Transmigration of Toxoplasma gondii across biological barriers Emily C. Ross Doctoral Thesis in Molecular Bioscience at Stockholm University, Sweden 2022
Transmigration of Toxoplasma gondii across biological barriers
Aug 24, 2022
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Transmigration of Toxoplasma gondii across biological barriers Emily C. Ross
Em ily C. Ross Tran
sm igration
Doctoral Thesis in Molecular Bioscience at Stockholm University, Sweden 2022
Department of Molecular Biosciences, The Wenner-Gren Institute
ISBN 978-91-7911-862-4
Transmigration of Toxoplasma gondii across biological barriers Emily C. Ross
Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Thursday 9 June 2022 at 09.30 in Vivi Täckholmsalen (Q-salen) NPQ-huset, Svante Arrhenius väg 20.
Abstract Toxoplasma gondii is an obligate intracellular parasite that can likely infect all warm-blooded vertebrates, with estimates of up to 30% of the global human population being infected. Although infection with T. gondii is usually asymptomatic or mild, in immunocompromised individuals infection can lead to lethal toxoplasmic encephalitis. Infection acquired during pregnancy can also lead to serious ocular or neurological damage and even death of the foetus. Following ingestion, the parasite is able to cross the first biological barrier it encounters, the gut epithelium and convert to the rapidly replicating tachyzoite stage. It can then disseminate throughout the body of the host, eventually reaching sites such as the brain, after crossing the blood-brain barrier (BBB). Previous findings have shown that T. gondii can use leukocytes, such as dendritic cells (DCs), for dissemination via a “Trojan horse”-type mechanism, but how T. gondii then crosses restrictive barriers such as the BBB is still not fully understood. The overall objective of this work has been to investigate how T. gondii crosses biological barriers and how infection impacts host cell signalling.
In paper I we demonstrate that T. gondii can cross polarised cell monolayers without significantly perturbing barrier integrity. Reduced phosphorylation of focal adhesion kinase (FAK) was observed in cell monolayers upon T. gondii challenge, and inhibition or gene silencing of FAK (Ptk2) facilitated transmigration of T. gondii across polarised cell monolayers. In paper II we found that upon T. gondii infection of DCs, secreted TIMP-1 induces hypermotility by activating β1 integrin-FAK signalling through interactions with CD63. In paper III we show that T. gondii can cross polarised endothelial cell monolayers inside DCs. We also report that parasitised DCs on endothelium do not display a hypermotile phenotype, switching to integrin-dependent motility. Blockade of β1 and β2 integrins or ICAM-1, and gene silencing of β1 (Itgb1) or talin (Tln1) restored infected-DC motility, and reduced the frequency of transmigration of T. gondii-challenged DCs across endothelium. In paper IV we demonstrate that, shortly after T. gondii inoculation in mice, parasites mainly localised to cortical capillaries of the brain. Early invasion to the brain parenchyma occurred in absence of a significant increase in BBB permeability, perivascular leukocyte cuffs or haemorrhage. Further, pharmacological inhibition or endothelial cell-specific knockout of FAK facilitated parasite transmigration to the brain parenchyma.
In paper V we report that DCs challenged with type II T. gondii transmigrate across polarised endothelial cell monolayers at a higher frequency than type I T. gondii, while type I infected DCs exhibited increased migratory velocities on endothelium. We also show that T. gondii-induced upregulation of ICAM-1 in DCs is genotype-dependent, and requires the T. gondii secreted effector GRA15. Finally, gene silencing of leukocyte ICAM-1 (Icam-1) or deletion of T. gondii GRA15 reduced transmigration across endothelial cell monolayers.
In summary, the work in this thesis provides novel insights into how T. gondii can potentially cross biological barriers on its journey to the brain. We find that T. gondii can cross polarised monolayers both as free parasites and using DCs as a “Trojan horse”, and identify new ways in which T. gondii can alter host cell dynamics to benefit its own dissemination.
Keywords: Apicomplexa, blood-brain barrier, leukocyte, immune cell, transendothelial migration, cell adhesion molecule, host-pathogen, FAK, integrin.
Stockholm 2022 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-204118
ISBN 978-91-7911-862-4 ISBN 978-91-7911-863-1
Stockholm University, 106 91 Stockholm
TRANSMIGRATION OF TOXOPLASMA GONDII ACROSS BIOLOGICAL BARRIERS
Emily C. Ross
Emily C. Ross
©Emily C. Ross, Stockholm University 2022 ISBN print 978-91-7911-862-4 ISBN PDF 978-91-7911-863-1 Printed in Sweden by Universitetsservice US-AB, Stockholm 2022
To Margareta
I
Thesis abstract Toxoplasma gondii is an obligate intracellular parasite that can likely infect all warm-blooded vertebrates, with estimates of up to 30% of the global human population being infected. Although infection with T. gondii is usually asymptomatic or mild, in immunocompromised individuals’ infection can lead to lethal toxoplasmic encephalitis. Infection acquired during pregnancy can also lead to serious ocular or neurological damage and even death of the foetus. Following ingestion, the parasite is able to cross the first biological barrier it encounters, the gut epithelium and convert to the rapidly replicating tachyzoite stage. It can then disseminate throughout the body of the host, eventually reaching sites such as the brain, after crossing the blood-brain barrier (BBB). Previous findings have shown that T. gondii can use leukocytes, such as dendritic cells (DCs), for dissemination via a “Trojan horse”-type mechanism, but how T. gondii then crosses restrictive barriers such as the BBB is still not fully understood. The overall objective of this work has been to investigate how T. gondii crosses biological barriers and how infection impacts host cell signalling. In paper I we demonstrate that T. gondii can cross polarised cell monolayers without significantly perturbing barrier integrity. Reduced phosphorylation of focal adhesion kinase (FAK) was observed in cell monolayers upon T. gondii challenge, and inhibition or gene silencing of FAK (Ptk2) facilitated transmigration of T. gondii across polarised cell monolayers. In paper II we found that upon T. gondii infection of DCs, secreted tissue inhibitor of metalloproteinase 1 (TIMP-1) induces hypermotility by activating b1 integrin-FAK signalling through interactions with CD63. In paper III we show that T. gondii can cross polarised endothelial cell monolayers inside DCs. We also report that parasitised DCs on endothelium do not display a hypermotile phenotype, switching to integrin-dependent motility. Blockade of b1 and b2 integrins or intercellular adhesion molecule 1 (ICAM-1), and gene silencing of b1 integrin (Itgb1) or talin (Tln1) restored infected-DC motility, and reduced the frequency of transmigration of T. gondii-challenged DCs across endothelium. In paper IV we demonstrate that, shortly after T. gondii inoculation in mice, parasites mainly localised to cortical capillaries of the brain. Early invasion to the brain parenchyma occurred in absence of a significant increase in BBB permeability, perivascular leukocyte cuffs or haemorrhage. Further, pharmacological inhibition or endothelial cell-specific knockout of FAK facilitated parasite transmigration to the brain parenchyma. In paper V we report that DCs challenged with type II T. gondii transmigrate across polarised endothelial cell monolayers at a higher frequency than type I T. gondii, while type I infected DCs exhibited increased migratory velocities on endothelium. We also show that T. gondii-induced upregulation of ICAM-1 in DCs is genotype- dependent, and requires the T. gondii secreted effector GRA15. Finally, gene silencing of leukocyte ICAM-1 (Icam-1) or deletion of the T. gondii effector GRA15 reduced transmigration across endothelial cell monolayers.
II
In summary, the work in this thesis provides novel insights into how T. gondii can potentially cross biological barriers on its journey to the brain. We find that T. gondii can cross polarised monolayers both as free parasites and using DCs as a “Trojan horse”, and identify new ways in which T. gondii can alter host cell dynamics to benefit its own dissemination. Keywords: Apicomplexa, blood-brain barrier, leukocyte, immune cell, trans- endothelial migration, cell adhesion molecule, host-pathogen, FAK, integrin
III
Populärvetenskaplig sammanfattning Den här avhandlingen handlar om parasiten Toxoplasma gondii, som enligt uppskattningar infekterar nästan en tredjedel av jordens befolkning. T. gondii finns i kontaminerad föda, till exempel kött, och tar sig in i människokroppen via tarmen. Personer som infekteras med T. gondii kommer troligen att leva med parasiten i resten av sina liv. Om personen är frisk för övrigt upplever hen vanligtvis inga symtom, men det finns situationer då T. gondii kan orsaka allvarlig sjukdom och till och med död. En sådan situation är förstagångsinfektion under graviditet, med risk för allvarliga skador på ögon och hjärna hos fostret. Hos individer med nedsatt immunsystem, till exempel HIV/AIDS, kan T. gondii orsaka livshotande hjärninflammation. Toxoplasmas resa i kroppen startar i tarmen och slutar i exempelvis hjärnan där cystor bildas. För att ta sig in i hjärnan måste parasiten passera flera så kallade biologiska barriärer som skyddar kroppen från infektion. Blod-hjärnbarriären skyddar hjärnan och är mycket restriktiv. Tidigare forskning har visat att T. gondii kan invadera immunceller för att sprida sig genom kroppen. Processen har liknats vid en trojansk häst. Men exakt hur T. gondii tar sig igenom blod- hjärnbarriären, och om den använder samma process för att göra det, är ännu oklart. Målet med mitt arbete har varit att förstå hur T. gondii tar sig igenom kroppens restriktiva barriärer och hur en cellsignalering påverkas av parasitinfektionen. Vi har kommit fram till att T. gondii sannolikt kombinerar strategier för att ta sig igenom kroppens barriärer. Det sker dels genom direkt invasion av parasiten och dels genom att parasiten använder sig av immunceller som en trojansk häst. Vi har också upptäckt att celler som infekterats med T. gondii förändrar signalering och bindningsmolekyler på deras yta på ett sätt som gynnar spridningen av parasiten och leder till passage igenom barriärerna. Forskning kring hur T. gondii tar sig igenom barriärer kommer att ge insikter om även hur andra typer av mikrober når skyddade platser som exempelvis hjärnan.
IV
List of papers The following papers are included in this thesis:
I. Ross EC, Olivera GC, Barragan A. Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers. Cellular Microbiology. 2019;21:e13048.
II. Ólafsson EB, Ross EC, Varas-Godoy M, Barragan A. TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63-ITGB1-FAK signaling. J Cell Sci. 2019 Feb 4;132(3):jcs225193.
III. Ross EC, Ten Hoeve AL, Barragan A. Integrin-dependent migratory switches regulate the translocation of Toxoplasma-infected dendritic cells across brain endothelial monolayers. Cell Mol Life Sci. 2021 Jun;78(12):5197-5212.
IV. Olivera GC, Ross EC, Peuckert C, Barragan A. Blood-brain barrier- restricted translocation of Toxoplasma gondii from cortical capillaries. Elife. 2021 Dec 8;10:e69182.
V. Ross EC, Ten Hoeve AL, Saeij JPJ, Barragan A. Toxoplasma effector- induced ICAM-1 expression by infected dendritic cells potentiates transmigration across polarised endothelium. Manuscript
Related publications (not included in the thesis) Ross EC, Olivera GC, Barragan A. Early passage of Toxoplasma gondii across the blood-brain barrier. Trends Parasitol. 2022 Feb 25:S1471-4922(22)00033-2.
V
Populärvetenskaplig sammanfattning ............................................................................ III
Introduction ........................................................................................................................ 1
Toxoplasma gondii ....................................................................................................... 1 Life cycle. ................................................................................................................. 1 Parasite lineages, virulence & toxoplasmosis. ......................................................... 2
Biological barriers of vertebrates ............................................................................... 2 The intestinal barrier. ............................................................................................... 2 The blood-brain barrier (BBB). ................................................................................ 2 The neurovascular unit (NVU). ................................................................................ 3 The choroid plexus and meninges. ........................................................................... 4 Immune cell trafficking at the CNS. ........................................................................ 4
Host-Parasite interactions ........................................................................................... 5 The invasion cascade. ............................................................................................... 5 Parasite effector molecules & their roles. ................................................................ 6 Immune response to T. gondii infection. .................................................................. 6 Dendritic cells. ......................................................................................................... 7
Cell migration .............................................................................................................. 7 Cell migration in 2D. ................................................................................................ 7 Cell migration in 3D. ................................................................................................ 8
Transmigration of T. gondii across biological barriers ............................................ 9 Transmigration of free parasites. .............................................................................. 9 Transmigration of parasitised-leukocytes. ............................................................... 9
Aims of this thesis ............................................................................................................ 11
Main hypothesis .......................................................................................................... 11
Specific aims ............................................................................................................... 11
Methods ............................................................................................................................ 12
Results .............................................................................................................................. 13 Paper I .................................................................................................................... 13 Paper II ................................................................................................................... 13 Paper III .................................................................................................................. 13 Paper IV .................................................................................................................. 14 Paper V ................................................................................................................... 14
Discussion ........................................................................................................................ 15
Acknowledgements ........................................................................................................... 19
References ........................................................................................................................ 20
Adherens junction Blood-Brain Barrier Blood-CSF Barrier Basement Membrane Cell Adhesion Molecule
CNS CP CSF
DC DNA EC
ECM FAK
GRA Dense Granule Protein HIV Human Immunodeficiency virus ICAM Intercellular Adhesion Molecule LPS MAT MBEC
Lipopolysaccharide Mesenchymal-to-amoeboid Transition Mouse Brain Endothelial Cell
MHC Major Histocompatibility Complex MIC MS
Microneme Protein Multiple Sclerosis
NF-κB Nuclear Factor κB PV Parasitophorous Vacuole ROP Rhoptry Protein shRNA TEM TgWIP TIMP TJ
Short Hairpin RNA Transendothelial Migration T. gondii-WAVE complex interacting protein Tissue Inhibitor of Matrix Metalloproteinases Tight junction
TLR Toll-like Receptor VCAM Vascular Cell Adhesion Molecule WT ZO
Wild-type Zonula Occludens
1
Introduction Toxoplasma gondii Toxoplasma gondii is an obligate intracellular protozoan parasite. It belongs to the phylum Apicomplexa, that includes other clinically relevant parasites such as Plasmodium falciparum and Cryptosporidium parvum, the causative agents of malaria and cryptosporidiosis, respectively [1]. T. gondii can likely infect all warm- blooded vertebrates, with estimates of up to 30% of the global human population being infected [2]. In healthy human adults, infection with T. gondii is usually asymptomatic or mild, and mimics other diseases such as flu. However, in immunocompromised individuals, infection can lead to lethal toxoplasmic encephalitis. Infection acquired during pregnancy can lead to serious ocular or neurological damage and even death of the foetus [3]. There is also increasing evidence to suggest that T. gondii cysts in the brain may play a role in psychiatric disorders, such as schizophrenia and depression [4, 5]. Gaining further insights into the biology that underlies Toxoplasma pathogenesis and persistence will help alleviate toxoplasmosis in at-risk groups and potentially contribute to understanding the role of Toxoplasma infection in psychiatric disorders. Life cycle. The life cycle of T. gondii is complex, as more than one infective form exists and it can be transmitted through various routes. The most common routes of infection in humans is by either ingesting undercooked meat containing T. gondii tissue cysts, or ingestion of food or water contaminated with oocysts shed from cat faeces [2]. Congenital transmission can also occur via the placenta in an infected mother. The three developmental stages that can infect a cell include tachyzoites (the rapidly replicating stage mostly found in acute infections); bradyzoites (the slow replicating stage, characteristic of chronic infection in tissue cysts); and sporozoites, which are only produced during sexual recombination in the definitive feline host [3]. In human hosts, following ingestion, tissue cyst bradyzoites or oocyst sporozoites excyst in the intestine and cross the first biological barrier they encounter, the gut epithelium. They then differentiate into the tachyzoite stage and disseminate throughout the host, where they can continue to successfully traverse other restrictive biological barriers such as the blood-retina barrier (BRB) and blood-brain barrier (BBB) [6]. Differentiation into bradyzoites is thought to occur following pressure from the immune system, or exposure to unique intracellular environments within neurons and skeletal muscle cells, both of which have been shown to initiate parasite stage conversion spontaneously in vitro [7, 8]. In vivo, bradyzoites are thought to persist in a semi-dormant state within tissue cysts in muscle tissue, the retina or the brain, for the lifetime of the host [9]. In this thesis, the tachyzoite stage of the parasite was used in all experiments.
2
Parasite lineages, virulence & toxoplasmosis. The genus Toxoplasma contains just one species, T. gondii, that is grouped into genotypes, the most common in Europe and North America being Type I, II and III [3, 10]. Of note, in South America, there is a high range of genetic diversity, with more than 150 lineages of T. gondii present [11, 12]. The types I, II and III differ in their virulence, their extracellular viability, replication rates, the host immune response they trigger and ultimately their role in the development of acute or chronic disease in mice [10, 11]. Type I parasites are considered to be the most virulent, which is defined by having a lethal dose (LD) of one single parasite in classical laboratory mice (LD100 = 1) [13], whereas type II and III are considered to be intermediate or low virulence strains in mice. The majority of toxoplasmosis cases in humans and livestock are caused by type II strains [11]. Treatments targeting the acute phase of infection, the tachyzoite stage, exist but so far, no drugs exist to target the bradyzoite stage, found in chronic infection. Although infection in healthy human hosts is generally asymptomatic, reactivation or primary infection of immunocompromised hosts, such as HIV/AIDS patients, can lead to lethal encephalitis. Primary infection of pregnant hosts can also lead to neurological or ocular damage of the foetus, and in some cases lead to abortion. Studies in mice have shown that infection with T. gondii can lead to immune-related changes in behaviour with reduced anxiety in infected mice, increased explorative behaviours, and altered predator aversion [11].
Biological barriers of vertebrates One major advantage that T. gondii tachyzoites have in their journey of dissemination within a host, is their ability to traverse biological barriers [14, 15]. These barriers are made up of a number of cell types, including endothelial or epithelial cells, that work to protect organs and tissues from physical, chemical or biological damage and maintain tissue homeostasis. The intestinal barrier. The semi-permeable intestinal barrier is the first line of defence against anything harmful that may have been ingested in food or drink. Underlying a mucous layer, that forms the first layer of protection, are epithelial cells that protect the host from incoming non-sterile substances. These epithelial cells express tight junctions (TJs), adherens junctions (AJs) and desmosomes that work to maintain cell-cell contacts, and provide a barrier between the inside of the body and the external environment [16, 17]. The lamina propria, underlying the epithelium, contains dendritic cells (DCs), macrophages (MFs) and natural killer (NK) cells [17, 18]. These are some of the first immune cells that invading pathogens breaching the intestinal epithelium will encounter, and they work to modulate the immune response. The blood-brain barrier (BBB). The BBB is a term used to describe the blood vessels that make up the vascular system of the central nervous system (CNS). The inner cellular lining of these blood vessels are made up of specialised
3
endothelial cells (ECs) that tightly regulate the movement of molecules, cells and infectious agents between the blood and the parenchyma [17, 19, 20], and provide stringent ionic homeostasis which is paramount for neuronal activity [21]. The cells of the BBB are polarised, meaning they have an abluminal surface that faces towards the brain and a luminal surface that faces towards the blood, and at each surface they express different transporters [22]. Like all tissues, BBB ECs express adherens junctions, such as cadherins and catenins, that make up the adhesions between ECs, that in turn support the integrity of the vascular tube. The ECs of the BBB are further specialised to restrict movement of solutes via their expression of TJs. These are cell adhesions that consist of transmembrane proteins that directly interact via their extracellular components, linking two cell membranes together [19]. They help to form a high-resistance electrical barrier with low permeability. TJs include occludin, claudins, junctional adhesion molecules (JAMs) and the cytoplasmic protein zonula occludens-1 (ZO-1), which binds intracellular TJs to the cytoskeleton. Claudin-5 (CLDN5) is the most abundant claudin at the BBB and Cldn5 knockout mice exhibit a leaky BBB [23]. Focal adhesion kinase (FAK) activity has been shown to be required for maintenance of endothelial cell barrier integrity [24] through vascular endothelial cadherin (VE-cadherin) phosphorylation, a major mediator of cell-cell adhesions in ECs [25].
Figure 1. Schematic representation of the blood-brain barrier (BBB). The blood vessels of the BBB are made up of vascular endothelial cells sealed by tight junctions. The basal lamina, astrocyte end-feet, pericytes, neurons and microglia (together with the endothelial cells) make up the neurovascular unit (NVU) and all play a role in the barrier properties of the BBB. Peripheral blood cells, including leukocytes, also interact with the BBB.
The neurovascular unit (NVU). The functional barrier properties of cerebral microvessels depend not only on the restrictive properties of the ECs. Brain ECs are in constant crosstalk with astrocytes, microglia, neurons, mast cells and pericytes, as well as circulating immune cells (Figure 1) [26]. Astrocytes and pericytes are especially important in helping to maintain the restrictive barrier properties of the BBB [27, 28]. The luminal surface of capillary ECs is also covered…
Em ily C. Ross Tran
sm igration
Doctoral Thesis in Molecular Bioscience at Stockholm University, Sweden 2022
Department of Molecular Biosciences, The Wenner-Gren Institute
ISBN 978-91-7911-862-4
Transmigration of Toxoplasma gondii across biological barriers Emily C. Ross
Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Thursday 9 June 2022 at 09.30 in Vivi Täckholmsalen (Q-salen) NPQ-huset, Svante Arrhenius väg 20.
Abstract Toxoplasma gondii is an obligate intracellular parasite that can likely infect all warm-blooded vertebrates, with estimates of up to 30% of the global human population being infected. Although infection with T. gondii is usually asymptomatic or mild, in immunocompromised individuals infection can lead to lethal toxoplasmic encephalitis. Infection acquired during pregnancy can also lead to serious ocular or neurological damage and even death of the foetus. Following ingestion, the parasite is able to cross the first biological barrier it encounters, the gut epithelium and convert to the rapidly replicating tachyzoite stage. It can then disseminate throughout the body of the host, eventually reaching sites such as the brain, after crossing the blood-brain barrier (BBB). Previous findings have shown that T. gondii can use leukocytes, such as dendritic cells (DCs), for dissemination via a “Trojan horse”-type mechanism, but how T. gondii then crosses restrictive barriers such as the BBB is still not fully understood. The overall objective of this work has been to investigate how T. gondii crosses biological barriers and how infection impacts host cell signalling.
In paper I we demonstrate that T. gondii can cross polarised cell monolayers without significantly perturbing barrier integrity. Reduced phosphorylation of focal adhesion kinase (FAK) was observed in cell monolayers upon T. gondii challenge, and inhibition or gene silencing of FAK (Ptk2) facilitated transmigration of T. gondii across polarised cell monolayers. In paper II we found that upon T. gondii infection of DCs, secreted TIMP-1 induces hypermotility by activating β1 integrin-FAK signalling through interactions with CD63. In paper III we show that T. gondii can cross polarised endothelial cell monolayers inside DCs. We also report that parasitised DCs on endothelium do not display a hypermotile phenotype, switching to integrin-dependent motility. Blockade of β1 and β2 integrins or ICAM-1, and gene silencing of β1 (Itgb1) or talin (Tln1) restored infected-DC motility, and reduced the frequency of transmigration of T. gondii-challenged DCs across endothelium. In paper IV we demonstrate that, shortly after T. gondii inoculation in mice, parasites mainly localised to cortical capillaries of the brain. Early invasion to the brain parenchyma occurred in absence of a significant increase in BBB permeability, perivascular leukocyte cuffs or haemorrhage. Further, pharmacological inhibition or endothelial cell-specific knockout of FAK facilitated parasite transmigration to the brain parenchyma.
In paper V we report that DCs challenged with type II T. gondii transmigrate across polarised endothelial cell monolayers at a higher frequency than type I T. gondii, while type I infected DCs exhibited increased migratory velocities on endothelium. We also show that T. gondii-induced upregulation of ICAM-1 in DCs is genotype-dependent, and requires the T. gondii secreted effector GRA15. Finally, gene silencing of leukocyte ICAM-1 (Icam-1) or deletion of T. gondii GRA15 reduced transmigration across endothelial cell monolayers.
In summary, the work in this thesis provides novel insights into how T. gondii can potentially cross biological barriers on its journey to the brain. We find that T. gondii can cross polarised monolayers both as free parasites and using DCs as a “Trojan horse”, and identify new ways in which T. gondii can alter host cell dynamics to benefit its own dissemination.
Keywords: Apicomplexa, blood-brain barrier, leukocyte, immune cell, transendothelial migration, cell adhesion molecule, host-pathogen, FAK, integrin.
Stockholm 2022 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-204118
ISBN 978-91-7911-862-4 ISBN 978-91-7911-863-1
Stockholm University, 106 91 Stockholm
TRANSMIGRATION OF TOXOPLASMA GONDII ACROSS BIOLOGICAL BARRIERS
Emily C. Ross
Emily C. Ross
©Emily C. Ross, Stockholm University 2022 ISBN print 978-91-7911-862-4 ISBN PDF 978-91-7911-863-1 Printed in Sweden by Universitetsservice US-AB, Stockholm 2022
To Margareta
I
Thesis abstract Toxoplasma gondii is an obligate intracellular parasite that can likely infect all warm-blooded vertebrates, with estimates of up to 30% of the global human population being infected. Although infection with T. gondii is usually asymptomatic or mild, in immunocompromised individuals’ infection can lead to lethal toxoplasmic encephalitis. Infection acquired during pregnancy can also lead to serious ocular or neurological damage and even death of the foetus. Following ingestion, the parasite is able to cross the first biological barrier it encounters, the gut epithelium and convert to the rapidly replicating tachyzoite stage. It can then disseminate throughout the body of the host, eventually reaching sites such as the brain, after crossing the blood-brain barrier (BBB). Previous findings have shown that T. gondii can use leukocytes, such as dendritic cells (DCs), for dissemination via a “Trojan horse”-type mechanism, but how T. gondii then crosses restrictive barriers such as the BBB is still not fully understood. The overall objective of this work has been to investigate how T. gondii crosses biological barriers and how infection impacts host cell signalling. In paper I we demonstrate that T. gondii can cross polarised cell monolayers without significantly perturbing barrier integrity. Reduced phosphorylation of focal adhesion kinase (FAK) was observed in cell monolayers upon T. gondii challenge, and inhibition or gene silencing of FAK (Ptk2) facilitated transmigration of T. gondii across polarised cell monolayers. In paper II we found that upon T. gondii infection of DCs, secreted tissue inhibitor of metalloproteinase 1 (TIMP-1) induces hypermotility by activating b1 integrin-FAK signalling through interactions with CD63. In paper III we show that T. gondii can cross polarised endothelial cell monolayers inside DCs. We also report that parasitised DCs on endothelium do not display a hypermotile phenotype, switching to integrin-dependent motility. Blockade of b1 and b2 integrins or intercellular adhesion molecule 1 (ICAM-1), and gene silencing of b1 integrin (Itgb1) or talin (Tln1) restored infected-DC motility, and reduced the frequency of transmigration of T. gondii-challenged DCs across endothelium. In paper IV we demonstrate that, shortly after T. gondii inoculation in mice, parasites mainly localised to cortical capillaries of the brain. Early invasion to the brain parenchyma occurred in absence of a significant increase in BBB permeability, perivascular leukocyte cuffs or haemorrhage. Further, pharmacological inhibition or endothelial cell-specific knockout of FAK facilitated parasite transmigration to the brain parenchyma. In paper V we report that DCs challenged with type II T. gondii transmigrate across polarised endothelial cell monolayers at a higher frequency than type I T. gondii, while type I infected DCs exhibited increased migratory velocities on endothelium. We also show that T. gondii-induced upregulation of ICAM-1 in DCs is genotype- dependent, and requires the T. gondii secreted effector GRA15. Finally, gene silencing of leukocyte ICAM-1 (Icam-1) or deletion of the T. gondii effector GRA15 reduced transmigration across endothelial cell monolayers.
II
In summary, the work in this thesis provides novel insights into how T. gondii can potentially cross biological barriers on its journey to the brain. We find that T. gondii can cross polarised monolayers both as free parasites and using DCs as a “Trojan horse”, and identify new ways in which T. gondii can alter host cell dynamics to benefit its own dissemination. Keywords: Apicomplexa, blood-brain barrier, leukocyte, immune cell, trans- endothelial migration, cell adhesion molecule, host-pathogen, FAK, integrin
III
Populärvetenskaplig sammanfattning Den här avhandlingen handlar om parasiten Toxoplasma gondii, som enligt uppskattningar infekterar nästan en tredjedel av jordens befolkning. T. gondii finns i kontaminerad föda, till exempel kött, och tar sig in i människokroppen via tarmen. Personer som infekteras med T. gondii kommer troligen att leva med parasiten i resten av sina liv. Om personen är frisk för övrigt upplever hen vanligtvis inga symtom, men det finns situationer då T. gondii kan orsaka allvarlig sjukdom och till och med död. En sådan situation är förstagångsinfektion under graviditet, med risk för allvarliga skador på ögon och hjärna hos fostret. Hos individer med nedsatt immunsystem, till exempel HIV/AIDS, kan T. gondii orsaka livshotande hjärninflammation. Toxoplasmas resa i kroppen startar i tarmen och slutar i exempelvis hjärnan där cystor bildas. För att ta sig in i hjärnan måste parasiten passera flera så kallade biologiska barriärer som skyddar kroppen från infektion. Blod-hjärnbarriären skyddar hjärnan och är mycket restriktiv. Tidigare forskning har visat att T. gondii kan invadera immunceller för att sprida sig genom kroppen. Processen har liknats vid en trojansk häst. Men exakt hur T. gondii tar sig igenom blod- hjärnbarriären, och om den använder samma process för att göra det, är ännu oklart. Målet med mitt arbete har varit att förstå hur T. gondii tar sig igenom kroppens restriktiva barriärer och hur en cellsignalering påverkas av parasitinfektionen. Vi har kommit fram till att T. gondii sannolikt kombinerar strategier för att ta sig igenom kroppens barriärer. Det sker dels genom direkt invasion av parasiten och dels genom att parasiten använder sig av immunceller som en trojansk häst. Vi har också upptäckt att celler som infekterats med T. gondii förändrar signalering och bindningsmolekyler på deras yta på ett sätt som gynnar spridningen av parasiten och leder till passage igenom barriärerna. Forskning kring hur T. gondii tar sig igenom barriärer kommer att ge insikter om även hur andra typer av mikrober når skyddade platser som exempelvis hjärnan.
IV
List of papers The following papers are included in this thesis:
I. Ross EC, Olivera GC, Barragan A. Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers. Cellular Microbiology. 2019;21:e13048.
II. Ólafsson EB, Ross EC, Varas-Godoy M, Barragan A. TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63-ITGB1-FAK signaling. J Cell Sci. 2019 Feb 4;132(3):jcs225193.
III. Ross EC, Ten Hoeve AL, Barragan A. Integrin-dependent migratory switches regulate the translocation of Toxoplasma-infected dendritic cells across brain endothelial monolayers. Cell Mol Life Sci. 2021 Jun;78(12):5197-5212.
IV. Olivera GC, Ross EC, Peuckert C, Barragan A. Blood-brain barrier- restricted translocation of Toxoplasma gondii from cortical capillaries. Elife. 2021 Dec 8;10:e69182.
V. Ross EC, Ten Hoeve AL, Saeij JPJ, Barragan A. Toxoplasma effector- induced ICAM-1 expression by infected dendritic cells potentiates transmigration across polarised endothelium. Manuscript
Related publications (not included in the thesis) Ross EC, Olivera GC, Barragan A. Early passage of Toxoplasma gondii across the blood-brain barrier. Trends Parasitol. 2022 Feb 25:S1471-4922(22)00033-2.
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Populärvetenskaplig sammanfattning ............................................................................ III
Introduction ........................................................................................................................ 1
Toxoplasma gondii ....................................................................................................... 1 Life cycle. ................................................................................................................. 1 Parasite lineages, virulence & toxoplasmosis. ......................................................... 2
Biological barriers of vertebrates ............................................................................... 2 The intestinal barrier. ............................................................................................... 2 The blood-brain barrier (BBB). ................................................................................ 2 The neurovascular unit (NVU). ................................................................................ 3 The choroid plexus and meninges. ........................................................................... 4 Immune cell trafficking at the CNS. ........................................................................ 4
Host-Parasite interactions ........................................................................................... 5 The invasion cascade. ............................................................................................... 5 Parasite effector molecules & their roles. ................................................................ 6 Immune response to T. gondii infection. .................................................................. 6 Dendritic cells. ......................................................................................................... 7
Cell migration .............................................................................................................. 7 Cell migration in 2D. ................................................................................................ 7 Cell migration in 3D. ................................................................................................ 8
Transmigration of T. gondii across biological barriers ............................................ 9 Transmigration of free parasites. .............................................................................. 9 Transmigration of parasitised-leukocytes. ............................................................... 9
Aims of this thesis ............................................................................................................ 11
Main hypothesis .......................................................................................................... 11
Specific aims ............................................................................................................... 11
Methods ............................................................................................................................ 12
Results .............................................................................................................................. 13 Paper I .................................................................................................................... 13 Paper II ................................................................................................................... 13 Paper III .................................................................................................................. 13 Paper IV .................................................................................................................. 14 Paper V ................................................................................................................... 14
Discussion ........................................................................................................................ 15
Acknowledgements ........................................................................................................... 19
References ........................................................................................................................ 20
Adherens junction Blood-Brain Barrier Blood-CSF Barrier Basement Membrane Cell Adhesion Molecule
CNS CP CSF
DC DNA EC
ECM FAK
GRA Dense Granule Protein HIV Human Immunodeficiency virus ICAM Intercellular Adhesion Molecule LPS MAT MBEC
Lipopolysaccharide Mesenchymal-to-amoeboid Transition Mouse Brain Endothelial Cell
MHC Major Histocompatibility Complex MIC MS
Microneme Protein Multiple Sclerosis
NF-κB Nuclear Factor κB PV Parasitophorous Vacuole ROP Rhoptry Protein shRNA TEM TgWIP TIMP TJ
Short Hairpin RNA Transendothelial Migration T. gondii-WAVE complex interacting protein Tissue Inhibitor of Matrix Metalloproteinases Tight junction
TLR Toll-like Receptor VCAM Vascular Cell Adhesion Molecule WT ZO
Wild-type Zonula Occludens
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Introduction Toxoplasma gondii Toxoplasma gondii is an obligate intracellular protozoan parasite. It belongs to the phylum Apicomplexa, that includes other clinically relevant parasites such as Plasmodium falciparum and Cryptosporidium parvum, the causative agents of malaria and cryptosporidiosis, respectively [1]. T. gondii can likely infect all warm- blooded vertebrates, with estimates of up to 30% of the global human population being infected [2]. In healthy human adults, infection with T. gondii is usually asymptomatic or mild, and mimics other diseases such as flu. However, in immunocompromised individuals, infection can lead to lethal toxoplasmic encephalitis. Infection acquired during pregnancy can lead to serious ocular or neurological damage and even death of the foetus [3]. There is also increasing evidence to suggest that T. gondii cysts in the brain may play a role in psychiatric disorders, such as schizophrenia and depression [4, 5]. Gaining further insights into the biology that underlies Toxoplasma pathogenesis and persistence will help alleviate toxoplasmosis in at-risk groups and potentially contribute to understanding the role of Toxoplasma infection in psychiatric disorders. Life cycle. The life cycle of T. gondii is complex, as more than one infective form exists and it can be transmitted through various routes. The most common routes of infection in humans is by either ingesting undercooked meat containing T. gondii tissue cysts, or ingestion of food or water contaminated with oocysts shed from cat faeces [2]. Congenital transmission can also occur via the placenta in an infected mother. The three developmental stages that can infect a cell include tachyzoites (the rapidly replicating stage mostly found in acute infections); bradyzoites (the slow replicating stage, characteristic of chronic infection in tissue cysts); and sporozoites, which are only produced during sexual recombination in the definitive feline host [3]. In human hosts, following ingestion, tissue cyst bradyzoites or oocyst sporozoites excyst in the intestine and cross the first biological barrier they encounter, the gut epithelium. They then differentiate into the tachyzoite stage and disseminate throughout the host, where they can continue to successfully traverse other restrictive biological barriers such as the blood-retina barrier (BRB) and blood-brain barrier (BBB) [6]. Differentiation into bradyzoites is thought to occur following pressure from the immune system, or exposure to unique intracellular environments within neurons and skeletal muscle cells, both of which have been shown to initiate parasite stage conversion spontaneously in vitro [7, 8]. In vivo, bradyzoites are thought to persist in a semi-dormant state within tissue cysts in muscle tissue, the retina or the brain, for the lifetime of the host [9]. In this thesis, the tachyzoite stage of the parasite was used in all experiments.
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Parasite lineages, virulence & toxoplasmosis. The genus Toxoplasma contains just one species, T. gondii, that is grouped into genotypes, the most common in Europe and North America being Type I, II and III [3, 10]. Of note, in South America, there is a high range of genetic diversity, with more than 150 lineages of T. gondii present [11, 12]. The types I, II and III differ in their virulence, their extracellular viability, replication rates, the host immune response they trigger and ultimately their role in the development of acute or chronic disease in mice [10, 11]. Type I parasites are considered to be the most virulent, which is defined by having a lethal dose (LD) of one single parasite in classical laboratory mice (LD100 = 1) [13], whereas type II and III are considered to be intermediate or low virulence strains in mice. The majority of toxoplasmosis cases in humans and livestock are caused by type II strains [11]. Treatments targeting the acute phase of infection, the tachyzoite stage, exist but so far, no drugs exist to target the bradyzoite stage, found in chronic infection. Although infection in healthy human hosts is generally asymptomatic, reactivation or primary infection of immunocompromised hosts, such as HIV/AIDS patients, can lead to lethal encephalitis. Primary infection of pregnant hosts can also lead to neurological or ocular damage of the foetus, and in some cases lead to abortion. Studies in mice have shown that infection with T. gondii can lead to immune-related changes in behaviour with reduced anxiety in infected mice, increased explorative behaviours, and altered predator aversion [11].
Biological barriers of vertebrates One major advantage that T. gondii tachyzoites have in their journey of dissemination within a host, is their ability to traverse biological barriers [14, 15]. These barriers are made up of a number of cell types, including endothelial or epithelial cells, that work to protect organs and tissues from physical, chemical or biological damage and maintain tissue homeostasis. The intestinal barrier. The semi-permeable intestinal barrier is the first line of defence against anything harmful that may have been ingested in food or drink. Underlying a mucous layer, that forms the first layer of protection, are epithelial cells that protect the host from incoming non-sterile substances. These epithelial cells express tight junctions (TJs), adherens junctions (AJs) and desmosomes that work to maintain cell-cell contacts, and provide a barrier between the inside of the body and the external environment [16, 17]. The lamina propria, underlying the epithelium, contains dendritic cells (DCs), macrophages (MFs) and natural killer (NK) cells [17, 18]. These are some of the first immune cells that invading pathogens breaching the intestinal epithelium will encounter, and they work to modulate the immune response. The blood-brain barrier (BBB). The BBB is a term used to describe the blood vessels that make up the vascular system of the central nervous system (CNS). The inner cellular lining of these blood vessels are made up of specialised
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endothelial cells (ECs) that tightly regulate the movement of molecules, cells and infectious agents between the blood and the parenchyma [17, 19, 20], and provide stringent ionic homeostasis which is paramount for neuronal activity [21]. The cells of the BBB are polarised, meaning they have an abluminal surface that faces towards the brain and a luminal surface that faces towards the blood, and at each surface they express different transporters [22]. Like all tissues, BBB ECs express adherens junctions, such as cadherins and catenins, that make up the adhesions between ECs, that in turn support the integrity of the vascular tube. The ECs of the BBB are further specialised to restrict movement of solutes via their expression of TJs. These are cell adhesions that consist of transmembrane proteins that directly interact via their extracellular components, linking two cell membranes together [19]. They help to form a high-resistance electrical barrier with low permeability. TJs include occludin, claudins, junctional adhesion molecules (JAMs) and the cytoplasmic protein zonula occludens-1 (ZO-1), which binds intracellular TJs to the cytoskeleton. Claudin-5 (CLDN5) is the most abundant claudin at the BBB and Cldn5 knockout mice exhibit a leaky BBB [23]. Focal adhesion kinase (FAK) activity has been shown to be required for maintenance of endothelial cell barrier integrity [24] through vascular endothelial cadherin (VE-cadherin) phosphorylation, a major mediator of cell-cell adhesions in ECs [25].
Figure 1. Schematic representation of the blood-brain barrier (BBB). The blood vessels of the BBB are made up of vascular endothelial cells sealed by tight junctions. The basal lamina, astrocyte end-feet, pericytes, neurons and microglia (together with the endothelial cells) make up the neurovascular unit (NVU) and all play a role in the barrier properties of the BBB. Peripheral blood cells, including leukocytes, also interact with the BBB.
The neurovascular unit (NVU). The functional barrier properties of cerebral microvessels depend not only on the restrictive properties of the ECs. Brain ECs are in constant crosstalk with astrocytes, microglia, neurons, mast cells and pericytes, as well as circulating immune cells (Figure 1) [26]. Astrocytes and pericytes are especially important in helping to maintain the restrictive barrier properties of the BBB [27, 28]. The luminal surface of capillary ECs is also covered…
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