Cerebral Toxoplasmosis

C H A P T E R

23 Cerebral Toxoplasmosis: Pathogenesis, Host Resistance and Behavioural Consequences Yasuhiro Suzuki*, Qila Sa*, Eri Ochiai*, Jeremi Mullins*, Robert Yolkeny, Sandra K. Halonen** *Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, USA yStanley Neurology Laboratory, Johns Hopkins University, Baltimore, Maryland, USA **Department of Microbiology, Montana State University, Bozeman, Montana, USA

O U T L I N E 23.1 Introduction

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23.2 Producers of Interleukin (IL)-12 Required for IFNg Production 23.2.1 Dendritic Cells 23.2.2 Macrophages 23.2.3 Neutrophils

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23.3 Producers of IFNg 23.3.1 Involvement of ‘Innate Immunity’ 23.3.1.1 Microglia and BloodDerived Macrophages 23.3.1.2 Gamma Delta (gd) T-Cells 23.3.1.3 NK-cells

Toxoplasma gondii, second edition http://dx.doi.org/10.1016/B978-0-12-396481-6.00023-4

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23.3.2 Importance of ‘Acquired Immunity’ Involving CD4þ and CD8þab T-Cells 23.4 Other Cytokines and Regulatory Molecules for Resistance 23.4.1 TNFa 23.4.2 Lymphotoxin 23.4.3 IL-4 23.4.4 IL-5 23.4.5 IL-6 23.4.6 IL-10 23.4.7 Lipoxin A4 23.4.8 IL-17 and IL-27 23.4.9 IL-33

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Copyright Ó 2014 Elsevier Ltd. All rights reserved.

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23.5 Involvement of Humoural Immunity in Resistance 23.6 IFNg Induced Effector Mechanisms 23.6.1 Nitric Oxide (NO)-Mediated Mechanism 23.6.2 Tryptophan Starvation Pathway 23.6.3 Immunity-Related GTPase (IRG) Family 23.6.4 Guanylate-Binding Proteins (GBPs or p65 GTPases) 23.6.5 Reactive Oxygen Intermediates (ROI) 23.6.6 Iron Deprivation 23.7 IFNg Effector Cells in the Brain with Activity Against Toxoplasma gondii 23.7.1 Microglial Cells 23.7.2 Astrocytes 23.7.3 Cerebral Microvascular Endothelial Cells 23.7.4 Dendritic Cells 23.8 The Role of Host Cells Harbouring Toxoplasma gondii in the Brain 23.8.1 T. gondii Infection in Neurons

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23.1 INTRODUCTION Toxoplasma gondii is a ubiquitous, obligate intracellular protozoan parasite in humans and animals. Chronic infection with this parasite is likely one of the most common infections of humans, affecting 10% to 25% of the world’s population. During the acute stage of infection, tachyzoites quickly proliferate within a variety of nucleated cells and spread throughout host tissues. Interferon-gamma (IFNg)-dependent cell-mediated immune responses, and humoural immune responses to a lesser extent, control the tachzyoite proliferation (reviewed in Chapters 24 and 25), but the parasite forms cysts in

23.8.2 Role of Astrocytes in Cerebral Toxoplasmosis 23.8.3 Microglia and AstrocyteeMicrogliaeNeuronal Interactions

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23.9 Immune Responses to the Cyst Stage of Toxoplasma gondii in the Brain 779 23.10 Host Genes Involved in Regulating Resistance

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23.11 Immune Effector Mechanisms in Ocular Toxoplasmosis

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23.12 Immune Effector Mechanisms in Congenital Toxoplasmosis

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23.13 Behavourial Consequences of Infection 23.13.1 Animal Studies 23.13.2 Human Studies

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23.14 Conclusions

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Acknowledgements

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References

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various organs, especially the brain, heart and skeletal muscle, and establishes a chronic infection. Acute acquired infection in immunocompetent individuals is usually unnoticed or causes a benign, self-limiting illness, and results in a chronic infection. Although such chronic infection has been considered ‘latent’, recent studies indicated a correlation of chronic T. gondii infection with cryptogenic epilepsy and schizophrenia. Another important aspect of chronic infection with this parasite is an occurrence of reactivation of the infection in immunocompromised individuals such as those with AIDS, organ transplants or cancer. The reactivation of chronic T. gondii infection is initiated by

23.2 PRODUCERS OF INTERLEUKIN (IL)-12 REQUIRED FOR IFNg PRODUCTION

disruption of cysts, followed by proliferation of tachyzoites which causes life-threatening toxoplasmic encephalitis (TE). Since TE occurs almost solely in immunocompromised individuals, it is clear that the immune response to T. gondii is crucial to prevent the disease. When an acute acquired infection occurs in pregnant women who have never been infected with the parasite before, the parasite can pass through the placenta and infect the foetus. The brain is the major organ affected by the congenital infection, as well. Murine models have mainly been used to analyse the mechanisms of the protective immunity to acute acquired infection and development of TE during the later stage of infection. Multiple types of cells are involved as producers of IFNg in resistance to infection, and a variety of cell types participate as effector cells that become activated by IFNg to control the parasite. Additionally, multiple cell types also contribute as producers of IL-12, which is required for induction of IFNg production.

23.2 PRODUCERS OF INTERLEUKIN (IL)-12 REQUIRED FOR IFNg PRODUCTION 23.2.1 Dendritic Cells IL-12 is the most important inducer of IFNg synthesis during the acute stage of infection. Neutralization of IL-12 with anti-IL-12 antibodies results in 100% mortality in mice following infection with an avirulent strain of T. gondii, and the mortality is associated with decreased IFNg production (Gazzinelli et al., 1994b). Dendritic cells were identified to be the source of IL-12 in the spleen in response to T. gondii (Reis e Sousa et al., 1997). All IL-12 positive cells in spleens of T. gondii-stimulated mice were found in T-cell areas and were CD8aþ, CD11cþ, DEC205þ dendritic cells. CCR5 expressed on the surface of dendritic cells is responsible for their migration

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into splenic T-cell areas following stimulation (Reis e Sousa et al., 1997). CCR5 signalling also plays an important role in activation of dendritic cells to produce IL-12 (Aliberti et al., 2000). CCR5-deficient mice are impaired in IL-12 production by dendritic cells and are highly susceptible to T. gondii infection. Cyclophilin-18 was identified as the principal molecule derived from the parasite that triggers IL-12 production through CCR5 (Aliberti et al., 2003). More recently, a profilin-like protein of the parasite was found to bind to Toll-like receptor 11 and stimulate IL-12 production by dendritic cells (Yarovinsky et al., 2005). Mice deficient in the gene encoding MyD88, an adaptor molecule essential for most TLR as well as IL-1 and IL-18 signalling, are susceptible to acute infection with an avirulent strain, and their susceptibility is associated with impaired IL-12 responses to the parasite (Scanga et al., 2002). The binding of IL12 to its receptor leads to the activation of signal transducer and activator of transcription (STAT) 4, and this signalling cascade is crucial for IFNg producing cells. In agreement with this, STAT4-deficient mice are susceptible to acute infection with T. gondii, and their mortality is associated with a defect in the ability to produce IFNg in response to infection (Cai et al., 2000). It was shown that lack of MyD88 in dendritic cells, but not in macrophages or neutrophils, resulted in high susceptibility to acute infection with T. gondii (Hou et al., 2011). During the chronic stage of infection, cells bearing the dendritic cell markers such as CD11c and 33D1 are located at inflammatory sites in the brains of mice (Fischer et al., 2000b). These brain dendritic cells are mature as indicated by high-level expression of MHC class II molecules, CD40, CD54, CD80 and CD86, and are able to trigger antigen-specific T-cell responses in vitro. The dendritic cells were revealed to be the major producers of IL-12 among mononuclear cells isolated from brains of infected animals (Fischer et al., 2000b). GMeCSF is suggested to be important for induction

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of the dendritic cells in primary brain cell cultures with T. gondii (Fischer et al., 2000b). Since IL-12 is important for the maintenance of IFNg production in T-cells mediating resistance to chronic infection (Yap et al., 2000), dendritic cells in the brains of the infected mice might play a role in maintaining IFNg production by T-cells in this organ. Dendritic cells also play a role in disseminating the parasite into the brain. Following oral infection with T. gondii, tachyzoites are preferentially detected in CD11cþ cells in the lamina propria on day 2 and in the mesenteric lymph nodes from days 3 to 7 of infection (Courret et al., 2006), suggesting that infected CD11cþCD11bþ/ dendritic cells (DC) are a carrier of the parasite, disseminating the infection from the lamina propria to mesenteric lymph nodes. Tachyzoiteinfected dendritic cells exhibit hypermotility in vitro (Lambert et al., 2006). When infected DC are injected intravenously into uninfected mice, the parasite can be detected in the brains of the recipients (Lambert et al., 2006; Courret et al., 2006).

23.2.2 Macrophages Macrophages produce IL-12 in response to tachyzoites or tachyzoite antigens in vitro (Gazzinelli et al., 1994b, 1996). T. gondii has three predominant genotypes; types I, II and III. Infection of murine macrophages with tachyzoites of a type II (avirulent to mice) strain in vitro resulted in a greater production of IL-12 than infection with tachyzoites of a type I (virulent to mice) strain (Robben et al., 2004). Therefore, the lower IL-12 production by macrophages may contribute to the acute virulence of type I parasite to mice. Kim et al. (Kim et al., 2006) reported that type I tachyzoites induce IL-12 production through MyD88-independent mechanisms, whereas type II tachyzoites do so by both MyD88-dependent and -independent mechanisms. Since macrophages infiltrate into the brains of mice following infection with T. gondii (Suzuki et al., 2005; Wilson et al., 2005), these cells, in addition to dendritic cells,

may be an important source of IL-12 in resistance against the parasite in the brain. As with dendritic cells, monocytes/macrophages appear to play a pathogenic role as well by assisting dissemination of the parasite during the acute stage of infection. Macrophages (Da Gama et al., 2004) have been shown to effectively disseminate tachyzoites into lymph nodes (Da Gama et al., 2004) in mice. In addition, CD11cCD11bþ monocytes are the major cell population that contains tachyzoites in the blood (Courret et al., 2006), suggesting an importance of this cell population in disseminating the infection to various organs, including the brain. In support of this possibility, at one day after an intravenous injection of CD11bþ blood cells from infected mice into uninfected animals, the parasite is detectable in mononuclear cells obtained from the brains of the recipient animals (Courret et al., 2006). This is in contrast to an intravenous injection of a small number of free tachyzoites, in which the parasite was not detected one day later. Treatment of infected mice with anti-CD11b mAb at six and seven days after infection resulted in 50% reduction in the number of the parasite in their brains detected at eight days after infection, suggesting an involvement of CD11b integrin in parasite dissemination to the brain.

23.2.3 Neutrophils Neutrophils rapidly infiltrate into the peritoneal cavity of mice following intraperitoneal infection with T. gondii. Approximately 85% of the neutrophils displayed intracellular storage of IL-12 (Bliss et al., 2000). Depletion of neutrophils during the first six days of infection resulted in increased mortality in mice in association with decreased production of IL-12 and IFNg by splenocytes (Bliss et al., 2001). Rapid infiltration of neutrophils into the site of infection appears to play an important role in induction of the protective Th1-type immune responses against the parasite during the early

23.3 PRODUCERS OF IFNg

stage of infection. The production of IL-12 by neutrophils in response to soluble tachyzoite antigens is in strict dependence upon functional MyD88 (Freund et al., 2001). JUNK2 mitogenassociated protein kinase was recently shown to be required for T. gondii-induced neutrophil IL-12 production (Aviles et al., 2008). However, it is unknown whether neutrophils are involved in the resistance in the brain to control T. gondii during the chronic stage of infection.

23.3 PRODUCERS OF IFNg 23.3.1 Involvement of ‘Innate Immunity’ 23.3.1.1 Microglia and Blood-Derived Macrophages Alphaebeta T-cells are essential to control T. gondii in both acute and chronic stages of infection (see Section 23.3.2). However, we found that, in addition to T-cells, IFNg production by cells other than T-cells is required for prevention of reactivation of T. gondii infection (TE) in the brain in chronically infected mice (Kang and Suzuki, 2001). Athymic nude, SCID and IFNg-deficient mice were infected with T. gondii and treated with sulphadiazine to establish chronic infection. After discontinuation of sulphadiazine, each of these animals developed severe TE due to reactivation of the chronic infection. When the animals received adoptive transfer of immune spleen or T-cells before discontinuation of sulphadiazine, infected athymic nude and SCID mice did not develop TE and survived. However, infected IFNg deficient mice still developed TE even after receiving cell transfers (Kang and Suzuki, 2001). Before cell transfer, IFNg mRNA was detected in brains of the nude and SCID mice but not in brains of the IFNg deficient mice. IFNg mRNA was also detected in brains of infected SCID mice depleted of NK-cells, and such animals did not develop TE after receiving immune T-cells

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(Kang and Suzuki, 2001). Thus, IFNg production by non-T-cells, in addition to T-cells, is required for prevention of reactivation of T. gondii infection in the brain. The IFNg producing nonT-cells do not appear to be NK-cells. In regard to the identity of the non-T-, nonNK-cells that produce IFNg in the brains of nude and SCID mice chronically infected with T. gondii, intracellular staining for IFNg followed by flow cytometry revealed that approximately 45%e60% of the cells expressing IFNg in their brains were positive for CD11b or F4/80 (markers for microglia/macrophages) on their surfaces (Suzuki et al., 2005). Smaller portions of the cells were positive for pan-NK marker. Further smaller portions were positive for CD11c (a marker for dendritic cells), and these cells were less than 5% of the IFNg expressing cells in brains of infected SCID mice. Large amounts of mRNA for IFNg were detected in CD11bþ cells purified from brains of infected mice, but it was not the case in the cells obtained from uninfected animals. In infected SCID mice depleted of NK-cells by treatment with antiasialoeGM1 antibody, cells expressing IFNg in their brains were all positive for CD11b, and the IFNg producing cells were detected in both CD45low and CD45high populations. These results suggest that CD11bþ CD45low microglia and CD11bþ CD45high blood-derived macrophages are the major non-T-, non-NK-cells which express IFNg in the brains of mice infected with T. gondii. Therefore, it is possible that IFNg production by microglia and/or macrophages plays an important role in prevention of TE in collaboration with ab T-cells. 23.3.1.2 Gamma Delta (gd) T-Cells During the acute stage of infection with T. gondii, increased numbers of T-cells expressing the gd T-cell receptor have been observed in the spleen and peritoneal cavity of mice and the peripheral blood of humans. Gd T-cells are cytotoxic to infected target cells and produce IFNg and TNFa in response to the parasite in

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vitro. Involvement of gd T-cells in resistance against acute infection with T. gondii has been shown in mice. Mice deficient in gd T-cells due to treatment with anti-TCR gd mAb (Hisaeda et al., 1995) or lack of the functional TCR d gene (Kasper et al., 1996) die earlier than control mice during the acute stage of infection. Gd T-cells may also be involved in prevention of TE during the late stage of infection, as gd T-cells are detectable in brains of chronically infected mice and rats. Of interest, the relative percentages of gd T-cells in lymphocyte preparations isolated from brains of infected mice are significantly higher than in their spleens (Suzuki et al., 1997). This suggests that gd T-cells preferentially infiltrate into the brain of T. gondiiinfected mice. In humans, a marked increase in gd T-cells was observed in the peripheral blood of a patient with CD40L defect (hyper-IgM syndrome) who had developed TE (Leiva et al., 1998). The patient responded well to antitoxoplasmic chemotherapy and to high dose immunoglobulin replacement therapy. Therefore, gd T-cells may have contributed to controlling the disease under the chemotherapy although their protective activity is not sufficient by itself to prevent development of TE. Lepage et al. (Lepage et al., 1998) suggested a possible role for gd T-cells to enhance the protective activity of CD8þ ab T-cells in their studies using adoptive transfer of the lymphocyte populations. 23.3.1.3 NK-cells NK-cells are an important source of IFNg in resistance against T. gondii during the early stage of infection. Depletion of NK-cells results in early or increased mortality in SCID and wild-type mice. In contrast to the early stage of infection, NK-cells do not appear to be crucial for prevention of TE during the late stage of infection. Depletion of NK-cells in SCID mice, which had received adoptive transfer of immune T-cells, did not abolish resistance of the recipient animals against

development of TE (Kang and Suzuki, 2001). In the depleted mice, NK-cells were undetectable by flow cytometry in their brains and spleens.

23.3.2 Importance of ‘Acquired Immunity’ Involving CD4D and CD8Dab T-Cells It is clear that ab T-cells are essential for resistance against T. gondii since athymic nude and SCID mice, which lack T-cells, succumb to acute infection and their mortality is associated with proliferation of large numbers of tachyzoites in various organs, including the brain. In resistance to acute infection in general, CD8þ T-cells are the major efferent limb of the protective cellular immunity although CD4þ T-cells are also involved. The protective activity of the T-cells is predominantly mediated by IFNg. IFNg also plays a critical role in prevention of TE during the late stage of infection in mice (Suzuki et al., 1989; Gazzinelli et al., 1992). Neutralization of the activity of IFNg in chronically infected mice by treatment with anti-IFNg monoclonal antibody (mAb) resulted in severe acute inflammation and development of large areas of necrosis in their brains (Suzuki et al., 1989). In the areas of acute inflammation and necrosis, tachyzoites and T. gondii antigens were detected, indicating that such inflammatory responses were caused by proliferation of tachyzoites. A marked increase in numbers of tachyzoites in brains of mice following treatment with anti-IFNg mAb was also demonstrated by detecting increased amounts of tachyzoite-specific SAG1 and SAG2 mRNA in their brains by the reverse transcriptasee polymerase chain reaction (RTePCR) (Gazzinelli et al., 1993). Thus, it is clear that IFNg is critical for prevention of proliferation of tachyzoites in the brains of mice. The same appears to be true in humans, since AIDS patients have an impaired ability to produce IFNg and they frequently develop TE.

23.3 PRODUCERS OF IFNg

Both CD4þ and CD8þ T-cells infiltrate the brain of mice following infection, and the T-cells are the main source of IFNg (Schluter et al., 1995; Suzuki et al., 1997; Hunter et al., 1994). CD8þ T-cells are known to be the major mediator of resistance, and this is consistent with evidence that the H-2Ld, a MHC Class I gene, confers resistance to TE in mice (Suzuki et al., 1994; Brown et al., 1995). The protective activity of the T-cells is through their production of IFNg (Wang et al., 2004). T-cells bearing T-cell receptor Vb8 are the most abundant population that produces IFNg in the brains of infected BALB/c mice genetically resistant to TE (Wang et al., 2005), and adoptive transfer of Vb8þ T-cells alone into infected nude mice prevents the development of TE (Wang et al., 2005; Kang et al., 2003). When immune Vb8þ T-cells were divided into CD4þ and CD8þ T-cell populations, the CD8þ population conferred much greater resistance to development of TE than did the CD4þ population (Wang et al., 2005). The protective activity of total Vb8þ T-cells was greater than that of CD8þVb8þ T-cells (Wang et al., 2005). Therefore, the CD8þ population plays a major role in the activity of Vb8þ immune T-cells against reactivation of infection in the brain although the CD4þ population works additively or synergistically with the CD8þ population. An importance of CD4þ T-cells for optimum IFNg production by cerebral CD8þ T-cells was also shown in CB6 (BALB/c  C57BL/6) mice infected with T. gondii (Chan et al., 2006). The mechanisms by which CD4þ T-cells enhance IFNg production and protective activity of CD8þ T-cells are unclear at this moment. One possible mechanism is that IL-4 produced by CD4þ T-cells upregulates IL-12 production of dendritic cells. IL-4 alone or together with IFNg efficiently enhances the production of bioactive IL-12 in mouse and human DC (Hochrein et al., 2000) (see Section 23.4.3 for additional information). Since IL-12 is important for the maintenance of IFNg production in T-cells mediating resistance to chronic infection (Yap

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et al., 2000), it is possible that IL-4 produced by CD4þ T-cells is involved in the activity of this T-cell population to enhance IFNg production by CD8þ T-cells during the chronic stage of T. gondii infection. This possibility is supported by evidence that STAT6, a molecule involved in IL-4 signalling, is important for activation of CD8þ T-cells and their IFNg production (Jin et al., 2009). In regard to the protective activity of CD4þ T-cells, it was previously shown that adoptive transfer of CD4þ immune T-cells conferred a partial protection against reactivation of infection in the recipient athymic nude mice whereas the same number of a total population of immune T-cells completely prevented the reactivation of infection (Kang and Suzuki, 2001). Therefore, a large number of CD4þ immune T-cells alone could confer a protection against reactivation of T. gondii infection. However, it is unclear how long the protective effect lasts in the absence of CD8þ T-cells. The presence of both T-cell subsets is critical for long-term maintenance of the latency of the chronic infection in the brain. Interactions of CD8þ T-cells with brain cells during T. gondii infection were recently visualized by the use of T. gondii transfected to express ovalbumin (OVA) and OT-1 CD8þ T-cells specific to OVA peptide in conjunction with two-photon microscopy of living brain tissue. The study showed that the antigen-specific cerebral CD8þ T-cells make transient contacts with granulomalike structures containing parasites and with individual CD11bþ antigen-presenting cells (Schaeffer et al., 2009). Another study showed that movement of brain infiltrating OT-1 T-cells is closely associated with an infection-induced reticular system of fibres (Wilson et al., 2009). In the study, seven to 14 days after a transfer of OT-1 cells into infected mice, a reduction in parasite burden in the brains of the recipients occurred (Wilson et al., 2009). However, the parasite burden gradually increased thereafter in association with an increase in PD-1 expression in the transferred OT-1 cells,

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suggesting that T-cells recruited to the brain during T. gondii infection down-regulate their ability to act as effector cells over time. A number of signalling molecules have been shown to be important for induction and/or maintenance of the protective T-cell responses during infection with T. gondii. In T-cell receptor signalling in response to the parasite, mice deficient in two Tec kinases, Rlk and Itk, had increased mortality associated with increased brain cyst numbers and decreased IFNg production by splenocytes following in vitro stimulation with a low dose of T. gondii antigens (Schaeffer et al., 1999). Protein kinase C-theta (PKC-q) is another enzyme involved in the signalling in the T-cell response. Infection of mice deficient in this enzyme (Pkcq/) resulted in impaired production of IFNg in both CD4þ and CD8þ T-cells, and the animals succumbed to necrotizing TE (Nishanth et al., 2010). The impaired IFNg production by T-cells in infected Pkcq/ mice is associated with decreased activation of transcription factors including nuclear factor (NF)kB, AP-1, and MAPK pathways. Tumour progression locus 2 (Tpl2), a serineethreonine kinase, and T-cell expression of myeloid differentiation factor 88 (MyD88) are also important for antigen-specific IFNg production by T-cells after infection (LaRosa et al, 2008; Walford et al, 2008). However, the mechanisms by which Tpl2 and MyD88 mediate the T-cell response remain to be determined. In regard to NFkB family transcription factors, RelB, c-Rel, NFkB1 and NFkB2 are all involved in regulating T-cell responses to T. gondii infection (Caamano et al., 1999, 2000; Mason et al., 2004; Harris et al., 2010). RelBdeficient (relB/) mice succumb to death after infection with T. gondii and T-cells from relB/ mice are defective in production of IFNg when stimulated with CD3 antibody in vitro (Caamano et al, 1999). Infected NFkB1deficient mice developed TE associated with

a local decrease in the number of CD8þ T-cells and IFNg production (Harris et al., 2010). A transfer of naive T-cells from the deficient animals to SCID mice conferred less protection against infection than the T-cells from wildtype animals. NFkB2-deficient mice have no defect in their ability to produce IL-12 and IFNg during the acute stage of infection. However, during the chronic stage of infection, the deficient mice succumbed to TE in association with a reduced capacity of production of IFNg by splenocytes. Apoptosis of T-cells appears to be involved in the reduced production of this cytokine (Caamano et al, 2000). C-Rel-deficient mice also survive the acute phase of infection but develop severe TE associated with decreased numbers of CD4þ T-cells and reduced production of IFNg in their brains during the later stage of infection (Mason et al., 2004). Bcl-3, a distinct member of the I-kB family, which functions as a positive regulator of nuclear factor NFkB activity, also plays a critical role in mounting a protective Th1 immune response to T. gondii (Franzoso et al., 1997). Bcl-3-deficient mice succumb to T. gondii infection due to the lack of a protective Th1 response. Recent studies determined multiple T. gondii epitopes recognized by CD8þ T-cells. H-2drestricted epitopes of GRA6, GRA4, ROP7, SAG1 and SAG3 of T. gondii have recently been identified in mice (Blanchard et al., 2008; Caetano et al., 2006; Frickel et al., 2008; Kirak et al., 2010). The reactivity of CD8þ T-cells to GRA4, GRA6 and ROP7 peaked 2, 4 and 6e8 weeks after infection (Blanchard et al., 2008; Frickel et al., 2008) and these changes would probably reflect changes in antigens available in association with conversion of T. gondii from the tachyzoite to the cyst stage during the course of infection. The GRA6 epitope, HF10, has a potent activity to stimulate IFNg production by CD8þ T-cells obtained from infected mice with H-2d haplotype, and an immunization with this epitope

23.4 OTHER CYTOKINES AND REGULATORY MOLECULES FOR RESISTANCE

peptide prevented mortality of B10.D2 (H-2d), but not C57BL/6 (H-2b) mice after challenge infection (Blanchard et al., 2008). Recently, an H-2b-restricted epitope of tgd057 was also identified (Wilson et al., 2010; Kirak et al., 2010). Tgd057-specific CD8þ T-cells obtained from ES-cloned mice following somatic cell nuclear transfer of individual nuclei from tgd057etetramerþ CD8þ T-cells into ES cells also mediated a significant protection to lethal challenge infection when transferred into recipient C57BL/6 mice (Kirak et al., 2010). A T. gondii epitope recognized by CD4þ T-cells of C57BL/6 mice has also been identified. This 15-mer epitope AS15 is derived from a T. gondii unknown protein CD4Ag28m, and presented by the H-2Ab molecule (Grover et al., 2012). An immunization of C57BL/6 mice with this peptide results in lower parasite burden in the brain of infected mice (Grover et al., 2012). In humans, HLA-A02, HLA-A03 and HLAB07 supertype-restricted CD8þ T-cell epitopes have been identified by screening predicted epitope peptides of T. gondii antigens for their activity to induce IFNg production by peripheral mononuclear cells from T. gondii seropositive individuals. Identified HLA-A02-restricted epitopes are those of SAG2C, SAG2D, SAG2X, SAG3, GRA6, GRA7, MIC1, MICA2P and SPA. The HLA-A03 supertype-restricted epitopes are those of SAG1, SAG2C, GRA6, GRA7 and SPA and the HLA-B07-restricted epitopes are those of GRA3 and GRA7 (Cong et al, 2010, 2011; Tan 2010). In addition, an immunization of transgenic mice expressing these human HLA Class I molecules (HLA-A0201 or HLA-A1101 (an HLA-A03 supertype allele)) with those identified epitope peptides conferred a protection associated with reduced parasite burden against challenge infection (Cong et al., 2010, 2011). Therefore, these epitopes appear to be promising candidates for human vaccine to induce the protective immune responses against T. gondii infection.

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23.4 OTHER CYTOKINES AND REGULATORY MOLECULES FOR RESISTANCE 23.4.1 TNFa Murine peritoneal macrophages become activated after treatment with a combination of IFNg and TNFa in vitro and the activated cells inhibit intracellular replication of tachyzoites through generation of NO by inducible NO synthase (NOS2) (Adams et al., 1990b). However, it was demonstrated that TNFa and NOS2 are not essential for controlling acute infection in vivo since mice lacking TNF receptor type 1 (R1) and type 2 (R2) and those lacking NOS2 control parasite growth in the peritoneal cavity following intraperitoneal infection (Yap et al., 1998; Deckert-Schluter et al., 1998; Scharton-Kersten et al., 1997b). Thus, the protective mechanism(s) which require IFNg but do not require TNFa or NOS2 is sufficient for mice to control parasite growth during the acute stage of the infection. Recently, IGTP and LRG-47, members of a new family of IFNg inducible genes, were shown to be required for the IFNg-mediated resistance against acute infection with T. gondii (see Section 23.6). In contrast to the acute stage of the infection, mice deficient in TNF R1/R2 or NOS2 succumbed to necrotizing TE during the late stage of the infection (Yap et al., 1998; Deckert-Schluter et al., 1998; Scharton-Kersten et al., 1997b). Their results are consistent with those of earlier studies; treatments of infected wild-type mice with anti-TNFa mAb or aminoguanididine, an NOS2 inhibitor, resulted in development of TE (Gazzinelli et al., 1993; Hayashi et al., 1996a). Thus, TNFa and NOS2 are critical for prevention of proliferation of tachyzoites in the brain. As mentioned earlier in this section, IFNg plays the central role in resistance of the brain against this parasite (Suzuki et al., 1989; Gazzinelli et al., 1992). Since neutralization of IFNg or TNFa results in decreased NOS2

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expression and development of severe TE (Gazzinelli et al., 1993), activation of NOS2 mediated by INFg and TNFa appears to play a key role in prevention of TE. Microglia and astrocytes are likely the effector cells involved in this protective mechanism (see Section 23.7). More recently, Collazo et al. (Collazo et al., 2002) reported an involvement of IGTP in the IFNg mediated protection against TE. In regard to induction of NOS2 in the brain, Yap et al. (Yap et al., 1998) reported that NOS2 induction in the brain was unimpaired in infected TNF R1/R2-deficient mice which are susceptible to TE, suggesting that TNF-dependent immune control of T. gondii expansion in the brain involves an effector function distinct from NOS2 activation. More recently, Deckert-Schluter et al. (Deckert-Schluter et al., 1998) reported that mice lacking TNF R1 but not those lacking TNF R2 developed necrotizing encephalitis following infection and that a remarkable reduction of NOS2 synthesis was observed in the brains of TNF R1-deficient animals as compared with TNF R2-deficient or control animals. They concluded that signalling through TNF R1, but not TNF R2, provides the stimulus required for the induction of NOS2 activation in the brain following infection (Deckert-Schluter et al., 1998). Thus, it appears that there are two pathways to activate NOS2 in the brain of T. gondiiinfected mice, one TNF-dependent and the other TNF-independent. Since different strains of T. gondii were employed in the studies mentioned above (Deckert-Schluter et al., 1998; Yap et al., 1998), the strain of the parasite may be an important factor affecting the activation pathway for iNOS. In regard to the role of TNFa and NOS2 in prevention of TE, Suzuki et al. (Suzuki et al., 2000a) reported that IFNg deficient mice infected and treated with sulphadiazine developed severe TE after discontinuation of sulphadiazine treatment, although these animals expressed equivalent amounts of mRNA for TNFa and iNOS in their brains when compared to control

animals. These results indicate that expression of TNFa and NOS2 in the absence of IFNg is insufficient for genetic resistance of BALB/c mice against TE.

23.4.2 Lymphotoxin Lymphotoxin (LT), in addition to TNFa, is the ligand of TNFR1. Schluter et al. (Schluter et al., 2003) reported that mice deficient in LT fail to control intracerebral T. gondii and succumbed to necrotizing TE following infection. IFNg expression in their brains was equivalent to those of control mice when the deficient animals had developed TE. Experiments with bone marrow chimera mice showed that hematopoietic cells need to express both LT and TNFa to control T. gondii in the brain.

23.4.3 IL-4 CD4þ T-cells are known to be heterogeneous (Tfh, Th1, Th2, Th17 and Treg) with regard to their function and cytokine secretion. Th1 cells preferentially secrete IL-2 and IFNg whereas Th2 cells preferentially produce IL-4, IL-5, IL-6 and IL-10. IL-4 has been reported to have a dominant effect on determining the pattern of cytokines (Th2-type) produced by CD4þ T-cells upon subsequent antigen stimulation in vitro. Since the role of IFNg is critical for prevention of development of TE as described above in Section 23.3, IL-4 was first expected to play a negative regulatory role in resistance to T. gondii infection. Surprisingly, IL-4 deficient (IL-4/) mice showed increased mortality compared to control animals (Suzuki et al., 1996; Roberts et al., 1996; Alexander et al., 1998). Therefore, IL-4 plays a protective role in resistance against T. gondii. However, the timing of the mortality and development of TE in IL-4/ is controversial between the studies. Suzuki et al. (Suzuki et al., 1996) reported that IL-4/ mice all died during the late stage (from six to 20 weeks) of infection whereas control mice all survived.

23.4 OTHER CYTOKINES AND REGULATORY MOLECULES FOR RESISTANCE

The mortality of IL-4/ mice was associated with greater numbers of cysts and areas of acute focal inflammation with tachyzoites in their brains (Suzuki et al., 1996). These results indicate that IL-4 is protective against development of TE by preventing formation of cysts and proliferation of tachyzoites in the brain. In these studies, at eight weeks after infection, spleen cells of wild-type mice produced significantly greater amounts of IFNg following stimulation in vitro with soluble T. gondii antigens than those of IL4/ mice (Suzuki et al., 1996). Therefore, IL-4 plays a role to enhance IFNg production during the late stage of infection, and the impaired ability of IL-4/ mice to produce IFNg likely contributes to their susceptibility for development of severe TE (Suzuki et al., 1996). In relation to these findings, it was reported that IL-4 enhances IFNg production by T-cells which have already been primed (differentiated) whereas it suppresses differentiation of unprimed T-cells to IFNg producing cells (Noble and Kemeny, 1995). During infection with T. gondii, IFNg production occurs earlier than IL-4 production (Beaman et al., 29e31 January 1993). Thus, it may be that IL-4 does not affect differentiation of unprimed T-cells to IFNg producing cells following T. gondii infection because of the absence (or very low production) of IL-4 in the early stage of the infection whereas it enhances IFNg production by differentiated T-cells in the late stage of the infection. In contrast, Roberts et al. (Roberts et al., 1996) reported that an increased mortality occurs in IL4/ mice during the acute stage of infection. However, in the later stage of infection, greater numbers of cysts and more severe pathology in the brain were observed in the control than IL4/ mice. During the acute stage of infection, which was the time that IL-4/ died, increased IFNg production was observed in spleen cells of IL-4/ mice when compared to those of control animals. Therefore, the authors suggested that IL-4 plays a protective role in preventing mortality by down-regulating

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pro-inflammatory cytokine production during the acute stage of infection. Reasons for the different outcomes in the studies described are unclear. However, since genetic backgrounds of mice and the strain of T. gondii differ between these two studies, these differences may have contributed to the different outcomes. In support of this possibility, genetic background of mice was shown to affect the outcome of IL-4/ mice following infection (Alexander et al., 1998). STAT6 is a molecule involved in IL-4 and ILe 13 signalling. In agreement with the observations in IL-4/ mice, significantly greater numbers of T. gondii cysts were recovered from the brains of STAT6-deficient (Stat6/) than from wild-type mice. CD8þ T-cells obtained from the cerebrospinal fluids and spleens of infected wild-type mice produced greater amounts of IFNg than T-cells from infected Stat6/ animals (Jin et al., 2009). In addition, transfer of CD8þ T-cells from wild-type to Stat6/ mice reduced cyst numbers in the brains of recipients. Transfer of splenic adherent cells from wild-type to Stat6/ mice induced activation of CD8þ T-cells and decreased brain cyst numbers. Therefore, STAT6 signalling is important in CD8þ T-cell activation after T. gondii infection, possibly through regulation of the activity of antigen presenting cells. In this regard, it has been shown that IL-4, alone or together with IFNg, efficiently enhances the production of bioactive IL-12 in mouse and human DCs (Hochein et al., 2000). The lack of this positive regulatory effect of IL-4 on IL-12 production by DCs most likely contributes to the reduced resistance of IL-4/ and Stat6/ mice to T. gondii infection described above. In addition to the regulatory effects of IL-4 on IFNg production, IL-4 has been shown to have an activity to modify intracellular replication of tachyzoites in murine macrophages (Swierczynski et al., 2000) and human fibroblast cell lines (Chaves et al., 2001). Therefore, IL-4 likely plays important regulatory roles in multiple stages of antimicrobial responses to T. gondii. More

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studies are needed to elucidate the role of IL-4 in the immunopathogenesis of toxoplasmosis.

23.4.4 IL-5 IL-5 enhances expression of IL-2 receptors on B-cells and promotes B-cell proliferation and differentiation (Swain et al., 1988; Takatsu et al., 1988). IL-5-deficient (IL-5/) mice showed increased mortality during the late stage of infection and their mortality was associated with greater numbers of cysts and tachyzoites in their brains when compared to infected control mice (Zhang and Denkers, 1999). IL-12 production by spleen cells from infected mice in response to tachyzoite antigens in vitro was markedly lower in IL-5/ than control animals, and this decrease correlated with a selective loss of B-cells during the culture. Therefore, IL-5 plays a protective role against T. gondii and its protective role is related to the production of IL-12.

23.4.5 IL-6 IL-6 is a multifunctional cytokine that regulates various aspects of the immune response, acute-phase reaction and haematopoiesis (Akira et al., 1993) and acts in the nervous system (Hirota et al., 1996). IL-6 mRNA is expressed in brains of mice infected with T. gondii (Gazzinelli et al., 1992; Deckert-Schluter et al., 1995; Hunter et al., 1992) and is detected in the CSF of those mice (Schluter et al., 1993). IL-6-deficient (IL-6/) mice formed significantly greater numbers of T. gondii cysts and areas of inflammation associated with tachyzoites in their brains than control mice (Suzuki et al., 1997; Jebbari et al., 1998). These results indicate that IL-6 is protective against development of TE by preventing formation of cysts and proliferation of tachyzoites in brains of infected mice. In brains of infected IL-6/ mice, the amounts of mRNA for IFNg detected by RTePCR were significantly less when compared to control mice, whereas the amounts of IL-10 mRNA were greater than in control

animals (Suzuki et al., 1997). In addition, lymphocyte preparations isolated from brains of infected IL-6/ mice had significantly lower ratios of gd T-cells and CD4þab T-cells but higher ratios of CD8þab T-cells than those of infected wild-type mice (Suzuki et al., 1997). Of interest, no differences were detected in the ratios of these T-cell subsets in spleens between these animals (Suzuki et al., 1997). In another study, serum IFNg levels were significantly greater in control than IL-6/ mice during the early stage of infection (Jebbari et al., 1998). Therefore, the protective activity of IL-6 against development of TE appears to be through its ability to stimulate IFNg production (systemic in the early stage of infection and in the brain in the later stage) and induce infiltration and accumulation of different T-cell subsets in brains of infected mice. In relation to the protective role of IL-6 against TE, it was reported that human foetal microglia treated with IL-6 inhibits intracellular replication of tachyzoites in vitro (Chao et al., 1994a) and that IL-6 acts synergistically with IFNg to inhibit proliferation of tachyzoites in murine astrocytes (Halonen et al., 1998b).

23.4.6 IL-10 IL-10 is an important negative regulator of inflammatory responses (Kuhn et al., 1993). IL-10-deficient (IL-10/) mice all die during the acute stage of infection with T. gondii and their mortality is associated with development of severe immunopathology mediated by Th1 immune responses in the liver (Gazzinelli et al., 1996) and intestine (Suzuki et al., 2000b). Therefore, IL-10 is crucial for down-regulating IFNg mediated immune responses and preventing development of pathology caused by the immune responses. When IL-10/ mice were treated with sulphadiazine to control proliferation of T. gondii in the early stage of infection, the animals survived the acute stage but developed lethal inflammatory responses in their brains in the later stage of infection (Wilson

23.4 OTHER CYTOKINES AND REGULATORY MOLECULES FOR RESISTANCE

et al., 2005). The importance of IL-10 dependent signalling for survival of mice during the acute and chronic stages of infection is confirmed by treating infected wild-type animals with antibodies against anti-IL-10 receptor (Jankovic et al., 2007). Therefore, IL-10 plays an important down-regulatory role to prevent immunopathology during the course of infection with T. gondii. Various populations of T-cells, such as Th2 and Tregs, are able to produce IL-10. However, conventional IFNg secreting T-betþFoxp3 Th1 cells were shown to be the main producers of IL-10 in T. gondii-infected mice. These IL-10þIFNgþCD4þ T-cells possess potent activity to prevent intracellular multiplication of tachyzoites within macrophages, while profoundly suppressing IL-12 production by antigen-presenting cells (Jankovic et al., 2007). In addition, these IL-10-producing T-cells are generated from IL-10IFNgþCD4þ T-cells after re-stimulation with tachyzoite antigens. These results indicate that IL-10 production by CD4þ T-cells after infection with T. gondii does not require a distinct regulatory Th subset, but can be generated in Th1 cells as part of the effector response to the parasite, which provides a crucial negative-feedback loop for prevention of pathogenic overstimulation of the Th1 response. There is more interesting evidence on the existence of a negative-feedback loop of Th1 response in T. gondii infection. Tyk2, a member of the Jak family of non-receptor tyrosine kinases, mediates the biological effects of IL-12 and IFNab and promotes IFNg production by Th1 cells, and Tyk2-null mice are susceptible to infection with the parasite. However, Tyk2 is also required for the production of IL-10 by immune CD4þ T-cells after challenge infection of vaccinated mice (Shaw et al., 2006). The Tyk2dependent production of IL-10 is mediated by IFNg, indicating negative autoregulation of the Th1 effector response in infection. Therefore, Tyk2 has a dual function mediating induction

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and suppression of the Th1 effector response, most likely for maximizing pathogen clearance while minimizing immunopathology.

23.4.7 Lipoxin A4 Lipoxin A4 (LXA4), an eicosanoid product generated from arachidonic acid, is an important down-regulator of IL-12 production to prevent pathogenic inflammatory responses in the brain during the chronic stage of T. gondii infection. Wild-type mice produced high levels of serum LXA4 beginning at the onset of chronic infection. 5-Lipoxygenase (5-LO) is an enzyme critical in the generation of LXA4, and mice deficient in 5-LO (Alox5/) succumbed to infection during the chronic stage displaying a marked inflammation in their brains (Aliberti et al., 2002). The increased mortality in the Alox5/ animals is not due to defective control of the parasite but due to enhanced inflammatory responses associated with elevations of IL-12 and IFNg, and their mortality is completely prevented by the administration of a stable LXA4 analogue. Therefore, LXA4 is important for down-regulation of proinflammatory responses during the chronic stage of T. gondii infection. Recent studies showed that lipoxins activate two receptors (AhR and LXAR) in dendritic cells, and that this activation triggers expression of suppressor of cytokine signalling (SOCS)-2. SOCS-2-deficient mice succumb to chronic infection with T. gondii in association with elevated IL-12 and IFNg responses and reduced brain cysts (Machado et al., 2006), as observed in Alox5/ animals (Machado et al., 2006).

23.4.8 IL-17 and IL-27 A unique subset of CD4þ T-cells, Th17, produces IL-17, and this T-cell population has been suggested to mediate inflammation in models of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. In infection with T. gondii, IL-17 has been shown to have

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both protective and pathogenic roles. IL-17receptor-deficient mice are more susceptible to acute infection with the parasite (Kelly et al., 2005). Their mortality was associated with increased parasite burden in the organs, including the brain, and with a defect in the migration of neutrophils to infected sites during the early stage of infection. Therefore, IL17-mediated induction of neutrophil migration to infected sites during the initial stage of infection appears to have an important role in resistance against acute infection with T. gondii. However, the activity of Th17 cells needs to be appropriately down-regulated by IL-27 to prevent the development of severe inflammatory changes in the brain during the later stage of infection. Chronically infected IL-27-receptordeficient mice developed severe inflammation in their brains mediated by CD4þ T-cells, and the pathology was associated with a prominent IL-17 response (Stumhofer et al., 2006). In addition, treatment of naive primary T-cells with IL-27 in vitro suppressed the development of Th17 cells induced by IL-6 and TGFb, and the suppressive effect was dependent on the intracellular signalling molecule STAT1 (Stumhofer et al., 2006). Therefore, IL-27 has a crucial role in the prevention of Th17-mediated inflammatory responses in the brain during the chronic stage of T. gondii infection.

23.4.9 IL-33 IL-33 is a member of the IL-1 family with the ability to down-regulate IFNg production by Th1 cells and up-regulate Th2 response. Mice deficient in T1/ST2, a component of the IL-33 receptor, demonstrated increased susceptibility to T. gondii infection that correlated with increased pathology and greater parasite burden in the brain (Jones et al., 2010). Real-time PCR analysis of cerebral cytokine levels revealed increased mRNA levels of IFNg, TNFa and NOS2 in infected T1/ST2-deficient animals. The mechanisms mediated by IL-33 receptor to

control T. gondii in the brain remain to be determined.

23.5 INVOLVEMENT OF HUMOURAL IMMUNITY IN RESISTANCE Antibodies are involved in resistance against T. gondii although cell-mediated immunity plays the major role as mentioned above. Frenkel and Taylor (Frenkel and Taylor, 1982) examined the effect of depletion of B-cells by treatment with anti-m antibody on toxoplasmosis in mice infected with a virulent strain and treated with sulphadiazine. They observed mortality associated with pneumonia, myocarditis and/ or encephalitis in infected anti-m-treated mice after discontinuation of sulphadiazine treatment. Administration of antisera to T. gondii reduces mortality in these animals. These results suggest that antibody production by B-cells may be important for controlling the latent persistent infection. However, these studies did not provide conclusive information because of the potential side effects of anti-m antibody treatment on the immune system. Kang et al. (Kang et al., 2000) reported the role of B-cells in resistance to T. gondii by using B-celldeficient (mMT) mice generated by disruption of one of the membrane exons of the m-chain gene. All B-cell-deficient mice died between three and four weeks after infection whereas no mortality was observed in wild-type mice until eight weeks after infection. At the stage during which mMT animals succumbed to the infection, large numbers of tachyzoites were detected only in their brains. Furthermore, treatment of infected mMT mice with anti-T. gondii IgG antibody reduced mortality and prolonged time to death. These results indicate that B-cells play an important role through production of specific antibodies in prevention of TE in mice. In regard to the protective role of antibodies, Johnson and Sayles (Johnson and Sayles, 2002) reported that

23.6 IFNg INDUCED EFFECTOR MECHANISMS

treatment of CD4-deficient mice with antiT. gondii sera prolonged their survival during the chronic stage of infection. In regard to antibody production to T. gondii in the later stage of infection, our recent study provided evidence indicating that tachyzoite proliferation in the brain of immunocompetent hosts during the chronic stage of infection induces production of IgG antibodies that recognize parasite antigens different from those recognized by the antibodies of infected hosts that do not have the tachyzoite growth (Hester et al., 2012). In this study, two groups of CBA/ J mice, which display continuous tachyzoite growth in their brains during the later stage of infection, were infected, and one group received treatment with sulphadiazine to prevent tachyzoite proliferation during the chronic stage of infection. T. gondii antigens recognized by the IgG antibodies from these two groups of mice were compared using immunoblotting following separation of tachyzoite antigens by two-dimensional gel electrophoresis. Although several antigens, including the microneme protein MIC2, the cyst matrix protein MAG1, the dense granule proteins GRA4 and GRA7, were commonly recognized by IgG antibodies from both groups of mice, there were multiple antigens recognized mostly by IgG antibodies of only one group of mice, either with or without cerebral tachyzoite growth. The antigens recognized only by or mostly by the antibodies of mice with cerebral tachyzoite growth include MIC6, the rhoptry protein ROP1, GRA2, one heat shock protein HSP90, one (putative) heat shock protein HSP70 and the myosin heavy chain. These results indicate that IgG antibody levels increase only to selected T. gondii antigens in association with cerebral tachyzoite proliferation (reactivation of infection) in immunocompetent hosts with chronic infection. Thus, humoural immune responses, in addition to IFNg dependent cell mediated immune responses, actively respond to cerebral tachzyoite growth, although the

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roles of these antibodies in controlling the tachyzoite need to be elucidated.

23.6 IFNg INDUCED EFFECTOR MECHANISMS Several mechanisms of IFNg induced antiToxoplasma activity in various host cells have been described. These anti-Toxoplasma effector mechanisms include nitric oxide (NO) production, tryptophan starvation, generation of reactive oxygen intermediates (ROI), iron deprivation, the immunity-related GTPases (IRG), and the p65 guanylate-binding proteins (Gbp). The actions of IFNg are initiated by the binding of IFNg with its IFNg receptor (IFNg-R) at the cell surface and the initiation of a signalling cascade, involving the JAK family of tyrosine kinases and STAT family of transcription factors. Upon IFNg stimulation, STAT1 dimerizes and translocates to the nucleus where it binds g-activated sequence elements in the promoter regions of IFNg-inducible genes and activates gene transcription. IFNg has been shown to induce expression of approximately 400e1200 host cell genes in immune effector cells such as macrophages and microglia but also non-professional immune effector cells such as fibroblasts, endothelial cells and astrocytes (Halonen et al., 2006; Moran et al., 2004; Boehm et al., 1997; Rock et al., 2005; MacMicking, 2004). Collectively these IFNg activated genes regulate the immune response, inducing expression in genes encoding for pro-inflammatory cytokines and components of the MHC-mediated antigen presentation pathway, cell adhesion molecules important in leukocyte activation and trafficking and genes with anti-microbial function (Boehm et al., 1997). IFNg-inducible genes, which are of particular importance for resistance to T. gondii, include genes involved in generation of NO, tryptophan degradation, reactive oxygen intermediates, the genes encoding for the IRG family of proteins

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and the p65 GBPs. Studies indicate that the expression of these anti-microbial mechanisms vary among the tissues, with NO synthesis, tryptophan starvation and the genes encoding for the IRG family shown to be of particular importance in controlling T. gondii in the brain. The importance of the IFNg-response genes in protection against Toxoplasma is illustrated by studies that have found STAT1-deficient mice (Stat/), which produce IFNg but cannot up-regulate anti-microbial effector functions, unable to control parasite replication (Gavrilescu et al., 2004; Lieberman et al., 2004).

23.6.1 Nitric Oxide (NO)-Mediated Mechanism Nitric oxide is one of the main IFNg induced anti-toxoplasmacidal mechanisms known to mediate resistance to T. gondii in mice (Hayashi et al., 1996b). IFNg induces synthesis of the enzyme inducible nitric oxide synthase (iNOS) that produces nitric oxide (NO) from L-arginine (Adams et al., 1991). The L-arginine dependent production of NO and subsequent conversion to reactive nitrogen species (RNS) has direct antimicrobial activity and results in parasite killing (James, 1995). T. gondii infection also induces apoptosis in non-infected macrophages due to the secretion of NO released by infected cells which may also serve to limit the spread of infection (Nishikawa et al., 2007). While NO-mediated toxoplasmacidal effects in macrophages limit parasite numbers it has not been found to be required for protection against the acute infection in mice, indicating other IFNg effector mechanisms, such as IRG proteins and likely other mechanisms, are also involved (Hayashi et al., 1996b, c; Scharton-Kersten et al., 1997a). Conversely, NO has been found to be essential for resistance to the chronic infection in mice (Scharton-Kersten et al., 1997a). IFNg activated microglia produce NO production that results in parasite killing (Chao et al., 1993a, c, 1994b). Microglia are the resident macrophage cell

population in the brain and likely major immune effector cells limiting tachyzoite replication in the brain via this NO-mediated mechanism. NO also induces bradyzoite differentiation, indicating NO may also play a role in inducing bradyzoite differentiation and cyst formation in the chronic infection (Bohne et al., 1994).

23.6.2 Tryptophan Starvation Pathway The IFNg induced inhibition of T. gondii via tryptophan starvation is a mechanism known to operate in many cell types including fibroblasts, epithelial cells, and endothelial cells, in a variety of host species including humans, rat, and mouse, amongst others (Dimier et al., 1992; Nagineni et al., 1996; Daubener et al., 1999; Pfefferkorn, 1984; Pfefferkorn and Guyre, 1984). The mechanism of inhibition is due to induction of the enzyme, idoleamine-2,3-dioxygenase (IDO) resulting in the degradation of tryptophan to kynurenine (Pfefferkorn et al., 1986; Pfefferkorn and Guyre, 1984). Inhibition of parasite growth is due to tryptophan starvation as tryptophan is an essential amino acid that the parasite derives from the host cell. Evidence indicates that the IDO pathway is important in host resistance to T. gondii early in infection and helps control dissemination into different tissues (Silva et al., 2002). For example, IFNg induction of the tryptophan/IDO pathway significantly inhibits parasite growth in epithelial and endothelial cells indicating this pathway may be important in limiting parasite growth in the intestinal phase of the infection and passage to the foetus in congenital toxoplasmosis (Dimier et al., 1992; Dimier and Bout, 1993, 1996b, 1997, 1998). Likewise, IFNg induction of the tryptophan/IDO pathway and inhibition of T. gondii has been found in human microvascular endothelial cells indicating this pathway may be of importance in restricting entrance of the parasite into the brain. The IFNg-induced tryptophan starvation pathway has also been found in human pigment epithelial cells (RPE) indicating

23.6 IFNg INDUCED EFFECTOR MECHANISMS

this pathway may also be of importance in controlling the replication of the parasite in ocular toxoplasmosis (Nagineni et al., 1996).

23.6.3 Immunity-Related GTPase (IRG) Family The immunity-related GTPases (IRGs) are a family of proteins induced by IFNg that are important in resistance against a wide variety of intravacuolar bacterial and parasitic pathogens, including T. gondii (Taylor et al., 2004, 2007; Zhao et al., 2009b). Of the hundreds of genes increased by IFNg, the IRG genes are amongst the most abundant. These proteins, formerly called the p47 GTPases, were first described in the 1990s and in the last decade numerous studies have established the role of IRG proteins in resistance to Toxoplasma (Hunn et al., 2011; Zhao et al., 2009b). Most of the work has involved the following seven IRG members: Irgm1 (LRG-47), Irgm2 (GTPI), Irgm3 (IGTP), Irga6 (IIGPI), Irgb6 (TGTP), Irgd (IRG-47) and Irgb10. Most of these IRG proteins have been found to be associated with inhibition of T. gondii in vitro, and of the four IRG genes that have been knocked out (Irgm1, Irgm3, Irga6 and Irgd), all have been found to significantly increase susceptibility to infection of T. gondii, thus establishing the role of IRG proteins in resistance to T. gondii in mice. The IRG proteins are 46e47 kDa GTPases, containing a Ras-like GTP-binding domain (termed G1). The IRG protein family consists of two subfamilies, based upon the nucleotidebinding domain within the G1 GTP-binding domain with one subfamily having a GMS amino acid motif and the other subfamily having a GKS motif. The three IRG members of the GMS subfamily include, Irgm1, Irgm2 and Irgm3 while IRG members, Irga6, Irgb6, Irgd and Irg10 belong to the GKS subfamily. The GMS IRG proteins are regulators of GKS proteins binding to the GKS IRG proteins and maintaining them in the inactivate state via a

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GDP-dependent interaction (Hunn et al., 2008). The IRG genes are present throughout the vertebrate phyla, being present in cephalochordates, amphibians, fish, reptiles and mammals. In mouse, the IRG family is diverse, encoding approximately 23 genes, 21 of which encode proteins (Bekpen et al., 2005). The IRG family, however, seems to have been repeatedly lost during evolution with no IRG genes present in any of the available bird genomes and the number of IRG genes in humans dramatically reduced with only two IRG genes, IRGC and IRGM, present (Bekpen et al., 2009, 2010). In IFNg stimulated host cells infected with Toxoplasma multiple IRG proteins localize to the Toxoplasma parasitophorous vacuole membrane within minutes of invasion, with the parasitophorous vacuolar membrane subsequently becoming vesiculated and finally disrupted, resulting in release of the parasite into the cytosol and degradation of the parasite (Martens et al., 2005; Ling et al., 2006; Melzer et al., 2008). In macrophages infected with Toxoplasma, destruction of the T. gondii is accompanied by inclusion of the parasite in autophagosomes and subsequent autophagomal delivery to the lysosomes (Ling et al., 2006; Butcher et al., 2005). IRG-mediated vacuolar disruption also occurs in IFNg-stimulated fibroblasts and astrocytes but the autophagy pathway was not found to be involved (Melzer et al., 2008; Zhao et al., 2009b; Martens et al., 2005). However, mice deficient in the autophagic regulator, atg5, are deficient in their ability to control T. gondii replication indicating the autophagic pathway is involved in some way (Konen-Waisman and Howard, 2007). Atg5 has been found to be necessary for delivery of IRG proteins to the PV, although this appears to operate by a mechanism independent of the normal autophagy pathway (Zhao et al., 2008). Finally, in IFNg-stimulated fibroblasts IRG-mediated PV disruption results in host cell necrosis, following release of the parasite into the host cytoplasm, indicating destruction of the host cell may be part of the IRG mechanism in some cell types (Zhao et al., 2009b).

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The IRG mechanism involves a coordinated loading of IRG GTPases on the Toxoplasma vacuole with at least six IRG proteins (Irgm2, Irgm3, Irga6, Irgb6, Irgd and Irg10) localizing to the Toxoplasma vacuole (Khaminets et al., 2010). The coating of the IRG proteins to the PV occurs within one hour of invasion and is hierarchical with Irgb6 and Irgb10 loading first. Upon infection with T. gondii, GKS proteins lose their interaction with GMS proteins and accumulate at the PV membrane (PVM) in the active GTP bound state leading to vesiculation and rupture of the PV (Hunn et al., 2008; Papic et al., 2008). Despite the large amount of information now understood about the molecular and biochemical aspects of IRG-mediated inhibition of T. gondii, the mechanisms involved in the vesiculation leading to PV disruption is still not understood. IRG proteins are related to the dynamin-type GTPases known to mediate vesicle formation and deformation of membranes and it has been suggested IRG protein acts in an analogous fashion mediating vesiculation of the PVM, although this has not been demonstrated (Hunn et al., 2011). The type I strains are resistant to IRG-mediated IFNg inhibition (Steinfeldt et al., 2010; Howard et al., 2011). This deficiency in IFNg mediated control is associated with a failure of accumulation of IRG proteins on the PVM (Zhao et al., 2009a). This has been found to be due largely to the polymorphic rhoptry kinase, ROP18, which in type I strains phosphorylates the GKS IRG proteins Irga6, Irgb6 and Irgb10, causing dissociation of IRG from the vacuole and inhibition of PV disruption (Zhao et al., 2009a; Steinfeldt et al., 2010; Fentress et al., 2010). Another rhoptry protein, ROP5, has been found to directly interact with IRG proteins, reducing IRG coating and inactivating IRG proteins (Fleckenstein et al., 2012; Niedelman et al., 2012). ROP5 can interact with IRGs in the absence of ROP18. However, rhoptry proteins, ROP5 and ROP18 while mediating inhibition in IFNg-activated murine cells, do not affect survival in IFNg activated human cells

(Niedelman et al., 2012). These results suggest that while ROP5 and ROP18 may have evolved to block the IRGs they may not have effects on parasite survival in species that do not have the IRG system, such as humans. Why the IRGs are such a large family of proteins in the murine genome and so reduced in humans, or if functional counterpart(s) exists in humans, is not yet clear.

23.6.4 Guanylate-Binding Proteins (GBPs or p65 GTPases) Stimulation of immune cells such as macrophages and dendritic cells by IFNg induces in gene expression of the IRG immunity-related GTPases but also the related the p65 guanylatebinding family proteins (GBPs) (Taylor et al., 2004). The GBPs have been shown to induce antibacterial responses involving phagocytic oxidases, autophagic effectors, and the inflammasome and they have recently also been found to play a critical role in host defence against T. gondii (Yamamoto et al., 2012). Mice deficient in a cluster of six GBP family genes (Gbp1, Gbp2, Gbp3, Gbp5, Gbp7 and Gbp2ps) on chromosome 3 (Gbpchr3) are highly susceptible to T. gondii infection with increased parasite burden in immune organs. Gbpchr3-deleted macrophages are defective in IFNg-mediated suppression of T. gondii intracellular growth and recruitment of IRG protein, Irgb6, to the parasitophorous vacuole (PV), suggesting GBPs may regulate Irgb6 recruitment to the PV. Another study of the behaviour of GBP1 in vitro in murine cells found GBP1 exerts its function in conjunction with GBP family members, GBP2 and GBP5, and that GBP1 recruitment correlates with virulence of the parasite strain (Virreira Winter et al., 2011). T. gondii proteins, ROP16, ROP18 and GRA15, were found to be partly responsible for the strain-specific accumulation of GBP1 at the PV. The IFNg-dependent host factors that regulate this complex process of GBP recruitment to the PV are unknown. However, it

23.7 IFNg EFFECTOR CELLS IN THE BRAIN WITH ACTIVITY AGAINST TOXOPLASMA GONDII

appears that along with the IRGs, GBPs are key factors mediating resistance to T. gondii. Interestingly, in contrast to the IRGs, the p65 GBPs are represented by 13 genes in the mouse genomes and are similarly represented by seven genes in the human genome indicating this system may also function in human cells (Kresse et al., 2008).

23.6.5 Reactive Oxygen Intermediates (ROI) IFNg activated production of reactive oxygen intermediates (ROI) has been demonstrated to induce anti-toxoplasmacidal activity in human macrophages (Murray et al., 1985a, b). The reactive oxygen intermediates generated include superoxide ion (O 2 ) and hydrogen peroxide (H2O2). ROI intermediates have also been found to mediate IFNg inhibition of T. gondii in murine dendritic cells but not murine microglia or astrocytes (Jun et al., 1993; Aline et al., 2002a; Halonen and Weiss, 2000b). It has been reported that the parasites are resistant to the oxygen metabolites produced in murine macrophages (Chang and Pechere, 1989) and p47phox-deficient mice (which lack an inducible oxidative burst) can control both the acute and chronic stages of T. gondii infection (Scharton-Kersten et al., 1997b). Thus the physiological significance of the ROI pathway still remains unclear, especially in mice.

23.6.6 Iron Deprivation IFNg has been shown to inhibit growth of T. gondii via iron deprivation in intestinal epithelial cells (Dimier and Bout, 1998; Bout et al., 1999). Cells usually acquire iron via the transferrin receptor pathway, in which iron bound to transferrin binds to the transferrin receptor at the cell surface and the receptoretransferrin complex is internalized via endocytosis with subsequent acidification of the endocytic vesicle causing the ferric ions to dissociate from transferrin. The iron ions are transported across the vesicle membrane into the cytoplasm and this

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intracellular pool of iron is available for metabolic processes. T. gondii replicates within the parasitophorous vacuole, a membranous compartment within the host cell that does not fuse with the lysosomes and is not contiguous with iron-transferrin as it passes through the endocytic pathway. Rather than acquiring iron from transferrin, experiments with intestinal epithelial cells indicate that IFNg can inhibit T. gondii replication by limiting the availability of intracellular iron for the parasite to scavenge from its host cell. The mechanism by which this occurs is not understood. Inhibition of T. gondii growth via IFNg induced iron deprivation has only been shown for enterocytes and may be an important anti-toxoplasmic mechanism on mucosal surfaces as a first line of defence against pathogen invasion. It is not clear if this IFNg induced iron dependent mechanism also occurs in other cell types and thus has a broader role in the control of Toxoplasma in the latent stage of infection and congenital toxoplasmosis.

23.7 IFNg EFFECTOR CELLS IN THE BRAIN WITH ACTIVITY AGAINST TOXOPLASMA GONDII The establishment of a chronic asymptomatic T. gondii infection requires the cytokine IFNg, as previously discussed, but additional resistance to T. gondii requires IFNg effector cells from haemopoietic and non-haemopoietic compartments (Yap and Sher, 1999; Suzuki et al., 1988). In the brain, these IFNg effector cells include infiltrating T-cells, dendritic cells and macrophages which stimulate the intracerebral immune responses, while activation of resident CNS IFNg effector cell populations such as microglia, cerebral microvascular endothelial cells and astrocytes are essential to control the parasite in the brain. The expression of the IFNg induced antimicrobial mechanisms varies between

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phagocytic cells such as macrophages and microglia, and non-phagocytic cells such as endothelial cells and astrocytes (Halonen et al., 2006; Moran et al., 2004; Rock et al., 2005; Kota et al., 2006; Indraccolo et al., 2007; Adams et al., 1990a). IFNg inhibition in macrophages is primarily mediated via a nitric oxidemediated mechanism while in non-phagocytic cells, IFNg inhibition is via non-NO mediated mechanisms such as IDO/tryptophan degradation, increases in reactive oxygen species, and induction of IRG proteins. The IRG-mediated mechanism is found in macrophages, as well as fibroblasts and astrocytes, and has been shown to be of particular importance in protection against T. gondii in mice (Zhao et al., 2009b; Martens et al., 2005). Most recently several members of the related family of IFNg-inducible GTPases, the guanylate-binding protein (GBP) family, have also been shown to play a role in host defence against T. gondii (Yamamoto et al., 2012).

subsequent T-cell immunity. Additionally, IFNg secretion by microglia may also be important in response to reactivated infections in which T-cell numbers are low. While IFNg activated microglia are clearly important effector cells against the parasite in the brain, it has recently been found that unstimulated microglia infected with T. gondii exhibit a hypermotility phenotype, similar to infected dendritic cells (Dellacasa-Lindberg et al., 2011). Thus, paradoxically, microglia cells may help disseminate the parasite within the brain. Furthermore, unstimulated microglia infected with T. gondii also exhibit increased sensitivity to T-cell mediated killing leading to parasite transfer to effector T-cells. Results of these in vitro studies indicate infected microglial might also transfer parasites between different cell populations such as cytotoxic T-cells, thus potentially facilitating dissemination of the parasite within the brain parenchyma in a ‘Trojan horse’ type mechanism in reactivated infections.

23.7.1 Microglial Cells Microglia are the resident brain tissue macrophage precursors that rapidly respond to infection in the brain (Nimmerjahn et al., 2005). IFNg activated microglia produce NO that is responsible for anti-Toxoplasma activity (Chao et al., 1993a, c, 1994b). IFNg activated microglia produce chemokines and cytokines including TNFa, IL-1b, IL-12 and IL-15, and induce expression of MHC I and II, LFA-1 and ICAM-1 indicating IFNg microglia also regulate infiltration of T-cells into the brain and act as an antigen-presenting cell in the brain (DeckertSchluter et al., 1999; Schluter et al., 2001). Microglia can also produce IFNg either in the presence or absence of T-cells (Wang and Suzuki, 2007). IFNg secretion by microglia may thus play a critical role in the early stage of infection in the brain, prior to the infiltration of T-cells, by stimulating IFNg effector cells to limit tachyzoite proliferation in the brain and initiating

23.7.2 Astrocytes Astrocytes are the dominant glial cell in the brain and numerous studies indicate they are central to the intracerebral immune response to T. gondii in the brain (Wilson and Hunter, 2004b). First, IFNg activated astrocytes significantly inhibit the growth of tachyzoites of T. gondii indicating they can function as an immune effector cells in the brain (Halonen et al., 2001b; Scheidegger et al., 2005). IFNg inhibition in astrocytes in mice is via the IRG-mediated mechanism while inhibition in human astrocytes is via nitric oxide or tryptophan degradation (Halonen et al., 1998a, 2001a; Halonen and Weiss, 2000a; Martens et al., 2005; Peterson et al., 1995; Oberdorfer et al., 2003). Additionally, in vitro studies indicate IFNg activated astrocytes can induce bradyzoite differentiation and support cyst formation indicating astrocytes may also foster

23.7 IFNg EFFECTOR CELLS IN THE BRAIN WITH ACTIVITY AGAINST TOXOPLASMA GONDII

bradyzoite differentiation/cyst formation in the brain and thus serve to help maintain the chronic infection (Jones et al., 1986b; Halonen et al., 1998c). In addition to having roles as IFNg effector cells controlling tachyzoite replication and as host cells to the cysts, astrocytes have other immune functions that are involved in the intracerebral immune response to T. gondii (Wilson and Hunter, 2004a). Upon infection with Toxoplasma there is a general inflammatory response in the brain characterized by cytokine production and astrocyte activation with astrocytes producing the chemokine’s IFNg inducible protein 10 (IP-10) and MCP-1, which attract activated T-cells and activated macrophages into the site of inflammation in the brain, respectively (Strack et al., 2002; Schluter et al., 1991). Studies in mice indicate astrocytes production of IP-10, precedes infiltration of T-cells indicating astrocytes play a key role in regulation of T-cell trafficking into the brain early in infection, while astrocyte production of MCP-1 may contribute to active recruitment of both macrophages and activated T-cells throughout the chronic infection (Strack et al., 2002; Wilson and Hunter, 2004b). Additionally, IFNg activated astrocytes are efficient in MHC I presentation and capable of cross-presentation, indicating they may be an important antigen presenting cell for T. gondii in the brain, capable of stimulating CD8þ T-cells (Dzierszinski et al., 2007). Finally, recent studies also indicate astrocytes play a crucial regulatory role reducing inflammation and serving to limit sites of parasite replication (Drogemuller et al., 2008), as discussed more fully below. Collectively, these studies indicate that astrocytes may play a pivotal role in the control of T. gondii in the brain, serving to limit replication of the tachyzoites, thus augmenting the more potent anti-microbicidal activity of microglia to clear the parasites from the brain, stimulating bradyzoite differentiation and cyst development while also serving as antigen-presenting cells

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and key regulators of T-cell trafficking into the brain during Toxoplasma infection.

23.7.3 Cerebral Microvascular Endothelial Cells IFNg stimulated human brain microvascular endothelial cells have been shown to induce inhibition of T. gondii via induction of IDO/tryptophan starvation pathway (Daubener et al., 2001). The anti-parasitic effect was enhanced by TNFa, although TNFa alone had no effect on parasite growth similar to IFNg inhibition observed in astrocytes. Since one of the first steps in the development of cerebral toxoplasmosis is penetration of the bloodebrain barrier, IFNg induced inhibition in these cells may be important in limiting the number of T. gondii parasites that enter the brain via a transcellular route. IFNg also induces expression of VCAM1 on endothelial cells that binds to an integrin on CD8þ T-cells, and thus is of importance for recruitment of T-cells into the brain during the chronic stage of T. gondii infection (Wang et al., 2007).

23.7.4 Dendritic Cells Dendritic cells (DCs) play a crucial role in the initiation of systemic immune responses to acute infection with T. gondii as discussed above (see Section 23.2.1). Evidence indicates DCs also play key roles in promoting resistance to cerebral toxoplasmosis. First, recent studies indicate infected DCs exhibit an enhanced migratory response, which facilitates DCs trafficking and may be involved in transporting single tachyzoites into the brain across the bloodebrain barrier via a Trojan horse-type mechanism (Courret et al., 2006; Unno et al., 2008; Bierly et al., 2008). Additionally, activated DCs are present within the brain during the chronic infection indicating DCs are important in the intracerebral immune response to Toxoplasma (Fischer et al., 1993, 2000a). Although the percentage of DCs in the brain is low in

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Toxoplasma infection (less than 10%), recent evidence indicates DCs can function as antigen-presenting cells (APCs) capable of processing and presenting antigen to naive T-cells (John et al., 2011). Additionally, both infected and uninfected DCs are able to interact with T-cells, indicating DCs are capable of presenting antigen via both direct- and cross-presentation pathways. This study also suggested a constant recruitment and retention of DCs into the CNS during the chronic infection. Real-time imaging studies found DCs are intimately associated with the parasite cysts suggesting a possible innate sensing of the cyst stage by DCs. Finally, in addition to these immune functions, DCs may also function as an IFNg effector cell as IFNg stimulation induces anti-toxoplasmacidal activity via reactive oxygen mediated mechanism (Aline et al., 2002b).

23.8 THE ROLE OF HOST CELLS HARBOURING TOXOPLASMA GONDII IN THE BRAIN T. gondii persists for the lifetime of the host within cysts located predominantly in the brain. Early in infection the cyst stage can occur in microglia, astrocytes and neurons but cysts persist predominantly in neurons during the chronic infection (Frenkel and Escajadillo, 1987; Sims et al., 1988, 1989a, 1989c; Fagard et al., 1999; Ferguson and Hutchison, 1987b). While neurons serve primarily as the host cell for the cyst stage, infected neurons may also participate in the regulation of the intracerebral immune response and, as neurons are in close proximity to astrocytes and microglia in the brain, infection and development in neurons are also likely affected by these neighbouring cells. Studies do indicate that a complex interplay exists between T. gondii cysts in neurons and activated astrocytes and microglial cells, which maintain the

chronic Toxoplasma infection in the brain and prevent neuropathology. Requirement of the immune system to maintain the latency of chronic infection is evident from reactivation of infection in immunocompromised patients, such as those with AIDS (Israelski and Remington, 1993; Wong and Remington, 1994). Tissue cysts are the most likely source of reactivation toxoplasmosis in immunocompromised patients. In reactivated infection (i.e. TE) bradyzoite to tachyzoite conversion is thought to occur and the resulting uncontrolled proliferation of the tachyzoite in the brain results in pathology. While the cysts appear to be located primarily in neurons and astrocytes, the tachyzoite may replicate in astrocytes, microglia and possibly neurons. The mechanisms and dynamics of tachyzoite-to-bradyzoite interconversion in the brain are not fully understood and are areas that are important in understanding the persistence of T. gondii in the brain in both immunocompetent and immunocompromised individuals and for devising treatments for cerebral toxoplasmosis.

23.8.1 T. gondii Infection in Neurons Neurons are a major host cell for Toxoplasma in the brain, supporting both growth of tachyzoite stage and serving as the predominant host cell for the cyst stage in the chronic infection (Ferguson and Hutchison, 1987a; Melzer et al., 2010; Sims et al., 1989b). In vitro studies using neuronal cultures indicate neurons are infected less efficiently than glial cells and that growth of tachyzoites is also slower in neurons than glial cells (Halonen et al., 1996; Creuzet et al., 1998; Fischer et al., 1997). The mechanism underlying the lower rates of infection could be due to various parameters such as absence or low level of expression of a cell-surface binding factor required for entry or the mitotic status of the host cell as attachment of Toxoplasma has been shown to be host cell-cycle-dependent and to decrease as cells enter the G2eM boundary

23.8 THE ROLE OF HOST CELLS HARBOURING TOXOPLASMA GONDII IN THE BRAIN

(Fagard et al., 1999; Grimwood et al., 1996; Dutta et al., 2000). The slow growth rate in neurons is not well understood but has been suggested to be due to the small cell size. In vitro studies using neuronal cultures also indicate stage conversion from the tachyzoite to the bradyzoite stage and cyst formation occurs spontaneously in neurons in the absence of IFNg or other factors such as NO (Luder et al., 1999a; Fischer et al., 1997). Thus, intrinsic factors of the neuronal host cell environment appear to play a role in inducing cyst formation, partially explaining the predominance of cysts in neurons. It has been suggested that encystation in neurons in the absence of T-cell-derived cytokines may provide a mechanism for a brain-internal spreading of bradyzoites which may help to sustain chronic infection (Fischer et al., 1997). However, neither the phenomenon of brain-internal spreading of bradyzoites in neurons nor the nature of intrinsic neuronal factors favouring cyst formation have been well studied or are well understood. Neither IFNg nor TNFa stimulated neurons, however, inhibit replication of T. gondii in neurons (Schluter et al., 2001). This is in marked contrast to the inhibition of growth that these cytokines induce in microglia and astrocytes (Chao et al., 1993b; Halonen et al., 1998a). Thus, IFNg and TNFa while important in controlling T. gondii in microglia and astrocytes are not sufficient to control replication of the parasite in neurons. However, the replication of the parasite in neurons is relatively slow and this in itself is indicative of some intrinsic ability of neurons to limit parasite growth. It has been suggested that astrocytes, as the dominant glial cell, intimately involved with neuronal processes and necessary to maintain functional integrity of neurons, may supply additional factors for the induction of inhibition of Toxoplasma in neurons, although this has not been well studied. Evidence indicates infected neurons can, however, participate in the regulation of the intracerebral immune response. For example, similar to infected astrocytes and microglia,

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neurons induce secretion of cytokines and chemokines in response to infection. Infected neurons secrete the chemokines MIP-1a and MIP-1b that attract inflammatory leukocytes, and the immunosuppressive cytokine, TGFb (Schluter et al., 2001). Intracellular parasites are commonly surrounded by an infiltrate of T-cells and granulocytes while the bradyzoite containing cysts are usually devoid of inflammatory cells, indicating that the neuronal production of chemokines may be dependent upon the stage of the parasite in neurons. Using synapsin-I (Syn)-Cre gp130/ mice, which lack gp130, the signaltransducing receptor for the IL-6 family of cytokines in neurons, it has recently been found that loss of gp130 in neurons results in loss of control T. gondii infection leading to hyperinflammation, lethal necrotizing TE and a progressive neuronal loss (Handel et al., 2012). IL-6 stimulation of neurons induced gp130-dependent TGF-b1, TGF-b2 and IL-27 production, and reduced death and apoptosis of infected neurons. These findings indicated a protective function of gp130expressing neurons in chronic toxoplasmosis. Collectively the above results indicate that T. gondii infected neurons contribute to the intracerebral immune response through the recruitment of inflammatory cells to the site of infection and via down-regulation of the intracerebral immune response resulting in prevention of neuronal loss in chronic toxoplasmosis. Finally, several recent studies indicate Toxoplasma infection affects neuronal functions. For example, an increase in dopamine levels in neurons containing cysts has recently been found (Prandovszky et al., 2011). The parasite contains a tyrosine hydroxylase gene that is able to synthesize L-DOPA, which is expressed only in bradyzoites and it has been proposed that this parasite enzyme is responsible for an increase in dopamine levels in infected neurons (Gaskell et al., 2009). The altered dopamine levels induced by T. gondii cysts in the brain could have significant consequences on brain functions, possibly leading to an array of

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behavioural changes and neurological malfunctions. Another recent study in chronically infected mice found parasite antigens in the host cell cytoplasm, including axons, indicating the parasite might directly interfere with neuronal function and found a decrease in the activity-dependent uptake of the potassium analogue thallium, indicating infected neurons were functionally silenced (Haroon et al., 2012). These studies indicate a functional impairment of cyst harbouring neurons and provide a possible mechanism that may lead to neurological effects and/or behavioural consequences in chronically infected hosts.

23.8.2 Role of Astrocytes in Cerebral Toxoplasmosis In addition to having a role as IFNg effector cells controlling tachyzoite replication in the brain, both in vivo and in vitro studies have found that astrocytes support the cyst stage. For example, cysts in astrocytes have been reported in human brain (Powell et al., 1978) and in vitro studies in astrocytes have found to be able to support cyst development (Jones et al., 1986a; Halonen et al., 1998b). Layers of glial intermediate-type filaments (GFAP) surround cysts in astrocytes and this type of GFAP layering of cysts may aid in stabilizing cysts, perhaps preventing cyst rupture (Halonen et al., 1998b; Luder et al., 1999b; Powell et al., 1978). The layers of glial filaments were observed to displace the host cell mitochondria from the surface of the parasitophorous vacuole and thus the glial filament wrapping has also been suggested to play a role in establishing the anaerobic environment helping to facilitate tachyzoite to bradyzoite formation (Halonen et al., 1998b). Studies indicate astrocytes are also crucial for effective control of inflammation and maintenance of the chronic infection in the brain. The importance of astrocytes in the intracerebral immune response against Toxoplasma has been illustrated in vivo in mice, in which astrocytes

deficient in the expression of their glial fibrillary protein (GFAP/), showed a reduced capacity to restrict T. gondii-associated inflammatory lesions, resulting in increased intracerebral load of the parasite and widespread areas of tachyzoiteinduced tissue necrosis (Stenzel et al., 2004). These studies involving GFAP/ mice indicate a host defence mechanism of astrocytes via restricting the pathogenic spread within the CNS. A more recent study in mice in which astrocytes were deficient in gp130 (GFAP-Cre-gp130/) was similarly found to be unable to contain sites of parasite replication resulting in inflammatory lesions in the brain leading to fatal toxoplasmic encephalitis (Drogemuller et al., 2008). The gp130 effect was not a direct anti-parasitic effect, as GFAP-Cre-gp130/ astrocytes were capable of IFNg-stimulated inhibition of parasite growth. Rather the effect appeared to be immunoregulatory as neighbouring uninfected GFAP-Cregp130/ astrocytes become apoptotic. Collectively these studies indicate a crucial role for astrocytes in the immunoregulatory response to Toxoplasma in the brain, reducing neuropathology and limiting spread of the parasite in the brain. Finally, astrocytes have been suggested to play a role in the behavioural effects of the chronic infection in the intermediate host. Activated astrocytes are a hallmark of toxoplasmic encephalitis with activated astrocytes prominent at sites of inflammation and parasite replication (Wilson and Hunter, 2004b). One of the immune effector functions associated with activated astrocytes in humans is induction of IDO, leading to tryptophan degradation, and parasite inhibition. One of the derived products of tryptophan metabolism is kynurenic acid (KYNA), which is released into the extracellular environment and inhibits glutamatergic and nicotinergic neurotransmission. The generation of KYNA in activated astrocytes has been suggested as a mechanism by which T. gondii infection possibly can affect the behaviour of the chronically-infected host (Schwarcz and Hunter, 2007). These and other hypotheses are an

23.9 IMMUNE RESPONSES TO THE CYST STAGE OF TOXOPLASMA GONDII IN THE BRAIN

active area of debate and further discussed in Section 23.13.

23.8.3 Microglia and Astrocytee MicrogliaeNeuronal Interactions Of the three main resident brain cells, microglia appears to function primarily to limit growth of T. gondii. IFNg activated murine microglia produce NO that is responsible for microbiostatic and microbicidal control of parasite growth, as described above in the Section 23.7. Recent studies, however, indicate a more complex interaction between microglia, astrocytes and neurons occurs in the control of T. gondii in the brain. For example, while Th1 cytokines are necessary for microglia activation and responsible for protection against T. gondii in the brain, products from activated microglia may also cause tissue damage and be detrimental to neuron functions. Nitric oxide (NO), one of the main mediators against T. gondii produced by microglia, is also one of the most noxious to the CNS cells. In chronic infections with T. gondii in an immunocompetent host in which the parasite persists in the brain, neuronal and tissue damage does not usually occur. Thus, immunomodulary mechanisms are thought to be involved in the prevention of neuronal degeneration and pathological alterations in the CNS during the latent phase of the infection. Several mechanisms involving downregulation of NO by microglia have been described. First, Toxoplasma-infected IFNg activated microglia inhibit iNOS expression and a corresponding decrease in NO production (Rozenfeld et al., 2005). Inhibition of iNOS only occurs in infected microglia indicating this inhibition is a parasite-mediated effect. Studies indicate the parasite triggers secretion of TGFb by infected microglia, with autocrine stimulation of TGFbereceptor signalling of the infected microglia resulting in a decrease in NO production (Rozenfeld et al., 2005). Additionally,

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Toxoplasma infection induces up-regulation of CD200 on blood vessel endothelial cells and the receptor, CD200R on microglia, the interaction of which down-regulates microglia activation (Deckert et al., 2006). In this study, inhibition of NO production and iNOS expression corresponded with protection of neurite outgrowth, indicating this T. gondii triggered mechanism is involved in the neuropreservation observed during immunocompetent host infections. A third mechanism known to down-modulate NO production by IFNg activated microglia involves release of prostaglandin, PGE2, by infected astrocytes (Rozenfeld et al., 2003). The presence of multiple and independent pathways in the CNS leading to NO inhibition and maintenance of neuronal viability during T. gondii infection likely reflect the importance of this neuroprotection mechanism in the brain during chronic toxoplasmosis.

23.9 IMMUNE RESPONSES TO THE CYST STAGE OF TOXOPLASMA GONDII IN THE BRAIN The basis of persistence of chronic infection with T. gondii is the tissue cyst, which remains largely quiescent for the life of host, but can reactivate and cause disease. This stage of the parasite is not affected by any of the current drug treatments and has been generally regarded as untouchable. However, our recent study revealed that the immune system can eliminate T. gondii cysts from the brains of infected hosts when immune T-cells are transferred into infected immunodeficient animals that have already developed large numbers of the cysts (Suzuki et al., 2010). This T-cell-mediated immune process was associated with accumulation of microglia and macrophages around tissue cysts. Since the accumulated phagocytes penetrate within the cyst, these cells appear to be the main effector cells that destroy the cysts

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and eliminate them from the brain after initiation of this process by immune T-cells. CD8þ immune T-cells possess a potent activity to remove the cysts. Of interest, the initiation of this process by CD8þ T-cells does not require their production of IFNg, the major mediator to prevent proliferation of tachyzoites during acute infection, but does require perforin. Perforin is the major molecule that mediates cytolysis of target cells by CD8þ cytotoxic T-cells. Therefore, our results suggest that CD8þ T-cells induce elimination of T. gondii cysts through their perforin-mediated cytotoxic activity. In relation to our observation, a study using two-photon microscopy in C57BL/6 mice with ovalbuminexpressing T. gondii showed that ovalbuminspecific CD8þ T-cells accumulated in regions containing isolated parasites (tachyzoites) but not around cysts in the brain. C57BL/6 mice are genetically susceptible to chronic infection with the parasite and continuous tachyzoite proliferation occurs in their brain. Therefore, it is possible that the activity of cerebral CD8þ T-cells of these animals is down-regulated in this environment and does not have a potent anti-cyst activity. Perforin-mediated cytolytic activity by T-cells in resistance against T. gondii infection was less appreciated before, when compared to the absolute requirement of IFNg to control tachyzoite proliferation during the acute stage of infection. Perforin knock-out mice survive acute infection (Wang et al., 2004; Denkers et al., 1997). In vitro studies demonstrated that the lysis of tachyzoiteinfected cells by cytotoxic CD8þ T-cells results in release of viable parasites (Yamashita et al., 1998). However, the situation of the parasite in the cyst stage seems different. Whereas the cyst resides within an infected cell (Ferguson and Hutchison, 1987c; Ghatak and Zimmerman, 1973) as do tachyzoites, bradyzoites within the cyst are surrounded by a thick cyst wall, which is unique to the cyst. Therefore, cytolysis of cystcontaining cells by perforin-mediated activity of CD8þ T-cells followed by quick accumulation of

large numbers of microglia and macrophages would probably provide the parasite little time to escape from the coordinated attack by the T-cells and phagocytes. A previous study (Denkers et al., 1997) reported a three- to fourfold increase in brain cyst numbers in perforin knock-out mice in the later stage of infection. The absence of perforin-dependent anti-cyst activity of CD8þ T-cells in these animals may have contributed to their observation.

23.10 HOST GENES INVOLVED IN REGULATING RESISTANCE Resistance against T. gondii is under genetic control in both acute and chronic stages of infection. Of interest, genes involved in resistance differ between these two stages. Susceptibility of inbred strains of mice to acute infection does not correlate with that to chronic infection (Suzuki et al., 1993). A minimum of five genes are involved in determining survival of mice during the acute stage (McLeod et al., 1989). One of these genes is linked to the major histocompatibility complex (H-2) (McLeod et al., 1989). During the chronic stage of infection, development of TE in mice is regulated by the gene(s) within the D region of the major histocompatibility complex (H-2) (Brown et al., 1995; Suzuki et al., 1991, 1994). Mice with the d haplotype in the D region are resistant to development of TE and those with the b or k haplotypes are susceptible. Freund et al. (Freund et al., 1992) found that polymorphisms in the Tnf-gene located in the D region of the H-2 complex correlate with resistance against development of TE and with levels of TNFa mRNA in brains of infected mice. However, more recent studies using deletion mutant mice (Suzuki et al., 1994) and transgenic mice (Brown et al., 1995) demonstrated that the Ld gene in the D region of the H-2 complex, but not the Tnf-gene, is important for resistance against development of TE. Resistance of mice

23.11 IMMUNE EFFECTOR MECHANISMS IN OCULAR TOXOPLASMOSIS

to development of TE is observed in association with resistance to formation of T. gondii cysts in the brain (Brown et al., 1995; Suzuki et al., 1991, 1994). McLeod et al. (McLeod et al., 1989) reported that although the Ld gene has the primary effect on numbers in the brain, the Bcg locus on chromosome 1 may also affect it. In humans, HLA-DQ3 was found to be significantly more frequent in white North American AIDS patients with TE than in the general white population or randomly selected control AIDS patients who had not developed TE (Suzuki et al., 1996). In contrast, the frequency of HLADQ1 was lower in TE patients than in healthy controls (Suzuki et al., 1996). Thus, HLA-DQ3 appears to be a genetic marker of susceptibility to development of TE in AIDS patients and DQ1 appears to be a resistance marker. HLADQ3 also appears to be a genetic marker of susceptibility to development of cerebral toxoplasmosis in congenitally infected infants since higher frequency of DQ3 was observed in infected infants with hydrocephalus than infected infants without hydrocephalus or normal controls (Mack et al., 1999). The role of the HLA-DQ3 and -DQ1 genes in regulation of the susceptibility/resistance of the brain to T. gondii infection is supported by the results from a transgenic mouse study (Mack et al., 1999). Expression of the HLA-DQ1 transgene conferred greater protection against parasite burden and necrosis in brains in mice than did the HLADQ3 transgene (Mack et al., 1999). Expression of the HLA-B27 and -Cw3 transgenes had no effects on the parasite burden (Brown et al., 1994). Since the Ld gene in mice and the HLADQ genes in humans are a part of the MHC which regulate the immune responses, the regulation of the responses by these genes appears to be important to determine the resistance/ susceptibility of the hosts to development of TE. Polymorphisms of the genes important for the protective immune responses to T. gondii, such as IFNg, toll-like receptor and Tyk2, have

781

been shown to affect susceptibility to the infection in humans and mice (Shaw et al., 2003; Peixoto-Rangel et al., 2009; Albuquerque et al., 2009).

23.11 IMMUNE EFFECTOR MECHANISMS IN OCULAR TOXOPLASMOSIS Ocular toxoplasmosis may result from congenital infection or after birth from acquired infections (reviewed in Chapter 5). The primary target of ocular toxoplasmosis is the neural retina, but infection may involve choroids, sclera, optic nerve and retinal pigment epithelial (RPE) cells (Norose et al., 2003b; Delair et al., 2011). The lesions are often necrotic, destroying the neural retina and often the choroid with retinochoroiditis, the most common lesion caused by infection. Ocular toxoplasmosis usually occurs from a reactivation of a latent infection but can also occur from newly acquired infections with T. gondii (Holland et al., 1988; Chakroun et al., 1990). Toxoplasmosis also causes retinochoroiditis in AIDS patients (Holland et al., 1988; Chakroun et al., 1990; Vallochi et al., 2008). The intraocular immune response to T. gondii is mediated primarily by CD4þ and CD8þ T-cell responses (Norose et al., 2011). Although the eye is an immune privileged site expressing low levels of MHC class I, infected mice show an increased expression of MHC Class I. Depletion of CD8þ cells results in increased lesion formation and cyst burden in a murine model, indicating CD8þ T-cells can also destroy parasiteinfected host cells in an MHC class I restricted manner in the eye (Gazzinelli et al., 1994a; Lyons et al., 2001) . B-cells may also contribute to the immune response to ocular toxoplasmosis by limiting tachyzoite proliferation in the eye (Lu et al., 2004). IFNg production has been associated with intraocular inflammation but numerous studies indicate IFNg is protective against ocular T. gondii infection (Norose et al., 2003a; Shen et al., 2001;

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Olle et al., 1996). Studies have shown, for example, increased parasite dissemination and load in the retina and other regions of the eye in IFNg-deficient mice, as compared to wildtype mice irrespective of host genetic background. Control of parasite proliferation in the eye is most likely through IFNg induced protective mechanisms. In human retinal pigment epithelial cells, an IDO-dependent mechanism was found to be involved in the mechanism of IFNg induced inhibition while in the murine model of ocular toxoplasmosis, IFNg induced inhibition was found to be via a NO-dependent mechanism (Shen et al., 2001; Nagineni et al., 1996). IFNg has also been shown to regulate T. gondii load and interconversion between the bradyzoite and tachyzoite stages in the murine eye (Norose et al., 2003a). Apoptosis is also an important method used by immunoprivileged sites, including the eye to maintain immune privilege utilizing the Fas/FasL system. Infection with T. gondii induces FasL expression on cells of the mouse retina indicating apoptosis is also a mechanism controlling the parasite in the eye (Vallochi et al., 2008). Clinical presentations of ocular toxoplasmosis vary amongst patients with some patients presenting with only one episode of inflammation while others have multiple recurrences leading to loss of eyesight. Recurrent lesions are usually identified at the borders of the retinochoroidal scars, typically found in clusters that have been attributed to the rupture of cysts within the old lesions (Holland, 2003, 2004). Variation in clinical presentation and severity of disease has been attributed to both host genetic heterogeneity and parasite genotype. A predominance of type I strains has been found to be associated with ocular infection in the US, Poland and Brazil (Grigg et al., 2001; Khan, 2006; Vallochi et al., 2005; Switaj et al., 2006). Ocular toxoplasmosis in southern Brazil is higher than in most other regions in the world and a more severe necrotizing retinochoroiditis is more common (Vallochi et al., 2008). In Brazil and South

America, in addition to a predominance of type I strains, divergent parasite genotypes are also present that also appear to be more virulent (Khan et al., 2006). These findings indicate that divergent genotypes likely contribute to severity of clinical outcomes although the underlying mechanisms affecting severity of T. gondii infection in the eye remain to be fully resolved.

23.12 IMMUNE EFFECTOR MECHANISMS IN CONGENITAL TOXOPLASMOSIS Congenital toxoplasmosis is the result of a primary infection with T. gondii during pregnancy in which transplacental passage of the tachyzoite stage occurs and infects the foetus (Montoya and Remington, 2008). Foetal involvement is most severe when maternal toxoplasmosis is contracted early in pregnancy leading to spontaneous abortion or severe neurological effects. Conversely, infection in the third trimester is often asymptomatic, with development of chorioretinitis commonly occurring later in life. Congenital transmission occurs almost solely in seronegative women who have acute infection during pregnancy and is not typically seen in women who are seropositive before pregnancy (Montoya and Rosso, 2005; Montoya and Remington, 2008). However, recently exceptions have been found in the case of infections with atypical genotypes, as discussed below. Additionally there have been occasional reports of congenital transmission in women with immune suppression who have reactivation of latent T. gondii during pregnancy (Jones et al., 2003). Studies in humans and murine models that mimic certain key aspects of human congenital toxoplasmosis have contributed to the understanding of congenital toxoplasmosis (Roberts and Alexander, 1992; Menzies et al., 2008). Evidence from these models indicates IFNg has a dual role, both protecting the foetus and enhancing transmission. For example, increased

23.13 BEHAVOURIAL CONSEQUENCES OF INFECTION

levels of IFNg levels can decrease maternal parasitemia and congenital transmission and enhance survival of pregnant mice (Abou-Bacar et al., 2004; Shirahata et al., 1992). Conversely, IFNg deficient mice have reduced abortion rates although these mice exhibit increased numbers of parasites in their uterus and the placentas as compared with WT mice (Shiono et al., 2007). Studies in human placenta villous explants indicate IFNg induces parasitized white blood cell adhesion to placental villous cells and IFNg may thus enhance the probability of vertical transmission (Ortiz-Alegria et al., 2010). Pregnancy has the ability to modulate the immune response and it has been suggested disruption of immunomodulatory mechanisms by T. gondii infection could have detrimental effects on pregnancy such as inducing abortion while pregnancy-mediated immunomodulation may also favour parasite replication and congenital transmission (Menzies et al., 2008). IFNg induced inhibition of T. gondii in umbilical endothelial cells is likely important in the prevention of transplacental transmission of congenital toxoplasmosis and in vitro studies investigating IFNg effector mechanisms using umbilical vein endothelial cells has been studied in several different species. In sheep, treatment of umbilical vein cells with IFNg blocks the growth of T. gondii via a NO and ROI independent mechanism (Dimier and Bout, 1996a). In human umbilical vein endothelial cells, IFNg induces inhibition of T. gondii via a NO, ROI and IDO independent mechanism (Woodman et al., 1991). A study in mice investigating the role of foetus versus maternal IFNg induced IDO and iNOS expressions following T. gondii infection found that IDO production is largely due to foetal expression while iNOS production is largely determined by the maternal phenotype (Pfaff et al., 2008). Placental trophoblast cells which form the border between maternal blood circulation and foetal tissue are also likely important in infection of the foetus. Studies in the BeWo model of human trophoblasts found these

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cells were refractory to stimulation by IFNg and TNFa but a lack of polyamine metabolic products via cross-regulation by nitric oxide production was found to limit parasite replication (Pfaff et al., 2005). These studies indicate multiple mechanisms may be involved in the host defence of the maternalefoetal interface, although further studies are clearly needed. Finally, while it has traditionally been thought that transmission of T. gondii to the foetus occurs predominantly in women who acquire the infection during gestation, a new concept of congenital toxoplasmosis is emerging due to an understanding of the influence of genotypes of T. gondii and virulence (Lindsay and Dubey, 2011). The three major genotypes of Toxoplasma differ in their pathogenicity in mice and prevalence in humans although some Toxoplasma isolates have been termed atypical because they do not belong to the three major clonal lineages (Su et al., 2006). The type II genotype is responsible for most congenital toxoplasmosis in Europe and the USA, but recently congenital toxoplasmosis in immune mothers infected by an atypical genotype has been found, indicating acquired immunity to the original infecting genotype may not protect against reinfection with an atypical strain (Elbez-Rubinstein et al., 2009). Additionally, congenital toxoplasmosis caused by atypical genotypes results in more severe clinical manifestation of congenital toxoplasmosis than infection caused by typical genotypes (Delhaes et al., 2010; Boughattas et al., 2011). Serological tests to distinguish between typical versus avirulent strains are being developed and may help resolve the underlying mechanisms contributing to severity of congenital toxoplasmosis.

23.13 BEHAVOURIAL CONSEQUENCES OF INFECTION One important aspect of Toxoplasma infection of intermediate hosts is its ability to alter host behaviour. Data regarding the effect of Toxoplasma on

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host behaviour come from two sources. One involves the experimental infection of laboratory animals, generally mice or rats, and the observation of the effects of infection on subsequent behaviour as measured by performance on defined cognitive and behavioural tests. The second set of studies involves the effect of Toxoplasma infections in human behaviour. Most of the studies of human behaviour involve the measurement of antibodies to Toxoplasma in sets of blood samples from case and control individuals who do or do not have the target behaviour. These studies can be divided into those which involve defined psychiatric diseases or conditions such as schizophrenia or suicide and those which involve the analysis of human personality traits. This section will focus on the effects of chronic Toxoplasma on immune competent hosts.

23.13.1 Animal Studies The first published studies of the effect of persistent Toxoplasma infection in learning and memory in mice and rats were reported in 1978 (Piekarski et al., 1978) and in 1979 (Witting, 1979). Since that time there have been multiple studies indicating that infection of mice and rats can result in deficits in learning and memory measured by a variety of standard laboratory measures such as mazes. However, the results of studies on mice and rats have been inconsistent with some studies showing little or no effect on memory or learning (Table 23.1) (Kannan and Pletnikov, 2012). Sources for this variability are likely to be related to differences in experimental methods, timing of infection, the genetic makeup of the infecting Toxoplasma organism and the gender and genetic makeup of the host (Kannan et al., 2010). A novel behavioural effect of Toxoplasma on host behaviour involves the apparent ability of Toxoplasma to lose its innate fear of felines, as measured by aversion to cat odours (Webster et al., 1994). This ‘fatal attraction‘ (Berdoy et al., 2000) effect of Toxoplasma infection on rodents

is postulated to have evolutionary significance since it would increase the likelihood of Toxoplasma in a secondary host such as a rodent completing its sexual cycle within the cat predator. The plausibility of this process is enhanced by the finding that the fear response is not lost when the smell is that of a non-feline predator such as mink (Lamberton et al., 2008) fitting in with the fact that the transfer of Toxoplasma from one intermediate host to another would not result in the completion of the sexual cycle. However, other studies have indicated that mice can have decreased novelty seeking independent of cat behaviour in a way that might result in increased general predation (Hutchison et al., 1980) suggesting that altered behaviour may also play a role in transmission of Toxoplasma among intermediate hosts (Afonso et al., 2012). Toxoplasma infection has also been reported to alter other behaviours in intermediate hosts which might have evolutional significance, including sexual attractiveness (Dass et al., 2011). These biological and molecular mechanisms by which Toxoplasma modulates host behaviour are not completely understood. Neurophysiological studies indicate that the effect is not mediated by overall changes in general behaviours such as olfaction fear, anxiety, olfaction, or nonaversive learning (Vyas et al., 2007), suggesting a high degree of specificity for some of the effects. However, other studies have documented more generalized effects of Toxoplasma infection on neuronal function (Haroon et al., 2012). The neuroanatomical correlates of this specificity are currently under investigation (Vyas and Sapolsky, 2010). There have also been a number of studies of the molecular basis of the Toxoplasma mediated host manipulation. Much of this research has centred on the ability of Toxoplasma infection to modulate the levels of dopamine within the host brain, due to the central importance of this neurotransmitter in animal behaviour. In 1985 it was reported that Toxoplasma infection can

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23.13 BEHAVOURIAL CONSEQUENCES OF INFECTION

TABLE 23.1

Learning and Memory in Rodents with Toxoplasma gondii Spatial Learning and Memory

Test Used

Rodent Tested

Effect

Reference

Labyrinth

Mouse (F), Rat (F)

Impaired

Piekarski et al., 1978

Mouse (F), Rat (F)

Impaired

Witting, 1979

Mouse (F)

Impaired

Witting, 1979

Rat (F)

None

Witting, 1979

Mouse (F)

Impaired

Kannan et al., 2010

Mouse (F)

None

Kannan et al., 2010

Mouse (F, M)

None

Hay et al., 1984

Mouse (M)

None

Hutchinson et al., 1980

Radial arm maze

Mouse (F)

Impaired

Hodkova et al., 2007

Morris water maze

Rat (M)

None

Vyas et al., 2007

Object recognition

Mouse (M)

None

Gulinello et al., 2010

Y maze

Olfactory Based Learning and Memory Test Used

Rodent Tested

Effect

Reference

Social transmission of food preference

Mouse (F)

None

Vyas et al., 2007

Mouse (F)

None

Xiao et al., 2012

Mouse (M)

Impaired

Xiao et al., 2012

Associative Learning and Memory Test Used

Rodent Tested

Effect

Reference

Passive avoidance

Mouse (UK)

Impaired

Wang et al., 2011

(M) male; (F) female; (UK) not stated in paper. Effect: Results related to learning and memory of each type assessed by the listed test. Table adapted from Kannan and Pletnikov, 2012.

result in an increase in dopamine concentrations in the brains of chronically infected mice (Stibbs, 1985), a finding which has been noted in some (Prandovszky et al., 2011), but not all (Goodwin et al., 2012), studies involving rodent brain infection with Toxoplasma. The possible role of dopamine is supported by findings indicating that dopamine uptake inhibitors can modulate the effect of Toxoplasma on rodent behaviour (Skallova et al., 2006) and that dopamine stimulates the growth of Toxoplasma organisms in brain

cell cultures (Strobl et al., 2012). The findings that the Toxoplasma genome contains DNA capable of encoding two genes homologous to tyrosine hydroxylase, the rate limiting step in the synthesis of dopamine, raises the possibility that Toxoplasma organisms may directly manipulate dopamine levels within the brain (Gaskell et al., 2009). However, the fact that immune modulators also change the level of dopamine in the brain (Capuron and Miller, 2011) raises the possibility that some of the alterations in

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neurotransmitters following Toxoplasma infection may be a by-product of the immune responses to that organism. Persistent Toxoplasma infection results in the altered transcription of a large number of genes within the brains of infected mice; the specific genes which are altered varies depending upon the strain of the infecting Toxoplasma organism and the sex of the animal host (Xiao et al., 2012). The effect of Toxoplasma infection within the brain can affect uninfected as well as infected cells perhaps due to rhoptry effector proteins. This phenomenon may explain the fact that Toxoplasma is capable of large scale effects on gene expression in the brain even when the number of cells infected is relatively small (Koshy et al., 2012). In conclusion, there is a great deal of evidence linking persistent Toxoplasma infection to behavioural alterations in experimentally infected mice and rats. The elucidation of the host and parasite biological and molecular mechanisms involved in these effects remains an important goal of research in this area. The investigation of the cognitive and behavioural effects of Toxoplasma should also be expanded to include additional species, such as primates, to allow for more relevant models of the effects of Toxoplasma infections in humans.

23.13.2 Human Studies There is a long history of reported psychiatric symptoms in immunocompetent individuals exposed to Toxoplasma. The earliest reported cases of psychiatric symptoms were those of laboratory workers accidently infected with Toxoplasma in 1951 (Strom, 1951) and 1953 (Sexton et al., 1953). Another early study of 114 individuals who acquired Toxoplasma infection between 1940 and 1964 found psychiatric symptoms in 24 (21.1%) of the cases at follow-up (Kramer, 1966). These and other studies led to a number of studies looking at the relationship

between serological evidence of Toxoplasma infection and schizophrenia. A recent metaanalysis reported on 38 studies which met inclusion criteria and found an odds ratio of approximately 2.5 linking schizophrenia and antibodies to Toxoplasma measured by several different methods (Kramer, 1966; Torrey et al., 2012) (Fig. 23.1). Increased levels of antibodies to Toxoplasma gondii have also been measured in individuals with bipolar disorder, which shares many features with schizophrenia (Pearce et al., 2012) as well as women with prenatal depression and anxiety (Groer et al., 2011). While most of these studies were retrospective in nature, there have been several prospective studies documenting that the increased level of Toxoplasma antibodies can be detected prior to the onset of symptoms (Pedersen et al., 2011). Furthermore, several studies have reported a significant relationship between Toxoplasma antibodies in the mother or in the neonate and the development of schizophrenia and psychosis in the offspring (Brown et al., 2005; Mortensen et al., 2007; Blomstrom et al., 2012) although this has not been noted in every population (Buka et al., 2001). One of the variations in terms of the susceptibility of offspring to schizophrenia following maternal exposure may be the serotype of the organism, with the offspring of mothers exposed to type I infections displaying the highest level of risk (Xiao et al., 2009). Another serious psychiatric behaviour associated with increased levels of exposure to T. gondii is suicide and related self-destructive behaviours. Several studies have documented an association between increased levels of antibodies to Toxoplasma and increased rates of suicide, suicide attempts, and other forms of self-directed violence with similar odds ratios (Okusaga et al., 2011; Ling et al., 2011; Yagmur et al., 2010; Arling et al., 2009; Zhang et al., 2012). One of the studies was prospective in nature with the Toxoplasma exposure being measured several years prior to the suicide or suicide attempt (Pedersen et al., 2012).

23.13 BEHAVOURIAL CONSEQUENCES OF INFECTION

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FIGURE 23.1 plot of 38 studies that address the question of the effect of Toxoplasma gondii infection on the risk of schizophrenia in humans. Figure reproduced with permission from Figure 1, Schizophrenia Bulletin 2012; 38(3): page 643 (Torrey et al., 2012).

Increased levels of suicide attempts have been noted in Toxoplasma seropositive individuals with schizophrenia, as well as in Toxoplasma seropositive individuals without a previous history of a psychiatric disorder (Okusaga et al., 2011). In addition, one study has documented a geographic association between risk of suicide and rates of Toxoplasma seropositivity within national populations in Europe (Lester, 2010). The mechanisms responsible for the association of Toxoplasma seropositivity and risk of suicide remain to be determined. It will be of interest to determine if the risk of suicide in humans is mechanistically related to the altered level of risk assessment found in Toxoplasma infected rodents. Increased levels of

antibodies to Toxoplasma have also been associated with increased rates of automobile (Flegr et al., 2009; Kocazeybek et al., 2009) and workplace (Alvarado-Esquivel et al., 2012) accidents as well as a range of personality traits (Fekadu et al., 2010). The association between Toxoplasma exposure and altered human behaviour is intriguing in light of the effects of experimental Toxoplasma on animal behaviour as noted above. However, it should be noted that there are a number of experimental limitations of the human studies. The most significant is that the human studies rely almost exclusively on the measurement of antibodies to Toxoplasma as markers of exposure. The levels measured in cross-sectional or

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longitudinal studies may thus reflect the robustness and genetic makeup of the host immune system and exposure to other antigens such as intestinally derived antigens (Severance et al., 2012) as well as to previous exposure to Toxoplasma. The timing of infection and the antigenic makeup of the infecting strains are additional variables which are difficult to disentangle in serological studies. The level of association between Toxoplasma exposure and behavioural alterations is also somewhat variable among populations, with some studies failing to find significant associations. Also, while there are large differences in prevalence of Toxoplasma in different geographic regions and among individuals based on geographical, demographic and socio-economic variables, these differences are generally not reflected in rates of schizophrenia and related psychiatric disorders, with the exception of suicide, noted above. For example, most areas of the world which have relatively high rates of Toxoplasma exposure, such as parts of Latin America, Eastern and Central Europe, the Middle East and Africa (Pappas et al., 2009), are not known to have particularly high rates of schizophrenia or bipolar disorder. Similarly, the declining rate of Toxoplasma noted in young adults in the USA (Jones et al., 2007) and Northern Europe (Hofhuis et al., 2011) does not seem to be associated with a concomitant decrease in rates of schizophrenia (Kirkbride et al., 2012), bipolar disorder (Yutzy et al., 2012), suicide (Hu et al., 2008) or accidents (Karch et al., 2011) in the same populations. The reasons for these discrepancies are not known with certainty but may be related to the effects of age of infection, the serotype and stage of the infecting strain, and the underlying genetic susceptibility of the human host. The availability of better assays to define these factors and to directly detect Toxoplasma tissue cysts within the brain and other organs would represent major steps forward in terms of defining the role of Toxoplasma in brain disorders in individuals and in populations and in

planning appropriate therapeutic and prophylactic interventions.

23.14 CONCLUSIONS Maintenance of latent infection in the normal host, reactivation of infection in the immunocompromised host and the neuropathologies in congenital infection and in the immunocompetent host are not fully understood although it is clear the immune response (especially IFNg) is largely responsible for controlling the growth of T. gondii in the CNS in all of these conditions. Control of the parasite in the brain involves a balance of these host immune protective mechanisms and immunomodulatory mechanisms that limit neuropathology. In the brain, the relevant IFNg effector cells include CD4þ and CD8þ T-cells, and dendritic cells, which are involved in the stimulation and maintenance of the intracerebral immune response, while microglia exert potent anti-Toxoplasma activity in the brain parenchyma and astrocytes play a crucial immunoregulatory role. Neurons serve as the main host cell for the cyst stage but recent studies indicate neurons also play a crucial immunoprotective role in the brain in chronic infections and that the infection significantly affects neuronal functions. Finally, although the chronic infection has been considered ‘latent’, increasing evidence indicates neuropsychiatric disorders, such as schizophrenia, and other neuropathologies, such as cryptogenic epilepsy, may be associated with the latent infection. A better understanding of the latent infection and the biology of T. gondii is of great importance for a greater understanding of the pathogenesis of cerebral and congenital toxoplasmosis and associated neuropathologies.

Acknowledgements This work is supported, in part, by National Institues of Health Grants AI078756 (Y.S.), AI095032 (Y.S.), and AI077887 (Y.S.) and a grant from the Stanley Medical Research Insitute 08R-2047 (Y.S.).

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