ABSTRACT
Toxoplasma gondii, a common neurotropic parasite, is increasingly being linked to neuropsychiatric disorders, including schizophrenia, Alzheimer’s disease, and Parkinson’s disease. However, the pathogenic mechanisms underlying these associations are not clear. Toxoplasma can reside in the brain for extensive periods in the form of tissue cysts, and this process requires a continuous immune response to prevent the parasite’s reactivation. Because neuroinflammation may promote the onset and progression of neurodegenerative diseases, we investigated neurodegeneration-associated pathological changes in a mouse model of chronic Toxoplasma infection. Under conditions of high-grade chronic infection, we documented the presence of neurodegeneration in specific regions of the prefrontal cortex, namely, the anterior cingulate cortex (ACC) and somatomotor cortex (SC). Neurodegeneration occurred in both glutamatergic and GABAergic neurons. Neurons that showed signs of degeneration expressed high levels of CX3CL1, were marked by profoundly upregulated complement proteins (e.g., C1q and C3), and were surrounded by activated microglia. Our findings suggest that chronic Toxoplasma infection leads to cortical neurodegeneration and results in CX3CL1, complement, and microglial interactions, which are known to mediate the phagocytic clearance of degenerating neurons. Our study provides a mechanistic explanation for the link between Toxoplasma infection and psychiatric disorders.
INTRODUCTION
It is increasingly evident that the brain is not an immune-privileged site, and inflammatory events occur during central nervous system (CNS) injury, exposure to toxic metabolites, infection, autoimmunity, and aging (1). This neuroinflammation acts as a protective mechanism for controlling neural insults. However, this protective response is often accompanied by diverse brain pathologies, such as synaptic loss, lesion, and neuronal cell death (2). Therefore, activation of the immune system within the CNS is widely found in psychiatric disorders, such as schizophrenia, and in neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease (3, 4). For instance, activation of microglia and the complement system are major components of inflammation in neurodegenerative diseases. Microglia have been postulated to clear degenerating neurons that are tagged by complement proteins via the fractalkine (CX3CL1) pathway (3, 5, 6).
Toxoplasma gondii is an intracellular parasite that infects approximately a billion people worldwide. Although initial infection with Toxoplasma is associated with few symptoms in immunocompetent individuals, the infection often becomes chronic and persists lifelong in the form of tissue cysts in the brain, retina, and muscles (7). Little is known about the effects of lifelong brain infections on the neurological health of people. Recent discoveries suggest that exposure to Toxoplasma increases the risk for neuropsychiatric disorders, such as schizophrenia (8), Alzheimer’s disease (9), and Parkinson’s disease (10). In the elderly, Toxoplasma seropositivity has been associated with alterations in cognitive functioning and overall cognitive decline (11). In animal studies, Toxoplasma infection has been reported to induce neophobia and to have behavioral effects on learning and memory, anxiety, and locomotion (12). Several studies have investigated neuropathological changes in Toxoplasma-infected brain. In mice with chronic toxoplasmosis, impaired fiber density and fiber continuity were revealed by analysis of neuronal connectivity in infected regions, particularly within somatosensory areas (13). Another study reported alterations in neuronal morphology (e.g., a reduction in dendritic spines) and network activity in brains of Toxoplasma-infected mice (14). Several lines of evidence support a causal role for Toxoplasma infection in disruption of glutamate signaling (14–16). In human studies, Toxoplasma-positive schizophrenic patients exhibited a significant reduction of gray matter volumein cortical regions compared with that of Toxoplasma-negative patients (17).
The mechanism by which Toxoplasma infection leads to neurodegenerative diseases is not clear. Neuroinflammation is postulated to be crucial in the onset and progression of neurodegenerative diseases (18). Studies show that Toxoplasma cysts can be found almost exclusively in neurons (19, 20) and are controlled mainly by cell-mediated immunity provided by resident CNS and infiltrating peripheral immune cells (21). Moreover, a low-grade brain inflammation has been documented in rodent models of chronic Toxoplasma infection characterized by microglia and astrocyte activation (22, 23), an increase in complement protein C1q (22, 24), and ventricular dilatation (22). These findings raise concerns about the possibility of neurodegeneration in Toxoplasma-infected brain, as neuroinflammatory responses might persist for years if infection is not resolved. In this study, we examined brains of Toxoplasma-infected mice for signs of neurodegeneration using Fluoro-Jade B (FJB), a known marker for degenerating neurons (25). We show that persistent Toxoplasma infection leads to cortical neurodegeneration affecting both glutamatergic and GABAergic neurons. Neurons that show signs of degeneration are characterized by complement deposition and overexpression of CX3CL1, a chemokine that binds to the CX3CR1 receptor on microglia (26). Activated microglia were found to accumulate at the site of degeneration, embracing and surrounding degenerative neurons. Our study identifies neurodegenerative effects as a possible mechanistic basis for the association of chronic Toxoplasma infection with neuropsychiatric diseases.
RESULTS
Cyst-associated neurodegeneration presents in the SC and ACC of mouse brain.We infected outbred CD-1 mice with a virulent type I strain of Toxoplasma. Mice were stratified into five groups based on antibody profiles, as follows: (i) an unexposed control group, (ii) an IgG-positive and highly matrix antigen 1 (MAG1)-positive (IgG+/MAG1+ high) group (with a high-grade infection and a MAG1 level of ≥0.5), (iii) an IgG+/MAG1+ low group (with a low-grade infection and a MAG1 level of <0.5), (iv) a Toxoplasma-exposed group that did not develop MAG1 antibody (IgG+/MAG1−), and (v) a Toxoplasma-exposed group that did not develop an antibody response (IgG−/MAG1−). Toxoplasma IgG is an indicator of exposure to the parasite, while MAG1 antibody serves as a serologic marker for cyst burden (27). We conducted staining with FJB, a well-known marker for neurons that undergo degeneration, of brain sections of the mice. Our characterization of neurodegeneration focused on the cortex and the striatum region based on studies implicating these regions in neurodegenerative diseases broadly (28, 29). Among the five groups, strong cortical FJB staining was found only in mice with high MAG1 antibody levels (Fig. 1). There were no FJB-positive cells in mice from the other groups (see Fig. S1 in the supplemental material). In MAG1+ high mice, cells positive for FJB were predominantly located in the somatomotor cortex (SC) and anterior cingulate cortex (ACC) (Fig. 1), while FJB-positive cells were scarce in the other regions of cortex. Higher magnification showed that FJB stained the cell bodies and processes of the cells (Fig. 1C, inset). We also performed FJB staining in combination with staining for the neuronal marker NeuN. Double-fluorescence staining showed the expected overlap between FJB and NeuN (Fig. 2, upper panels), confirming that the degenerating cells were neurons. To see if degenerating cells are associated with parasite antigens, we performed double staining with Toxoplasma antigen and FJB. However, there was no parasite-positive staining in degenerating cells (Fig. S2).
Brain examination revealed neurodegeneration in the anterior cingulate cortex (ACC) and somatomotor cortex (SC) of mice with high-grade chronic Toxoplasma infection. (A) Anatomical diagram illustrating the ACC and SC. STR, striatum; SS, somatosensory system; VS, ventricular system. Coronal sections of Toxoplasma- and sham-infected mice at 5 months postinfection are shown. FJB staining was performed to evaluate neuronal degeneration. (B) No FJB-positive cells were observed in the ACC and SC of the sham-infected mice. (C) Numerous FJB-positive cells were visualized in the ACC and SC of the MAG1 high mice (arrows in the inset white box at ×40 magnification). The presence of IgG is indicative of exposure to Toxoplasma, while MAG1 antibody serves as a serologic marker for cyst burden. MAG1 high, optical density of >0.5. Scale bar, 100 μm or 20 μm (insets). Images are representative of staining for 3 mice/per group.
The FJB-positive degenerating cells were glutamatergic and GABAergic neurons. Coronal sections of MAG1 high mice were subjected to dual immunofluorescence staining of FJB with NeuN, NMDAR1, and GAD67 at the anterior cingulate and somatomotor cortex. DAPI was used in order to identify nuclei (blue). Images of cells were obtained by bright-field light (fourth column). To visualize better the NMDAR1, GAD67, and FJB staining colocalized into the cell, we superimposed the bright-field images upon merged images of fluorescence (fifth column). (Upper panels) Double staining of NeuN (red) and FJB (green) showed that all FJB-positive cells were colocalized with NeuN (yellow fluorescence, arrows), confirming that the degenerating cells were neurons. (Middle panels) Double staining of NMDAR1 (red) and FJB (green) showed that most NMDAR1-stained glutamatergic neurons were colocalized with FJB (yellow arrows), though some were not (white arrows). (Lower panels) Double-labeling of GAD67 (red) and FJB (green) showed that most GAD67-stained GABAergic neurons were colocalized with FJB (yellow arrows), though some were not (white arrows). Scale bar = 20 μm for all panels. Images are representative of staining from 3 animals.
Both glutamatergic and GABAergic neurons display degeneration.There are two dominant families of neurons in the cortex, excitatory glutamatergic neurons and inhibitory GABAergic neurons. To ascertain whether Toxoplasma-induced neurodegeneration is cell type specific, neurons were double-fluorescence FJB stained with markers of glutamatergic or GABAergic neurons. Fluorescence immunohistochemistry was carried out for NMDA receptor 1 (NMDAR1), a marker of glutamatergic neurons, and glutamic acid decarboxylase 67 (GAD67), a marker of GABAergic neurons, as described previously (30, 31). Images of cells were obtained by bright-field light. In brains of MAG1 high mice, most NMDAR1-stained glutamatergic neurons were colocalized with FJB (Fig. 2, middle panels, yellow arrows), though some were not (white arrows). Similarly, FJB staining is present in many but not all GAD67-stained glutamatergic neurons (Fig. 2, lower panels). These results suggest that chronic Toxoplasma infection causes degeneration of both glutamatergic and GABAergic neurons.
Cyst-associated elevation of cerebral complement components.Recent evidence demonstrates that the classical complement system is active in various neurodegenerative and age-related diseases (3). Here, we evaluated whether the complement cascade is upregulated in brains when parasite-driven neurodegeneration was present. We measured levels of several complement components in the prefrontal cortex from Toxoplasma- and sham-infected mice, including initiators of the classical pathway C1q and C1r, the central component C3, which also initiates the alternative pathway, and the downstream complement component C4.
The mRNA levels of these complement components were substantially and significantly elevated in MAG1 high mice relative to levels in MAG1 low or control mice, as revealed by real-time reverse transcription-PCR (RT-PCR). As depicted in Fig. 3, mRNA expression was increased 3.8-fold for C1q, 7.8-fold for C1r, 40.2-fold for C3, and 15.9-fold for C4 in mice with high levels of MAG1 antibodies compared to those of controls (8 mice/group). We next measured complement proteins in homogenates of prefrontal cortex to confirm the increases found at the RNA level. In mouse brain, C1q antibodies specifically detected a band at 30 to ∼40 kDa (Fig. 4A, corresponding to the C1q A chain), C1r antibodies detected a band at 60 to ∼80 kDa (Fig. 4B), and C4 antibodies detected a band at 80 to ∼110 kDa (Fig. 4D). Using a monoclonal anti-C3 antibody, we detected the α-chain (115 kDa), the β-chain (70 kDa), and C3c alpha chain fragment 2 (43 kDa) (Fig. 4C). We found that these proteins were profoundly upregulated in MAG1 high mice, as evidenced by the 34-fold increase for C1qA, the 2.7- to ∼4.5-fold increase for C3 subtypes, and an increase from undetectable to detectable levels for C1r and C4, in a comparison with controls (4 mice/group). These protein results are highly consistent with the mRNA data.
Cyst-associated upregulation of cortical complement components. Outbred CD-1 mice were infected with a virulent type I strain of Toxoplasma for 5 months. mRNA levels of C1q, C1r, C3, and C4 were elevated in brains of MAG1 high mice compared to levels in MAG1 low and control mice (8 mice per group). The box and whisker plot represents the ΔΔCT distribution, the bottom and top of the box are the first and third quartiles, and the band inside the box is the median. The ends of the whiskers represent the minimum and maximum values of the data. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Cyst-associated elevation of cortical complement proteins. Outbred CD-1 mice were infected with a virulent type I strain of Toxoplasma for 5 months, and Western blot analysis of complement proteins C1q (A), C1r (B), C3 (C), and C4 (D) in cortical extracts from MAG1 high, MAG1 low, and control mice, alongside β-actin loading controls, was performed. Histograms indicate densitometric analysis of blots, expressed as means ± SEM. Analysis was performed in three independent experiments. The circles show individual values from one representative experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Complement C1q and C3 deposited on the surfaces of FJB-positive cells.Complement proteins C1q and C3 appear to be particularly important in tagging weak or unnecessary synapses for removal via microglial complement receptor-mediated phagocytosis (32). Given the activation of the complement cascade based on our RNA and protein studies, we next visualized the extent to which these complement components were deposited on degenerating neurons. Sections containing the FJB-positive cells were immunostained for C1q and C3 and analyzed by confocal microscopy. As shown in Fig. 5, we found that C1q and C3 staining were associated with FJB-positive cells, whereas deposition of C1q and C3 were detected on the surfaces of cell bodies (arrows) as well as along cell processes (arrowhead).
Confocal microscopy showed that complement proteins deposited on the surfaces of degenerative neurons. Coronal sections of MAG1 high mice were subjected to dual-immunofluorescence staining of FJB with complements C1q (A) and C3 (B). The staining of C1q (red) and C3 (red) was associated with FJB-positive (green) cells; deposition was detected on the cell bodies (arrows) as well as along cell processes (arrowhead). Scale bar = 10 μm for all panels.
Degenerating neurons were surrounded and infiltrated by activated microglia.Microglia are resident phagocytes in the brain that sense pathological tissue alterations, such as synapse loss and lesion. They have been recognized as key players in neurodegenerative lesions (4). To determine whether microglia are associated with cortical neurodegeneration, we performed FJB staining in combination with a marker of microglia, IBA1, and sections were imaged by confocal microscopy (Fig. 6D , E, and F represent Z-stacks of 3 confocal scans, with a total thickness of 5 μm). Microglial activation can be identified by changes in shape and density. In sham-infected animals, the prefrontal cortex contained a few resident microglia displaying small perikarya that projected thin processes (Fig. 6A). In contrast, the level of IBA1-labeled microglia at the site of neurodegeneration increased markedly in MAG1 high mice (Fig. 6D). Moreover, IBA1-positive cells displayed activated cell morphology showing enlarged perikarya and thicker processes (Fig. 6D). IBA1 and FJB coimmunostaining showed an intimate interrelationship between microglia and neurodegenerating cells. As shown in Fig. 6F, IBA1-positive microglia can be seen completely engulfing FJB-positive cells (arrows).
Confocal microscopy showed activated microglia surrounding degenerate neurons. Coronal sections of MAG1 high and control mice were subjected to dual-immunofluorescence staining of FJB (green) with IBA1 (red) at the anterior cingulate and somatomotor cortex. An increased microglial population density with activated cell morphology in MAG1 high mice was observed (E, arrows) compared to that of uninfected controls (B). The activated microglial cells were in close vicinity of degenerating neurons, embracing and engulfing them (F, arrows). Scale bar =100 μm (inset) or 20 μm for all panels.
Fractalkine (CX3CL1) increased significantly in degenerating neurons.It has been proposed that fractalkine (CX3CL1) signaling mediates the communication between neurons and microglia (26), because CX3CL1 is produced by neurons and its sole receptor is expressed on microglia. Given the elevated number of activated microglia at the site of neurodegeneration, we wondered whether CX3CL1 would be upregulated by degenerating neurons. In MAG1 high mice, cellular CX3CL1 was highly expressed compared to expression levels in sham-infected mice (Fig. S3). Neurons that positively stained with CX3CL1 colocalized with neurons that positively stained with FJB (Fig. 7), suggesting that neurons that undergo degeneration express high levels of CX3CL1.
CX3CL1 was upregulated and localized to degenerating neurons. Coronal sections of MAG1 high mice were subjected to dual-immunofluorescence staining of FJB (green) with CX3CL1 (red). Representative microscopy images showed that CX3CL1-positive cells were colocalized to the FJB-positive cells (merge), suggesting that neurons that are undergoing degeneration express high levels of CX3CL1. DAPI staining of nuclei (blue). Scale bar = 20 μm for all panels.
DISCUSSION
Synapse loss and neuroinflammation characterize many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis (3, 4). Toxoplasma has been implicated in several neurodegenerative diseases (33), but the mechanisms underlying such associations are not known. We set out to test the effect of chronic Toxoplasma infection on neuronal degeneration in mice and to explore the potential mechanisms that are involved. We show that high-grade chronic Toxoplasma infection caused neurodegeneration in specific regions of the prefrontal cortex, namely, the anterior cingulate cortex (ACC) and somatomotor cortex (SC). Both glutamatergic and GABAergic neurons were affected, indicating nonspecific neuropathology. Neurons that showed signs of degeneration expressed high levels of CX3CL1 and were tagged by complement proteins; both processes are known to recruit and activate nearby microglia. Indeed, activated microglia were amply present at sites of neurodegeneration and closely interacted with degenerate neurons. Our experimental mouse model of Toxoplasma infection simulated many aspects of the initiation and pathogenesis of neurodegenerative diseases and may provide a mechanistic explanation for the involvement of chronic Toxoplasma infection in neurodegenerative diseases.
Neurodegeneration, identified by FJB-labeled neurons, was present in MAG1 high mice but not in MAG1 low or MAG1-seronegative mice. This result suggests that parasite burden is a major determinant for neuronal degeneration, as we have previously shown that the MAG1 antibody response is a reliable measure of cyst burden (27). However, parasite antigens were not found in degenerating cells. Therefore, it is likely that neurodegeneration is an indirect effect of the parasite infection. Several studies found that pathogen burden was positively correlated with the degree of neuroinflammation. In a study reported by Evans et al. (23), activated microglia were found in all Toxoplasma-infected rats with cysts but not in Toxoplasma-infected rats without cysts. Using a murine model of human African trypanosomiasis, Rodgers et al. (34) found a concomitant increase in the degree of neuroinflammation as the infection progressed in the brain. Given that neuroinflammation has been implicated in the etiology of neurodegenerative diseases (18), the brain inflammation generated by the presence of cysts might be a critical factor driving neural degeneration. We did not detect neurodegeneration in mice with low-grade infection (MAG1 low), presumably due to an insufficient level of cyst-derived antigens; these antigens are needed to stimulate a robust inflammatory response. Our finding that different Toxoplasma serological profiles are associated with the presence or absence of neurodegenerative pathology may be important in interpreting the conflicting epidemiological studies reporting associations of Toxoplasma infection and neurodegenerative disorders (9, 11, 35, 36). In these studies, chronic Toxoplasma infection is often confirmed only by seropositivity to the whole organism, and the presence or burden of the organism is not determined.
Inflammatory processes that effectively control parasite reactivation are potential candidates triggering neurodegeneration; however, the molecular cascades leading to neurodegeneration are unclear. Evidence of an inflammatory response in Toxoplasma-infected brain includes activation of microglia and astrocyte, an increase in cytokines and chemokines (e.g., CXCL9, CXCL10, and CCL5), and recruitment of immune cells (22, 23, 37). Given that the infection lasted a long period of time (5 months postinfection) and has a chronic progressive course, we do not have data for the evolution of the CNS inflammatory profile. Therefore, inflammatory factors that initiate and cause the neurodegenerative processes to progress are difficult to identify. Indeed, how the inflammatory response affects neuronal populations and contributes to neurodegenerative disease in general remains a critical and unanswered question (38). We showed here that, after the neuron degenerated, activated microglia engaged in phagocytosis by engulfment of synapses, likely via a complement-dependent mechanism, as well as in response to receptor-mediated recognition of specific molecular tags at the synapses.
Degenerating neurons were prominently present in specific regions of the prefrontal cortex, including in the ACC and SC, two areas that are also prominently associated with cognitive function (39, 40). The reason for selective involvement of these regions is unclear. Although tissue cysts are randomly distributed throughout the brain, several studies have found that inflammation and parasite burden are greater in the cortex than in other areas of the brain (22, 23), which may provide an explanation for the particular targeting. In Alzheimer’s disease, neurons of the hippocampus and entorhinal cortex are the first to degenerate, whereas in Parkinson’s disease, dopaminergic neurons in the substantia nigra degenerate (18). Therefore, it is commonly recognized that different diseases affect different neuronal populations, though the mechanisms for their selective targeting remain poorly understood. It is of note that neurodegenerative diseases can progress over time, involving one brain region followed by another and then another (41). It is possible that Toxoplasma-induced degenerative pathology over time may involve other areas of the brain.
There are two dominant families of neurons in the cortex, GABAergic and glutamatergic neurons, which establish inhibitory and excitatory synapses, respectively. We found that both types of neurons exhibited degeneration. Previously, GABAergic and glutamatergic synapses and signaling have been reported to be altered upon Toxoplasma infection (14–16, 42). Neurons that show signs of degeneration can potentially lead to changes in neuronal connectivity and synaptic plasticity. As observed by previous studies, alterations of GABAergic signaling have been linked to seizures, Alzheimer’s disease, temporal lobe epilepsy, and schizophrenia (14). Altered glutamate signaling are linked to many neurological conditions, such as schizophrenia, Parkinson’s disease, cognition, addiction, anxiety, and depression (43). With respect to the pathology of neurodegenerative diseases, our findings support the involvement of Toxoplasma in these diseases.
We previously reported that levels of C1q are increased in the brains of MAG1 high mice and can be found as punctate patterns, which are indicative of synapses along neuronal cell processes (24). The present study extends these previous findings by showing that additional complement components (C1r/C3/C4) are activated and deposited on degenerative neurons. We demonstrated, at both the RNA and protein levels, a dramatic upregulation of components of the complement cascade in MAG1 high mice. Although the blood brain barrier normally protects the brain from plasma-derived complement, brain-resident neurons and glial cells, most often in response to injury or inflammatory signals, can locally synthesize many complement components (44, 45). We found that C1q and the downstream complement protein, C3, target degenerating neurons. Given complement’s well-ascribed role in synaptic pruning (3), the function of these immune molecules appears to be to mark dysfunctional neurons for removal.
There is significant microglia recruitment and activation at the site of neurodegeneration, possibly recruited by increased levels of the chemokine CX3CL1. As described before, CX3CL1 binds to the CX3CR1 receptor on microglia, a crucial receptor regulating microglial activation in the CNS and cellular communication between microglia and neurons (26). Microglial activation may also be triggered by upregulated complement C3, as C3 receptors are expressed on microglia (46). This binding promotes phagocytosis. Accumulating evidence suggests that microglia have a well-ascribed role in clearing out dying cells in the aged and diseased brain (47). Here, we found that microglial branches surrounded the degenerating neurons in an intimate manner, the purpose of which may be to remove degenerate neurons that are tagged by complement proteins and to control inflammatory processes. These results are in accordance with recent literature where complement-mediated phagocytosis is involved in the pathology of the ageing brain and neurodegenerative diseases (3, 4).
Our study suggests that neurodegenerative processes are involved in the pathogenesis of murine toxoplasmosis and may contribute to changes in neuronal connectivity, synaptic plasticity, and behavior. This mouse model raises questions of whether Toxoplasma can cause or contribute to degenerative pathology in the brains of humans. In our model, fractalkine was highly expressed by the degenerative neurons and played a role in microglial recruitment. However, whether this translates to humans is unclear, because the functions of the fractalkine/CX3CR1 axis differ in humans and mice (48). The link between Toxoplasma and mechanisms associated with neurodegenerative diseases is potentially important because this parasite infects approximately a billion people worldwide. Our result also suggests that decreasing antigen availability, through gradual resolution of infections or intervention strategies that promote parasite control, might help to alleviate or cure symptoms of neurodegeneration. In a recent study (49), we showed that immune checkpoint blockade directed against the programmed death 1 (PD-1) pathway results in a significant reduction in brain cyst number (77% lower), which offers a novel method to treat chronic toxoplasmosis. Another study reported increased expression of PD-L1 in brain tissue of Toxoplasma-infected mice (22).
MATERIALS AND METHODS
Chronic Toxoplasma type I infection.All mouse specimens were collected as part of a previously published study (27); no additional animals were sacrificed for the present study. As described before (27), 7- to 9-week-old female outbred CD-1 mice (ICR-Harlan Sprague) were infected intraperitoneally (i.p.) with 500 Toxoplasma GT1 strain tachyzoites (type I, virulent). Control mice received vehicle only (phosphate-buffered saline [PBS]). The type I strain was chosen for study in light of our previous epidemiological studies, where we found that virulent strains, such as those of type I, are associated with increased frequency and severity of human toxoplasmosis (50–52). To establish a chronic infection with this virulent strain, both control and infected mice were treated with anti-Toxoplasma chemotherapy (sulfadiazine sodium) in drinking water (400 mg/liter; Sigma) from days 5 to 30. Mice were sacrificed at 5 months postinfection. Brains were harvested and divided sagittally. Half of the brain was used for immunofluorescence staining, and the other half was used for dissection of the prefrontal cortex for mRNA and protein analysis.
This model generates varied outcomes ranging from aborted to severe infections, i.e., various degrees of cyst burden (27). Exposure to Toxoplasma was confirmed using serological measurement of anti-Toxoplasma antibodies with enzyme-linked immunosorbent assays (ELISAs) (IBL America). Cyst burden was measured with matrix antigen 1 (MAG1) antibody by using previously developed cyst MAG1 assays (27, 53). According to antibody profiles, mice were stratified into five groups: (i) non-Toxoplasma-exposed control mice (IgG−/MAG1−), (ii) exposed mice with a high MAG1 antibody level (IgG+/MAG1+ high [MAG1 level of ≥0.5]); (iii) exposed mice with a low MAG1 antibody level (IgG+/MAG1+ low [with a MAG1 level of <0.5]), (iv) exposed mice that did not develop MAG1 antibody (IgG+/MAG1−), and (v) exposed mice that did not develop an antibody response (IgG−/MAG1−).
Fluoro-Jade B staining.Fluoro-Jade B (FJB) is a fluorescent dye that specifically labels degenerating neurons (25). To perform FJB staining, paraffin-embedded specimens were cut into 5-μm sections. After deparaffinization and rehydration, the sections were immersed in a solution containing 0.06% potassium permanganate for 10 min with gentle shaking. Brain sections were then placed in a 0.0004% FJB solution for 20 min. After being washed in distilled water, the sections were mounted with prolong diamond antifade mountant (ThermoFisher Scientific).
Immunofluorescence staining.Immunohistochemistry for all antibodies was performed using standard protocols. Paraffin wax-embedded sections were cut coronally from the hemisphere at a thickness of 5 μm. Serial sections (3 mice per group; 5 sections per mouse) were treated in xylene and a graduated alcohol series and rinsed in distilled water. Antigen retrieval was performed by boiling the sections in antigen unmasking solution (Vector Laboratories; item H-3300) for 30 min. Sections were first blocked with 10% normal donkey serum for 30 min and then with 0.1 mg/ml Fab fragment donkey anti-mouse IgG (Jackson ImmunoResearch; item 715-007-003) for 1 h. Sections were stained overnight using the following primary antibodies: anti-IBA1 antibody (polyclonal, item 019-19741, 1:200; Wako), anti-C1q antibody (monoclonal, item ab71089, 1:100; Abcam), anti-C3 antibody (monoclonal, item ab225539, 1:2,000; Abcam), anti-CX3CL1 antibody (monoclonal, item ab25088, 1:500; Abcam), anti-GLUN2B antibody (monoclonal, item ab93610, 1:250; Abcam), anti-NeuN antibody (monoclonal, item ab104224, 1:1,000; Abcam), anti-GAD67 (monoclonal, item ab213508; Abcam), and anti-Toxoplasma (polyclonal, item PU125-5UP, 1:100; BioGenex). Double-fluorescence staining was performed for these markers in different combinations.
Secondary antibodies were purchased from Life Technologies, Inc. Finally, the sections were stained with DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich) and mounted with prolong diamond antifade mountant (ThermoFisher Scientific). Images were visualized using an Olympus BX41 microscope and a reflected fluorescence system. A confocal laser microscope (Zeiss LSM700) was used to verify the colocalization or intimate associations.
Immunoblot analyses.Total protein was extracted from the prefrontal cortex from the MAG1 high, MAG1 low, and control groups, as described before (54). In brief, prefrontal cortex was homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma) containing protease inhibitors, sonicated for 5 min at 4°C, and centrifuged for 5 min at 10,000 × g. Proteins were probed with primary antibodies for C1q (monoclonal, item ab71089, 1:1,000; Abcam), C1r (polyclonal, item ab205546, 1:2,000; Abcam), C3 (monoclonal, item ab225539, 1:1,000; Abcam), and C4 (polyclonal, item ab11863, 1:100; Abcam). Bands were visualized using enhanced chemiluminescence (ECL Prime Western blotting detection reagent; GE Healthcare Life Sciences). Protein values were normalized for corresponding values of β-actin. Relative optical densities were assessed using Scanalytics image analysis software (Bio-Rad).
Quantitative PCR.RNA was isolated from mouse prefrontal cortex from the MAG1 high, MAG1 low, and control groups, as described before (55). Quantitative PCR was performed according to the manufacturer’s protocol using inventoried TaqMan mRNA assays (Life Technologies), as previously described (56). Fold changes between groups were evaluated using relative quantification (ΔΔCT method, where CT is threshold cycle); β-actin was the endogenous control. We examined the mRNA expression of four complement proteins, including C1q (C1qa; TaqMan assay identifier Mm00432142_m1), C1r (Mm00517767_m1), C3 (Mm01232779_m1), and C4 (C4b; Mm00437893_g1). Quantitative PCR analyses were repeated at least three times to confirm expression levels, and only results that were consistent across all three analyses were considered valid.
Statistical analyses.Data are presented as means ± standard errors of the means (SEM). Analysis of variance (ANOVA) was performed to compare means between the three MAG1-designated groups by quantitative PCR and immunoblotting experiments. The Bonferroni multiple-comparison test was applied. Statistical analyses were conducted in Graph-Pad Prism V7.03. Significance was denoted as a P of <0.05.
ACKNOWLEDGMENT
This work was supported by the Stanley Medical Research Institute.
FOOTNOTES
- Received 19 February 2019.
- Returned for modification 5 April 2019.
- Accepted 31 May 2019.
- Accepted manuscript posted online 10 June 2019.
Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00139-19.
- Copyright © 2019 American Society for Microbiology.
