An important function of the blood–brain barrier is to exclude pathogens from the central nervous system, but some microorganisms benefit from the ability to enter this site. It has been proposed that Toxoplasma gondii can cross biological barriers as a motile extracellular form that uses transcellular or paracellular migration, or by infecting a host cell that then crosses the blood–brain barrier. Unexpectedly, analysis of acutely infected mice revealed significant numbers of free parasites in the blood and the presence of infected endothelial cells in the brain vasculature. The use of diverse transgenic parasites combined with reporter mice and intravital imaging demonstrated that replication in and lysis of endothelial cells precedes invasion of the central nervous system, and highlight a novel mechanism for parasite entry to the central nervous system.

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Interleukin-1β (IL-1β) functions as a key regulator of inflammation and innate immunity. The protozoan parasite Toxoplasma gondii actively infects human blood monocytes and induces the production of IL-1β; however, the host and parasite factors that mediate IL-1β production during T. gondii infection are poorly understood. We report that T. gondii induces IL-1β transcript, processing/cleavage, and release from infected primary human monocytes and THP-1 cells. Treating monocytes with the caspase-1 inhibitor Ac-YVAD-CMK reduced IL-1β release, suggesting a role for the inflammasome in T. gondii-induced IL-1β production. This was confirmed by performing short hairpin RNA (shRNA) knockdown of caspase-1 and of the inflammasome adaptor protein ASC. IL-1β induction required active parasite invasion of monocytes, since heat-killed or mycalolide B-treated parasites did not induce IL-1β. Among the type I, II, and III strains of T. gondii, the type II strain induced substantially more IL-1β mRNA and protein release than did the type I and III strains. Since IL-1β transcript is known to be induced downstream of NF-κB signaling, we investigated a role for the GRA15 protein, which induces sustained NF-κB signaling in a parasite strain-specific manner. By infecting human monocytes with a GRA15-knockout type II strain and a type I strain stably expressing type II GRA15, we determined that GRA15 is responsible for IL-1β induction during T. gondii infection of human monocytes. This research defines a pathway driving human innate immunity by describing a role for the classical inflammasome components caspase-1 and ASC and the parasite GRA15 protein in T. gondii-induced IL-1β production.

Toxoplasma gondii is a highly successful global pathogen that is remarkable in its ability to infect nearly any nucleated cell in any warm-blooded animal. Infection with T. gondii typically occurs through the ingestion of contaminated food or water, but the parasite then breaches the intestinal epithelial barrier and spreads from the lamina propria to a large variety of other organs in the body. A key feature of T. gondii pathogenesis is the parasite's ability to cross formidable biological barriers in the infected host and enter tissues such as the brain, eye and placenta. The dissemination of T. gondii into these organs underlies the severe disease that accompanies human toxoplasmosis. In this review, we will focus on seminal studies as well as exciting recent findings that have shaped our current understanding of the cellular and molecular mechanisms by which T. gondii journeys throughout the host and enters organs to cause disease.

Toxoplasma gondii actively infects circulating immune cells, including monocytes and DCs, and is thought to use these cells as Trojan horses for parasite dissemination. To investigate the interactions of T. gondii-infected human monocytes with vascular endothelium under conditions of shear stress, we developed a fluidic and time-lapse fluorescence microscopy system. Both uninfected and infected monocytes rolled, decelerated, and firmly adhered on TNF-α-activated endothelium. Interestingly, T. gondii-infected primary human monocytes and THP-1 cells exhibited altered adhesion dynamics compared with uninfected monocytes: infected cells rolled at significantly higher velocities (2.5- to 4.6-fold) and over greater distances (2.6- to 4.8-fold) than uninfected monocytes, before firmly adhering. During monocyte searching, 29-36% of infected monocytes compared with 0-11% of uninfected monocytes migrated >10 μm from the point where they initiated searching, and these "wandering" searches were predominantly in the direction of flow. As infected monocytes appeared delayed in their transition to firm adhesion, we examined the effects of infection on integrin expression and function. T. gondii did not affect the expression of LFA-1, VLA-4, or MAC-1 or the ability of Mn(2+) to activate these integrins. However, T. gondii infection impaired LFA-1 and VLA-4 clustering and pseudopod extension in response to integrin ligands. Surprisingly, a single intracellular parasite was sufficient to mediate these effects. This research has established a system for studying pathogen modulation of human leukocyte adhesion under conditions of physiological shear stress and has revealed a previously unappreciated effect of T. gondii infection on ligand-dependent integrin clustering.