of MNT versus SYN is represented by the ratio of green fluorescence per unit area for each cell type. Each symbol represents the average of green fluorescence per unit area in six random microscopic fields (20×), bar represents median.

Figure 3. Actin cytoskeleton of murine syncytiotrophoblast (SYN) contributes to its elastic strength.

A. Microrheology with an atomic force microscope was used to measure elastic strength of mouse trophoblast stem cells (TSC) and SYN. Photos depict microscopic cantilever positioned above cultured live cells prior to measurement. B. The elastic modulus (Young's modulus) of SYN is significantly higher than that of TSC (p = 4.7×10⁻⁵ by Student's T-test). Elastic modulus of SYN was measured in the exact same spot prior to and after treatment with Cyto-D for 40–60 min. Disruption of the actin cytoskeleton with Cyto-D significantly decreased the elastic modulus of SYN (p = 0.001 by Student's T-test). Bars represent median values. Graph is based on three independent experiments performed in triplicate. C. Immunofluorescence images of the actin (red) in mSYN show that the characteristic actin meshwork (i–iii) is disrupted by 1 hr treatment with Cyto-D (iv–vi). Nuclei are shown in white. Bars in panels i and iv are 50 um. Panels ii–iii and v–vi are representative close-up images of untreated and Cyto-D treated mSYN, respectively. Panels iii and vi show just the actin channel of ii and v respectively. Aggregation of microfilaments in distinct puncta are observed upon treatment. Bars = 10 um.

Figure 4. Cell-to-cell spread of L. monocytogenes (LM) into mouse syncytiotrophoblast (SYN) is enhanced by syncytial actin network disruption.

Untreated and Cyto-D treated SYN was incubated with LM-infected murine macrophages and foci of bacterial spread were quantified. Bacterial foci were included in the analysis when multiple bacteria were observed unbounded by the outline of a macrophage membrane; many such foci were surrounded by actin clouds. A. Panel i: Representative immunofluorescence image of untreated differentiated TSCs shows bacterial infection of mononuclear trophoblasts (MNT) versus SYN. SYN is outlined and marked by blue star. Panel ii: One hour of Cyto-D treatment increases the number of bacterial foci in SYN. Arrowheads indicate adherence of infected macrophages to trophoblasts; arrows indicate spread events. Actin is shown in red, nuclei in blue, LM in green. Bars = 50 um. B. Quantification of bacterial foci per unit area in untreated versus Cyto-D treated SYN at 5 hours p.i. with macrophages. Each data point represents the average of bacterial foci in ten random fields (20×) with at least 50% syncytium. Treatment with Cyto-D significantly increases infection of SYN via cell-to-cell spread (p = 0.03 by Student's T-test); treatment does not significantly change infection of MNT (p = 0.33). Bars represent median.

Figure 5. Cell-to-cell spread of L. monocytogenes (LM) into human syncytiotrophoblast (SYN) is enhanced by syncytial actin network disruption.

A. Immunofluorescence image of sectioned primary human placenta showing diffuse actin structure in the syncytium (outlined and marked by blue star.) Bar = 10 um. B. Untreated and 1 hr Cyto-D treated human placental organ cultures were incubated with LM-infected macrophages and foci of spread observed and quantified. Panels i and ii show representative images showing cell-to-cell spread occurs almost exclusively at the extravillous trophoblasts (EVT) in untreated placenta; Cyto-D treatment increases incidence of spread into SYN. Bar = 100 um. Panel iii shows representative image of bacterial presence in the syncytium in Cyto-D treated placenta. Bar = 10 um. LM is shown in green, nuclei in white, SYN (b-hCG staining) is shown in red. C. Quantification of cell-to-cell spread into SYN. Each data point represents average co-localization of LM with b-hCG from ten microscopic fields (10×); bar represents median, graph is based on seven independent experiments. Cyto-D treatment significantly increases bacterial cell-to-cell spread into placental syncytium (p = 0.04 by Student's T-test).