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

doi:10.21203/rs.3.rs-5121481/v1

Download PDF

Article

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

https://doi.org/10.21203/rs.3.rs-5121481/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

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. Here, we addressed the mechanisms of BBB crossing by intracarotid delivery of parasites and parasite-infected leukocytes into the cerebral circulation of mice. Unexpectedly, parasitized dendritic cells (DCs) and other peripheral blood mononuclear cells persistently sequestered in 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. Secreted parasite effectors TgWIP and GRA15, implicated in leukocyte hypermigration and inflammatory activation, strain genotype-dependently elevated numbers of sequestered parasitized DCs in capillaries and cerebral parasite loads. The data unveil that sequestration of parasitized leukocytes in cortical capillaries, with posterior parasite transmigration across the BBB upon egress, constitutes a mechanism for the rapid reach of T. gondii to cortical neurons during primary infection.

Biological sciences/Microbiology/Parasitology/Parasite host response

Health sciences/Diseases/Infectious diseases/Parasitic infection

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 CNS ⁹.

The microvascular lumen of the BBB is covered by highly specialized endothelial cells characterized by low permeability, low pinocytic activity and high transcellular electrical resistance ¹⁰. Intercellular tight junction proteins restrict the paracellular flux of hydrophilic molecules and regulates the migration of cells across the endothelial barrier ¹¹. 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 neuroinflammation ¹⁴, are crucial to eradicate pathogens that succeed in translocating across the restrictive CNS barriers ². However, the generated inflammation can also perturb BBB functions ¹ and alter neuronal function ¹⁵.

CNS infections by microbial pathogens are among the most devastating infectious diseases worldwide, often with highly limited therapeutic options ¹⁶. 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 infected ¹⁷. From its point of entry in the intestine, T. gondii disseminates systemically via the blood circulation and rapidly achieves latent infection in the CNS ¹⁸. 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 neonates ¹⁹.

T. gondii tachyzoites are obligate intracellular and use gliding motility to actively invade cells where they replicate ²⁰. Gliding motility also facilitates transmigration of tachyzoites across polarized endothelium in vitro ^{21, 22} and provides an effective mechanism of propulsion in the microenvironment inside tissues ²³. In their journey to form chronic cysts, the tachyzoites cross the cellular brain barriers to establish latent infection in the CNS ²⁴. 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⁺ leukocytes ²⁵. Dendritic cells (DCs) and other phagocytes can act as Trojan horses for systemic dissemination of T. gondii in mice ²⁶, in a parasite genotype-related fashion ²⁷. Upon active invasion by T. gondii, DCs are induced to migrate via activation of non-canonical GABAergic signaling and MAP kinase activation ^{28, 29, 30, 31}. This migratory activation, termed hypermigratory phenotype ³², implicates secreted parasite effectors ^{33, 34, 35} and chemotactic activation of parasitized phagocytes ³⁶.

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 ^{23, 24}. 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 ^{37, 38}. 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.

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 ^{37, 38} entail entering the systemic circulation with entrapment of circulating parasites in peripheral organs, especially the lungs and liver (Fig. S1B, C, D, 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 organs ³⁸. 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⁺ cells with replicating tachyzoites 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 and type II-infected DCs (Fig. 1I). In contrast, higher numbers of type II-infected DCs compared with type I-infected DCs were found in cortex (Fig. 1J), consistent with elevated adhesion to polarized endothelial monolayers (Fig. 1K). We conclude that parasitized CD45⁺ leukocytes, including DCs, can sequester in cortical capillaries, with variations between T. gondii strains.

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 ^{37, 38} 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 28h (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.

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 ^{37, 38}, 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 a higher relative frequency 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), jointly likely reflecting a superior invasive capacity by type I tachyzoites ²³. 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 equal numbers of free tachyzoites yielded dramatically 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 strains ²⁷ and the higher transmigration frequencies in vitro by extracellular type I tachyzoites ²³. 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.

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, D, 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, G, H). Interestingly, upon ICA injection, significantly higher numbers on sequestered infected DCs were detected in pre-infected animals (Fig. 4F, G, H, 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.

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 antibodies ³⁹. Importantly, both treatment with anti-ICAM-1^{40, 41} and anti-CD18 ⁴², 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 ^{35, 36, 43}, 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, H, I, 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. S5 C). Next, we determined the long-term impact 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). Finally, 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. S5D). We conclude that T. gondii effectors TgWIP and GRA15 exacerbate DC sequestration in the brain, with an impact on parasite loads.

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 ^{23, 38}.

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 ^{40, 41, 42}, 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 ^{44, 45} and also because it mediates adhesion and transmigration of free tachyzoites in vitro ²². Thus, the data indicates that enhanced expression of ICAM-1 in cerebral endothelium upon T. gondii challenge facilitates adhesion of parasitized leukocytes first, and, later, transmigration of egressing tachyzoites. Interestingly, in cerebral Plasmodium falciparum malaria, ICAM-1 has been implicated in the sequestration of parasitized erythrocytes in the vasculature via parasite-derived variant adhesins ⁴⁶.

We demonstrate that two T. gondii effectors modulate the sequestration of parasitized DCs in cortical capillaries. Specifically, GRA15 elevated leukocyte adhesion to the brain vasculature thereby facilitating parasite transmigration upon egress, in line with data implicating GRA15 in ICAM-1-mediated adhesion of phagocytes to endothelium in vitro ^{43, 44}. 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 dissemination ²⁷ and the inferior sequestration by type I strain RH, which lacks a functional GRA15 ⁴⁷. Also, because GRA15 activates NF-kB ⁴⁸ and endothelial NF-kB mediates neuronal activation via TLR/Myd88 signaling ⁴⁹, it is plausible that GRA15-mediated pro-inflammatory NF-kB activation facilitates colonization ⁵⁰. Further, TgWIP elevated sequestration and cerebral parasite loads, in line with its attributed roles in hypermotility and adhesion of DCs ^{35, 43, 51}. Jointly, because the type I strain RH expresses a non-functional form of GRA15 ⁴⁷, 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 ^{36, 50}. The BBB endothelium constitutively expresses the chemokine CCL19 ¹⁴ and, its receptor CCR7 has been suggested to mediate the migration of circulating T cells across the BBB ⁵². 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 effects ⁵¹, 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 capacity ²³ 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 capillaries ⁵³. Taking into consideration the ability of freshly egressed tachyzoites to transmigrate in vitro and migrate ex vivo in tissues ^{23, 22, 21}, 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 ^{37, 38} 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 IgM ⁵⁴. 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 ^{23, 38} to the BBB in vivo. 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 ^{37, 38}. 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 ^{55, 56}, but also to capillaries ⁵⁷. 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 capillaries ⁵⁸. We report a surprisingly rapid (24 h) inflammatory activation of the cerebral microvasculature after intraperitoneal T. gondii infection, prior to the dramatically elevated systemic cytokine responses later during infection ⁵⁹. In contrast, despite the very early neuronal infection, no evidence of inflammatory leukocyte infiltration, hemorrhage or disruption of the BBB ⁶⁰ was found at these early timepoints, though transient focal elevations of BBB permeability ³⁸ cannot be fully excluded. Importantly, these findings match well with the stealthy -clinically silent- CNS colonization during primary T. gondii infection, in the absence of focal neurologic signs ¹⁹. 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 elucidated ².

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 organs ³⁸. 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 ^{61, 62}.

In recent years, work in the field has described dissemination pathways for T. gondii and possible routes for CNS colonization ^{28, 29, 34, 35, 37, 38, 63, 64, 65}. Here, we add that the sequestration of parasitized leukocytes in cortical capillaries constitutes a remarkable mechanism facilitating translocation of T. gondii to the cerebral parenchyma and, ultimately, neuronal infection. In our experimental system, direct transportation of T. gondii into the parenchyma (Trojan horse) was a rare event compared with the higher frequency of CD18/ICAM-1-dependent sequestration of leukocytes. Thus, sequestered leukocytes, alike endothelium ^{37, 38}, constituted a replicative niche that facilitated direct transmigration of tachyzoites upon egress. Importantly, parasite loads as a consequence of sequestration outnumbered loads after inoculation with free tachyzoites. 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.

Competing interests

The authors declare no competing interests.

Acknowledgments

We are grateful to Drs. Jeroen Saeij, Ali Hakimi, Chris Hunter and David Bzik for parasite lines. 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.).

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).

Parasite culture and cell lines

T. gondii tachyzoites of type I: RH-GFP, RHΔTgWIP-GFP ³⁵, RH CPS-mCherry ⁶⁶ and type II: ME49-RFP ⁶⁷, PRU-GFP ⁴⁸ and PRUΔTgGRA15-GFP ⁴⁸ 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 described ⁶³. Bone marrow cells were purified from 4–8 weeks old C57BL/6NCrl mice (Charles River) 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 weeks 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 ^{36, 63}. Briefly, DCs or splenocytes were pre-labelled 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) for 4 h to obtain an infection frequency of ∼50%. Cell suspensions were then washed thrice and spun at 60-70xg to minimize free tachyzoites in the inoculum. Total number 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 min − 1 h from collection. Total number of colony-forming units (cfu) injected into animals was confirmed by plaquing assays.

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

C57BL/6NCrl mice were inoculated ip with 2x10⁵ 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) was injected iv (0,4 mg/Kg) 10 min before euthanasia. For adoptive transfer assays, mice were inoculated iv with 20x10⁶ cfu of T. gondi-challenged DCs and sacrificed 24 hpi.

Microsurgery, intracarotid artery inoculations and treatments

C57BL/6NCrl 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), anti-mo LFA-1 beta (eBioscience, Cat# 14-0181-85) or isotype (eBioscience, Cat# 14-4321-85) (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, 2mM EDTA in PBS). Challenged DCs were stained with PE-Cyanine7 anti-mo CD11c (eBioscience, Cat# 25-0114-82) PE anti-mo CD18 (eBioscience, Cat# 101407) and Super Bright 702 anti-mo MHCII I-A/I-E (eBioscience, Cat# 67-5321-82) 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™ (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 focus 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 ) and anti-GPAF (Invitrogen, Cat# 13–0300 ) diluted 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; Molecular probes, Cat# A21471) diluted 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 described ³⁸. 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 4500xg for 30 min at 4°C.

Plaquing and adhesion assays

Plaquing assays were performed as described by ⁶³. Briefly, brain, liver, and spleen from infected mice were extracted and homogenized using 70 µm cell strainers. 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.

For adhesion assays, DCs were pre-labelled with CMTMR or CFSE and challenged with freshly egressed GFP- or RFP-expressing tachyzoites (MOI 1 for type I, MOI 2 for type II). 1-5x10³ 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 µg/ 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.

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

There is NO Competing Interest.

Tableprimers.docx
Table S1. Primer sequences for qPCR
Sup1.pdf
Figure S1. Flow cytometry gatings, sequestration and parasite load in lungs and liver A. Representative flow cytometry plots show CD45 expression on PBMCs in control conditions. PBMCs were identified based on scatter-A/side scatter-A (left panel), followed by single cell (middle panel) analysis of CD45 expression (right panel, MFI normalized to mode). Mice were injected iv with 0.4 mg/kg anti-CD45-647. After 10 min, blood was collected. B. CFSE pre-labelled DCs were challenged in vitro with T. gondii (type II ME49-RFP) to obtain a DC infection frequency of ~50%. Infected DCs (40x10⁶DCs / 20x10⁶ cfu Tg) were inoculated iv in mice and organs were collected 24 hpi. C. Representative micrographs show T. gondii (ME49-RFP)-infected DCs (CFSE⁺, RFP⁺) in liver, brain and lung. Scale bars are shown. D. Experimental set up. Freshly egressed GFP-expressing T. gondii (Tg) tachyzoites (PRU-GFP, 2x10⁵cfu) were inoculated ip in mice and organs were collected 3 or 6 dpi. E. Representative micrographs show T. gondii (PRU-GFP) foci in liver, brain and lung. Scale bar: 50 µm.
S2dockingCPS.pdf
Figure S2. Sequestration in the absence of replication A. CMTMR or CFSE pre-labelled DCs were challenged in vitro with T. gondii RH-WT (GFP⁺, MOI 1) or RH-CPS (mCherry⁺, MOI 1), respectively, to obtain a DC infection frequency of ~50%. Infected DCs (20x10⁶DCs / 10x10⁶ cfu Tg) were inoculated in the ICA and brains collected 16 or 48 hpi. Confocal micrographs show intravascularly located (Evans blue, cyan) RH-CPS infected DCs (CFSE⁺; mCherry⁺) at 16 and 48 hpi (left panels) and extravascular RH-WT tachyzoites (GFP⁺) at 48 hpi (right panel). Arrows indicate infected DCs. Asterisk indicates DC CMTMR⁺ remnant (“ghost”, red). Scale bars: 10 µm.
s3.pdf
Figure S3. Association of T. gondii with astrocytes and vascular/parenchymal localization after inoculation of free (extracellular) tachyzoites A. Infected CMTMR pre-labelled DCs (20x10⁶DCs / 10x10⁶ cfu Tg) were inoculated in the ICA and brains extracted 48 hpi. Confocal micrographs (left panels) show the localization of RH tachyzoites (GFP⁺, green) and astrocytes (GAFP⁺, red) in relation to the vascular marker Evans blue (grey). Arrows indicate infected GAFP⁺ cell magnified in inset (right panel). Right panel: Confocal 3D surface analysis shows GAFP⁺ cell-associated tachyzoite. Scale bar: 10 µm. B. Confocal micrographs and corresponding 3D surfaces illustrate intravascular and extravascular localization of tachyzoites (type I RH, GFP⁺ green and type II ME49, RFP⁺, cyan) in relation to the vascular marker Evans blue (red). 20x10⁶ cfu of freshly egressed tachyzoites were inoculated in the ICA and brains were collected 16 hpi. Scale bar: 50 µm.
Sup4.pdf
Figure S4. Role of the inflammatory response on sequestration A. Graph shows the relative attachment frequency of T. gondii type II PRU-infected DCs to polarized brain endothelial cells (bEnd.3) in vitro, in control (Tg), heparin or LPS pre-treatment conditions. Data expressed as mean (± SEM) from three independent experiments. B. Graph shows the absolute number (mean ± SEM) of T. gondii type I RH-GFP and type II ME49-RFP-infected DCs per cm² of cortical tissue at 16 hpi. Mice were inoculated in the ICA with 20x10⁶DCs /10x10⁶ cfu Tg in control (Tg) and LPS pre-treated conditions. Data are from 20-30 cortical sections per condition from three independent experiments (n= 3 mice). C.D. Graph shows the absolute number (mean ± SEM) of T. gondii (type II, PRU-GFP)-infected splenocytes per cm² of cortex tissue at 16 hpi (C) and the relative expression (qPCR) of TgB1 gene in brain tissue (D), respectively. Mice were inoculated in the ICA with 20x10⁶ DCs/10x10⁶ cfu Tg in control (Tg) and LPS pre-treatment conditions. Data from 30 cortical sections from 2 independent experiments (n= 2 mice). E. Representative micrographs show T. gondii (type II, PRU-GFP)-infected splenocytes (CMTMR⁺, GFP⁺) in cortical sections of LPS pre-treated mice at 16 hpi. Insets (a) shows magnification of the white box and asterisks indicate infected splenocytes (CMTMR⁺ GFP⁺, red/green). Scale bar: 20 µm. F. Experimental set up. Mice were inoculated ip with 2x10⁵ cfu of freshly egressed tachyzoites (type II, ME49-RFP) or control medium. After 48 h, brain, liver and lung were collected. G. Graphs show the relative expression (qPCR) of TgB1 gene (mean± SEM) in brain, liver and lung tissues of control (unchallenged) or infected mice (Tg) at 48 hpi. Data from three independent experiments (n= 3 mice). H. Graph shows the relative expression of TgB1 gene (mean± SEM) in brain tissue of pre- ip infected mice (pre-ip Tg) and pre- ip infected mice plus inoculation of infected DCs in the ICA (pre- ip Tg + ICA DC/Tg). Mice were inoculated ip with 2x10⁵ cfu freshly egressed tachyzoites (ME49-RFP, pre-ip Tg). After 48h, infected DCs (10x10⁶ DCs/5x10⁶ cfu Tg, PRU-GFP) were inoculated in the ICA (ICA DC/Tg) and brains collected 1 hpi. I. Confocal micrographs and corresponding 3D surfaces show the extravascular localization (Evans blue, cyan) of T. gondii (PRU-GFP)-infected DC (CMTMR⁺ GFP⁺, red/green) of LPS pre-treated mice. Asterisks indicate infected CMTMR⁺ cells. Distance to the nearest vessel is shown. Scale bars: 10 µm. J. Relative mRNA expression (qPCR) of TIMP-1, CCL2, CCL5, and CXCR3 in brain micro-vessels. Mice were inoculated with freshly egressed RFP-expressing T. gondii (Tg) tachyzoites (ME49-RFP, 2x10⁵ cfu) or control medium and brains micro-vessels were purified at 24, 48 and 72 hpi. Data expressed as mean ± SEM from three interdependent experiments (n= 3 mice). K. Graphs show relative expression (MFI) of CD18 in T. gondii WT, ΔGRA15 or ΔTgWIP – infected DCs at 5 hours post challenge, assessed by flow cytometry. DCs were challenged with GFP-expressing type I RH (WT or ΔTgWIP, MOI 1) or type II (PRU WT or ΔGRA15, MOI 2) tachyzoites. Expression of CD18 (mean ± SEM) in T. gondii- infected (inf) and bystander (byst) CD11c^-, CD11c⁺/MHCII^Lo and CD11c⁺/MHCII^Hi cells is shown. Data from three interdependent experiments. L. Bar graph shows the relative attachment frequency of T. gondii type II (PRU-GFP) infected DCs to polarized brain endothelial cells (bEnd.3) in vitro, in control (isotype) of anti-ICAM 1 pre-treatment conditions. Data are expressed as mean ± SEM from three independent experiments. (A, G, H, L) Unpaired student’s t-test. (B, C) Mann-Whitney test-U. (K) One-way ANOVA followed Bonferroni’s multiple comparison test. * p < 0,05; ** p < 0,01, *** p < 0,001, **** p < 0,0001, ns: non-significant.
Sup5.pdf
Figure S5. Localization of DCs infected with TgWIP and GRA15 mutants in the cortical microvasculature, in vitro adhesion and sequestration of DCs challenged with GRA15 mutant A. Experimental set up. CMTMR pre-labelled DCs were challenged with GFP-expressing wild type (WT), ΔGRA15 or ΔTgWIP tachyzoites followed by inoculation in the ICA (20x10⁶DCs / 10x10⁶ cfu Tg). Brains were collected 16 hpi. B. Representative micrographs show infected DCs (CMTMR⁺, GFP⁺) per cm² cortical tissue for PRU-WT versus PRUΔGRA15 (upper panels) and RH-WT versus RHΔTgWIP (lower panels), respectively. Arrows indicate infected DCs (CMTMR⁺ GFP⁺, red/green) magnified in the insets. Scale bars: 20 µm. C. Bar graph shows the relative attachment frequency of T. gondii type II (PRU-WT versus PRUΔGRA15)-infected DCs to polarized brain endothelial cells (bEnd.3) in vitro. Data are expressed as mean (± SEM) from three independent experiments. D. Relative expression (qPCR) of T. gondii TgB1 gene (mean ± SEM) in brain tissue of mice inoculated with DC/Tg PRU-WT versus PRUΔGRA15. Infected DCs (2x10⁴DCs / 1x10⁴ cfu Tg) were inoculated in the ICA of mice in control (Tg), heparin or LPS-pre-treated conditions. Brains were extracted 7 dpi. Data from three independent experiments (n= 4 mice).
videoS1speed.mp4
Movie S1. Sequestration of infected DC in the vascular branching point Confocal micrograph shows the intravascular localization of T. gondii type I RH (GFP⁺, green)-infected DCs (CMTMR, red) in relation to the vascular marker Evans blue (cyan). Infected CMTMR pre-labelled DCs (20x10⁶DCs / 10x10⁶ cfu Tg) were inoculated in the ICA and brains extracted at 16 hpi.
S2Docking.mp4
Movie S2. After egressing from the infected DC, tachyzoites transmigrate across the BBB Confocal micrographs and corresponding 3D surface analysis show the localization of type I RH tachyzoites (GFP⁺, green) and DCs remnant (“ghost”, CMTMR⁺, grey) in relation to the vascular marker Evans blue (red). Infected CMTMR pre-labelled DCs (20x10⁶DCs / 10x10⁶ cfu Tg) were inoculated in the ICA and brains extracted at 16 hpi.
S3InfectedNeuron16hpi.mp4
Movie S3. T. gondii tachyzoites confined inside neuron after egressing from infected DC Confocal micrographs and corresponding 3D surface analysis show the localization of type I RH tachyzoites (GFP⁺, green) and neurons (NeuN⁺, grey) in relation to the vascular marker Evans blue (red). Infected CMTMR pre-labelled DCs (20x10⁶DCs / 10x10⁶ cfu Tg) were inoculated in the ICA and brains extracted at 16 hpi.
S4InfectedNeuron28hpi.mp4
Movie S4. T. gondii tachyzoite confined inside neuron after egressing from infected DC Confocal micrographs and corresponding 3D surface analysis show the localization of GFP-expressing RH tachyzoites (GFP⁺, green), DCs (CMTMR⁺, blue) and neurons (NeuN⁺, grey) in relation to the vascular marker Evans blue (red). Infected CMTMR pre-labelled DCs (20x10⁶DCs / 10x10⁶ cfu Tg) were inoculated in the ICA and brains extracted at 28 hpi.
S5RHfree1hpi.mp4
Movie S5. T. gondii tachyzoite confined in the brain parenchyma Confocal micrographs and corresponding 3D surfaces show the extravascular localization of type I RH wild type (GFP⁺, green) tachyzoites in relation to the vascular marker Evans blue (red). 20x10⁶cfu of freshly egressed tachyzoites were inoculated in the ICA and brains extracted 1 hpi.
S6CPSfree1hpi.mp4
Movie S6. Non-replicative T. gondii tachyzoite (CPS) confined in the brain parenchyma Confocal micrographs and corresponding 3D surfaces show the extravascular localization of non-replicative type I RH-CPS (mCherry⁺, red) tachyzoites in relation to the vascular marker Evans blue (grey). 20x10⁶cfu of freshly egressed tachyzoites were inoculated via the ICA and brains were extracted 1 hpi.
S7PARENCHYMALPS.mp4
Movie S7. T. gondii-infected DC confined in the brain parenchyma Confocal micrograph shows the extravascular localization of T. gondii type II PRU (GFP⁺, green)-infected DCs (CMTMR, red) in relation to the vascular marker Evans blue (cyan). 20x10⁶DCs / 10x10⁶ cfu Tg were inoculated in the ICA of LPS pre-treated mice and brains extracted at 16 hpi.

Download PDF

Version 1

posted

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

Introduction

Results

Parasitized CD45⁺ leukocytes sequester in cerebral capillaries

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

Discussion

Declarations

Competing interests

Acknowledgments

Methods

References

Additional Declarations

Supplementary Files

Status:

Version 1

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

Status:

Version 1

Abstract

Figures

Introduction

Results

Parasitized CD45+ leukocytes sequester in cerebral capillaries

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

Discussion

Declarations

Competing interests

Acknowledgments

Methods

References

Additional Declarations

Supplementary Files

Status:

Version 1

Parasitized CD45⁺ leukocytes sequester in cerebral capillaries