New role of sertraline against Toxoplasma gondii-induced depression-like behaviours in mice.

Toxoplasma gondii (T. gondii) is a neurotropic protozoan parasite, which can cause mental and behavioural disorders. The present study aimed to elucidate the effects and underlying molecular mechanisms of sertraline (SERT) on T. gondii-induced depression-like behaviours. In the present study, a mouse model and a microglial cell line (BV2 cells) model were established by infecting with the T. gondii RH strain. In in vivo and in vitro experiments, the underlying molecular mechanisms of SERT in inhibiting depression-like behaviours and cellular perturbations caused by T. gondii infection were investigated in the mouse brain and BV2 cells. The administration of SERT significantly ameliorated depression-like behaviours in T. gondii-infected mice. Furthermore, SERT inhibited T. gondii proliferation. Treatment with SERT significantly inhibited the activation of microglia and decreased levels of pro-inflammatory cytokines such as tumour necrosis factor-alpha, and interferon-gamma, by down-regulating tumour necrosis factor receptor 1/nuclear factor-kappa B signalling pathway, thereby ameliorating the depression-like behaviours induced by T. gondii infection. Our study provides insight into the underlying molecular mechanisms of the newly discovered role of SERT against T. gondii-induced depression-like behaviours.

Reduction of Amyloid Burden by Proliferated Homeostatic Microglia in Toxoplasma gondii-Infected Alzheimer's Disease Model Mice.

In this study, we confirmed that the number of resident homeostatic microglia increases during chronic Toxoplasma gondii infection. Given that the progression of Alzheimer's disease (AD) worsens with the accumulation of amyloid ß (Aß) plaques, which are eliminated through microglial phagocytosis, we hypothesized that T. gondii-induced microglial proliferation would reduce AD progression. Therefore, we investigated the association between microglial proliferation and Aß plaque burden using brain tissues isolated from 5XFAD AD mice (AD group) and T. gondii-infected AD mice (AD + Toxo group). In the AD + Toxo group, amyloid plaque burden significantly decreased compared with the AD group; conversely, homeostatic microglial proliferation, and number of plaque-associated microglia significantly increased. As most plaque-associated microglia shifted to the disease-associated microglia (DAM) phenotype in both AD and AD + Toxo groups and underwent apoptosis after the lysosomal degradation of phagocytosed Aß plaques, this indicates that a sustained supply of homeostatic microglia is required for alleviating Aß plaque burden. Thus, chronic T. gondii infection can induce microglial proliferation in the brains of mice with progressed AD; a sustained supply of homeostatic microglia is a promising prospect for AD treatment.

Molecular chaperone function of stress inducible Hsp70 is critical for intracellular multiplication of Toxoplasma gondii.

Intracellular pathogens like Toxoplasma gondii often target proteins and pathways critical for host cell survival and stress response. Molecular chaperones encoded by the evolutionary conserved Heat shock proteins (Hsps) maintain proteostasis and are vital to cell survival following exposure to any form of stress. A key protein of this family is Hsp70, an ATP-driven molecular chaperone, which is stress inducible and often indiscernible in normal cells. Role of this protein with respect to intracellular survival and multiplication of protozoan parasite like T. gondii remains to be examined. We find that T. gondii infection upregulates expression of host Hsp70. Hsp70 selective inhibitor 2-phenylethynesulfonamide (PES) attenuates intracellular T. gondii multiplication. Biotinylated PES confirms selective interaction of this small molecule inhibitor with Hsp70. We show that PES acts by disrupting Hsp70 chaperone function which leads to dysregulation of host autophagy. Silencing of host Hsp70 underscores its importance for intracellular multiplication of T. gondii, however, attenuation achieved using PES is not completely attributable to host Hsp70 indicating the presence of other intracellular targets of PES in infected host cells. We find that PES is also able to target T. gondii Hsp70 homologue which was shown using PES binding assay. Detailed molecular docking analysis substantiates PES targeting of TgHsp70 in addition to host Hsp70. While establishing the importance of protein quality control in infection, this study brings to the fore a unique opportunity of dual targeting of host and parasite Hsp70 demonstrating how structural conservation of these proteins may be exploited for therapeutic design.

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection.

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

Toxoplasma infection induces microglia-neuron contact and the loss of perisomatic inhibitory synapses.

Infection and inflammation within the brain induces changes in neuronal connectivity and function. The intracellular protozoan parasite, Toxoplasma gondii, is one pathogen that infects the brain and can cause encephalitis and seizures. Persistent infection by this parasite is also associated with behavioral alterations and an increased risk for developing psychiatric illness, including schizophrenia. Current evidence from studies in humans and mouse models suggest that both seizures and schizophrenia result from a loss or dysfunction of inhibitory synapses. In line with this, we recently reported that persistent T. gondii infection alters the distribution of glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes GABA synthesis in inhibitory synapses. These changes could reflect a redistribution of presynaptic machinery in inhibitory neurons or a loss of inhibitory nerve terminals. To directly assess the latter possibility, we employed serial block face scanning electron microscopy (SBFSEM) and quantified inhibitory perisomatic synapses in neocortex and hippocampus following parasitic infection. Not only did persistent infection lead to a significant loss of perisomatic synapses, it induced the ensheathment of neuronal somata by myeloid-derived cells. Immunohistochemical, genetic, and ultrastructural analyses revealed that these myeloid-derived cells included activated microglia. Finally, ultrastructural analysis identified myeloid-derived cells enveloping perisomatic nerve terminals, suggesting they may actively displace or phagocytose synaptic elements. Thus, these results suggest that activated microglia contribute to perisomatic inhibitory synapse loss following parasitic infection and offer a novel mechanism as to how persistent T. gondii infection may contribute to both seizures and psychiatric illness.

Toxoplasma Infection Induces Sustained Up-Regulation of Complement Factor B and C5a Receptor in the Mouse Brain via Microglial Activation: Implication for the Alternative Complement Pathway Activation and Anaphylatoxin Signaling in Cerebral Toxoplasmosis.

Toxoplasma gondii is a neurotropic protozoan parasite, which is linked to neurological manifestations in immunocompromised individuals as well as severe neurodevelopmental sequelae in congenital toxoplasmosis. While the complement system is the first line of host defense that plays a significant role in the prevention of parasite dissemination, Toxoplasma artfully evades complement-mediated clearance via recruiting complement regulatory proteins to their surface. On the other hand, the details of Toxoplasma and the complement system interaction in the brain parenchyma remain elusive. In this study, infection-induced changes in the mRNA levels of complement components were analyzed by quantitative PCR using a murine Toxoplasma infection model in vivo and primary glial cells in vitro. In addition to the core components C3 and C1q, anaphylatoxin C3a and C5a receptors (C3aR and C5aR1), as well as alternative complement pathway components properdin (CFP) and factor B (CFB), were significantly upregulated 2 weeks after inoculation. Two months post-infection, CFB, C3, C3aR, and C5aR1 expression remained higher than in controls, while CFP upregulation was transient. Furthermore, Toxoplasma infection induced significant increase in CFP, CFB, C3, and C5aR1 in mixed glial culture, which was abrogated when microglial activation was inhibited by pre-treatment with minocycline. This study sheds new light on the roles for the complement system in the brain parenchyma during Toxoplasma infection, which may lead to the development of novel therapeutic approaches to Toxoplasma infection-induced neurological disorders.

Transcriptome analysis of the effect of C-C chemokine receptor 5 deficiency on cell response to Toxoplasma gondii in brain cells.

BACKGROUND: Infection with Toxoplasma gondii is thought to damage the brain and be a risk factor for neurological and psychotic disorders. The immune response-participating chemokine system has recently been considered vital for brain cell signaling and neural functioning. Here, we investigated the effect of the deficiency of C-C chemokine receptor 5 (CCR5), which is previously reported to be associated with T. gondii infection, on gene expression in the brain during T. gondii infection and the relationship between CCR5 and the inflammatory response against T. gondii infection in the brain. RESULTS: We performed a genome-wide comprehensive analysis of brain cells from wild-type and CCR5-deficient mice. Mouse primary brain cells infected with T. gondii were subjected to RNA sequencing. The expression levels of some genes, especially in astrocytes and microglia, were altered by CCR5-deficiency during T. gondii infection, and the gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed an enhanced immune response in the brain cells. The expression levels of genes which were highly differentially expressed in vitro were also investigated in the mouse brains during the T. gondii infections. Among the genes tested, only Saa3 (serum amyloid A3) showed partly CCR5-dependent upregulation during the acute infection phase. However, analysis of the subacute phase showed that in addition to Saa3, Hmox1 may also contribute to the protection and/or pathology partly via the CCR5 pathway. CONCLUSIONS: Our results indicate that CCR5 is involved in T. gondii infection in the brain where it contributes to inflammatory responses and parasite elimination. We suggest that the inflammatory response by glial cells through CCR5 might be associated with neurological injury during T. gondii infection to some extent.

Assessment of Parasite-Microglia Interactions In Vitro.

An extensive number of parasites are able to invade the central nervous system (CNS) and cause a plethora of pathologies. Microglia, the resident macrophages of nervous tissue, are responsible for the protection against intruders, and therefore, they are an important line of defense against parasites. The phagocytosis is one of the weapons in the microglia's arsenal to fight against parasites. Several prior studies of microglia-parasite interactions and phagocytosis have been performed using microscopic techniques. As this methodology allows only a limited number of cells to be analyzed, additional approaches are required to provide a more complete picture of how microglia interact with these pathogens. Here, we describe a protocol based on flow cytometry to analyze single-celled parasites/microglia interactions in thousands of events in an accurate and reliable way. We use Trypanosoma brucei as a model organism, as it is a well-known parasite causing primary meningoencephalitis. However, the interaction/phagocytosis assay can be applied to other single-celled parasites as well.

Establishment of BV2 microglia polarization model and its effect on Toxoplasma gondii proliferation.

Toxoplasma gondii is an intracellular opportunistic, parasitic protozoan. Microglia have been classified into two main types: M1 (classically activated macrophages) and M2 (alternatively activated macrophages). BV2 cells were used in this study, together with lipopolysaccharide (LPS) and interferon (IFN)-γ or interleukin (IL)-4, which were used to induce resting microglia. Expression levels of M1/M2 markers were determined at both mRNA and protein levels, using PCR, western blot, and flow cytometry. Furthermore, cells were infected with T. gondii PLK strain, and the dynamic changes in M1/M2 marker expression levels were determined. An in vitro polarization model was successfully established. Expression of Nos2 and M1-associated markers was significantly upregulated at 12 h post-infection in BV2 cells. Further, the JAK/STAT1 and NF-κB signaling pathways were also activated following T. gondii infection. This demonstrated that T. gondii infection induces M1-type microglial polarization in vitro. The present study demonstrated that T. gondii infection affects microglial activation in vitro and elucidated the effects of activated microglia on T. gondii proliferation. This data may serve as a useful reference for more detailed elucidation of interactions between T. gondii and the innate immune system.

Neuronal impairment following chronic Toxoplasma gondii infection is aggravated by intestinal nematode challenge in an IFN-γ-dependent manner.

BACKGROUND: It has become increasingly evident that the immune and nervous systems are closely intertwined, relying on one another during regular homeostatic conditions. Prolonged states of imbalance between neural and immune homeostasis, such as chronic neuroinflammation, are associated with a higher risk for neural damage. Toxoplasma gondii is a highly successful neurotropic parasite causing persistent subclinical neuroinflammation, which is associated with psychiatric and neurodegenerative disorders. Little is known, however, by what means neuroinflammation and the associated neural impairment can be modulated by peripheral inflammatory processes. METHODS: Expression of immune and synapse-associated genes was assessed via quantitative real-time PCR to investigate how T. gondii infection-induced chronic neuroinflammation and associated neuronal alterations can be reshaped by a subsequent acute intestinal nematode co-infection. Immune cell subsets were characterized via flow cytometry in the brain of infected mice. Sulfadiazine and interferon-γ-neutralizing antibody were applied to subdue neuroinflammation. RESULTS: Neuroinflammation induced by T. gondii infection of mice was associated with increased microglia activation, recruitment of immune cells into the brain exhibiting Th1 effector functions, and enhanced production of Th1 and pro-inflammatory molecules (IFN-γ, iNOS, IL-12, TNF, IL-6, and IL-1ß) following co-infection with Heligmosomoides polygyrus. The accelerated cerebral Th1 immune response resulted in enhanced T. gondii removal but exacerbated the inflammation-related decrease of synapse-associated gene expression. Synaptic proteins EAAT2 and GABAAα1, which are involved in the excitation/inhibition balance in the CNS, were affected in particular. These synaptic alterations were partially recovered by reducing neuroinflammation indirectly via antiparasitic treatment and especially by application of IFN-γ-neutralizing antibody. Impaired iNOS expression following IFN-γ neutralization directly affected EAAT2 and GABAAα1 signaling, thus contributing to the microglial regulation of neurons. Besides, reduced CD36, TREM2, and C1qa gene expression points toward inflammation induced synaptic pruning as a fundamental mechanism. CONCLUSION: Our results suggest that neuroimmune responses following chronic T. gondii infection can be modulated by acute enteric nematode co-infection. While consecutive co-infection promotes parasite elimination in the CNS, it also adversely affects gene expression of synaptic proteins, via an IFN-γ-dependent manner.

The impairment in the NLRP3-induced NO secretion renders astrocytes highly permissive to T. cruzi replication.

Trypanossoma cruzi (T. cruzi), the causative protozoan of Chagas disease (CD) invades many cell types, including central nervous system (CNS) cells triggering local lesions and neurological impact. Previous work from our group described NLRP3 inflammasomes as central effectors for the parasite control by macrophages. Recent evidences demonstrate that NLRP3 can be activated in CNS cells with controversial consequences to the control of infections and inflammatory pathologies. However, the relative contribution of NLRP3 in different cell types remains to be elucidated. In this article, we described an effector response mediated by NLRP3 that works on microglia but not on astrocytes to control T. cruzi infection. Despite T. cruzi ability to invade astrocytes and microglia, astrocytes were clearly more permissive to parasite replication. Moreover, the absence of NLRP3 renders microglia but not astrocytes more permissive to T. cruzi replication. In fact, microglia but not astrocytes were able to secrete NLRP3-dependent IL-1ß and NO in response to T. cruzi. Importantly, the pharmacological inhibition of iNOS with aminoguanidine resulted in a significant increase in the numbers of amastigotes found in microglia from wild-type but not from NLRP3-/- mice, indicating the importance of NLRP3-mediated NO secretion to the infection control by these cells. Taken together, our findings revealed that T. cruzi differentially activates NLRP3 inflammasomes in astrocytes and microglia and established a role for these platforms in the control of a protozoan infection by glial cells from CNS.

Cerebral inducible nitric oxide synthase protein expression in microglia, astrocytes and neurons in Trypanosoma brucei brucei-infected rats.

To study the anatomo-biochemical substrates of brain inflammatory processes, Wistar male rats were infected with Trypanosoma brucei brucei. With this reproducible animal model of human African trypanosomiasis, brain cells (astrocytes, microglial cells, neurons) expressing the inducible nitric oxide synthase (iNOS) enzyme were revealed. Immunohistochemistry was achieved for each control and infected animal through eight coronal brain sections taken along the caudorostral axis of the brain (brainstem, cerebellum, diencephalon and telencephalon). Specific markers of astrocytes (anti-glial fibrillary acidic protein), microglial cells (anti-integrin alpha M) or neurons (anti-Neuronal Nuclei) were employed. The iNOS staining was present in neurons, astrocytes and microglial cells, but not in oligodendrocytes. Stained astrocytes and microglial cells resided mainly near the third cavity in the rostral part of brainstem (periaqueductal gray), diencephalon (thalamus and hypothalamus) and basal telencephalon. Stained neurons were scarce in basal telencephalon, contrasting with numerous iNOS-positive neuroglial cells. Contrarily, in dorsal telencephalon (neocortex and hippocampus), iNOS-positive neurons were plentiful, contrasting with the marked paucity of labelled neuroglial (astrocytes and microglial) cells. The dual distribution between iNOS-labelled neuroglial cells and iNOS-labelled neurons is a feature that has never been described before. Functionalities attached to such a divergent distribution are discussed.

Toxoplasma-Induced Hypermigration of Primary Cortical Microglia Implicates GABAergic Signaling.

Toxoplasma gondii is a widespread obligate intracellular parasite that causes chronic infection and life-threatening acute infection in the central nervous system. Previous work identified Toxoplasma-infected microglia and astrocytes during reactivated infections in mice, indicating an implication of glial cells in acute toxoplasmic encephalitis. However, the mechanisms leading to the spread of Toxoplasma in the brain parenchyma remain unknown. Here, we report that, shortly after invasion by T. gondii tachyzoites, parasitized microglia, but not parasitized astrocytes, undergo rapid morphological changes and exhibit dramatically enhanced migration in 2-dimensional and 3-dimensional matrix confinements. Interestingly, primary microglia secreted the neurotransmitter γ-aminobutyric acid (GABA) in the supernatant as a consequence of T. gondii infection but not upon stimulation with LPS or heat-inactivated T. gondii. Further, microglia transcriptionally expressed components of the GABAergic machinery, including GABA-A receptor subunits, regulatory molecules and voltage-dependent calcium channels (VDCCs). Further, their transcriptional expression was modulated by challenge with T. gondii. Transcriptional analysis indicated that GABA was synthesized via both, the conventional pathway (glutamate decarboxylases GAD65 and GAD67) and a more recently characterized alternative pathway (aldehyde dehydrogenases ALDH2 and ALDH1a1). Pharmacological inhibitors targeting GABA synthesis, GABA-A receptors, GABA-A regulators and VDCC signaling inhibited Toxoplasma-induced hypermotility of microglia. Altogether, we show that primary microglia express a GABAergic machinery and that T. gondii induces hypermigration of microglia in a GABA-dependent fashion. We hypothesize that migratory activation of parasitized microglia by Toxoplasma may promote parasite dissemination in the brain parenchyma.

Morphological changes, nitric oxide production, and phagocytosis are triggered in vitro in microglia by bloodstream forms of Trypanosoma brucei.

The flagellated parasite Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT). By a mechanism not well understood yet, trypanosomes enter the central nervous system (CNS), invade the brain parenchyma, and cause a fatal encephalopathy if is not treated. Trypanosomes are fast dividing organisms that, without any immune response, would kill the host in a short time. However, infected individuals survive either 6-12 months or more than 3 years for the acute and chronic forms, respectively. Thus, only when the brain defense collapses a lethal encephalopathy will occur. Here, we evaluated interactions between trypanosomes and microglial cells, which are the primary immune effector cells within the CNS. Using co-cultures of primary microglia and parasites, we found clear evidences of trypanosome phagocytosis by microglial cells. Microglia activation was also evident; analysis of its ultrastructure showed changes that have been reported in activated microglia undergoing oxidative stress caused by infections or degenerative diseases. Accordingly, an increase of the nitric oxide production was detected in supernatants of microglia/parasite co-cultures. Altogether, our results demonstrate that microglial cells respond to the presence of the parasite, leading to parasite's engulfment and elimination.

Granulocyte macrophage colony-stimulating factor alone reduces Toxoplasma gondii replication in microglial culture by superoxide and nitric oxide, without IFN-γ production: a preliminary report.

In vitro studies have demonstrated that GM-CSF in combination with other stimulatory factors induces a microbicidal response that control T. gondii infection. We assessed whether GM-CSF alone can control T. gondii replication in murine microglial cultures. Microglia were collected and cultured with or without GM-CSF and the half of each group was infected with T. gondii. We determined the T. gondii infectivity, cytokines levels, NO and superoxide detection. GM-CSF alone primes microglia, which after infection induces the production of TNF-α and IL-6, leading to NO and superoxide production, without any stimulus from IL-12p70 and IFN-γ.

Investigation of tissue cysts in the retina in a mouse model of ocular toxoplasmosis: distribution and interaction with glial cells.

The conversion of tachyzoites into bradyzoites is a way for Toxoplasma gondii to establish a chronic and asymptomatic infection and achieve lifelong persistence in the host. The bradyzoites form tissue cysts in the retina, but not much is known about the horizontal distribution of the cysts or their interactions with glial cells in the retina. A chronic ocular toxoplasmosis model was induced by per oral administration of T. gondii Me49 strain cysts to BALB/c mice. Two months after the infection, retinas were flat-mounted and immunostained to detect cysts, ganglion cells, Müller cells, astrocytes, and microglial cells, followed by observation under fluorescence and confocal microscope. The horizontal distribution showed a rather clustered pattern, but the clusters were not restricted to certain location of the retina. Axial distribution was confined to the inner retina, mostly in ganglion cell layer or the inner plexiform layer. Both ganglion cells, a type of retinal neurons, and Müller cells, predominant retinal glial cells, could harbor cysts. The cysts were spatially separated from astrocytes, the most abundant glial cells in the ganglion cell layer, while close spatial distribution of microglial cells was observed in two thirds of retinal cysts. In this study, we demonstrated that the retinal cysts were not evenly distributed horizontally and were confined to the inner retina axially. Both neurons and one type of glial cells could harbor cysts, and topographic analysis of other glial cells suggests role of microglial cells in chronic ocular toxoplasmosis.

AAH2 gene is not required for dopamine-dependent neurochemical and behavioral abnormalities produced by Toxoplasma infection in mouse.

Infection with the protozoan parasite, Toxoplasma gondii (T. gondii), has been associated with the increased risk for several psychiatric disorders. The exact mechanisms of a hypothesized contribution of T. gondii infection are poorly understood. The T. gondii genome contains two aromatic amino acid hydroxylase genes (AAH1 and AAH2) that encode proteins that can produce L-DOPA. One popular hypothesis posits that these encoded enzymes might influence dopamine (DA) production and hence DA synaptic transmission, leading to neurobehavioral abnormalities in the infected host. Prior studies have shown that deletion of these genes does not alter DA levels in the brain or exploratory activity in infected mice. However, possible effects of AAH gene deficiency on infection-induced brain and behavior alterations that are directly linked to DA synaptic transmission have not been evaluated. We found that chronic T. gondii infection of BALB/c mice leads to blunted response to amphetamine or cocaine and decreased expression of Dopamine Transporter (DAT) and Vesicular Monoamine Transporter 2 (VMAT2). Deletion of AAH2 had no effects on these changes in infected mice. Both wild type and Δaah2 strains produced comparable levels of neuroinflammation. Our findings demonstrate that AAH2 is not required for T. gondii infection-produced DA-dependent neurobehavioral abnormalities.

Characteristics of Infection Immunity Regulated by Toxoplasma gondii to Maintain Chronic Infection in the Brain.

To examine the immune environment of chronic Toxoplasma gondii infection in the brain, the characteristics of infection-immunity (premunition) in infection with T. gondii strain ME49 were investigated for 12 weeks postinfection (PI). The results showed that neuronal cell death, microglia infiltration and activation, inflammatory and anti-inflammatory cytokine expression, Stat1 phosphorylation, and microglia activation and inflammatory gene transcripts related to M1 polarization in the brain were increased during the acute infection (AI) stage (within 6 weeks PI), suggesting that innate and cellular inflammatory response activation and neurodegeneration contributed to excessive inflammatory responses. However, these immune responses decreased during the chronic infection (CI) stage (over 6 weeks PI) with reductions in phosphorylated STAT1 (pSTAT1) and eosinophilic neurons. Notably, increases were observed in transcripts of T-cell exhaustion markers (TIM3, LAG3, KLRG1, etc.), suppressor of cytokines signaling 1 protein (SOCS1), inhibitory checkpoint molecules (PD-1 and PD-L1), and Arg1 from the AI stage (3 weeks PI), implying active immune intervention under the immune environment of M1 polarization of microglia and increases in inflammatory cytokine levels. However, when BV-2 microglia were stimulated with T. gondii lysate antigens (strain RH or ME49) in vitro, nitrite production increased and urea production decreased. Furthermore, when BV-2 cells were infected by T. gondii tachyzoites (strain RH or ME49) in vitro, nitric oxide synthase and COX-2 levels decreased, whereas Arg1 levels significantly increased. Moreover, Arg1 expression was higher in ME49 infection than in RH infection, whereas nitrite production was lower in ME49 infection than in RH infection. Accordingly, these results strongly suggest that immune triggering of T. gondii antigens induces M1 polarization and activation of microglia as well as increase NO production, whereas T. gondii infection induces the inhibition of harmful inflammatory responses, even with M1 polarization and activation of microglia and Th1 inflammatory responses, suggesting a host-parasite relationship through immune regulation during CI. This is a characteristic of infection immunity in infection with T. gondii in the central nervous system, and SOCS1, a negative regulator of toxoplasmic encephalitis, may play a role in the increase in Arg1 levels to suppress NO production.

Apoptosis and necroptosis of mouse hippocampal and parenchymal astrocytes, microglia and neurons caused by Angiostrongylus cantonensis infection.

BACKGROUND: Angiostrongylus cantonensis has been the only parasite among Angiostrongylidae to cause human central nervous system infection characterized by eosinophilic meningitis or meningoencephalitis. The mechanism of the extensive neurological impairments of hosts caused by A. cantonensis larvae remains unclear. The aim of the present study was to investigate apoptosis, necroptosis and autophagy in the brains of mice infected with A. cantonensis, which will be valuable for better understanding the pathogenesis of angiostrongyliasis cantonensis. METHODS: Functional and histological neurological impairments of brain tissues from mice infected with A. cantonensis were measured by the Morris water maze test and haematoxylin and eosin (H&E) staining, respectively. The transcriptional and translational levels of apoptosis-, necroptosis- and autophagy-related genes were quantified by quantitative real-time polymerase chain reaction (RT-PCR), and assessed by western blot and immunohistochemistry (IHC) analysis. Apoptotic and necroptotic cells and their distributions in infected brain tissues were analysed by flow cytometry and transmission electron microscopy (TEM). RESULTS: Inflammatory response in the central nervous system deteriorated as A. cantonensis infection evolved, as characterized by abundant inflammatory cell infiltration underneath the meninges, which peaked at 21 days post-infection (dpi). The learning and memory capacities of the mice were significantly decreased at 14 dpi, indicating prominent impairment of their cognitive functions. Compared with those of the control group, the mRNA levels of caspase-3, -4, -6, and RIP3 and the protein levels of caspase-4, cleaved caspase-3, cleaved caspase-6, RIP3, and pRIP3 were obviously elevated. However, no changes in the mRNA or protein levels of FADD, Beclin-1 or LC3B were evident, indicating that apoptosis and necroptosis, but not autophagy, occurred in the brain tissues of mice infected with A. cantonensis. The quantitative RT-PCR, western blot, IHC, flow cytometry and TEM results further revealed the apoptotic and necroptotic microglia, astrocytes and neurons in the parenchymal and hippocampal regions of infected mice. CONCLUSIONS: To our knowledge, we showed for the first time that A. cantonensis infection causes the apoptosis and necroptosis of microglia and astrocytes in the parenchymal and hippocampal regions of host brain tissues, further demonstrating the pathogenesis of A. cantonensis infection and providing potential therapeutic targets for the management of angiostrongyliasis.

Uptake of parasite-derived vesicles by astrocytes and microglial phagocytosis of infected erythrocytes may drive neuroinflammation in cerebral malaria.

Astrocytes and microglia are activated during cerebral malaria (CM) and contribute to the production and release of several mediators during neuroinflammatory processes. Whether these changes are the consequence of a direct crosstalk between glial cells and the malarial parasite and how these cells participate in the pathogenesis of CM is not yet clear. We therefore examined the interaction of astrocytes and microglia with Plasmodium berghei ANKA-infected red blood cells using primary cell cultures derived from newborn C57BL/6 mice. We observed a dynamic transfer of vesicles from the parasite to astrocytes within minutes of contact, and the phagocytosis of infected red blood cells by microglia. Differential gene expression studies using the Affymetrix GeneChip® microarray, and quantitative PCR analyses showed the increase in expression of the set of genes belonging to the immune response network in parasite activated astrocytes and microglia. Interestingly, expression of these genes was also significantly upregulated in brains of mice dying from CM compared with uninfected mice or infected mice that did not develop the neuropathology. Accumulation of parasite-derived vesicles within astrocytes, and the phagocytosis of infected red blood cells by microglia induced a subsequent increase in interferon gamma inducible protein 10 (IP10) in both the brain and plasma of infected mice at the onset of CM, confirming a role for this molecule in CM pathogenesis. Altogether, these observations point to a possible role for glial cells in the neuropathological processes leading to CM. GLIA 2016 GLIA 2017;65:75-92.