ICAM-1/CD18-mediated sequestration of parasitized phagocytes in cortical capillaries promotes neuronal colonization by Toxoplasma gondii

Rodriguez, Matias E.; Hassan, Ali; Linaroudis, Nikolaos; Harryson-Oliveberg, Felix; ten Hoeve, Arne L.; Barragan, Antonio

doi:10.1038/s41467-025-58655-z

Download PDF

Article
Open access
Published: 14 April 2025

ICAM-1/CD18-mediated sequestration of parasitized phagocytes in cortical capillaries promotes neuronal colonization by Toxoplasma gondii

Nature Communications volume 16, Article number: 3529 (2025) Cite this article

4895 Accesses
1 Citations
44 Altmetric
Metrics details

Subjects

Abstract

Microbial translocation across the blood-brain barrier (BBB) is a prerequisite for colonization of the central nervous system. The obligate intracellular parasite Toxoplasma gondii chronically infects the brain parenchyma of humans and animals, in a remarkably stealthy fashion. We investigated the mechanisms of BBB traversal by T. gondii (genotypes I, II, III) and T. gondii-infected leukocytes, using intracarotid arterial delivery into the cerebral circulation of mice. Unexpectedly, parasitized dendritic cells (DCs) and other peripheral blood mononuclear cells were found to persistently sequester within cortical capillaries. Post-replicative egress of T. gondii from sequestered DCs was followed by rapid parasite localization within cortical neurons. Infection-induced microvascular inflammation dramatically elevated the sequestration of parasitized DCs, while treatments targeting the ICAM-1/CD18 leukocyte adhesion axis with blocking antibodies strongly reverted sequestration. The parasite effectors TgWIP and GRA15, known to promote leukocyte hypermigration and inflammatory activation, further increased both the capillary sequestration of infected DCs and cerebral parasite loads in a strain-dependent manner. These findings reveal that the sequestration of parasitized leukocytes in cortical capillaries, with subsequent BBB traversal following parasite egress, provides a mechanism for T. gondii’s rapid access to cortical neurons during primary infection.

Immunotherapy targeting the PD-1 pathway alleviates neuroinflammation caused by chronic Toxoplasma infection

Article Open access 23 January 2023

The spatiotemporal transcriptional profiling of murine brain during cerebral malaria progression and after artemisinin treatment

Article Open access 11 February 2025

Reduced neural progenitor cell count and cortical neurogenesis in guinea pigs congenitally infected with Toxoplasma gondii

Article Open access 27 November 2023

Introduction

The blood-brain barrier (BBB) protects the vertebrate central nervous system (CNS) from most infectious insults, with maintained homeostasis and regulated passage of cells to the brain parenchyma^1,2. Anatomically localized to the cerebral microvasculature, the BBB is constituted by arterioles, capillaries and venules^3,4. Two internal carotid arteries (ICAs) and 2 vertebral arteries merge at the base of the brain in the circle of Willis to supply the brain with blood. From the circle of Willis, inter-cerebral and pial arteries lead the blood to arterioles, which penetrate the parenchyma and lead to the capillary network^5,6. With an estimated total length of 600 km (370 miles) and a luminal surface area of 15–25 m² (50–82 sq. ft.) in the adult human, the BBB vasculature is dominated by capillaries of luminal diameter <10 µm, surrounded by a neurovascular unit (NVU) wall of <10 µm and an average cortical inter-capillary distance of 40 µm^7,8. It is becoming increasingly clear that neuro-vascular interactions are crucial to maintain homeostasis in the CNS⁹.

The microvascular lumen of the BBB is covered by highly specialized endothelial cells characterized by low permeability, low pinocytic activity and high transcellular electrical resistance¹⁰. Intercellular tight junction proteins restrict the paracellular flux of hydrophilic molecules and regulates the migration of cells across the endothelial barrier¹¹. Basal lamina, pericytes and astrocyte ‘end-feet’ also contribute to the restricted permeability of the NVU^12,13. Immune activity by resident cells and infiltrating leukocytes, in the absence or presence of neuroinflammation¹⁴, are crucial to eradicate pathogens that succeed in translocating across the restrictive CNS barriers². However, the generated inflammation can also perturb BBB functions¹ and alter neuronal function¹⁵.

CNS infections by microbial pathogens are among the most devastating infectious diseases worldwide, often with highly limited therapeutic options¹⁶. The Apicomplexan parasite Toxoplasma gondii infects a broad range of warm-blooded vertebrates, with an estimated 1/3 of the global human population being chronically infected¹⁷. Three clonal lineages of T. gondii (type I, II, III) predominate in humans and animals in Europe and North America¹⁸. From its point of entry in the intestine, T. gondii disseminates systemically via the blood circulation and rapidly achieves latent infection in the CNS¹⁹. While chronic infection is generally considered asymptomatic, reactivated or acute infection can lead to life-threatening encephalitis in immune-compromised individuals and to severe neurological disorders in neonates²⁰.

The rapidly replicating invasive stage of T. gondii -the tachyzoite- is obligate intracellular and uses gliding motility to actively invade cells where it replicates²¹. Gliding motility also facilitates transmigration of tachyzoites across polarized endothelium in vitro^22,23 and provides an effective mechanism of propulsion in the microenvironment inside tissues²⁴. In their journey to form chronic cysts, the tachyzoites cross the cellular brain barriers to establish latent infection in the CNS²⁵. T. gondii also exploits the trafficking of mononuclear phagocytes for dissemination. Systemic spread from the intestine to peripheral organs via the blood circulation is largely mediated by parasitized CD11c⁺ / CD11b⁺ leukocytes²⁶. Dendritic cells (DCs) and other phagocytes can act as Trojan horses for systemic dissemination of T. gondii in mice²⁷, in a parasite genotype-related fashion²⁸. Upon active invasion by T. gondii, DCs are induced to migrate via activation of non-canonical GABAergic signaling and MAP kinase activation^29,30,31,32. This migratory activation, termed hypermigratory phenotype³³, implicates secreted parasite effectors^34,35,36 and chemotactic activation of parasitized phagocytes³⁷.

Thus, a number parasite dissemination pathways and mechanisms of passage to the brain have been proposed in recent years. Yet, the relative contribution or importance of the different putative pathways mediating passage across the BBB have remained unresolved^24,25. Also, studies have been partly hampered by the low parasite numbers reaching the brain parenchyma early during infection, forcing characterizations at later time points with exacerbated systemic infection^38,39. To overcome these limitations and gain novel insights in the early mechanisms of passage, we developed an experimental system with delivery of T. gondii and parasitized phagocytes directly into the cerebral circulation.

Results

Parasitized CD45⁺ leukocytes sequester in cerebral capillaries

While it is well established that mononuclear phagocytes mediate systemic and tissue dissemination of T. gondii, their association to the appearance of tachyzoites in the brain parenchyma has remained unresolved. To address this, we first performed intraperitoneal (ip) infections with T. gondii and detected increased numbers of peripheral blood mononuclear cell (PBMC)-associated tachyzoites in blood over time (Fig. 1A, B, Fig. S1A). Interestingly, analyses of brain cortices detected CD45⁺ leukocytes with replicating GFP⁺ tachyzoite vacuoles contained within cortical capillaries (Fig. 1C). However, experimental approaches in mice with delivery of T. gondii via the tail vein or ip^38,39 entail entering the systemic circulation with entrapment of circulating parasites in peripheral organs, especially the lungs and liver (Fig. S1B–E). The restrictiveness of the BBB makes early quantifications of parasite numbers reaching the cerebral circulation difficult prior to replication and expansion locally, compared with other organs³⁹. To overcome this known limitation, T. gondii-infected CFSE- or CMTMR-labeled DCs were injected directly into the cerebral circulation via the ICA (Fig. 1D), and the localization of parasitized cells was assessed in the frontal cortex 16 h post-inoculation (hpi) (Fig. 1E). Interestingly, GFP⁺CMTMR⁺, RFP⁺CFSE⁺ or CFSE⁺ CMTMR⁺ cells with replicating type I, II or III tachyzoites, respectively, were readily detected in the vasculature (Fig. 1F, G; Movie S1) with luminal diameter <10 μm (Fig. 1H), preferentially in the vicinity of vascular branching points and similarly for T. gondii type I-, II- and III-infected DCs (Fig. 1I). In contrast, higher numbers of type II-infected DCs compared with type I- and III-infected DCs were found in cortex (Fig. 1J), consistent with elevated adhesion of type II-infected DCs to polarized endothelial monolayers (Fig. 1K). We conclude that parasitized CD45⁺ leukocytes, including DCs, can sequester in cortical capillaries, with measurable variations between T. gondii strains from clonal lineages I, II and III.

**Fig. 1: Sequestration of infected PBMCs and DCs in the brain microvasculature.**

Sequestration of parasitized DCs in cortical capillaries is rapidly followed by parasite egress and translocation of tachyzoites across the BBB

To determine the immediate fate of T. gondii tachyzoites contained in sequestered DCs, the brains of mice injected in the ICA were extracted 4–48 hpi (Fig. 2A) and the localization and numbers of infected DCs and T. gondii foci were microscopically assessed in relation to the vasculature. By 4 hpi, tachyzoites (GFP⁺) were exclusively associated with DCs (CMTMR⁺) within capillaries (Fig. 2B). Importantly, tachyzoites were found extra-vascularly by 16 hpi (Fig. 2B; Movie S2), indicating egress from DCs and passage across the endothelium. Expectedly, localization and replication within endothelial cells^38,39 with appearance of DC membrane remnants (ghosts) also occurred by 28 hpi (Fig. 2B; Movie S2). By 48 hpi, larger extravascular foci with replicating tachyzoite vacuoles appeared (Fig. 2B). Interestingly, while absolute numbers of infected DCs in the microvasculature decreased over time (Fig. 2C), numbers of non-DC-associated tachyzoites conversely increased (Fig. 2D), yielding a dramatic change in the ratio between the two (Fig. 2E). In contrast, numbers of non-infected DCs remained stable for 28 h (Fig. 2F), with maintained ratios related to infected DCs (Fig. 2G, H). Further, DCs infected with non-replicating T. gondii (CPS) remained adhered to the microvasculature for up to 48 hpi, in absence of replication or egress from DCs and strongly contrasting with the appearance of invasive parasitic foci upon T. gondii-WT challenge (Fig. 2I, J; Fig. S2). Jointly, the data show that replicating T. gondii tachyzoites egress from infected DCs sequestered in cortical capillaries and either transmigrate across endothelium or invade endothelial cells.

**Fig. 2: Replication and egress of *T. gondii* from sequestrated DCs precedes parenchymal localization.**

T. gondii tachyzoites egressing from sequestered DCs are rapidly retrieved in cortical neurons

To address the fate of T. gondii following egress from infected DCs, tissues were stained for the neuronal nuclear and perinuclear cytoplasmatic marker NeuN. By 16–28 hpi, replicating tachyzoites were detected in NeuN⁺ cells (Fig. 3A, B; Movies S3, 4), evidencing a rapid translocation to neurons. Tachyzoites were also detected in the vicinity of astrocytes (GFAP⁺), albeit in the absence of major replication vacuoles (Fig. S3A). Next, to address if egressed free tachyzoites possess the ability to directly transmigrate from the vascular lumen across the BBB, freshly egressed tachyzoites were injected in the ICA and their localizations were assessed at 1 or 16 hpi. Both single WT tachyzoites and non-replicating CPS tachyzoites were retrieved in extravascular locations at 1 hpi, showing the rapidity of the tissue invasion or extravasation processes and that replication was not imperatively necessary (Fig. 3C; Movie S5, 6). While the vast majority of tachyzoites were located intravascularly or associated with the endothelium, in line with previous observations^38,39, tachyzoite vacuoles were also identified at locations >10 μm from the capillary (Fig. 3D; Fig. S3B), implying extravascular localization in the parenchyma^7,8. We also measured higher relative numbers of type I tachyzoites in cortical tissue compared with type II tachyzoites (Fig. 3E), with similar localization to capillaries (Fig. 3F; Fig. S3B) but longer distances to branching points in the vasculature (Fig. 3G). Typically, a 3–5 fold higher inoculum dose of type II was needed to reach comparable numbers of parasite foci in the brain (Fig. S3C), likely reflecting a superior invasive capacity²⁴ and extracellular viability (Fig. S3D) by type I tachyzoites. In a similar fashion, we compared the relative tachyzoite numbers upon delivery of infected DCs or delivery as free tachyzoites, for both type I and type II strains. Consistent with microscopy analyses (Fig. 3E), inoculation of comparable cfu of free tachyzoites yielded higher parasite loads for type I (Fig. 3H). Further, while delivery in infected DCs yielded higher parasite loads for both type I and type II compared with free tachyzoites (Fig. 3I), type II loads exhibited a superior relative increase upon delivery in DCs, related to delivery as free type II tachyzoites (Fig. 3J). Jointly, the data align, and extend to the BBB, both the ascribed higher relative dependence on leukocytes for systemic dissemination by type II strains²⁸ and the higher transmigration frequencies in vitro by extracellular type I tachyzoites²⁴. We conclude that tachyzoites egressing from sequestered DCs or delivered into the cerebral circulation as free tachyzoites can transmigrate across the BBB strain/type-dependently and in the absence of replication, thereby expediting the invasion of neurons.

**Fig. 3: Following sequestration of infected DCs, *T. gondii* is retrieved inside neurons.**

Sequestration of parasitized DCs depends on ICAM-1/CD18 and is exacerbated by a rapid systemic microvascular inflammation upon T. gondii infection

To determine the fate of T. gondii under inflammatory conditions in the CNS, infected DCs were injected in the ICA of animals pre-treated with pro-inflammatory LPS. Alternatively, heparin, which has anti-inflammatory and anti-adhesive effects, was administered (Fig. 4A). Pre-injection with LPS or heparin treatment dramatically elevated and reduced, respectively, the numbers of sequestered DCs in the vasculature (Fig. 4B, C). Interestingly, LPS-treatment elevated DC numbers in capillaries but not in post-capillary venules or arterioles (Fig. 4D, E), indicating selective effects. LPS and heparin, respectively, had similar opposite effects in vitro on endothelial monolayers, corroborating direct impacts on the adhesion frequencies of parasitized DCs (Fig. S4A). In vivo, LPS treatment elevated adhesion for both type I- and type II-infected DCs (Fig. S4B) and for T. gondii-challenged splenocytes (Fig. S4C–E), indicating that the pro-inflammatory effects were not highly dependent on parasite strain or host cell/leukocyte type. Next, we assessed if pre-infection with T. gondii ip also impacted numbers of sequestered DCs upon ICA injection. First, we confirmed that pre-infection ip yielded undetectable parasite loads in the brain at 48 hpi, yet quantifiable loads in lungs and liver (Fig. S4F–H). Interestingly, upon ICA injection, significantly higher numbers on sequestered infected DCs were detected in pre-infected animals (Fig. 4F–I), similar to the effects of pre-treatment with LPS. Of note, while the quantitatively predominant phenomenon was sequestration of parasitized DCs in the microvasculature, extravascular localization of infected DCs in the parenchyma was also demonstrated (Fig. S4I; Movie S7). However, the low frequency of this event precluded consistent quantification.

**Fig. 4: *T. gondii*-induced BBB inflammation promotes the sequestration of parasitized DCs.**

Next, we investigated the inflammatory response. A surprisingly rapid (<24 h) transcriptional upregulation of adhesion molecules, including ICAM-1, and inflammatory markers in freshly-isolated cerebral microvessels from T. gondii ip-challenged mice (Fig. 4K; Fig. S4J). Reciprocally, parasitized DCs maintained CD18 expression (Fig. S4K). These findings, together with the paramount role of the ICAM-1/CD18 axis in leukocyte vascular adhesion, motivated treatments with blocking antibodies⁴⁰. Importantly, both treatment with anti-ICAM-1^41,42 and anti-CD18⁴³, but not isotype control-treatments, dramatically reduced numbers of sequestered DCs (Fig. 4M, N; Fig. S4L) and parasite loads (Fig. 4O, P). We conclude that, upon T. gondii infection, a systemic inflammatory response rapidly activates the cerebral endothelium which facilitates sequestration of parasitized leukocytes in cortical capillaries, with an important implication of the ICAM-1/CD18 adhesion axis.

Secreted parasite effectors impact the sequestration of infected DCs and cerebral parasite loads

Because T. gondii effectors TgWIP and GRA15 modulate the migratory features and inflammatory activation of parasitized leukocytes in vitro and in vivo^36,37,44, we assessed their putative impact on the sequestration of parasitized DCs in cerebral capillaries. Interestingly, mice challenged with Δgra15 or ΔTgWIP-infected DCs (Fig. 5A), consistently exhibited lower numbers of sequestered parasitized DCs in capillaries (Fig. 5B, C) and total parasite loads (Fig. 5D, E). Next, to determine the role of inflammation in this process, mice were pre-challenged with WT T. gondii ip and brains were extracted 1 hpi (Fig. 5F; Fig. S5A). Consistent with previous data (Fig. 4), total numbers of sequestered DCs increased dramatically under inflammatory condition, with maintained differences between WT and mutants (Fig. 5G–J; Fig. S5B), indicating direct effects on sequestration by TgWIP and GRA15. Consistently, Δgra15-infected DCs exhibited reduced adhesion to endothelial cell monolayers in vitro, compared with WT-infected DCs (Fig. S5C). Similarly, a re-elevation of sequestration was measured upon challenge with a complemented mutant (Δgra15 + GRA15), reaching sequestration levels similar to WT (Fig. S5D). Next, we determined the long-term impact of GRA15 on parasite loads in the brain. Plaquing assays confirmed significantly lower cerebral parasite loads in mice challenged with Δgra15-infected DCs at 7 dpi, with non-significant differences in the spleen (Fig. 5K, L). Further, we confirmed that pro-inflammatory treatment (LPS) and anti-inflammatory treatment (heparin) exacerbated and reduced, respectively, cerebral parasite loads at 7 dpi (Fig. 5M, N; Fig. S5E). Finally, we evaluated whether anti-ICAM-1 treatment affected cerebral parasite loads in mice at 7 dpi. Since pre-inoculation ip with wild-type T. gondii could contribute to total cerebral parasite loads at this stage, we instead used the non-replicative CPS strain. Prior to this, we confirmed that CPS induced an inflammatory response in the cerebral endothelium, similar to wild-type T. gondii (Fig. S5F, G). Infected DCs were then administered into the ICA ± anti-ICAM-1 treatment (Fig. 5O). Treated animals exhibited lower cerebral parasite loads compared to isotype control-treated mice, whereas splenic parasite loads remained unchanged (Fig. 5P). Altogether, we conclude that the T. gondii effectors TgWIP and GRA15 intensify ICAM-1-dependent sequestration of infected DCs in the brain vasculature, with an impact on cerebral parasite loads.

**Fig. 5: *T. gondii* effectors TgWIP and GRA15 modulate the sequestration of parasitized DCs.**

Discussion

Stemming from the finding of sequestered parasitized CD45⁺ leukocytes in cortical capillaries, we undertook an experimental approach with controlled delivery of T. gondii into the cerebral circulation to assess early translocation events across the BBB. Strikingly, parasite replication within sequestered DCs was followed by egress and rapid intracellular localization in cortical neurons, with an impact on total cerebral parasite loads. This implied a sequential process with (i) sequestration of parasitized leukocytes, (ii) niched intracellular replication in the sequestered leukocyte, (iii) egress and rapid extracellular transmigration across the BBB and (iv) subsequent infection of neurons (Fig. 6). We propose that this mechanism maximizes the transmigration efficiency and, consequently, facilitates the passage of T. gondii to the brain parenchyma. Indeed, it is upon egress from host cells that T. gondii tachyzoites peak in gliding motility and invasiveness, while these abilities decay over time^24,39.

**Fig. 6: Sequestration of *T. gondii*-infected leukocytes at the BBB facilitates CNS colonization.**

The data establish that a persevering sequestration of parasitized leukocytes takes place in the cerebral microvasculature during T. gondii infection. Further, the sequestration of parasitized DCs was dramatically exacerbated by the inflammatory response to infection and by secreted parasitic effectors. Indeed, pre-infection in the peritoneum or LPS-treatment ip very rapidly activated the cerebral endothelium, elevating transcriptional expression of cell adhesion molecules, such as ICAM-1, in microvasculature. Importantly, targeting ICAM-1 or its integrin counterpart CD18 with blocking antibodies^41,42,43, dramatically reduced sequestration and parasite loads, identifying a prime role for these two ligands in the sequestration of T. gondii within cortical capillaries. ICAM-1 came into focus due to its implication in transmigration of parasitized leukocytes in vitro^45,46 and also because it mediates adhesion and transmigration of free tachyzoites in vitro²³. Along these lines, in cerebral Plasmodium falciparum malaria, ICAM-1 has been implicated in the sequestration of parasitized erythrocytes in the vasculature via parasite-derived variant adhesins⁴⁷. Thus, the data indicates that the rapidly enhanced expression of ICAM-1 in cerebral endothelium upon T. gondii challenge facilitates adhesion of parasitized leukocytes first, and, later, transmigration of egressing tachyzoites. Importantly, de-sequestration treatment by ICAM-1 blockade had an immediate (1- 24 h) and longer term (7 days) impact on cerebral parasite loads.

We demonstrate that two T. gondii effectors modulate the sequestration of parasitized DCs in cortical capillaries. Specifically, GRA15 elevated leukocyte sequestration in the microvasculature thereby facilitating parasite transmigration upon egress. This relative abundant and persistent sequestration, in the absence of a measurable passage of infected DCs to the parenchyma, is partly contrasting with the recent implication of both GRA15 and ICAM-1 in elevated transmigration of phagocytes across polarized endothelium in vitro^44,45. The data also indicate that the adhesion to the vasculature by infected leukocytes does not automatically imply transmigration and underscore the importance of cautious extrapolations of in vitro phenotypes to conditions in vivo⁴⁸. One possibility is that, very early during infection, parenchymal inflammatory cues that may promote leukocyte extravasation are not present. Further, the elevated sequestration by GRA15-expressing type II T. gondii is also consistent with the relatively higher dependency of type II strains on parasitized leukocytes for systemic dissemination²⁸ and the inferior sequestration by type I strain RH, which lacks a functional GRA15⁴⁹. Also, because GRA15 activates NF-kB⁵⁰ and endothelial NF-kB mediates neuronal activation via TLR/Myd88 signaling⁵¹, it is plausible that GRA15-mediated pro-inflammatory NF-kB activation facilitates colonization⁵². Further, TgWIP elevated sequestration and cerebral parasite loads, in line with its attributed roles in hypermotility and adhesion of DCs^36,44,53. Jointly, because the type I strain RH expresses a non-functional form of GRA15⁴⁹, TgWIP deficiency in type I RH may phenotypically reflect a combined GRA15/TgWIP deficiency in this respect. This likely explains the overall inferior sequestration frequency of ΔTgWIP-infected DCs compared with ΔGRA15-infected DCs at very early time points (1 hpi), that is, before parasite egress or significant replication sets in. Finally, the recent discovery that a set of T. gondii effectors, including GRA15, induce CCR7-mediated chemotaxis of parasitized phagocytes elicits speculation^37,52. The BBB endothelium constitutively expresses the chemokine CCL19¹⁴ and, its receptor CCR7 has been suggested to mediate the migration of circulating T cells across the BBB⁵⁴. However, direct evidence for endothelial CCL19 in mediating immune cell trafficking to the CNS in the absence of neuroinflammation is lacking and awaits further investigations. Altogether, the findings highlight that the diversity and redundancy of polymorphic effectors, including co-operative effects⁵¹, need to be taken into account when studying phenotypes linked to dissemination and colonization of the CNS by T. gondii. The data also gives rise to the intriguing possibility that type II tachyzoites compensate their inferior invasive capacity²⁴ of cerebral endothelium with a superior sequestration of infected leukocytes in the microvasculature.

We report that cortical neurons can contain replicating T. gondii as early as 16–28 h after intracarotid inoculation. Rapidly, tachyzoites were detected within the parenchyma, preferentially, but not exclusively, associated with neurons located in the immediate vicinity of the vasculature. The BBB (NVU wall) is <10 µm thick, with an average cortical inter-capillary distance of 40 µm^7,8 and an average distance of 15 µm between neuronal somata and capillaries⁵⁵. Taking into consideration the ability of freshly egressed tachyzoites to transmigrate in vitro and migrate ex vivo in tissues²⁴^,²³^,²², the present results extend in vivo the ability of T. gondii to translocate across non-permissive biological barriers, specifically the BBB. We confirm previous data of infection of endothelial cell linings^38,39 and add that endothelial cells and neurons can become infected simultaneously in time, indicating that replication within endothelium is common but not imperative for brain colonization. However, while circulating leukocytes facilitate systemic dissemination of T. gondii, it remains unclear whether free/extracellular tachyzoites in the blood contribute to dissemination, especially early during infection with low parasitemia, and given their sensitivity to neutralization by the complement system and IgM⁵⁶. Here, we also provide evidence that extracellular tachyzoites and parasitized DCs, when delivered in the cerebral circulation, can directly enter the brain parenchyma, albeit at lower frequency, compared with tachyzoites entering after egress from sequestered DCs. Moreover, we extend previous findings that extracellular tachyzoites of type I (RH) transmigrate at higher frequencies than type II strains in vitro^24,39 to the BBB in vivo. Inherent differences between strains in gliding motility, invasiveness, and extracellular viability of tachyzoites likely contribute to these phenotypes²⁴^,. The data indicate that direct transmigration across the BBB by circulating extracellular tachyzoites and direct transmigration by parasitized leukocytes represent relatively minor, but contributing, pathways to the initial colonization of the CNS.

The preferential localization of T. gondii in cortical capillaries compared with other vascular segments after intracarotid inoculation is consistent with findings in infections ip or iv^38,39. This localization contrasts somewhat with observations in human and experimental cerebral malaria. Malaria-parasitized erythrocytes bind preferentially to postcapillary venules and larger venules, with components of inflammatory leukocytes^57,58, but also to capillaries⁵⁹. Further, the preferential localization of T. gondii-infected DCs to vascular branching points is intriguing and reminiscent of the arrest of metastatic cancer cells in branching points of brain capillaries⁶⁰. We report a surprisingly rapid (24 h) inflammatory activation of the cerebral microvasculature after intraperitoneal T. gondii infection, preceding the dramatically elevated systemic cytokine responses later during infection⁶¹ and in line with the recently reported early inflammation of choroid plexus endothelium by day 3 post-infection⁶². In contrast, despite the very early neuronal infection, no evidence of inflammatory leukocyte infiltration, hemorrhage or disruption of the BBB⁶³ was found at these early timepoints, though transient focal elevations of BBB permeability³⁹ cannot be fully excluded. Importantly, these findings match well with the stealthy CNS colonization during primary T. gondii infection, which is typically clinically silent or accompanied by mild symptomatology in the absence of focal neurologic signs²⁰. In sharp contrast, vascular colonization during meningococcal meningitis includes endothelial alterations, inflammation and purpuric lesions, and paracellular translocation of Neisseria meningitidis, although the fate of internalized bacteria has not yet been elucidated².

On a technical note, unilateral occlusion of the ICA can unlikely explain the observed sequestration pattern because circulation is rapidly compensated by collateral vessels and redistribution of blood at the circle of Willis^5,6, confirmed by the sequestration of parasitized DCs in the contralateral cerebral hemisphere and by a lack of focal symptomatology in mice post-procedure. Regardless of inoculation route, the colonization of the brain parenchyma by T. gondii consists of low-frequency events, related to the total parenchymal infiltration in other organs³⁹. The undertaken approach allowed spatiotemporal quantitative analyses of extensive cortical areas at high resolution to measure low-frequency early events, which is limited using other approaches. The immediate effects of blocking antibodies avoided the adaptation and compensatory mechanisms in mutant mice owing to partly overlapping or redundant functions of CAMs and integrins^64,65.

In recent years, work in the field has described dissemination pathways for T. gondii and possible routes for CNS colonization^{29,30,35,36,38,39,66,67,68}. Here, we add that the sequestration of parasitized leukocytes within cortical capillaries represents a remarkable mechanism enabling T. gondii translocation to the cerebral parenchyma and subsequent neuronal infection. In our experimental system, direct migration of T. gondii-infected DCs into the parenchyma was rare, occurring far less frequently than the CD18/ICAM-1-dependent sequestration of DCs. Thus, sequestered leukocytes, alike endothelium^38,39, constituted a replicative niche that facilitated direct transmigration of T. gondii tachyzoites upon egress. To better understand the dissemination of T. gondii, we argue that it is essential to distinguish between (i) the role of leukocytes in facilitating systemic dissemination to peripheral organs, including their transport to the brain organ^26,27,66, and (ii) the mechanisms that mediate the pathogen’s crossing of the BBB into the brain parenchyma itself^19,48. The findings presented here establish a crucial pathogenetic link between systemic dissemination and CNS invasion in toxoplasmosis. Specifically, the data highlight how leukocyte sequestration acts as a bridge between these two critical stages of infection, ultimately facilitating neuronal colonization.

Importantly, cerebral parasite loads as a consequence of sequestration outnumbered loads after inoculation with free tachyzoites for type I and II strains. It is reasonable to speculate that a rapid invasion of neurons minimizes the risk of elimination by the immune system, permits a swift initiation of bradyzoite cyst formation and avoids excessive host-detrimental inflammation that lytic processes at the BBB entail. The ensuing immune response, which follows the initial parasite invasion to the parenchyma, likely neutralizes excessive replicating tachyzoites around vasculature and a few cysts can develop in neurons. We propose therefore that the adhesion of parasitized leukocytes to the cerebral vasculature provides, from the pathogen’s perspective, a safe and cost-effective mechanism for passage to the CNS. From an evolutionary perspective, this relatively silent process may favor chronic asymptomatic carriage in the CNS of intermediate hosts, to ultimately ensure transmission to the feline definitive host upon predation. Future prophylactic or therapeutic approaches targeting cerebral toxoplasmosis need to take into consideration the diverse mechanisms used by T. gondii for colonizing the CNS.

Methods

Ethical considerations

The Regional Animal Research Ethical Board, Stockholm, Sweden, approved experimental procedures in mice and protocols involving extraction of cells from mice (permit number 16403-2022), following proceedings described in EU legislation (Council Directive 2010/63/EU). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https://arriveguidelines.org).

Mice

All experiments were performed using male and female C57BL/6NCrl mice (strain code 027, Charles River), aged 4 to 10 weeks, housed in a ventilated facility, provided with unrestricted access to tap water and food, and kept under a 12-h light/dark cycle at a temperature of 20–22 °C.

Parasite culture and cell lines

T. gondii tachyzoites of type I: RH-GFP, RHΔTgWIP-GFP³⁶, RH CPS-mCherry⁶⁹, type II: ME49-RFP⁷⁰, PRU-GFP⁵⁰, PRUΔTgGRA15-GFP⁵⁰, PRUΔTgGRA15 + GRA15-GFP⁵⁰ and type III: CTG (ATCC, 50842^TM) were routinely maintained by serial 48 h passaging in human foreskin fibroblasts, HFFs (ATCC, CRL-2088). HFFs and murine brain endothelial cells, bEnd.3 (ATCC, CRL-2299), were cultured in High glucose Dulbecco’s modified Eagle’s medium (DMEM, VWR, Cat# 392-0415) supplemented with 10% heat inactivated fetal bovine serum (FBS, HyClon, Cat# SV30160.03), 20 μg/ml gentamicin (Gibco, Cat# 15710-049) and 10 mM HEPES (HyClone, Cat# SH30237.01). Cell cultures and parasites were cultured at 37 °C, 5% CO₂ in a humidified atmosphere. All cultures were regularly tested for Mycoplasma.

Primary DCs and splenocytes

Murine bone marrow-derived DCs were generated as previously described⁶⁶. Bone marrow cells were purified from 4–8 week-old mice and cultured in RPMI 1640 medium (VWR, Cat# 392-0427) supplemented with 10% FBS, 20 μg/ml gentamicin, 10 mM HEPES and 10 ng/ml recombinant GM-CSF (PeproTech, Cat# 1479-1407). DCs (loosely adherent cells) were harvested after 6 days. Spleens were isolated from 4–8 week-old mice and mashed using a 40 µm cell strainer. Splenocytes were collected and RBC lysed with RBC-lysis buffer (Invitrogen, Cat# 00-4300-54).

Preparations of T. gondii-infected cells and extracellular tachyzoites

Inoculations with infected cells were performed as previously described^37,66. Briefly, DCs or splenocytes were pre-labeled with CFSE (Invitrogen, Cat# C34554) or CMTMR (Invitrogen, Cat# C2927) and challenged with freshly egressed T. gondii tachyzoites at multiplicity of infection (MOI) 1 (type I, RH) or MOI 2 (type II, PRU, ME49; type III, CTG) for 4 h to obtain an infection frequency of ∼50%. CTG was pre-labeled with CFSE. Cell suspensions were then washed thrice and spun at 60–70 x g to minimize free tachyzoites in the inoculum. Total numbers of colony-forming units (cfu) injected into animals was confirmed by plaquing assays. Host cell number and viability was assessed by ocular hemocytometry and flow cytometry, respectively.

For inoculations with free tachyzoites, tachyzoites were collected from monolayers with maximum 50% HFF lysis, passed twice through a 27-gauge needle, washed twice by centrifugation at 870xg, kept at RT and inoculated in mice within 30–60 min from collection. Total numbers of cfu injected into animals was confirmed by plaquing assays.

Intraperitoneal (ip) and intravenous (iv) inoculations in mice

Six to 8 week-old mice were inoculated ip with 0,2–10 × 10⁶ cfu of T. gondii tachyzoites. To assess parasite loads, animals were sacrificed at 2, 3 or 6 dpi. When indicated, anti-mo CD45 Alexa fluor 647-conjugated antibody (R&D Systems, Cat# FAB3507R, Clone 319211, Lot# 1726103) was injected iv (0,4 mg/kg) 10 min before euthanasia. For adoptive transfer assays, mice were inoculated iv with 20 × 10⁶ cfu of T. gondi-challenged DCs and sacrificed 24 hpi.

Microsurgery, intracarotid artery inoculations and treatments

Six to 8-week-old mice were anesthetized with 2% isoflurane (Sigma-Aldrich, Cat# CDS019936) and the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were exposed under a dissecting microscope (Leica M205 FA). The left CCA was carefully separated from the vague nerve using micro-forceps. A 6–0 silk ligature was tied around the CCA and the ECA. A new loosely ligature was placed in the CCA below the bifurcation site. Using micro-scissors, a small cut in the CCA was made between the first and loose ligatures. A 32gauge/.8Fr catheter (Instech, Cat# BTPU-010) fitted on a syringe with 100 μl of medium containing cell/parasite suspensions was inserted into the CCA, guided to the bifurcation site and tied with a new ligature. The loose ligature was opened and the fluid was slowly injected over 5 min into the ICA. Then, the loose ligature was tightened and the catheter removed. Finally, fat tissue and muscles were replaced, the skin sutured and analgesic (Temgesic, Eumedica Pharmaceuticals) administrated. When indicated, 100 U/kg of heparin (ThermoFisher Scientific) was injected iv 2 h after ICA inoculation. Alternatively, 2 mg/kg of LPS (Sigma-Aldrich) was injected ip 4 h prior the surgical procedure. For blocking assays, anti-mo CD54 (eBioscience, Cat# 14-0541-85, Clone YN1/1.7.4, Lot# 2089516), anti-mo LFA-1 beta (eBioscience, Cat# 14-0181-85, Clone M18/2, Lot# 2577324) or isotype (eBioscience, Cat# 14-4321-85, Clone eBR2a, Lot# 2777082) (all 0,2 mg/kg) were mixed with the cell/parasite suspensions 5-10 min prior to ICA inoculation.

Flow cytometry

DCs were challenged with freshly egressed tachyzoites for 5 h, washed in PBS and stained in FACS buffer (0,5% BSA, 2 mM EDTA in PBS). Challenged DCs were stained with 1/50 (v/v) PE-Cyanine7 anti-mo CD11c (eBioscience, Cat# 25-0114-82, Clone N418, Lot# 2142958), 1/50 (v/v) PE anti-mo CD18 (eBioscience, Cat# 101407, Clone M18/2. Lot# B313003) and 1/ 50 (v/v) Super Bright 702 anti-mo MHCII I-A/I-E (eBioscience, Cat# 67-5321-80, Clone M5/114.15.2, Lot# 2020037) antibodies and analyzed on a LSR Fortessa cell cytometer (BD Biosciences). Blood was collected by cardiac puncture into heparinized tubes and peripheral blood mononuclear cells (PBMCs) were purified with Lymphoprep^TM (STEMCELL Technologies, Cat# 07801). Cells were fixed in 4% PFA (Histolab, Cat# 02176), resuspended in FACS buffer and analyzed on LSR Fortessa. Data were analyzed with FlowJo software v10.

Quantification of T. gondii foci in cortical brain sections

To visualize cerebral blood vessels, mice were injected iv with 50 μl of PBS 3% Evans blue (Sigma, Cat# E2129-106) and 5% BSA (Sigma-Aldrich) prior brain extraction. Brains were washed in PBS and fixed in 4% PFA for 48 h at 4 °C. Then, PFA was replaced by PBS 10% sucrose (Sigma-Aldrich) for 2 d at 4 °C. Fixed brains were frozen in liquid nitrogen and 50 μm thick cryosections were collected on glass slides. To quantify the number of infected DC/T. gondii foci, the prefrontal cortex area of the brain was imaged using epifluorescence microscopy (Leica DMi8). A T. gondii foci was defined as a single replicating vacuole or a localized group of tachyzoites that was not confined inside or associated with CMTMR⁺ DCs. Data were expressed as the number of T. gondii foci or infected DCs per cm² of tissue. Images were manually quantified with Fiji/ImageJ software. Data tables were exported and plotted using GraphPad Prism v.10 software.

3D Immunohistochemistry and image processing

Brain sections (50 μm thick) were transferred into 2 ml U-bottom tubes (5 sections/ tube) and washed 3 times with PBS. Then, samples were permeabilized and blocked in 1 ml of blocking buffer (1% Triton X-100 and 10% FBS in PBS) for 2 h at RT. Then, samples were incubated with 0,2 ml of primary antibodies anti-NeuN (Abcam, Cat# ab177487, Clone EPR12763, Lot# GR3275122-10) and anti-GPAF (Invitrogen, Cat# 13-0300, Clone 2.2B10, Lot# VH307339) diluted 1/250 (v/v) in blocking buffer for 48 h at 4 °C with gentle shaking. Sections were washed in PBS 0.1% Triton X-100 (3 times x 1 h) and incubated with 0,2 ml of Alexa Fluor 594-conjugated secondary antibodies (Invitrogen, Cat# A21442, Lot# 2906378; Molecular probes, Cat# A2147, Lot# 52521 A) diluted 1/1000 (v/v) in blocking buffer for 24 h at 4 °C with gentle shaking. After washing with PBS 0,1% Triton X-100, samples were mounted for imaging with LSM 800 Airyscan, Zeiss confocal microscope.

To quantify the localization of T. gondii in relation to blood vessels, DCs, neurons and astrocytes, brain sections were imaged using laser lines 488 nm (for GFP and CFSE), 561 nm (for RFP, CMTMR and Alexa 594) and 640 nm (For Evans blue and Alexa 647). Z-stacks from brain sections were collected with 40x objective from 0 to 50 µm in depth with 0.5-1 µm interval in the vertical z-axis and processed with IMARIS v.10.1 software. The Surface rendering tool was used to automatically define brain vasculature, DCs, T. gondii, neurons and astrocytes. The diameter (μm) of the vessels, distance to the branching points, and distance to the nearest vessel were manually defined and data tables were exported and plotted with GraphPad Prism v.10 software. Infected DCs/neurons/astrocytes were defined with the Surface-Surface Colocalization tool.

RT-Quantitative Polymerase Chain Reaction (PCR)

Total genomic DNA was purified from brain, liver and lung using DNeasy blood & Tissue kit (Qiagen, Cat# 69506) according to manufacturer’s protocols. Organs were homogenized in 4 ml of sterile PBS on 70 µm cell strainers. 0.5-1 ml of tissue homogenate was pelleted in 1.5 ml tubes for gDNA purification. gDNA concentration was measured using a spectrophotometer (Nano Drop 1000, ThermoFisher Scientific). Real-time PCR was performed using 100 ng gDNA, forward and reverse primers (200 nM) targeting TgB1 gene or mGAPDH gene and SYBR green PCR master mix (Kappa Biosystem, Cat# KK4602) with a QuantStudio™ 5 real-time PCR system (ThermoFisher). GAPDH was used as a house-keeping gene to generate ∆Ct values in order to calculate relative expression (2^^-ΔΔCT)^.

For brain micro-vessels, total RNA was extracted using RNeasy mini kit (Qiagen, Cat# 74104) according manufacture’s protocol. The RNA was quantified by spectrophotometry (NanoDrop 1000). cDNA was synthesized with Maxima first strand cDNA synthesis kit (ThermoFisher, Cat# EP0753). Real-time PCR as performed using 10–100 ng cDNA, 200 nM forward and reverse primers and SYBR green PCR master kit (Kapa Biosystem). Primers are listed in Table S1. GAPDH and HRT were used as house-keeping genes to generate ∆Ct values in order to calculate relative expression (2^^-ΔΔCT).

Isolation of cerebral micro-vessels

Brain micro-vessels were isolated as previously described³⁹. Brains were homogenized by passage through a 23-gauge needle. Homogenates were diluted 1:1 (v/v) with 30% dextran solution (MW 70.000; Sigma-Aldrich, Cat# 31390-25) and centrifuged at 10.000xg for 15 min at 4 °C. The myelin layer was discarded and the pellet resuspended in DMEM 10% FBS. The suspension was passed through a 40-µm cell strainer and the vessels fragments retrieved were washed with PBS. Finally, the cell strainer was back-flushed with PBS and the micro-vessels pelleted by centrifugation at 4500 x g for 30 min at 4 °C.

Plaquing and adhesion assays

For plaquing assays⁶⁶, brains, livers, and spleens from infected mice were extracted and homogenized using cell strainers (70 μm pore size). Homogenates were diluted in DMEM 10% FBS at 1/10 to 1/1000 (v/v) and added to confluent HFF monolayers in 24-well plates. After 24 h, medium was replaced. The numbers of viable parasites per g of tissue were determined by counting plaque formation after 3–5 days. To assess parasite viability, freshly egressed tachyzoites were resuspended in DMEM with 10% FBS (800 tachyzoites/ml), 250 µl were added to confluent HFF monolayers in 24-well plates at 0, 2, and 4 h after harvest and the number of viable parasites was determined by plaque formation.

For adhesion assays, DCs were pre-labeled with CMTMR or CFSE and challenged with freshly egressed GFP- or RFP-expressing tachyzoites (MOI 1 for type I, MOI 2 for type II). 1–5 × 10³ cfu of T. gondii challenged DCs were added to confluent bEnd.3 monolayers for 1 h. Then, monolayers were washed 5 times x 5 min with PBS under shaking (300 rpm), fixed 5 min in PFA 4%, washed with PBS and the number of infected DCs per well was quantified by epifluorescence microscopy. When indicated, monolayers were pre-treated with LPS (100 ng/ml) overnight. Before the adhesion assay, LPS-treated monolayers were washed 5 times with DMEM 10% FBS. Alternatively, heparin (100 U/ml) was mixed with the challenged DCs immediately before adding them to the monolayers. For blocking assays, anti-mo ICAM-1 or isotype (0,5 g/ ml) were mixed with the cell suspensions and immediately added to the monolayers.

Statistical analyses

Statistical analyses were performed with GraphPad Prism v. 10. For multiple sample comparisons, one-way ANOVA followed Bonferroni’s multiple comparison test were carried out on data sets with normal distribution, and Kruskal–Wallis test followed Dunn’s post-hoc test was performed on data sets with non-normal distribution. For data sets from 2 samples with normal distribution, two-tailed Student’s t-test was performed. For data sets from 2 samples with non-normal distribution, Mann–Whitney U-test was performed. A p-value < 0.05 was defined as significant differences for all statistical tests.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Source data are provided in this paper. All data needed to support the conclusions are presented in the paper, the Supplementary Information and the Source Data file. Source data are provided with this paper.

References

Abbott, N. J., Ronnback, L. & Hansson, E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat. Rev. Neurosci. 7, 41–53 (2006).
Article CAS PubMed Google Scholar
Coureuil, M., Lecuyer, H., Bourdoulous, S. & Nassif, X. A journey into the brain: insight into how bacterial pathogens cross blood-brain barriers. Nat. Rev. Microbiol 15, 149–159 (2017).
Article CAS PubMed Google Scholar
Wilhelm, I., Nyul-Toth, A., Suciu, M., Hermenean, A. & Krizbai, I. A. Heterogeneity of the blood-brain barrier. Tissue Barriers 4, e1143544 (2016).
Article PubMed PubMed Central Google Scholar
Gould, I. G., Tsai, P., Kleinfeld, D. & Linninger, A. The capillary bed offers the largest hemodynamic resistance to the cortical blood supply. J. Cereb. Blood Flow. Metab. 37, 52–68 (2017).
Article CAS PubMed Google Scholar
Hossmann, K. A. Pathophysiology and therapy of experimental stroke. Cell Mol. Neurobiol. 26, 1057–1083 (2006).
Article PubMed Google Scholar
Polycarpou, A. et al. Adaptation of the cerebrocortical circulation to carotid artery occlusion involves blood flow redistribution between cortical regions and is independent of eNOS. Am. J. Physiol. Heart Circ. Physiol. 311, H972–H980 (2016).
Article PubMed Google Scholar
Duvernoy, H., Delon, S. & Vannson, J. L. The vascularization of the human cerebellar cortex. Brain Res Bull. 11, 419–480 (1983).
Article CAS PubMed Google Scholar
Wong, A. D. et al. The blood-brain barrier: an engineering perspective. Front Neuroeng. 6, 7 (2013).
Article CAS PubMed PubMed Central Google Scholar
Kaplan, L., Chow, B. W. & Gu, C. Neuronal regulation of the blood-brain barrier and neurovascular coupling. Nat. Rev. Neurosci. 21, 416–432 (2020).
Article CAS PubMed PubMed Central Google Scholar
Stamatovic, S. M., Keep, R. F. & Andjelkovic, A. V. Brain endothelial cell-cell junctions: how to “open” the blood brain barrier. Curr. Neuropharmacol. 6, 179–192 (2008).
Article CAS PubMed PubMed Central Google Scholar
Alon, R. & van Buul, J. D. Leukocyte Breaching of Endothelial Barriers: The Actin Link. Trends Immunol. 38, 606–615 (2017).
Article CAS PubMed Google Scholar
Daneman, R. & Prat, A. The blood-brain barrier. Cold Spring Harb. Perspect. Biol. 7, a020412 (2015).
Article PubMed PubMed Central Google Scholar
Armulik, A. et al. Pericytes regulate the blood-brain barrier. Nature 468, 557–561 (2010).
Article ADS CAS PubMed Google Scholar
Marchetti, L. & Engelhardt, B. Immune cell trafficking across the blood-brain barrier in the absence and presence of neuroinflammation. Vasc. Biol. 2, H1–H18 (2020).
Article CAS PubMed PubMed Central Google Scholar
Dando, S. J. et al. Pathogens penetrating the central nervous system: infection pathways and the cellular and molecular mechanisms of invasion. Clin. Microbiol Rev. 27, 691–726 (2014).
Article PubMed PubMed Central Google Scholar
Le Govic, Y., Demey, B., Cassereau, J., Bahn, Y. S. & Papon, N. Pathogens infecting the central nervous system. PLoS Pathog. 18, e1010234 (2022).
Article PubMed PubMed Central Google Scholar
Pappas, G., Roussos, N. & Falagas, M. E. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. Int J. Parasitol. 39, 1385–1394 (2009).
Article PubMed Google Scholar
Sibley, L. D., Khan, A., Ajioka, J. W. & Rosenthal, B. M. Genetic diversity of Toxoplasma gondii in animals and humans. Philos. Trans. R. Soc. Lond. 364, 2749–2761 (2009).
Article Google Scholar
Schluter, D. & Barragan, A. Advances and challenges in understanding cerebral Toxoplasmosis. Front Immunol. 10, 242 (2019).
Article PubMed PubMed Central Google Scholar
Montoya, J. G. & Liesenfeld, O. Toxoplasmosis. Lancet 363, 1965–1976 (2004).
Article CAS PubMed Google Scholar
Dobrowolski, J. M. & Sibley, L. D. Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 84, 933–939 (1996).
Article CAS PubMed Google Scholar
Ross, E. C., Olivera, G. C. & Barragan, A. Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers. Cell Microbiol 21, e13048 (2019).
Article PubMed Google Scholar
Barragan, A., Brossier, F. & Sibley, L. D. Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2. Cell Microbiol 7, 561–568 (2005).
Article CAS PubMed Google Scholar
Barragan, A. & Sibley, L. D. Transepithelial migration of Toxoplasma gondii is linked to parasite motility and virulence. J. Exp. Med. 195, 1625–1633 (2002).
Article CAS PubMed PubMed Central Google Scholar
Matta, S. K., Rinkenberger, N., Dunay, I. R. & Sibley, L. D. Toxoplasma gondii infection and its implications within the central nervous system. Nat. Rev. Microbiol 19, 467–480 (2021).
Article CAS PubMed Google Scholar
Courret, N. et al. CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain. Blood 107, 309–316 (2006).
Article CAS PubMed PubMed Central Google Scholar
Lambert, H., Hitziger, N., Dellacasa, I., Svensson, M. & Barragan, A. Induction of dendritic cell migration upon Toxoplasma gondii infection potentiates parasite dissemination. Cell Microbiol. 8, 1611–1623 (2006).
Article CAS PubMed Google Scholar
Lambert, H., Vutova, P. P., Adams, W. C., Lore, K. & Barragan, A. The Toxoplasma gondii-shuttling function of dendritic cells is linked to the parasite genotype. Infect. Immun. 77, 1679–1688 (2009).
Article CAS PubMed PubMed Central Google Scholar
Bhandage, A. K. et al. A motogenic GABAergic system of mononuclear phagocytes facilitates dissemination of coccidian parasites. Elife 9, e60528 (2020).
Article CAS PubMed PubMed Central Google Scholar
Kanatani, S. et al. Voltage-dependent calcium channel signaling mediates GABAA receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii. PLoS Pathog. 13, e1006739 (2017).
Article PubMed PubMed Central Google Scholar
Olafsson, E. B., Ross, E. C., Varas-Godoy, M. & Barragan, A. TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63-ITGB1-FAK signaling. J. Cell Sci. 132, jcs230920 (2019).
Article PubMed Google Scholar
Olafsson, E. B. et al. Convergent met and voltage-gated Ca(2+) channel signaling drives hypermigration of Toxoplasma-infected dendritic cells. J. Cell Sci. 134, jcs241752 (2020).
Article PubMed Google Scholar
Weidner, J. M. & Barragan, A. Tightly regulated migratory subversion of immune cells promotes the dissemination of Toxoplasma gondii. Int J. Parasitol. 44, 85–90 (2014).
Article CAS PubMed Google Scholar
Weidner, J. M. et al. Migratory activation of parasitized dendritic cells by the protozoan Toxoplasma gondii 14-3-3 protein. Cell Microbiol. 18, 1537–1550 (2016).
Drewry, L. L. et al. The secreted kinase ROP17 promotes Toxoplasma gondii dissemination by hijacking monocyte tissue migration. Nat. Microbiol. 4, 1951–1963 (2019).
Article CAS PubMed PubMed Central Google Scholar
Sangare, L. O. et al. In vivo CRISPR screen identifies TgWIP as a Toxoplasma modulator of dendritic cell migration. Cell Host Microbe 26, 478–492 e478 (2019).
Article CAS PubMed PubMed Central Google Scholar
Ten Hoeve, A. L. et al. The Toxoplasma effector GRA28 promotes parasite dissemination by inducing dendritic cell-like migratory properties in infected macrophages. Cell Host Microbe 30, 1570–1588.e1577 (2022).
Article PubMed PubMed Central Google Scholar
Konradt, C. et al. Endothelial cells are a replicative niche for entry of Toxoplasma gondii to the central nervous system. Nat. Microbiol. 1, 16001 (2016).
Article CAS PubMed PubMed Central Google Scholar
Olivera, G. C., Ross, E. C., Peuckert, C. & Barragan, A. Blood-brain barrier-restricted translocation of Toxoplasma gondii from cortical capillaries. Elife 10, e69182 (2021).
Article CAS PubMed PubMed Central Google Scholar
Sagar, D. et al. Mechanisms of dendritic cell trafficking across the blood-brain barrier. J. Neuroimmune Pharm. 7, 74–94 (2012).
Article Google Scholar
Zhao, Y. J. et al. Blockade of ICAM-1 improves the outcome of polymicrobial sepsis via modulating neutrophil migration and reversing immunosuppression. Mediators Inflamm. 2014, 195290 (2014).
Article PubMed PubMed Central Google Scholar
Yu, T. et al. M-MDSCs mediated trans-BBB drug delivery for suppression of glioblastoma recurrence post-standard treatment. J. Control Release 369, 199–214 (2024).
Article CAS PubMed Google Scholar
Jain, P., Coisne, C., Enzmann, G., Rottapel, R. & Engelhardt, B. Alpha4beta1 integrin mediates the recruitment of immature dendritic cells across the blood-brain barrier during experimental autoimmune encephalomyelitis. J. Immunol. 184, 7196–7206 (2010).
Article CAS PubMed Google Scholar
Ross, E. C., Hoeve, A. L. T., Saeij, J. P. J. & Barragan, A. Toxoplasma effector-induced ICAM-1 expression by infected dendritic cells potentiates transmigration across polarised endothelium. Front Immunol. 13, 950914 (2022).
Article CAS PubMed PubMed Central Google Scholar
Ueno, N. et al. Real-time imaging of Toxoplasma-infected human monocytes under fluidic shear stress reveals rapid translocation of intracellular parasites across endothelial barriers. Cell Microbiol. 16, 580–595 (2014).
Article CAS PubMed Google Scholar
Ross, E. C., Ten Hoeve, A. L. & Barragan, A. Integrin-dependent migratory switches regulate the translocation of Toxoplasma-infected dendritic cells across brain endothelial monolayers. Cell Mol. Life Sci. 78, 5197–5212 (2021).
Article CAS PubMed PubMed Central Google Scholar
Adams, Y. et al. Plasmodium falciparum erythrocyte membrane protein 1 variants induce cell swelling and disrupt the blood-brain barrier in cerebral malaria. J. Exp. Med. 218, e20201266 (2021).
Article CAS PubMed PubMed Central Google Scholar
Ross, E. C., Olivera, G. C. & Barragan, A. Early passage of Toxoplasma gondii across the blood-brain barrier. Trends Parasitol. 38, 450–461 (2022).
Article PubMed Google Scholar
Yang, N. et al. Genetic basis for phenotypic differences between different Toxoplasma gondii type I strains. BMC Genomics 14, 467 (2013).
Article CAS PubMed PubMed Central Google Scholar
Rosowski, E. E. et al. Strain-specific activation of the NF-kappaB pathway by GRA15, a novel Toxoplasma gondii dense granule protein. J. Exp. Med. 208, 195–212 (2011).
Article CAS PubMed PubMed Central Google Scholar
Gosselin, D. & Rivest, S. MyD88 signaling in brain endothelial cells is essential for the neuronal activity and glucocorticoid release during systemic inflammation. Mol. Psychiatry 13, 480–497 (2008).
Article CAS PubMed Google Scholar
Ten Hoeve, A. L. et al. Hypermigration of macrophages through the concerted action of GRA effectors on NF-kappaB/p38 signaling and host chromatin accessibility potentiates Toxoplasma dissemination. mBio 15, e0214024 (2024).
Article PubMed Google Scholar
Morales, P. et al. The Toxoplasma secreted effector TgWIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2. Cell Mol. Life Sci. 81, 294 (2024).
Article CAS PubMed PubMed Central Google Scholar
Kivisakk, P. et al. Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin. Proc. Natl Acad. Sci. USA 100, 8389–8394 (2003).
Article ADS PubMed PubMed Central Google Scholar
Tsai, P. S. et al. Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels. J. Neurosci. 29, 14553–14570 (2009).
Article CAS PubMed PubMed Central Google Scholar
Couper, K. N., Roberts, C. W., Brombacher, F., Alexander, J. & Johnson, L. L. Toxoplasma gondii-specific immunoglobulin M limits parasite dissemination by preventing host cell invasion. Infect. Immun. 73, 8060–8068 (2005).
Article CAS PubMed PubMed Central Google Scholar
Haldar, K., Murphy, S. C., Milner, D. A. & Taylor, T. E. Malaria: mechanisms of erythrocytic infection and pathological correlates of severe disease. Annu Rev. Pathol. 2, 217–249 (2007).
Article CAS PubMed Google Scholar
Nacer, A. et al. Experimental cerebral malaria pathogenesishemodynamics at the blood brain barrier. PLoS Pathog. 10, e1004528 (2014).
Article PubMed PubMed Central Google Scholar
Strangward, P. et al. A quantitative brain map of experimental cerebral malaria pathology. PLoS Pathog. 13, e1006267 (2017).
Article PubMed PubMed Central Google Scholar
Kienast, Y. et al. Real-time imaging reveals the single steps of brain metastasis formation. Nat. Med. 16, 116–122 (2010).
Article CAS PubMed Google Scholar
Mordue, D. G., Monroy, F., La Regina, M., Dinarello, C. A. & Sibley, L. D. Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines. J. Immunol. 167, 4574–4584 (2001).
Article CAS PubMed Google Scholar
Figueiredo, C. A. et al. Immune response and pathogen invasion at the choroid plexus in the onset of cerebral toxoplasmosis. J. Neuroinflammation 19, 17 (2022).
Article CAS PubMed PubMed Central Google Scholar
Galea, I. The blood-brain barrier in systemic infection and inflammation. Cell Mol. Immunol. 18, 2489–2501 (2021).
Article CAS PubMed PubMed Central Google Scholar
Gorina, R., Lyck, R., Vestweber, D. & Engelhardt, B. beta2 integrin-mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood-brain barrier. J. Immunol. 192, 324–337 (2014).
Article CAS PubMed Google Scholar
Lehmann, J. C. et al. Overlapping and selective roles of endothelial intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafficking. J. Immunol. 171, 2588–2593 (2003).
Article CAS PubMed Google Scholar
Fuks, J. M. et al. GABAergic signaling is linked to a hypermigratory phenotype in dendritic cells infected by Toxoplasma gondii. PLoS Pathog. 8, e1003051 (2012).
Article CAS PubMed PubMed Central Google Scholar
Schneider, C. A. et al. Imaging the dynamic recruitment of monocytes to the blood-brain barrier and specific brain regions during Toxoplasma gondii infection. Proc. Natl. Acad. Sci. USA 116, 24796–24807 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Schneider, C. A. et al. Toxoplasma gondii dissemination in the brain is facilitated by infiltrating peripheral immune cells. mBio 13, e0283822 (2022).
Article PubMed Google Scholar
Fox, B. A. & Bzik, D. J. De novo pyrimidine biosynthesis is required for virulence of Toxoplasma gondii. Nature 415, 926–929 (2002).
Article ADS CAS PubMed Google Scholar
Wang, Y. et al. Three Toxoplasma gondii dense granule proteins are required for induction of Lewis rat macrophage pyroptosis. mBio 10, e02388–18 (2019).
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We thank Drs. Jeroen Saeij, Ali Hakimi, Chris Hunter and David Bzik for parasite lines, Dr. Raad Askar for expert veterinary advice, the Imaging Facility Stockholm University (IFSU), the In Vivo Imaging Facility Stockholm University (IVIMSU) and the Experimental Core Facility (ECF). The studies were funded by the Swedish Research Council, grants 2018-02411 (A.B.), 2022-00520 (A.B.), The Swedish Brain Foundation (Hjärnfonden, grant FO2024-0022-HK-19 to A.B.), Åhlen Foudation grant 223020 (A.B.) and the Sven and Lily Lawski Foundation (M.E.R./A.B.).

Funding

Open access funding provided by Stockholm University.

Author information

Authors and Affiliations

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 10691, Stockholm, Sweden
Matias E. Rodriguez, Ali Hassan, Nikolaos Linaroudis, Felix Harryson-Oliveberg, Arne L. ten Hoeve & Antonio Barragan

Authors

Matias E. Rodriguez
View author publications
Search author on:PubMed Google Scholar
Ali Hassan
View author publications
Search author on:PubMed Google Scholar
Nikolaos Linaroudis
View author publications
Search author on:PubMed Google Scholar
Felix Harryson-Oliveberg
View author publications
Search author on:PubMed Google Scholar
Arne L. ten Hoeve
View author publications
Search author on:PubMed Google Scholar
Antonio Barragan
View author publications
Search author on:PubMed Google Scholar

Contributions

M.E.R. and A.B. developed the concept. M.E.R. developed method and performed microsurgery. M.E.R., A.H., N.L., F. H-O. and A.L.H. performed experiments, image processing and analyzed data. M.E.R. and A.B. wrote the manuscript.

Corresponding author

Correspondence to Antonio Barragan.

Ethics declarations

Competing interests

The authors declare that no competing interests exist.

Peer review

Peer review information

Nature Communications thanks Adrian Hehl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Description of Additional Supplementary Files

Movie S1

Movie S2

Movie S3

Movie S4

Movie S5

Movie S6

Movie S7

Reporting Summary

Transparent Peer Review file

Source data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Rodriguez, M.E., Hassan, A., Linaroudis, N. et al. ICAM-1/CD18-mediated sequestration of parasitized phagocytes in cortical capillaries promotes neuronal colonization by Toxoplasma gondii. Nat Commun 16, 3529 (2025). https://doi.org/10.1038/s41467-025-58655-z

Download citation

Received: 20 September 2024
Accepted: 27 March 2025
Published: 14 April 2025
DOI: https://doi.org/10.1038/s41467-025-58655-z