Infected Dendritic Cells Facilitate Systemic Dissemination and Transplacental Passage of the Obligate Intracellular Parasite Neospora caninum in Mice

The obligate intracellular parasite Neospora caninum disseminates across the placenta and the blood-brain barrier, to reach sites where it causes severe pathology or establishes chronic persistent infections. The mechanisms used by N. caninum to breach restrictive biological barriers remain elusive. To examine the cellular basis of these processes, migration of different N. caninum isolates (Nc-1, Nc-Liverpool, Nc-SweB1 and the Spanish isolates: Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9) was studied in an in vitro model based on a placental trophoblast-derived BeWo cell line. Here, we describe that infection of dendritic cells (DC) by N. caninum tachyzoites potentiated translocation of parasites across polarized cellular monolayers. In addition, powered by the parasite's own gliding motility, extracellular N. caninum tachyzoites were able to transmigrate across cellular monolayers. Altogether, the presented data provides evidence of two putative complementary pathways utilized by N. caninum, in an isolate-specific fashion, for passage of restrictive cellular barriers. Interestingly, adoptive transfer of tachyzoite-infected DC in mice resulted in increased parasitic loads in various organs, e.g. the central nervous system, compared to infections with free parasites. Inoculation of pregnant mice with infected DC resulted in an accentuated vertical transmission to the offspring with increased parasitic loads and neonatal mortality. These findings reveal that N. caninum exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues. The findings are indicative of conserved dissemination strategies among coccidian apicomplexan parasites

The Inflammatory Kinase MAP4K4 Promotes Reactivation of Kaposi's Sarcoma Herpesvirus and Enhances the Invasiveness of Infected Endothelial Cells

Kaposi's sarcoma (KS) is a mesenchymal tumour, which is caused by Kaposi's sarcoma herpesvirus (KSHV) and develops under inflammatory conditions. KSHV-infected endothelial spindle cells, the neoplastic cells in KS, show increased invasiveness, attributed to the elevated expression of metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). The majority of these spindle cells harbour latent KSHV genomes, while a minority undergoes lytic reactivation with subsequent production of new virions and viral or cellular chemo- and cytokines, which may promote tumour invasion and dissemination. In order to better understand KSHV pathogenesis, we investigated cellular mechanisms underlying the lytic reactivation of KSHV. Using a combination of small molecule library screening and siRNA silencing we found a STE20 kinase family member, MAP4K4, to be involved in KSHV reactivation from latency and to contribute to the invasive phenotype of KSHV-infected endothelial cells by regulating COX-2, MMP-7, and MMP-13 expression. This kinase is also highly expressed in KS spindle cells in vivo. These findings suggest that MAP4K4, a known mediator of inflammation, is involved in KS aetiology by regulating KSHV lytic reactivation, expression of MMPs and COX-2, and, thereby modulating invasiveness of KSHV-infected endothelial cells. © 2013 Haas et al

Syndromics: A Bioinformatics Approach for Neurotrauma Research

Substantial scientific progress has been made in the past 50 years in delineating many of the biological mechanisms involved in the primary and secondary injuries following trauma to the spinal cord and brain. These advances have highlighted numerous potential therapeutic approaches that may help restore function after injury. Despite these advances, bench-to-bedside translation has remained elusive. Translational testing of novel therapies requires standardized measures of function for comparison across different laboratories, paradigms, and species. Although numerous functional assessments have been developed in animal models, it remains unclear how to best integrate this information to describe the complete translational “syndrome” produced by neurotrauma. The present paper describes a multivariate statistical framework for integrating diverse neurotrauma data and reviews the few papers to date that have taken an information-intensive approach for basic neurotrauma research. We argue that these papers can be described as the seminal works of a new field that we call “syndromics”, which aim to apply informatics tools to disease models to characterize the full set of mechanistic inter-relationships from multi-scale data. In the future, centralized databases of raw neurotrauma data will enable better syndromic approaches and aid future translational research, leading to more efficient testing regimens and more clinically relevant findings

Interferon-Induced Cell Membrane Proteins, IFITM3 and Tetherin, Inhibit Vesicular Stomatitis Virus Infection via Distinct Mechanisms▿

Tetherin and IFITM3 are recently identified interferon-induced cellular proteins that restrict infections by retroviruses and filoviruses and of influenza virus and flaviviruses, respectively. In our efforts to further explore their antiviral activities against other viruses and determine their antiviral mechanisms, we found that the two antiviral proteins potently inhibit the infection of vesicular stomatitis virus (VSV), a prototype member of the Rhabdoviridae family. Taking advantage of this well-studied virus infection system, we show that although both tetherin and IFITM3 are plasma membrane proteins, tetherin inhibits virion particle release from infected cells, while IFITM3 disrupts an early event after endocytosis of virion particles but before primary transcription of incoming viral genomes. Furthermore, we demonstrate that both the N-terminal 21 amino acid residues and C-terminal transmembrane region of IFITM3 are required for its antiviral activity. Collectively, our work sheds light on the mechanisms by which tetherin and IFITM3 restrict infection with rhabdoviruses and possibly other pathogenic viruses

Malaria‐derived hemozoin exerts early modulatory effects on the phenotype and maturation of human dendritic cells

Plasmodium falciparum (P. falciparum)-induced effects on the phenotype of human dendritic cells (DC) could contribute to poor induction of long-lasting protective immunity against malaria. DC ability to present antigens to naïve T cells, thus initiating adaptive immune responses depends on complex switches in chemokine receptors, production of soluble mediators and expression of molecules enabling antigen-presentation and maturation. To examine the cellular basis of these processes in the context of malaria, we performed detailed analysis of early events following exposure of human monocyte-derived DC to natural hemozoin (nHZ) and the synthetic analog of its heme core, β-hematin. DC exposed to either molecule produced high levels of the inflammatory chemokine MCP-1, showed continuous high expression of the inflammatory chemokine receptor CCR5, no upregulation of the lymphoid homing receptor CCR7 and no cytoskeletal actin redistribution with loss of podosomes. DC partially matured as indicated by increased expression of major histocompatibility complex (MHC) class II and CD86 following nHZ and β-hematin exposure, however there was a lack in expression of the maturation marker CD83 following nHZ but not β-hematin exposure. Overall our data demonstrate that exposure to nHZ partially impairs the capacity of DC to mature, an effect in part differential to β-hematin

GABAergic Signaling Is Linked to a Hypermigratory Phenotype in Dendritic Cells Infected by Toxoplasma gondii

During acute infection in human and animal hosts, the obligate intracellular protozoan Toxoplasma gondii infects a variety of cell types, including leukocytes. Poised to respond to invading pathogens, dendritic cells (DC) may also be exploited by T. gondii for spread in the infected host. Here, we report that human and mouse myeloid DC possess functional γ-aminobutyric acid (GABA) receptors and the machinery for GABA biosynthesis and secretion. Shortly after T. gondii infection (genotypes I, II and III), DC responded with enhanced GABA secretion in vitro. We demonstrate that GABA activates GABA(A) receptor-mediated currents in T. gondii-infected DC, which exhibit a hypermigratory phenotype. Inhibition of GABA synthesis, transportation or GABA(A) receptor blockade in T. gondii-infected DC resulted in impaired transmigration capacity, motility and chemotactic response to CCL19 in vitro. Moreover, exogenous GABA or supernatant from infected DC restored the migration of infected DC in vitro. In a mouse model of toxoplasmosis, adoptive transfer of infected DC pre-treated with GABAergic inhibitors reduced parasite dissemination and parasite loads in target organs, e.g. the central nervous system. Altogether, we provide evidence that GABAergic signaling modulates the migratory properties of DC and that T. gondii likely makes use of this pathway for dissemination. The findings unveil that GABA, the principal inhibitory neurotransmitter in the brain, has activation functions in the immune system that may be hijacked by intracellular pathogens

Constitutional Court of the Republic of Latvia : constitutional regulation, competence and content of the petitions

During acute infection in human and animal hosts, the obligate intracellular protozoan Toxoplasma gondii infects a variety of cell types, including leukocytes. Poised to respond to invading pathogens, dendritic cells (DC) may also be exploited by T. gondii for spread in the infected host. Here, we report that human and mouse myeloid DC possess functional γ-aminobutyric acid (GABA) receptors and the machinery for GABA biosynthesis and secretion. Shortly after T. gondii infection (genotypes I, II and III), DC responded with enhanced GABA secretion in vitro. We demonstrate that GABA activates GABA(A) receptor-mediated currents in T. gondii-infected DC, which exhibit a hypermigratory phenotype. Inhibition of GABA synthesis, transportation or GABA(A) receptor blockade in T. gondii-infected DC resulted in impaired transmigration capacity, motility and chemotactic response to CCL19 in vitro. Moreover, exogenous GABA or supernatant from infected DC restored the migration of infected DC in vitro. In a mouse model of toxoplasmosis, adoptive transfer of infected DC pre-treated with GABAergic inhibitors reduced parasite dissemination and parasite loads in target organs, e.g. the central nervous system. Altogether, we provide evidence that GABAergic signaling modulates the migratory properties of DC and that T. gondii likely makes use of this pathway for dissemination. The findings unveil that GABA, the principal inhibitory neurotransmitter in the brain, has activation functions in the immune system that may be hijacked by intracellular pathogens

Identification of Five Interferon-Induced Cellular Proteins That Inhibit West Nile Virus and Dengue Virus Infections▿

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-α for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-α is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles

Gliding motility by <i>N. caninum</i> tachyzoites.

<p><b>A</b>. <i>N. caninum</i> deposited surface membrane trails during gliding on a solid substrate. Trails were visualized by staining with anti-SAG1 mAb. Representative micrographs of Nc-1, Nc-Spain 3H and Nc-Liverpool trails are shown. Scale bar: 5 µm. <b>B</b>. <i>N. caninum</i> isolates exhibit significant differences in gliding motility (P<0.0001; one-way ANOVA test), being highest for Nc-Spain 9 (Nc-Spain 9 <i>versus</i> Nc-1, Nc-SweB1, Nc-Spain 3H and Nc-Spain 7; P<0.05-0.0001; Bonferroni's post-test), and lowest for Nc-Spain 3H (Nc-Spain 3H <i>versus</i> all isolates; P<0.05-0.0001; Bonferroni's post-test). The mean length of trails formed by Nc-Liverpool was also superior to that of Nc-1 (P<0.05; Bonferroni's post-test). Trails were measured as relative parasite body lengths. Ten to fifteen trails were measured per strain per experiment. Mean (±SEM) of the determinations from three independent experiments is represented. Asterisks indicate isolates which showed significant differences.</p