Theroles of C5a Receptor 1 and Chemokine Receptor 5 In

Home , C5a receptor, Chemokine receptor, CXCR5

From the Institute of Systemic Inflammation Research of the University of Lübeck Director: Prof. Dr. med. Jörg Köhl

The roles of C5a receptor 1 and chemokine receptor 5 in Toxoplasma gondii infection

Dissertation for Fulfillment of the Requirements for the Doctoral Degree of the University of Lübeck

From the Department of Natural Sciences

Submitted by Daria Briukhovetska from Dnipropetrovsk

Lübeck 2017 First referee: Prof. Dr. med. Jörg Köhl

Second referee: Prof. Dr. Rainer Duden

Date of oral examination: 29.08.2017 Approved for printing: 29.08.2017 List of contents

Summary ...... VI

Zusammenfassung ...... VIII

1 INTRODUCTION ...... 1

1.1. Innate and adaptive immunity as two important parts of the immune system ...... 1

1.2. The complement system is a major player of humoral immunity ...... 2

1.2.1. Canonical activation of the complement cascade ...... 2

1.2.2. Non-canonical activation of the complement components ...... 4

1.2.3. Regulation of the complement system ...... 5

1.2.4. The anaphylatoxin C5a and its cognate receptors...... 6

1.2.4.1. The anaphylatoxin C5a ...... 7

1.2.4.2. Receptors for C5a ...... 7

1.2.4.3. Expression of C5aR1 ...... 8

1.2.5. Crosstalk between the C5a receptors and Toll-like receptors ...... 9

1.2.6. Dimerization of C5aR1 with other G-protein coupled receptors ...... 9

1.3. Dendritic cells as a key component of the pathogen recognition system...... 10

1.3.1. Dendritic cell subpopulations in humans and mice ...... 10

1.3.2. Chemokine receptors of dendritic cells ...... 13

1.3.3. Interleukin-12 family cytokines ...... 13

1.4. Biology and immune recognition of Toxoplasma gondii ...... 15

1.4.1. Recognition of the T. gondii parasite by the immune system ...... 16

1.4.2. Innate immune responses to Toxoplasma gondii infection ...... 18

1.4.3. Adaptive immune responses to Toxoplasma gondii infection ...... 21

1.4.3.1. B cell response ...... 21

1.4.3.2. T cell responses ...... 22

1.4.1. Latent T. gondii infection in the brain ...... 22

I List of contents

1.4.2. The intracellular niche of T. gondii that allows parasite survival and spreading in the host...... 23

1.4.3. Resolution of inflammation in response to T. gondii infection...... 24

1.4.4. Activation of the complement system during T. gondii infection ...... 25

1.5. Hypothesis and specific aims of the project ...... 26

2 MATERIAL ...... 27

2.1. Material ...... 27

2.1.1. Mouse strains ...... 27

2.1.2. Parasites and cell lines ...... 27

2.1.3. Chemicals and reagents ...... 27

2.1.4. Buffers, solutions and media ...... 29

2.1.5. Antibodies for flow cytometry ...... 32

2.1.6. Plastic ware and disposable items ...... 34

2.1.7. Commercially available kits ...... 35

2.2. Equipment and software ...... 35

2.2.1. Laboratory equipment ...... 35

2.2.2. Computer software...... 37

3 METHODS ...... 38

3.1. Protein expression in bacterial culture ...... 38

3.1.1. Heat-shock transformation of chemically competent bacteria ...... 38

3.1.2. Plasmid preparation ...... 39

3.1.3. Purification of GST-tagged protein from bacterial biomass ...... 39

3.1.4. Endotoxin removal from the recombinant protein preparation ...... 40

3.1.5. Determination of endotoxin content ...... 40

3.1.6. Analysis of protein homogeneity by SDS-PAGE ...... 40

3.1.7. Staining protein gels with Coomassie blue ...... 40

3.1.8. Staining protein gels with silver staining...... 40

3.1.9. Peptidyl-prolyl isomerase activity assay ...... 41

3.2. Immune cell isolation from mouse tissues ...... 41

II List of contents

3.2.1. Isolation of cells from mouse peritoneal cavity...... 42

3.2.2. Isolation of cells from spleen ...... 42

3.2.3. Isolation of cells from mesenteric lymph node ...... 42

3.2.4. Isolation of immune cells from mouse brain ...... 42

3.2.5. Determination of cell numbers ...... 43

3.2.6. Magnetic-activated cell sorting ...... 43

3.3. Receptor labelling and confocal microscopy...... 44

3.3.1. Generation of dendritic cells from bone marrow culture (BMDCs) ...... 44

3.3.2. Transfection of BMDC culture ...... 44

3.3.3. Laser scanning confocal microscopy ...... 45

3.4. Maintenance of T. gondii tachyzoites in vitro ...... 45

3.4.1. Vero cell culture ...... 45

3.4.2. Preparation of soluble toxoplasma antigen (STAg) ...... 46

3.5. T. gondii mouse infection model...... 46

3.5.1. Determination of brain cyst numbers in T. gondii-infected mice ...... 46

3.5.2. Intraperitoneal T. gondii infection ...... 47

3.6. Determination of serum cytokines in T. gondii-infected mice ...... 48

3.6.1. Determination of cytokine production by enzyme-linked immunosorbent assay (ELISA) ...... 48

3.6.2. Determination of serum cytokine concentrations using the Bio-Plex ProTM (BioRad) assay...... 49

3.6.3. Determination of serum cytokine concentrations using the Multiplex (MSD) assay ...... 49

3.6.4. Determination of serum Alanine amino transferase (ALT) activity ...... 50

3.7. Flow cytometry...... 50

3.7.1. Intracellular cytokine staining ...... 51

3.7.2. Tetramer staining ...... 51

3.8. Statistical analysis ...... 52

4 RESULTS...... 53

III List of contents

4.1. Impact of C5aR1 and CCR5 activation on the dendritic cell response to Toxoplasma gondii antigens in vitro ...... 53

4.1.1. Expression of recombinant Toxoplasma gondii cyclophilin-18 ...... 53

4.1.2. Interleukin-12 is secreted by dendritic cells upon challenge with Toxoplasma gondii antigens in vitro ...... 55

4.1.3. Impact of C5a, CCL5 stimulation or T. gondii tachyzoite infection on C5aR1 or CCR5 internalization in DCs ...... 59

4.2. The role of C5aR1 and CCR5 activation in mouse survival during intraperitoneal T. gondii infection ...... 63

4.2.1. Parasite load in the brain of chronically infected mice ...... 66

4.3. Impact of C5aR1 or CCR5 activation on cytokine response to acute T. gondii infection...... 67

4.3.1. Early cytokine response to soluble toxoplasma antigen in vivo ...... 67

4.3.2. Serum cytokine response following acute T. gondii infection ...... 68

4.4. Impact of the C5a/C5aR1 axis on the cellular response during acute T. gondii infection...... 71

4.4.1. Cellular responses in the spleen against T. gondii infection ...... 72

4.4.1.1. IL-12 secretion by spleen cDCs ...... 72

4.4.1.2. IFN-γ production by NK, NKT and T-cells ...... 73

4.4.1.3. Assessment of the mononuclear and polymorphonuclear phagocyte compartments ...... 75

4.4.2. Cellular response against T. gondii infection in the peritoneal cavity ...... 77

4.4.2.1. Change in peritoneal B-1 cell composition in response to acute T. gondii infection ...... 79

4.4.3. Analysis of the cellular responses in the mesenteric lymph nodes to T. gondii infection ...... 81

4.4.3.1. DC subsets in the MLNs of T. gondii-infected mice ...... 83

4.5. Impact of the C5a/C5aR1 axis on the cellular response to the acute and chronic T. gondii infection in the brain...... 85

4.6. T-cell immune response during chronic T. gondii infection ...... 87

4.6.1. Phenotype of T-cells in the brain of chronically infected mice ...... 87

IV List of contents

4.6.2. T. gondii-specific T-cells in spleen and brain at day 30 after infection ...... 89

5 DISCUSSION...... 92

5.1. The C5aR1/C5a axis controls TLR-dependent IL-12 production from DCs in response to T. gondii-derived stimuli in vitro...... 92

5.2. Cognate ligands but not T. gondii tachyzoites induce internalization of C5aR1 and CCR5 from the surface of BMDCs ...... 93

5.3. C5aR1 signaling is crucial for the survival of T. gondii-infected mice and the control of parasite load in the brain of chronically infected mice ...... 94

5.4. C5aR1/C5a axis activation modulates T H1 cytokine and chemokine production in response to T. gondii infection in vivo ...... 95

5.5. C5aR1/C5a axis in the cellular response to intraperitoneal T. gondii infection in mice ...... 97

5.6. C5aR1/C5a axis has an impact on the amount of the antigen-specific T-cells in the spleen the during chronic stage of T. gondii infection ...... 100

References ...... 101

APPENDIX...... 119

Abbreviations ...... 120

Figures ...... 123

Tables ...... 131

Congress contributions...... 132

Curriculum Vitae ...... 133

List of publications ...... 135

Acknowledgments ...... 136

Summary

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is spread globally in humans and animals. The infection is of medical importance in immunocompromised individuals such as patients with AIDS, and can cause severe vertically transmitted infection during pregnancy. In otherwise healthy adults, T. gondii is usually controlled by the immune system and remains in a dormant state by forming cysts within the brain or muscles.

In mice, the immune response against T. gondii is mainly induced through recognition of T. gondii-derived profilin by the Toll-like receptors (TLR) 11 and 12 expressed in splenic CD8α+ dendritic cells (DCs). It was also discovered that MyD88-independent mechanism of T. gondii sensing exists in CD8α+ DCs. More specifically, the G-protein coupled chemokine receptor CCR5 is activated upon binding of T. gondii-derived antigen cyclophilin-18 (TgCyp18). Further, CCR5 was shown to form heterodimers with the receptor for the complement cleavage product C5a, i.e. C5a receptor 1 (C5aR1), which plays important roles in infection with the parasite Trypanosoma cruzi and HIV. Previously, it was shown that T. gondii causes complement cascade activation and fixation of C3 cleavage fragments.

Based on these findings, I hypothesized that TgCyp18 binds to C5aR1/CCR5 heterodimers, which serve as a novel MyD88-independent sensing mechanism for T. gondii. I further hypothesized that such cross-linking of C5aR1/CCR5 heterodimers on DCs drives IL-12 as an important mechanism to induce protective TH1 immunity.

To test these hypotheses, I first purified recombinant TgCyp18 from E. coli and tested its ability to drive IL-12 production from spleen-derived DCs. In contrast to previous findings, TgCyp18 did not induce IL-12 production, whereas soluble T. gondii antigen (STAg) was a strong inductor of IL-12 by an MyD88-, C5aR1-, and CCR5-dependent mechanism. Infection of bone marrow-derived DCs (BMDC) with T. gondii tachyzoites did not result in C5aR1 or CCR5 internalization suggesting that the parasite does not use these receptors for entry.

Next, I assessed the role of C5aR1 and CCR5 activation in an experimental model of intraperitoneal T. gondii infection. This model allows to evaluate the complex interplay of immune cells during the induction of innate immune response. I found a significantly decreased survival of C5aR1- but not C5aR2-, CCR5- or C5aR1xCCR5-double-deficient mice 30 days after infection. Further, C5aR1-, C5aR2- and C5aR1xCCR5-double-deficient suffered from higher numbers of brain cysts. In search for mechanisms underlying the higher mortality of C5aR1-deficient mice, I observed significantly reduced systemic IL-12 or IFN-γ production 5 or 7 days after T. gondii

Summary infection in C5aR1-deficient mice when compared to WT animals. Also, STAg-induced serum IL- 12 and IFN-γ production was almost completely absent in mice lacking C5aR1 expression suggesting a crucial role of the C5a/C5aR1 in early IL-12-mediated IFN-γ production. The reduced potency of C5aR1-deficient mice to mount an appropriate early IL12/IFN-γ response was associated with increased cellular infiltration in the peritoneal cavity, spleen, brain and the mesenteric lymph nodes 5 days after infection.

My findings provide first detailed insights into the regulatory roles of the C5a/C5aR axes in experimental T. gondii infection. They clearly demonstrate that activation of the C5a/C5aR1 axis in DCs is important for IL-12/IFN-γ production during the initial phase of parasite infection. The decreased survival rate of C5aR1-deficient mice associated with the increased parasite burden in the brain 30 days after infection point towards a critical role of C5aR1-regulated early IL-12/IFN-g production to control T. gondii infection. Further studies are needed to explore the role of the C5a/C5aR1 axis regarding the cross-talk between DCs and NK cells during early T. gondii infection.

VII Summary Zusammenfassung

Toxoplasma gondii (T. gondii) ist ein obligat intrazellulärer einzelliger Parasit, der weltweit in Mensch und Tier vorkommt. Die Infektion ist von medizinscher Relevanz in immunkompromittierten Individuen wie z.B. Patienten, die an AIDS leiden und kann schwere Infektionen des Fetus verursachen bei Erstinfektion der Mutter in der Schwangerschaft. In gesunden Individuen wird die Infektion durch das Immunsystem kontrolliert und mündet in eine latente Infektion, bei der die Parasiten in Form von Gewebszysten im Gehirn oder der Muskulatur überdauern.

In der Maus wird die Immunantwort gegen T. gondii vor allem durch die Erkennung des von T. gondii-exprimierten Profilin durch die Toll-like-Rezeptoren (TLR) 11 und 12 auf CD8a+ dendritischen Zellen (DZ) der Milz induziert. Es wurde zudem gezeigt, dass DZ T. gondii auch durch MyD88-unabhängige Mechanismen erkennen. Spezifisch wurde gezeigt, dass der G- Protein-gekoppelte Chemokinrezeptor CCR5 durch Bindung des T. gondii Antigens Cyclophilin- 18 (TgCyp18) aktiviert wird. CCR5 kann Heterodimere bilden mit dem Rezeptor für das Komplementspaltprodukt C5a, dem C5a Rezeptor 1 (C5aR1), der eine wichtige Rolle spielt bei der Infektion mit dem Parasiten Trypanosoma cruzi und HIV spielt. Zuvor wurde zudem gezeigt, dass T. gondii die Komplementkaskade aktiviert und C3 Spaltprodukte bindet.

Basierend auf diesen Befunden stellte ich die Hypothese auf, dass TgCyp18 an das C5aR1/CCR5 Heterodimer bindet, welches einen neuen, MyD88-unabhängigen Erkennnungsmechanismus für T. gondii darstellt. Darüber hinaus postulierte ich, dass das Cross- Linking des C5aR1/CCR5 Heterodimers auf DZ zur Freisetzung von IL-12 führt als wichtiger, initialer Mechanismus für die Induktion der protektiven TH1 Immunantwort.

Um diese Hypothesen zu prüfen, reinigte ich zunächst rekombinantes TgCyp18 aus E. coli auf und testete seine Fähigkeit, IL-12 aus DZ der Milz freizusetzen. Im Gegensatz zu den Vorbefunden induzierte TyCyp18 keine IL-12 Produktion, wohingegen lösliches T. gondii Antigen (STAg) eine starke Induktion von IL-12 bewirkte, dessen Freisetzung abhängig war von der MyD88, C5aR1 und CCR5 Expression. Die Infektion von DZ, die aus dem Knochenmark generiert wurden mit Tachyzoiten von T. gondii führte allerdings nicht zur Internalisierung von C5aR1 oder CCR5, was nahelegt, dass der Parasit diese Rezeptoren nicht für das Eindringen in die Zelle verwendet.

Im nächsten Schritt untersuchte ich die Bedeutung der Aktivierung von C5aR1 und von CCR5 in dem experimentellen T. gondii Modell der intraperitonealen Infektion. Dieses Modell erlaubt die Untersuchung des komplexen Zusammenspiels von Immunzellen während der Aktivierung des angeborenen Immunsystems. Ich konnte ein signifikant vermindertes Überleben der C5aR1-, VIII Summary nicht jedoch der C5aR2, CCR5 oder C5aR1xCCR5-doppel-defizienten Mäuse 30 Tage nach der Infektion beobachten. Zudem waren die C5aR1-, C5aR2-, CCR5 und die C5aR1xCCR5-doppel- defizienten Mäuse durch eine höhere Anzahl von Gewebszysten im Gehirn beeinträchtigt. Auf der Suche nach Mechanismen, die der erhöhten Mortalität in den C5aR1-defizienten Mäusen zugrunde liegen, konnte ich eine signifikant reduzierte Produktion von Serum IL-12 oder IFN-γ in C5aR1-defizienten Mäusen im Vergleich zu WT Tieren an Tag 5 oder 7 nach T. gondii Infektion beobachten. Zudem, war die STAg-induzierte IL-12 und IFN-γ Produktion fast komplett abwesend in Mäusen, die keinen C5aR1 exprimierten, was auf eine wesentliche Rolle der C5a/C5aR1 Achse für die frühe IL-12-induzierte IFN-γ Produktion spricht. Die verminderte Potenz der C5aR1- defizienten Mäuse eine adäquate IL-12/IFN-γ Antwort zu induzieren war assoziiert mit einer verstärkten zellulären Infiltration der Bauchhöhle, der Milz, des Gehirns und der mesenterialen Lymphknoten 5 Tage nach der Infektion.

Zusammenfassend geben meine Befunde erste detaillierte Einblicke in die regulatorische Funktion der C5a/C5aR Achsen im experimentellen Modell der T. gondii Infektion. Sie zeigen, dass die Aktivierung der C5a/C5aR1 Achse in DZ wichtig ist für die IL-12/INF-γ Produktion währende der frühen Phase der T. gondii Infektion. Die verminderte Überlebensrate der C5aR1- defizienten Mäuse assoziiert mit der erhöhten Parasitenbeladung im Gehirn 30 Tage nach der Infektion spricht für eine wichtige Rolle der C5aR1-regulierten frühen IL-12/IFN-γ Produktion, die den späteren Verlauf der Infektion kontrolliert. Weiterführende Untersuchungen sind notwendig, um die die Rolle der C5a/C5aR1 Achse für den Cross-talk zwischen DZ und NK Zellen während der frühen Phase der T.gondii Infektion zu untersuchen.

1 INTRODUCTION

1.1. Innate and adaptive immunity as two important parts of the immune system

The immune system is composed of cells and molecules that act together to protect the body from a variety of infectious agents and eliminate damaged, transformed or senescent cells, thus maintaining the functional integrity and homeostasis of an organism. Pathogen invasion is counteracted by physiological barriers and action of the innate immune system. It elicits immediate defense against exogenous and endogenous threats. Infectious agents that evade innate immune responses are eliminated through specific activation of the adaptive immune system. In this case, defined molecules specifically recognize microbial and non-microbial substances resulting in the elimination of the threat [1].

The way, how innate immunity responds to pathogens, the product of injured cells and the nature of the response are similar, even in case of repetitive challenges. This system has evolved early in the evolution of multicellular organisms and comprises a panel of defined components that build the essential part of innate immunity. Those include anatomical and chemical barriers such as the skin and mucosal surfaces and antimicrobial agents. These initial defense mechanisms protect from infection, prevent exposure of internal tissue to microbes and keep them out of the body. When the integrity of these barriers is breached or pathogens evade the initial lines of defense, humoral and cellular components of innate immunity come into play. Phagocytic cells (granulocytes and macrophages), dendritic cells (DCs), mast cells, natural killer cells (NK-cells) and innate lymphoid cells (ILCs) as well as T and B lymphocytes with limited antigen receptor specificities act together to eliminate a wide range of the infectious agents. Together, they comprise the cellular part of innate immunity. The complement system is composed of a group of plasma proteins that act together and possess important effector activity and contribute to both innate and adaptive immune responses. Together with other mediators of inflammation and cytokines that regulate and coordinate the cellular responses, these soluble factors comprise the humoral components of innate immunity [2].

To detect and respond to different groups of pathogens, the innate immune system has several sensing systems that rely on recognizing common structural features associated with different classes of microorganisms. The multiple mechanisms for the induction of immune responses are based on common principles, where cells sense the infectious agents and produce

1 1 Introduction distinct set of cytokines/chemokines to induce appropriate immune responses and in turn activate effector mechanisms [3].

The immune response is a reaction of the immune system that is initiated by pathogen components and known as pathogen-associated molecular patterns (PAMPs). The immune system also recognizes endogenous molecules that are produced or released from damaged or dying cells of the host organism named damage-associated molecular patterns (DAMPs). The innate immune system includes a wide range of cellular receptors and soluble molecules able to recognize PAMPs and DAMPs. These receptors are expressed extra- and intracellularly by cells of the innate immune system and are named pattern-recognition receptors (PRRs). Detection of PAMPS and DAMPS by PRRs of the innate immune system triggers different classes of the effector responses [2].

1.2. The complement system is a major player of humoral immunity

The humoral innate immune response consists of multiple components, including naturally occurring antibodies, pentraxins and the complement cascade. As soluble plasma components, these innate proteins provide key elements in the prevention and control of diseases [4].

Proteins of the complement system appeared early in the evolution and provide protection from pathogens and facilitate their recognition by cells with phagocytic activity [5]. Starting with C3, the complement system has evolved and includes more than 30 different plasma proteins as well as soluble and membrane-bound receptors. Most of the circulatory proteins are enzymes that are synthetized in the liver and are present in the serum in an inactive form as so called proenzymes or zymogens. There are several pathways to activate the complement cascade, such as the presence of pathogens or immune complexes. It also acts as another link between innate and adaptive immunity [2].

Canonical activation of the complement cascade Activation of the complement system may occur through three different pathways: the classical, the lectin and the alternative pathway. Classical pathway activation occurs, when plasma protein C1 binds to antibodies or directly to components of the microbial surface as it was shown that this molecule is evolutionary older than the adaptive immune system [6]. C1 consists of the hexameric recognition protein C1q associated with the proenzymes C1r and C1s. The potency of antibodies to fix and activate complement varies between different subclasses in mice and humans [7]. IgM antibodies are most potent, as they have pentameric or hexameric

2 1 Introduction structures, which are physically more suitable to bind the C1q molecule. Activation of the lectin pathway is dependent on mannose binding lectin (MBL) and ficolins. These soluble recognition molecules form complexes with MBL-associated serine proteases MASP-1 and MASP-2 and can bind to carbohydrate residues on microbial surfaces. Activated C1s of the classical pathway or MASP-2 of the lectin pathway cleaves C4 into 2 fragments – C4a and C4b. The latter binds to the pathogen surface and acquires proteolytic activity to release C2b from C2 and form C4bC2a complex known as the classical C3 convertase of both the lectin and the classical pathways.

Activation of the alternative pathway is dependent on the property of C3 in plasma to undergo spontaneous hydrolysis and constantly generates low levels of C3a and C3b that are quickly degraded. When C3b covalently binds to microbial surface, it activates plasma protein factor D to cleave factor B into Ba and Bb and forms the complex C3bBb known as the alternative pathway C3 convertase [2]. Regardless of the pathway of complement activation, both C3 convertases cleave its substrate C3 into C3a and C3b and bind the larger fragment C3b to form the C5 convertase (C4bC2aC3b or C3b2Bb), which cleaves C5 into C5a and C5b. Both C3b and C5b fragments are active serine proteases. When they are attached to pathogens, they exert several important functions: C3b acts as an important opsonin and enhances the phagocytosis of pathogens, while C5b initiates the assembly of the terminal complement proteins (C6-C9) and formation of membrane attack complex (MAC). The MAC induces pore formation in the cell membrane of the pathogen and subsequently leads to its elimination. The C3a and C5a complement peptides, released from C3 and C5 during the activation of the complement cascade, are important mediators of inflammation and together are referred to as anaphylatoxins [2] (Figure 1).

3 1 Introduction

Figure 1: Activation of the complement system. The complement system is activated via one of the three pathways (classical, lectin or alternative) and leads to the generation of C3 and then C5 convertases. It results in the assembly of the membrane attack complex and generation of the anaphylatoxins C3a and C5a, which are potent inflammatory mediators. Adapted from [8]. Non-canonical activation of the complement components Hepatocytes serve as the primary source of most of complement components [9]. However, there is evidence that many cell types including immune cells can generate and release significant amounts of complement components constitutively or upon activation of cytokine receptors, TLRs or FcγRs [9]. At the same time, local complement release and activation can 4 1 Introduction modulate cytokine production [10, 11]. In the central nervous system (CNS), local production is the main source of the complement [12]. Further, activation of locally produced C3 or C5 components is mediated not only by conventional convertases [13]. First, it was described that in addition to conventional convertases, trypsin causes proteolysis of C5 resulting in the generation of C5a [14]. Since then, multiple extrinsic mechanisms of anaphylatoxin generation were discovered and reported. Some of these mechanisms serve as a cross-talk between the coagulation and complement cascades and are mediated by thrombin, plasmin, factors Xa/XIa (reviewed in [15]), factor VII-activating protease [16] or kallikrein-related peptidase 14 [17]. Complement activation can occur also intracellularly. It was shown that lysosomal endopeptidase cathepsin L cleaves C3 and is important for T-cell homeostasis [18]. Intracellular C5 cleavage in

T-cells promotes inflammasome assembly and TH1 immune responses [19]. Interestingly, release of the lysosomal endopeptidases such as cathepsin D from apoptotic cells under trauma conditions causes generation of C5a [20]. Also, immune cells were found to secrete peptidases like β-tryptase of mast cells [21] or granzyme B [22] with the same activity. Phagocytic cells, such as neutrophils and macrophages can generate C5a from C5 using serine proteases [23]. There is also an evidence that local generation of C5a by cancer cell can contribute the disease development [24]. On the other hand, not only host enzymes are known to cause extrinsic generation of anaphylatoxins. Degradation of the complement components through release of specific proteases is one of the strategies known for pathogens to evade complement-mediated killing. This can also lead to release of biologically active C3a and C5a peptides that can contribute pathogen virulence [25]. Additionally, house dust mite feces contain proteases that cause release of C3a and C5a and may contribute to the pathogenesis of allergic diseases caused by this allergen [26].

Taken together, non-canonical activation of the complement components acts as a mechanism of local generation of biologically relevant amounts of the complement peptides. This mechanism is not only used by cells of the host under various conditions, but also by pathogens to exploit and evade the defense mechanisms of the immune system [28].

Regulation of the complement system Upon activation of the complement cascade, different effector peptides are generated that bind to the pathogen surface or are released into the liquid phase, where they exert their biological activity. In addition, complement components are activated at a low rate under steady state condition. In both cases, regulatory mechanisms are necessary to balance activation and resolution of the system to protect the host from detrimental and excessive activation of effector mechanisms. The complement cascade activation follows a specific sequence that can be subdivided into following steps: initiation, formation of C3 convertase and amplification, formation 5 1 Introduction of C5 convertase and assembly of the MAC. Each part of the complement system is controlled by regulators and inhibitors. An overview of these regulators is provided in Table 1. Dysregulation or imbalance of the complement system can lead to pathological states and diseases [27, 28].

Regulator Ligand Function

Stabilizes alternative pathway Properdin C3 convertase Factor H C3b and C3d Cofactor for factor I, displaces Bb

Factor I C3b, C4b Cleaves C3b and C4b Blocks serine proteases C1r/s and C1 inhibitor (C1INH) C1r, C1s, MASP2 MASP2

С4-binding protein (C4BP) C4b Cofactor for factor I, displaces C2a

Inactivation or modulation of Carboxypeptidase N (CPN1) C3a, C4a, C5a anaphylatoxin function

S-protein (Vitronectin) C5b-7 Inhibits MAC formation CRIg (Complement receptor C3b, iC3b and Inhibits activation of alternative pathway of the immunoglobulin family) C3c

Cofactor for factor I, regulation of C3 CR1 (Complement receptor C3b, iC3b, C4b convertases decay, clearance of immune 1, CD35) and C1q complexes, enhancement of phagocytosis DAF (Decay-acceleration C3 convertase Acceleration of C3 convertases decay factor, CD55)

MCP (Membrane cofactor C3 degradation, cofactor for factor I and C3b, C4b protein, CD46) factor H Protectin (CD59) C8 Inhibits MAC formation

The anaphylatoxin C5a and its cognate receptors Activation of the complement cascade results in generation of the anaphylatoxins C3a and C5a. These peptides play critical roles as proinflammatory mediators. Further. they are important immunoregulators [30].

6 1 Introduction

1.2.4.1. The anaphylatoxin C5a C5a is a small polypeptide that consists of 74-79 amino acids depending on the species [8]. Upon release, C5a is quickly modified by serum carboxypeptidases that remove the C- terminal arginine residue turning C5a into C5a des-Arg [31-35]. Conventionally, this modification is considered as a mechanism for deactivation of C5a as the desarginated state shows reduced potencies to activate cells in several in vitro assays [36]. However, recent evidence suggests that C5a des-Arg can induce strong activation of cell lines and primary cells through C5aR1 [37]. C5a and its desarginated form can be cleared from the fluid phase via receptor-mediated endocytosis [38].

C5a is a strong pro-inflammatory mediator and has a number of biological effects that are important for host defense and pathogen clearance (reviewed in [39]) including chemotaxis of inflammatory cells, increased vascular permeability [40], release of cytokines and chemokines and phagocytosis [41]. Initially C5a was described to cause anaphylaxis as it causes contraction [42] and Ca2+ influx in smooth muscle cells [43]. It is also capable of stimulating the secretion of histamine from mast cells [44] and basophils [45]. Now it is recognized as a molecule with pleiotropic functions that modulates the activity of many cell types, with a broad range of biological functions both inside and outside of the immune system. Upon release, C5a serves as a powerful chemoattractant for neutrophils [46], macrophages [47] including microglia [48], monocytes [49], eosinophils [50], mast cells [51], basophils [52], activated B [53] and T cells [54]. Additionally, it can trigger oxidative burst in phagocytic cells [55, 56]. In addition, C5a can modulate cytokine production and release from different cells [57-59], change the expression of adhesion molecules [60-62], blood-brain barrier permeability [63, 64] and regulates the formation of complex glycosphingolipids [65].

1.2.4.2. Receptors for C5a C5a binds to two seven-transmembrane domain receptors namely C5aR1 (C5aR, CD88) [66] and C5aR2 (C5L2, gpr77) [67, 68], expressed on a wide variety of cells but particularly on the surface of immune cells. Most abundant expression was shown for cells of myeloid origin, such as neutrophils, eosinophils and basophils, monocytes/macrophages, mast cells and DCs [69-72] (reviewed in [73]). Expression of C5aR1 in mouse cells is well studied using GFP-reporter animals [74, 75]. Expression of C5aR1 on cells of lymphoid origin in mice and humans remain a matter of controversy, as it was found in some studies [19, 76, 77] but not confirmed using GFP- reporter mouse models [74, 75].

C5aR1 belongs to the superfamily of G-protein coupled receptors (GPCR). Signal transduction depends on activation of heterotrimeric G-proteins and [78]. In most experimental

7 1 Introduction models, the affinity of C5a towards C5aR1 is 10- to 100-fold higher than that of C5a-desArg [79]. Upon ligand binding, signaling is mainly induced through coupling to the pertussis toxin sensitive protein Gαi2 [80] or the pertussis toxin insensitive Gα16 [81]. Interestingly, the chemotactic property of C5aR1 is dependent on Gαi2 signaling that also occurs in chemokine receptor CCR2 [82]. C5a binding to C5aR1 causes Ca2+ influx from intracellular stores and the extracellular medium. After activation, β-arrestins 1 and 2 bind to C5aR1 and mediate receptor internalization [83]. Binding of arrestins depends on phosphorylation of the C-terminus of the receptor by G-protein coupled receptor kinases (GRKs). These kinases also interact with other compo nents of intracellular signaling such as mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase gamma (PI3Kγ)/Akt (reviewed in [84]).

C5aR2 is a second receptor for C5a. It shares 58% sequence identity with C5aR1 and has a similar structure [85]. Similar to C5aR1, it is expressed in various immune cells [86-88]. C5aR2 binds C5a des-Arg with a higher affinity than C5aR1 [89]. However, due to replacement of arginine residue in so-called DRY sequence in the third transmembrane domain, it is unable to couple to G-proteins and induce Ca2+ mobilization. Initially, C5aR2 was described as a decoy receptor that is important for C5a recycling and has anti-inflammatory properties [90]. However, C5aR2 was also shown to induce β-arrestin-mediated signaling [91-93] and to have pro- inflammatory properties through modulation of C5aR1-induced ERK phosphorylation [94].

1.2.4.3. Expression of C5aR1 C5aR1 expression in mice was studied in different cell types using monoclonal antibodies and GFP-reporter transgenic animals [74, 75, 95]. Extra- and intracellular expression was reported differently for C5aR1 and C5aR2 depending on the cell type and species [19, 68, 96]. In mice, C5aR1 is predominantly expressed in cells of myeloid origin such as granulocytes, monocytes, macrophages and DCs. Mouse neutrophils were first described to highly express C5aR1 and exert multiple biological responses when stimulated by C5a [70, 97]. C5aR1 expression was shown in mouse lung eosinophils and human basophils [75, 98]. Mouse macrophages from the lung, airways, lamina propria of the small intestine, peritoneal cavity and spleen red pulp express C5aR1 [75]. In the central nervous system, receptor expression was found on microglia but not on neurons [99]. Controversial reports exist concerning C5aR1 expression on murine DCs. One study reported that CD11b+, CD8α+ cDCs and CD64+ moDC but not CD103+ cDCs express C5aR1 [75], whereas other studies found no expression of C5aR1 on CD11c+ cells [74, 95]. Most human DC subsets were reported to express this receptor [100]. No receptor expression was found on NK, T cells and B2 cells in mice under steady state conditions [74, 75, 95]. Only a small fraction of spleen NKT cells expressed C5aR1 [75]. However, NK cells may upregulate C5aR1 in E. coli-induced sepsis [101]. Steady state expression was reported in 8 1 Introduction human NK and T cells [54, 102]. Although C5aR1 expression was not detected on human tonsillar B cells using a monoclonal antibody, such cells can migrate towards C5a suggesting that they express C5aR1 at low numbers [53, 98]. Less is known about expression of C5aR2. It was demonstrated on neutrophils, immature DCs, macrophages, lymphocytes, and monocytes. C5aR2 was shown to be localized mostly intracellularly in monocytic cells [68].

Crosstalk between the C5a receptors and Toll-like receptors C5a induces signaling through inflammatory pathways via its receptors C5aR1 and C5aR2. The involvement of signaling molecules such as NF-kB, MAPK, and c-Jun N-terminal kinase (JNK) in their downstream pathways suggests a potential crosstalk with other intracellular signaling pathways, such as the TLRs (reviewed in [103, 104]). Indeed, complement influences TLR-induced inflammation [105]. However, the impact of the anaphylatoxins on TLR-induced responses is cell type-dependent [104]. In studies with macrophages and monocyte-derived DCs, it was reported that C5a negatively regulates TLR-induced cytokine production and T H1 and TH17 inflammatory responses [59, 106, 107], whereas opposite effects were reported on monocytes and DCs [108, 109]. Also, the absence of C5aR1 on splenic DCs was associated with induction of Tregs in response to OVA [110]. Potential intersection points of TLR and C5aR1 signaling pathways are activation of cAMP and ERK1/2 phosphorylation, both of which differentially modulate cell responses (reviewed in [104, 111]). Cross-talk between C5aR2 and TLRs has also been demonstrated. C5aR2 was shown to inhibit C5aR1-induced signaling through ERK1/2 phosphorylation in macrophages [96, 112], whereas TLR activation was shown to reduce C5aR2 activity, thereby decreasing the negative modulation of C5aR1 and enhancing pro-inflammatory responses [113].

Dimerization of C5aR1 with other G-protein coupled receptors C5aR1 can form homo- and heterodimers with C5aR2 and other GPCRs [8]. It is well appreciated that GPCRs exist as homodimers and heterodimers and that their oligomeric assembly has important functional roles [114]. C5aR1 homodimerization was shown to appear early in receptor biosynthesis [115, 116], but can also be induced by C5a and is important for receptor internalization [117].

C5aR1 was shown to associate with C5aR2, which can shift downstream signaling from G protein-dependent to β-arrestin-mediated events supporting the idea of a regulatory role of C5aR2 [96, 112]. Modulation of signaling downstream of C5aR1 has been shown in response to C5aR2 stimulation [118-121].

9 1 Introduction

C5aR1 is known to form heterodimers with chemokine receptor CCR5 followed by β- arrestin-mediated internalization [122]. Recent studies have revealed a role of C5aR1 in CCR5- dependent HIV entry into the cell and induction of cell signaling [123]. A similar effect was observed for C5aR1 and bradykinin receptor 2B that regulates innate and adaptive responses to the intracellular parasite Trypanosoma cruzi [124].

1.3. Dendritic cells as a key component of the pathogen recognition system

Cells of the innate immune system recognize PAMPs and DAMPs and produce antimicrobial molecules and cytokines. They are specialized in killing pathogens and infected cells and inducing adaptive immune responses. This group includes several sell types of myeloid and lymphoid origin such as phagocytes, DCs, NK-cells, mast cells, T and B lymphocytes with limited antigen receptor specificities and innate lymphoid cells [1].

DCs are a widely distributed, migratory group of bone marrow-derived leukocytes of myeloid origin. In 2011, Ralph Steinman was awarded the Nobel Prize in Physiology or Medicine for the discovery of DCs and the important role they have in initiating the adaptive immune response [125]. DCs are sentinel cells that bridge the innate and adaptive immune systems. They are specialized in uptake, transport, processing and presentation of antigens to T cells. Immature DCs reside in peripheral tissues continuously sampling the environment for the presence of antigens. Encounter with microbial products or tissue damage initiates the migration of DCs to lymph nodes (LNs). The sampled antigen is then processed into peptides and transported to the DC surface. There they are presented as a complex with major histocompatibility complex (MHC) molecules. At the same time, DCs also upregulate co-stimulatory molecules that are required for effective interaction with T cells and secrete different classes of cytokines. In the LNs, they efficiently trigger an immune response by T cells that have receptors specific for the peptide-MHC complexes on the DC surface [126].

Dendritic cell subpopulations in humans and mice DCs differ in the regulatory signals they transmit to T cells. The nature of these signals defines different types of effector responses or tolerance. The different DC subtypes arise from distinct developmental pathways. Their development and function are modulated by exogenous factors [126].

There are several markers that are commonly used to define DC subsets, i.e. CD11c,

MHCII, CD11b, CD103, CD8α, CD64, CD172a, XCR1 and CX3CR1 [127]. Several studies have 10 1 Introduction used different marker combinations [128-133], which resulted in heterogeneity of flow cytometry- based identification strategies for the distinction of the different DC subsets. The phenotypes of the main murine and human DC subsets that are present in tissues or lymphoid organs, are shown in Table 2.

Table 2: Major subsets of DCs in humans and mice. BATF3, basic leucine zipper transcription factor ATF-like 3; BDCA, blood dendritic cell antigen; BST2, bone marrow stromal antigen 2; CLEC9A, C-type lectin domain family 9 member A; CX3CR1, CX3C-chemokine receptor 1; ESAM, endothelial cell-selective adhesion molecule; IRF4, interferon-regulatory factor 4; ND, not described; SIGLEC, sialic acid-binding immunoglobulin-like lectin; SIRPα, signal- regulatory protein-α; TCF4, transcription factor 4; XCR1, XC-chemokine receptor 1. Modified from [134].

Phenotype Specific Specific antigen DC subsets transcription Location presentation Mice Humans factors capacities CD11cint, CD11c–, Present and MHC class MHC class cross-present Lymphoid Plasmacytoid TCF4 (also known IIint, B220, IIint, CD123, peptides only organs DC as E2-2) BST2 and BDCA2 and after SIGLEC‑H BDCA4 activation Cross- CD11chi, CD11cint, present MHC class IIhi, MHC class peptides on Lymphoid CD8α+ cDC XCR1, IIhi, XCR1, BATF3 MHC class I organs CLEC9A and CLEC9A and molecules to CD8α BDCA3 CD8+ T cells Cross- CD11chi, CD11chi, present MHC class MHC class IIhi, peptides on Peripheral CD103+ cDC IIhi, XCR1, BATF3 CD103, XCR1 MHC class I tissues CLEC9A and and CLEC9A molecules to BDCA3 CD8+ T cells CD11chi, Present CD11chi, Lymphoid MHC class peptides on MHC class IIhi, organs, CD11b+ cDC IIhi, CD11b, IRF4 and Notch 2 MHC class II CD11b and peripheral SIRPα and molecules to CD24 tissues BDCA1 CD4+ T cells CD11chi, MHC Present Inflamed Monocyte- class IIhi, CD11chi, peptides on lymph derived DC CD11b, CD64, MHC class IIhi ND MHC class II nodes and CX3CR1 and and CD11b molecules to tissues CD209 CD4+ T cells

Initiation of the immune response by these cells starts upon recognition of DAMPs by pattern recognition receptors (PRRs). DCs are armed with several types of intra- and extracellular PRRs that enable recognition of pathogens through PAMPs or DAMPs. DCs comprise 3 major subsets: conventional (cDC), monocyte-derived (moDC) and plasmacytoid (pDC) cells.

11 1 Introduction

Each subset can be differentiated phenotypically. It exerts its specific function with specific sets of receptors (Table 3). Upon activation, pDCs rapidly produces type 1 interferon (IFN) through nucleic acid-sensing TLRs, such as TLR 7 and TLR 9. Mouse cDCs can be categorized into two distinct subsets: CD11b+ and CD8α+ (similar to CD103+ cDCs in tissues). The CD8α+ DC subset is highly efficient at mediating antigen cross-presentation to cytotoxic T-cells (reviewed in [135]), whereas CD11b+ DCs preferentially present MHC class II‑restricted antigens to CD4+ T cells. It was proposed to subdivide cDCs into two main linages based on their ontogeny: cDC1, that include CD8α+ and CD103+ DCs, and cDC2, that include CD11b+ and CD172a+ DCs [136].

Table 3: Expression patterns of innate immune receptors in mouse and human DC subsets. +, expressed; −, low or no expression; ?, expression level is unknown; BDCA, blood DC antigen; CLEC, C-type lectin domain family member; NLRP3, NOD-, LRR- and pyrin domain-containing 3; NOD1, nucleotide-binding oligomerization domain- containing protein 1; TLR, Toll-like receptor; XCR1, XC-chemokine receptor 1. Modified from [137].

Mouse DC subsets Human DC subsets Receptor CD8α+ BDCA1+ or CD11b+ pDC BDCA1+ pDC or XCR1+ XCR1+

TLR3 - + - - + -

TLR4 -/+ - - + - -

TLR7 - - + + - +

TLR9 + + + - - +

NLRP3 + - - + ? ?

NOD1 + - - + ? ?

The family of TLRs is the best-characterized PRR. They sense invading pathogens outside the cell and intracellularly in endosomes and lysosomes. There are 10 TLRs that have been identified in humans and 13 in mice with similar TLRs 1-9 in both species.

TLR1, TLR2, TLR4, TLR5 and TLR6 are located on the cell membrane, whereas TLR3, TLR7, TLR8, and TLR9 are found intracellularly. The different mouse and human DC subsets express distinct sets of TLRs and other PRRs (Table 3). TLRs have a dimeric structure and require specific adaptor molecules to facilitate signaling: TIRAP (TIR domain-containing adaptor protein), MyD88 (Myeloid differentiation primary response gene 88) and/or TRIF (TIR-domain- containing adaptor inducing IFN-β) and TRAM (TRIF-related adaptor molecule). The initiated signaling cascades result in activation of nuclear factor kappa b (NF-κB), mitogen-activated protein kinase (MAPK) and interferon regulatory factors 1, 3, 5 and 7 (IRF-3, -5 and -7). In turn, activation of the transcription factors drives expression of cytokines, interferons and chemokines, influences cell maturation and survival [138]. 12 1 Introduction

Chemokine receptors of dendritic cells DCs sense the microenvironment, which determines the profile of their cytokine secretion and the general program of their gene expression. DCs need to receive several migration signals to migrate to peripheral and lymphoid tissues, where they present processed antigens to T cells. Such migratory signals are provided by chemokines that form a large group of polypeptides with defined structural properties. They mediate their functions through activation of defined chemokine receptors, which also belong to the large group of GPCRs (Table 4) [139].

Table 4: Chemokine receptors and their ligands that are involved in dendritic cell migration. BCA, B cell attracting chemokine 1; BLC, B lymphocyte chemoattractant; ELC, EBV induced molecule 1 ligand chemokine; IP, IFN- γ-induced protein-10; LARC, liver and activation-regulated chemokine; MCP, monocyte chemoattractant protein; MIP, macrophage inhibitory protein; SLC, secondary lymphoid-tissue chemokine; SDF, stromal cell derived factor. Adapted from [139].

Receptor Chemokine ligand

MIP-1α ССR1 MIP-1β CCL5/Rantes CCR2 CCL2/MCP-1 MIP-1α CCR5 MIP-1β Rantes CCR6 CCL20/MIP-3α/LARC

CCL19/ELC/MIP-3β CCR7 CCL21/SLC/6Ckine

CXCR3 CXCL10/IP-10 CXCR4 CXCL12/SDF-1α

CXCR5 CXCL13/BLC/BCA-1 Interleukin-12 family cytokines An important feature of cDCs is to produce IL-12 family cytokines, which include IL-12, IL- 23, IL-27, and IL-35 (reviewed in [140]). IL-12 family cytokines form heterodimers that consist of an α-chain (p19, p28 or p35) and a β-chain (p40 or Ebi3) (Figure 2). Pairing of p40 chain with p35 or p19 forms IL-12 and IL-23, respectively, whereas heterodimers of Ebi3 with p28 or p35 result in IL-27 and IL-35. IL-12 family cytokine receptors have heterodimeric structures sharing receptor subunits such as IL-12Rβ1 or gp130 (Figure 2). Signaling of these receptors is mediated by the members of Jak-STAT family involving STAT 1, 3 and 4 (Figure 2).

IL-12 and IL-23 are mainly pro-inflammatory and pro-stimulatory cytokines with key roles in the development of the TH1 and TH17 subsets of helper T cells, respectively. IL-27 is reported to have mainly inhibitory activity and often generated during the resolution phase of an immune

13 1 Introduction response by APCs. IL-35 is a potent inhibitory cytokine expressed mainly by Treg cells. It controls the development of TH1 and TH17 cells and inhibits TH1 cell proliferation (reviewed in [141]).

Figure 2: IL-12 family cytokines, their receptors and signaling components. The IL-12 family comprises the heterodimeric cytokines IL-12, IL-23, IL-27 and IL-35 that consist of an α-chain (p19, p28 or p35) and a β-chain (p40 or Ebi3). These cytokines exert their functions upon binding to heterodimeric receptors and involve distinct JAK-STAT signaling partners. The bottom bar reflects their functional spectrum ranging from most pro-inflammatory (IL-23) to most inhibitory (IL-35). From [141].

14 1 Introduction 1.4. Biology and immune recognition of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that infects virtually any nucleated cell and is present in a wide range of hosts. It has a sexual cycle in members of the felidae family and a two-stage asexual cycle in secondary hosts including humans (Figure 3).

Figure 3: Life cycle of Toxoplasma gondii. Intermediate hosts such as humans, pigs, sheep, cattle and chickens become infected through ingestion of oocytes shed by members of the feline family (definitive host). Humans, as well as felines can also become infected after ingestion of poorly cooked meat containing tissue cysts. From [142]. In addition to its widespread distribution in warm-blooded animals serving as secondary hosts [143], it occurs in virtually all classes of animals. It comprises three clonal lineages (I, II and III). In the acute phase of infection, tachyzoites multiply in cells and causes different degrees of tissue destruction. With the onset of the immune response, tachyzoites are transformed into bradyzoites that multiply slowly in cells to produce tissue cysts [144]. Members of the cat family (Felidae) are the only known definitive hosts for the sexual stages of T. gondii and a reservoir for infection. Cats shed oocysts that are resistant to disinfectants, freezing and drying and can survive in the environment for several months [145].

Even though T. gondii infection is relatively common in the human population, i.e. approximately 30% of the global population has been exposed depending on age and environment, clinical manifestation of the disease is relatively rare. Those particularly at risk of developing clinical illness include pregnant women, as the parasite can be a serious threat to the unborn child, if the mother is infected for the first time during pregnancy, and to individuals, who are immunosuppressed such as tissue transplant and AIDS patients as well as those undergoing certain forms of cancer therapy. These individuals are at risk of developing acute lethal infection

15 1 Introduction is untreated. In a few cases, people without immunodeficiency may develop symptoms such as general malaise, fever and lymphadenopathy. However, toxoplasmosis is considered as a leading cause of death attributed to foodborne illness in the United States and one of the Neglected Parasitic Infections, a group of five parasitic diseases that have been targeted by Centers for Disease Control and Prevention (USA) for public health action [143]. The most likely sources of human infection are consumption of raw or undercooked meat containing live T. gondii tissue cysts, raw or undercooked vegetables contaminated with oocysts or exposure to oocysts from cat feces, which may be encountered in gardens and children’s sand pits [146].

Recognition of the T. gondii parasite by the immune system T. gondii infection causes rapid recruitment of monocytes, neutrophils and DCs to the site of infection and these cell types have been implicated in resistance to this parasite [147-151]. One of the most critical functions of the innate immune system is the ability of DCs to sense the pathogen, produce the cytokine IL-12 and promote adaptive TH1 immune responses [152]. Numerous studies have aimed to define cell types that contribute to the production of IL-12 in vivo and have identified neutrophils, inflammatory monocytes and macrophages as alternative sources (reviewed in [153, 154]).

The production of IL-12 during toxoplasmosis requires sensing of the parasite by the host. TLRs appear to have a key role in this process. In fact, mice deficient in the adapter molecule MyD88, which is required for downstream signaling of most TLRs, are highly susceptible to toxoplasmosis [155]. Specific TLRs implicated in the immune response to T. gondii include TLRs 2, 4, 9, 7, 11 and 12 [156]. TLR11 and TLR12 respond to a profilin-like molecule conserved among protozoan parasites and have a central role in innate immune recognition of toxoplasma in rodents [157, 158]. These two receptors were shown to form functional heterodimers in DCs and TLR12 can also form homodimers, which appear to be important in the process of profilin recognition [156, 159, 160]. TLR7 and TLR9 sense nucleic acids and have endosomal localization in the host cell [161, 162]. TLR2 functions as a general innate immune receptor for the glycosylphosphatidylinositol (GPI) anchors that cover the cell surface of multiple protozoan parasites, including T. gondii. In addition to TLR2, TLR4 can also be activated by T. gondii GPI in vitro [161]. Both TLR2 and TLR4 can also be activated by T. gondii-derived heat shock protein 70 (HSP70) [163]. Following oral infection with T. gondii, bacterial antigens from the gut can trigger TLRs 2, 4, and 9 responding to the microbial stimuli [164-167]. Quadruple knockout mice deficient in TLRs 3, 7, 9 and 11 show complete loss in resistance to toxoplasma infection [156]. These receptors require common the chaperone molecule UNC93B1 that is required for translocation from endoplasmic reticulum to the endocytic system [168, 169].

16 1 Introduction

Unlike rodents, human cells lack functional TLR11 and TLR12 genes. Although a homolog of the TLR11 gene is found on human chromosome 1, it contains three stop codons and does not encode a functional protein. Further, the TRL12 gene is completely absent in the human genome [170, 171]. Therefore, mechanism of toxoplasma-derived profilin recognition by the innate immune system as shown in rodents does not apply to humans (reviewed in [172]). However, studies performed in humans support data obtained in rodents that TH1/TH17-based immune responses and T-cell-derived IFN-γ are critical in parasite control [173]. Despite that, a detailed model of T. gondii recognition by the human innate immune system is still missing. In one study, phylogenetic analysis has shown that human TLR5 is an evolutionary close member of the TLR family of mouse tlr11. Human peripheral CD14+ blood monocytes express TLR5 and secrete pro- inflammatory cytokines in response to profilin challenge. Importantly, monocytes from donors with TLR5 gene polymorphism R392X that lack functional TLR5 failed to produce cytokines in vitro in response to antigen challenge. However, this study did not investigate the impact of this polymorphism on the pathogenesis and epidemiology of the disease [174]. In another study, a major role for CD16+ peripheral blood monocytes and the CD1c+ subset of DCs in cytokine production in humans upon T. gondii challenge has been described [175]. Soluble tachyzoite extracts failed to induce IL-12 production arguing against a role of profilin for the induction of T. gondii-specific immune responses. These authors proposed that phagocytosis of live tachyzoites rather than host cell invasion is required for cytokine production by human myeloid cells [175]. In addition, endosomal acidification and maturation upon phagocytosis of live parasites was shown to be important for cytokine secretion [175, 176]. Additionally, it was reported that inhibitors of endosomal TLRs 3,7 and 9 failed to suppress the cytokine respo nse of human monocytes to tachyzoites. Thus, immune recognition of this parasite in humans remains enigmatic [172].

Despite the critical importance of MyD88, protective immunity can be induced in MyD88- deficient mice and is not completely abolished in the absence of MyD88 [155, 167, 177]. T. gondii antigen 18-kDa cyclophilin, TgCyp18, which is released by extracellular tachyzoites, was shown to trigger IL-12 production from DCs through binding to chemokine receptor CCR5 independently of MyD88 [178-181]. This effect appears to occur by a pathway unique to T. gondii that involves triggering of CCR5 in DCs and macrophages. TgCyp18 is a peptidyl-prolyl cis-trans isomerase (PPIase) and it appears to induce IL-12 production by interacting directly with CCR5, an effect that is blocked by the addition of cyclosporine A (CsA). Moreover, recent studies demonstrated that treatment with TgCyp18 enhanced expression of CCR5 on macrophages and spleen cells. TgCyp18 was also shown to influence proliferation and migration of macrophages and induce nitric oxide production [182-184].

17 1 Introduction

Another way to detect pathogen ligands in the cytosolic compartment linked to T. gondii resistance is the inflammasome. Inflammasome activation in response to T. gondii infection is mediated via NOD-like receptor (NLR) family members NLRP1 and NLRP3 [185, 186]. Downstream activation of caspases 1 and 11 results in pyroptotic cell death and prevents intracellular parasite replication. Caspase 1 processes IL-1β and IL-18 into their active forms that are involved in host defense against the parasite [187, 188].

Innate immune responses to Toxoplasma gondii infection The effectiveness of T. gondii as a parasite is defined by its ability to regulate pro- and anti-inflammatory signaling in a way that maximizes parasite multiplication and spread, while maintaining host survival. Parasite invasion triggers IL-12-mediated IFN-γ production necessary

+ + for appropriate induction of adaptive TH1 responses by CD4 and CD8 T-cells which is critical for host survival (Figure 4) (reviewed in [189-192]).

Secretion of IL-12 by DCs is essential to trigger IFN-γ-dependent immune responses and host resistance against T. gondii. IL-12p70 consists of the two subunits IL-12p35 and IL-12p40. It has been shown that pharmacological blocking or genetic knockout of each subunit leads to high susceptibility to T. gondii infection [152, 193-195]. In mouse models, the CD8α+ DC subset was identified as a main source of TLR-driven IL-12 production [196]. However, other DC subsets, macrophages and neutrophils also contribute to the IL-12 pool upon priming with IFN-γ [149, 195, 197, 198].

Transcription factors basic leucine zipper transcriptional factor ATF-like 3 (BATF3) and IFN-regulatory factor 8 (IRF8) are necessary for the development of CD8α+ DCs [199]. Mice that are deficient in IRF8 [200] or BATF3 [201] lack the CD8α+ DC subset. These animals failed to trigger IL-12 production in response to T. gondii infection confirming the key role of this subset in sensing and initiation of the adaptive immune response. Induction of TLR signaling and IL-12 production in DCs is necessary to induce early IFN-γ secretion from NK cells that is crucial for control of the parasite replication. A conditional knockout model of MyD88 showed that absence of TLR signaling in DCs does not alter IFN-γ secretion from T-cells but from NK cells [202].

18 1 Introduction

Figure 4: Immune cell interplay during T. gondii infection. Parasite invasion triggers TLR-dependent IL-12 production from DCs that induces IFN-γ secretion from NK cells and neutrophils. Priming of cells with IFN-γ leads to the elimination of intracellular parasites. This is critical for host survival during the acute phase of infection and results + + in adaptive TH1-based CD4 and CD8 T-cell-mediated immune responses. From [188]. Neutrophils contain pre-stored IL-12 and secret it in response to T. gondii [149, 203-205]. Additionally, neutrophils can release IFN-γ independent of TLR-11 signaling [206]. The amount of IFN-γ secreted by each neutrophil is low compared to NK or T-cells, but they accumulate in the large numbers at the site of acute infection [204, 207, 208]. Neutrophils exert several effector functions to control parasite spreading including phagocytosis and the release of reactive chemical species [209, 210]. Accumulated neutrophils also contribute to parasite control by release of DNA-based extracellular traps (NETs) [211]. Neutropenia significantly reduces recruitment of inflammatory monocytes that are important for the development of resistance to T. gondii infection [151, 212]. Depletion of neutrophils leads to elevated parasite burden and increased susceptibility during the initial stage of infection [151, 213].

Monocytes are critical for infection control and survival during toxoplasmosis [151, 214]. CCR2+ monocytes have been shown to control T. gondii infection in the CNS and regulate mucosal immunity [150, 215-217]. They contribute to IL-12 and IL-1 family cytokine production during toxoplasmosis, which is necessary to induce proper IFN-γ responses [218-221]. An important monocyte effector function is the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO), which inhibits parasite replication [150, 215, 222, 223]. iNOS- deficient mice survive acute challenge with T. gondii, but cannot control persistent infection [224]. It has been shown that human monocytes produce high levels of ROS that kill intracellular parasites [225]. Monocytes develop into moDCs capable of inducing adaptive immune response or macrophages that control T. gondii infection through GTPase-mediated mechanisms [166, 226, 227]. 19 1 Introduction

Macrophages were one of first cell types described to control toxoplasmosis [228, 229]. Dead or opsonized parasites that are phagocytosed by macrophages are degraded in lysosomal compartments, whereas live parasites that invade host cell are protected in nonfusogenic parasitophorous vacuoles (PV) [176, 230]. Activation by IFN-γ drives anti-microbial effector mechanisms to limit intra-vacuolar parasite replication. In combination with TNF-α, it triggers upregulation of iNOS and stimulates production of NO, which is important for parasite control in the brain [224, 231-233]. Additionally, IFN-γ induces expression of 47-48 kDa immune GTPases (IGTP, LRG47, IRG47) that are essential for acute and chronic resistance to the parasite by limiting its replication [234-237]. However, there is also evidence for IFN-γ independent antimicrobial activity in macrophages involving TNF-α, and CD40/CD40L signaling [238, 239]. Activated macrophages contribute to IL-12 production [195] that is regulated by TRAF6- dependent phosphorylation of the MAPK family members p38 and ERK1/2 in response to T. gondii infection [240].

NK cells serve as an early source of IFN-γ critical for resistance to T. gondii infection [241- 244]. In mice lacking CD8+ T cells, the number of NK cells expands and augments IFN-γ production [198, 245]. DCs play critical roles in NK cell activation as an early source of IL-12 [198]. Inflammatory cytokines IFNα/β, IL-2, IL-15, IL-18 and TNF-α synergize with activating receptor signals or stimulate NK cell activation and survival (reviewed in [246]). DCs, do not only produce activating cytokines, but upregulate cell-surface expression of co-stimulatory and adhesion molecules that are important to stablish the immunological synapse, and, depending on the stimuli received, might also express ligands for NK cell activating receptors (Figure 5) (reviewed in [247]). Although the main contribution of NK cells in parasite control is mediated by IFN-γ, they can also be directly cytotoxic for cells infected with T. gondii [248, 249]. NK cells are also a source of IL- 10 in systemic T. gondii infection counterbalancing the inflammatory response during acute T, gondii infection [250].

20 1 Introduction

Figure 5: DC-mediated NK cell activation. DC-mediated activation of NK cells results in increased NK-cell cytolytic activity and IFN-γ production. Both, cell contact-dependent interactions and soluble cytokine signals are involved in DC-NK cell crosstalk. NKG2D (NK group 2, member D) ligands that are expressed by DCs in response to microbial stimuli. A role for adhesion molecules has also been indicated by the finding that LFA1 (lymphocyte function-associated antigen 1)–ICAM1 (intercellular adhesion molecule 1) interactions are important for DC-mediated activation of NK cells. The relevance of interactions mediated by CD70–CD27, CD48–2B4 and CLRB (C-type-lectin-related B)–NKR-P1B (NK-cell receptor protein 1B) or NKR- P1D requires assessment. Cytokine signals are essential for NK cell activation, and several cytokines, such as IFN-α, IFN-β, IL-2, IL-12, IL-15 and IL-8 are involved. Reciprocally, NK cells can affect DC functions and lead to DC activation. From [247]. Adaptive immune responses to Toxoplasma gondii infection Patients with primary or acquired defects in adaptive immune responses are susceptible to cerebral toxoplasmosis demonstrating the importance of adaptive immune responses for the resistance to chronic T. gondii infection [251]. The adaptive immune responses towards T. gondii infection are mediated by both B and T cells.

1.4.3.1. B cell response B cell response mediates parasite elimination and control thought production of parasite- specific antibodies (reviewed in [252]). B-1 cells are an important source of naturally occurring IgM antibodies that have been shown to enhance phagocytosis and tachyzoite killing by neutrophils and act as a potent complement activator [210, 253]. T. gondii-specific IgM has been shown to prevent cellular invasion and limit systemic dissemination of tachyzoites [254]. IgG is a second antibody class that is secreted in response to T. gondii infection [255]. It was shown that B cell-deficient animals have normal levels of IFN-γ, IL-10, and iNOS and are able to survive the acute stage of infection with T. gondii, but succumb to death 3-4 weeks after parasite infection. Production of IgG against T. gondii by B cells is critical for prevention of mortality in mice during the chronic stage of infection [256, 257]. Both types of parasite-specific IgA are induced during toxoplasma infection [255]. Secretory IgA can be found in the digestive tract and tears [258]. Titers of toxoplasma-specific IgM, IgG2, IgA and IgE are important diagnostic criteria to diagnose T. gondii infection [259-262]. Protective effects of toxoplasma-specific antibodies are mediated through parasite opsonization and phagocytosis, blocking of invasion and activation of the classic complement pathway [263-266]. B-cells have been described to amplify IFN-γ production from CD4+ T cells independent of antibody secretion in a contact-dependent fashion [267].

21 1 Introduction

1.4.3.2. T cell responses During T. gondii infection, the presentation of parasite-derived antigens to CD4+ and CD8+ T cells is essential for long-term resistance to this pathogen [268]. Critical roles of CD4+ T cells for resistance during toxoplasmosis was studied in HIV-infected patients and CD4-deficient animals [269, 270]. CD4+ T cells serve as a main source of IL-12-dependent IFN-γ production [271]. They mediate optimal B and CD8+ T cell responses and activate effector functions of macrophages through CD40L-CD40 interaction [270, 272, 273].

T-cell responses are initiated when naïve CD4+ or CD8+ T cells encounter antigen presenting cells bearing their cognate antigen in the context of MHCII and MHCI molecules, respectively [274]. IL-12 together with CD28 or ICOS signaling is necessary to promote T-cell proliferation and IFN-γ secretion [275, 276]. pDCs and CD8α+ DCs were shown to be efficient in antigen presentation to CD4+ T cells during toxoplasmosis [277, 278].

CD8+ T cells control T. gondii infection through the production of IFN-γ, CD40/CD40L interactions and perforin-mediated cytolysis of infected host cells and free tachyzoites [279-281]. CD8+ T-cell-induced apoptosis in cells infected with T. gondii mediate tachyzoite release from the cell. Futher, extracellular parasites are directly killed by CD8+ cytotoxic effector cells [279, 282, 283].

Various peptides derived from T. gondii are presented in the context of different MHCI alleles to prime naïve CD8+ T cells [284, 285]. Resident CD8α+ DCs have been shown to be most efficient at cross-presentation of antigens to CD8+ T-cells [286, 287]. In recent studies, transgenic parasites were used to better understand antigen processing and presentation by DCs in T. gondii infection. It was observed that IL-12 is mostly produced by non-infected DCs that did not phagocytose parasites and that the mechanism of cytokine secretion relies on remote sensing of parasite-derived antigens [288]. However, it was also shown that infected DCs are required for the development of CD4+ and CD8+ T cell responses to T. gondii [268, 289-291].

Latent T. gondii infection in the brain Properly mounted immune responses lead to clearance of T. gondii from the body tissues, but parasites persist as a chronic latent infection in the form of intraneuronal cysts that are controlled, but not eliminated by the immune system (reviewed in [292]). T. gondii utilizes CD11c- CD11b+ monocytes as Trojan horse to deliver single parasites intracellularly through the blood- brain barrier (BBB) into the brain [293, 294]. In the CNS, tachyzoites infect microglia, neurons and astrocytes, but only microglia and astrocytes can clear the parasites upon priming with T- and NK cell-derived IFN-γ [295-297]. The exact mechanisms of T. gondii-mediated immune evasion

22 1 Introduction inside neurons remains unclear. CD4+ and CD8+ T cells secrete IFN-γ in the brain that is important for parasite control and clearance.

Recruitment of monocytes, CD4+ and CD8+ T-cells begins during the acute stage of infection from the periphery [280]. Mechanisms of T-cell entry into the CNS depend on the integrin receptor vascular cell adhesion molecule (VCAM)-1 that is upregulated on the vascular endothelium of T. gondii-infected mice and its ligand, the α4β1 integrin (VLA-4) that is expressed on activated CD4+ and CD8+ T-cells [298]. Brain-infiltrating T-cells show low or no proliferative capacity in comparison to peripheral T-cells [299]. Both, CD4+ and CD8+ T-cells play a critical roles in controlling toxoplasma-mediated encephalitis in C57Bl/6 mice [280, 300]. In mice, T-cell were found in the brain in granuloma-like structures that surround isolated parasites and contain CD11b+ and CD11c+ cells [301]. CD11b+ cells in the brain include macrophages, monocytes, DC and microglia, which play significant roles as APCs by fine-tuning T cell functions. Resident microglia cells produce pro- and anti-inflammatory cytokines such as IL-1β, IL-10, TNF, IL-12 and IL-15 [302-304]. Several subsets of DCs infiltrate the brain upon T. gondii infection such as CD11b+ cDCs, CD8α+ cDCs, and PDCA+B220+ plasmacytoid DCs. They are the main source of IL-12 and co-stimulatory signaling to maintain IFN-γ production by T cells in the brain [305, 306]. Ly6ChiCCR2+ inflammatory monocytes are recruited to the T. gondii-infected brain. They produce high levels of pro-inflammatory mediators, such as IL-1α, IL-1β, IL-6, iNOS, TNF-α and ROS and are essential for parasite control [147, 151].

The effector mechanisms inhibiting parasite replication in neurons have not been identified yet. In murine astrocytes, the IFN-γ-inducible GTPases IGTP and IIGP1 inhibit tachyzoite replication by disruption of the PV [237, 307, 308]. In human astrocytes, the enzyme indoleamine 2,3-dioxygenase degrades tryptophan and causes tryptophan starvation of the parasite [309, 310]. Guanylate binding proteins (GBPs) are highly induced in cells upon stimulation with IFN-γ. Mouse guanylate binding protein 2 (mGBP2) seems to be is important to control T. gondii in astrocytes and macrophages in vitro and in vivo [311].

The intracellular niche of T. gondii that allows parasite survival and spreading in the host T. gondii is protected inside the cell within the PV that acts as an interface between the parasite and host cytoplasm. The parasite has a machinery to subvert signaling pathways of the host cell and manipulate host immune responses to either enhance or inhibit its specific components to survive and spread in the host. For this purpose, the parasite releases rhoptry proteins (ROPs) and dense granule proteins (GRAs) into the PV after invasion (reviewed in [312]). In vitro experiments revealed that T. gondii can increase the levels of activated MEK1/2 and 23 1 Introduction

Erk1/2 in infected macrophages and splenic cells in a PI3K and Gi protein-coupled receptor- dependent manner [313]. The recently found protein GRA24 directly interacts with the mitogen- activated protein (MAP) kinase p38α (MAPK14), leading to activation of inflammatory responses that control the progress of the parasite infection [314]. Activation of STAT3 signaling induced in infected macrophages suppresses secretion of IL-12 and TNFα [315]. T. gondii infection is known to interfere with STAT1-induced expression of interferon regulatory factor 1 (IRF1) and STAT6 activation [316, 317].

T. gondii tachyzoites use the host cell to spread through the host and evade immune response within the PV. Cell invasion leads to a rapid and prolonged migratory enhancement of DCs, neutrophils and NK cells [207, 318, 319]. Additionally, killing of infected DCs by NK cells promotes parasite spreading and NK cell invasion [320].

Resolution of inflammation in response to T. gondii infection The success of an intracellular parasite to survive the hostile cell environment is defined by its ability to control the host immune response and limit the development of immune pathology. In this regard, IL-10 is an essential cytokine for resolution of inflammation. It is produced by a number of cell types, including macrophages, NK, T- and B-cells, and functions by inhibiting the activation of accessory cells and adaptive immune responses [321]. When infected with T. gondii, IL-10-deficient mice succumb to lethal inflammatory responses mediated by CD4+ T-cells and marked overproduction of IL-12, IFN-γ and TNFα [322]. In turn, IL-10 decreases the potency of macrophages to kill intracellular parasites, thereby promoting parasite survival [323]. The major cellular source of IL-10 during acute toxoplasmosis are CD4+ T cells but not regulatory T cells (Tregs) [324]. However, Tregs contribute to IL-10 production and use IL-2-mediated mechanisms of immunosuppression to control CD4+ and CD8+ T-cell proliferation [325]. Additionally, T. gondii infection induces CD1dhighCD5+ regulatory B cells (Bregs) as a source of IL-10. It has been shown that Bregs during T. gondii infection are associated with cyst formation in the host brain and the establishment of chronic infection [326].

Further, lipoxin A4 (LXA4) is a lipid mediator, that is important for the resolution of inflammation. It inhibits STAg-induced DC migration and IL-12 production in vivo and in vitro independently of IL-10 [327]. Interestingly, T. gondii expresses the enzyme 15-lipoxygenase that participates in LXA4 biosynthesis, an evasion mechanism from the LXA4-downregulated immune responses [328].

24 1 Introduction

Activation of the complement system during T. gondii infection Antibody-dependent complement-mediated killing of T. gondii tachyzoites in immune serum was discovered in 1948 and used as a serological test procedure named Sabin–Feldman dye test [329]. It served for the detection of T. gondii specific antibodies long time before the actual mechanism underlying this phenomenon was described [266, 330]. In 1980, the classical complement activation pathway was described to be the main route of complement activation in response to T. gondii infection as complement factors B, D and properdin, necessary for the alternative pathway activation, were shown to be dispensable for complement-mediated killing of tachyzoites using T. gondii-specific immune serum [266]. Evasion of the parasite from alternative pathway activation can be explained by the limited capacity of C3b deposition on the cellular membrane of tachyzoites and its fast degradation into its inactive form iC3b [331, 332]. Recent studies demonstrated presence of the complement activation during the chronic stage of infection in the brain. Degeneration and rupture of brain cysts causes C1q production and fixation as an initial step of classical complement activation [333]. It is reported that treatment of infected mice with anti-C3R antibody resulted in the deficient CD4+ and CD8+ T-cell response and high susceptibility to the acute T. gondii infection [334]. Similarly, mice deficient in C3aR and C5aR1 were highly susceptible to T. gondii infection due to decreased IL-12 and IFN-γ production pointing to the importance of the complement activation in the resistance to the parasite [76].

25 1 Introduction 1.5. Hypothesis and specific aims of the project Previous reports from the Aliberti laboratory showed that T. gondii infection drives IL-12 production through a CCR5-dependent mechanism [178]. Further, several studies from the Köhl laboratory demonstrated that the C5a/C5aR1 axis controls IL-12 and IL-23 cytokine production from DCs upon TLR stimulation [110] or in response to parasite infection [124]. Also, previous reports from our collaborator Julio Aliberti (CCHMC) suggested that TgCyp18 drives heterodimerization between CCR5 and C5aR1, which serves a novel danger sensing mechanism in T. gondii infection. Based on such data, I hypothesized that this dimerization promotes IL-12 production from CD8α+ splenic DCs by a MyD88-independent pathway as a critical pathway that induces protective TH1 immune responses against T. gondii. To test these hypotheses, I aimed to pursue the following specific aims: 1. Define the impact of soluble Toxoplasma antigen (STAg) and TgCyp18 on IL-12 production from splenic dendritic cells in vitro. 2. Delineate the receptor pathways underlying the STAg- and TyCyp18-driven IL-12 production from splenic dendritic cells in vitro using cells from wild type, C5aR1, CCR5 and MyD88-deficient mice. 3. Assess the cross-talk between C5aR1 and CCR5 in response to cognate ligand interaction and T. gondii tachyzoite infection in vitro. 4. Delineate the role of C5aR1 and CCR5 activation in mouse survival using the intraperitoneal T. gondii infection model. 5. Delineate the impact of C5aR1 and CCR5 activation on systemic cytokine/chemokine production in response to acute T. gondii infection in vivo. 6. Determine the role of the C5a/C5aR1 axis for the cellular response in the peritoneum and the spleen in response to acute T. gondii infection. 7. Define the role of C5aR1 and CCR5 for the development of protective immunity in the brain during the chronic stage of T. gondii infection. 8. Determine the role of the C5a/C5aR1 axis in the development of protective adaptive immunity to T. gondii infection.

2 MATERIAL

2.1. Material Mouse strains

Table 5: Mouse strains. B6 = C57BL/6, cg = congenic, tg = transgenic, tm = targeted mutation.

Mouse line Nomenclature Strain Supplier B6.Cg(B10.D2)-Hc0H2dH2- C5-/- C57BL/6 Internal breeding T18c/oSnj C5aR1 GFP+/fl B6.C5aR1tm1JKo C57BL/6 Internal breeding C5aR1-/- B6.129S4-C5ar1tm1Cge C57BL/6 Internal breeding C5aR1-/- x CCR5-/- C5ar1tm1Cge-Ccr5tm1Kuz C57BL/6 Internal breeding C5aR2-/- B6.Cg-Gpr77tm1Cge C57BL/6 Internal breeding CCR5-/- B6.129P2-Ccr5tm1Kuz/J C57BL/6 Internal breeding MyD88-/- B6.129-Myd88tm1Aki C57BL/6 Internal breeding NMRI Crl:NMRI(Han) NMRI Charles River Wild type B6.JRj C57BL/6 Janvier

All animal studies were reviewed and approved by local authorities of the Animal Care and Use Committee (Ministerium für Landwirtschaft, Energiewende, Umwelt und Ländliche Räume, Kiel, Germany) according to permission number 39 (36-3/15) and Institutional Animal Care and Use Committee (IACUC) according to the protocol number 2013-0144 (CCHMC, Cincinnati, OH, USA). Animal were used at 8 to 12 weeks of age, control and knockout groups were matched by age and sex. Mixed gender groups were used except where stated otherwise.

Parasites and cell lines T. gondii cysts of the ME49 type II were maintained through passages in NMRI mice, T. gondii tachyzoites type II Prugniaud strain expressing GFP were kindly provided by Dirk Schlüter (Otto von Guericke University Magdeburg, Magdeburg) and were maintained in monkey kidney adherent epithelial cells (Vero-B4 cell line, ACC 33, DSMZ, Braunschweig). T. gondii tachyzoites type I RH strain were provided by Julio Aliberti (CCHMC, Cincinnati, OH, USA) and were maintained on human foreskin fibroblasts Hs27 (ATCC® CRL-1634™, Manassas, Virginia, USA).

Chemicals and reagents

Table 6: Chemicals and reagents.

Substance Manufacturer

1-StepTM Ultra TMB ELISA-Substrate solution Thermo Scientifc Inc., Waltham, USA

Agar, Bacto BD Medical Systems, Erembodegem, Belgium

27 2 Materials

Ammonium chloride (NH4Cl) Sigma-Aldrich Chemie GmbH, Steinheim

Ampicillin - anhydrous Sigma-Aldrich Chemie GmbH, Steinheim

Adenosine triphosphate (ATP) Sigma-Aldrich Chemie GmbH, Steinheim

Auto MACS Rinsing-Solution Miltenyi Biotec GmbH, Bergisch Gladbach

BD Cytofix™ fixation buffer BD Biosciences Europe, Erembodegem, Belgium

BD FACS Flow Sheath Fluid BD Biosciences Europe, Erembodegem, Belgium

Bovine serum albumin (BSA) Sigma-Aldrich Chemie GmbH, Steinheim

Brefeldin A, 1000x solution eBioscience, Vienna, Austria

C5a, human, Recombinant Hycult Biotech, Uden, Netherlands

Calcium chloride (CaCl2) Sigma-Aldrich Chemie GmbH, Steinheim

CCL5 (RANTES), human Peprotech, Hamburg

CD11c MicroBeads Milteny Biotec GmbH, Bergisch Gladbach

Compensation beads (anti rat/hamster) BD Biosciences Europe, Erembodegem, Belgium

Coomassie Brilliant Blue R-250 Bio-Rad Laboratories GmbH, München

CpG ODN 1668 InvivoGen, San Diego, USA

CSF-2 (GM-CSF), recombinant murine Peprotech Corporation, Rocky Hill, USA

Dimethyl sulfoxide (DMSO) Sigma-Aldrich Chemie GmbH, Steinheim

Dithiothreitol (DTT) Thermo Scientifc Inc., Waltham, USA

Dulbecco's Phosphate Buffered Saline (DPBS) Gibco® by Life Technologies Corporation, Carlsbad, USA

E. coli, NEB 5 alpha Competent New England Biolabs, Ipswich, USA

Ethanol, 70% denaturated Carl Roth GmbH & Co. KG, Karlsruhe

Ethanol, 96% denaturated Carl Roth GmbH & Co. KG, Karlsruhe

Ethanol, absolute J. T. Baker, Deventer, Netherlands Ethylene glycol-bis(β-aminoethyl ether)- Sigma-Aldrich Chemie GmbH, Steinheim N,N,N',N'-tetraacetic acid (EGTA)

Ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich Chemie GmbH, Steinheim

Fetal calf serum (FCS) PAA Laboratories GmbH, Pasching, Östereich

Formaldehyde solution, 37% Sigma-Aldrich Chemie GmbH, Steinheim

Glacial acetic acid Merck, KGaA, Darmstadt

Glycerol Merck, KGaA, Darmstadt

Glycine Calbiochem/Merck, Darmstadt

Hepes buffer solution 1M Life technologies Corporation, Carlsbad, USA

Isopropanol Otto Fishar GmbH & Ko. KG, Saarbrücken

Isopropyl-1-thio-β-D-galactoside (IPTG) Sigma-Aldrich Chemie GmbH, Steinheim

L-glutamine (200 mM concentrate) Life technologies Corporation, Carlsbad, USA

Lysogeny broth (LB) powder Carl Roth GmbH & Co. KG, Karlsruhe

Lysozyme Sigma-Aldrich Chemie GmbH, Steinheim 28 2 Materials

MACS BSA stock solution Milteny Biotec GmbH, Bergisch Gladbach

Magnesium chloride (MgCl₂ · 6H₂O) Millipore, Billerica, USA

Magnesium sulfate (MgSO4·7H2O) Sigma-Aldrich Chemie GmbH, Steinheim

Methanol (CH3OH) Sigma-Aldrich Chemie GmbH, Steinheim

N-succinyl-ALA-ALA-PRO-PHE-P-Nitroanilide Sigma-Aldrich Chemie GmbH, Steinheim (N-succinyl-AAPF-p-nitroanilide)

Penicillin-Streptomycin, 100x Liquid Gibco® by Life Technologies Corporation, Carlsbad, USA

Potassium bicarbonate (KHCO3) Sigma-Aldrich Chemie GmbH, Steinheim

Potassium carbonate (Na2CO3) Sigma-Aldrich Chemie GmbH, Steinheim

Potassium chloride (KCl) Carl Roth GmbH & Co. KG, Karlsruhe

Potassium dihydrogen phosphate (KH2PO4) Carl Roth GmbH & Co. KG, Karlsruhe

Prestained marker, broad range Bio-Rad Laboratories GmbH, München

RPMI 1640 Gibco® by Life Technologies Corporation, Carlsbad, USA

Silver nitrate concentrate (AgNO3), for 0,5M Sigma-Aldrich Chemie GmbH, Steinheim solution

Sodium chloride (NaCl) Carl Roth GmbH & Co. KG, Karlsruhe

Sodium dihydrogen phosphate (NaH2PO4) Carl Roth GmbH & Co. KG, Karlsruhe

Sodium dodecyl sulfate (SDS) AppliChem GmbH, Darmstadt

Sodium hydrogen phosphate (Na2HPO4) Sigma-Aldrich Chemie GmbH, Steinheim

Sodium pyruvate Life technologies Corporation, Carlsbad, USA

Sulfuric acid (H2SO4) Sigma-Aldrich Chemie GmbH, Steinheim

Thrombin GE Healthcare Life Science, Chicago, USA

Tris-aminomethane (Tris) Sigma-Aldrich Chemie GmbH, Steinheim

Triton™ X-100 Sigma-Aldrich Chemie GmbH, Steinheim

Trypan blue Life technologies Corporation, Carlsbad, USA

Trypsin-EDTA 0,5%, 10x Life technologies Corporation, Carlsbad, USA

Tween®20 Sigma-Aldrich Chemie GmbH, Steinheim

α-Chymotrypsin from bovine pancreas Sigma-Aldrich Chemie GmbH, Steinheim

Buffers, solutions and media

Table 7: Buffers, solutions and media.

Buffer/Solution/Medium Formulation

RPMI 1640 10% FBS, heat inactivated Complete RPMI medium 100 Units/ml Penicillin 100 µg/ml Streptomycin

29 2 Materials

2mM L-Glutamine

Aqua destillata Destaining solution for Coomassie staining 40% Methanol 10% Acetic acid

Aqua destillata 13,7 mM NaCl 0,27 mM KCl Dulbecco's Phosphate-Buffered Saline (DPBS) 0,81 mM Na2HPO4

0,15 mM KH2PO4 pH 7,2-7,4

ELISA wash buffer 0,05% Tween®20 in DPBS

FC buffer, ELISA block buffer 1% BSA in DPBS

50mM Tris Glutathione agarose binding buffer 150mM NaCl pH 8,0

50mM Tris 150mM NaCl Glutathione agarose elution buffer 10mM Reduced Glutathione pH 8,0

Aqua destillata Lysogeny broth (LB) agar 25 g/l LB powder 1,5% Agar

Aqua destillata Lysogeny broth (LB) medium 25 g/l LB powder

Auto MACS Rinsing-Solution MACS buffer MACS BSA stock solution 1:20

Aqua destillata

155 mM NH4Cl

Red blood cell lysis buffer (RBC) 10 mM KHCO3 0,1 mM EDTA pH 7,2, sterile

Aqua destillata 2% SDS SDS-PAGE loading buffer (2x) 10% Glycerol 50 mM TRIS-HCl 200 mM DTT 0,01% Bromophenol blue

Aqua destillata SDS-PAGE running buffer 25 mM TRIS-HCl 192 mM Glycin 0,1% SDS 30 2 Materials

Silver staining solutions for SDS-PAGE

6g Na2CO3 50µl 37% Formaldehyde Developing solution 2ml Sodium thiosulfate solution Ad 100ml Aqua destillata

100ml Stop solution Fixation solution 50µl 37% Formaldehyde

2,35ml 0,5M AgNO3 (0,2%) Silver nitrate solution 75µl 37% Formaldehyde Ad 100ml Aqua destillata

20mg Sodium thiosulfate Sodium thiosulfate solution Ad 100ml Aqua destillata

Aqua destillata Stop solution 30% Ethanol 10% Acetic acid

Aqua destillata 50mM Tris Sonication buffer 50mM NaCl 1mM EDTA 1mM DTT

Aqua destillata 2% Trypton 0,5% Yeast extract 10mM NaCl Super Optimal broth with Catabolite repression (SOC) 2,5mM KCl

10mM MgCl2

10mM MgSO4

20mM Glucose

Aqua destillata

10mM KH2PO4 2mM EGTA 5mM MgCl2 Transfection buffer for electroporation 25nM HEPES 0,5mM CaCI2 5mM Glutathione 2mM ATP

Aqua destillata Tris buffer 500 mM NaCl

20 mM TRIS-HCl

31 2 Materials

Antibodies for flow cytometry

Table 8: Antibodies. AF = Alexa Fluor®, APC = Allophycocyanin, BV = Brilliant Violet, Cy = Cyanine, FITC = Fluorescein Isothiocyanate, PE = Phycoerythrin, PerCP = Peridinin-chlorophyll-protein complex.

Stock Dilution Epitope Dye Clone Species Manufacturer concentration used

R&D Systems CCR2 APC 475301 Rat - 1:400 Minneapolis, USA

PerCP- Armenian BioLegend Europe, CD103 E7 0,2mg/ml 1:800 Cy5.5 hamster London, UK

BioLegend Europe, CD11b BV510 M1/70 Rat 50μg/ml 1:400 London, UK

eBioscience CD11b AF700 M1/70 Rat 0,2mg/ml 1:400 Vienna, Austria

BD Biosciences Armenian Europe, CD11c PE HL3 0,2mg/ml 1:400 hamster Erembodegem, Belgium

Armenian eBioscience CD11c APC N418 0,2mg/ml 1:400 hamster Vienna, Austria

eBioscience CD16/32 - 93 Rat 1 mg/mL 1:100 Vienna, Austria

eBioscience CD19 eFluor450 eBio1D3 Rat 0,2mg/ml 1:400 Vienna, Austria

eBioscience CD3 eFluor450 17A2 Rat 0,2mg/ml 1:400 Vienna, Austria

PerCP- Armenian eBioscience CD3e 145-2C11 0,2mg/ml 1:400 Cy5.5 hamster Vienna, Austria

BD Biosciences Armenian Europe, CD3e FITC 145-2C11 0,5mg/ml 1:400 hamster Erembodegem, Belgium

eBioscience CD4 FITC GK1.5 Rat 0,5mg/ml 1:400 Vienna, Austria

eBioscience CD4 PE-Cy7 RM4-5 Rat 0,2mg/ml 1:400 Vienna, Austria

BD Biosciences Europe, CD43 BV421 S7 Rat 0,2mg/ml 1:400 Erembodegem, Belgium

32 2 Materials

eBioscience CD44 eFluor450 IM7 Rat 0,2 mg/mL 1:400 Vienna, Austria

PerCP- eBioscience CD45 30-F11 Rat 0,2 mg/mL 1:100 Cy5.5 Vienna, Austria

eBioscience CD45R APC RA3-6B2 Rat 0,5 mg/mL 1:400 Vienna, Austria

eBioscience CD5 PE 53-7.3 Rat 0,2mg/ml 1:400 Vienna, Austria

BioLegend Europe, CD62L APC MEL-14 Rat 0,2 mg/mL 1:100 London, UK BioLegend Europe, CD64 PE X54-5/7.1 Mouse 0,2 mg/mL 1:800 London, UK

BioLegend CD8α BV510 53-6.7 Rat 50μg/ml 1:400 San Diego, USA

eBioscience F4/80 PE-Cy7 BM8 Rat 0,2mg/ml 1:400 Vienna, Austria

eBioscience IFN-γ PE XMG1.2 Rat 0,2mg/ml 1:100 Vienna, Austria eBioscience IgM PE-Cy7 Il/41 Rat 0,2mg/ml 1:400 Vienna, Austria

BD Biosciences Europe, IL-12p40/70 PE C15.6 Rat 0,2 mg/ml 1:100 Erembodegem, Belgium

PerCP- eBioscience Ly6C HK1.4 Rat 0,2mg/ml 1:400 Cy5.5 Vienna, Austria

BD Biosciences Europe, Ly6C PE-Cy7 AL-21 Rat 0,2mg/ml 1:400 Erembodegem, Belgium

BioLegend Europe, Ly6G APC-Cy7 1A8 Rat 0,2mg/ml 1:400 London, UK

APC- eBioscience MHCII M5/114.15.2 Rat 0,2mg/ml 1:1000 eFluor780 Vienna, Austria BioLegend Europe, NK1.1 AF700 PK136 Rat 0,5mg/ml 1:400 London, UK

33 2 Materials

Plastic ware and disposable items

Table 9: Plastic ware and disposable items.

Material Manufacturer

BD Microtainer tube BD Biosciences Europe, Erembodegem, Belgium

Blunt fill needle, 18G BD Biosciences Europe, Erembodegem, Belgium

Cell strainer 40 µm BD Biosciences Europe, Erembodegem, Belgium

Glaswarenfabrik Karl Hecht GmbH & Co KG, Cover glass, 20x20mm, thickness 1 Sondheim/Rhön

Cover glass, circular, 25mm, borosilicate galss VWR, International GmbH, Darmstadt thickness 1

ELISA-reservoir, 25 ml VWR International GmbH, Darmstadt

Falcon Tube 15 ml, 50 ml Sarstedt AD & Co., Nümbrecht

Filter tip 10 µl, 100 µl, 1000 µl Sarstedt AD & Co., Nümbrecht

Gene Pulser Cuvettes, 0,2cm, green Bio-Rad Laboratories GmbH, München

MACS Separation MS Column Milteny Biotec GmbH, Bergisch Gladbach

Microscope slide, 76x26mm Gerhard Menzel GmbH, Braunschweig

Microtiter plate, 6-well Sarstedt Inc., Newton, USA

Microtiter plate, 96-well, high bind Corning, Corning, USA

Microtiter plate, 96-well, with lid (F bottom) Greiner Bio-One GmbH, Frickenhausen

Microtiter plate, 96-well, with lid (U bottom) Greiner Bio-One GmbH, Frickenhausen

Needle hypodermic,26G BD Biosciences Europe, Erembodegem, Belgium

Nitrile Powder-Free Examination Gloves Ansell Healthcare GmbH, Munich

Petri dish, 60x15 mm Greiner Bio-One GmbH, Frickenhausen

Pipette tip, 10 µl, 100 µl, 1000 µl Sarstedt AD & Co., Nümbrecht

Pipette with tip, 5 ml, 10 ml, 25 ml Greiner Bio-One GmbH, Frickenhausen

Reaction tube 0.5 ml; 1.5 ml; 2 ml; Sarstedt AD & Co., Nümbrecht

Reaction tube 0.5 ml; 1.5 ml; 2 ml; 5ml Eppendorf AG, Hamburg

Single use cannula 21G, blunt B. Braun Melsungen AG, Melsungen

Spatula VWR International GmbH, Darmstadt

Syringe 1ml, 5 ml, 10 ml, Luer-LokTM BD Biosciences Europe, Erembodegem, Belgium

Syringe, 1 ml B. Braun Melsungen AG, Melsungen

Syringe, 5 ml, 10 ml BD Biosciences Europe, Erembodegem, Belgium

34 2 Materials

Tube 5 ml, 75x12mm, PS unsterile for FC Sarstedt AD & Co., Nümbrecht

Weighing dish Greiner Bio-One GmbH, Frickenhausen

Commercially available kits

Table 10: Commercially available kits

Kit Manufacturer

BD Cytofix/Cytoperm™ kit BD Biosciences Europe, Erembodegem, Belgium

Bio-Plex Pro Mouse Cytokine 23-Plex Panel Bio-Rad Laboratories GmbH, München

EndoTrap Red® Endotoxin removal kit Hyglos GmbH, Bernried am Starnberger See

EndoZyme® Endotoxin detection assay Hyglos GmbH, Bernried am Starnberger See

Glutathione Chromatography Cartridges Thermo Fisher Scientific, Waltham, USA

Mouse IFN-gamma DuoSet ELISA R&D Systems, Minneapolis, USA

Mouse IL-12 p70 DuoSet ELISA R&D Systems, Minneapolis, USA

Mouse IL-12/IL-23 p40 Non Allele-specific R&D Systems, Minneapolis, USA DuoSet ELISA

NucleoBond® Xtra Midi prep kit Macherey-Nagel, Düren

V-PLEX Proinflammatory Panel 1 (mouse) Kit Meso Scale Diagnostics, Rockville, USA

2.2. Equipment and software Laboratory equipment

Table 11: Laboratory equipment

Equipment Manufacturer

Biological Safety Cabinets Nuaire Inc., Plymouth, USA

Centrifuge 5424 Eppendorf AG, Hamburg

Centrifuge 5424R Eppendorf AG, Hamburg

Centrifuge 5810R Eppendorf AG, Hamburg

Centrifuge Rotofix 32A Hettich Lab Technology, Tuttlingen

Chemical hood Waldner Laboreinrichtungen GmbH & Co KG, Wangen

Confocal Microscope FluoView 1000 Olympus, Hamburg

Dissecting scissors WPI Deutschland GmbH, Berlin

Electrophoresis Cells Mini Protean Tetra Bio-Rad Laboratories GmbH, Munich

35 2 Materials

ELISA-Reader Fluostar Omega 0415 BMG Labtech GmbH, Ortenberg

ELISA-Washer Nunc-ImmunoTM Wash 12 Thermo Fisher Scientific Inc., Waltham, USA

Flow cytometer BD LSR II Beckton Dickinson GmbH, Heidelberg

Forceps WPI Deutschland GmbH, Berlin

Liebherr-International Deutschland GmbH, Biberach an der Fridge, 4 °C and -20 °C combined Riß

Gel documentation system ImageQuant 350 GE Healthcare Europe GmbH, Freiburg

Gene Pulser II Electroporation System Bio-Rad Laboratories GmbH, München

Ice-machine AF-10 Scotsman, Milan, Italy

IR Direct Heat CO2 Incubator Nuaire Inc., Plymouth, USA

MACS Magnetic Cell Separator Milteny Biotec GmbH, Bergisch Gladbach

Magnet stirrer C-Mag MS7 IKA, Stauten

MESO Quick Plex SQ120 MSD, Rockville, USA

Microscope BA310 LED Motic, Wetzlar

Microscope camera Leica EC3 Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany

Microscope camera Moticam Motic, Wetzlar

Microscope Fluovert FS Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany

Microscope Leica DM IL LED Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany

Multichannel pipette Biohit M300 Sartorius Biohit Liquid Handling Oy, Helsinki, Finland

Neubauer counting chamber, improved VWR International GmbH, Darmstadt pH-Meter Seven Easy PH S20-K Mettler Toledo, Schwerzenbach, Switzerland

Pipetboy pipette controller Integra Biosciences AG, Zizers, Switzerland

Pipette (0,1-2,5 µl; 0,5-10 µl; 10-100 µl; 20-200 Eppendorf AG, Hamburg µl; 100-1000 µl)

Precision balance LC6200S Sartorius AG, Gottingen

Precision balance EMB 1000-2 Kern & Sohn, Balingen

Pump Peristaltic P-1 Pharmacia, Uppsala, Sweden

Pump, VacuGene Pharmacia, Belgium

Pure water system Nanopure Diamond Thermo Fisher Scientific GmbH, Bremen D11931

Shaker Polymax 1040 Heidolph Instruments GmbH & Co. KG, Schwabach

WEBECO Hygiene in Medizin und Labor GmbH & Co. KG, Steam sterilizer E14 Hydromat Selmsdorf

36 2 Materials

Suction system Vacusafe 158310 Integra Biosciences GmbH, Fernwald

Table centrifuge Carl Roth GmbH & Co. KG, Karlsruhe

Tablet Acer Iconia Tab Acer, Xizhi, New Taipei, Taiwan

Thermomixer 5436 Eppendorf, Hamburg

Ultra-low temperature freezer, -80 °C SANYO Electrics Co., Japan

Vortex-Genie 2 Scientific Industries Inc., New York, USA

Water bath GFL, Burgwedel

Computer software

Table 12 : Computer software

Software Developer

Adobe Acrobat Reader DC Adobe Systems Inc., San Jose, USA BD FACSDiva 7.0 BD Biosciences, San Jose, USA

FlowJo X FlowJo, LLD, Ashland, USA

Fluo View FV1000 2.0b Olympus, Hamburg

GraphPad Prism 6.0 Graph Pad Software Inc., LaJolla, USA Imaris 7.2.3 Bitplane AG, Zurich, Switzerland

Leica Aquire 1.0 Leica Microsystems CMS GmbH, Wetzlar

Microsoft Office 365 Microsoft Corporation, Redmond, USA

MARS – Data Analysis Software 2.30 R2 BMG Labtech GmbH, Ortenberg

Omega 1.30 BMG Labtech GmbH, Ortenberg

3 METHODS

3.1. Protein expression in bacterial culture

The plasmid construct that encodes recombinant protein TgCyp18 was kindly provided by Y. Nishikawa (Obihiro University of Agriculture and Veterinary Medicine, Japan) [182]. Briefly, they made a construct that was based on the bacterial expression vector pGEX-4T1 (GE Healthcare, Buckinghamshire, England) (Figure 6). Its sequence encodes a fusion protein of wild-type recombinant TgCyp18 consisting of 163 amino acids without the signal peptide (amino acids 1 to 17) and a GST-tag. The DNA and amino acid sequences of TgCyp18 are stored in the GenBank database under accession number U04633.1. TgCyp18 cDNA was inserted into the vector using BamHI and EcoRI restriction sites to generate plasmid pGEX-TgCyp18 [182, 183].

Figure 6: Expression vector pGEX-T1 used to produce the GST-TgCyp18 fusion protein. BamHI and EcoRI restriction sites were used to insert the TgCyp18 encoding DNA sequence. Heat-shock transformation of chemically competent bacteria Chemically competent E. coli NEB5α (New England Biolabs, USA) cells were transformed with the pGEX-TgCyp18 plasmid. Provided aliquots that contain 50µl of bacterial cells were thawed and 300ng of plasmid DNA in 2µl solution was added to the tube. The mixture was pre-incubated on ice for 30min. For transformation, the tube was heated in a water bath for 30sec at 42°C and then instantly cooled for 2min on ice. Transformed bacteria were then transferred into a culture tube containing 950µl of SOC medium and incubated for 60min at 37°C with shaking. After incubation, the culture was diluted 1:10 and 1:100 in LB, 100µl of each dilution was spread on agar plates containing 100µg/ml ampicillin and incubated overnight at 37°C. Individual colonies were counted and plates were sealed and stored at 4°C or used for plasmid preparation culture.

38 3 Methods

Plasmid preparation Single colonies were picked to inoculate 100ml of LB medium + 100µg/ml ampicillin and then incubated overnight at 37°C with shaking. Glycerol stocks were created to preserve bacteria that express TgCyp18. For this purpose, 1ml of overnight culture was mixed with 300µl of 76% glycerol, frozen and stored at -80°C. Cells from the bacterial culture were harvested by centrifugation at 5.000g for 10min at 4°C, the supernatant was discarded and plasmid DNA was extracted from the cell pellet using the NucleoBond Xtra Midi prep kit following the manufacturer’s instructions. The DNA pellet was resuspended in 200µl of Tris buffer, frozen and stored at -20°C for subsequent use. The DNA concentration and quality was determined by NanoDrop spectrophotometer using Nucleic Acid program by measuring the light absorbance at a wavelength of 260nm and 280nm. Absorbance at 260nm was used to determine the DNA concentration. The ratio of absorbance at 260 nm and 280 nm was used to assess the purity of DNA. DNA samples used in experiments had a 260/280 ratio of ~1,8 or higher.

Purification of GST-tagged protein from bacterial biomass The bacterial culture was prepared by adding 5µl of the glycerol stock to 20ml LB broth + 50µg/ml ampicillin and incubating overnight at 37°C with shaking. Then the pre-culture was added to 800ml fresh LB broth + 50µg/ml ampicillin and cultured for 4h at 37°C with shaking. Finally, IPTG was added at a final concentration of 1mM to induce protein expression and cultured for additional 6h. The bacterial biomass was harvested by centrifugation at 5.000g for 15min at 4°C. The bacterial pellet was resuspended in 20ml of sonication buffer with addition of lysozyme at a final concentration 500µg/ml and incubated on ice for 30min. Then, 2 ml of 10% Triton X-100/PBS was added to the mixture and incubated for an additional hour. Eventually the mixture was placed on ice and sonicated 5x until the suspension became clear. Then it was centrifuged at 12.000g for 15 min at 4°C and supernatant was collected.

In the next step, the sample was mixed at a ratio of 1:1 with the degassed glutathione agarose binding buffer and run over the cartridge containing glutathione resin at a flow rate of 1ml/min overnight. Then, the cartridge was first washed with binding buffer (15ml) followed by with DPBS (10ml). Thrombin solution (240 units in 1ml) was loaded onto the cartridge and incubated at a room temperature for 16h. Then, the TgCyp18 protein was eluted with DPBS and 250µl per fraction was collected. The protein concentration was determined by NanoDrop at 280nm. Fractions that contained protein were pooled and subjected to SDS-PAGE analysis. GST-tag and uncleaved protein were eluted from the column by glutathione agarose elution buffer and collected separately.

39 3 Methods

Endotoxin removal from the recombinant protein preparation Pre-packed EndoTrap® (Hyglos GmbH, Bernried am Starnberger See) affinity chromatography columns were used for endotoxin removal according to the manufacturer’s instructions. Briefly, columns were washed two times with 3ml of regeneration buffer provided with the kit, then two times with 3ml of sterile endotoxin-free DPBS. Subsequently, the sample was added and allowed to recirculate through the column using a peristaltic pump at a flow rate of 0,2ml/min overnight at 4°C. Eventually, the TgCyp18 protein preparation was tested for the recovery and endotoxin concentration.

Determination of endotoxin content The concentration of LPS in the prepared TgCyp18 isolate was determined before and after purification using EndoZyme® (Hyglos GmbH, Bernried am Starnberger See), recombinant factor C endotoxin detection assay. This is a homogeneous enzymatic assay, which uses the LPS receptor recombinant factor C of the horseshoe crab blood clotting cascade in combination with a fluorogenic substrate. The assay was performed according to the manufacturer’s instructions.

Analysis of protein homogeneity by SDS-PAGE Protein samples were analyzed using SDS-PAGE. For this purpose, samples were mixed at a ratio of 1:1 with 2xSDS-loading buffer and incubated for 10min at 95°C, cooled on ice, centrifuged at 4.000g for 10min at 4°C and the supernatant was used for analysis. The electrophoresis chamber was loaded with a pre-cast 4-14% acrylamide gel and filled with electrophoresis buffer. The comb was removed and gel pockets were washed. Then, 10µl of sample or standard were loaded. The gel was run for 50min at 120V. Then it was removed from the electrophoresis chamber and used for subsequent analysis.

Staining protein gels with Coomassie blue Coomasie R250 staining solution was used to detect the protein bands. The detection limit of this staining is around 40ng/band [335]. The gel was stained with Coomassie Blue R250 for one hour with shaking at RT to visualize the protein bands. Then it was destained for 2 hours with rocking at RT in destain solution with at least two changes of this solution until the background became transparent.

Staining protein gels with silver staining Polyacrylamide gels can be impregnated with soluble silver ions (Ag+) and developed by treatment with a reducing agent [336]. Macromolecules in the gel promote the reduction of silver ion to metallic silver (Ag0), which is insoluble and visible, allowing bands containing protein or nucleic

40 3 Methods acid to be seen. The initial deposition of metallic silver promotes further deposition in an autocatalytic process, resulting in exceptionally high sensitivity at round 1ng per protein band.

The gel was fixed in fixation solution for 2h at RT and washed with 50% ethanol 3 times for 20min. Then, the gel was incubated for 1min in sodium thiosulfate solution and washed 3 times for 20sec with A. dest., then incubated for 20 min in silver nitrate solution, washed 2 times for 20sec with A. dest. and developed for 1 min in developing solution, washed 2 times for 2min and the reaction was stopped by incubating for 10min in the stop solution. Then the gel was sealed and image was acquired using gel documentation system ImageQuant 350 (GE Healthcare Europe GmbH).

Peptidyl-prolyl isomerase activity assay The prolyl cis-trans-isomerase activity of the TgCyp18 protein was evaluated by a photometric assay based on the isomerization of the N-succinyl-AAPF-p-nitroanilide described in the following scheme:

N-Suc-Ala-Ala-cis-Pro-Phe-p-NA Peptidyl-prolyl isomerase Suc-Ala-Ala-trans-Pro-Phe-p-NA

Chymotrypsin N-Suc-Ala-Ala-trans-Pro-Phe-p-NA + H2O Suc-Ala-Ala-trans-Pro-Phe + p-NA

In brief, isomerization of the substrate N-succinyl-AAPF-p-nitroanilide by TgCyp18, STAg or mouse serum was determined by hydrolysis of the trans-prolyl isomerization product with α- chymotrypsin at RT in 50mM Tris buffer (pH=7,5) followed by the measurement of p-nitroaniline absorbance at 390nm as described in [337]. To determine the enzymatic activity, 25µl of the sample was added to 95µl Tris buffer in clear flat-bottom 96-well plate and preincubated with 25µl α- chymotrypsin (5mg/ml) for 10min. Then 5µl of the substrate (1mg/ml) was added to the reaction with a final volume of 200µl and the absorbance at 390nm was measured for 5min using ELISA-Reader Fluostar Omega. The change in absorbance over the course of hydrolysis was fitted to a single exponential curve, and values for the observed first-order rate constant (Ko) were compared for fresh mouse serum sample and the recombinant protein.

3.2. Immune cell isolation from mouse tissues

All animal experiments were conducted according to animal research plan approved by ethical committee (detailed in section 2.1). Mice were euthanized by CO2 inhalation followed by cervical dislocation.

41 3 Methods

Isolation of cells from mouse peritoneal cavity For isolation of peritoneal cells, mice were euthanized and pinned down with needles. A small incision was made in the outer skin of peritoneum, then the skin was gently pulled back to expose the inner skin lining of the peritoneal cavity. Then, 5ml of ice-cold DPBS were injected into the peritoneal cavity using a 27g needle. The peritoneum was then gently massaged to release attached cells into the peritoneal cavity. The cell suspension was carefully aspirated with the same syringe and transferred into 15ml falcon tube. The tube was centrifuged for 5min at 450g, 4°C. The supernatant was discarded and the cell pellet was used for analysis. Cells were kept on ice between manipulations.

Isolation of cells from spleen The spleen was removed and transferred into a 1,5ml reaction tube containing 1ml of DPBS and placed on ice. To obtain single cell suspension, it was transferred to a nylon cell strainer (pore size 40µm), which was placed in a 50ml falcon tube. Then cells were isolated from the spleen by grinding it with a plunger from a 5ml syringe. The cell strainer was washed twice with 5ml DPBS. Then, the cell suspension was centrifuged for 5 min at 350g, 4°C. Finally, the pellet was resuspended in RBC-lysis buffer and incubated for 3 min at RT. After that time, the reaction was stopped by the addition of 42ml DPBS. A 10µl aliquot was removed for cell counting. Cells were washed again (5 min, 350g, 4°C) and used for subsequent assays.

Isolation of cells from mesenteric lymph node Mesenteric lymph nodes were removed and placed into 1,5ml reaction tube containing 1ml DPBS and placed on ice. To obtain a single cell suspension, the same procedure as described for splenic cell isolation was used except that the red blood cell lysis was omitted.

Isolation of immune cells from mouse brain

Animals were euthanized by inhalation of CO2 and cervical dislocation. To eliminate any contamination of brain tissue with blood cells, the heart was perfused with 20ml ice-cold DPBS prior to the brain removal. The head was wetted with 70% ethanol and the skin was incised to expose the skull. Using a pair of small curved scissors, the skull was opened and the brain exposed. The brain was removed with forceps and placed into a 5ml reaction tube containing 1ml of sterile ice-cold DPBS.

A single cell suspension of brain tissue was obtained by repeated aspiration and ejection from a 5ml luer lock syringe connected to an 18g, 21g and finally a 26g needle. For isolation of immune cells from the homogenized brain, cells were separated on a discontinuous 70%/30%

42 3 Methods

Percoll gradient. The homogenate was then filtered through a cell strainer (40µm) placed on a 50ml falcon tube and washed with 40ml of sterile ice-cold DPBS. The suspension was centrifuged (310g, 10 min, 4°C), the supernatant was discarded and the pellet was resuspended in 2,5ml 30% Percoll (pink) and transferred into a fresh 15ml falcon tube, to which 2,5ml 70% Percoll (clear) was added underneath. The tubes were centrifuged in a swing-bucket rotor for 30min at 1350g, 4°C without brake. Then ~2ml of the interface, which contained the desired cells, was collected into a fresh tube and washed with 13ml of DPBS for 10 min at 700g, 4°C to remove traces of Percoll. The cell pellet was then resuspended and used for subsequent applications.

Determination of cell numbers To determine the number of cells, an aliquot of the cell suspension was taken and mixed with the same amount of trypan blue. Using a Neubauer counting chamber, the living cells were counted under the microscope. The number of cells was calculated according to the following formula:

cells counted x dilution factor x 104 cells/ml = number of counted big squares

Magnetic-activated cell sorting Magnetic-activated cell sorting (MACS) is a method for separation or isolation of specific cell populations using MACS MicroBeads (Miltenyi Biotec) coated with antibodies. Cells positive for a specific antigen will bind those beads and be magnetically retained on the column placed in a strong magnetic field, while all other cells will flow through.

To enrich sDC culture, single cell suspension was prepared from the mouse spleen as described in section 3.2.2. The cell pellet was resuspended in 400µl of MACS buffer, 100µl of CD11c MicroBeads were added and the cell suspension was incubated for 15min at 4°C. Then, cells were washed with 10ml of MACS buffer at 350g for 5min, 4°C, and resuspended in 500µl of MACS buffer. MACS Separation MS Column was placed in MACS Magnetic Cell Separator and equilibrated with 500µl of MACS buffer. The cell suspension was applied to the column and was allowed to drain by gravity flow. Then the column was washed 3x with 500µl of MACS buffer, removed from the separator and placed on a 1,5ml reaction tube. Finally, bound cells were eluted with 1ml of MACS buffer using a plunger provided with the column. A 10µl aliquot was removed for cell counting. Cells were pelleted for 5 min at 350g, 4°C, resuspended at a concentration of 107cells/ml in complete RPMI medium and used for culture.

43 3 Methods 3.3. Receptor labelling and confocal microscopy Generation of dendritic cells from bone marrow culture (BMDCs) For isolation of bone marrow, the femora and tibiae were taken out and cleaned using paper and scissors. The bones were transferred into a 6-well plate under the sterile hood and rinsed with 70% ethanol followed by a wash step using sterile DPBS. The epiphyses of the bones were cut off and the bone marrow was flushed out with cold DPBS into a 50ml falcon tube fitted with a 40μm strainer. The residual tissue was pressed trough a mesh with a 5ml syringe plunger and washed with 5ml DPBS. Then the strainer was discarded and the cell suspension was centrifuged for 5 min at 450g, 4°C). In the next step the pellet was resuspended in RBC-lysis buffer and incubated for 3 min at RT, then reaction was stopped by addition of 42ml DPBS. An aliquot was taken for cell counting. Finally, cells were pelleted for 5 min at 350g, 4°C and used for cell culture.

BM cells (106 cells/ ml) were cultured in complete RPMI medium supplemented with 20 ng/ml recombinant murine GM-CSF in a 6-well plate (4ml per well). The culture was incubated at

37 °C, 5% CO2. On days 2, 4, 6 and 8, half of the medium was replaced with fresh complete RPMI medium containing 40 ng/ml of recombinant murine GM-CSF. Cells were cultured for 9 days and harvested for subsequent use.

Transfection of BMDC culture To visualize C5aR1 or CCR5 expression in BMDCs, such cells were transiently transfected by electroporation with plasmids encoding fusion proteins of either C5aR1 or CCR5, which were N- terminally labelled with the fluorescent protein GFP. The plasmid constructs that encode fusion protein of CCR5 or C5aR1 and GFP were kindly provided by Irina Majoul (Institute for Biology, University of Lübeck) [122, 338].

For transfection, 1,2·107 BMDCs were washed in DPBS at 350g for 5 min, 4°C, and resuspended in 200µl of transfection buffer and transferred into Bio-Rad Gene Pulser cuvette (green cap). Then, ~3µg of endotoxin-free plasmid DNA in 20 µl of solution was added to the cuvette and incubated for 10min (37°C, 5%CO2). The mixture was pulsed using the Bio-Rad Gene Pulser Electroporation System with an exponential decay program at 0,7kV, 250µF and a resulting time constant of 1,44-1,64 for successfully transfected cells. Then, the cell suspension was immediately incubated for ~20min (37°C, 5%CO2) until cells settled to the bottom of the cuvette. After that, 70µl of the suspension containing ~4·106 cells were transferred into the well of a 6-well plate containing 4ml of pre-warmed complete RPMI medium and a circular cover glass. Cells were incubated ON

(37°C, 5%CO2) and visualized using LSM confocal microscopy [339].

44 3 Methods

Laser scanning confocal microscopy Coverslips with adherent BMDCs were mounted using ring coverslip holder cell chamber (custom-made by I. Majoul). An assembled chamber was filled with 1ml of the complete RPMI medium and visualized using Olympus FluoView™ FV1000 LSM confocal microscope equipped with a temperature- and CO2-controlled chamber with temperature set to 37°C. The image was acquired using a 60x oil immersion objective and the GFP signal was detected using Ex488/Em520nm channel with Kalman filter mode. Eventually, the acquired images were analyzed using the Imaris 7.2.3 (Bitplane) software.

3.4. Maintenance of T. gondii tachyzoites in vitro Vero cell culture Tachyzoites of T. gondii were cultured in vitro within mammalian cells. For this purpose, I used the Vero-B4 cell line, a kidney epithelial cell line from African green monkey that was purchased from Deutsche Sammlung von Microorganismen und Zellkulturen GmbH (DSMZ). Such cells were cultured in complete RPMI medium in 75-cm2 tissue culture flasks. When the cells reached confluence, the medium was aspirated from the flask, the cell monolayer was washed with DPBS to remove traces of FCS and detached with 2ml of 0,5% Trypsin-EDTA solution for 10min at 37°C. The reaction was stopped by addition of 13ml complete RPMI medium, after which the suspension was transferred into a 50ml Falcon tube. An aliquot was taken for cell counting. Cells were pelleted for 5 min at 450g, 4°C, resuspended at density of 106/ml and 1ml of the cell suspension was transferred into a 75-cm2 tissue culture flask containing 19ml complete RPMI medium and allowed to rest for 2h

(5% CO2, 37°C) for adhesion.

3.4.2 T. gondii tachyzoite culture

T. gondii Pru-GFP tachyzoites were added to the Vero culture in 1ml of complete RPMI medium at an MOI of 1:1. The next day, the culture medium was changed to remove non-viable cells and parasites. The monolayers were examined daily using an inverted phase-contrast microscope under 100-fold magnification (Figure 7). After the monolayer was destroyed by the parasite, the culture medium containing the extracellular tachyzoites was collected, parasites were counted, pelleted (900g, 10 min, 4°C), resuspended in complete RPMI medium at a density of ~106 parasites per ml and used to infect a new flask containing Vero cells at a MOI of 1:1 or for co-culture with DCs.

45 3 Methods

Figure 7 : In vitro culture of T.gondii tachyzoites visualized by phase contrast light microscopy 100x.

Preparation of soluble toxoplasma antigen (STAg) To prepare STAg, T. gondii RH tachyzoites were used. They were provided by J. Aliberti (CCHMC, Cincinnati, OH, USA) and maintained on human foreskin fibroblasts Hs27 (ATCC® CRL- 1634™, Manassas, Virginia, USA) following the protocols outlined in section 3.4.1. Lysed cells containing tachyzoites were transferred into a 50ml Falcon tube and pelleted (900g, 10min, 4°C). Then, cells were washed 3 times to remove medium traces by resuspension in 10ml fresh DPBS and centrifuged at 900g for 10min, 4°C. Cell pellets were frozen and stored at -80°C until 12 pellets were collected. For preparation of STAg, pellets were combined and resuspended in ice-cold DPBS to obtain a concentration of 5·108 cells/ml, placed into an ice-bath and sonicated 4x for 20sec. Intact parasites and cell debris were removed by centrifugation for 20 min at 900g, 4°C, the supernatant was collected, sterile-filtered and used in experiments. The protein concentration was quantified by spectrophotometry using Nanodrop. STAg was aliquoted and stored at -80°C.

3.5. T. gondii mouse infection model To obtain a stock of T. gondii ME49 tissue cysts, NMRI were infected with brain tissue cysts i.p. as described below in section 3.5.2. NMRI mice do not develop toxoplasmic encephalitis and survive the latent infection. They can be used for passaging of tissue cysts [340]. To obtain larger number of cysts, the stock was expanded in C57Bl/6 mice 4 weeks prior to the experiments [142].

Determination of brain cyst numbers in T. gondii-infected mice

Animals were euthanized by inhalation of CO2 and cervical dislocation. Then the head was wetted with 70% ethanol and the skin was incised to expose the skull. Using a pair of small curved scissors, the skull was opened to expose the brain, which was removed with forceps and placed into

46 3 Methods a 5ml reaction tube containing 1ml of sterile ice-cold DPBS. Brain tissue was homogenized using repeated aspiration and ejection with a 5ml luer lock syringe connected to either 18g, 21g or 26g needles. The number of tissue cysts in the brain suspension was determined under the phase- contrast light microscope at a 40-fold magnification. For this purpose, the sample was vortexed, 20µl of suspension was transferred onto the microscope slide and covered with a 20x20mm coverslip. The number of tissue cysts was counted in the entire sample and used to calculate the concentration of cysts per ml of brain suspension (Figure 8).

Figure 8: T. gondii tissue cyst in mouse brain homogenate visualized by phase contrast light microscopy (40x).

Intraperitoneal T. gondii infection For intraperitoneal T. gondii infection, a brain suspension in DPBS containing tissue cysts was prepared and the number of cysts was determined as described in the previous section. Then, the brain suspension was diluted with DPBS to adjust the final concentration of tissue cyst to 250 cysts/ml. 0,2ml of this solution containing 50 cysts was administered intraperitoneally to C57Bl/6 mice. Then, the progression was assessed by measuring the severity score of the disease, survival time, absolute and relative weight changes. Score and specific termination criteria were set in compliance with Section 31(1) sentence 2(1d) of Animal Protection Ordinance (Tierschutz- Versuchstierverordnung). If one of the following criteria was met, the affected animal was euthanized by inhalation of CO2 and cervical dislocation:

1. Weight loss of more than 20% of the initial weight at the beginning of the experiment. Relative

weight was calculated using following formula:

current weight, g relative weight, % = × 100% initial weight, g

2. If the score of 5 was reached. Severity score was determined as follows:

47 3 Methods

- Score 1: coat, appetite, gait, posture is normal. Mouse is alert and responds normally to the external stimuli (escape behavior, reactivity); - Score 2: Mouse is active, but moves slower than normal, back slightly humped, fur starts to become dull; - Score 3: Score 2 plus tilted head, ataxia and delayed righting reflex; - Score 4: Mouse is quiet, alert and responsive, but shows a strongly curved back, coarse fur, reacts with movement only to external stimuli; - Score 5: Mouse no longer responds to stimuli and is apathetic, lays on the side.

The leukocyte distribution was assessed by flow cytometry during the acute stage of T. gondii infection at day 5 post-infection in peritoneum, spleen, mesenteric lymph nodes and brain and during the chronic stage at day 30 in the brain. Serum was sampled during the acute stage of infection to measure cytokine levels (Figure 9) [142].

Figure 9: Experimental design for the analysis of mice infected i.p. with T. gondii.

3.6. Determination of serum cytokines in T. gondii-infected mice

Blood was obtained from mice through submandibular venipuncture [341] and collected in BD Microtainer tube. Tubes were inverted, incubated for 30min at RT for complete blood clotting and centrifuged at 12.500g for 5min at RT. Separated serum was collected in 0,5ml reaction tubes and stored at -20°C for further analysis.

Determination of cytokine production by enzyme-linked immunosorbent assay (ELISA) An enzyme-linked immunosorbent assay (ELISA) was used to determine cytokine levels in cell culture supernatants and serum samples. The amounts of IL-12/23p40, IL-12p70 and IFN-γ were determined using DuoSet ELISA-Kits from R&D Systems according to manufacturer’s

48 3 Methods recommendations. The reaction volume was reduced to 50µl without loss of sensitivity and reproducibility.

Determination of serum cytokine concentrations using the Bio- Plex ProTM (BioRad) assay The Bio-Plex ProTM Mouse Cytokine 23-Plex Panel kit (Bio-Rad Laboratories GmbH, München) was used to assess the cytokine content in mouse serum. It comprises quantitative determination of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70 and TNF-α in 12,5µl of serum sample. It is based on a sandwich immunoassay principle that uses 6,5µm magnetic beads as the substrate for the antibody sandwich. Beads are colored internally with two different fluorescent dyes (red and infrared). Different concentrations of red and infrared dyes are used to generate 23 distinct bead regions that are discriminated by Bio-Plex 200 flow cytometry-based reader. The assay was performed according to manufacturer’s instructions. Briefly, the capture antibody-coupled beads were first incubated with antigen standards or samples. Then the plate was washed to remove unbound materials, followed by incubation with biotinylated detection antibodies. After washing the unbound biotinylated antibodies, the beads were incubated with a reporter streptavidin-phycoerythrin conjugate (SA-PE). Following removal of excess SA-PE, the beads were passed through the Bio- Plex 200 reader, which measures the fluorescence of the bound SA-PE.

Determination of serum cytokine concentrations using the Multiplex (MSD) assay Serum samples were analyzed using the V-PLEX Proinflammatory panel 1 (mouse) kit from MSD. Briefly, this assay uses a sandwich immunoassay principle, where the microtiter plate is provided pre-coated with capture antibodies on 10 independent spots. Capture antibodies of the multiplex assay plates are immobilized on the working electrode surface and bind the analyte from the sample. Detection antibodies conjugated with electrochemiluminescent labels (MSD SULFO- TAGTM) are recruited by the analyte and complete the sandwich assay. Subsequently, the reading buffer, provided in the kit, is applied to ensure the appropriate chemical environment. The plate is inserted into the MSD instrument, where a voltage is applied to the plate electrodes that causes the captured labels to emit light. The instrument measures the intensity of emitted light to provide a quantitative measure of the analytes in the sample.

The assay was performed according to manufacturer’s protocol. Briefly, 50µl of each sample (1:4 dilutions with assay buffer) or standard were applied to the wells and incubated for 2h with shaking at RT. The plate was washed 3 times with MSD wash buffer, the detection antibody mix was applied and the plate was incubated for an additional 2h. After that, plate was washed 3 times with

49 3 Methods

MSD wash buffer and 150µl of reading buffer was added into wells. Finally, the plate was analyzed in the MSD instrument.

Determination of serum Alanine amino transferase (ALT) activity Alanine Aminotransferase (ALT), also known as serum glutamic-pyruvic transaminase (SGPT), is a pyridoxalphosphate-dependent enzyme that catalyzes the reversible transfer of an amino group from alanine to -ketoglutarate, generating pyruvate and glutamate. ALT is found primarily in liver and injury often results in an increase of serum ALT levels, therefore, it can be used as a marker for liver injury. ALT activity was determined using the Alanine Aminotransferase Activity Assay Kit (Sigma-Aldrich Chemie GmbH, Steinheim). This is a coupled enzyme assay, which results in a colorimetric (570nm) product, proportional to the pyruvate generated. The procedure was performed in 96-well plate according to manufacturer’s instructions using 6µl of each serum sample per well. The absorbance at 570nm was measured using the ELISA-Reader Fluostar Omega. One unit of ALT is defined as the amount of enzyme that generates 1,0mole of pyruvate per minute at 37C.

3.7. Flow cytometry

The different immune cells located in the spleen, peritoneum, MLNs and brain were identified phenotypically using flow cytometry. For this purpose, 105-5·106 cells were resuspended in 100µl of FC buffer and incubated with 10µg/ml anti-CD16/CD32 antibody for 15min at 4°C to block Fcγ receptors and prevent unspecific binding of antibodies. For the specific staining, antibodies listed in Table 8 were used at the given dilutions for the preparation of an antibody master mix using FC buffer as diluent. Then cells were washed with 1ml of FC buffer at 350g for 5min, RT, resuspended in 100µl of master mix and incubated for 20min at 4°C. Then cells were washed with 1ml of FC buffer, resuspended in 300µl of BD Cytofix™ buffer and incubated for 10min at 4°C and washed again with 1ml of FC buffer. Finally, the cell pellet was resuspended in 300µl of FC buffer and analyzed using the BD LSRII flow cytometer. To analyze cell subsets, up to 1·106 cells were recorded with a threshold set at 5,000-10,000 to exclude small particles and cell debris. Cell debris was

50 3 Methods excluded from the analysis based on the SSC/FSC signal and doublets were excluded according to FSC-A/FSC-H signal ratio as shown in Figure 10.

Figure 10: Typical plots showing the gating strategy to exclude cell debris (A) and doublets (B) from analysis. Single cells were defined to have an 1:1 FSC-A/FSC-H signal ratio.

Intracellular cytokine staining Intracellular cytokine staining was performed in fixed and permeabilized cells using the BD Cytofix/Cytoperm™ kit. A minimum of 107 cells were pelleted in a 50ml falcon tube (350g, 5min, 4°C) and resuspended in 10ml of complete RPMI medium with addition of 10µl Brefeldin A (1000x stock, final conc. 3,0µg/ml.) to keep accumulation of cytokines inside of cells. The cell suspension was incubated for 2h (5% CO2, 37°C). After that, cells were pelleted (350g, 5min, 4°C), resuspended in 1ml FACS buffer and ~3·106 cells were transferred into separate 1,5ml reaction tube. Cells were first stained for surface markers as described above and then the BD Cytofix/Cytoperm™ kit was used for intracellular labelling. Briefly, the cell pellet was resuspended in 300µl of Fixation/Permeabilization solution and incubated for 20min at 4°C, then washed with 1ml of BD Perm/Wash™ buffer (350g, 5min, 4°C) and resuspended in 100µl of 1x Perm/Wash™ buffer containing fluorescently labelled antibodies (see Table 8), incubated for 30min at 4°C to allow penetration and binding of the antibodies to their intracellular targets. After incubation, unbound antibodies were removed by washing with 1ml of BD Perm/Wash™ buffer (350g, 5min, 4°C) and the pellet was resuspended in 300µl of FACS buffer. Finally, cells were analyzed using the BD LSRII flow cytometer as described above.

Tetramer staining T. gondii-specific tetramers were provided by J. Aliberti (obtained from NIH Tetramer Core Facility). APC-conjugated TGME49 tetramers were used to determine T. gondii specific CD4+ T-cells and PE-conjugated TGD057 for CD8+ T-cells in the spleen and brain. For tetramer staining, CD3/CD4/CD8 stained cells in 100 µl of FC buffer were incubated with 10 µl of each tetramer solution

51 3 Methods for 1h at 37°C, then washed with 1ml of FC buffer, resuspended in 300 µl of FC buffer and analyzed using BD LSRII as described above. The following two tetramers were used:

Tetramer-PE Toxoplasma gondii TGD057 H-2 Kb (SVLAFRRL) Tetramer-APC Toxoplasma gondii TGME49 I-A(b) (AVEIHRPVPGTAPPS)

3.8. Statistical analysis

Statistical analysis was performed using the GraphPad Prism version 6. The data are presented as scatter plots that show the individual values and the mean ± standard error of the mean (SEM). Outliers were excluded from analysis using ROUT method (robust regression and outlier removal) with Q=1%. Multiple group comparison was done using one-way analysis of variance (one- way ANOVA) with Dunnett multiple comparison test. Analysis of groups with two different independent variables was done using two-way ANOVA with Sidak multiple comparison test. The log-rank (Mantel-Cox) test was used to compare the survival curves of two groups. P-values ≤ 0,05 were considered statistically significant.

4 RESULTS

4.1. Impact of C5aR1 and CCR5 activation on the dendritic cell response to Toxoplasma gondii antigens in vitro Expression of recombinant Toxoplasma gondii cyclophilin-18 Recombinant Toxoplasma gondii cyclophilin-18 (TgCyp18) was expressed as a glutathione S-transferase (GST) fusion protein in bacterial culture as described in section 3.1. Samples obtained at the different stages of protein expression were analyzed using SDS-PAGE electrophoresis to verify the presence and size of the target products (Figure 11).

Figure 11: Monitoring of the expression of TgCyp18 in the bacterial culture. Samples were analyzed by SDS-PAGE electrophoresis on a 4-15% acrylamide gel followed by Coomasie R250 staining. 1. Standard ladder, 2. Bacterial culture before protein induction, 3. After addition of IPTG, 4. Culture supernatant, 5. Bacterial culture lysate after sonication, 6. Lysate after recombinant protein extraction, 7. Recombinant protein after on-column cleavage, 8. Fraction that contains GST-tag and traces of uncleaved protein eluted from the column.

I purified recombinant TgCyp18 from the bacterial homogenate using glutathione agarose affinity columns and eluted the protein after on-column cleavage of the GST-tag by thrombin. I collected the fractions that contained the protein and analyzed them by SDS-PAGE electrophoresis to assess the composition of the eluted protein as described in section 3.1.6. I found a band with an approximate molecular weight of 18kDa on the gel that corresponds to the molecular mass of TgCyp18 (Figure 12A) [178]. I also stained the gel with a silver solution to increase the sensitivity of the assay and to assess the purity (Figure 12B). Fractions that contained recombinant TgCyp18

53 4 Results were pooled and the protein concentration was determined spectrophotometrically by NanoDrop. The total yield of the recombinant protein was 6,74mg.

Figure 12: Recombinant TgCyp18 fractions obtained after on-column cleavage of the GST-tag. Samples were analyzed using SDS-PAGE electrophoresis on a 4-15% acrylamide gel followed by (A) Coomasie R250 or (B) silver staining. 1. Standard ladder, 2-4, 6-8. Different elution fractions collected from the column, 5,9. Fractions that contain the GST-tag.

Previously, it was reported that low-dose LPS (1ng/ml) is not sufficient to induce robust IL-12 production but has the potential to prime DCs and to enhance the effect of other ligands like TNF-α and IFN-γ [342] and modulate signaling networks responsible for IL-12 production [343]. A similar effect has been described for IL-6 production from macrophages [344]. These findings suggest that even low amounts of LPS change immune cell responses response during in vitro stimulation experiments. Thus, I aimed to remove the LPS contamination from the TgCyp18 protein preparation.

To get rid of LPS, I loaded TgCyp18 protein onto an EndoTrap® Red (Hyglos GmbH, Bernried am Starnberger See) affinity chromatography column as described in section 3.1.4. The efficiency of the LPS removal was confirmed using the EndoZyme® (Hyglos GmbH, Bernried am Starnberger See) recombinant factor C endotoxin detection assay as described in section 3.1.5. The final preparation contained ~0,23EU of endotoxin (~0,23-0,46ng) per microgram of recombinant protein. The final concentration of the detoxified TgCyp18 was 0,8mg/ml (44,4µmol/l) in DPBS.

TgCyp18 is an enzyme with peptidyl-prolyl isomerase activity, which catalyzes the isomerization of peptide bonds from the trans to cis isoform at proline residues and facilitates protein folding [345]. I performed a peptidyl-prolyl isomerization assay as described in section 3.1.9. The change in the optical density was analyzed by non-linear regression analysis and fitted to an exponential decay curve with calculation of the first-order rate constant of the reaction for the recombinant protein, STAg and mouse serum (Figure 13). Recombinant TgCyp18 had no enzymatic activity in this assay. Its reaction constant was ~200 times lower than the one of the fresh mouse serum used as a positive

54 4 Results control. The STAg preparation also showed no-to-low enzymatic activity that might be explained by time-dependent decay [337].

Figure 13: Peptidyl-prolyl isomerization assay. (A) Exponential curve fit of values obtained in the assay; (B) first-order rate constants of mouse serum, STAg and recombinant TgCyp18.

It was described previously that the enzymatic activity is not necessary for binding of

TgCyp18 to CCR5, but it is required for the induction of IL-12 [180]. Despite the low K0 determined for STAg and TgCyp18, I decided to assess the potency of the prepared TgCyp18 to promote IL-12 production from spleen-derived DCs.

Interleukin-12 is secreted by dendritic cells upon challenge with Toxoplasma gondii antigens in vitro I determined IL-12 production in vitro using MACS-enriched primary mouse spleen DCs as it was shown that CD8α+ cells from the mouse spleen serve as the main source of IL-12 and are a crucial part of innate immune sensing of this parasite [278]. Primary DCs from mouse spleen were prepared using anti-CD11c MACS beads as described in section 3.2.6. The composition of the final cell preparation was assessed using flow-cytometry. The enriched cells contained ~80% of CD11c+ including ~15% of CD8+CD11c+ cells (Figure 14). I stimulated the enriched DCs using recombinant TgCyp18 prepared as described in the previous section. Another recombinant TgCyp18 and soluble tachyzoite antigen (STAg) were obtained from our collaboration partner J. Aliberti (CCHMC, Cincinnati, OH, USA) and TLR9 antagonist CpG ODN1668 was purchased from InvivoGen, San Diego, USA. The amount of IL-12 p40 and p70 was determined by ELISA.

55 4 Results

Figure 14: Composition of MACS-enriched DC culture from mouse spleen as determined by flow cytometry. A. Mouse spleen cell suspension before (A) and after (B) positive CD11c selection. Numbers in the gates represent the frequencies within the 4 quadrants.

I found no IL-12 production from DCs in response to treatment with recombinant TgCyp18 (50µg/ml), whereas CpG and STAg (conc. 1µg/ml) induced a strong IL-12p40 response (Figure 15 left). STAg also induced a strong IL-12p70 production, whereas the CpG-induced IL-12p70 production was minor (Figure 15 right).

Figure 15: Production of IL-12p40 and p70 by sDC culture in response to T. gondii-derived ligands. Concentration of IL-12 p40 (left) and p70 (right) was determined upon addition of the TLR9 antagonist CpG 1668 (1µg/ml), STAg (1µg/ml), recombinant TgCyp18-1 (50µg/ml) or recombinant TgCyp18-2 provided by the Aliberti lab (50µg/ml) as determined by ELISA. The dotted line represents the lower detection limit of the assay. n=3, bars shown are the mean ± SEM. Statistical significant differences are indicated by * as determined by ANOVA, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001.

To test whether STAg induces TLR-independent IL-12 production, I used spleen DCs from MyD88-deficient animals. These cells are unable to respond to TLR stimulation as MyD88 serves as a central adapter protein used by most TLRs (except TLR 3) in a signaling cascade that activates the transcription factor NF-κB (reviewed in [346]). 56 4 Results

STAg (0,01-50µg/ml) induced IL-12 production in a dose-dependent manner from WT but not from MyD88-deficicent cells (Figure 16).

Figure 16: Production of IL-12 in WT and MyD88-/- sDC culture in response to STAg. Levels of IL-12 p40 (left) and p70 (right) secreted in response to STAg (0,01-50µg/ml) in WT (closed circle) or MyD88-/- (open circles) cells. IL-12 levels were determined by ELISA, n=2.

This finding was surprising as it did not recapitulate the result of studies showing MyD88- independent IL-12 induction from DCs using recombinant TgCyp18 or STAg [181]. Next, I tested whether the absence of CCR5 or C5aR1 signaling may have an impact on MyD88-mediated signaling induced by toxoplasma antigens as cross-talk between GPCR and TLR signaling has been shown [106]. I found that in the absence of C5aR1 and/or CCR5, IL-12p70 production in response to CpG and STAg was significantly decreased (Figure 17A, B). C5aR1-deficiency also caused

decrease in IL-12p40 secretion in response to STAg (Figure 17B left). In contrast, the C5aR1 and/or CCR5 signaling in sDCs did not alter the amount of IL-12p40 or IL-12p70 secreted in response to T.gondii tachyzoites when stimulated at a MOI 1:1 for 24h in vitro in comparison to WT DCs (Figure 17C).

57 4 Results

Figure 17: Production of IL-12 from C5aR1 and/or CCR5-deficient sDCs in vitro. Spleen DCs from WT, C5aR1-/-, CCR5-/- and C5aR1xCCR5-/- were stimulated with (A) CpG ODN1668, (B) STAg or (C) tachyzoites at a MOI 1:1 for 24h and the amount of IL-12p40 (left) and IL-12p70 (right) was measured by ELISA. n=5-6, bars shown are the mean ± SEM. Statistical significant differences are indicated by * as determined by ANOVA. * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001.

58 4 Results

Impact of C5a, CCL5 stimulation or T. gondii tachyzoite infection on C5aR1 or CCR5 internalization in DCs I examined whether C5aR1/CCR5 heterodimer formation facilitates receptor co- internalization after heterologous receptor activation. First, I aimed to show that stimulation of both receptors with their cognate ligands C5a or CCL5 (RANTES) results in homologous internalization of C5aR1 or CCR5 from the surface of transfected cells. Labelled C5aR1 or CCR5 were expressed in BMDCs or peritoneal macrophages by transfection with a plasmid that encodes a GFP fusion protein of the receptors. Then they were visualized using a laser-scanning confocal microscope with an incubator chamber as described in section 3.3

C5aR1 and CCR5 receptor clustering and internalization from the surface of the cells occurred as soon as 5 min and had increased until ~20 min upon addition of the cognate ligands (Figure 18 B, D). Imaging of the cells for the same period without stimulation did not show any alterations in the receptor distribution (Figure 18 A, C). Based on these findings, I conclude that C5aR1 and CCR5 responded to their cognate ligands and were functional in transfected cells.

Next, I tested whether addition of C5a changed the distribution of the GFP-labelled CCR5 in the cells. There was no change in CCR5 localization in the cells upon addition of C5a at the final concentration 1µg/ml during the observation period of 30min (Figure 19 A). Addition of 1µg/ml of CCL5 to the same chamber resulted in theCCR5 polarization and internalization (Figure 19 D). This finding confirms receptor functionality. It further show that C5a and CCL5 do not drive C5aR1 or CCR5 cross-internalization in BMDCs as has been shown for RBL cells.

The presence of T.gondii tachyzoites to the culture at an MOI 1:1 did not result in a change of C5aR1 or CCR5 localization during the observation period of 30 min (Figure 20 A, C). As a positive control, C5a was added to the chamber with DCs and T. gondii parasites. Under these conditions, I found a clear evidence for receptor internalization into vesicles confirming their functional capability to respond to the cognate ligand (Figure 20 B) .

59 4 Results

Figure 18: Homologous internalization of C5aR1 and CCR5 in BMDCs. C5aR1-GFP localization in steady state (A) and internalization upon addition of 1µg/ml of C5a (B); CCR5-GFP localization in steady state (C) and internalization upon addition of 1µg/ml of CCL5 (D). Cells were visualized using a LSM confocal microscope for ~30min at 37°C, 5% CO2 as described in section 3.3.3. White arrows indicate points or receptor clustering and internalization.

60 4 Results

Figure 19: Cross-internalization of CCR5 in BMDCs. Dynamics of CCR5 localization upon addition of (A) C5a (1µg/ml) and (B) C5a (1µg/ml) + CCL5 (1µg/ml). Cells were visualized using a LSM confocal microscope for ~30min at 37°C, 5% CO2 as described in section 3.3.3. White arrows indicate points or receptor clustering and internalization.

61 4 Results

Figure 20: T. gondii-induced internalization of C5aR1 and CCR5 in BMDCs. Localization of C5aR1 (A) and CCR5 (C) was visualized upon addition of T. gondii tachyzoites at a MOI 1:1 and (B) C5a (1µg/ml). Cells were visualized using a LSM confocal microscope for ~30min at 37°C, 5% CO2 as described in section 3.3.3. White arrows indicate points or receptor clustering and internalization.

62 4 Results 4.2. The role of C5aR1 and CCR5 activation in mouse survival during intraperitoneal T. gondii infection

To study the impact of C5aR1 and CCR5 activation on mouse survival, I used the intraperitoneal T. gondii infection model described in section 3.5.2. WT, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice were infected with 50 T. gondii ME49 cysts i.p. and observed for survival for 30 days. I found a significant decrease in the survival of C5aR1-/- mice in comparison to WT (Figure 21 A). In contrast, I observed no significant differences between the survival rates of CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- and WT mice (Figure 21 B, C, D, E).

Figure 21: Survival of WT, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice infected with T. gondii. WT, C5aR1-/- (A), CCR5-/- (B), C5aR1xCCR5-/- (C), C5aR2-/- (D) and C5-/- (E) mice were infected and evaluated for survival until day 30 p.i. Statistical significant differences are indicated by * as determined by Log-rank (Mantel-Cox) test, n=9-40, * P ≤ 0,05.

It was reported previously that male and female mice show differences in their susceptibility to T. gondii infection that is mediated by difference in the amount and kinetic of cytokine production [347]. It has also been reported that female C57Bl/6 mice have lower levels of terminal complement components [348]. Therefore, I determined potential differences in the resistance of male and female WT and C5aR1-/- mice to T. gondii infection. I observed a significantly higher survival rate of male WT in comparison to male C5aR1-/- mice, while there was no significant difference in female mice (Figure 22).

63 4 Results

Figure 22: Survival of male and female WT and C5aR1-/- mice infected with T. gondii. Male (A) and female (B) WT (solid line) and C5aR1-/- (dashed line) mice were infected and evaluated for survival until day 30 p.i. Statistical significant differences are indicated by * as determined by Log-rank (Mantel-Cox) test, n=12-21, * P ≤ 0,05.

I also assess the impact of T. gondii infection on animal weight. I observed a significantly higher weight of C5aR1-/- animals in comparison to WT mice at the beginning and during the first 9 days after T. gondii infection, whereas in other knockout groups the weight was similar to WT mice (Figure 23). This observation is in agreement with a previous report showing that C5aR1-/- have a higher weight under normal diet [349].

Figure 23: Absolute weight of WT, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice infected with T. gondii. WT, C5aR1-/- (A), CCR5-/- (B), C5aR1xCCR5-/- (C), C5aR2-/- (D) and C5-/- (E) mice were infected and weight was measured daily until day 30 p.i. Values shown are the mean ± SEM, n=8-35 Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05.

There was a similar kinetic of the weight loss in WT and knockout mice. I observed a steep decrease of ~15% of the relative weight between days 5 and 10 after infection. At day 10, I found a significantly lower relative weight of C5aR1-/- in comparison to WT mice, whereas the weight loss in the other groups was similar to WT mice (Figure 24 A). Further, I observed a slight recovery and weight gain of ~5% between days 10 and 15 after infection in all groups except C5aR1xCCR5-/-mice, where this effect was less pronounced (Figure 24 C). From day 15 after T. gondii infection, the relative weight gradually decreased in all groups of mice (Figure 24).

64 4 Results

Figure 24: Relative weight of WT, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice infected with T. gondii. WT, C5aR1-/- (A), CCR5-/- (B), C5aR1xCCR5-/- (C), C5aR2-/- (D) and C5-/- (E) mice were infected and relative weight was calculated daily until day 30 p.i. as described in section 3.5.2. Values shown are the mean ± SEM, n=8-35 Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05.

Next, I determined the severity score of T. gondii infection as described in section 3.5.2. I observed clinical signs of the disease starting from day 8 in WT and all knockout groups (Figure 25 A, B, C, D, E). Mice that survived the acute stage of systemic infection showed an improved clinical score at ~21 day after infection and developed latent infection in the brain. Eventually, all C57Bl/6 mice developed T. gondii-induced encephalitis at ~60 days after infection (data not shown). I observed significantly higher mean of the severity score in C5aR1-/-, CCR5-/- and C5aR2-/- animals in comparison to WT mice, whereas C5aR1xCCR5-/- and C5-/- mice had a score similar to the WT group (Figure 25 F).

Figure 25: Severity score of T. gondii infection in WT, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice. WT, C5aR1-/- (A), CCR5-/- (B), C5aR1xCCR5-/- (C), C5aR2-/- (D) and C5-/- (E) mice were infected and the severity score of the disease was determined daily until day 30 p.i. as described in section 3.5.2. Values shown are the mean ± SEM, n= 3-18. Statistical significant differences of the mean severity score (F) are indicated by * as determined by ANOVA, ** P ≤ 0,01, *** P ≤ 0,001.

65 4 Results

Parasite load in the brain of chronically infected mice It has been shown that mice that are unable to control the parasite replication in the brain succumb to infection [216]. T. gondii cysts cause pathological changes in the mouse brain contribute the pathogenesis of the disease [350-353]. I determined the number of tissue cysts in the CNS at day 30 after infection in mice that survived the acute stage of T. gondii infection. The numbers of brain cysts were significantly increased in C5aR1-/-, C5aR1xCCR5-/- and C5aR2-/- mice in comparison to WT animals, whereas the parasite burden in the brain of CCR5-/- and C5-/- mice was similar to WT (Figure 26 left graph).

Figure 26: Number of T. gondii tissue cysts in mouse brain. Tissue cysts in the brain were counted at day 30 after intraperitoneal infection in WT, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice (left). Right graph represents cyst number in female (open bar) and male (filled bar) mice. Values shown are the mean ± SEM, n=9-35. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001.

In line with previous observations, female mice were more susceptible to the infection and had higher parasite burden in the brain than male mice (Figure 26 right graph). In general, the increase in cyst numbers over WT was more pronounced in C5aR1-/- and C5aR2-/- male than in female animals. This findings might be explained by more potent activation of terminal complement components in male animals [348].

66 4 Results 4.3. Impact of C5aR1 or CCR5 activation on cytokine response to acute T. gondii infection

I used mouse STAg injection and intraperitoneal infection models to assess the impact of C5aR1 or CCR5 activation serum cytokine production in response to the parasite in vivo. Injection with STAg gives insights into the initial immune recognition and early responses to toxoplasma antigens. The intraperitoneal infection model allows to study the complex cellular interplay of innate and adaptive immunity during acute and chronic stages of infection and minimizes the impact of gut microbiota on the induction of the immune response.

Early cytokine response to soluble toxoplasma antigen in vivo Mice were injected with 100µg of STAg in 0,2ml of DPBS i.p. and blood was collected 6h after the injection as described in section 3.6. Recognition of the antigen by the innate immune system resulted in systemic secretion of Th1 cytokines into the bloodstream that were detected using ELISA. High amounts of IL-12p40, IL-12p70 and IFN-γ were found in serum as soon as 6h after administration of the antigen in wild type animals. In contrast, neither IL-12p40 nor p70 increased in C5aR1-/- mice, whereas I found a clear systemic increase in IL-12p40 and p70 levels in C5aR1xCCR5-/- mice that was similar to that observed in WT mice (Figure 27 left, middle graphs). Further, IFN-γ levels in C5aR1-/- mice increased only slightly and no IFN-γ secretion was induced in C5aR1xCCR5-/- mice. Thus, signaling through both, C5aR1 and CCR5, seem to be of major importance for the induction of IFN-γ in response to STAg (Figure 27 right graphs).

Figure 27: Serum cytokine levels in response to STAg injection in WT, C5aR1-/- and C5aR1xCCR5-/- mice. 100µg of STAg in 0,2ml DPBS were administered i.p.; 6h after the injection blood was collected and serum cytokine levels were determined by ELISA. Values shown are the mean ± SEM. Statistical significant differences are indicated by * as determined by ANOVA, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001.

67 4 Results

Serum cytokine response following acute T. gondii infection Wild type, C5aR1-/-, CCR5-/- and C5aR1xCCR5-/- mice were injected i.p. with 50 brain cysts as described in section 3.5 to assess the early immune response to T. gondii. Blood was collected and serum separated from uninfected animals, 1, 3, 5 and 7 days after infection. I found a similar kinetic of IL-12p40 secretion in the WT, C5aR1-/-and C5aR1xCCR5-/- groups at the indicated time points. IL-12p40 levels increased on day 3 and reached a maximum on day 5 which remained similar on day 7. C5aR1-/- mice had slightly lower IL-12p40 levels at day 5 after infection, but were like WT at day 7. At day 7 the IL-12p40 levels in CCR5-/- mice were significantly lower as compared with all other groups. IFN-γ levels increased strongly in all groups at day 5 and were highest in CCR5-/- mice. At day 7, the IFN-γ levels increased strongly in WT, whereas the increase was not as pronounced in C5aR1-/- and C5aR1xCCR5-/- mice and absent in CCR5-/- mice.

Figure 28: Early kinetics of IL-12p40 and IFN-γ levels in T. gondii-infection. Wild type, C5aR1-/-, CCR5-/- and C5aR1xCCR5-/- mice were infected with 50 brain cysts i.p. Serum was sampled from animals 1, 3, 5 and 7 days after infection. The amount of IL-12p40 and IFN-γ in the serum was determined by ELISA. Values shown are the mean ± SEM, n=5-33. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, *** P ≤ 0,001.

To study the regulation of other cytokines by C5aR1, levels of IL-1β, IL-2, IL4, IL-5, IL-6, IL- 10, TNFα and KC were analyzed in the serum of wild type and C5aR1-/- animals at day 7 after infection using Bio-Plex ProTM (Bio-Rad) as described in section 3.6.2. Serum levels of IL-6, IL-10, IL-12p70, IFN-γ, KC, MCP and MIP-1α were significantly decreased in C5aR1-deficient animals. G- 68 4 Results

CSF and GM-CSF showed a similar tendency. CCL5 (RANTES), which is a cognate ligand for CCR5, was increased in the absence of C5aR1 (Figure 29).

Figure 29: Serum cytokine levels in WT and C5aR1-deficient mouse 7 days after T. gondii infection as determined by Bio-Plex ProTM assay. Serum from wild type and C5aR1-/- mice was sampled at day 7 after i.p. T. gondii infection. The amount of IL-1α, IL-1β, IL-2, IL-3, IL-5, IL-6, IL-10, IL12-p40, IL-12p70, IL-13, IL-17, eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MCP, MIP-1α, MIP-1b, RANTES and TNFα in serum was determined by Bio-Plex Pro Mouse Cytokine 23-Plex Panel (Bio- Rad Laboratories GmbH, München). n=3, values shown are the mean ± SEM. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001.

The findings suggest that IL-12p70 production is controlled by C5aR1 activation during the initial stage of T. gondii infection and this deficiency in IL-12 production causes the decrease in IFN- γ secretion. Next, I determined whether CCR5, C5aR2 or C5 had a similar impact on the IL-12p70 levels. For that purpose, I used WT, C5-/-, C5aR1-/-, C5aR2-/-, CCR5-/- and C5aR1xCCR5-/- mice. Blood was sampled from these mice 7 days after i.p. T. gondii infection. All groups showed similar levels of IL-12p40. In sharp contrast, C5aR1-/-, C5aR1xCCR5-/- and C5aR2-/- animals showed a massive decrease in IL-12p70 and IFN-γ, whereas in C5-/- I observed only a drop in IL-12p70 levels (Figure 30).

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Figure 30: Regulation of IL-12p40, IL-12p70 and IFN-γ in the serum 7 days after T.gondii infection. Wild type, C5aR1- /-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice were infected with 50 brain cysts i.p. and serum was drawn 7 days after infection. The amount of IL-12p40 and IFN-γ in the serum was determined by ELISA; The amount of IL-12p70 was determined by Multiplex analysis (MSD) as described in section 3.6.3. n=5-26, values shown are the mean ± SEM. Statistical significant differences are indicated by * and were determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001.

It was described previously that T. gondii infection induces liver damage, lipid change and hepatic steatosis that is increased in the absence of CCR5 [354]. Additionally, IFN-γ exacerbated liver damage, the hepatic progenitor cell response and fibrosis in a mouse model of chronic liver injury [355]. Therefore, I investigated whether increased systemic IFN-γ level correlated with a change in the serum level of alanine amino transferase (ALT), an enzyme localized in hepatocyte cytoplasm and commonly used as a marker for liver damage [356].

I observed an upregulation of ALT levels in the serum of WT and C5aR1-/- mice that confirms T. gondii-induced liver damage. However, there was no significant difference in ALT levels observed in the serum of the infected WT, C5-/-, C5aR1-/-, C5aR2-/-, CCR5-/- or C5aR1xCCR5-/- mice (Figure 31).

Figure 31: Levels of alanine amino transferase (ALT) in the serum of WT, C5-/-, C5aR1-/-, C5aR2-/-, CCR5-/- and C5aR1xCCR5-/- mice 7 days after T.gondii infection. Control ALT levels were measured in the serum of uninfected WT and C5aR1-/- mice. Analysis was performed by ALT Activity Assay kit (Sigma) as described in section 3.6.4. Values shown are the mean ± SEM. Statistical significant differences are indicated by * as determined by ANOVA, *** P ≤ 0,001.

70 4 Results 4.4. Impact of the C5a/C5aR1 axis on the cellular response during acute T. gondii infection

I analyzed, how activation of the C5a/C5aR1 axis on different immune cells regulates cellular responses during the acute stage of T. gondii infection. For this purpose, mice were sacrificed 5 days after i.p. T. gondii infection and the cellular response in the peritoneal cavity, spleen, mesenteric lymph node and the brain was analyzed using flow cytometry.

I found that the number of cells isolated from the peritoneal cavity was increased in WT and C5aR1-/- mice. Surprisingly, the increase in C5aR1-/- mice was significantly higher than in WT animals. Whereas the cell number did not increase in the spleen and a MLN in WT mice, I observed a significant increase in the assessed organs of C5aR1-/- animals. Also in the brain, I noticed a tendency towards a higher cell number in C5aR1-/- as compared with WT mice after infection (Figure 32).

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Cellular responses in the spleen against T. gondii infection 4.4.1.1. IL-12 secretion by spleen cDCs Spleen cDCs are divided into two major fractions according to their expression of CD8α+ and CD11b+. CD8α+ DCs initiate the immune response against T.gondii through IL-12 production as reviewed in section 1.4.2. I analyzed the spleen DC compartment before and 5 days after T. gondii infection. The gating strategy to define CD8α+ and CD11b+ cDCs is shown in Figure 33.

Figure 33: Gating strategy to identify mouse spleen DC subsets that produce IL-12. The CD8α+ subset of splenic DCs was identified as a CD3-CD11c+CD11b-CD8+ cell population, the CD11b+ subset was identified as a CD3- CD11c+CD11b+CD8+ cell population. IL-12 production in DCs was determined using intracellular staining. Shown are cells from uninfected mice (A) and mice 5 days after T. gondii infection (B) as described in section 3.7.1. Numbers shown in the gate represent the frequencies of the parent population.

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Wild type mice have similar numbers of CD8α+ and CD11b+ DCs before and after infection, whereas numbers of both populations are elevated in C5aR1-deficient animals at day 5 p.i. (Figure 34 A, B left graph). Within the CD8α+ DC population, only cells from C5aR1-/- but not from WT mice showed a significant increase in IL-12 production (Figure 34 A, middle and right graphs). In contrast, I found a significant increase in IL-12 producing CD11b+ DCs in WT and C5aR1-/- mice after infection (Figure 34 B, middle and right graphs).

Figure 34: IL-12 production from CD8α+ and CD11b+ sDCs during acute T.gondii infection. Numbers of CD8α+ (A, left panel) and CD11b+ (B, left panel) cDCs in the spleen from uninfected (open circles) and infected (closed circles) mice 5 days after T. gondii infection. Graphs represent total numbers of cell subset (left), frequency (middle) and number of IL- 12+ cells (right). Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA. * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001. 4.4.1.2. IFN-γ production by NK, NKT and T-cells NK cells are known to be the main IFN-γ producing cell type during the initial stage of T.gondii infection, whereas T-cells become the major IL-12-secreting population at later time points [242]. NK, NKT and T-cells were defined according to the gating strategy shown in Figure 35.

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Figure 35: Gating strategy to identify NK, NKT and T-cells in the mouse spleen that produce IFN-γ. Contour plots represent the gating strategy to identify NK (I. NK1.1+CD3-), NKT (II. NK1.1+CD3+) and T-cells (III. NK1.1-CD3+) in the mouse spleen. Cells from (A) uninfected mice and (B) mice infected for 5 days were analyzed for IFN-γ production by intracellular staining as described in section 3.7.1.

In this experiment, the numbers of NK and NKT-cells did not change significantly in the infected WT and C5aR1-/- mice (Figure 36 A, left and middle graph). However, C5aR1-/- animals had higher number of T-cells in the spleen after infection (Figure 36 A, right graph). Further in WT and C5aR1-/- animals, NK, NKT and T-cells upregulated IFN-γ+ production after infection. I observed the higher frequency of IFN-γ+ cells in the NK cell population (~10%). Importantly, the frequency of IFN-

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γ+ NK cells was significantly decreased in C5aR1-/- mice (Figure 36 B, left graph). Total numbers of IFN-γ+ NK, NKT and T-cells isolated from the spleen of WT and C5aR1-/- animals are elevated upon infection (Figure 36, C). There was a significantly higher number of IFN-γ+ NKT cells in the spleen of C5aR1-/- mice after infection (Figure 36 C, middle graph). Similarly, I observed a higher tendency of INF-γ+ T-cells in C5aR1-deficient mice.

Figure 36: Production of IFN-γ by spleen NK, NKT an T-cells at day 5 after T. gondii infection. Total cell numbers (A), frequency (B) and number of IFN-γ+ (C) cells among NK (left), NKT (center) and T-cells (right). Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001.

4.4.1.3. Assessment of the mononuclear and polymorphonuclear phagocyte compartments Important functions of neutrophils and macrophages in T. gondii infection include parasite clearance through their effector mechanisms. In addition to DCs, neutrophils and macrophages are also known to contribute to IL-12 production and release in response to parasite infection as reviewed in section 1.4.2. Following the gating strategy shown below, I identified neutrophils, macrophages, Ly6Chi pro-inflammatory and Ly6Clo resident monocytes in the spleen of WT and C5aR1-/- uninfected mice and 5 days after T. gondii infection. Ly6Chi pro-inflammatory monocytes 75 4 Results express CCR2 that couples to the same Gαi2 adaptor molecule as C5aR1 and mediates the migratory properties of the cells [82]. Therefore, I determined the levels of CCR2 expression on Ly6Chi cells before and after infection and assessed whether it affected receptor expression or turnover (Figure 37).

Figure 37: Identification of neutrophils and monocyte/macrophage populations in the spleen of uninfected and infected animals. NK1.1+ and CD11c+ cells were analyzed in the previous section and excluded from this gating strategy. Representative contour plots for identification of (a) Ly6G+ neutrophils, (b) F4/80+CD11bint macrophages, (c) F4/80- /intCD11b+Ly6CloCCR2- resident monocytes and (d) F4/80-/intCD11b+Ly6ChiCCR2+ pro-inflammatory monocytes. Histograms on the right show CCR2 expression in Ly6Chi monocytes. Dashed line show isotype control staining. Lin marker included CD3 and CD19. Cells were analyzed in the uninfected WT and C5aR1-/- animals (A) and 5 days after infection (B). Numbers represent the frequencies of the parent population. I observed that T. gondii infection caused significant infiltration of neutrophils into the spleen to a similar level in WT and C5aR1-deficient animals (Figure 38a). There was no significant change in the numbers of F4/80+ macrophages in WT and C5aR1-/- mice after infection, however, C5aR1-/- animals had slightly higher numbers of macrophages after infection than WT animals (Figure 38b). In contrast, I found significant decrease in numbers of Ly6Clo and Ly6Chi spleen monocytes in WT but not in C5ar1-/- animals after infection (Figure 38c, d). Also, there was a significantly higher number of Ly6Chi monocytes in the spleen of C5aR1-/- than WT mice after infection (Figure 38d, left graph). However, CCR2 expression on Ly6Chi cells equally increased in infected in comparison to uninfected state in both, WT and C5aR1-/- mice suggesting that it is not responsible for the difference in monocyte migration (Figure 38d, right graph). 76 4 Results

In the first step, T- and B-cells were excluded from analysis by gating on CD3-CD19- cells. Neutrophils were identified as CD3-CD19-Ly6G+ cells and NK cells as CD3-CD19-Ly6G-NK1.1+ cells. Further, I gated on CD11b+ cells to assess the mononuclear phagocyte compartment. In this compartment, the Ly6C-F4/80hi population is considered as large peritoneal macrophages (LPM) and Ly6C-F4/80int as small peritoneal macrophages (SPM) [357]. CD11b+Ly6C+F4/80int cells are monocyte precursors that appear after infection and are designated to replenish the SPM population [358]. The rest of the CD11b+ cells comprise Ly6ChiCCR2+ and Ly6CloCCR2- monocytes [359] (Figure 39).

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Figure 39: Gating strategy to identify cell populations in the peritoneal cavity of uninfected and infected animals Contour plots depict gating strategy to identify following cell subsets: (a) neutrophils (Ly6G+), (b) NK cells (Ly6G-NK1.1+), (c) large peritoneal macrophages (CD11b+F4/80hiLy6C-), (d) small peritoneal macrophages (CD11b+F4/80loLy6C-), (e) inflammatory monocytes (CD11b+F4/80intLy6ChiCCR2+), (f) Ly6C monocytes (CD11b+F4/80-Ly6C+), (g) CCR2+ inflammatory monocytes (CD11b+F4/80-Ly6C+CCR2+). Numbers shown in the gate represent frequencies of the parent population. Lin marker includes CD3 and CD19. Cells were analyzed in the uninfected animals (A) and 5 days after infection (B) in WT and C5aR1-/- mice. Numbers represent the frequencies of the parent population. I observed significant infiltration of neutrophils to the site of infection in WT and C5aR1-/- animals (Figure 40a). Both, WT and C5aR1-deficcient mice showed equal influx of NK cells suggesting that their recruitment does not depend on C5aR1 signaling (Figure 40b). When I analyzed macrophage compartment, I observed the absence of LPMs in infected mice (Figure 40c). This effect is also described for other infection models and upon stimulation with various antigens and described as macrophage disappearance reaction [227, 360]. It can be explained by migration of macrophages rather than by cell death. Similar numbers of SPMs occurred in WT and C5aR1-/- mice before and after infection (Figure 40d). Phenotypically SPMs did not express Ly6C. Further, there was a similar influx of inflammatory monocytes that acquire F4/80 in the peritoneal cavity after infection (Figure 40e) which contribute to both SPM and LPM populations [358]. These cells also express CCR2 and have previously been shown to be indispensable in maintaining the immune responce to T.gondii [151, 361]. In addition, F4/80- cells that vary in expression of Ly6C are also upregulated after infection

78 4 Results in both WT and C5aR1-/- mice (Figure 40f). However, in the absence of C5aR1, the numbers of Ly6ChiCCR2+ inflammatory monocytes were makedly decreased (Figure 40g).

Figure 40: Change in cell composition of the peritoneal compartment during the acute stage of T. gondii infection. Graphs represent total numbers of (a) neutrophils; (b) NK cells, (c) large peritoneal macrophages, (d) small peritoneal macrophages, (e) F4/80+Ly6C+ monocytes/macrophages, (f) F4/80-Ly6C+ monocytes and (g) Ly6C+CCR2+ inflammatory monocytes in WT and C5aR1-/- uninfected mice and 5 days after T. gondii infection. Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001.

4.4.2.1. Change in peritoneal B-1 cell composition in response to acute T. gondii infection The peritoneal cavity also serves as a reservoir for B-1 cells [362]. The Köhl laboratory found that migration of these cells in response to TLR activation is dependent on C5a/C5aR1 signaling (PhD Thesis of K. Bröker). It has been described that the depletion of B-1 cells causes high susceptibility to T. gondii infection [363]. The role of peritoneal B-1 cells in the resistance to parasites is mainly mediated by IgM production that is necessary for complement activation, preventing host invasion and parasite clearance [254]. Thus, I analyzed B-1 cells in the peritoneal compartment before and 5 days after T. gondii infection using the gating strategy described below.

First, macrophages and T-cells were excluded based on their expression of CD11b and CD5. Interestingly, CD11bhi cells, previously identified as LPMs, disappear after infection ( Figure 41 C). Conventional B1-cells are defined as CD43+IgMhi population, migratory trans- B-1 cells are defined as CD43+IgMint population. Further, these two populations were subdivided into B-1a and B-1b cells based on their expression of CD5 ( Figure 41) [364].

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Figure 41: Gating strategy to identify B-1a and B-1b cells in the peritoneal cavity of uninfected and T.gondii- infected animals. Representative contour plots for identification of resident (IgMhi) and migratory (IgMlo) B-1a (CD11bneg to loCD43+IgM+CD5+) and B-1b cells (CD11bneg to loCD43+IgM+CD5-). Cells were analyzed in the uninfected WT and C5aR1- /- animals (A) and 5 days after infection (B). Numbers represent the frequencies of the parent population.

B-1 cells have been shown to downregulate surface expression of IgM upon TLR stimulation and gain a migratory phenotype. This allows trans-B-1 cells to leave the peritoneal cavity and migrate into the spleen where they become antibody-producing cells. I observed similar numbers of B-1 and trans-B-1 cells in the peritoneum of WT and C5aR1-/- under steady state conditions. However, 5 days after infection, I detected ~3-fold increase in the number of B-1 cells and ~2-fold increase in the number of IgMlow B-1 cells in C5aR1-/- mice (Figure 42 A, B left graph). In contrast, there was only a slight increase in the number of B-1 and IgMlow B-1 cells in WT animals (Figure 42 A, B left graph). There was a significant upregulation of B-1a and to an even greater extent of B-1b cells in T. gondii- infected C5aR1-/- animals in comparison to uninfected WT mice (Figure 42 A middle and right graphs). Further, I observed increased numbers of IgMlow B-1b but not IgMlow B-1a cells in the peritoneum of WT and C5aR1-/- mice 5 days after T. gondii infection (Figure 42 B middle and right 80 4 Results graphs). Also, I observed a significantly higher number of IgMlow B-1b cells in T. gondii-infected C5aR1-/- mice than in WT animals (Figure 42 B right graph).

Figure 42: B-1 cell compartment in the peritoneal cavity of uninfected and T. gondii-infected animals. Numbers of resident (A) and migratory IgMlow (B) B-1 (left), B-1a (middle) and B-1b (right) cells before and 5 days after T. gondii infection in WT and C5aR1-/- mice. Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001. Analysis of the cellular responses in the mesenteric lymph nodes to T. gondii infection MLNs are the draining lymph nodes for the small intestine are also considered as sentinel lymph nodes for the peritoneal cavity in rodents [365]. The role of MLN cell subsets in the immune responses to T. gondii infection was primarily studied in the context of the oral infection model [166]. Here, I investigated the impact of C5aR1/C5a axis on the cellular composition of the MLNS at day 5 after intraperitoneal T. gondii infection.

To study NK cells and the phagocyte compartment, T- and B-cells were excluded from the analysis by gating on CD3-CD19- cells. NK cells were identified as CD11c-NK1.1+ cells. Neutrophils were identified as CD11c-NK1.1-F4/80-Ly6G+ cells and macrophages were identified as CD11c- NK1.1- Ly6G-F4/80+ cells. Further, I gated on CD11b+ cells to assess the monocyte compartment, where I defined Ly6Chi and Ly6Clo monocyte subpopulations (Figure 43).

I observed a significant increase in the number of NK cells in MLNs of T. gondii infected C5aR1-/- mice in comparison to uninfected animals, whereas the increase in NK cells in WT mice was less pronounced (Figure 44a). WT and C5aR1-/- showed a similar ~3-fold increase in the 81 4 Results numbers of neutrophils in the MLNs at day 5 after infection arguing against the significant role of C5aR1/C5a axis in the migration of these cells under the conditions studied (Figure 44b). Further, I observed only a slight increase in the number of macrophages, Ly6Chi and Ly6Clo monocytes in the MLNs of T. gondii-infected animals that was similar in WT and C5aR1-/- mice (Figure 44c, d, e).

Figure 43: Gating strategy to identify cell populations in the mesenteric lymph nodes of uninfected and infected animals Contour plots depict gating the strategy to identify the following cell subsets: (a) NK cells (Lin-CD11c-NK1.1+), (b) neutrophils (Lin-NK1.1-Ly6G+), (c) macrophages (Lin-NK1.1-Ly6G-F4/80+), (d) Ly6Chi monocytes (Lin-CD11c-NK1.1-Ly6G- F4/80-CD11b+Ly6Chi) and (e) Ly6Clo monocytes (Lin-CD11c-NK1.1-Ly6G-F4/80-CD11b+Ly6Clo). Lin marker includes CD3 and CD19. Cells were analyzed in uninfected animals (A) and 5 days after infection (B) in WT and C5aR1-/- mice. Numbers represent the frequencies of the parent population.

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Figure 44: Change in cell composition in the lymph node during the acute stage of T. gondii infection. Graphs represent total numbers of (a) NK cells, (b) neutrophils, (c) macrophages, (d) Ly6Chi monocytes and (e) Ly6Clo monocytes in WT and C5aR1-/- uninfected mice and 5 days after T. gondii infection. Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01.

4.4.3.1. DC subsets in the MLNs of T. gondii-infected mice CD8α+ DC in the spleen are the essential source of IL-12 during i.p. T. gondii infection [278]. However, CD103+CD11b− and CD103−CD11b− MLN DC subsets also greatly contribute to IL-12p40 production in response to oral T. gondii infection [166]. Here, I investigated the DC subset composition during acute i.p. T. gondii infection.

First, T- and B-cells were excluded from analysis by gating on the CD3-CD19- population. Then, DCs were defined as MHCII+CD11c+ cells. DC subpopulations were defined as following: CD103+CD11b−cDCs, CD103-CD11b+ cDCs and CD103-CD11b- cDCs. Further, I differentiated cDC and moDC subpopulations of CD103-CD11b+ cells according to their expression of CD64 (Figure 45).

I observed no significant difference in the total DC numbers at day 5 after T. gondii infection in WT and C5aR1-/- mice in comparison to the uninfected state (Figure 46a). Further, there was no significant difference in the numbers of CD103+CD11b−, CD103-CD11b+ and CD103-CD11b- cDCs in uninfected mice and 5 days after T. gondii infection (Figure 46c, d, e). However, I found a significant

83 4 Results infiltration of CD11b+ moDCs in the lymph node of WT mice after infection that was lower in C5aR1- /- mice (Figure 46f).

Figure 45: Gating strategy to identify DC populations in the mesenteric lymph nodes of uninfected and infected animals. Contour plots depict gating strategy to identify following cell subsets: MHCII+CD11c+ DCs (a), CD103-CD11b+ (b), CD103+CD11b- (c) and CD103-CD11b- (d) cDC subsets. CD103-CD11b+ cells were further differentiated into CD64- cDCs (e) and CD64+ moDCs (f). Lin marker includes CD3 and CD19. Cells were analyzed in the uninfected animals (A) and 5 days after infection (B) in WT and C5aR1-/- mice. Numbers represent the frequencies of the parent population.

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Figure 46: DC subsets in the MLNs during the acute stage of T. gondii infection. Graphs represent total DC numbers (a), CD103-CD11b+ (b), CD103+CD11b- (c) and CD103-CD11b- (d) cDCs, CD103-CD11b+ cDCs (e) and moDCs (f) in WT and C5aR1-/- uninfected mice and 5 days after T. gondii infection. Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05.

4.5. Impact of the C5a/C5aR1 axis on the cellular response to the acute and chronic T. gondii infection in the brain Upon reaching the brain, T. gondii persists lifelong within tissue cysts when controlled by the host immune system [292]. Brain resident immune cells such as microglia and astrocytes become activated, displaying significant antiparasitic properties and induce IFN-γ-dependent immune responses during the chronic stage of infection [366]. At this stage of T. gondii infection, immune cells from the periphery migrate into the brain. The adaptive immune responses in the CNS play an important role in local parasite control [268].

I isolated immune cells from the brain of uninfected mice, 5 and 30 days after T. gondii infection as described in section 3.2.4. Microglia was defined as CD45intCD11b+ population and other brain leukocytes fall into CD45hiCD11bneg to int gate (Figure 47). There was a significant increase in the number of cells isolated from the brain at day 30 after infection in comparison to the uninfected state and at day 5 after infection in WT and C5aR1-/- mice. Moreover, there was a significantly higher number of cells isolated from the brain of C5aR1-/- mice at day 30 in comparison to WT animals (Figure 48 left).

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Figure 47: Gating strategy to identify immune cells in the brain of WT and C5aR1-/- mice chronically infected with T. gondii. Contour plots depict the gating strategy to identify the following cell subsets: CD45intCD11b+ microglia and CD45hiCD11bneg to lo brain leukocytes. Cells were analyzed in uninfected animals (A), 5 (B) and 30 (C) days after T. gondii infection in WT and C5aR1-/- mice. Numbers represent the frequencies of the parent population. I observed a gradual increase in the number of microglia cells through the course of T. gondii infection in WT and C5aR1-/- mice with a significant difference between the uninfected state and at the day 30 after infection (Figure 48 middle). There was a significant increase in the number of the CD45hi cell population, which comprises innate and adaptive immune cells infiltrating the brain to control chronic T. gondii infection at day 30 in both WT and C5aR1-/- mice. However, C5aR1-/- mice have significantly lower number of infiltrating CD45hiCD11bneg to lo leukocytes in the brain at day 30 after T. gondii infection (Figure 48 right).

Figure 48: Immune cells in the brain of WT and C5aR1-/- mice during the acute and chronic stage of T. gondii infection. Graphs represent total number of immune cells (left), microglia (middle) and other leukocytes (right) in the brain of WT and C5aR1-/- mice in uninfected animals (open circles), 5 (half closed circles) and 30 (closed circles) days after T. gondii infection. Values shown are the mean ± SEM, n=4-12. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001. 86 4 Results 4.6. T-cell immune response during chronic T. gondii infection Phenotype of T-cells in the brain of chronically infected mice It has been shown that the control of latent T. gondii infection in the mouse brain depends on CD4+ and CD8+ T cells [280]. T cells control the parasite mainly by their production of IFN-γ [303, 367]. Further, large numbers of antigen-specific T cells are recruited from the periphery into the brain upon chronic T. gondii infection. These T cells phenotypically resemble activated T cells as they express high levels of CD44 and are mostly CD62L negative [368].

Here, I investigated whether the C5a/C5aR axes and CCR5 signaling has an impact on the recruitment and phenotype of CD4+ and CD8+ T cells into the brain of mice during the chronic stage of T. gondii infection. I isolated immune cells from the brain of chronically infected mice as described in section 3.2.4. T cells were defined as CD45hiCD3+ population divided according to their expression of CD4 and CD8. Further, T cells were differentiated into effector/effector memory (CD44+CD62L-) and central memory (CD44+CD62L+) subpopulations (Figure 49) [368].

I observed similar numbers of total T cells, CD4+ and CD8+ T cells in the brain of WT, C5aR1- /-, CCR5-/- and C5aR2-/- mice 30 days after T. gondii infection (Figure 50, upper row). There were similar numbers of CD4+ and CD8+ cells within the T cell population, which is consistent with previous reports where it has been shown that both T cell populations are necessary for the resistance to T. gondii infection [280].

Furthermore, most of the CD4+ and CD8+ T cells in WT, C5aR1-/-, CCR5-/- and C5aR2-/- had effector/effector memory phenotype (CD44+CD62L-) which is consistent with previous reports showing that T cells are recruited from the periphery into the brain to exert their effector functions (Figure 50, lower row) [368]. A minor fraction of central memory (CD44+CD62L+) cells was also detected in the CD4+ and CD8+ T cell populations, which was similar in all experimental groups of mice (Figure 50, lower row).

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Figure 49: Gating strategy to identify CD4+ and CD8+ T-cells and their phenotype in the brain of WT, C5aR1-/-, CCR5- /- and C5aR2-/- mice at day 30 after T. gondii infection. First, the leukocyte population was pre-gated as CD45hi cells as shown in the previous section and T cells were defined by the expression of CD3. Expression of CD44 and CD62L was determined in CD4+ and CD8+ T cell populations. The CD44+CD62L+ population was defined as central memory (upper gate) and CD44+CD62L- (lower gate) as effector T cells. Cells were isolated from the mouse brain at day 30 after T. gondii infection from WT and C5aR1-/- mice. Numbers represent the frequencies of the parent population.

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+ + -/- -/- - Figure 50: Number of CD4 TH and CD8 T cells and their subsets in the brain of WT, C5aR1 , CCR5 and C5aR2 /- + + mice chronically infected with T. gondii. Total numbers of T cells, CD4 TH cells, CD8 T cells, effector/effector + + + + memory CD4 TH cells, central memory CD4 TH cells, central memory CD8 T cells, effector/effector memory CD8 T cells in the brain of WT (closed circles), C5aR1-/- (open circles), CCR5-/- (open squares) and C5aR2-/- (open triangles) mice at day 30 after T. gondii infection. Values shown are the mean ± SEM, n=2-5. Statistical differences were determined by ANOVA. T. gondii-specific T-cells in spleen and brain at day 30 after infection Next, I determined the specificity of CD8+ and CD4+ T cells in the spleen and brain of WT, C5aR1-/- and C5aR1xCCR5-/- mice with chronical infection using MHCI and MHCII tetramers in complex with T. gondii-specific peptides [369, 370]. Cells from spleen and brain were prestained with CD3, CD4 and CD8 to differentiate between CD8+ and CD4+ T cells, labelled with tetramers as described in section 3.7.2 and analyzed using the gating strategy shown in Figure 51.

I observed similar numbers of CD4+ T cells in the spleen and brain of WT, C5aR1-/- and C5aR1xCCR5-/- mice at day 30 after T. gondii infection. Importantly, I found a ~200-fold higher frequency of CD4+ T cells specific for the T. gondii peptide in the brain of infected mice as compared with the spleen (Figure 52 A, B right graphs). This finding strongly supports the idea of antigen- specific recruitment of T cells into the brain from the periphery. Further, I found a similar frequency of CD8+ T cells in the brain of WT, C5aR1-/- and C5aR1xCCR5-/- animals (Figure 52 A left graph). Of note, I detected a significantly lower percentage of antigen-specific CD8+ T cells in the spleen of C5aR1-/- mice in comparison to WT mice at day 30 after T. gondii infection (Figure 52 B left graph).

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Figure 51: Gating strategy to identify T. gondii-specific T-cells in the spleen and brain of WT, C5aR1-/- and C5aR1xCCR5-/- mice at day 30 after infection. Cells from the brain (A) and spleen (B) were stained for CD3, CD4 and CD8 to gate on T cell populations. Antigen-specific cells were stained using T. gondii-specific tetramers as described in section 3.7.2. Numbers represent the frequencies of the parent population.

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Figure 52: T. gondii-specific CD4+ and CD8+ T-cells in the spleen and brain of chronically infected WT, C5aR1-/- and C5aR1xCCR5-/- mice. Percentage of antigen-specific cells among the CD8+ (left) and CD4+ (right) T cells in the brain (A) and spleen (B) of WT, C5aR1-/- and C5aR1xCCR5-/- mice at day 30 after T. gondii infection. Values shown are the mean ± SEM, n=4-5 Statistical significant differences are indicated by * as determined by ANOVA, *** P ≤ 0,001.

5 DISCUSSION

5.1. The C5aR1/C5a axis controls TLR-dependent IL-12 production from DCs in response to T. gondii-derived stimuli in vitro

Previous findings from our collaborator J. Aliberti (CCHMC, USA) suggested that TgCyp18 induces IL-12 production from DCs in MyD88-independent manner through activation of CCR5 [178- 181]. CCR5 forms heterodimers with C5aR1 which is important for entry of HIV into macrophages [123]. Further, it has been shown that the cognate ligands of C5aR1 and CCR5, i.e. C5a and CCL5, promote not only the internalization of their cognate receptor but induce cross-internalization of either C5aR1 or CCR5 [122]. In a first set of experiments, I assessed the impact of C5aR1 and CCR5 signaling on the induction of IL-12 from splenic cDCs. To test the hypothesis of GPCR-dependent IL-12 induction, I expressed recombinant TgCyp18 in E. coli as reported before [182-184]. For in vitro tests, I also used recombinant TgCyp18 provided by J. Aliberti [178].

Surprisingly, I found no IL-12 secretion form the splenic DC culture upon stimulation with recombinant TgCyp18 proteins, whereas stimulation with the TLR9 agonist CpG or STAg induced strong IL-12 production (Figure 15). In addition, neither recombinant TgCyp18 nor STAg, from which TgCyp18 was primarily isolated and described, did not show enzymatic activity using a peptidyl- prolyl isomerization assay (Figure 13). Previous findings demonstrated that such enzymatic activity of TgCyp18 is required for CCR5-dependent IL-12 induction but not for the binding of the protein to the receptor [180]. This discrepancy might be explained by the improper folding of the eukaryotic recombinant protein in the bacterial expression system. However, the recombinant TgCyp18 provided by J. Aliberti was chemically refolded [178]. In this case, the long storage of the cyclophilin even at -70°C may have resulted in the loss of the enzymatic activity as has been reported [337]. Additionally, it was shown that low levels of LPS can modulate the signaling networks driving IL-12 production [343]. Therefore, I reduced the endotoxin contamination from the recombinant TgCyp18 preparation below the level reported for LPS-induced IL-12 production using affinity chromatography.

Furthermore, I observed no IL-12 secretion from MyD88-deficient splenic DC culture upon stimulation with STAg demonstrating that T. gondii sensing was primarily mediated by TLR signaling (Figure 16). However, also splenic DCs from C5aR1-/-, CCR5-/- and C5aR1xCCR5-/- mice produced significantly lower amounts of IL-12p70 in vitro in response to CpG and STAg in comparison to DCs from WT animals (Figure 17). Splenic DCs from C5aR1-/- and C5aR1xCCR5-/- mice expressed also

92 5 Discussion lower levels of IL-12p40 when stimulated with STAg (Figure 17). These findings suggest a role of GPCRs in modulation of TLR-induced cytokine secretion from splenic DCs in response to T. gondii- derived antigens rather than a direct effect on parasite sensing. This view is supported by previous reports showing that C5aR1/C5a signaling modulates IL-12 production from DCs in response to microbial stimuli [110]. Also, I found no significant difference between WT, C5aR1-/-, CCR5-/- and C5aR1xCCR5-/- mice with regard to IL-12 levels in DC-tachyzoite co-cultures (Figure 17), which might be explained by cell-parasite interaction [315]. In fact, using transgenic T. gondii strain it has been shown that splenic cDCs remotely sense the parasite through parasite-derived antigens and initiate the innate immune responses without encountering living tachyzoites [288].

In summary, I could show that the induction of IL-12 from splenic cDC in vitro in response to T. gondii-derived ligands was mediated by TLRs and modulated by C5aR1 and CCR5 signaling.

5.2. Cognate ligands but not T. gondii tachyzoites induce internalization of C5aR1 and CCR5 from the surface of BMDCs

Next, I investigated whether the stimulation C5aR1 and CCR5 with C5a or CCL5 induces cross-internalization of the receptors in DCs. For this purpose, I transiently transfected BMDCs to express C5aR1-GFP and CCR5-GFP as described in section 3.3 and visualized receptor internalization by LSM confocal microscopy. First, I found that the transiently expressed C5aR1- and CCR5-GFP reporter receptors were functional as the showed homologous internalization when stimulated with their cognate ligands (Figure 18).

Cross-internalization of C5aR1 and CCR5 was shown previously using a receptor- transfected rat basophilic leukemia (RBL) cell line [122]. Further, co-expression was shown using human monocyte-derived macrophages [123]. Surprisingly, I did not detect heterologous internalization of CCR5 in BMDCs when stimulated with C5a (Figure 19). Furthermore, I observed no internalization of C5aR1 or CCR5 when cells were co-cultured with T. gondii tachyzoites. It was described previously that T. gondii tachyzoites secrete TgCyp18 into the medium and that T. gondii antigens are released into the culture supernatant in response to spontaneous lysis [178]. I found clear evidence of C5aR1 internalization, when C5a was added to the tachyzoite-DC co-culture (Figure 20). The difference between my findings and previous results might be explained by differences in the signaling pathways used by C5aR1 and/or CCR5 in DCs as compared to macrophages or the RBL cell line. Further experiments will be needed to explore this possibility. 93 5 Discussion 5.3. C5aR1 signaling is crucial for the survival of T. gondii- infected mice and the control of parasite load in the brain of chronically infected mice

Further, I investigated the role C5a/C5aR1 axis and CCR5 signaling on the resistance to T. gondii infection. I used the intraperitoneal infection model described in section 3.5.2 [142]. I observed a decreased survival of C5aR1-deficient animals 30 days after infection (Figure 21). Additionally, this difference was more pronounced in male mice than in female despite the fact that male mice are generally more resistant to the parasite because of stronger and earlier IL-12 and IFN-γ induction (Figure 22) [371]. The differences regarding the effect of C5aR1-deficiency in T. gondii-infected male and female mice might be due to the naturally lower levels of terminal complement components in female mice in comparison to those in males [348]. Additionally, C5aR1-deficient mice had a higher weight at the beginning of the experiment and therefore a higher relative weight loss at the early stage of infection (Figure 23 and Figure 24). The influence of complement receptors on glucose metabolism under normal diet conditions has been reported earlier [349, 372]. Furthermore, C5aR1- /-, CCR5-/- and C5aR2-/- mice suffered from a significantly higher average disease severity score when compared to WT animals (Figure 25).

All animals were sacrificed at day 30 after infection and the brain cyst count was determined as described in section 3.5.1. I observed a higher parasite load in the brain of C5aR1-/-, C5aR1xCCR5-/- and C5aR2-/- mice than in WT animals. It was described earlier that the increased number of cysts in the mouse brain most likely results from increased numbers of parasites shuttled into the brain by CD11b+CD11c+ DCs rather than local replication of parasites [293]. Additionally, infected cells acquire a hypermotile phenotype that contributes to parasite spreading [207, 373-377]. These cells are also able to transport parasites across endothelial barriers [378].

In general, the parasite load is higher in female than in male mice which is due to the lower

-/- -/- TH1 cytokine production is females [371]. Differences between WT and C5aR1 , C5aR1xCCR5 , C5aR2-/- mice were more pronounced in male mice, which might also be explained by the differences in the amount of terminal complement components [348]. Of note, the survival of C5-/- mice did not reflect the phenotype observed in C5aR1-/- and C5aR2-/- mice regarding survival, disease severity score or parasite load. This might be explained by findings from the Colten lab showing that serum C5-deficiency in these animals results in a defect in C5 secretion, whereas C5 fragments were found within macrophages [379].

94 5 Discussion

5.4. C5aR1/C5a axis activation modulates TH1 cytokine and chemokine production in response to T. gondii infection in vivo

In search for mechanisms that may account for the higher mortality in C5aR1-deficient mice and their higher parasite burden in the brain, I determined the impact of C5aR1- and/or CCR5- deficiency on the early production of cytokines in response to T. gondii infection in vivo. For this purpose, I injected STAg into WT, C5aR1-/- and C5aR1xCCR5-/- mice and analyzed serum IL-12 and IFN-γ levels 6h later. Intriguingly, I observed significantly lower levels of IL-12p70 and consequently IFN-γ in the serum of these mice (Figure 27).

It is known that IL-12 is necessary and sufficient to trigger IFN-γ production [190]. Despite the fact that C5aR1-/- mice produced less IFN-γ than WT animals, they induced similar levels of the

IL-12p40 subunit, which is shared by IL-12p70 and IL-23 (Figure 27,Figure 28Figure 30) [141]. In contrast, C5aR1-deficiency resulted in decreased production of IL-12p70 that is composed of the p40 and p35 subunits and was necessary to trigger IFN-γ production from NK and T-cells (Figure 30) [190]. Data from our laboratory provide evidence that NK and T-cells do not express C5aR1 on the cell surface suggesting that the reduced IFN-γ production results from an indirect effect mediated through the impact of C5a/C5aR1 signaling in APCs, which is critical to mount an appropriate IL- 12p70 response in response to PRR activation [75]. These finding further imply that C5aR1 activation is critical for the migration and crosstalk of APCs with NK and T-cells. In support of this view, it is well appreciated that IFN-γ secretion serves as a positive feedback loop promoting increased IL-12 production from DCs and macrophages [380-382].

Given the importance of C5aR1 activation for mouse survival, parasite burden and cytokine production, I assessed the impact of C5aR1/C5a axis on the cellular innate and adaptive immune responses to T. gondii infection. In earlier study it was shown that simultaneous deficiency of C3aR and C5aR1 leads to a diminished TH1 response and increased susceptibility to T. gondii infection [76]. However, the exact contribution and mechanisms underlying the impaired adaptive immune response has not been determined in this study.

First, I focused on the DC compartment, as CD8α+ DC in spleen are the main source of IL- 12 during T. gondii infection [181]. Therefore, I determined IL-12 production in DCs and IFN-γ production in NK, NKT and T cells using intracellular cytokine staining (Figure 34, Figure 36). I observed IL-12 production by both, CD11b+ and CD8α+ cDCs in the spleen. Surprisingly, there was a higher number of CD8α+IL-12+ cDCs in the spleen of C5aR1-/- mice than in WT animals. Moreover, there were similar numbers of IFN-γ+ NK and T cells in WT and C5aR1-/- mice and higher numbers 95 5 Discussion of IFN-γ+ NKT cells in the spleen of C5aR1-/- animals than in WT mice. Interestingly, the frequency of IFN-γ+ NK cells was lower in C5aR1-/- than in WT mice, however, this decreased frequency was compensated by higher total cell numbers in the spleen as compared with WT animals. Intracellular IL-12 and IFN-γ staining provides information about the number and frequency of cells expressing these cytokines. It is important to consider that the data provide no information about the amount of cytokines secreted by such cells. Additionally, I determined whether the observed differences in IFN- γ production and parasite replication in WT and C5aR1-deficient mice affected the liver function in these mice. For this purpose, I determined serum levels of ALT as an indicator for the liver damage [355, 356]. I detected elevated but similar levels of ALT in the serum of WT, C5aR1-/-, C5aR2-/-, C5- /-, CCR5-/- and C5aR1xCCR5-/- mice indicating that differences in serum IFN-γ levels did not lead to differences in the liver pathology in these mice (Figure 31).

Further, I determined whether C5aR1/C5a axis signaling influences the secretion of other

TH1 and TH2 cytokines and chemokines using a Bioplex cytokine assay as described in section 3.6.2. In addition to IL-12p70 and IFN-γ described earlier, I observed significantly lower levels of IL-10, G- CSF, GM-CSF, KC, MCP and MIP-1α in the sera of C5aR1-/- in comparison to WT mice at day 7 after T. gondii infection (Figure 29). IL-10 has anti-inflammatory properties and is essential for the control of the inflammation and mouse survival during T. gondii infection [322, 383, 384]. It is surprising that the IL-10 downregulation in C5aR1-/- mice in comparison to WT goes along with decreased IL-12 and IFN-γ inflammatory responses in those animals, as IL-10 and IL-12 typically exert reciprocal counterbalancing effects [385]. G-CSF and GM-CFS were discovered to be secreted in response to T. gondii by human fibroblasts and are essential for delayed neutrophil apoptosis necessary for the control of T. gondii infection [386, 387]. In experiments with human epithelial cells and fibroblasts infected with T. gondii, they secreted KC (CXCL1) and MCP-1, which are potent chemoattractants and activators of neutrophils and macrophages [388]. Interestingly, C5aR1-/- mice had significantly higher level of RANTES (CCL5), a cognate ligand for CCR5, which might be explained by a regulatory role of C5aR1/CCR5 heterodimers, which is absent in the C5aR1-deficient background (Figure 29).

Both, WT and C5aR1-/- had similarly high levels of IL-1α, IL-1β, IL-12p40, IL-13, eotaxin and TNF-α in serum at day 7 after T. gondii infection (Figure 29). IL-1 family cytokines together with IL- 12 are known to be essential in the activation of NK cells and IFN-γ induction during T. gondii infection [220, 389]. TNF-α is an essential cytokine for T. gondii killing and conversion of infection into the latent phase that utilizes signaling pathways different from the one induced by IFN-γ [390- 392]. Elevated levels of eotaxin and IL-13 are usually connected with helminth invasions and allergic reactions. However, it is known that T. gondii spreads into the lung during acute stage of infection where it can cause pulmonary toxoplasmosis [393-397]. Lung inflammation can lead to an increase 96 5 Discussion in the level of these cytokines, however, not much is known regarding their roles in the resistance to parasite. The levels of IL-2, IL-3, IL-5, IL-6, IL-17 and MIP-1β in the serum of WT and C5aR1-/- mice were very low suggesting that pathways that drive the production of such cytokines and chemokines are not strongly activated during the early phase of the infection (Figure 29). Finally, I found significantly lower serum levels of IL-6 in C5aR1-/- mice in comparison to WT. This finding is of interest as it is known that IL-6 promotes NK cell-dependent IL-17 production during toxoplasmosis [398].

5.5. C5aR1/C5a axis in the cellular response to intraperitoneal T. gondii infection in mice

Given the strong impact of C5aR1-deficiency on survival, parasite burden and systemic cytokine and chemokine production, I aimed to determine the impact of C5aR1/C5a axis signaling on the cellular composition in organs essential for the resistance to intraperitoneal T. gondii infection [397]. I compared the cell numbers of different immune cell subsets in C5aR1-/- and WT animals at day 5 after T. gondii infection. The results are summarized in Table 13.

I found significantly elevated cell numbers in the spleen and peritoneum of C5aR1-/- in comparison to WT mice, whereas cell numbers in MLNs and brain were similar in those animals at day 5 after infection. Despite the major role of C5aR1 in neutrophil migration, the numbers of neutrophils were similar in peritoneum, spleen and MLNs of WT and C5aR1-/- animals [399]. Also, the absence of C5a/C5aR1 signaling did not alter NK cell trafficking, which could be explained by the lack of C5aR1 expression on mouse NK cells [75].

Surprisingly, C5aR1-deficient animals had significantly higher T cell number in the spleen 5 days after T. gondii infection, which is most likely due to an indirect effect on T cell proliferation and activation by C5aR1-expressing APCs (Figure 35). Furthermore, C5aR1-/- mice showed elevated numbers of CD8α+ cDCs in the spleen in comparison to WT animals after infection (Figure 34). It is known that CD8α+ cDCs can cross-present antigens to CD8+ T-cells [400].

Further, C5aR1-/- mice had elevated numbers of F4/80+ macrophages and Ly6Chi monocytes in the spleen but lower numbers of the Ly6Chi monocytes in the peritoneum than WT mice at day 5 after T. gondii infection. It is known that deficiency in Ly6Chi monocyte but not in neutrophil recruitment leads to the acute susceptibility during early and latent T. gondii infection [150, 151, 216].

In addition, C5aR1-/- mice had higher numbers of B-1 cells in the peritoneum than WT animals. Previous findings from our laboratory demonstrated that the C5aR1/C5a axis orchestrates

97 5 Discussion the migration of B-1 cells from the peritoneal cavity into the spleen upon antigen stimulation (PhD thesis K. Bröker). Such egress of B-1 cells from the peritoneum into the spleen is necessary for the differentiation of B-1 into plasma cells that secrete antigen-specific IgM and IgG antibodies. B-1 cells bridge innate and adaptive immunity and make an optimal transition between the two immune responses by producing the first wave of IgM antibodies required for pathogen clearance [401]. It has been also shown that T. gondii specific IgM is essential for parasite killing and also limits entrance of free tachyzoites into APCs [253, 254]. However, at this point it is unknown whether C5aR1-/- mice have an altered T. gondii-specific IgM production. This issue should be addressed in future studies.

I also observed elevated numbers of immune cells isolated from the brain of C5aR1-/- mice in comparison to WT animals at day 30 after T. gondii infection (Figure 48). There was slight elevation in the numbers of microglia in the brain of C5aR1-/- mice in comparison to WT. Those animals also had significantly lower numbers of CD45hi cells which are mainly cells that are recruited from the circulation and are necessary to suppress parasite replication in the brain [292]. However, similar numbers of CD8+ and CD4+ T-cells, which are responsible for parasite control in the brain, were present in the brain of infected WT and C5aR1-/- mice at day 30 [272, 280]. Further, I found that CD8+ and CD4+ T-cells in WT and C5aR1-/- mice were predominantly effector T cells necessary to control parasite during the chronic stage of infection [368].

98 5 Discussion

Table 13: Differences in the cell number of immune cell subsets in T.gondii-infected C5aR1-/- mice as compared with WT mice 5 and 30 days after infection. ↑ = higher; − = no significant difference; ↓ = lower; ND = not determined; NA = not applicable.

Organ Day 5 after infection Day 30 after infection

Spleen Peritoneum MLNs Brain Brain Cell type Number of isolated cells ↑ ↑ − − ↑ DCs ↑ ND − ND ND Neutrophils − − − ND ND NK cells − − − ND ND Macrophages ↑ − − ND ND Microglia NA NA NA − −

Ly6Clo monocytes − − − ND ND

Ly6Chi monocytes ↑ ↓ − ND ND T cells ↑ ND ND ND − CD4 T cells ND ND ND ND − CD8 T cells ND ND ND ND − B-1 cells ND ↑ ND ND ND B-1a cells ND ↑ ND ND ND B-1b cells ND ↑ ND ND ND

99 5 Discussion 5.6. C5aR1/C5a axis has an impact on the amount of the antigen-specific T-cells in the spleen the during chronic stage of T. gondii infection

Finally, I determined the amount of antigen-specific CD8+ and CD4+ T-cells in the spleen and brain of WT and C5aR1-/- mice at day 30 after T. gondii infection (Figure 52). I observed that both, WT and C5aR1-/- expressed similar frequencies of T. gondii-specific CD8+ and CD4+ T-cells in the brain. However, C5aR1-/- mice had a significantly lower frequency of antigen-specific CD8+ T cells in the spleen. This finding is interesting as antigen-specific CD8+ T cells are necessary to prevent T. gondii reactivation in the brain and are recruited from the periphery in antigen-dependent manner [402, 403]. Exhaustion of the CD8+ T cell population have been shown to cause reactivation of latent infection during later phases of chronic toxoplasmosis [404, 405].

Using parasite extract-pulsed DCs it has been demonstrated that such cells induce proliferation of antigen-specific CD8+ T cells in the spleen, which plays a major role to control the number of intracerebral T. gondii cysts [406]. My findings suggest that activation of the C5aR1/C5a axis signaling on spleen DCs might be important in this regard. However, the exact mechanisms need to be explored in future studies.

100

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APPENDIX

Abbreviations

-/- knock-out °C degree Celsius % percent A Ampere AF Alexa Fluor® APC Allophycocyanin APC antigen-presenting cell BMDC bone marrow derived DC BSA bovine serum albumine C5aR1 C5a receptor 1 C5aR2 C5a receptor 2 CCL CC-chemokine ligand CCR C-C chemokine receptor CD cluster of differentiation cDC conventional DC CFSE carboxyfluorescein succinimidyl ester CSF-2 Colony Stimulating Factor 2 (also known as GM-CSF) Da Dalton DAMP danger-associated molecular pattern DC dendritic cell E. coli Escherichia coli EDTA ethylenediaminetetraacetate ELISA Enyzme-linked immunosorbent assay et al. et alii FACS fluorescence activated cell sorting FBS fetal bovine serum FCS fetal calf serum FITC fluorescein isothiocyanate FSC forward scatter g gram (unit) g gravity GFP green fluorescent protein GM-CSF granulocyte-macrophage colony-stimulating factor GPCR G-protein coupled receptor h hour

hi high HRP horseradish peroxidase i.e. in example IL interleukin int intermediate IVC individually ventilated cage i.p. intraperitoneal IPTG Isopropyl β-D-1-thiogalactopyranoside KC/GRO keratinocyte chemoattractant /human growth-regulated oncogene lo low LPM large peritoneal macrophages LPS Lipopolysaccharide MACS magnetic associated cell sorting MFI mean fluorescence intensity mg milligram MHC major histocompatibility complex min minute MOI multiplicity of infection µl microliter ml milliliter moDC monocyte-derived DC MyD88 myeloid differentiation primary response gene 88 NA nitroanilide NK natural killer PAMP pathogen-associated molecular pattern PBS phosphate buffered saline PCR polymerase chain reaction PE phycoerythrin PerCP peridinin chlorophyll PPI peptidyl-prolyl isomeraze rec recombinant rpm rounds per minute RT room temperature SD standard deviation SDS-PAGE sodium dodecyl sulfate - polyacrylamide gel electrophoresis SEM standard error of the mean SPM small peritoneal macrophages

SSC side scatter STAg soluble toxoplasma antigen TgCyp18 Toxoplasma gondii cyclophilin 18 T. gondii Toxoplasma gondii TLR toll-like receptor TM trademark TNF tumor necrosis factor USA United States of America V Volt WT wild type

Figures

Figure 1: Activation of the complement system. The complement system is activated via one of the three pathways (classical, lectin or alternative) and leads to the generation of C3 and then C5 convertases. It results in the assembly of the membrane attack complex and generation of the anaphylatoxins C3a and C5a, which are potent inflammatory mediators. Adapted from [8]...... 4 Figure 2: IL-12 family cytokines, their receptors and signaling components. The IL-12 family comprises the heterodimeric cytokines IL-12, IL-23, IL-27 and IL-35 that consist of an α-chain (p19, p28 or p35) and a β-chain (p40 or Ebi3). These cytokines exert their functions upon binding to heterodimeric receptors and involve distinct JAK-STAT signaling partners. The bottom bar reflects their functional spectrum ranging from most pro-inflammatory (IL-23) to most inhibitory (IL-35). From [141]...... 14 Figure 3: Life cycle of Toxoplasma gondii. Intermediate hosts such as humans, pigs, sheep, cattle and chickens become infected through ingestion of oocytes shed by members of the feline family (definitive host). Humans, as well as felines can also become infected after ingestion of poorly cooked meat containing tissue cysts. From [142]...... 15 Figure 4: Immune cell interplay during T. gondii infection. Parasite invasion triggers TLR- dependent IL-12 production from DCs that induces IFN-γ secretion from NK cells and neutrophils. Priming of cells with IFN-γ leads to the elimination of intracellular parasites. This is critical for host

+ + survival during the acute phase of infection and results in adaptive T H1-based CD4 and CD8 T- cell-mediated immune responses. From [188]...... 19 Figure 5: DC-mediated NK cell activation. DC-mediated activation of NK cells results in increased NK-cell cytolytic activity and IFN-γ production. Both, cell contact-dependent interactions and soluble cytokine signals are involved in DC-NK cell cross-talk. NKG2D (NK group 2, member D) ligands that are expressed by DCs in response to microbial stimuli. A role for adhesion molecules has also been indicated by the finding that LFA1 (lymphocyte function-associated antigen 1)–ICAM1 (intercellular adhesion molecule 1) interactions are important for DC-mediated activation of NK cells. The relevance of interactions mediated by CD70–CD27, CD48–2B4 and CLRB (C-type-lectin-related B)–NKR-P1B (NK-cell receptor protein 1B) or NKR-P1D requires assessment. Cytokine signals are essential for NK cell activation, and several cytokines, such as IFN-α, IFN-β, IL-2, IL-12, IL-15 and IL-8 are involved. Reciprocally, NK cells can affect DC functions and lead to DC activation. From [247]...... 21 Figure 6: Expression vector pGEX-T1 used to produce the GST-TgCyp18 fusion protein. BamHI and EcoRI restriction sites were used to insert the TgCyp18 encoding DNA sequence...... 38 Figure 7 : In vitro culture of T.gondii tachyzoites visualized by phase contrast light microscopy 100x...... 46

Figure 8: T. gondii tissue cyst in mouse brain homogenate visualized by phase contrast light microscopy (40x)...... 47 Figure 9: Experimental design for the analysis of mice infected i.p. with T. gondii. ... 48 Figure 10: Typical plots showing the gating strategy to exclude cell debris (A) and doublets (B) from analysis. Single cells were defined to have an 1:1 FSC-A/FSC-H signal ratio...... 51 Figure 11: Monitoring of the expression of TgCyp18 in the bacterial culture. Samples were analyzed by SDS-PAGE electrophoresis on a 4-15% acrylamide gel followed by Coomasie R250 staining. 1. Standard ladder, 2. Bacterial culture before protein induction, 3. After addition of IPTG, 4. Culture supernatant, 5. Bacterial culture lysate after sonication, 6. Lysate after recombinant protein extraction, 7. Recombinant protein after on-column cleavage, 8. Fraction that contains GST- tag and traces of uncleaved protein eluted from the column...... 53 Figure 12: Recombinant TgCyp18 fractions obtained after on-column cleavage of the GST-tag. Samples were analyzed using SDS-PAGE electrophoresis on a 4-15% acrylamide gel followed by (A) Coomasie R250 or (B) silver staining. 1. Standard ladder, 2-4, 6-8. Different elution fractions collected from the column, 5,9. Fractions that contain the GST-tag...... 54 Figure 13: Peptidyl-prolyl isomerization assay. (A) Exponential curve fit of values obtained in the assay; (B) first-order rate constants of mouse serum, STAg and recombinant TgCyp18...... 55 Figure 14: Composition of MACS-enriched DC culture from mouse spleen as determined by flow cytometry. A. Mouse spleen cell suspension before (A) and after (B) positive CD11c selection. Numbers in the gates represent the frequencies within the 4 quadrants...... 56 Figure 15: Production of IL-12p40 and p70 by sDC culture in response to T.gondii- derived ligands. Concentration of IL-12 p40 (left) and p70 (right) was determined upon addition of the TLR9 antagonist CpG 1668 (1µg/ml), STAg (1µg/ml), recombinant TgCyp18-1 (50µg/ml) or recombinant TgCyp18-2 provided by the Aliberti lab (50µg/ml) as determined by ELISA. The dotted line represents the lower detection limit of the assay. n=3, bars shown are the mean ± SEM. Statistical significant differences are indicated by * as determined by ANOVA, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001...... 56 Figure 16: Production of IL-12 in WT and MyD88-/- sDC culture in response to STAg. Levels of IL-12 p40 (left) and p70 (right) secreted in response to STAg (0,01-50µg/ml) in WT (closed circle) or MyD88-/- (open circles) cells. IL-12 levels were determined by ELISA, n=2...... 57 Figure 17: Production of IL-12 from C5aR1 and/or CCR5-deficient sDCs in vitro. Spleen DCs from WT, C5aR1-/-, CCR5-/- and C5aR1xCCR5-/- were stimulated with (A) CpG ODN1668, (B) STAg or (C) tachyzoites at a MOI 1:1 for 24h and the amount of IL-12p40 (left) and IL-12p70 (right) was measured by ELISA. n=5-6, bars shown are the mean ± SEM. Statistical significant differences are indicated by * as determined by ANOVA. * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001...... 58

Figure 18: Homologous internalization of C5aR1 and CCR5. C5aR1-GFP localization in steady state (A) and internalization upon addition of 1µg/ml of C5a (B); CCR5-GFP localization in steady state (C) and internalization upon addition of 1µg/ml of CCL5 (D). Cells were visualized using a LSM confocal microscope for ~30min at 37°C, 5% CO2 as described in section 3.3.3. White arrows indicate points or receptor clustering and internalization...... 60 Figure 19: Cross-internalization of CCR5. Dynamics of CCR5 localization upon addition of (A) C5a (1µg/ml) and (B) C5a (1µg/ml) + CCL5 (1µg/ml). Cells were visualized using a LSM confocal microscope for ~30min at 37°C, 5% CO2 as described in section 3.3.3. White arrows indicate points or receptor clustering and internalization...... 61 Figure 20: T.gondii-induced internalization of C5aR1 and CCR5. Localization of C5aR1 (A) and CCR5 (C) was visualized upon addition of T. gondii tachyzoites at a MOI 1:1 and (B) C5a (1µg/ml). Cells were visualized using a LSM confocal microscope for ~30min at 37°C, 5% CO2 as described in section 3.3.3. White arrows indicate points or receptor clustering and internalization...... 62 Figure 21: Survival of WT, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice infected with T. gondii. WT, C5aR1-/- (A), CCR5-/- (B), C5aR1xCCR5-/- (C), C5aR2-/- (D) and C5-/- (E) mice were infected and evaluated for survival until day 30 p.i. Statistical significant differences are indicated by * as determined by Log-rank (Mantel-Cox) test, n=9-40, * P ≤ 0,05...... 63 Figure 22: Survival of male and female WT and C5aR1-/- mice infected with T. gondii. Male (A) and female (B) WT (solid line) and C5aR1-/- (dashed line) mice were infected and evaluated for survival until day 30 p.i. Statistical significant differences are indicated by * as determined by Log- rank (Mantel-Cox) test, n=12-21, * P ≤ 0,05...... 64 Figure 23: Absolute weight of WT, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice infected with T. gondii. WT, C5aR1-/- (A), CCR5-/- (B), C5aR1xCCR5-/- (C), C5aR2-/- (D) and C5-/- (E) mice were infected and weight was measured daily until day 30 p.i. Values shown are the mean ± SEM, n=8-35 Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05...... 64 Figure 24: Relative weight of WT, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice infected with T. gondii. WT, C5aR1-/- (A), CCR5-/- (B), C5aR1xCCR5-/- (C), C5aR2-/- (D) and C5-/- (E) mice were infected and relative weight was calculated daily until day 30 p.i. as described in section 3.5.2. Values shown are the mean ± SEM, n=8-35 Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05...... 65 Figure 25: Severity score of T. gondii infection in WT, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/- , C5aR2-/- and C5-/- mice. WT, C5aR1-/- (A), CCR5-/- (B), C5aR1xCCR5-/- (C), C5aR2-/- (D) and C5-/- (E) mice were infected and the severity score of the disease was determined daily until day 30 p.i. as described in section 3.5.2. Values shown are the mean ± SEM, n= 3-18. Statistical significant

differences of the mean severity score (F) are indicated by * as determined by ANOVA, ** P ≤ 0,01, *** P ≤ 0,001...... 65 Figure 26: Number of T. gondii tissue cysts in mouse brain. Tissue cysts in the brain were counted at day 30 after intraperitoneal infection in WT, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice (left). Right graph represents cyst number in female (open bar) and male (filled bar) mice. Values shown are the mean ± SEM, n=9-35. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001.... 66 Figure 27: Serum cytokine levels in response to STAg injection in WT, C5aR1-/- and C5aR1xCCR5-/- mice. 100µg of STAg in 0,2ml DPBS were administered i.p.; 6h after the injection blood was collected and serum cytokine levels were determined by ELISA. Values shown are the mean ± SEM. Statistical significant differences are indicated by * as determined by ANOVA, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001...... 67 Figure 28: Early kinetics of IL-12p40 and IFN-γ levels in T.gondii-infection. Wild type, C5aR1-/-, CCR5-/- and C5aR1xCCR5-/- mice were infected with 50 brain cysts i.p. Serum was sampled from animals 1, 3, 5 and 7 days after infection. The amount of IL-12p40 and IFN-γ in the serum was determined by ELISA. Values shown are the mean ± SEM, n=5-33. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, *** P ≤ 0,001...... 68 Figure 29: Serum cytokine levels in WT and C5aR1-deficient mouse 7 days after T.gondii infection as determined by Bio-Plex ProTM assay. Serum from wild type and C5aR1-/- mice was sampled at day 7 after i.p. T. gondii infection. The amount of IL-1α, IL-1β, IL-2, IL-3, IL-5, IL-6, IL-10, IL12-p40, IL-12p70, IL-13, IL-17, eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MCP, MIP-1α, MIP-1b, RANTES and TNFα in serum was determined by Bio-Plex Pro Mouse Cytokine 23-Plex Panel (Bio-Rad Laboratories GmbH, München). n=3, values shown are the mean ± SEM. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001...... 69 Figure 30: Regulation of IL-12p40, IL-12p70 and IFN-γ in the serum 7 days after T.gondii infection. Wild type, C5aR1-/-, CCR5-/-, C5aR1xCCR5-/-, C5aR2-/- and C5-/- mice were infected with 50 brain cysts i.p. and serum was drawn 7 days after infection. The amount of IL-12p40 and IFN-γ in the serum was determined by ELISA; The amount of IL-12p70 was determined by Multiplex analysis (MSD) as described in section 3.6.3. n=5-26, values shown are the mean ± SEM. Statistical significant differences are indicated by * and were determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001...... 70 Figure 31: Levels of alanine amino transferase (ALT) in the serum of WT, C5-/-, C5aR1- /-, C5aR2-/-, CCR5-/- and C5aR1xCCR5-/- mice 7 days after T.gondii infection. Control ALT levels were measured in the serum of uninfected WT and C5aR1-/- mice. Analysis was performed by ALT Activity Assay kit (Sigma) as described in section 3.6.4. Values shown are the mean ± SEM. Statistical significant differences are indicated by * as determined by ANOVA, *** P ≤ 0,001...... 70

Figure 32: Number of cells isolated from different organs of WT and C5aR1-deficient mice before and 5 days after i.p. T.gondii infection. The graphs represent the number of cells obtained from the peritoneal cavity (upper left), mesenteric lymph node (upper right), spleen (lower left) and brain (lower right) of uninfected (open circles) and infected mice 5 days after administration of T. gondii (closed circles) animals. Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, **** P ≤ 0,0001. 71 Figure 33: Gating strategy to identify mouse spleen DC subsets that produce IL-12. The CD8α+ subset of splenic DCs was identified as a CD3-CD11c+CD11b-CD8+ cell population, the CD11b+ subset was identified as a CD3-CD11c+CD11b+CD8+ cell population. IL-12 production in DCs was determined using intracellular staining. Shown are cells from uninfected mice (A) and mice 5 days after T. gondii infection (B) as described in section 3.7.1. Numbers shown in the gate represent the frequencies of the parent population...... 72 Figure 34: IL-12 production from CD8α+ and CD11b+ sDCs during acute T.gondii infection. Numbers of CD8α+ (A, left panel) and CD11b+ (B, left panel) cDCs in the spleen from uninfected (open circles) and infected (closed circles) mice 5 days after T. gondii infection. Graphs represent total numbers of cell subset (left), frequency (middle) and number of IL-12+ cells (right). Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA. * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001...... 73 Figure 35: Gating strategy to identify NK, NKT and T-cells in the mouse spleen that produce IFN-γ. Contour plots represent the gating strategy to identify NK (I. NK1.1+CD3-), NKT (II. NK1.1+CD3+) and T-cells (III. NK1.1-CD3+) in the mouse spleen. Cells from (A) uninfected mice and (B) mice infected for 5 days were analyzed for IFN-γ production by intracellular staining as described in section 3.7.1...... 74 Figure 36: Production of IFN-γ by spleen NK, NKT an T-cells at day 5 after T. gondii infection. Total cell numbers (A), frequency (B) and number of IFN-γ+ (C) cells among NK (left), NKT (center) and T-cells (right). Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001...... 75 Figure 37: Identification of neutrophils and monocyte/macrophage populations in the spleen of uninfected and infected animals. NK1.1+ and CD11c+ cells were analyzed in the previous section and excluded from this gating strategy. Representative contour plots for identification of (a) Ly6G+ neutrophils, (b) F4/80+CD11bint macrophages, (c) F4/80- /intCD11b+Ly6CloCCR2- resident monocytes and (d) F4/80-/intCD11b+Ly6ChiCCR2+ pro-inflammatory monocytes. Histograms on the right show CCR2 expression in Ly6Chi monocytes. Dashed line show isotype control staining. Lin marker included CD3 and CD19. Cells were analyzed in the uninfected WT and C5aR1-/- animals (A) and 5 days after infection (B). Numbers represent the frequencies of the parent population...... 76

Figure 38: Neutrophils, monocytes and macrophages in the spleen of uninfected and infected animals. Graphs represent total numbers of (a) neutrophils, (b) macrophages, (c) Ly6Clo and (d) Ly6Chi monocytes (left) and MFI of CCR2 in Ly6Chi cells (right). Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01...... 77 Figure 39: Gating strategy to identify cell populations in the peritoneal cavity of uninfected and infected animals Contour plots depict gating strategy to identify following cell subsets: (a) neutrophils (Ly6G+), (b) NK cells (Ly6G-NK1.1+), (c) large peritoneal macrophages (CD11b+F4/80hiLy6C-), (d) small peritoneal macrophages (CD11b+F4/80loLy6C-), (e) inflammatory monocytes (CD11b+F4/80intLy6ChiCCR2+), (f) Ly6C monocytes (CD11b+F4/80-Ly6C+), (g) CCR2+ inflammatory monocytes (CD11b+F4/80-Ly6C+CCR2+). Numbers shown in the gate represent frequencies of the parent population. Lin marker includes CD3 and CD19. Cells were analyzed in the uninfected animals (A) and 5 days after infection (B) in WT and C5aR1-/- mice. Numbers represent the frequencies of the parent population...... 78 Figure 40: Change in cell composition of the peritoneal compartment during the acute stage of T. gondii infection. Graphs represent total numbers of (a) neutrophils; (b) NK cells, (c) large peritoneal macrophages, (d) small peritoneal macrophages, (e) F4/80+Ly6C+ monocytes/macrophages, (f) F4/80-Ly6C+ monocytes and (g) Ly6C+CCR2+ inflammatory monocytes in WT and C5aR1-/- uninfected mice and 5 days after T. gondii infection. Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001...... 79 Figure 41: Gating strategy to identify B-1a and B-1b cells in the peritoneal cavity of uninfected and T.gondii-infected animals. Representative contour plots for identification of resident (IgMhi) and migratory (IgMlo) B-1a (CD11bneg to loCD43+IgM+CD5+) and B-1b cells (CD11bneg to loCD43+IgM+CD5-). Cells were analyzed in the uninfected WT and C5aR1-/- animals (A) and 5 days after infection (B). Numbers represent the frequencies of the parent population...... 80 Figure 42: B-1 cell compartment in the peritoneal cavity of uninfected and T. gondii- infected animals. Numbers of resident (A) and migratory IgMlow (B) B-1 (left), B-1a (middle) and B- 1b (right) cells before and 5 days after T. gondii infection in WT and C5aR1-/- mice. Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001...... 81 Figure 43: Gating strategy to identify cell populations in the mesenteric lymph nodes of uninfected and infected animals Contour plots depict gating the strategy to identify the following cell subsets: (a) NK cells (Lin-CD11c-NK1.1+), (b) neutrophils (Lin-NK1.1-Ly6G+), (c) macrophages (Lin-NK1.1-Ly6G-F4/80+), (d) Ly6Chi monocytes (Lin-CD11c-NK1.1-Ly6G-F4/80-CD11b+Ly6Chi) and (e) Ly6Clo monocytes (Lin-CD11c-NK1.1-Ly6G-F4/80-CD11b+Ly6Clo). Lin marker includes CD3 and

CD19. Cells were analyzed in uninfected animals (A) and 5 days after infection (B) in WT and C5aR1- /- mice. Numbers represent the frequencies of the parent population...... 82 Figure 44: Change in cell composition in the lymph node during the acute stage of T. gondii infection. Graphs represent total numbers of (a) NK cells, (b) neutrophils, (c) macrophages, (d) Ly6Chi monocytes and (e) Ly6Clo monocytes in WT and C5aR1-/- uninfected mice and 5 days after T. gondii infection. Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01...... 83 Figure 45: Gating strategy to identify DC populations in the mesenteric lymph nodes of uninfected and infected animals. Contour plots depict gating strategy to identify following cell subsets: MHCII+CD11c+ DCs (a), CD103-CD11b+ (b), CD103+CD11b- (c) and CD103-CD11b- (d) cDC subsets. CD103-CD11b+ cells were further differentiated into CD64- cDCs (e) and CD64+ moDCs (f). Lin marker includes CD3 and CD19. Cells were analyzed in the uninfected animals (A) and 5 days after infection (B) in WT and C5aR1-/- mice. Numbers represent the frequencies of the parent population...... 84 Figure 46: DC subsets in the MLNs during the acute stage of T. gondii infection. Graphs represent total DC numbers (a), CD103-CD11b+ (b), CD103+CD11b- (c) and CD103-CD11b- (d) cDCs, CD103-CD11b+ cDCs (e) and moDCs (f) in WT and C5aR1-/- uninfected mice and 5 days after T. gondii infection. Values shown are the mean ± SEM, n=6. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05...... 85 Figure 47: Gating strategy to identify immune cells in the brain of WT and C5aR1-/- mice chronically infected with T. gondii. Contour plots depict the gating strategy to identify the following cell subsets: CD45intCD11b+ microglia and CD45hiCD11bneg to lo brain leukocytes. Cells were analyzed in uninfected animals (A), 5 (B) and 30 (C) days after T. gondii infection in WT and C5aR1-/- mice. Numbers represent the frequencies of the parent population...... 86 Figure 48: Immune cells in the brain of WT and C5aR1-/- mice during the acute and chronic stage of T. gondii infection. Graphs represent total number of immune cells (left), microglia (middle) and other leukocytes (right) in the brain of WT and C5aR1-/- mice in uninfected animals (open circles), 5 (half closed circles) and 30 (closed circles) days after T. gondii infection. Values shown are the mean ± SEM, n=4-12. Statistical significant differences are indicated by * as determined by ANOVA, * P ≤ 0,05, ** P ≤ 0,01, *** P ≤ 0,001, **** P ≤ 0,0001...... 86 Figure 49: Gating strategy to identify CD4+ and CD8+ T-cells and their phenotype in the brain of WT, C5aR1-/-, CCR5-/- and C5aR2-/- mice at day 30 after T. gondii infection. First, the leukocyte population was pre-gated as CD45hi cells as shown in the previous section and T cells were defined by the expression of CD3. Expression of CD44 and CD62L was determined in CD4+ and CD8+ T cell populations. The CD44+CD62L+ population was defined as central memory (upper gate) and CD44+CD62L- (lower gate) as effector T cells. Cells were isolated from the mouse brain at day 30 after T. gondii infection from WT and C5aR1-/- mice. Numbers represent the frequencies of the parent population...... 88 + + Figure 50: Number of CD4 TH and CD8 T cells and their subsets in the brain of WT, C5aR1-/-, CCR5-/- and C5aR2-/- mice chronically infected with T. gondii. Total numbers of T cells, + + + + CD4 TH cells, CD8 T cells, effector/effector memory CD4 TH cells, central memory CD4 TH cells, central memory CD8+ T cells, effector/effector memory CD8+ T cells in the brain of WT (closed circles), C5aR1-/- (open circles), CCR5-/- (open squares) and C5aR2-/- (open triangles) mice at day 30 after T. gondii infection. Values shown are the mean ± SEM, n=2-5. Statistical differences were determined by ANOVA...... 89 Figure 51: Gating strategy to identify T. gondii-specific T-cells in the spleen and brain of WT, C5aR1-/- and C5aR1xCCR5-/- mice at day 30 after infection. Cells from the brain (A) and spleen (B) were stained for CD3, CD4 and CD8 to gate on T cell populations. Antigen-specific cells were stained using T. gondii-specific tetramers as described in section 3.7.2. Numbers represent the frequencies of the parent population...... 90 Figure 52: T. gondii-specific CD4+ and CD8+ T-cells in the spleen and brain of chronically infected WT, C5aR1-/- and C5aR1xCCR5-/- mice. Percentage of antigen-specific cells among the CD8+ (left) and CD4+ (right) T cells in the brain (A) and spleen (B) of WT, C5aR1-/- and C5aR1xCCR5-/- mice at day 30 after T. gondii infection. Values shown are the mean ± SEM, n=4-5 Statistical significant differences are indicated by * as determined by ANOVA, *** P ≤ 0,001...... 91

Tables

Table 1: Regulators of the complement system. Regulatory soluble proteins and receptors of the complement system. MASP2, Mannan-binding lectin serine protease 2; MAC, membrane attack complex; C, complement component. Modified from [27, 29] ...... 6 Table 2: Major subsets of DCs in humans and mice. BATF3, basic leucine zipper transcription factor ATF-like 3; BDCA, blood dendritic cell antigen; BST2, bone marrow stromal antigen 2; CLEC9A, C-type lectin domain family 9 member A; CX3CR1, CX3C-chemokine receptor 1; ESAM, endothelial cell-selective adhesion molecule; IRF4, interferon-regulatory factor 4; ND, not described; SIGLEC, sialic acid-binding immunoglobulin-like lectin; SIRPα, signal-regulatory protein- α; TCF4, transcription factor 4; XCR1, XC-chemokine receptor 1. Modified from [134]...... 11 Table 3: Expression patterns of innate immune receptors in mouse and human DC subsets. +, expressed; −, low or no expression; ?, expression level is unknown; BDCA, blood DC antigen; CLEC, C-type lectin domain family member; NLRP3, NOD-, LRR- and pyrin domain- containing 3; NOD1, nucleotide-binding oligomerization domain-containing protein 1; TLR, Toll-like receptor; XCR1, XC-chemokine receptor 1. Modified from [137]...... 12 Table 4: Chemokine receptors and their ligands that are involved in dendritic cell migration. BCA, B cell attracting chemokine 1; BLC, B lymphocyte chemoattractant; ELC, EBV induced molecule 1 ligand chemokine; IP, IFN-γ-induced protein-10; LARC, liver and activation- regulated chemokine; MCP, monocyte chemoattractant protein; MIP, macrophage inhibitory protein; SLC, secondary lymphoid-tissue chemokine; SDF, stromal cell derived factor. Adapted from [139]...... 13 Table 5: Mouse strains. B6 = C57BL/6, cg = congenic, tg = transgenic, tm = targeted mutation...... 27 Table 6: Chemicals and reagents...... 27 Table 7: Buffers, solutions and media...... 29 Table 8: Antibodies. AF = Alexa Fluor®, APC = Allophycocyanin, BV = Brilliant Violet, Cy = Cyanine, FITC = Fluorescein Isothiocyanate, PE = Phycoerythrin, PerCP = Peridinin-chlorophyll- protein complex...... 32 Table 9: Plastic ware and disposable items...... 34 Table 10: Commercially available kits ...... 35 Table 11: Laboratory equipment ...... 35 Table 12 : Computer software...... 37 Table 13: Difference in the cell number of immune cell subsets in T.gondii-infected C5aR1-/- mice over WT mice 5 and 30 days after infection. ↑ = higher; − = no significant difference; ↓ = lower; ND = not determined; NA = not applicable...... 99 Congress contributions Poster Presentations

The role of the C5aR1/CCR5 axes in Toxoplasma gondii infection International Cluster Symposium, CAU Kiel (2015)

The role of the C5aR1/CCR5 axes in Toxoplasma gondii infection 1st International Symposium Allergy meets Infection, Lübeck (IRTG1911 Retreat) (2015)

Oral Presentations Novel role for C5aR1 in clearance of Toxoplasma gondii infection 26th International Complement Workshop, Kanazawa, Japan (2016)

Immune sensing of Toxoplasma gondii infection by C5aR1 and CCR5 pairing International Cluster Symposium, CAU Kiel (2015)

Immune sensing of Toxoplasma gondii infection by C5aR and CCR5 pairing IRTG1911 retreat, Neumünster (2013) List of publications

Quell KM, Karsten CM, Kordowski A, Almeida L, Briukhovetska D, Wiese AV, Sun J, Ender F, Antoniou K, Schröder T, Schmudde I, Berger JL, König P, Vollbrandt T, Laumonnier Y, Köhl J. Monitoring C3aR expression using a floxed tdTomato-C3aR1 reporter knock-in mouse. J. Immunol., 2017 (in revision)

Karsten CM, Laumonnier Y, Mey F, Figge J, Schmudde I, Kordowski A, Woodruff T, Briukhovetska D, Wiese AV, Sun J, Ender F, Almeida L, Vollbrandt T, Köhl J. Characterization of a novel tdTomato-C5aR2 reporter knock-in mouse. J. Immunol., 2017 (submitted)

Briukhovetska D, Ohm B, Mey F, Aliberti J, Karsten CM, Köhl J. The role of C5a receptor 1 in acute and chronic experimental T. gondii infection. (Manuscript in preparation) Acknowledgments

I would like to express my deep sense of gratitude to all the people that made this work possible and especially:

Prof. Dr. med. Jörg Köhl for the chance to carry out this work at his institute, his supervision and patience. Dr. Christian Karsten and Dr. Julia Figge for their inspiring and thoughtful discussions, their personal attention to everyone and the atmosphere they created in the lab. Dr. Julio Aliberti for his co-supervision, warm hosting in Cincinnati and sharing his experience. Prof. Dr. Rainer Duden and PD Dr. Irina Majoul provided plasmids for C5aR1 and CCR5 labeling, shared their vast experience in live cell imaging and welcomed me in their lab. Also I thank Prof. Reiner Duden for his kind agreement to review my doctoral thesis. Prof. Dr. Norbert Tautz for being a head of examination board. Prof. Dr. Jan Rupp for taking part in yearly reports as the 3rd supervisor of this project. Prof. Dr. med. Dirk Schlüter from Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Magdeburg University for parasite cultures and experimental design consultations. Assoc. Prof. Yoshifumi Nishikawa from Obihiro University of Agriculture and Veterinary medicine, Japan for TgCyp18 plasmid and protein purification protocol. Dr. Yves Laumonnier for his valuable advices. Fabian Mey and Birte Ohm, my fellow students and collaborators for all the work we’ve done side by side, their patience and coffee brakes. Lana Pohle and Nate Shryock for their excellent technical assistance and friendly atmosphere. Also Gabriele Köhl and Esther Strerath for their technical assistance and animal management. Claudia Delfs for assistance with paperwork. To all past and present ISEF colleagues Katharina, Fanny, Anna, Olga, Larissa, Balint and Kristin that made my work there pleasant and memorable.

Special thanks to my parents Viktoria and Aleksandr Briukhovetski for their enormous support, encouragement and understanding during the whole time of my PhD.