Toxoplasma gondii Dysregulates Barrier Function and Mechanotransduction Signaling in Human Endothelial Cells.

Toxoplasma gondii is a foodborne parasite that infects virtually all warm-blooded animals and can cause severe disease in individuals with compromised or weakened immune systems. During dissemination in its infected hosts, T. gondii breaches endothelial barriers to enter tissues and establish the chronic infections underlying the most severe manifestations of toxoplasmosis. The research presented here examines how T. gondii infection of primary human endothelial cells induces changes in cell morphology, barrier function, gene expression, and mechanotransduction signaling under static conditions and under the physiological conditions of shear stress found in the bloodstream. Understanding the molecular interactions occurring at the interface between endothelial cells and T. gondii may provide insights into processes linked to parasite dissemination and pathogenesis.

IMPORTANCE Toxoplasma gondii is a foodborne parasite that infects virtually all warm-blooded animals and can cause severe disease in individuals with compromised or weakened immune systems. During dissemination in its infected hosts, T. gondii breaches endothelial barriers to enter tissues and establish the chronic infections underlying the most severe manifestations of toxoplasmosis. The research presented here examines how T. gondii infection of primary human endothelial cells induces changes in cell morphology, barrier function, gene expression, and mechanotransduction signaling under static conditions and under the physiological conditions of shear stress found in the bloodstream. Understanding the molecular interactions occurring at the interface between endothelial cells and T. gondii may provide insights into processes linked to parasite dissemination and pathogenesis. with actin (3). In this role, VE-cadherin is a major regulator of vascular permeability, and its dysregulation is linked to human pathologies (2). VE-cadherin, actin, and associated binding proteins form a force-sensitive complex that enables rapid, global changes in endothelial cells in response to mechanical forces in the microenvironment (4). The Hippo pathway is highly conserved in mammals, and its signaling regulates cell growth, differentiation, and survival by inhibiting the transcriptional coactivator Yes-associated protein 1 (YAP) (5). Forces, such as the tension generated by shear stress, are detected by the adherens junctions and cytoskeleton and activate the Ste20-like protein kinases Mst1/2 (6). Large tumor suppressor kinase 1 (LATS1) is activated by Mst1/2 via phosphorylation at threonine residue 1079, resulting in YAP cytoplasmic retention and inhibition of YAP target gene expression (7,8). Toll-like receptor (TLR) signaling has also been shown to activate the Hippo pathway in Drosophila (9). Interestingly, YAP is now appreciated as a key regulator of mammalian endothelial activation and inflammation (10), indicating that Hippo signaling is critical for endothelial cells to respond to vascular perturbations, such as coagulation, infection, or injury.
Toxoplasma gondii is an obligate intracellular parasite that infects an estimated one-third of the global population and causes significant morbidity and mortality in immunocompromised individuals (11). Humans are typically infected by consuming food or water contaminated with parasite cysts or through vertical transmission from mother to fetus. During dissemination in its host, T. gondii crosses formidable biological barriers, such as the blood-brain barrier (BBB), to exit the bloodstream and infect tissues where the parasite establishes a lifelong chronic infection (12). Current research suggests that T. gondii may leave the circulation to enter tissues inside motile immune cells that extravasate from the bloodstream or by directly infecting and lysing vascular endothelial cells (13). Indeed, T. gondii tachyzoites can adhere to and invade human vascular endothelium under shear stress conditions (14), and T. gondii can replicate in human retinal vascular endothelial cells (15). Recent evidence indicates that endothelial cells of the blood-brain barrier provide a replicative niche for T. gondii and facilitate parasite crossing of the BBB and entry into the central nervous system (CNS) (16). Despite a growing appreciation for the importance of endothelial infection in T. gondii pathogenesis, the molecular interactions occurring at this host-pathogen interface remain poorly defined.
In the present study, we investigated the morphological and functional consequences of T. gondii infection of primary human umbilical vein endothelial cells (HUVEC). We found that T. gondii infection dysregulated endothelial cell barrier function and remodeled the endothelial cell actin cytoskeleton. By conducting a global transcriptome analysis of infected endothelial cells, we identified gene expression changes associated with mechanotransduction and show that T. gondii infection activated Hippo signaling, as evidenced by LATS1 phosphorylation, and altered the subcellular localization of YAP, a protein that plays a critical role in sensing mechanical force and linking biomechanical stresses to gene expression changes in the cell.

RESULTS
T. gondii infection dysregulates endothelial cell barrier integrity and function. T. gondii can infect endothelial cells to exit the bloodstream and enter host tissues, such as the lung and CNS (16). To examine the effect of infection on vascular endothelial barrier integrity, electrical cell-substrate impedance sensing (ECIS) assays were used. HUVEC were seeded into fibronectin-coated wells in an ECIS plate and cultured to confluence for 72 h (see Fig. S1 in the supplemental material). The cells were then mock infected with fresh media, infected with green fluorescent protein (GFP)-expressing type II (Prugniaud strain) T. gondii at a multiplicity of infection (MOI) of 1 or 2, or treated with interleukin-1beta (IL-1␤) as a positive control to induce barrier permeability. Impedance measurements were made prior to treatment and continuously every 15 min for at least 18 h (Fig. 1A). Mock-infected HUVEC showed no significant changes in transendothelial electrical resistance (TEER) throughout the time frame of analysis, whereas HUVEC treated with IL-1␤ exhibited a reduction in TEER beginning at 3 h, which progressed to 6 h. Interestingly, T. gondii infection of HUVEC reduced TEER as early as 6 hpi. The reduction in TEER persisted over time, and this effect was dose dependent, as TEER levels decreased with increasing parasite MOI. Decreased TEER measurements in infected cells did not appear to be the result of cell lysis, as visual inspection by microscopy confirmed intact monolayers at 18 hpi with the dose and strain of T. gondii used in all experiments (see Fig. 2 to 4). These data suggest that T. gondii infection of host endothelial cells reduced barrier integrity within 6 h after infection.
To further test barrier function in endothelial cells, we examined whether T. gondii infection of endothelial monolayers altered permeability to a low-molecular-weight polymer in a transwell assay. HUVEC were seeded onto fibronectin-coated 0.4-m porous membrane inserts in the apical chamber and cultured to confluence for 72 h. Confluent monolayers were mock infected, infected with T. gondii (MOI of 2), or treated with IL-1␤ as a positive control for 18 h. For the final 30 min of culture, 40-kDa fluorescein isothiocyanate (FITC)-dextran was added to the apical chamber, and the contents of the basolateral chamber were collected to evaluate endothelial barrier permeability (Fig. 1B). As expected, IL-1␤ treatment increased the amount of FITCdextran passing through the HUVEC monolayer compared to mock-infected cells. T. gondii infection also increased monolayer permeability 1.99-fold at 18 h postinfection T. gondii infection leads to decreased VE-cadherin localization to the cell periphery. Adherens junctions are critical for the formation and maintenance of endothelial barriers, and VE-cadherin is the main determinant of the integrity of these junctions (2,17,18). To investigate whether the observed changes in barrier permeability and function coincided with altered levels of VE-cadherin in endothelial cells following infection, HUVEC were cultured to confluence for 72 h on fibronectin. They were then mock infected with fresh media or infected with GFP-expressing T. gondii. At 18 hpi, the monolayers were analyzed for VE-cadherin signal by immunofluorescence microscopy ( Fig. 2A). Mock-infected monolayers showed a zipper-like VE-cadherin  staining pattern between adjacent cells that is characteristic of mature adherens junctions. Continuity of VE-cadherin staining was quantified on an individual cell basis by examining immunofluorescent signal of VE-cadherin at the cell periphery and defining gaps of Ն2.5 m as discontinuous signal (Fig. S2). Continuous VE-cadherin staining was observed in 83% of mock-infected HUVEC over multiple experiments (Fig. 2B). In contrast, continuous VE-cadherin was observed in only 27% T. gondiiinfected HUVEC (Fig. 2B), as infection led to a marked reduction in VE-cadherin at the cell periphery, gaps in VE-cadherin staining, and partial loss of VE-cadherin signal in some infected cells ( Fig. 2A). By examining total VE-cadherin protein levels from endothelial cell lysates, an 18.7% reduction in VE-cadherin protein levels was observed in infected cells at 18 hpi ( Fig. 2D and E). The cytoplasmic tail of VE-cadherin interacts with ␤-catenin to promote stability of endothelial junctions (19). Therefore, we also examined whether T. gondii infection dysregulated ␤-catenin localization during infection. Continuous ␤-catenin staining was observed in 80% of mock-infected HUVEC compared to 16% of infected HUVEC (Fig. 2C). We observed that ␤-catenin localization to the cell periphery appeared dysregulated in infected HUVEC in a manner similar to VE-cadherin localization ( Fig. 2A). However, Western blotting did not reveal changes in total protein levels of ␤-catenin at 18 hpi ( Fig. 2D and F).
T. gondii infection alters cell morphology and F-actin stress fiber abundance. Adherens junctions are linked to the actin cytoskeleton to maintain stability and cell shape (2,19). In addition to detecting changes in VE-cadherin at the cell periphery, we also observed changes in the morphology of T. gondii-infected endothelial cells. At 18 hpi, more nuclei were observed per field of view (FOV) (Fig. 3A), and the maximal cell length was decreased by 11% in T. gondii-infected HUVEC compared to mock-infected HUVEC (Fig. 3B). To determine whether these subtle changes in morphology were associated with cytoskeletal rearrangements, we investigated whether infection altered the actin cytoskeleton by staining the cells with fluorescently conjugated phalloidin and examining the cells by confocal microscopy (Fig. 3C). Quantification of the filamentous actin (F-actin) area per FOV revealed that F-actin abundance was significantly reduced in infected monolayers (Fig. 3D). Notably, the cytoplasmic network of F-actin stress fibers appeared to be rearranged in infected HUVEC by 18 hpi. By examining F-actin on an individual cell level, we determined that T. gondii infection reduced cytoplasmic stress fiber abundance by 34.15% in directly infected cells and by 17.3% in bystander cells (Fig. 3E). The effect on F-actin was observed in cells with both large and small vacuoles ( Fig. 3C and Fig. S3). These data suggest that T. gondii modulates actin cytoskeleton organization in HUVEC during infection.
Planar cell polarity is disrupted in T. gondii-infected endothelial cells. In the bloodstream, the mechanical forces caused by fluidic shear stress alter cell morphology and the arrangement of the cytoskeleton (20)(21)(22)(23). To further investigate the effect of T. gondii infection on the endothelial cell actin cytoskeleton, we determined whether the changes in F-actin detected in static cultures were also observed under more physiological shear stress conditions. HUVEC were seeded in ibidi microfluidic chambers and cultured under unidirectional continuous flow for 72 h at 5.5-dyne/cm 2 shear stress to simulate conditions in postcapillary venules (24). As previously reported (22), HUVEC adopted a polarized morphology under flow conditions, in which the cells aligned in the direction of flow (Fig. 4A). The cells were then mock infected or infected with GFP-expressing T. gondii. After 18 h, the monolayers were stained with phalloidin to detect F-actin. Consistent with the findings in static conditions, T. gondii also disrupted cytoplasmic F-actin organization in infected HUVEC under shear stress conditions ( Fig. 4A and B). Cytoplasmic stress fiber abundance was reduced by 35.7% in directly infected cells and by 8.5% in bystander cells compared to mock-infected HUVEC (Fig. 4C).
We next determined whether the observed disruption in F-actin impacted HUVEC planar cell polarity. The position of the Golgi apparatus relative to the nucleus provides an indication of planar cell polarity (25). When the Golgi position was evaluated in mock-infected monolayers by staining for gm130 as a marker of the Golgi apparatus, 79% of the cells displayed Golgi localization consistent with a polarized phenotype ( Fig. 5A and B). In contrast, there was reduced planar cell polarity in T. gondii-infected cultures, in which 54% and 59% of the cells were polarized in directly infected and bystander cells, respectively ( Fig. 5A and B). In addition, it was noted that T. gondii caused Golgi fragmentation under shear stress conditions (Fig. 5A), as previously reported for static culture conditions (26). Interestingly, Golgi fragmentation was significantly increased in directly infected HUVEC compared to both mock-infected or bystander cells (Fig. 5C). Collectively, these data demonstrate that T. gondii infection of endothelial cells led to altered F-actin distribution and abundance, as well as reduced planar cell polarity in cells cultured under microfluidic shear stress conditions.

T. gondii infection induces gene expression changes in endothelial cells.
To further investigate T. gondii-induced changes in cell morphology and barrier function, we examined changes in the gene expression profiles of mock-infected and T. gondiiinfected HUVEC at 18 hpi using RNA sequencing (RNA-Seq). Infection resulted in a greater number of upregulated genes than downregulated genes (Fig. 6A). Among the most abundant transcripts at 18 hpi were CCL1 (chemokine ligand 1), CCL20 (chemokine ligand 20), HAS2 (hyaluronan synthase 2), and SELE (selectin E), which are markers of endothelial activation in response to inflammatory stimuli (Fig. 6A). We identified 214 protein-coding genes as differentially expressed between mock-infected and T. gondiiinfected HUVEC that met the following criteria: fold change Ͼ 2, false-discovery rate (FDR) Ͻ 0.05, and reads per kilobase per million (RPKM) Ͼ 5. Among these differentially expressed genes (DEGs), 181 were increased in transcript abundance and 33 were decreased in transcript abundance (Fig. 6B). Functional enrichment using Metascape revealed that gene sets with increased transcript abundance mapped to Gene Ontology (GO) pathways associated with cytokine-mediated signaling, extracellular structure reorganization, and regulation of cell death (Fig. 6C). Many of the genes in these pathways are involved in inflammatory responses and signaling, as has been previously reported during T. gondii infection of other cell types (27)(28)(29). On the other hand, gene sets with decreased transcript abundance during infection mapped to GO terms related to the regulation of receptor signaling, cell adhesion, and regulation of mitogen- activated protein kinase (MAPK) signaling and branching structures (Fig. 6D). Interestingly, T. gondii infection reduced transcript abundance of ANKRD1 (ankyrin repeat domain 1), CTGF (connective tissue growth factor), and CYR61 (cysteine-rich angiogenic inducer 61), three genes that are associated with mechanotransduction signaling pathways linked to changes in the actin cytoskeleton (30-32) (Fig. 6E).

YAP activity is altered in T. gondii-infected endothelial cells.
Mechanotransduction plays an integral role in the ability of cells to sense the external environment and respond to mechanical force to regulate cell proliferation and shape (30). In endothelial cells, the actin cytoskeleton forms a mechanosensitive complex through protein interactions with the adherens junctions to detect changes in extracellular matrix (ECM) stiffness, cell-cell contacts, and shear stress (4). Signals from the plasma membrane and changes in F-actin stress fiber organization modulate the activity of the transcriptional coactivator Yes-associated protein (YAP), a key downstream effector of canonical Hippo signaling. In response to mechanical stress or disruptions in cell adhesion, Hippo signaling induces Mst1/2-mediated phosphorylation of LATS1, leading to YAP nuclear export and repression of YAP target gene transcription (33). The expression of YAP target genes ANKRD1, CTGF, and CYR61 was reduced in infected HUVEC compared to mock-infected endothelial cells (Fig. 6E). Using the same mRNA samples as in the RNA-Seq experiments, we validated the RNA-Seq results for these genes using quantitative PCR (qPCR) with primers specific for ANKRD1, CTGF, and CYR61 and confirmed reduced mRNA transcript abundance for each of these genes in T. gondii-infected endothelial cells compared to mock-infected endothelial cells (Fig. 7A).
To investigate whether T. gondii infection affected YAP subcellular localization, we performed immunofluorescence microscopy for YAP in HUVEC during infection. Mockinfected HUVEC expressed strong nuclear YAP signal and weak cytoplasmic signal, consistent with these cells being at confluence on a relatively stiff surface of fibronectin on glass (30). Interestingly, YAP nuclear localization appeared reduced in T. gondii- infected HUVEC (Fig. 7B). Quantification of nuclear YAP signal revealed a 22% reduction in mean fluorescence intensity (MFI) at 18 hpi (Fig. 7C). We did not detect any statistically significant changes in YAP mRNA by RNA-Seq or YAP protein expression by Western blot analysis ( Fig. 7D and E), suggesting that YAP was most likely regulated at the level of its subcellular localization in infected HUVEC. We next determined whether T. gondii infection induced the Hippo signaling pathway in endothelial cells by examining phosphorylation of the kinase LATS1, which is upstream of the Hippo effector YAP. Western blotting revealed no changes in total LATS1 protein expression, but we observed increased phosphorylation of LATS1 at Thr1079 in T. gondii-infected cells at 18 hpi compared to mock-infected cells (Fig. 7F and G). 12-O-Tetradecanoylphorbol-13-acetate (TPA) was used as a positive control to induce phospho-LATS1 (Fig. 7F and G). These findings support a model whereby T. gondii infection modulates pathways relevant to mechanical stress in endothelial cells through the Hippo signaling pathway, leading to phosphorylation of LATS1 and cytoplasmic retention of YAP, thereby reducing YAP nuclear activity and target gene expression.

DISCUSSION
Actin is the most abundant, highly conserved protein in mammalian cells and constitutes about 10% of total protein in endothelial cells (34,35). The dynamic polymerization and depolymerization of actin filaments are important for organelle position, intracellular trafficking, ECM sensing, and cell shape (36)(37)(38). Filamentous actin interacts with catenins to anchor interendothelial cell junctions and stabilize endothelial barrier integrity (37). Actin, VE-cadherin, and associated binding proteins form a mechanosensing complex in endothelial cells (4). As a result, changes in the external environment, such as mechanical forces, are transduced through the actin cytoskeleton into cellular responses (33).
As the cells that line the blood vessel walls, vascular endothelial cells are exposed to the shear stress of rapidly flowing blood. Shear stress induces profound morphological and functional adaptations via mechanotransduction signaling in vascular endothelial cells (20)(21)(22). Planar cell polarity, which is the alignment of cells in a tissue plane, is induced by shear stress (23). This polarity provides directionality to intracellular trafficking, and if dysregulated, can contribute to a variety of developmental diseases, as well as vascular pathologies, such as polycystic kidney cancer (23,39,40). In examining the extent to which T. gondii infection altered the actin cytoskeleton of endothelial cells under continuous shear stress conditions, we found that infection disrupted planar cell polarity and led to Golgi fragmentation, indicating potential dysregulation of vesicle trafficking. T. gondii is reported to fragment the host cell Golgi apparatus and reposition the functional ministacks around its parasitophorous vacuole to modulate vesicular trafficking and scavenge sphingolipids in nonendothelial cell types (26). In endothelial cells, vesicular transport is important for VE-cadherin localization to the plasma membrane and adherens junction stability (41). As a result, T. gondii disruption of vesicular transport may disrupt VE-cadherin trafficking to cell-cell junctions in infected HUVEC, thereby contributing to barrier dysregulation and increased paracellular permeability. This may explain the modest reduction in VEcadherin total protein levels observed during infection, despite the dramatic difference in localization at the plasma membrane that was observed by confocal microscopy. Interestingly, a recent study by Ross et al. demonstrated that extracellular parasites can transmigrate across brain endothelial cells without perturbing barrier integrity (42). T. gondii decreased phosphorylation of focal adhesion kinase (FAK), a regulator of endothelial tight junctions, to transiently destabilize endothelial barriers without lysing the host cell (42). This adds to growing evidence that live infection with T. gondii affects FAK regulation, perhaps aiding the parasite in dissemination (43,44).
A variety of obligate intracellular pathogens modulate endothelial cell biology and function during infection (45). Gram-negative bacteria, such as pathogenic Rickettsia species and Chlamydia trachomatis alter cell morphology and function in infected endothelial cells to facilitate intracellular motility, cell-to-cell dissemination, and nutrient acquisition by intercepting vesicular transport from the host cell Golgi apparatus (46)(47)(48). Both Rickettsia species and C. trachomatis disrupt adherens junctions and increase vascular permeability during infection by inducing VE-cadherin phosphorylation (49,50). Furthermore, intracellular replication of these pathogens within endothelial cells leads to an activated, proinflammatory endothelial phenotype, which may also contribute to increased barrier permeability, since inflammatory cytokine signaling is known to both upregulate adhesion molecules and reduce barrier integrity.
T. gondii infection results in a proinflammatory gene expression profile in several cell types (27)(28)(29)51). Consistent with these previous reports, our RNA-Seq data confirm that T. gondii induces proinflammatory genes in human endothelial cells during infection. Functional enrichment revealed upregulated DEGs associated with the immune response and immune cell adhesion, such as ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), CXCL3 (chemokine ligand 3), and SELE (selectin E). By analyzing the F-actin area in directly infected cells compared to bystander cells in the same culture, we observed that cells that were directly infected by T. gondii had the greatest reduction in F-actin area compared to mock-infected cells; however, the uninfected bystander cells had an intermediate phenotype, suggesting that soluble inflammatory factors acting in a paracrine manner may contribute to this bystander effect, which was more apparent under static culture conditions than under conditions of fluidic shear stress. Although HUVEC barrier integrity was reduced in response to IL-1␤ treatment, as demonstrated by TEER assays, the RNA-Seq data demonstrated that T. gondii infection of HUVEC did not induce IL-1␤ transcripts, suggesting that other inflammatory mediators may contribute to the bystander effect in the infected HUVEC culture.
T. gondii is also reported to induce quiescent cells into S phase, thereby facilitating host cell cycle progression (52)(53)(54). Interestingly, we detected increased expression of genes related to cell proliferation, such as G0S2 (G 0 /G 1 switch 2) and EGR1 (early growth response 1), which may help to explain the increased number of nuclei per FOV detected in the infected-cell cultures.
YAP plays a central role in mechanotransduction by enabling cells to detect perturbations in actin cytoskeleton organization caused by mechanical forces and transducing these signals into changes in gene transcription (30,31,33). Our RNA-Seq analysis revealed a subset of downregulated DEGs that were associated with receptor signaling activity, cell-substrate adhesion, and regulation of cellular proliferation. Among these DEGs, we observed reduced mRNA transcripts for three YAP target genes: ANKRD1, CTGF, and CYR61. ANKRD1 is a transcriptional cofactor that negatively regulates matrix metalloproteinase transactivation and plays a vital role in wound healing (55). CTGF and CYR61 are matricellular proteins of the CCN (connective tissue growth factor, cysteine-rich protein, and nephroblastoma overexpressed gene) family that are secreted into the ECM to regulate cell adhesion, cell proliferation, and vascular remodeling (56,57). YAP lacks DNA-binding activity and must interact with a DNA-binding protein to enter the nucleus and modulate expression of its target genes (58). YAP interacts with TEAD (TEA/ATTS domain) transcription factors to drive expression of ANKRD1, CTGF, CYR61, and other genes important for cell proliferation. Alterations in the activity of YAP and expression of YAP target genes were correlated with upstream phosphorylation of LATS1 at Thr1079, suggesting that T. gondii infection of endothelial cells activates a Hippo signaling cascade, likely due to changes in mechanical stress on the host cell.
YAP activity depends on its subcellular localization, which can be modulated through phosphorylation at serine residue 127 via Hippo signaling and subsequent interactions with 14-3-3 proteins, which anchor YAP in the cytoplasm (7). 14-3-3 proteins are a family of proteins involved in a multitude of diverse signaling pathways. Interestingly, T. gondii secretes its own 14-3-3 protein (Tg14-3-3) into host cells, which interacts with host 14-3-3 proteins at the parasitophorous vacuole membrane (PVM) and contributes to the hypermotility of T. gondii-infected dendritic cells (59). Although YAP does not appear to localize at the PVM in T. gondii-infected endothelial cells, the possibility of cross talk between host and parasite-secreted 14-3-3 proteins in modulating YAP activity may be an interesting avenue for investigation.
The current research provides evidence that T. gondii remodels components of the endothelial cell cytoskeleton and alters endothelial cell barrier function during infection. Because the actin cytoskeleton, through its interactions with binding partners and junction proteins, lies at the heart of mechanotransduction signaling, these findings also provide new insights into host cell biomechanical sensing of intracellular T. gondii infection.

MATERIALS AND METHODS
Mammalian and parasite cell culture. Human umbilical vein endothelial cells (HUVEC) (Lonza, Allendale, NJ) were cultured in endothelial growth medium 2 (EGM-2) with EGM-2 SingQuot supplements and growth factors (Lonza) or in EGM (R&D Systems, Minneapolis, MN). GFP-expressing and tdTomatoexpressing type II T. gondii tachyzoites (Prugniaud strain) were maintained in human foreskin fibroblasts (HFF) and syringe lysed, and washed immediately before experimentation as previously described (60). Briefly, T. gondii-infected HFF monolayers were scraped with a cell scraper, and the resulting cell suspension was syringe lysed with a 27 1/2 gauge syringe before bringing to a final volume of 15 ml with fresh D-10% medium and spinning at 1,500 rpm for 7 min at room temperature (RT). Next, the parasite pellet was resuspended with 3 ml fresh D-10% before passage through a 0.5-m filter to remove cellular debris. The filtrate was brought to a final volume of 15 ml with fresh D-10% before spinning at 1,500 rpm for 7 min at RT. Lysed and filtered parasites were resuspended in 1 ml fresh EGM and counted with a hemocytometer. HUVEC were infected at an MOI of 1 or 2. Mock-infected cells were those to which prewarmed fresh medium was added in the place of parasites. All parasite and human cell cultures were routinely tested for Mycoplasma contamination and confirmed to be negative.
HUVEC TEER assays. A 96-well electrode array comprised of 20 interdigitated electrode fingers with a total area of 3.92 mm 2 (Applied Biophysics, Troy, NY) was coated with 20 g/ml fibronectin, and 4 ϫ 10 4 to 5 ϫ 10 4 HUVEC were seeded into each well. Immediately after seeding, the array was placed on an ECIS Z (Applied Biophysics) and maintained at 37°C and 5% CO 2 . The impedance in each well was measured every 15 min at multiple alternating current (AC) frequencies. HUVEC were provided fresh media at 24 and 48 h after seeding. At 72 h after initial seeding, 0.5 ng/ml IL-1␤, syringe-lysed T. gondii tachyzoites at an MOI of 1 or 2, or fresh media were added to the test wells, and the assay was conducted for an additional 24 h. Impedance measured at 4,000 Hz and 64,000 Hz was used to calculate resistance and capacitance, respectively. Resistance was then multiplied by the surface area of the electrical cellsubstrate impedance sensing (ECIS) electrodes to calculate transendothelial electrical resistance (TEER) values (61).
Microfluidics. Microfluidic experiments were performed using the ibidi pump system (ibidi GmbH, Martinsried, Germany). For shear stress conditions, 2.5 ϫ 10 5 HUVEC were seeded into ibiTreated -slide I 0.4 Luer chambers and cultured under static conditions at 37°C with 5% CO 2 for 1 to 3 h. The -slide was then connected to the ibidi pump system, and HUVEC monolayers were cultured to confluence under 5.5 dyne/cm 2 of continuous flow for 72 h. After the cells reached confluence, flow was paused, and syringe-lysed tachyzoites or fresh media were added to monolayers and cultured under static conditions for 1 h. The -slide was then reconnected to flow and cultured under shear conditions for an additional 17 h.
Immunofluorescence microscopy. For static cultures, 2.5 ϫ 10 5 HUVEC were seeded onto fibronectin-coated coverslips and cultured to confluence at 37°C and 5% CO 2 for 72 h. Syringe-lysed tachyzoites or fresh media were added, and HUVEC were cultured for an additional 18 h. For shear stress conditions, the -slide containing mock-infected or T. gondii-infected HUVEC (described above) was disconnected from the flow pump. Coverslips or -slides were then washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA), blocked with PBS containing 5% normal goat serum (NGS) (Southern Biotech, Birmingham, AL), and permeabilized with 0.1% Triton X-100 (Sigma, St. Louis, MO).
HUVEC were stained with antibodies recognizing VE-cadherin (BV9; Biolegend, San Diego, CA), ␤-catenin (Cell Signaling Technologies, Danvers, MA), YAP (G-6; Santa Cruz Biotech, Dallas, TX), and gm130 (Sigma) and with Hoechst dye (Life Technologies, Carlsbad, CA). Alexa Fluor 488 (AF 488)conjugated goat anti-mouse IgG, AF 594-conjugated goat anti-mouse IgG, AF 647-conugated goat anti-mouse IgG, and AF 594-conjugated anti-rabbit IgG, or AF 647-conjugated goat anti-rabbit IgG (all from Life Technologies) were used as secondary antibodies. For visualization of F-actin, monolayers were incubated with AF 594-conjugated phalloidin (Life Technologies). Coverslips were mounted using Vectashield with or without DAPI (Vector Laboratories, Burlingame, CA). For shear stress conditions, -slides were sealed with ibidi's mounting medium. Confocal microscopy was performed using a Leica SP-8 microscope with a 63ϫ objective, and data were analyzed using ImageJ software. Prism (GraphPad Software, La Jolla, CA) was used to graph data and perform statistics.
Quantification of fluorescence microscopy images. Quantification of cellular structures was performed on confocal images from at least three independent experiments per analysis. Images were analyzed using ImageJ. Discontinuity in VE-cadherin or ␤-catenin staining was defined by the absence of fluorescence signal at the cell periphery measuring a distance 2.5 m or greater. To quantify cell numbers and morphology, DAPI-stained nuclei per 63ϫ field of view (FOV) were counted, and only nuclei represent the absolute normalized expression (transcripts per million [TPM]); the range of colors is based on scaled and centered TPM values of the entire set of genes (red represents high expression, whereas blue represents low expression).
Statistics. Statistical analysis was performed using Prism software. Student's t test was used for comparisons between mock-infected and T. gondii-infected samples. Two-way analysis of variance (ANOVA) with a Bonferroni posthoc test was used for TEER data. One-way ANOVA with a Tukey posthoc test was used for all experiments with multiple comparisons.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.