The vertebrate central nervous system (CNS) is protected by restrictive cellular barriers that maintain homeostasis and regulate passage of molecules and cells to the brain parenchyma (Abbott et al., 2006). Three main cellular barrier systems protect neurons from blood-borne external insults, such as infection: the blood-brain barrier (BBB), the blood-cerebrospinal fluid barriers and the meningeal barriers (Coureuil et al., 2017). Of these, the BBB exerts the greatest immediate impact on the cerebral microenvironment.

The BBB is anatomically localized to the cerebral microvasculature, which is constituted by capillaries with a luminal diameter (Ø) of <10 μm, arterioles and venules (~10–100 μm Ø) (Wilhelm et al., 2016; Gould et al., 2017). The microvascular lumen is covered by highly specialized endothelial cells characterized by low permeability, low pinocytic activity and high transcellular electrical resistance (TCER) (Stamatovic et al., 2008). Basal lamina, astrocyte ‘end-feet’ and pericytes also contribute to restrict permeability (Daneman and Prat, 2015; Armulik et al., 2010). Intercellular tight junction (TJ) proteins seal the spaces between adjacent endothelial cells and separate the apical from the basolateral side. This restricts the paracellular flux of hydrophilic molecules and regulates the migration of cells across the endothelial barrier (Alon and van Buul, 2017). Focal adhesion kinase (FAK), also named protein tyrosine kinase 2 (PTK2), regulates signaling pathways involved in cell-cell adhesion and focal adhesion complexes in endothelial cells (Lee et al., 2010; Siu et al., 2009). The turnover of FAK has been shown to regulate barrier function, and dysregulation of FAK impacts the integrity of cellular barriers (Ivey et al., 2009) by increasing paracellular permeability, which can imply disruption of the barrier function (Ma et al., 2013).

Inflammation can cause impairment of the blood-CNS barrier functions (Abbott et al., 2006). Immune activity by resident cells and infiltrating leukocytes are crucial to eradicate pathogens that succeed in translocating across the restrictive CNS barriers (Coureuil et al., 2017), but this response can also alter neuronal function (Dando et al., 2014). Therefore, striking a balance between controlling infection and limiting excessive inflammation is crucial (Klein and Hunter, 2017).

The obligate intracellular parasite Toxoplasma gondii infects a broad range of warm-blooded vertebrates, with an estimated 1/3 of the global human population being chronically infected (Pappas et al., 2009). From the point of entry in the intestinal tract, T. gondii rapidly achieves systemic dissemination and establishes latent infection in the CNS (Schlüter and Barragan, 2019). While chronic infection is generally considered asymptomatic, reactivated or acute infection can lead to life-threatening encephalitis in immune-compromised individuals and to neurological disorders in neonates (Montoya and Liesenfeld, 2004). T. gondii tachyzoites use gliding motility to actively invade cells where they replicate (Dobrowolski and Sibley, 1996). Gliding motility also facilitates transmigration of tachyzoites across the endothelium in vitro (Ross et al., 2019; Barragan et al., 2005) and provides an effective mechanism of propulsion in the microenvironment inside tissues (Barragan and Sibley, 2002). In their journey to form chronic cysts, the tachyzoites cross the cellular brain barriers to establish latent infection in the CNS (Feustel et al., 2012).

In recent years, work in the field has focused on parasite dissemination pathways and mechanisms of passage to the brain (Konradt et al., 2016; Drewry et al., 2019; Kanatani et al., 2017; Bhandage et al., 2020; Schneider et al., 2019; Sangaré et al., 2019). Yet, the roles played by the blood-CNS barriers have remained unresolved. Here, we address the implication of the BBB in the early passage of T. gondii tachyzoites to the CNS. Specifically, we assess the loci of passage of T. gondii across the cerebral cellular barriers and the impacts of BBB integrity, dysregulation and inflammation on the access of tachyzoites to the parenchyma. We identify a role for the TJ regulator FAK in the passage of T. gondii to the brain parenchyma.

Passage across the cellular blood-CNS barriers is a prerequisite for the establishment of latent cerebral toxoplasmosis in humans and rodents. Here, we studied the crucial interaction of T. gondii with the brain vasculature in mice.

Our study establishes that the principal point of entry of T. gondii from the blood circulation into the brain parenchyma is at the level of cortical capillaries, similarly in three separate mouse strains and for prototypic T. gondii type I and type II strains. The data evidence preferential localization of parasites to capillaries with low α5 laminin/P-glycoprotein expression ratio, a relative less frequent localization to post-capillary venules, and scarce or absence of localization to penetrating arterioles, pre-capillary arterioles and penetrating venules.

We report that the BBB integrity is paramount to restrict T. gondii infection of the brain parenchyma. First, from day one post-inoculation, the accumulation of T. gondii tachyzoites in the brain was dramatically inferior to that observed in lung and liver, indicating a relative lower permissiveness of the brain vasculature to adhesion or invasive events by T. gondii. Second, the initial vascular localization with replicating parasites within brain endothelium (Konradt et al., 2016), was rapidly accompanied by appearance of vacuoles with replicating parasites in the perivascular spaces and in the cerebral parenchyma. Third, perturbations of BBB permeability by pro-inflammatory, anti-inflammatory stimuli or high doses of T. gondii resulted in elevated parasite loads in the CNS. Finally, pharmacological inhibition or induced gene ablation of the endothelial BBB intercellular junction regulator FAK facilitated parasite passage to the brain parenchyma.

In natural oral infections, relatively low numbers of T. gondii cysts ultimately form in the CNS indicating that passage across the BBB is a relatively rare event, compared with the initial parasite loads in the parenchyma of other peripheral organs, for example, lungs, liver, spleen, or in musculature. We validated an experimental approach that allowed controlled assessment of T. gondii localization in the CNS after intraperitoneal or intravenous inoculation and that yielded parasite load distributions comparable with oral and other intraperitoneal infection models (Konradt et al., 2016; Kanatani et al., 2017; Zenner et al., 1998; Mordue et al., 2001). While the approaches utilized in this and other studies permit reproducible quantifications, they may also entail limitations. An impact on the relative localization of parasites or kinetics of passage cannot be excluded. However, similar BBB localization and invasion patterns were observed for both inoculation routes, irrespective of mouse strain, parasite strain or dose. Importantly, for the vast majority of parasite foci, the clear vicinity to one capillary indicated the putative translocation site to the brain parenchyma. Further, the observation of perivascular parasite vacuoles beneath the vascular endothelium is interesting and the identification of the hosting cells remains to be investigated.

Our data show that T. gondii tachyzoites very early (1–3 dpi) interact primarily with the endothelium of cortical capillaries, confirming and extending the earlier reported replication of parasites in brain endothelium at later timepoints (7–9 dpi) (Konradt et al., 2016). This localization contrasts with findings in cerebral malaria, where parasitized erythrocytes preferentially bind to post-capillary venules and larger venules with components of inflammatory leukocytes (Haldar et al., 2007; Nacer et al., 2014). The capillary bed offers the largest hemodynamic resistance to the cortical blood supply and has lower flow speeds (Gould et al., 2017). Hypothetically, this together with the restricted diameter of capillaries may facilitate the adhesion, invasion and crossing of T. gondii tachyzoites. In vitro, ICAM-1 and integrins are implicated in adhesion and transmigration of T. gondii tachyzoites (Barragan et al., 2005) and of parasitized leukocytes (Ueno et al., 2014; Ross et al., 2021). Among other inflammatory parameters and cell adhesion molecules (CAMs), here we report elevated expression of ICAM-1 and VCAM-1 in infected brains and, specifically, in purified brain microvessels from challenged mice. Jointly with the finding of elevated TIMP-1 expression in microvessels, and its recently attributed functions in maintenance of FAK-related BBB integrity (Tang et al., 2020) and T. gondii dissemination (Ólafsson et al., 2018; Ólafsson et al., 2019), future investigations need to address the implication of CAMs in endothelial and parenchymal invasion.

We provide evidence that T. gondii can invade the CNS across the choroid plexus (CP) and meningeal vessels, albeit in lower relative numbers compared with total parasite numbers entering across the extensive parenchymal vascular network. However, taking into account the relative smaller endothelial cell surface area and tissue volume of the CP and meningeal vessels related to cortical vasculature, the data indicate that passage is facilitated at the CP. Hypothetically, this may implicate the fenestrated choroidal endothelium, a known locus minoris resistentiae for brain invasion by the causative agent of sleeping sickness Trypanosoma brucei (Schultzberg et al., 1988). Similarly, bacteria and viruses can translocate across meninges and CP (Schwerk et al., 2015) or by disrupting the parenchymal BBB (Doran et al., 2016; Spindler and Hsu, 2012). The data is also in line with the reported localization of T. gondii reactivation foci in grey matter, with few reactivation foci in the CP and the meninges (Dellacasa-Lindberg et al., 2007). Further, human cerebral toxoplasmosis is associated with encephalitis and seldomly associated with meningeal inflammation (Schlüter and Barragan, 2019), while detection of T. gondii in the cerebrospinal fluid is used in clinical practice (Mesquita et al., 2010). Jointly, this indicates that, while parenchymal invasion occurs primarily across cortical capillaries, passage across the CP and meningeal vessels provides T. gondii access to the cerebrospinal fluid circulatory system, which may contribute to parasite dissemination within the CNS.

We report that early passage of T. gondii to the brain parenchyma occurs in absence of measurable BBB leakage, perivascular cuffing or prominent leukocyte infiltration, similarly in different mouse strains and for T. gondii type I and type II strains. In fact, measurable leukocyte infiltration in the vicinity of parasite foci was absent early during infection (< day 5). In contrast, infiltration was abundant by day 10, in line with recent findings (Schneider et al., 2019). Thus, perivascular cuffing or significant leukocyte infiltration does not appear to be a requisite for initial T. gondii passage to the parenchyma (< day 5). However, an inflammatory activation of the BBB endothelium by the infection was evidenced by elevated expression of CAMs but also inflammation-dampening TIMP-1 (Ólafsson et al., 2018; Ólafsson et al., 2019). Pro-inflammatory pre-treatment with LPS yielded elevations of parenchymal parasite loads and significantly larger cerebral parasite foci. This indicates that initial parasite passage was facilitated by LPS-induced increased BBB permeability (Banks et al., 2015), which may implicate regulation via FAK (Guo et al., 2015), and by exacerbated inflammation (Haruwaka et al., 2019). Conversely, anti-inflammatory pre-treatment with hydrocortisone led to elevated total parasite loads in the brain but also in other organs, likely secondary to a hampered immune response. Jointly, though these treatments have pleiotropic effects with a likely impact on parasitemia, the data is indicative that passage of T. gondii across the BBB is not only restricted by the inflammatory response. Along these lines, unnaturally high doses of T. gondii tachyzoites were required to induce BBB leakage similar to LPS. This strongly advocates against a generalized BBB dysfunction early in natural T. gondii infections, but does not exclude the occurrence of focalized dysregulations (Estato et al., 2018). In fact, focalized permeability elevations were detected in the vicinity of parasite foci, albeit in low numbers and without increased relative numbers over time. This is indicative of BBB permeability variations over time following the penetration of parasites to the parenchyma or that the BBB is transiently dysregulated. Thus, future investigations need to determine if an elevated BBB permeability around a given focus is consistently present, albeit transient, or if translocation can also occur in the absence of elevated permeability. Also, taking into consideration the alternative pathways of translocation discussed below, these two possibilities are not necessarily mutually exclusive. Noteworthy, despite the infection of endothelium and expected natural lysis of parasitized cells after replication, we found no evidence of irreversible vascular disruption with petechial hemorrhages, as described in cerebral malaria (Haldar et al., 2007) and meningococcal disease (Coureuil et al., 2014). Yet, this does not rule out that single translocation events of T. gondii or egress of tachyzoites from single endothelial cells transiently destabilize the BBB below the detection threshold for the permeability tracer EB. In in vitro models using polarized primary brain endothelium, extracellular tachyzoites and parasitized dendritic cells rapidly translocated in absence of endothelial cell lysis, with a modest perturbation of the transcellular electrical resistance (TCER) but, importantly, in absence of leakage of a low-molecular weight permeability tracer (Ross et al., 2019; Ross et al., 2021). In sharp contrast, cellular depolarization events or lysis of cells in the monolayers following parasite replication at later time-points led to significant decreases in TCER and dramatic elevations of permeability tracer concentration in vitro (Ross et al., 2019; Ross et al., 2021). Altogether, these findings motivated an exploration of the BBB regulatory mechanisms in vivo.

We demonstrate a role for the BBB junction regulator FAK in the restricted passage of T. gondii to the brain parenchyma. Despite being embryonic lethal (Ilić et al., 1995), unchallenged adult mice with conditional deletion of endothelial FAK normally exhibit wildtype phenotype and apparently normal BBB morphology (Lee et al., 2010; Weis et al., 2008), consistent with the maintained morphology and the absence of leakage of blood tracer observed here. However, upon T. gondii challenge, FAK-deficient mice carried higher numbers of parasite foci and foci of larger size in the parenchyma, indicative of increased permissiveness compared to wildtype. Interestingly, pharmacological treatment with FAK inhibitor had a more pronounced effect on parasite loads with elevated BBB permeability, compared with conditional ablation of endothelial FAK. This difference may be due to a number of factors. First, conditional deletion of endothelial FAK is partially functionally compensated (Weis et al., 2008). Second, although the FAK inhibitor defactinib appears to be well tolerated, immunomodulatory effects may also contribute to the higher parasite loads in peripheral organs, and, thus, elevated numbers of parasites may enter the cerebral circulation (Osipov et al., 2019). Additionally, inhibition of FAK may interfere with parasite dissemination in infected leukocytes (Ólafsson et al., 2019; Cook et al., 2018) and parasite survival in endothelium (Portillo et al., 2017). Regardless, induced ablation of FAK in endothelium, with maintained expression of FAK in bone-marrow-derived cells, yielded higher numbers and size of parasite foci in brain parenchyma, demonstrating endothelium-related effects. Further, we recently reported that altered FAK phosphorylation upon T. gondii infection facilitates parasite passage across highly polarized primary brain endothelial cell monolayers (Ross et al., 2019). Importantly, this transient dysregulation of FAK occurs without disruption of monolayer polarization or increased permeability to low-molecular weight tracer, consistent with in vivo observations here. Thus, FAK inhibition or ablation appears to have a destabilizing, but non-disruptive, effect on the BBB. Jointly, the data show that endothelial FAK-regulated BBB restrictiveness is one of the determinants of T. gondii passage across the BBB.

Alternative routes have been proposed for the translocation of T. gondii across restrictive biological barriers (Lambert and Barragan, 2010), including the BBB (Schlüter and Barragan, 2019), and it is likely that more than one mechanism mediates entry into the CNS, including growth across endothelium, direct penetration and trafficking of leukocytes (Matta et al., 2021). The paracellular entry route (Ross et al., 2019; Barragan et al., 2005; Barragan and Sibley, 2002; Furtado et al., 2012; Weight et al., 2015) implies passing cellular TJs, and the transcellular route (Konradt et al., 2016; Lambert and Barragan, 2010; Dubey, 1997) implies apical parasite invasion and basolateral exit from the cells after replication. Additionally, leukocytes traffic into the brain parenchyma during toxoplasmosis (John et al., 2011) and parasitized hypermigratory leukocytes may aid in direct translocation (Bhandage et al., 2020; Courret et al., 2006; Lachenmaier et al., 2011) or by depositing parasites on the endothelium (Lambert and Barragan, 2010; Baba et al., 2017). Although the relative importance of the alternative pathways of passage is not specifically addressed here, the data are relevant for these alternatives or combinations of pathways because focal invasion events with an impact on TJ and BBB barrier integrity are likely to occur, for example, upon egress of tachyzoites from endothelium (Konradt et al., 2016). In these settings, we demonstrate that a non-replicating T. gondii line (CPS) transmigrates across polarized endothelium and epithelium in vitro, with elevated transmigration frequencies upon FAK inhibition. Thus, despite the reduced gliding motility and transmigration abilities of this mutant, the data provide additional evidence that FAK impacts the endothelial process of tachyzoite transmigration, in absence of monolayer disruption and independently of parasite replication in vitro. In mice, surprisingly few CPS parasites were detected in the brain and other peripheral organs shortly after inoculation, consistent with the absence of symptomatology in mice despite the inoculation of doses that are imminently lethal with wildtype ( > 10⁸) and with scarce localization of single CPS tachyzoites intravascularly, and extravascularly in the brain parenchyma. Partly contrasting with in vitro results, elevated total numbers of CPS parasites were not detected in the brain upon FAK inhibition or ablation in vivo. Given the high TCER of the BBB, this may be related to the expected overall reduced invasive and transmigration abilities of this mutant, in combination with a sensitivity limitation of the assay due to scarce total invasion events. Importantly, the low numbers of intravascular CPS parasites compared with wildtype (RH) precluded direct comparisons with wildtype for numbers of translocation events in vivo. Together, the reduced gliding and TCER-related transmigration abilities of this auxotroph mutant upon uracil deprivation indicate that metabolic stress (Blondy et al., 2020) negatively impacts invasion events in vitro and in vivo and, that this mutant is rapidly neutralized in vivo, possibly as a consequence of its generally reduced invasion capability.

Jointly, the present study identifies FAK-regulated TJ stability as a determinant of T. gondii translocation across cortical capillaries. We postulate that the infection-related destabilization of the BBB by FAK dephosphorylation (Ross et al., 2019) facilitates active transmigration of parasites across the BBB. The impact of transient and focal BBB dysregulation on the alternative pathways of parasite passage to the parenchyma, together with the roles of the non-endothelial cellular constituents of the neurovascular unit, need to be addressed in future investigations.

In humans and mice, primary infection with T. gondii entails invasion of the CNS with passage of the parasite across the BBB. Natural infections in humans normally present mild or no symptomatology (Montoya and Liesenfeld, 2004). Thus, our data supports the concept that passage of T. gondii to the brain parenchyma is an early and clinically silent process in absence of focal neurological symptoms or cardinal signs of inflammation. As the immune response develops, tachyzoites are neutralized and developmental switch to bradyzoite stages takes place for chronic perseverance in the brain parenchyma.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Gabriela C Olivera

   Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

2. Emily C Ross

   Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

3. Christiane Peuckert

   Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

   Contribution

   Data curation, Formal analysis, Methodology, Validation, Visualization

   Competing interests

   No competing interests declared

4. Antonio Barragan

   Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   antonio.barragan@su.se

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7746-9964

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