Modulation of mononuclear phagocyte function in the dissemination of Toxoplasma gondii Arne L. ten Hoeve Doctoral Thesis in Molecular Bioscience at Stockholm University, Sweden 2022
Modulation of mononuclear phagocyte function in the dissemination of Toxoplasma gondii
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Modulation of mononuclear phagocyte function in the dissemination of Toxoplasma gondii Arne L. ten Hoeve
Arne L. ten H oeve M
odu lation
Doctoral Thesis in Molecular Bioscience at Stockholm University, Sweden 2022
Department of Molecular Biosciences, The Wenner-Gren Institute
ISBN 978-91-7911-824-2
Modulation of mononuclear phagocyte function in the dissemination of Toxoplasma gondii Arne L. ten Hoeve
Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Friday 6 May 2022 at 09.30 in Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20.
Abstract Toxoplasma gondii is an obligate intracellular Apicomplexan parasite that could possibly infect all warm-blooded animals. Acute infections with T. gondii produce generally mild symptoms in healthy individuals, but infections of the fetus during pregnancy and infections in those that are immunocompromised can cause severe and life-threatening pathology. The parasite gains entry to the host by crossing the biological barriers of the intestine or placenta. Mononuclear phagocytes (MPs) and other leukocytes at these barriers become infected by T. gondii. Previous work has established that upon T. gondii infection, dendritic cells (DCs) undergo morphological and phenotypical changes and display enhanced migration. Components of this ‘hypermigratory phenotype’ have been confirmed in other MPs, such as monocytes, macrophages and microglia. The mechanisms underlying the hypermigratory phenotype are the subject of this thesis.
In paper I, we describe a non-canonical extended upregulation of the transcription factor Egr1 in T. gondii-infected DCs. While the rapid and transient canonical induction of Egr1 depends on the ERK1/2-pathway, the extended upregulation was dependent on p38 MAPK and p38-activating parasite-derived effector GRA24. The hypermotility component of the hypermigratory phenotype did not depend on GRA24/p38, but on ERK1/2. We determined that EGR1 acts as an inhibitor of phenotypic maturation and that GRA24 stimulates Il2 and Il12p40 expression in T. gondii-infected DCs.
In paper II, we characterize actors upstream of ERK1/2 in hypermotility of T. gondii-infected DCs. Two axes that output on the ERK1/2 pathway were found to be required for hypermotility. The first involves Ca2+ influx through voltage- gated calcium channel Cav1.3, resulting in activation of calcium/calmodulin-dependent protein kinase II (CaMKII) via Ca2+ sensor calmodulin (CaM). The other axis relies on hepatocyte growth factor (HGF), which is secreted by DCs, and its receptor Met. Both axes converge on the ERK1/2 pathway via the GTPase Ras.
In paper III, we study the migratory behavior of T. gondii-infected DCs on and across endothelial cell monolayers. Particularly infected DCs transmigrated across endothelial cell monolayers, but were, unlike on 2D surfaces, not hypermotile on endothelial cells. We characterize the differential involvement of β1 and β2 integrins, cell adhesion molecules ICAM-1 and PECAM-1 and pan-integrin-cytoskeleton linker talin in transmigration across endothelial cells and in migration on endothelial cells and 2D surfaces.
Finally, we report in paper IV that T. gondii imparts a DC-like transcriptional signature on infected macrophages. Infected macrophages upregulate chemokine receptor CCR7 and display chemotaxis to CCR7-ligand CCL19, like DCs. Concomitantly, these macrophages upregulate the expression of transcription factors associated with DCs and of immune activation-related genes and markers. T. gondii-infected macrophages thus display a remarkable transcriptional and functional plasticity. We identify GRA28 as the primary T. gondii-derived effector protein responsible for these phenotypes, with parasite-derived ROP16 having partially opposing effects.
Altogether, my thesis identifies novel aspects of the hypermigratory phenotype in T. gondii-infected MPs and provides insights into the molecular components and signaling that underlie them.
Keywords: Apicomplexa, dendritic cell, macrophage, intracellular signaling, parasite-derived effector protein, chemotaxis, leukocyte migration, transcription factor.
Stockholm 2022 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-202986
ISBN 978-91-7911-824-2 ISBN 978-91-7911-825-9
Stockholm University, 106 91 Stockholm
MODULATION OF MONONUCLEAR PHAGOCYTE FUNCTION IN THE DISSEMINATION OF TOXOPLASMA GONDII
Arne L. ten Hoeve
Modulation of mononuclear phagocyte function in the dissemination of Toxoplasma gondii
Arne L. ten Hoeve
©Arne L. ten Hoeve, Stockholm University 2022 ISBN print 978-91-7911-824-2 ISBN PDF 978-91-7911-825-9 Printed in Sweden by Universitetsservice US-AB, Stockholm 2022 Cover art by D.L. Elling: Artistic impression of a Toxoplasma gondii-infected cell with two tachyzoites present in a parasitophorous vacuole. Streams of different parasite-derived proteins are visible in the host cell cytoplasm.
“Somewhere, something incredible is waiting to be known.” – Sharon Begley (commonly misattributed to Carl Sagan)
I
Thesis abstract Toxoplasma gondii is an obligate intracellular Apicomplexan parasite that could possibly infect all warm-blooded animals. Acute infections with T. gondii produce generally mild symptoms in healthy individuals, but infections of the fetus during pregnancy and infections in those that are immunocompro- mised can cause severe and life-threatening pathology. The parasite gains en- try to the host by crossing the biological barriers of the intestine or placenta. Mononuclear phagocytes (MPs) and other leukocytes at these barriers become infected by T. gondii. Previous work has established that upon T. gondii in- fection, dendritic cells (DCs) undergo morphological and phenotypical changes and display enhanced migration. Components of this ‘hypermigratory phenotype’ have been confirmed in other MPs, such as monocytes, macro- phages and microglia. The mechanisms underlying the hypermigratory phe- notype are the subject of this thesis.
In paper I, we describe a non-canonical extended upregulation of the tran- scription factor Egr1 in T. gondii-infected DCs. While the rapid and transient canonical induction of Egr1 depends on the ERK1/2-pathway, the extended upregulation was dependent on p38 MAPK and p38-activating parasite-de- rived effector GRA24. The hypermotility component of the hypermigratory phenotype did not depend on GRA24/p38, but on ERK1/2. We determined that EGR1 acts as an inhibitor of phenotypic maturation and that GRA24 stim- ulates Il2 and Il12p40 expression in T. gondii-infected DCs.
In paper II, we characterize actors upstream of ERK1/2 in hypermotility of T. gondii-infected DCs. Two axes that output on the ERK1/2 pathway were found to be required for hypermotility. The first involves Ca2+ influx through voltage-gated calcium channel Cav1.3, resulting in activation of calcium/cal- modulin-dependent protein kinase II (CaMKII) via Ca2+ sensor calmodulin (CaM). The other axis relies on hepatocyte growth factor (HGF), which is se- creted by DCs, and its receptor Met. Both axes converge on the ERK1/2 path- way via the GTPase Ras.
In paper III, we study the migratory behavior of T. gondii-infected DCs on and across endothelial cell monolayers. Particularly infected DCs transmi- grated across endothelial cell monolayers, but were, unlike on 2D surfaces, not hypermotile on endothelial cells. We characterize the differential involve- ment of β1 and β2 integrins, cell adhesion molecules ICAM-1 and PECAM-1 and pan-integrin-cytoskeleton linker talin in transmigration across endothelial cells and in migration on endothelial cells and 2D surfaces.
Finally, we report in paper IV that T. gondii imparts a DC-like transcriptional signature on infected macrophages. Infected macrophages upregulate chemo- kine receptor CCR7 and display chemotaxis to CCR7-ligand CCL19, like
II
DCs. Concomitantly, these macrophages upregulate the expression of tran- scription factors associated with DCs and of immune activation-related genes and markers. T. gondii-infected macrophages thus display a remarkable tran- scriptional and functional plasticity. We identify GRA28 as the primary T. gondii-derived effector protein responsible for these phenotypes, with para- site-derived ROP16 having partially opposing effects.
Altogether, my thesis identifies novel aspects of the hypermigratory pheno- type in T. gondii-infected MPs and provides insights into the molecular com- ponents and signaling that underlie them.
Keywords: Apicomplexa, dendritic cell, macrophage, intracellular signal- ing, parasite-derived effector protein, chemotaxis, leukocyte migration, tran- scription factor
III
Popular scientific summary Toxoplasma gondii (T. gondii) is a parasite that can cause the disease toxoplasmo-
sis in all warm-blooded animals, including humans. The parasite generally enters the body through the gut. An acute infection by T. gondii often has mild symptoms among healthy individuals, but can also cause severe and life-threatening symptoms. For in- stance, the parasite can cause infections in the eyes and brain, as well as miscarriage through the crossing of the placenta. In addition, T. gondii can cause a chronic infec- tion where a number of parasites remain in the brain, where they are less active and cannot replicate as much.
T. gondii can only survive and multiply inside cells, as it is a so-called obligate intracellular parasite. The infection takes place first among cells in the gut. Different types of white blood cells, such as mononuclear phagocytes, migrate through the body and transport the parasites through the whole body. Mononuclear phagocytes are a collection of cells, which include dendritic cells and macrophages. Different types of mononuclear phagocytes have different abilities and functions. For example, dendritic cells move with higher speeds compared with macrophages. Additionally, stimulated dendritic cells can receive signals from different organs of the immune system (so- called lymphoid organs) and migrate to these organs to stimulate immunity. On the other hand, macrophages do not have these abilities and functions or are considerably worse at it.
Previous research has established that infected mononuclear phagocytes migrate more efficiently compared with non-infected cells (this is the so-called hypermigra- tory phenotype). In addition, this phenotype includes morphological and phenotypical changes to the infected cells. The studies in this thesis explore the underlying mecha- nisms of the hypermigratory phenotype.
In the studies included in this thesis, T. gondii modulates signals that cause some genes in infected cells to be more active compared with non-infected cells. In Study I, we found a parasite protein that activates a gene called Egr1, which generated an- other protein that reduced the ability of dendritic cells to stimulate immunity. At the same time, this parasite protein also stimulated inflammation in the infected cells. Study II describes two signals that are activated by T. gondii in infected dendritic cells and that together are required for faster-moving infected cells compared with non-infected cells. In a model of the so-called blood-brain barrier (the border between the blood and the brain), infected dendritic cells cross more often than non-infected cells. Study III describes which molecules on infected dendritic cells recognize the blood-brain barrier and which molecules would be required for infected dendritic cells to cross this barrier. In Study IV, infected macrophages became more motile and were suddenly able to respond to the signal from lymphoid organs. This behavior is differ- ent from the normal behavior of macrophages, as they are normally stationary and do not receive this signal from lymphoid organs, but instead have the role of cleaning up microorganisms. The different behavior of macrophages is similar to the behavior of dendritic cells, because infected macrophages could migrate to lymphoid organs and consequently help the dissemination of T. gondii throughout the body.
In summary, this thesis has described novel aspects of the hypermigratory pheno- type in infected mononuclear phagocytes. The thesis gives insight into the involved components and underlying mechanisms in T. gondii and infected cells on a molecular level.
IV
Populärvetenskaplig sammanfatning Toxoplasma gondii (T. gondii) är en parasit som kan orsaka sjukdomen
toxoplasmos hos alla varmblodiga djur, inklusive människor. Parasiten kommer vanligtvis in till kroppen genom tarmkanalen. En akutsmitta på grund av T. gondii har oftast milda symptom bland friska individer men kan också orsaka allvarliga och livshotande effekter. Exempelvis kan parasiten orsaka ögoninfektion, hjärninflammation och missfall bland gravida genom korsning av moderkakan. Dessutom kan T. gondii orsaka en kronisksmitta där ett antal parasiter lever kvar i hjärnan då de är mindre aktiva och inte kan föröka sig mycket.
För att T. gondii ska kunna överleva och föröka sig måste smittan ske inom cellen (s.k. obligatorisk intracellulär). Detta är anledningen till varför smittan sker först inom celler i tarmkanalen. Olika typer av vita blodkroppar, t.ex. mononukleära fagocyter, flyter igenom kroppen och kan därmed bära parasiterna igenom hela kroppen. Mononukleära fagocyter är en samling av celler som inkluderar bl.a. dendritceller och makrofager. Olika typer av mononukleära fagocyter har olika förmågor och funktioner. Till exempel, har dendritceller jämförelsevis högre hastighet än makrofager. Dessutom kan stimulerade dendritceller ta emot signaler från olika organ i immunsystemet (s.k. lymfatiska organ) och förflytta sig till dessa organ för att kunna stimulera immunitet. Makrofager däremot har inte likadana förmåga och funktion.
Tidigare forskning har fastställt att smittade mononukleära fagocyter flyttar ännu effektivare (s.k. hypermigratoriskfenotyp) jämfört med icke smittade celler. Dessutom inkluderar denna fenotyp morfologiska och fenotypiska förändringar till de smittade cellerna. Studierna i denna avhandling undersöker de underliggande mekanismerna i denna hypermigratoriskfenotyp.
I de studierna som inkluderades i denna avhandling omvandlade T. gondii signaler vilket införde mer aktiva gener hos smittade celler jämfört med icke smittade celler. I Studie I fann vi ett parasitprotein som aktiverar en av dessa gener som heter Egr1 vilket frambringade ett annat protein som minskar dendritcellernas förmåga att stimulera immuniteten. Parasitproteinet stimulerade också inflammation hos smittade celler. Studie II beskrev två signaler som aktiverades av T. gondii hos smittade dendritceller vilket tillsammans medför snabbare celler hos de smittade jämfört med icke smittade celler. I en modellering av den s.k. blod-hjärnbarriären (gränsen mellan blodet och hjärnan) korsar smittade dendritceller oftare jämfört med icke smittade celler. Studie III därför beskrev vilka molekyler hos de smittade dendritcellerna igenkänner blod-hjärnbarriären och vilka molekyler är nödvändiga för smittade dendritceller att korsa denna barriär. I Studie IV, blev de smittade makrofagerna rörligare och kunde plötsligt svara på signalen från lymfatiska organ. Detta beteende avviker från makrofagernas normala beteende där de normalt är stillasittande celler som inte tar emot den signalen från lymfatiska organ då den brukar ha rollen att städa undan främmande varelser såsom mikroorganismer. Avvikelsen i makrofagernas beteende i denna studie liknar dendritceller då smittade makrofager kunde möjligtvis förflytta sig till lymfatiska organ och därmed hjälpa till med smittspridningen igenom kroppen.
Sammanfattningsvis har denna avhandling sammanställt de hypermigratorisk- fenotypa aspekter av smittade mononukleära fagocyter som inte har beskrivits förut. Avhandlingen tillhandahåller insyn till de komponenter och underliggande mekanismer av T. gondii och smittade celler på molekylärnivå.
V
Populairwetenschappelijke samenvatting Toxoplasma gondii (T. gondii) is een parasiet die de ziekte toxoplasmose kan
veroorzaken in alle warmbloedige dieren, waaronder mensen. De parasiet komt normaal gesproken het lichaam binnen via het darmkanaal. Een acute infectie met T. gondii heeft vaak milde symptomen in gezonde individuen, maar kan ook ernstige en levensbedreigende effecten hebben. De parasiet kan bijvoorbeeld oog- en herseninfecties en, via de placenta, een miskraam veroorzaken. Verder is T. gondii de veroorzaker van een chronische infectie, waarbij een aantal parasiten in de hersenen achterblijft, die daar minder actief zijn en zich minder vermenigvuldigen.
Als een obligaat intracellulaire parasiet moet T. gondii cellen infecteren om te overleven en zich te vermenigvuldigen. De infectie vindt daarom eerst plaats in cellen in het darmkanaal. Verschillende soorten witte bloedcellen, waaronder zogenaamde mononucleaire fagocyten, migreren door het lichaam en transporteren de parasiet door het hele lichaam. Mononucleaire fagocyten zijn een verzameling van celtypen, waaronder dendritische cellen en macrofagen. Verschillende soorten mononucleaire fagocyten hebben verschillende competenties en functies. Dendritische cellen bewegen zich sneller voort dan macrofagen bijvoorbeeld. Bovendien kunnen dendritische cellen signalen ontvangen van organen van het immuunsysteem (zogheten lymfoïde organen) en migreren naar die organen om immuniteit op te wekken. Makrofagen kunnen dit niet of zijn er veel slechter in.
Eerder onderzoek wijst uit dat geïnfecteerde mononucleaire fagocyten meer efficiënt migreren dan niet-geïnfecteerde cellen (het zogeheten hypermigratie- fenotype). Dit fenotype omvat ook morfologische en fenotypische veranderingen aan geïnfecteerde cellen. De studies in dit proefschrift gaan over de onderliggende mechanismen van dit hypermigratiefenotype.
In de studies in dit proefschrift, moduleert T. gondii signalen zo, dat sommige genen in geïnfecteerde cellen meer actief zijn dan in niet-geïnfecteerde cellen. In studie I identificeren we een parasitair eiwit dat een gen dat Egr1 heet activeert. Van dit gen komt een ander eiwit dat het vermogen van dendritische cellen om immuniteit op te wekken remt. Daarentegen stimuleert het parasitaire eiwit ook een ontstekingsreactie in geïnfecteerde cellen. Studie II beschrijft twee signalen die door T. gondii worden geactiveerd in geïnfecteerde dendritische cellen. Zij zijn beiden noodzakelijk voor de snellere beweging van geïnfecteerde cellen in vergelijking met niet-geïnfecteerde. In een model van de bloed-hersenbarrière (de grens tussen het bloed en de hersenen), steken geïnfecteerde dendritische cellen de barrière vaker over dan niet-geïnfecteerde cellen. Studie III beschrijft welke moleculen op geïnfecteerde cellen noodzakelijk zijn voor de herkenning en het oversteken van de barrière. In studie IV waren geïnfecteerde macrofagen meer bewegelijk dan niet-geïnfecteerde en konden zij opeens op het signaal van de lymfoïde organen reageren. Dit is anders dan het gebruikelijke gedrag van macrofagen: normaal gesproken zitten zij stil en ontvangen het signaal van de lymfoïde organen niet. Daarentegen zijn zij er voor het opruimen van micro-organismen. Het veranderde gedrag lijkt op het gedrag van dendritische cellen en geïnfecteerde macrofagen kunnen zo migreren naar lymfoïde organen en de verspreiding van de parasiet door het lichaam bevorderen.
Samengevat beschrijft dit proefschrift nieuwe aspecten van het hypermigratie- fenotype in geïnfecteerde mononucleaire fagocyten en geeft daarbij inzicht in de componenten en onderliggende mechanismen in T. gondii en de geïnfecteerde cellen op een moleculair niveau.
VI
List of scientific papers The following papers are included in this thesis:
I. Ten Hoeve AL, Hakimi MA, Barragan A. Sustained Egr-1 Response via
p38 MAP Kinase Signaling Modulates Early Immune Responses of Den-
dritic Cells Parasitized by Toxoplasma gondii. Front Cell Infect Micro-
biol. 2019 Oct 11;9:349.
II. Ólafsson EB, Ten Hoeve AL, Li Wang X, Westermark L, Varas-Godoy
M, Barragan A. Convergent Met and voltage-gated Ca2+ channel signal-
ing drives hypermigration of Toxoplasma-infected dendritic cells. J Cell
Sci. 2020 Apr 21;134(5):jcs24175.
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. Ten Hoeve AL, Olivera GC, Saeij JPJ, Hakimi MA, Barragan A. The
Toxoplasma effector GRA28 promotes parasite dissemination by inducing
dendritic cell-like migratory properties in infected macrophages. Manu-
script
VII
Contents
List of scientific papers .....................................................................VI
List of abbreviations ........................................................................ VIII
Introduction ........................................................................................ 1 Toxoplasma gondii ................................................................................ 1 Biological barriers and cellular immunity ............................................... 2
Mononuclear phagocytes (MPs): origin and functions ..................... 2 Growth factors and transcription factors regulating MPs.................. 6
Host-parasite interactions and T. gondii dissemination ...................... 10 Parasite-derived proteins modulate immune responses ................ 11 The ‘Trojan horse’ hypothesis ......................................................... 12 From dissemination to chronic infection ......................................... 15
Aims of this thesis ............................................................................ 16 Main hypothesis ................................................................................... 16 Specific aims ....................................................................................... 16
Summary of papers ......................................................................... 17 Paper I ................................................................................................. 17 Paper II ................................................................................................ 17 Paper III ............................................................................................... 18 Paper IV ............................................................................................... 18
Discussion and conclusions ............................................................. 20 Macrophages and T. gondii: change of cell identity? .......................... 20 Activation of familiar pathways ............................................................ 23 An attempt to take control of immunity ................................................ 24
Acknowledgements ......................................................................... 25
References ...................................................................................... 27
BATF Basic leucine zipper ATF-like transcription factor
BMDC Bone marrow-derived dendritic cell
BMDM Bone marrow-derived macrophage
CAM Cell adhesion molecule
cMoP Common monocyte progenitor
CMP Common myeloid progenitor
EZH2 Enhancer of zeste homolog 2
FLT3 FMS-like tyrosine kinase 3
FLT3L FMS-like tyrosine kinase 3 ligand
GABA γ-aminobutyric acid
GRA Dense Granule protein
HGF Hepatocyte growth factor
ICAM Intercellular adhesion molecule
IX
MEK MAP/ERK kinase
pDC Plasmacytoid dendritic cell
PRR Pattern recognition receptor
STING Stimulator of interferon gene
TEEGR Toxoplasma E2F4-associated EZH2-inducing gene regulator
TLR Toll-like receptor
VGCC Voltage-gated calcium channel
1
Introduction
Toxoplasma gondii Toxoplasma gondii is an obligate intracellular Apicomplexan parasite that
occurs nearly worldwide [1]. It could possibly infect all warm-blooded ani-
mals as intermediate hosts, while felids, such as domestic cats, that predate
intermediate hosts, form its definitive host. In intermediate hosts, such as hu-
mans and rodents, the development of the parasite goes through two main
phases. During the first phase, the rapidly replicating tachyzoite form multi-
plies inside nucleated cells within membrane-limited vacuoles – the para-
sitophorous vacuoles (PVs) – through repeated endodyogeny. The pressure of
cell-mediated immunity initiates the second phase, in which slowly replicating
bradyzoites reside in tissue cysts. These infectious cysts are located primarily
in the central nervous system, as well as eyes and skeletal and cardiac muscu-
lature. It is thought that once infected, the hosts will remain infected…
Arne L. ten H oeve M
odu lation
Doctoral Thesis in Molecular Bioscience at Stockholm University, Sweden 2022
Department of Molecular Biosciences, The Wenner-Gren Institute
ISBN 978-91-7911-824-2
Modulation of mononuclear phagocyte function in the dissemination of Toxoplasma gondii Arne L. ten Hoeve
Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Friday 6 May 2022 at 09.30 in Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20.
Abstract Toxoplasma gondii is an obligate intracellular Apicomplexan parasite that could possibly infect all warm-blooded animals. Acute infections with T. gondii produce generally mild symptoms in healthy individuals, but infections of the fetus during pregnancy and infections in those that are immunocompromised can cause severe and life-threatening pathology. The parasite gains entry to the host by crossing the biological barriers of the intestine or placenta. Mononuclear phagocytes (MPs) and other leukocytes at these barriers become infected by T. gondii. Previous work has established that upon T. gondii infection, dendritic cells (DCs) undergo morphological and phenotypical changes and display enhanced migration. Components of this ‘hypermigratory phenotype’ have been confirmed in other MPs, such as monocytes, macrophages and microglia. The mechanisms underlying the hypermigratory phenotype are the subject of this thesis.
In paper I, we describe a non-canonical extended upregulation of the transcription factor Egr1 in T. gondii-infected DCs. While the rapid and transient canonical induction of Egr1 depends on the ERK1/2-pathway, the extended upregulation was dependent on p38 MAPK and p38-activating parasite-derived effector GRA24. The hypermotility component of the hypermigratory phenotype did not depend on GRA24/p38, but on ERK1/2. We determined that EGR1 acts as an inhibitor of phenotypic maturation and that GRA24 stimulates Il2 and Il12p40 expression in T. gondii-infected DCs.
In paper II, we characterize actors upstream of ERK1/2 in hypermotility of T. gondii-infected DCs. Two axes that output on the ERK1/2 pathway were found to be required for hypermotility. The first involves Ca2+ influx through voltage- gated calcium channel Cav1.3, resulting in activation of calcium/calmodulin-dependent protein kinase II (CaMKII) via Ca2+ sensor calmodulin (CaM). The other axis relies on hepatocyte growth factor (HGF), which is secreted by DCs, and its receptor Met. Both axes converge on the ERK1/2 pathway via the GTPase Ras.
In paper III, we study the migratory behavior of T. gondii-infected DCs on and across endothelial cell monolayers. Particularly infected DCs transmigrated across endothelial cell monolayers, but were, unlike on 2D surfaces, not hypermotile on endothelial cells. We characterize the differential involvement of β1 and β2 integrins, cell adhesion molecules ICAM-1 and PECAM-1 and pan-integrin-cytoskeleton linker talin in transmigration across endothelial cells and in migration on endothelial cells and 2D surfaces.
Finally, we report in paper IV that T. gondii imparts a DC-like transcriptional signature on infected macrophages. Infected macrophages upregulate chemokine receptor CCR7 and display chemotaxis to CCR7-ligand CCL19, like DCs. Concomitantly, these macrophages upregulate the expression of transcription factors associated with DCs and of immune activation-related genes and markers. T. gondii-infected macrophages thus display a remarkable transcriptional and functional plasticity. We identify GRA28 as the primary T. gondii-derived effector protein responsible for these phenotypes, with parasite-derived ROP16 having partially opposing effects.
Altogether, my thesis identifies novel aspects of the hypermigratory phenotype in T. gondii-infected MPs and provides insights into the molecular components and signaling that underlie them.
Keywords: Apicomplexa, dendritic cell, macrophage, intracellular signaling, parasite-derived effector protein, chemotaxis, leukocyte migration, transcription factor.
Stockholm 2022 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-202986
ISBN 978-91-7911-824-2 ISBN 978-91-7911-825-9
Stockholm University, 106 91 Stockholm
MODULATION OF MONONUCLEAR PHAGOCYTE FUNCTION IN THE DISSEMINATION OF TOXOPLASMA GONDII
Arne L. ten Hoeve
Modulation of mononuclear phagocyte function in the dissemination of Toxoplasma gondii
Arne L. ten Hoeve
©Arne L. ten Hoeve, Stockholm University 2022 ISBN print 978-91-7911-824-2 ISBN PDF 978-91-7911-825-9 Printed in Sweden by Universitetsservice US-AB, Stockholm 2022 Cover art by D.L. Elling: Artistic impression of a Toxoplasma gondii-infected cell with two tachyzoites present in a parasitophorous vacuole. Streams of different parasite-derived proteins are visible in the host cell cytoplasm.
“Somewhere, something incredible is waiting to be known.” – Sharon Begley (commonly misattributed to Carl Sagan)
I
Thesis abstract Toxoplasma gondii is an obligate intracellular Apicomplexan parasite that could possibly infect all warm-blooded animals. Acute infections with T. gondii produce generally mild symptoms in healthy individuals, but infections of the fetus during pregnancy and infections in those that are immunocompro- mised can cause severe and life-threatening pathology. The parasite gains en- try to the host by crossing the biological barriers of the intestine or placenta. Mononuclear phagocytes (MPs) and other leukocytes at these barriers become infected by T. gondii. Previous work has established that upon T. gondii in- fection, dendritic cells (DCs) undergo morphological and phenotypical changes and display enhanced migration. Components of this ‘hypermigratory phenotype’ have been confirmed in other MPs, such as monocytes, macro- phages and microglia. The mechanisms underlying the hypermigratory phe- notype are the subject of this thesis.
In paper I, we describe a non-canonical extended upregulation of the tran- scription factor Egr1 in T. gondii-infected DCs. While the rapid and transient canonical induction of Egr1 depends on the ERK1/2-pathway, the extended upregulation was dependent on p38 MAPK and p38-activating parasite-de- rived effector GRA24. The hypermotility component of the hypermigratory phenotype did not depend on GRA24/p38, but on ERK1/2. We determined that EGR1 acts as an inhibitor of phenotypic maturation and that GRA24 stim- ulates Il2 and Il12p40 expression in T. gondii-infected DCs.
In paper II, we characterize actors upstream of ERK1/2 in hypermotility of T. gondii-infected DCs. Two axes that output on the ERK1/2 pathway were found to be required for hypermotility. The first involves Ca2+ influx through voltage-gated calcium channel Cav1.3, resulting in activation of calcium/cal- modulin-dependent protein kinase II (CaMKII) via Ca2+ sensor calmodulin (CaM). The other axis relies on hepatocyte growth factor (HGF), which is se- creted by DCs, and its receptor Met. Both axes converge on the ERK1/2 path- way via the GTPase Ras.
In paper III, we study the migratory behavior of T. gondii-infected DCs on and across endothelial cell monolayers. Particularly infected DCs transmi- grated across endothelial cell monolayers, but were, unlike on 2D surfaces, not hypermotile on endothelial cells. We characterize the differential involve- ment of β1 and β2 integrins, cell adhesion molecules ICAM-1 and PECAM-1 and pan-integrin-cytoskeleton linker talin in transmigration across endothelial cells and in migration on endothelial cells and 2D surfaces.
Finally, we report in paper IV that T. gondii imparts a DC-like transcriptional signature on infected macrophages. Infected macrophages upregulate chemo- kine receptor CCR7 and display chemotaxis to CCR7-ligand CCL19, like
II
DCs. Concomitantly, these macrophages upregulate the expression of tran- scription factors associated with DCs and of immune activation-related genes and markers. T. gondii-infected macrophages thus display a remarkable tran- scriptional and functional plasticity. We identify GRA28 as the primary T. gondii-derived effector protein responsible for these phenotypes, with para- site-derived ROP16 having partially opposing effects.
Altogether, my thesis identifies novel aspects of the hypermigratory pheno- type in T. gondii-infected MPs and provides insights into the molecular com- ponents and signaling that underlie them.
Keywords: Apicomplexa, dendritic cell, macrophage, intracellular signal- ing, parasite-derived effector protein, chemotaxis, leukocyte migration, tran- scription factor
III
Popular scientific summary Toxoplasma gondii (T. gondii) is a parasite that can cause the disease toxoplasmo-
sis in all warm-blooded animals, including humans. The parasite generally enters the body through the gut. An acute infection by T. gondii often has mild symptoms among healthy individuals, but can also cause severe and life-threatening symptoms. For in- stance, the parasite can cause infections in the eyes and brain, as well as miscarriage through the crossing of the placenta. In addition, T. gondii can cause a chronic infec- tion where a number of parasites remain in the brain, where they are less active and cannot replicate as much.
T. gondii can only survive and multiply inside cells, as it is a so-called obligate intracellular parasite. The infection takes place first among cells in the gut. Different types of white blood cells, such as mononuclear phagocytes, migrate through the body and transport the parasites through the whole body. Mononuclear phagocytes are a collection of cells, which include dendritic cells and macrophages. Different types of mononuclear phagocytes have different abilities and functions. For example, dendritic cells move with higher speeds compared with macrophages. Additionally, stimulated dendritic cells can receive signals from different organs of the immune system (so- called lymphoid organs) and migrate to these organs to stimulate immunity. On the other hand, macrophages do not have these abilities and functions or are considerably worse at it.
Previous research has established that infected mononuclear phagocytes migrate more efficiently compared with non-infected cells (this is the so-called hypermigra- tory phenotype). In addition, this phenotype includes morphological and phenotypical changes to the infected cells. The studies in this thesis explore the underlying mecha- nisms of the hypermigratory phenotype.
In the studies included in this thesis, T. gondii modulates signals that cause some genes in infected cells to be more active compared with non-infected cells. In Study I, we found a parasite protein that activates a gene called Egr1, which generated an- other protein that reduced the ability of dendritic cells to stimulate immunity. At the same time, this parasite protein also stimulated inflammation in the infected cells. Study II describes two signals that are activated by T. gondii in infected dendritic cells and that together are required for faster-moving infected cells compared with non-infected cells. In a model of the so-called blood-brain barrier (the border between the blood and the brain), infected dendritic cells cross more often than non-infected cells. Study III describes which molecules on infected dendritic cells recognize the blood-brain barrier and which molecules would be required for infected dendritic cells to cross this barrier. In Study IV, infected macrophages became more motile and were suddenly able to respond to the signal from lymphoid organs. This behavior is differ- ent from the normal behavior of macrophages, as they are normally stationary and do not receive this signal from lymphoid organs, but instead have the role of cleaning up microorganisms. The different behavior of macrophages is similar to the behavior of dendritic cells, because infected macrophages could migrate to lymphoid organs and consequently help the dissemination of T. gondii throughout the body.
In summary, this thesis has described novel aspects of the hypermigratory pheno- type in infected mononuclear phagocytes. The thesis gives insight into the involved components and underlying mechanisms in T. gondii and infected cells on a molecular level.
IV
Populärvetenskaplig sammanfatning Toxoplasma gondii (T. gondii) är en parasit som kan orsaka sjukdomen
toxoplasmos hos alla varmblodiga djur, inklusive människor. Parasiten kommer vanligtvis in till kroppen genom tarmkanalen. En akutsmitta på grund av T. gondii har oftast milda symptom bland friska individer men kan också orsaka allvarliga och livshotande effekter. Exempelvis kan parasiten orsaka ögoninfektion, hjärninflammation och missfall bland gravida genom korsning av moderkakan. Dessutom kan T. gondii orsaka en kronisksmitta där ett antal parasiter lever kvar i hjärnan då de är mindre aktiva och inte kan föröka sig mycket.
För att T. gondii ska kunna överleva och föröka sig måste smittan ske inom cellen (s.k. obligatorisk intracellulär). Detta är anledningen till varför smittan sker först inom celler i tarmkanalen. Olika typer av vita blodkroppar, t.ex. mononukleära fagocyter, flyter igenom kroppen och kan därmed bära parasiterna igenom hela kroppen. Mononukleära fagocyter är en samling av celler som inkluderar bl.a. dendritceller och makrofager. Olika typer av mononukleära fagocyter har olika förmågor och funktioner. Till exempel, har dendritceller jämförelsevis högre hastighet än makrofager. Dessutom kan stimulerade dendritceller ta emot signaler från olika organ i immunsystemet (s.k. lymfatiska organ) och förflytta sig till dessa organ för att kunna stimulera immunitet. Makrofager däremot har inte likadana förmåga och funktion.
Tidigare forskning har fastställt att smittade mononukleära fagocyter flyttar ännu effektivare (s.k. hypermigratoriskfenotyp) jämfört med icke smittade celler. Dessutom inkluderar denna fenotyp morfologiska och fenotypiska förändringar till de smittade cellerna. Studierna i denna avhandling undersöker de underliggande mekanismerna i denna hypermigratoriskfenotyp.
I de studierna som inkluderades i denna avhandling omvandlade T. gondii signaler vilket införde mer aktiva gener hos smittade celler jämfört med icke smittade celler. I Studie I fann vi ett parasitprotein som aktiverar en av dessa gener som heter Egr1 vilket frambringade ett annat protein som minskar dendritcellernas förmåga att stimulera immuniteten. Parasitproteinet stimulerade också inflammation hos smittade celler. Studie II beskrev två signaler som aktiverades av T. gondii hos smittade dendritceller vilket tillsammans medför snabbare celler hos de smittade jämfört med icke smittade celler. I en modellering av den s.k. blod-hjärnbarriären (gränsen mellan blodet och hjärnan) korsar smittade dendritceller oftare jämfört med icke smittade celler. Studie III därför beskrev vilka molekyler hos de smittade dendritcellerna igenkänner blod-hjärnbarriären och vilka molekyler är nödvändiga för smittade dendritceller att korsa denna barriär. I Studie IV, blev de smittade makrofagerna rörligare och kunde plötsligt svara på signalen från lymfatiska organ. Detta beteende avviker från makrofagernas normala beteende där de normalt är stillasittande celler som inte tar emot den signalen från lymfatiska organ då den brukar ha rollen att städa undan främmande varelser såsom mikroorganismer. Avvikelsen i makrofagernas beteende i denna studie liknar dendritceller då smittade makrofager kunde möjligtvis förflytta sig till lymfatiska organ och därmed hjälpa till med smittspridningen igenom kroppen.
Sammanfattningsvis har denna avhandling sammanställt de hypermigratorisk- fenotypa aspekter av smittade mononukleära fagocyter som inte har beskrivits förut. Avhandlingen tillhandahåller insyn till de komponenter och underliggande mekanismer av T. gondii och smittade celler på molekylärnivå.
V
Populairwetenschappelijke samenvatting Toxoplasma gondii (T. gondii) is een parasiet die de ziekte toxoplasmose kan
veroorzaken in alle warmbloedige dieren, waaronder mensen. De parasiet komt normaal gesproken het lichaam binnen via het darmkanaal. Een acute infectie met T. gondii heeft vaak milde symptomen in gezonde individuen, maar kan ook ernstige en levensbedreigende effecten hebben. De parasiet kan bijvoorbeeld oog- en herseninfecties en, via de placenta, een miskraam veroorzaken. Verder is T. gondii de veroorzaker van een chronische infectie, waarbij een aantal parasiten in de hersenen achterblijft, die daar minder actief zijn en zich minder vermenigvuldigen.
Als een obligaat intracellulaire parasiet moet T. gondii cellen infecteren om te overleven en zich te vermenigvuldigen. De infectie vindt daarom eerst plaats in cellen in het darmkanaal. Verschillende soorten witte bloedcellen, waaronder zogenaamde mononucleaire fagocyten, migreren door het lichaam en transporteren de parasiet door het hele lichaam. Mononucleaire fagocyten zijn een verzameling van celtypen, waaronder dendritische cellen en macrofagen. Verschillende soorten mononucleaire fagocyten hebben verschillende competenties en functies. Dendritische cellen bewegen zich sneller voort dan macrofagen bijvoorbeeld. Bovendien kunnen dendritische cellen signalen ontvangen van organen van het immuunsysteem (zogheten lymfoïde organen) en migreren naar die organen om immuniteit op te wekken. Makrofagen kunnen dit niet of zijn er veel slechter in.
Eerder onderzoek wijst uit dat geïnfecteerde mononucleaire fagocyten meer efficiënt migreren dan niet-geïnfecteerde cellen (het zogeheten hypermigratie- fenotype). Dit fenotype omvat ook morfologische en fenotypische veranderingen aan geïnfecteerde cellen. De studies in dit proefschrift gaan over de onderliggende mechanismen van dit hypermigratiefenotype.
In de studies in dit proefschrift, moduleert T. gondii signalen zo, dat sommige genen in geïnfecteerde cellen meer actief zijn dan in niet-geïnfecteerde cellen. In studie I identificeren we een parasitair eiwit dat een gen dat Egr1 heet activeert. Van dit gen komt een ander eiwit dat het vermogen van dendritische cellen om immuniteit op te wekken remt. Daarentegen stimuleert het parasitaire eiwit ook een ontstekingsreactie in geïnfecteerde cellen. Studie II beschrijft twee signalen die door T. gondii worden geactiveerd in geïnfecteerde dendritische cellen. Zij zijn beiden noodzakelijk voor de snellere beweging van geïnfecteerde cellen in vergelijking met niet-geïnfecteerde. In een model van de bloed-hersenbarrière (de grens tussen het bloed en de hersenen), steken geïnfecteerde dendritische cellen de barrière vaker over dan niet-geïnfecteerde cellen. Studie III beschrijft welke moleculen op geïnfecteerde cellen noodzakelijk zijn voor de herkenning en het oversteken van de barrière. In studie IV waren geïnfecteerde macrofagen meer bewegelijk dan niet-geïnfecteerde en konden zij opeens op het signaal van de lymfoïde organen reageren. Dit is anders dan het gebruikelijke gedrag van macrofagen: normaal gesproken zitten zij stil en ontvangen het signaal van de lymfoïde organen niet. Daarentegen zijn zij er voor het opruimen van micro-organismen. Het veranderde gedrag lijkt op het gedrag van dendritische cellen en geïnfecteerde macrofagen kunnen zo migreren naar lymfoïde organen en de verspreiding van de parasiet door het lichaam bevorderen.
Samengevat beschrijft dit proefschrift nieuwe aspecten van het hypermigratie- fenotype in geïnfecteerde mononucleaire fagocyten en geeft daarbij inzicht in de componenten en onderliggende mechanismen in T. gondii en de geïnfecteerde cellen op een moleculair niveau.
VI
List of scientific papers The following papers are included in this thesis:
I. Ten Hoeve AL, Hakimi MA, Barragan A. Sustained Egr-1 Response via
p38 MAP Kinase Signaling Modulates Early Immune Responses of Den-
dritic Cells Parasitized by Toxoplasma gondii. Front Cell Infect Micro-
biol. 2019 Oct 11;9:349.
II. Ólafsson EB, Ten Hoeve AL, Li Wang X, Westermark L, Varas-Godoy
M, Barragan A. Convergent Met and voltage-gated Ca2+ channel signal-
ing drives hypermigration of Toxoplasma-infected dendritic cells. J Cell
Sci. 2020 Apr 21;134(5):jcs24175.
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. Ten Hoeve AL, Olivera GC, Saeij JPJ, Hakimi MA, Barragan A. The
Toxoplasma effector GRA28 promotes parasite dissemination by inducing
dendritic cell-like migratory properties in infected macrophages. Manu-
script
VII
Contents
List of scientific papers .....................................................................VI
List of abbreviations ........................................................................ VIII
Introduction ........................................................................................ 1 Toxoplasma gondii ................................................................................ 1 Biological barriers and cellular immunity ............................................... 2
Mononuclear phagocytes (MPs): origin and functions ..................... 2 Growth factors and transcription factors regulating MPs.................. 6
Host-parasite interactions and T. gondii dissemination ...................... 10 Parasite-derived proteins modulate immune responses ................ 11 The ‘Trojan horse’ hypothesis ......................................................... 12 From dissemination to chronic infection ......................................... 15
Aims of this thesis ............................................................................ 16 Main hypothesis ................................................................................... 16 Specific aims ....................................................................................... 16
Summary of papers ......................................................................... 17 Paper I ................................................................................................. 17 Paper II ................................................................................................ 17 Paper III ............................................................................................... 18 Paper IV ............................................................................................... 18
Discussion and conclusions ............................................................. 20 Macrophages and T. gondii: change of cell identity? .......................... 20 Activation of familiar pathways ............................................................ 23 An attempt to take control of immunity ................................................ 24
Acknowledgements ......................................................................... 25
References ...................................................................................... 27
BATF Basic leucine zipper ATF-like transcription factor
BMDC Bone marrow-derived dendritic cell
BMDM Bone marrow-derived macrophage
CAM Cell adhesion molecule
cMoP Common monocyte progenitor
CMP Common myeloid progenitor
EZH2 Enhancer of zeste homolog 2
FLT3 FMS-like tyrosine kinase 3
FLT3L FMS-like tyrosine kinase 3 ligand
GABA γ-aminobutyric acid
GRA Dense Granule protein
HGF Hepatocyte growth factor
ICAM Intercellular adhesion molecule
IX
MEK MAP/ERK kinase
pDC Plasmacytoid dendritic cell
PRR Pattern recognition receptor
STING Stimulator of interferon gene
TEEGR Toxoplasma E2F4-associated EZH2-inducing gene regulator
TLR Toll-like receptor
VGCC Voltage-gated calcium channel
1
Introduction
Toxoplasma gondii Toxoplasma gondii is an obligate intracellular Apicomplexan parasite that
occurs nearly worldwide [1]. It could possibly infect all warm-blooded ani-
mals as intermediate hosts, while felids, such as domestic cats, that predate
intermediate hosts, form its definitive host. In intermediate hosts, such as hu-
mans and rodents, the development of the parasite goes through two main
phases. During the first phase, the rapidly replicating tachyzoite form multi-
plies inside nucleated cells within membrane-limited vacuoles – the para-
sitophorous vacuoles (PVs) – through repeated endodyogeny. The pressure of
cell-mediated immunity initiates the second phase, in which slowly replicating
bradyzoites reside in tissue cysts. These infectious cysts are located primarily
in the central nervous system, as well as eyes and skeletal and cardiac muscu-
lature. It is thought that once infected, the hosts will remain infected…
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