Regulation of Proinflammatory Enzymes by Peroxisome Proliferator-Activated Receptor Gamma in Astroglia Infected with Toxoplasma gondii.

Peroxisome proliferator-activated receptor gamma (PPARγ) regulates neuroinflammation, and its agonists act as neuroprotective agents. This study aims to investigate the correlation between PPARγ and proinflammatory enzyme expression in astroglia infected with Toxoplasma gondii tachyzoite in vitro. Our results showed that matrix metalloprotease (MMP)-2, MMP-9, cyclooxygenase-2 (COX-2), prostaglandin (PGE)-2, inducible nitric-oxide synthase (iNOS), and nitric oxide (NO) were significantly increased in T. gondii-infected astroglia. Furthermore, the expression levels of MMP-2, MMP-9, COX-2, PGE-2, iNOS, and NO were significantly decreased by rosiglitazone-a PPARγ agonist. By contrast, the treatment with GW9662, a PPARγ antagonist, efficiently increased the expression levels of MMP-2, MMP-9, COX-2, PGE-2, iNOS, and NO. These results suggested that the treatment with rosiglitazone offers a potential strategy for controlling the inflammatory factors in T. gondii infection.

The excretory/secretory products of fifth-stage larval Angiostrongylus cantonensis induces autophagy via the Sonic hedgehog pathway in mouse brain astrocytes.

Angiostrongyliasis is induced by the nematode Angiostrongylus cantonensis and leads to eosinophilic meningitis and meningoencephalitis in humans. Excretory-secretory products (ESPs) are important investigation targets for studying the relationship between hosts and nematodes. These products assist worms in penetrating the blood-brain barrier and avoiding the host immune response. Autophagy is a catabolic process that is responsible for digesting cytoplasmic organelles, proteins, and lipids and removing them through lysosomes. This process is essential to cell survival and homeostasis during nutritional deficiency, cell injury and stress. In this study, we investigated autophagy induction upon treatment with the ESPs of the fifth-stage larvae (L5) of A. cantonensis and observed the relationship between autophagy and the Shh pathway. First, the results showed that A. cantonensis infection induced blood-brain barrier dysfunction and pathological changes in the brain. Moreover, A. cantonensis L5 ESPs stimulated autophagosome formation and the expression of autophagy molecules, such as LC3B, Beclin, and p62. The data showed that upon ESPs treatment, rapamycin elevated cell viability through the activation of the autophagy mechanism in astrocytes. Finally, we found that ESPs induced the activation of the Sonic hedgehog (Shh) signaling pathway and that the expression of autophagy molecules was increased through the Shh signaling pathway. Collectively, these results suggest that A. cantonensis L5 ESPs stimulate autophagy through the Shh signaling pathway and that autophagy has a protective effect in astrocytes.

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.

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.

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.

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.

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.

Stimulation of IL-1ß and IL-6 through NF-κB and sonic hedgehog-dependent pathways in mouse astrocytes by excretory/secretory products of fifth-stage larval Angiostrongylus cantonensis.

BACKGROUND: Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. Previous studies have shown that the Sonic hedgehog (Shh) signaling pathway may reduce cell apoptosis by inhibiting oxidative stress in A. cantonensis infection. In this study, we investigated the relationship between cytokine secretion and Shh pathway activation after treatment with excretory/secretory products (ESP) of fifth-stage larval A. cantonensis (L5). RESULTS: The results showed that IL-1ß and IL-6 levels in mouse astrocytes were increased. Moreover, ESP stimulated the protein expression of Shh pathway molecules, including Shh, Ptch, Smo and Gli-1, and induced IL-1ß and IL-6 secretion. The transcription factor nuclear factor-κB (NF-κB) plays an important role in inflammation, and it regulates the expression of proinflammatory genes, including cytokines and chemokines, such as IL-1ß and TNF-α. After ESP treatment, NF-κB induced IL-1ß and IL-6 secretion in astrocytes by activating the Shh signaling pathway. CONCLUSIONS: Overall, the data presented in this study showed that ESP of fifth-stage larval A. cantonensis stimulates astrocyte activation and cytokine generation through NF-κB and the Shh signaling pathway.

Astrocyte Apoptosis and HIV Replication Are Modulated in Host Cells Coinfected with Trypanosoma cruzi.

The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease. In immunosuppressed individuals, as it occurs in the coinfection with human immunodeficiency virus (HIV), the central nervous system may be affected. In this regard, reactivation of Chagas disease is severe and often lethal, and it accounts for meningoencephalitis. Astrocytes play a crucial role in the environment maintenance of healthy neurons; however, they can host HIV and T. cruzi. In this report, human astrocytes were infected in vitro with both genetically modified-pathogens to express alternative fluorophore. As evidenced by fluorescence microscopy and flow cytometry, HIV and T. cruzi coexist in the same astrocyte, likely favoring reciprocal interactions. In this context, lower rates of cell death were observed in both T. cruzi monoinfected-astrocytes and HIV-T. cruzi coinfection in comparison with those infected only with HIV. The level of HIV replication is significantly diminished under T. cruzi coinfection, but without affecting the infectivity of the HIV progeny. This interference with viral replication appears to be related to the T. cruzi multiplication rate or its increased intracellular presence but does not require their intracellular cohabitation or infected cell-to-cell contact. Among several Th1/Th2/Th17 profile-related cytokines, only IL-6 was overexpressed in HIV-T. cruzi coinfection exhibiting its cytoprotective role. This study demonstrates that T. cruzi and HIV are able to coinfect astrocytes thus altering viral replication and apoptosis.

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.

STAT1 Signaling in Astrocytes Is Essential for Control of Infection in the Central Nervous System.

The local production of gamma interferon (IFN-γ) is important to control Toxoplasma gondii in the brain, but the basis for these protective effects is not fully understood. The studies presented here reveal that the ability of IFN-γ to inhibit parasite replication in astrocytes in vitro is dependent on signal transducer and activator of transcription 1 (STAT1) and that mice that specifically lack STAT1 in astrocytes are unable to limit parasite replication in the central nervous system (CNS). This susceptibility is associated with a loss of antimicrobial pathways and increased cyst formation in astrocytes. These results identify a critical role for astrocytes in limiting the replication of an important opportunistic pathogen. IMPORTANCE: Astrocytes are the most numerous cell type in the brain, and they are activated in response to many types of neuroinflammation, but their function in the control of CNS-specific infection is unclear. The parasite Toxoplasma gondii is one of the few clinically relevant microorganisms that naturally infects astrocytes, and the studies presented here establish that the ability of astrocytes to inhibit parasite replication is essential for the local control of this opportunistic pathogen. Together, these studies establish a key role for astrocytes as effector cells and in the coordination of many aspects of the protective immune response that operates in the brain.

Nitric oxide and cytokine production by glial cells exposed in vitro to neuropathogenic schistosome Trichobilharzia regenti.

BACKGROUND: Helminth neuroinfections represent a serious health problem, but host immune mechanisms in the nervous tissue often remain undiscovered. This study aims at in vitro characterization of the response of murine astrocytes and microglia exposed to Trichobilharzia regenti which is a neuropathogenic schistosome migrating through the central nervous system of vertebrate hosts. Trichobilharzia regenti infects birds and mammals in which it may cause severe neuromotor impairment. This study was focused on astrocytes and microglia as these are immunocompetent cells of the nervous tissue and their activation was recently observed in T. regenti-infected mice. RESULTS: Primary astrocytes and microglia were exposed to several stimulants of T. regenti origin. Living schistosomulum-like stages caused increased secretion of IL-6 in astrocyte cultures, but no changes in nitric oxide (NO) production were noticed. Nevertheless, elevated parasite mortality was observed in these cultures. Soluble fraction of the homogenate from schistosomulum-like stages stimulated NO production by both astrocytes and microglia, and IL-6 and TNF-α secretion in astrocyte cultures. Similarly, recombinant cathepsins B1.1 and B2 triggered IL-6 and TNF-α release in astrocyte and microglia cultures, and NO production in astrocyte cultures. Stimulants had no effect on production of anti-inflammatory cytokines IL-10 or TGF-ß1. CONCLUSIONS: Both astrocytes and microglia are capable of production of NO and proinflammatory cytokines IL-6 and TNF-α following in vitro exposure to various stimulants of T. regenti origin. Astrocytes might be involved in triggering the tissue inflammation in the early phase of T. regenti infection and are proposed to participate in destruction of migrating schistosomula. However, NO is not the major factor responsible for parasite damage. Both astrocytes and microglia can be responsible for the nervous tissue pathology and maintaining the ongoing inflammation since they are a source of NO and proinflammatory cytokines which are released after exposure to parasite antigens.

Neurons are the Primary Target Cell for the Brain-Tropic Intracellular Parasite Toxoplasma gondii.

Toxoplasma gondii, a common brain-tropic parasite, is capable of infecting most nucleated cells, including astrocytes and neurons, in vitro. Yet, in vivo, Toxoplasma is primarily found in neurons. In vitro data showing that interferon-γ-stimulated astrocytes, but not neurons, clear intracellular parasites suggest that neurons alone are persistently infected in vivo because they lack the ability to clear intracellular parasites. Here we test this theory by using a novel Toxoplasma-mouse model capable of marking and tracking host cells that directly interact with parasites, even if the interaction is transient. Remarkably, we find that Toxoplasma shows a strong predilection for interacting with neurons throughout CNS infection. This predilection remains in the setting of IFN-γ depletion; infection with parasites resistant to the major mechanism by which murine astrocytes clear parasites; or when directly injecting parasites into the brain. These findings, in combination with prior work, strongly suggest that neurons are not incidentally infected, but rather they are Toxoplasma's primary in vivo target.

Cutting Edge: IFN-γ Produced by Brain-Resident Cells Is Crucial To Control Cerebral Infection with Toxoplasma gondii.

In vitro studies demonstrated that microglia and astrocytes produce IFN-γ in response to various stimulations, including LPS. However, the physiological role of IFN-γ production by brain-resident cells, including glial cells, in resistance against cerebral infections remains unknown. We analyzed the role of IFN-γ production by brain-resident cells in resistance to reactivation of cerebral infection with Toxoplasma gondii using a murine model. Our study using bone marrow chimeric mice revealed that IFN-γ production by brain-resident cells is essential for upregulating IFN-γ-mediated protective innate immune responses to restrict cerebral T. gondii growth. Studies using a transgenic strain that expresses IFN-γ only in CD11b(+) cells suggested that IFN-γ production by microglia, which is the only CD11b(+) cell population among brain-resident cells, is able to suppress the parasite growth. Furthermore, IFN-γ produced by brain-resident cells is pivotal for recruiting T cells into the brain to control the infection. These results indicate that IFN-γ produced by brain-resident cells is crucial for facilitating both the protective innate and T cell-mediated immune responses to control cerebral infection with T. gondii.

Interferon-gamma promotes infection of astrocytes by Trypanosoma cruzi.

The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ+ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.

Persistence of Toxoplasma gondii in the central nervous system: a fine-tuned balance between the parasite, the brain and the immune system.

Upon infection of humans and animals with Toxoplasma gondii, the parasites persist as intraneuronal cysts that are controlled, but not eliminated by the immune system. In particular, intracerebral T cells are crucial in the control of T. gondii infection and are supported by essential functions from other leukocyte populations. Additionally, brain-resident cells including astrocytes, microglia and neurons contribute to the intracerebral immune response by the production of cytokines, chemokines and expression of immunoregulatory cell surface molecules, such as major histocompatibility (MHC) antigens. However, the in vivo behaviour of these individual cell populations, specifically their interaction during cerebral toxoplasmosis, remains to be elucidated. We discuss here what is known about the function of T cells, recruited myeloid cells and brain-resident cells, with particular emphasis on the potential cross-regulation of these cell populations, in governing cerebral toxoplasmosis.

Altering the motility of Trypanosoma cruzi with rabbit polyclonal anti-peptide antibodies reduces infection to susceptible mammalian cells.

Trypanosoma cruzi's trypomastigotes are highly active and their incessant motility seems to be important for mammalian host cell infection. The kinetoplastid membrane protein-11 (KMP-11) is a protein expressed in all parasite stages, which induces a cellular and humoral immune response in the infected host, and is hypothesized to participate in the parasite's motility. An N-terminal peptide from KMP-11, termed K1 or TcTLE, induced polyclonal antibodies that inhibit parasitic invasion of Vero cells. The goal of this study was to evaluate the motility and infectivity of T. cruzi when exposed to polyclonal anti-TcTLE antibodies. Rabbits were immunized with TcTLE peptide along with FIS peptide as an immunomodulator. ELISA assay results showed that post-immunization sera contained high titers of polyclonal anti-TcTLE antibodies, which were also reactive against the native KMP-11 protein and live parasites as detected by immunofluorescence and flow cytometry assays. Trypomastigotes of T. cruzi were incubated with pre- or post-immunization sera, and infectivity to human astrocytes was assessed by Giemsa staining/light microscope and flow cytometry using carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled parasites. T. cruzi infection in astrocytes decreased approximately by 30% upon incubation with post-immunization sera compared with pre-immunization sera. Furthermore, trypomastigotes were recorded by video microscopy and the parasite's flagellar speed was calculated by tracking the flagella. Trypomastigotes exposed to post-immunization sera had qualitative alterations in motility and significantly slower flagella (45.5 µm/s), compared with those exposed to pre-immunization sera (69.2 µm/s). In summary, polyclonal anti-TcTLE serum significantly reduced the parasite's flagellar speed and cell infectivity. These findings support that KMP-11 could be important for parasite motility, and that by targeting its N-terminal peptide infectivity can be reduced.

Electron microscopic features of brain edema in rodent cerebral malaria in relation to glial fibrillary acidic protein expression.

The mechanisms leading to cerebral malaria (CM) are not completely understood. Brain edema has been suggested as having an important role in experimental CM. In this study, CBA/CaH mice were infected with Plasmodium berghei ANKA blood-stage and when typical symptoms of CM developed on day 7, brain tissues were processed for electron-microscopic and immunohistochemical studies. The study demonstrated ultrastructural hallmarks of cerebral edema by perivascular edema and astroglial dilatation confirming existing evidence of vasogenic and cytogenic edema. This correlates closely with the clinical features of CM. An adaptive response of astrocytic activity, represented by increasing glial fibrillary acidic protein (GFAP) expression in the perivascular area and increasing numbers of large astrocyte clusters were predominately found in the CM mice. The presence of multivesicular and lamellar bodies indicates the severity of cerebral damage in experimental CM. Congestion of the microvessels with occluded white blood cells (WBCs), parasitized red blood cells (PRBCs) and platelets is also a crucial covariate role for CM pathogenesis.

Astrocytic TGF-ß signaling limits inflammation and reduces neuronal damage during central nervous system Toxoplasma infection.

The balance between controlling infection and limiting inflammation is particularly precarious in the brain because of its unique vulnerability to the toxic effects of inflammation. Astrocytes have been implicated as key regulators of neuroinflammation in CNS infections, including infection with Toxoplasma gondii, a protozoan parasite that naturally establishes a chronic CNS infection in mice and humans. In CNS toxoplasmosis, astrocytes are critical to controlling parasite growth. They secrete proinflammatory cytokines and physically encircle parasites. However, the molecular mechanisms used by astrocytes to limit neuroinflammation during toxoplasmic encephalitis have not yet been identified. TGF-ß signaling in astrocytes is of particular interest because TGF-ß is universally upregulated during CNS infection and serves master regulatory and primarily anti-inflammatory functions. We report in this study that TGF-ß signaling is activated in astrocytes during toxoplasmic encephalitis and that inhibition of astrocytic TGF-ß signaling increases immune cell infiltration, uncouples proinflammatory cytokine and chemokine production from CNS parasite burden, and increases neuronal injury. Remarkably, we show that the effects of inhibiting astrocytic TGF-ß signaling are independent of parasite burden and the ability of GFAP(+) astrocytes to physically encircle parasites.