Toxoplasma effector-induced ICAM-1 expression by infected dendritic cells potentiates transmigration across polarised endothelium.

The obligate intracellular parasite Toxoplasma gondii makes use of infected leukocytes for systemic dissemination. Yet, how infection impacts the processes of leukocyte diapedesis has remained unresolved. Here, we addressed the effects of T. gondii infection on the trans-endothelial migration (TEM) of dendritic cells (DCs) across polarised brain endothelial monolayers. We report that upregulated expression of leukocyte ICAM-1 is a feature of the enhanced TEM of parasitised DCs. The secreted parasite effector GRA15 induced an elevated expression of ICAM-1 in infected DCs that was associated with enhanced cell adhesion and TEM. Consequently, gene silencing of Icam-1 in primary DCs or deletion of parasite GRA15 reduced TEM. Further, the parasite effector TgWIP, which impacts the regulation of host actin dynamics, facilitated TEM across polarised endothelium. The data highlight that the concerted action of the secreted effectors GRA15 and TgWIP modulate the leukocyte-endothelial interactions of TEM in a parasite genotype-related fashion to promote dissemination. In addition to the canonical roles of endothelial ICAM-1, this study identifies a previously unappreciated role for leukocyte ICAM-1 in infection-related TEM.

Early passage of Toxoplasma gondii across the blood-brain barrier.

The blood-brain barrier (BBB) efficiently protects the central nervous system (CNS) from infectious insults. Yet, the apicomplexan parasite Toxoplasma gondii has a remarkable capability to establish latent cerebral infection in humans and other vertebrates. In addition to the proposed mechanisms for access to the brain parenchyma, recent findings highlight a paramount role played by the BBB in restricting parasite passage and minimizing parasite loads in the brain. Consistent with clinically asymptomatic primary infections in humans, mounting evidence indicates that the original colonization of the brain by T. gondii encompasses previously unappreciated, nondisruptive translocation processes that precede the onset of parasite-limiting immune responses.

Blood-brain barrier-restricted translocation of Toxoplasma gondii from cortical capillaries.

The cellular barriers of the central nervous system proficiently protect the brain parenchyma from infectious insults. Yet, the single-celled parasite Toxoplasma gondii commonly causes latent cerebral infection in humans and other vertebrates. Here, we addressed the role of the cerebral vasculature in the passage of T. gondii to the brain parenchyma. Shortly after inoculation in mice, parasites mainly localized to cortical capillaries, in preference over post-capillary venules, cortical arterioles or meningeal and choroidal vessels. Early invasion to the parenchyma (days 1-5) occurred in absence of a measurable increase in blood-brain barrier (BBB) permeability, perivascular leukocyte cuffs or hemorrhage. However, sparse focalized permeability elevations were detected adjacently to replicative parasite foci. Further, T. gondii triggered inflammatory responses in cortical microvessels and endothelium. Pro- and anti-inflammatory treatments of mice with LPS and hydrocortisone, respectively, impacted BBB permeability and parasite loads in the brain parenchyma. Finally, pharmacological inhibition or Cre/loxP conditional knockout of endothelial focal adhesion kinase (FAK), a BBB intercellular junction regulator, facilitated parasite translocation to the brain parenchyma. The data reveal that the initial passage of T. gondii to the central nervous system occurs principally across cortical capillaries. The integrity of the microvascular BBB restricts parasite transit, which conversely is exacerbated by the inflammatory response.

Integrin-dependent migratory switches regulate the translocation of Toxoplasma-infected dendritic cells across brain endothelial monolayers.

Multiple cellular processes, such as immune responses and cancer cell metastasis, crucially depend on interconvertible migration modes. However, knowledge is scarce on how infectious agents impact the processes of cell adhesion and migration at restrictive biological barriers. In extracellular matrix, dendritic cells (DCs) infected by the obligate intracellular protozoan Toxoplasma gondii undergo mesenchymal-to-amoeboid transition (MAT) for rapid integrin-independent migration. Here, in a cellular model of the blood-brain barrier, we report that parasitised DCs adhere to polarised endothelium and shift to integrin-dependent motility, accompanied by elevated transendothelial migration (TEM). Upon contact with endothelium, parasitised DCs dramatically reduced velocities and adhered under both static and shear stress conditions, thereby obliterating the infection-induced amoeboid motility displayed in collagen matrix. The motility of adherent parasitised DCs on endothelial monolayers was restored by blockade of ß1 and ß2 integrins or ICAM-1, which conversely reduced motility on collagen-coated surfaces. Moreover, parasitised DCs exhibited enhanced translocation across highly polarised primary murine brain endothelial cell monolayers. Blockade of ß1, ß2 integrins, ICAM-1 and PECAM-1 reduced TEM frequencies. Finally, gene silencing of the pan-integrin-cytoskeleton linker talin (Tln1) or of ß1 integrin (Itgb1) in primary DCs resulted in increased motility on endothelium and decreased TEM. Adding to the paradigms of leukocyte diapedesis, the findings provide novel insights in how an intracellular pathogen impacts the migratory plasticity of leukocytes in response to the cellular environment, to promote infection-related dissemination.

Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers.

The apicomplexan parasite Toxoplasma gondii invades tissues and traverses non-permissive biological barriers in infected humans and other vertebrates. Following ingestion, the parasite penetrates the intestinal wall and disseminates to immune-privileged sites such as the brain parenchyma, after crossing the blood-brain barrier. In the present study, we have established a protocol for high-purification of primary mouse brain endothelial cells to generate stably polarised monolayers that allowed assessment of cellular barrier traversal by T. gondii. We report that T. gondii tachyzoites translocate across polarised monolayers of mouse brain endothelial cells and human intestinal Caco2 cells without significantly perturbing barrier impermeability and with minimal change in transcellular electrical resistance. In contrast, challenge with parasite lysate or LPS increased barrier permeability by destabilising intercellular tight junctions (TJs) and accentuated transmigration of T. gondii. Conversely, reduced phosphorylation of the TJ-regulator focal adhesion kinase (FAK) was observed dose-dependently upon challenge of monolayers with live T. gondii but not with parasite lysate or LPS. Pharmacological inhibition of FAK phosphorylation reversibly altered barrier integrity and facilitated T. gondii translocation. Finally, gene silencing of FAK by shRNA facilitated transmigration of T. gondii across epithelial and endothelial monolayers. Jointly, the data demonstrate that T. gondii infection transiently alters the TJ stability through FAK dysregulation to facilitate transmigration. This work identifies the implication of the TJ regulator FAK in the transmigration of T. gondii across polarised cellular monolayers and provides novel insights in how microbes overcome the restrictiveness of biological barriers.

TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63-ITGB1-FAK signaling.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) exerts pleiotropic effects on cells including conferring metastatic properties to cancer cells. As for metastatic cells, recent paradigms of leukocyte migration attribute important roles to the amoeboid migration mode of dendritic cells (DCs) for rapid locomotion in tissues. However, the role of TIMP-1 in immune cell migration and in the context of infection has not been addressed. We report that, upon challenge with the obligate intracellular parasite Toxoplasma gondii, primary DCs secrete TIMP-1 with implications for their migratory properties. Using a short hairpin RNA (shRNA) gene silencing approach, we demonstrate that secreted TIMP-1 and its ligand CD63 are required for the onset of hypermotility in DCs challenged with T. gondii Further, gene silencing and antibody blockade of the ß1-integrin CD29 (ITGB1) inhibited DC hypermotility, indicating that signal transduction occurred via ITGB1. Finally, gene silencing of the ITGB1-associated focal adhesion kinase (FAK, also known as PTK2), as well as pharmacological antagonism of FAK and associated kinases SRC and PI3K, abrogated hypermotility. The present study identifies a TIMP-1-CD63-ITGB1-FAK signaling axis in primary DCs, which T. gondii hijacks to drive high-speed amoeboid migration of the vehicle cells that facilitate its systemic dissemination.

Developmental histories of perceived racial discrimination and diurnal cortisol profiles in adulthood: A 20-year prospective study.

Perceived racial discrimination (PRD) has been associated with altered diurnal cortisol rhythms in past cross-sectional research. We investigate whether developmental histories of PRD, assessed prospectively, are associated with adult diurnal cortisol profiles. One-hundred and twelve (N=50 Black, N=62 White) adults from the Maryland Adolescent Development in Context Study provided saliva samples in adulthood (at approximately age 32 years) at waking, 30min after waking, and at bedtime for 7 days. Diurnal cortisol measures were calculated, including waking cortisol levels, diurnal cortisol slopes, the cortisol awakening response (CAR), and average daily cortisol (AUC). These cortisol outcomes were predicted from measures of PRD obtained over a 20-year period beginning when individuals were in 7th grade (approximately age 12). Greater average PRD measured across the 20-year period predicted flatter adult diurnal cortisol slopes for both Black and White adults, and a lower CAR. Greater average PRD also predicted lower waking cortisol for Black, but not White adults. PRD experiences in adolescence accounted for many of these effects. When adolescent and young adult PRD are entered together predicting cortisol outcomes, PRD experiences in adolescence (but not young adulthood) significantly predicted flatter diurnal cortisol slopes for both Black and White adults. Adolescent, but not young adult PRD, also significantly predicted lower waking and lower average cortisol for Black adults. Young adult PRD was, however, a stronger predictor of the CAR, predicting a marginally lower CAR for Whites, and a significantly larger CAR for Blacks. Effects were robust to controlling for covariates including health behaviors, depression, income and parent education levels. PRD experiences interacted with parent education and income to predict aspects of the diurnal cortisol rhythm. Although these results suggest PRD influences on cortisol for both Blacks and Whites, the key findings suggest that the effects are more pervasive for Blacks, affecting multiple aspects of the cortisol diurnal rhythm. In addition, adolescence is a more sensitive developmental period than adulthood for the impacts of PRD on adult stress biology.