Immunosuppression is a characteristic feature of Toxoplasma gondii-infected murine hosts. The present study aimed to determine the effect of the immunosuppression induced by T. gondii infection on the pathogenesis and progression of Alzheimer's disease (AD) in Tg2576 AD mice. Mice were infected with a cyst-forming strain (ME49) of T. gondii, and levels of inflammatory mediators (IFN-γ and nitric oxide), anti-inflammatory cytokines (IL-10 and TGF-β), neuronal damage, and β-amyloid plaque deposition were examined in brain tissues and/or in BV-2 microglial cells. In addition, behavioral tests, including the water maze and Y-maze tests, were performed on T. gondii-infected and uninfected Tg2576 mice. Results revealed that whereas the level of IFN-γ was unchanged, the levels of anti-inflammatory cytokines were significantly higher in T. gondii-infected mice than in uninfected mice, and in BV-2 cells treated with T. gondii lysate antigen. Furthermore, nitrite production from primary cultured brain microglial cells and BV-2 cells was reduced by the addition of T. gondii lysate antigen (TLA), and β-amyloid plaque deposition in the cortex and hippocampus of Tg2576 mouse brains was remarkably lower in T. gondii-infected AD mice than in uninfected controls. In addition, water maze and Y-maze test results revealed retarded cognitive capacities in uninfected mice as compared with infected mice. These findings demonstrate the favorable effects of the immunosuppression induced by T. gondii infection on the pathogenesis and progression of AD in Tg2576 mice.

Funding: This work was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2009-0076262 & No. 2011-0013824), and by the Seoul National University Bunding Hospital Research Fund (SNUBH No. 03-2010-016). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In the present study, we undertook to determine whether T. gondii infection is involved in the neuroinflammation and neurodegeneration mechanisms of dementia. We hypothesized that latent T. gondii infection in the mouse brain may induce immunosuppression to escape host immune attack, and thus, act to suppress the pathogenesis of AD. Thus, the objective of the present study was to investigate the effect of the inhibition of neuroinflammation by T. gondii on the progression of AD. For this purpose, we used Tg2576 mice (an accepted model of AD) infected with an avirulent strain (ME49). Degrees of neuronal protection in brains were determined histologically, and immune responses were examined in brain tissues and BV-2 microglial cells. In addition, the water and Y-maze behavioral tests were used to quantify learning and memory abilities and cognitive functions.

Investigations on the possible link between T. gondii infection and degenerative central nervous system (CNS) disease in man have been mainly limited in the serology testing of T. gondii-specific antibody [16] , [17] , [18] . Recently, such studies have been conducted in AD patients, PD patients, and patients with other psychiatric conditions. For example, in one study, the seropositivity rate for T. gondii-specific IgG was found to be more in patients with AD than in healthy controls (44.1% versus 24.3%) [16] . In another study, 42 patients with obsessive-compulsive disorder (OCD) and 52 patients with Parkinson's disease (PD) also showed significantly higher titers in seropositivity than controls [17] , [18] . As a result, the authors suggested that T. gondii infection may be involved in the pathogenetic mechanisms of CNS diseases [16] , [18] . However, in those study the anti-T. gondii IgG titers shows IgG levels examined at that time, and thus, do not suggest that a causal relationship exists between toxoplasmosis and the etiology of psychiatric illness and dementia [19] . Because the causal relationship between toxoplasmosis and the etiology of CNS diseases cannot be simply determined, experimentation is needed under known conditions, that is, with information on times between infection and disease onset.

Microglial cells also augment inflammatory responses by releasing various mediators, such as, cytokines, reactive oxygen species, complement factors, neurotoxic secretary products, and free radicals [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] , [15] , and many of these mediators are known to stimulate amyloid precursor protein (APP) deposition and contribute to neuronal death in AD [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] , [15] . Eventually, microglial cell activation can establish a vicious cycle of inflammatory mediator release and the stimulation of APP production [15] . In this respect, we considered that it would be interesting to probe the relationship between host immune response against the long-lived and latent pathogen T. gondii in the brain and the progression of age-related neurodegenerative disorder.

The neuronal degeneration induced by neuroinflammation is known to play a key role in the pathogenesis of chronic neurodegenerative diseases [9] , [10] , and in particular, Alzheimer's disease (AD) is the most common cause of dementia in the elderly causing progressive and permanent reductions in learning, memory, and cognitive abilities [10] . The pathogenesis of AD is characterized by widespread neuronal degeneration, involving synaptic and neuronal loss, and the formations of extracellular neuritic plaques containing β-amyloid peptides and intracellular neurofibrillary tangles [10] . Histologically, AD brain tissues show increased numbers of reactive microglia [11] , which when exposed to inflammatory stimuli express the inducible form of NO synthase (iNOS), and thus, increase NO production [12] . Moreover, adjacent neuronal cells are extremely susceptible to the toxic effects of NO, and this sensitivity plays a central role in the pathogenesis of neurodegenerative diseases [12] , [13] .

Notably, the neuronal degeneration induced by neuroinflammation is not a common finding in the brains of mice chronically infected with T. gondii [7] . Similarly, chronic or latent infections by infectious agents in the CNS may delay the onset of neurodegenerative changes related to nerve inflammation [8] . Regarding neuroinflammation in T. gondii infection, NO production is known to be an important regulator and indicator. For example, NO production was found to be significantly down-regulation when conditioned medium of a T. gondii-infected astrocyte culture was added to a microglia culture [7] , which suggests that the immune responses triggered by T. gondii infection can reduce inflammatory response in the host brain and prevent neuronal degeneration.

Immune responses to T. gondii infection differ during the proliferative (acute phase) and dormant (chronic and latent phase) stages and depend on the virulence of the parasite strain, for example, RH is a highly virulent strain (type I) whereas ME49 is avirulent (type II). The acute phase is characterized by marked elevations in serum Th1 cytokine levels, such as, of IFN-γ, TNF-α, IL-12, and IL-18, and is followed by a lethal outcome in mice. On the other hand, non-lethal infection is characterized by modest elevations in Th1 cytokine levels that led to the control of T. gondii infection and minimal damage to the host [3] . In particular, ME49 is an avirulent strain that can exist in the brain for a considerable time by suppressing immune responses in the central nervous system (CNS) [3] , [4] . During the course of T. gondii infection, the levels of anti-inflammatory cytokines, like IL-10 and TGF-β, increase in brain tissues, whereas the productions of inflammatory mediators, such as, nitric oxide (NO), decrease [4] , [5] , [6] . Furthermore, these anti-inflammatory responses may prevent tissue injury and establish a chronic state of host-parasite equilibrium [4] , [5] , [6] .

Toxoplasma gondii (T. gondii) is a protozoan parasite that commonly infects humans and animals [1] . Humans are generally infected by ingesting oocysts released in cat feces or by consuming undercooked meat containing tissue cysts. Following ingestion, bradyzoites and sporozoites released from cysts and oocysts invade intestinal cells and convert to tachyzoites [1] , which when disseminated via blood or the lymphatic system to remote organs induce acute or chronic inflammatory responses. Furthermore, during the chronic stage, the brain is the most commonly affected site [2] . T. gondii is a serious pathogen that can invade vital organs, but usually the infection is mild and asymptomatic in immunocompetent hosts. Possible clinical manifestations include lymphadenopathy, myocarditis, hepatitis, sepsis syndrome, retinochoroiditis, and encephalitis [1] . However, normally the infection becomes chronic, remains latent in the brain, and elicits life-long immunity against toxoplasmosis [1] .

The passive avoidance test was performed to confirm the effect of T. gondii infection on wild type and Tg2576 mice ( Figure S1 ). Times taken to enter the dark chamber (latencies) were measured for 300 sec (retention trial). T. gondii-infected Tg2576 mice were found to have greater latencies ( Figure S1 ), which means the T. gondii infection in AD model mice (Tg2576) inhibited memory deterioration, as was observed for the Morris water maze and Y-maze behavioral tests.

The Y-maze test was also conducted to evaluate learning and memory functions, as described previously [23] ( Figure 5 ). The success rate of T. gondii-infected mice in the Y-maze test was significantly greater than for uninfected mice (p<0.01, by one-way ANOVA and Tukey's post hoc test). Accordingly, Morris water maze and Y-maze testing showed that T. gondii infection inhibited spontaneous memory functional impairments in Tg2576 mice ( Figure 5 ).

Differences in learning and memory between wild type (WT) and Tg2576 mice (TG) at 9 mo of age were examined using the Morris water maze test. (A) In a 60 s probe trial, the ability of uninfected (PBS-treated)-Tg2576 mice (TG+PBS) to find the training quadrant (zone 4, which contained the platform) was significantly less than those of mice in the other experimental groups (A; p<0.0001). On the other hand, T. gondii-infected Tg2576 mice (TG+ME49) performed as well as wild type mice (B). Representative swimming paths of mice during the probe trial (platform removed) were as follows (C); TG+PBS mice seemed unaware of the platform position, whereas TG+ME49 mice and wild-type mice (WT+PBS and WT+ME49) remained in the vicinity of the platform. The yellow box in the figure indicates the hidden platform.

Based on these findings, we used the Morris water maze and the Y-maze behavioral tests to quantify learning and memory functions, as previously described [22] . In the water maze test, Tg2576 mice infected with T. gondii were found to differ significantly from uninfected (PBS-treated) mice (p = 0.0055, F = 4.49; Figure 4A ). However, no difference was found between T. gondii-infected and uninfected wild type mice ( Figure 4A ). In the probe test at 48 hours after final spatial training, T. gondii-infected Tg2576 mice stayed significantly longer in zone 4 than in other zones (zones 1–3) ( Figure 4A ). Mean durations in each zone for T. gondii-infected Tg2576 mice in the water maze test were as follows; zone 4 (platform), 26.77 s; zone 1, 16.36 s; zone 2, 9.87 s; and zone 3, 6.98 s ( Figure 4A ). However, uninfected Tg2576 mice exhibited a different pattern, suggesting loss of memory (zone 1, 12.69 s; zone 2, 10.04 s; zone 3, 12.58 s; and zone 4, 11.97 s). This result was confirmed by measuring the mean times required to reach the platform (a measure of learning and memory) ( Figure 4B ), and by investigating swimming paths in zone 4 during the last probe trial (platform removed) ( Figure 4C ).

T. gondii infection was found to induce the productions of IL-10 and TGF-β in brain and to suppress nitrite production in response to TLA, indicating the local inductions of anti-inflammatory responses in brain. In addition, β-amyloid plaque depositions were found to be reduced in the cortex and hippocampus of infected mice. These findings raised the question as to whether progressive impairments of learning and memory in Tg2576 mice could be reduced by the anti-inflammatory responses induced by T. gondii infection.

Differences between β-amyloid deposit levels in T. gondii-infected and non-infected mice (PBS-treated mice) were examined in the cortex and hippocampus regions using Congo red and by immunohistochemical staining with 6E10 antibody. Congo red staining was performed on; uninfected (PBS-treated) wild type (A1), T. gondii-infected wild type (A2), uninfected Tg2576 (A3), and infected Tg2576 (A4) mouse groups. Wild type mice with or without T. gondii infection showed no amyloid plaque (A1, A2) at 9 mo after birth, whereas PBS-treated Tg2576 mice showed many Congo red-stained regions in the cortex (A3). However, no amyloid plaque was observed in T. gondii infected Tg2576 mouse brain tissues (A4). Immunostaining results of cortex and hippocampus concurred with Congo red findings (A5, A6, A7, A8). (×40, Scale bar = 200 µm).). In addition, numbers of plaques in cortex and hippocampus were counted using a color digital camera attached to a microscope and Image J software, and the results obtained showed that numbers of plaques were significantly less in the hippocampus and cortex of T. gondii-infected Tg2576 mice (B).

Similarly, anti-inflammatory cytokines and NO levels were measured after treating BV2 cells with TLA ( Figure 2E, F, G ). The addition of LPS increased nitrite production by cells, whereas the addition of TLA, IL-10, or TGF-β decreased nitrite levels ( Figure 2G ). Observed decreases in nitrite production were similar when cells were treated with TLA, IL-10, or TGF-β. However, when they were co-treated with LPS, the reduced nitrite level elicited by TLA was significantly greater than the levels elicited by IL-10 and TGF-β ( Figure 2G ; p<0.05). The above results show that the productions of anti-inflammatory cytokines (IL-10 and TGF-β) and reduced nitrite production were observed under all experimental conditions (T. gondii-infected Tg2576 mice and TLA stimulated primary cultured microglia and BV-2 cells).

When exposed to inflammatory stimuli, microglial cells produce the inducible form of NO synthase (iNOS), and the NO subsequently produced is critically required to construct protective host responses against foreign pathogens. However, this immune response eventually damages host tissues, and in particular, NO is a key mediator of glia-induced neuronal death [12] . In the present study, primary cultured microglial cells were prepared from wild type mice (C57BL/6), and incubated with TLA, recombinant IL-10, or recombinant TGF-β ( Figure 2D ). As was expected, 100 ng/ml LPS increased nitrite production by cells, whereas the addition of 50 µg/ml TLA, recombinant IL-10, or recombinant TGF-β decreased nitrite production. Furthermore, the effect of TLA on nitrite production was also observed in the presence of LPS ( Figure 2D ). Microglial cells from primary cultured brain tissues showed significantly less nitrite production when TLA was added, and the addition of TLA decreased nitrite production under conditions of LPS stimulation ( Figure 2D ).

Because the anti-inflammatory cytokines, IL-10 and TGF-β, are produced by microglial cells for neuroprotection after traumatic injury or stroke [20] , we examined the secretion of these cytokines using T. gondii-infected Tg2576 mouse brain tissues ( Figure 2B, C ) and BV2 cells (a microglial cell line; Figure 2E, F ). IL-10 and TGF-β cytokine levels were found to be significantly higher in infected Tg2576 mouse brain tissues (TG+ME49) than in uninfected control mouse tissues (TG+PBS) (p<0.05) ( Figure 2B–C ). Furthermore, levels of IL-10 and TGF-β in BV2 cells cultured in vitro were found to be increased remarkably by the addition of T. gondii lysate antigen (TLA), which concurred with our findings in T. gondii-infected mouse brains ( Figure 2B–C ).

(A) The mRNA expressions of inflammation-suppressing cytokines (IL-10 and TGF-β) were higher in T. gondii (Me49)-infected Tg2576 mice than in their uninfected littermates (A). mRNA levels are presented as percentages of cytokine levels in infected mice versus uninfected mice. Furthermore, IL-10 and TGF-β cytokines were significantly higher in the brain tissues of infected mice than in those of uninfected mice (P<0.05) (B & C). Nitrite levels suggested the production of nitric oxide (NO), an inflammatory mediator related to neuronal death. Primary cultured microglial cells were prepared from wild type mice (C57BL/6 mice) and tested for nitrite production (D). Microglial cells cultured from brain tissues showed a significant decrease in nitrite production when TLA or anti-inflammatory cytokines were added (D). To investigate the possibility that these cytokines were produced by microglial cells, BV-2 cells were cultivated with LPS and/or T. gondii lysate antigen (TLA) (E & F). BV-2 microglia treated with TLA significantly increased the productions of IL-10 (E) and TGF-β (F) in the presence or absence of LPS. BV-2 cells were incubated for 12 h in the presence of TLA, LPS, IL-10, or TGF-β, and the supernatants obtained were analyzed for nitrite (G). The results obtained showed that nitrite concentrations were lower in TLA-, IL-10-, or TGF-β-treated cells than in non-treated control cells. In particular, the nitrite concentration increase induced by LPS was significantly lowered by co-treatment with TLA (P<0.05).

Because neurodegeneration is related to immune balance between inflammatory mediators and inflammation-suppressing cytokines, the mRNA levels of IFN-γ, IL-10, and TGF-β were examined in T. gondii-infected Tg2576 mice by quantitative real-time PCR ( Figure 2A ). IFN-γ mRNA levels were found to be no different in infected and uninfected Tg2576 mice, but IL-10 and TGF-β mRNA levels were higher in infected mice than in uninfected control mice ( Fure 2A ).

Histological changes in the hippocampal formation of Tg2576 AD mice were observed by H&E staining, and changes in T. gondii-infected (B; ME49) and uninfected mice were compared (A; PBS). Neuronal death, represented by eosinophilic neurons (PBS, ×100), was remarkably lower in the hippocampal region of infected Tg2576 mice (B; ME49) than in Tg2576 mice (A; PBS), which showed spontaneous neuronal degeneration (×100). Scale bar = 100 µm. Results are represented as percentages of degenerative cells among all cells in dentate gyrus of hippocampal formation (C).

To determine the neuronal damage caused by T. gondii infection, histopathologic changes in the hippocampal region were examined by hematoxylin and eosin (H-E) staining in Tg2576 AD mice infected or not infected by T. gondii. As shown in Figure 1 , neuronal death (eosinophilic neurons) was remarkable in the hippocampal region of phosphate-buffered saline (PBS)-treated uninfected mice ( Figure 1A , progressive AD mice as a control), whereas infected Tg2576 mice ( Figure 1B ) and infected wild type mice (data not shown) exhibited few eosinophilic neurons in the same region. Progressive AD mice (TG+PBS) showed numerous dead neurons ((57.7±2.5)%) in the dentate gyrus at 9 months after birth ( Figure 1C ), whereas T. gondii-infected Tg2576 mice (TG+ME49) showed relatively few dead neurons ((29.2±0.81)%) ( Figure 1C )

Discussion

T. gondii is a zoonotic protozoan that can infect many vertebrates. It has a worldwide distribution, and is the causative agent of human and animal toxoplasmosis [2]. According to a survey conducted in the USA in 1988–1994, T. gondii seroprevalence in the overall age-adjusted population was 22.5%, and it showed a gradual increase with age [24]. Furthermore, France and several other European countries, Latin America, and sub-Saharan Africa have even higher seroprevalences than the USA [25].

T. gondii has two developmental stages in man, that is, tachyzoites (trophozoites during acute infection) and bradyzoites (cysts during chronic infection), and these have different pathogenic consequences [2]. Tachyzoites disseminated via blood or the lymphatic system to different organs during acute stage disease cause toxoplasmosis, which is characterized by lymphadenopathy and reticular cell hyperplasia [2]. However, during the chronic, latent stage, the brain is the most commonly affected [2]. Furthermore, during this stage, the infection is likely to be accompanied by nerve degeneration, which progresses with age. In this respect, T. gondii is an important infectious agent during the course of neuronal degenerative diseases, such as, AD. However, the relationships between chronic, latent T. gondii infection and age-related neuronal degenerative diseases have not been determined. Since most cases of primary T. gondii infection are asymptomatic and the parasite has established immune privilege in chronically infected host tissues, such as, in the brain [2], the relationships between neurodegenerative diseases, such as, AD, and T. gondii infection have been overlooked.

T. gondii is classified into three clonal lineages by virulence [3]. The type I genotype is highly virulent to mice, whereas the lethalities of the type II and III strains are substantially lower. Type II strains, which include ME49, are highly prevalent in animals, and are also associated with toxoplasmosis in man [3]. The lethality of T. gondii in mice is largely determined by strain genotype. Lethal infections are mediated by a strong Th1 cytokine response, such as, by IFN-γ, IL-12, TNF-α, and IL-18, whereas non-lethal type II ME49 infections are controlled by the modest inductions of Th1 cytokines [3]. The overstimulation of host immune responses that are normally required for protection is an important immunological feature in acute toxoplasmosis [3]. In contrast, during latent infections, T. gondii contributes to the control of host immune response in a manner that results in immunosuppression [5], [7], [26]. In fact, several in vitro studies have shown that T. gondii triggered immune responses reduce inflammatory responses and prevent neuronal degeneration [5], [7]. Thus, the reduction of neuroinflammation by immune modulation could alter the disease onset and progression of AD [9], [10], [12], [13], [14], [15]. However, although the authors of these reports suggested that T. gondii infection in the brain reduces neuroinflammation, the effects of T. gondii infection in the development of neurodegenerative disorders, such as, AD, have not been examined.

In recent studies, it has been found that more than 40% of patients with a severe CNS disease have anti-T. gondii IgG [16], [17], [18]. In a Turkish study of such patients, T. gondii infection was screened for based on seropositivity against T. gondii immunoglobulin regardless of symptom onset. Accordingly, results would be interpreted to mean that psychiatric and dementia patients have life styles that differ from those of healthy controls [19], for example, patients with impaired social and occupational functions are more likely to stay at home, and if they owned cats, would be at greater risk of being exposed to T. gondii [19]. Thus the causal relationship between toxoplasmosis and the etiology of certain CNS diseases, such as, dementia, cannot be easily determined [19]. To overcome these types of problems associated with human research, animal research is required because it can provide information regarding temporal relations between infection and disease onset.

In the present study, we focused on the effects of chronic, latent Toxoplasma infection in the brains of Tg 2576 mice on neurodegenerative changes and inflammation induced genetically. Histopathologic studies showed that the hippocampal region of non-infected Tg 2576 mice exhibited many eosinophilic β-amyloid peptide plaques, and showed that these plaques were less present in the brains of T. gondii-infected Tg2576 mice or wild type mice. These results convince us that the neuroprotection observed was due to T. gondii infection. Furthermore, our results also suggest that the neuroprotective effects of T. gondii extend to learning and memory impairments in Tg2576 mice and possibly to the progression of AD.

In the CNS, microglial cells, astrocytes, and neurons are susceptible to T. gondii infection, and in chronic infections, latent cysts are produced in the CNS [27]. Although microglial cells are largely responsible for preventing T. gondii proliferation in the brain, they also sometimes produce the anti-inflammatory cytokine IL-10, and facilitate parasite persistence by suppressing immune responses in the CNS [28], [29]. IL-10 is produced mainly by brain mononuclear cells, and inhibits the productions of IL-12, IL-6, IFN-γ, and TNF-α, suggesting that IL-10 has an ameliorative effect on the severe inflammation caused by toxoplasmic encephalitis [30].

Notably, an intraperitoneal injection of lipopolysaccharide (LPS), an inflammation inducing endotoxin, was found to result in memory impairments in mice [31]. In addition, repeated injections of LPS enhanced β-amyloid generation, and conversely, treatment with anti-inflammatory agents suppressed LPS-induced amyloidogenesis, memory dysfunction, and neuronal cell death [31]. In this study, it was also found that inflammation resulted in an accumulation of β-amyloid 1–42 via increased β- and γ-secretase activities accompanied by elevated APP expression and astrocyte activation [31].

In the AD brain, various neuroinflammatory mediators, including complement activators and inhibitors, chemokines, cytokines, radical oxygen species, and inflammatory enzyme systems, are released by microglia, astrocytes, and neurons [15]. Accordingly, anti-inflammatory cytokines, such as, IL-10 and TGF-β, are required to prevent immunopathological effects by inhibiting the chemotactic migrations of these microglial cells toward β-amyloid aggregates and by directly inducing anti-inflammatory responses [15], [32]. Convincing evidence shows that Th2 cytokines have beneficial immunomodulatory effects in toxoplasmosis [28], [29], [30]. Actually, IL-10 is known to play vital roles in the control of inflammatory responses during acute toxoplasmosis, and to inhibit tissue damage during the chronic phase [28], [29], [30]. Furthermore, the high susceptibility of IL-10-knockout mice to T. gondii infection has been shown to be mediated by an exacerbated inflammatory process not caused by parasite proliferation [33]. The inflammation process is an important countermeasure to infection, but it also damages host tissues. In the long term, harmful inflammations may be eliminated by pathogen's strategy to inhibit host cellular immunity [28], [29], [30]. T. gondii provides a good example of this effect.

In the present study, T. gondii was found to induce an anti-inflammatory effect by up-regulating IL-10 and TGF-β. Furthermore, in vitro treatment of primary microglial cells and BV-2 cells with TLA reduced NO production, which is known to play important roles during neuroinflammation and the pathogenesis of AD. Furthermore, inflammation in the brain may be an important component of the mechanism of dementia and cognitive decline in the elderly. Accordingly, it has been suggested that the inhibition of inflammatory cascades may attenuate amyloidogenic processes, such as, those of AD [34]. Furthermore, because the suppression of NO synthesis was found to prevent cell death and restore lymphocyte proliferation in T. gondii (ME49)-infected mice [4], our results support the notion that the inhibition of neuroinflammation by T. gondii attenuates the progression of amyloidogenesis in Tg2576 mice.

TGF-β1 and IL-10 are important cytokines that can induce tolerance and suppress exaggerated immune response. In particular, in one study, elevated levels of TGF-ß1 were found to be related to amyloid plaque reductions in the parenchyma, hippocampus, and neocortex of the hAPP Tg mouse brain, and to reduced levels of dystrophic neuritis in aged hAPP Tg mice [35]. Similarly, a decrease in TGF-β signaling in cultured neuroblastoma cells was found to cause neuronal degeneration and to increase levels of secreted Aβ and β-secretase-cleaved soluble APP [36].

Our results also show that T. gondii-infected Tg2576 mice exhibited higher levels of the anti-inflammatory cytokines, IL-10 and TGF-β, in brain tissues, and less neuronal death, amyloid plaque deposition, and neurodegeneration than non-infected mice. In one previous study, it was found that T. gondii-triggered immune regulations, which included prostaglandin E2 secretion by astrocytes and cAMP-dependent IL-10 secretion by microglia, reduced NO production, and as a result, the authors suggested that T. gondii reduced tissue inflammation in the host brain [7]. An in vivo study also demonstrated a remarkable increase of IL-10 expression in the brains of T. gondii-infected mice [37]. In the present study, T. gondii antigen decreased NO production by primary cultured microglia and BV-2 cells, which also suggests that T. gondii prevented neuron degeneration in Tg2576 AD model mice.

One interesting observation made in the present study was that T. gondii infection attenuated impairments in learning and memory functions in Tg2576 mice, as demonstrated by the water- and Y-maze tests. Furthermore, these results well matched with the observed reduction in NO production and the expressional upregulations of anti-inflammatory cytokines. It is known that Tg2576 mice exhibit an increase in ß-amyloid deposition and memory loss at the time of the appearance of detergent-insoluble ß-amyloid aggregates (ß-amyloid insol ) 6 months after birth [22]. In the present study, T. gondii-infected Tg2576 mice were found to retain the memory capacities of non-AD wild type mice (control mice) according to water- and Y-maze test results. In this respect, our findings agree with those of other investigators, who found that C57BL/6 mice infected with the avirulent ME49 strain showed normal cognitive behaviors despite widespread brain pathologies and sensorimotor deficits [38].

The present study demonstrates effects of chronic, latent T. gondii infection on progressive neurodegenerative disease in an AD transgenic mouse model, whereas previous studies has been performed at the early infection stage (within 2 months of infection) [3], [4], [28], [29], [37]. The age of the mice used in the present study and the infection period seem to be more relevant, because T. gondii has a long latency period in the host brain [1]. Thus, our results describe the long-lasting (at least 6 months after infection) effects of T. gondii infection on mice. In addition, our study highlights the possibility that the neuroprotection induced by T. gondii infection before the onset of AD could inhibit the β-amyloid accumulation and neurodegeneration that probably diminish cognitive abilities. Furthermore, we suggest that our results, which obtained using a mouse AD model, are helpful in terms of determining the relationship between T. gondii infection and human AD, because most cases of human toxoplasmosis are caused by genotype II, to which ME49 used in the present study belongs [39], [40].