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  • 1.
    Barragan, Antonio
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Weidner, Jessica M.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Jin, Z.
    Korpi, E. R.
    Birnir, B.
    GABAergic signalling in the immune system2015In: Acta Physiologica, ISSN 1748-1708, E-ISSN 1748-1716, Vol. 213, no 4, p. 819-827Article, review/survey (Refereed)
    Abstract [en]

    The GABAergic system is the main inhibitory neurotransmitter system in the central nervous system (CNS) of vertebrates. Signalling of the transmitter c-aminobutyric acid (GABA) via GABA type A receptor channels or G-protein-coupled type B receptors is implicated in multiple CNS functions. Recent findings have implicated the GABAergic system in immune cell functions, inflammatory conditions and diseases in peripheral tissues. Interestingly, the specific effects may vary between immune cell types, with stage of activation and be altered by infectious agents. GABA/GABA-A receptor-mediated immunomodulatory functions have been unveiled in immune cells, being present in T lymphocytes and regulating the migration of Toxoplasma-infected dendritic cells. The GABAergic system may also play a role in the regulation of brain resident immune cells, the microglial cells. Activation of microglia appears to regulate the function of GABAergic neurotransmission in neighbouring neurones through changes induced by secretion of brain-derived neurotrophic factor. The neurotransmitter-driven immunomodulation is a new but rapidly growing field of science. Herein, we review the present knowledge of the GABA signalling in immune cells of the periphery and the CNS and raise questions for future research.

  • 2.
    Bhandage, Amol K.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Calling in the CaValry-Toxoplasma gondii Hijacks GABAergic Signaling and Voltage-Dependent Calcium Channel Signaling for Trojan horse-Mediated Dissemination2019In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 9, article id 61Article, review/survey (Refereed)
    Abstract [en]

    Dendritic cells (DCs) are regarded as the gatekeepers of the immune system but can also mediate systemic dissemination of the obligate intracellular parasite Toxoplasma gondii. Here, we review the current knowledge on how T. gondii hijacks the migratory machinery of DCs and microglia. Shortly after active invasion by the parasite, infected cells synthesize and secrete the neurotransmitter gamma-aminobutyric acid (GABA) and activate GABA-A receptors, which sets on a hypermigratory phenotype in parasitized DCs in vitro and in vivo. The signaling molecule calcium plays a central role for this migratory activation as signal transduction following GABAergic activation ismediated via the L-type voltage-dependent calcium channel (L-VDCC) subtype Ca(V)1.3. These studies have revealed that DCs possess a GABA/L-VDCC/Ca(V)1.3 motogenic signaling axis that triggers migratory activation upon T. gondii infection. Moreover, GABAergic migration can cooperate with chemotactic responses. Additionally, the parasite-derived protein Tg14-3-3 has been associated with hypermigration of DCs andmicroglia. We discuss the interference of T. gondii infection with host cell signaling pathways that regulate migration. Altogether, T. gondii hijacks non-canonical signaling pathways in infected immune cells to modulate their migratory properties, and thereby promote its own dissemination.

  • 3.
    Bhandage, Amol K.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    GABAergic signaling by cells of the immune system: more the rule than the exception2021In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 78, no 15, p. 5667-5679Article, review/survey (Refereed)
    Abstract [en]

    Gamma-aminobutyric acid (GABA) is best known as an essential neurotransmitter in the evolved central nervous system (CNS) of vertebrates. However, GABA antedates the development of the CNS as a bioactive molecule in metabolism and stress-coupled responses of prokaryotes, invertebrates and plants. Here, we focus on the emerging findings of GABA signaling in the mammalian immune system. Recent reports show that mononuclear phagocytes and lymphocytes, for instance dendritic cells, microglia, T cells and NK cells, express a GABAergic signaling machinery. Mounting evidence shows that GABA receptor signaling impacts central immune functions, such as cell migration, cytokine secretion, immune cell activation and cytotoxic responses. Furthermore, the GABAergic signaling machinery of leukocytes is implicated in responses to microbial infection and is co-opted by protozoan parasites for colonization of the host. Peripheral GABA signaling is also implicated in inflammatory conditions and diseases, such as type 1 diabetes, rheumatoid arthritis and cancer cell metastasis. Adding to its role in neurotransmission, growing evidence shows that the non-proteinogenic amino acid GABA acts as an intercellular signaling molecule in the immune system and, as an interspecies signaling molecule in host-microbe interactions. Altogether, the data raise the assumption of conserved GABA signaling in a broad range of mammalian cells and diversification of function in the immune system.

  • 4.
    Bhandage, Amol K.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Friedrich, Laura M.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kanatani, Sachie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Jakobsson-Björkén, Simon
    Escrig-Larena, J. Ignacio
    Wagner, Arnika K.
    Chambers, Benedict J.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    GABAergic signaling in human and murine NK cells upon challenge with Toxoplasma gondii2021In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 110, no 4, p. 617-628Article in journal (Refereed)
    Abstract [en]

    Protective cytotoxic and proinflammatory cytokine responses by NK cells impact the outcome of infections by Toxoplasma gondii, a common parasite in humans and other vertebrates. However, T. gondii can also sequester within NK cells and downmodulate their effector functions. Recently, the implication of GABA signaling in infection and inflammation-related responses of mononuclear phagocytes and T cells has become evident. Yet, the role of GABAergic signaling in NK cells has remained unknown. Here, we report that human and murine NK cells synthesize and secrete GABA in response to infection challenge. Parasitized NK cells secreted GABA, whereas activation stimuli, such as IL-12/IL-18 or parasite lysates, failed to induce GABA secretion. GABA secretion by NK cells was associated to a transcriptional up-regulation of GABA synthesis enzymes (glutamate decarboxylases [GAD65/67]) and was abrogated by GAD inhibition. Further, NK cells expressed GABA-A receptor subunits and GABA signaling regulators, with transcriptional modulations taking place upon challenge with T. gondii. Exogenous GABA and GABA-containing supernatants from parasitized dendritic cells (DCs) impacted NK cell function by reducing the degranulation and cytotoxicity of NK cells. Conversely, GABA-containing supernatants from NK cells enhanced the migratory responses of parasitized DCs. This enhanced DC migration was abolished by GABA-A receptor antagonism or GAD inhibition and was reconstituted by exogenous GABA. Jointly, the data show that NK cells are GABAergic cells and that GABA hampers NK cell cytotoxicity in vitro. We hypothesize that GABA secreted by parasitized immune cells modulates the immune responses to T. gondii infection.

  • 5.
    Bhandage, Amol K.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kanatani, Sachie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Toxoplasma-Induced Hypermigration of Primary Cortical Microglia Implicates GABAergic Signaling2019In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 9, article id 73Article in journal (Refereed)
    Abstract [en]

    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 gamma-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.

  • 6.
    Bhandage, Amol K.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Olivera, Gabriela C.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kanatani, Sachie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Thompson, Elizabeth
    Loré, Karin
    Varas-Godoy, Manuel
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    A motogenic GABAergic system of mononuclear phagocytes facilitates dissemination of coccidian parasites2020In: eLIFE, E-ISSN 2050-084X, Vol. 9, article id e60528Article in journal (Refereed)
    Abstract [en]

    Gamma-aminobutyric acid (GABA) serves diverse biological functions in prokaryotes and eukaryotes, including neurotransmission in vertebrates. Yet, the role of GABA in the immune system has remained elusive. Here, a comprehensive characterization of human and murine myeloid mononuclear phagocytes revealed the presence of a conserved and tightly regulated GABAergic machinery with expression of GABA metabolic enzymes and transporters, GABA-A receptors and regulators, and voltage-dependent calcium channels. Infection challenge with the common coccidian parasites Toxoplasma gondii and Neospora caninum activated GABAergic signaling in phagocytes. Using gene silencing and pharmacological modulators in vitro and in vivo in mice, we identify the functional determinants of GABAergic signaling in parasitized phagocytes and demonstrate a link to calcium responses and migratory activation. The findings reveal a regulatory role for a GABAergic signaling machinery in the host-pathogen interplay between phagocytes and invasive coccidian parasites. The co-option of GABA underlies colonization of the host by a Trojan horse mechanism.

  • 7. García-Sanchez, Marta
    et al.
    Jiménez-Pelayo, Laura
    Horcajo, Pilar
    Regidor-Cerrillo, Javier
    Ólafsson, Einar B.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Bhandage, Amol K.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Werling, Dirk
    Miguel Ortega-Mora, Luis
    Collantes-Fernández, Esther
    Differential Responses of Bovine Monocyte-Derived Macrophages to Infection by Neospora caninum Isolates of High and Low Virulence2019In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 10, article id 915Article in journal (Refereed)
    Abstract [en]

    Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, represents one of the main causes of abortion in cattle. Macrophages (Mempty sets) are mediators of the innate immune response against infection and likely one of the first cells encountered by the parasite during the host infection process. In this study, we investigated in vitro how high or low virulent isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively) interact with bovine monocyte-derived Mempty sets and the influence of the isolate virulence on the subsequent cellular response. Both isolates actively invaded, survived and replicated in the Mempty sets. However, Nc-Spain7 showed a higher invasion rate and a replication significantly faster, following an exponential growth model, whereas Nc-Spain1H presented a delayed replication and a lower growth rate without an exponential pattern. N. caninum infection induced a hypermigratory phenotype in bovine Mempty sets that was characterized by enhanced motility and transmigration in vitro and was accompanied by morphological changes and abrogated extracellular matrix degradation. A significantly higher hypermotility was observed with the highly virulent isolate Nc-Spain7. Nc-Spain1H-infected Mempty sets showed elevated reactive oxygen species (ROS) production and IL12p40 expression, which also resulted in increased IFN-gamma release by lymphocytes, compared to cells infected with Nc-Spain7. Furthermore, IL-10 was upregulated in Mempty sets infected with both isolates. Infected Mempty sets exhibited lower expression of MHC Class II, CD86, and CD1b molecules than uninfected Mempty sets, with non-significant differences between isolates. This work characterizes for the first time N. caninum replication in bovine monocyte-derived Mempty sets and details isolate-dependent differences in host cell responses to the parasite.

  • 8.
    Hildebrandt, Franziska
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Andersson, Alma
    Saarenpää, Sami
    Larsson, Ludvig
    Van Hul, Noémi
    Kanatani, Sachie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Masek, Jan
    Ellis, Ewa
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mollbrink, Annelie
    Andersson, Emma R.
    Lundeberg, Joakim
    Ankarklev, Johan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the mouse liver2021In: Nature Communications, E-ISSN 2041-1723, Vol. 12, no 1, article id 7046Article in journal (Refereed)
    Abstract [en]

    Reconstruction of heterogeneity through single cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we apply Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology.

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  • 9.
    Hildebrandt, Franziska
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mohammed, Mubasher
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Dziedziech, Alexis
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Institut Pasteur, France.
    Bhandage, Amol K.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Divne, Anna-Maria
    Barrenäs, Fredrik
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Henriksson, Johan
    Ankarklev, Johan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Uppsala University, Sweden.
    scDual-Seq of Toxoplasma gondii-infected mouse BMDCs reveals heterogeneity and differential infection dynamics2023In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 14, article id 1224591Article in journal (Refereed)
    Abstract [en]

    Dendritic cells and macrophages are integral parts of the innate immune system and gatekeepers against infection. The protozoan pathogen, Toxoplasma gondii, is known to hijack host immune cells and modulate their immune response, making it a compelling model to study host-pathogen interactions. Here we utilize single cell Dual RNA-seq to parse out heterogeneous transcription of mouse bone marrow-derived dendritic cells (BMDCs) infected with two distinct genotypes of T. gondii parasites, over multiple time points post infection. We show that the BMDCs elicit differential responses towards T. gondii infection and that the two parasite lineages distinctly manipulate subpopulations of infected BMDCs. Co-expression networks define host and parasite genes, with implications for modulation of host immunity. Integrative analysis validates previously established immune pathways and additionally, suggests novel candidate genes involved in host-pathogen interactions. Altogether, this study provides a comprehensive resource for characterizing host-pathogen interplay at high-resolution.

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  • 10. Idro, Richard
    et al.
    Ogwang, Rodney
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Raimondo, Joseph Valentino
    Masocha, Willias
    Neuroimmunology of Common Parasitic Infections in Africa2022In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 13, article id 791488Article, review/survey (Refereed)
    Abstract [en]

    Parasitic infections of the central nervous system are an important cause of morbidity and mortality in Africa. The neurological, cognitive, and psychiatric sequelae of these infections result from a complex interplay between the parasites and the host inflammatory response. Here we review some of the diseases caused by selected parasitic organisms known to infect the nervous system including Plasmodium falciparumToxoplasma gondiiTrypanosoma brucei spp., and Taenia solium species. For each parasite, we describe the geographical distribution, prevalence, life cycle, and typical clinical symptoms of infection and pathogenesis. We pay particular attention to how the parasites infect the brain and the interaction between each organism and the host immune system. We describe how an understanding of these processes may guide optimal diagnostic and therapeutic strategies to treat these disorders. Finally, we highlight current gaps in our understanding of disease pathophysiology and call for increased interrogation of these often-neglected disorders of the nervous system.

  • 11.
    Kanatani, Sachie
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Toxoplasma-induced hypermigration of primary cortical microglia implicates an alternative GABA synthesis pathwayManuscript (preprint) (Other academic)
  • 12.
    Kanatani, Sachie
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Fuks, Jonas M.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Olafsson, Einar B.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Westermark, Linda
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Chambers, Benedict
    Varas-Godoy, Manuel
    Uhlén, Per
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Voltage-dependent calcium channel signaling mediates GABA(A) receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii2017In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 13, no 12, article id e1006739Article in journal (Refereed)
    Abstract [en]

    The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon T. gondii-infection,. Upsilon-aminobutyric acid (GABA)/GABAA receptor signaling triggers a hypermigratory phenotype in dendritic cells (DCs) by unknown signal transduction pathways. Here, we demonstrate that calcium (Ca2+) signaling in DCs is indispensable for T. gondii-induced DC hypermotility and transmigration in vitro. We report that activation of GABAA receptors by GABA induces transient Ca2+ entry in DCs. Murine bone marrow-derived DCs preferentially expressed the L-type voltage-dependent Ca2+ channel (VDCC) subtype Cav1.3. Silencing of Cav1.3 by short hairpin RNA or selective pharmacological antagonism of VDCCs abolished the Toxoplasma-induced hypermigratory phenotype. In a mouse model of toxoplasmosis, VDCC inhibition of adoptively transferred Toxoplasma-infected DCs delayed the appearance of cell-associated parasites in the blood circulation and reduced parasite dissemination to target organs. The present data establish that T. gondii-induced hypermigration of DCs requires signaling via VDCCs and that Ca2+ acts as a second messenger to GABAergic signaling via the VDCC Cav1.3. The findings define a novel motility-related signaling axis in DCs and unveil that interneurons and DCs share common GABAergic motogenic pathways. T. gondii employs GABAergic non-canonical pathways to induce host cell migration and facilitate dissemination.

  • 13.
    Kanatani, Sachie
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Uhlén, Per
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Infection by Toxoplasma gondii Induces Amoeboid-Like Migration of Dendritic Cells in a Three-Dimensional Collagen Matrix2015In: PLOS ONE, E-ISSN 1932-6203, Vol. 10, no 9, article id e0139104Article in journal (Refereed)
    Abstract [en]

    Toxoplasma gondii, an obligate intracellular parasite of humans and other warm-blooded vertebrates, invades a variety of cell types in the organism, including immune cells. Notably, dendritic cells (DCs) infected by T. gondii acquire a hypermigratory phenotype that potentiates parasite dissemination by a 'Trojan horse' type of mechanism in mice. Previous studies have demonstrated that, shortly after parasite invasion, infected DCs exhibit hypermotility in 2-dimensional confinements in vitro and enhanced transmigration in transwell systems. However, interstitial migration in vivo involves interactions with the extracellular matrix in a 3-dimensional (3D) space. We have developed a collagen matrix-based assay in a 96-well plate format that allows quantitative locomotion analyses of infected DCs in a 3D confinement over time. We report that active invasion of DCs by T. gondii tachyzoites induces enhanced migration of infected DCs in the collagen matrix. Parasites of genotype II induced superior DC migratory distances than type I parasites. Moreover, Toxoplasma-induced hypermigration of DCs was further potentiated in the presence of the CCR7 chemotactic cue CCL19. Blocking antibodies to integrins (CD11a, CD11b, CD18, CD29, CD49b) insignificantly affected migration of infected DCs in the 3D matrix, contrasting with their inhibitory effects on adhesion in 2D assays. Morphological analyses of infected DCs in the matrix were consistent with the acquisition of an amoeboid-like migratory phenotype. Altogether, the present data show that the Toxoplasma-induced hypermigratory phenotype in a 3D matrix is consistent with integrin-independent amoeboid DC migration with maintained responsiveness to chemotactic and chemokinetic cues. The data support the hypothesis that induction of amoeboid hypermigration and chemotaxis/chemokinesis in infected DCs potentiates the dissemination of T. gondii.

  • 14. Mulas, Floriana
    et al.
    Wang, Xu
    Song, Shanshan
    Nishanth, Gopala
    Yi, Wenjing
    Brunn, Anna
    Larsen, Pia-Katharina
    Isermann, Berend
    Kalinke, Ulrich
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Naumann, Michael
    Deckert, Martina
    Schlüter, Dirk
    The deubiquitinase OTUB1 augments NF-κB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC132021In: Cellular & Molecular Immunology, ISSN 1672-7681, E-ISSN 2042-0226, Vol. 18, p. 1512-1527Article in journal (Refereed)
    Abstract [en]

    Dendritic cells (DCs) are indispensable for defense against pathogens but may also contribute to immunopathology. Activation of DCs upon the sensing of pathogens by Toll-like receptors (TLRs) is largely mediated by pattern recognition receptor/nuclear factor-kappa B (NF-kappa B) signaling and depends on the appropriate ubiquitination of the respective signaling molecules. However, the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood. Here, we reveal that the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) is upregulated in DCs upon murine Toxoplasmagondii infection and lipopolysaccharide challenge. Stimulation of DCs with the TLR11/12 ligand T. gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-kappa B activation in OTUB1-competent cells, resulting in elevated interleukin-6 (IL-6), IL-12, and tumor necrosis factor (TNF) production, which was also observed upon the specific stimulation of TLR2, TLR3, TLR7, and TLR9. Mechanistically, OTUB1 promoted NF-kappa B activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13, resulting in increased K63-linked ubiquitination of IRAK1 (IL-1 receptor-associated kinase 1) and TRAF6 (TNF receptor-associated factor 6). Consequently, DC-specific deletion of OTUB1 impaired the production of cytokines, in particular IL-12, by DCs over the first 2 days of T. gondii infection, resulting in the diminished production of protective interferon-gamma (IFN-gamma) by natural killer cells, impaired control of parasite replication, and, finally, death from chronic T.encephalitis, all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection. In contrast, impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology. Collectively, these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.

  • 15.
    Olivera, Gabriela Carina
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ross, Emily Charlotte
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Peuckert, Christiane
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Blood-brain barrier-restricted translocation of Toxoplasma gondii from cortical capillaries2021In: eLIFE, E-ISSN 2050-084X, Vol. 10, article id e69182Article in journal (Refereed)
    Abstract [en]

    The cellular barriers of the central nervous system proficiently protect the brain parenchyma from infectious insults. Yet, the single-celled parasite Toxoplasma gondii commonly causes latent cerebral infection in humans and other vertebrates. Here, we addressed the role of the cerebral vasculature in the passage of T. gondii to the brain parenchyma. Shortly after inoculation in mice, parasites mainly localized to cortical capillaries, in preference over post-capillary venules, cortical arterioles or meningeal and choroidal vessels. Early invasion to the parenchyma (days 1-5) occurred in absence of a measurable increase in blood-brain barrier (BBB) permeability, perivascular leukocyte cuffs or hemorrhage. However, sparse focalized permeability elevations were detected adjacently to replicative parasite foci. Further, T. gondii triggered inflammatory responses in cortical microvessels and endothelium. Pro- and anti-inflammatory treatments of mice with LPS and hydrocortisone, respectively, impacted BBB permeability and parasite loads in the brain parenchyma. Finally, pharmacological inhibition or Cre/loxP conditional knockout of endothelial focal adhesion kinase (FAK), a BBB intercellular junction regulator, facilitated parasite translocation to the brain parenchyma. The data reveal that the initial passage of T. gondii to the central nervous system occurs principally across cortical capillaries. The integrity of the microvascular BBB restricts parasite transit, which conversely is exacerbated by the inflammatory response.

  • 16.
    Ross, Emily C.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Olivera, Gabriela C.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers2019In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 21, no 9, article id e13048Article in journal (Refereed)
    Abstract [en]

    The apicomplexan parasite Toxoplasma gondii invades tissues and traverses non-permissive biological barriers in infected humans and other vertebrates. Following ingestion, the parasite penetrates the intestinal wall and disseminates to immune-privileged sites such as the brain parenchyma, after crossing the blood-brain barrier. In the present study, we have established a protocol for high-purification of primary mouse brain endothelial cells to generate stably polarised monolayers that allowed assessment of cellular barrier traversal by T. gondii. We report that T. gondii tachyzoites translocate across polarised monolayers of mouse brain endothelial cells and human intestinal Caco2 cells without significantly perturbing barrier impermeability and with minimal change in transcellular electrical resistance. In contrast, challenge with parasite lysate or LPS increased barrier permeability by destabilising intercellular tight junctions (TJs) and accentuated transmigration of T. gondii. Conversely, reduced phosphorylation of the TJ-regulator focal adhesion kinase (FAK) was observed dose-dependently upon challenge of monolayers with live T. gondii but not with parasite lysate or LPS. Pharmacological inhibition of FAK phosphorylation reversibly altered barrier integrity and facilitated T. gondii translocation. Finally, gene silencing of FAK by shRNA facilitated transmigration of T. gondii across epithelial and endothelial monolayers. Jointly, the data demonstrate that T. gondii infection transiently alters the TJ stability through FAK dysregulation to facilitate transmigration. This work identifies the implication of the TJ regulator FAK in the transmigration of T. gondii across polarised cellular monolayers and provides novel insights in how microbes overcome the restrictiveness of biological barriers.

  • 17.
    Ross, Emily C.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    ten Hoeve, Arne L.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Integrin-dependent migratory switches regulate the translocation of Toxoplasma-infected dendritic cells across brain endothelial monolayers2021In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 78, p. 5197-5212Article in journal (Refereed)
    Abstract [en]

    Multiple cellular processes, such as immune responses and cancer cell metastasis, crucially depend on interconvertible migration modes. However, knowledge is scarce on how infectious agents impact the processes of cell adhesion and migration at restrictive biological barriers. In extracellular matrix, dendritic cells (DCs) infected by the obligate intracellular protozoan Toxoplasma gondii undergo mesenchymal-to-amoeboid transition (MAT) for rapid integrin-independent migration. Here, in a cellular model of the blood–brain barrier, we report that parasitised DCs adhere to polarised endothelium and shift to integrin-dependent motility, accompanied by elevated transendothelial migration (TEM). Upon contact with endothelium, parasitised DCs dramatically reduced velocities and adhered under both static and shear stress conditions, thereby obliterating the infection-induced amoeboid motility displayed in collagen matrix. The motility of adherent parasitised DCs on endothelial monolayers was restored by blockade of β1 and β2 integrins or ICAM-1, which conversely reduced motility on collagen-coated surfaces. Moreover, parasitised DCs exhibited enhanced translocation across highly polarised primary murine brain endothelial cell monolayers. Blockade of β1, β2 integrins, ICAM-1 and PECAM-1 reduced TEM frequencies. Finally, gene silencing of the pan-integrin-cytoskeleton linker talin (Tln1) or of β1 integrin (Itgb1) in primary DCs resulted in increased motility on endothelium and decreased TEM. Adding to the paradigms of leukocyte diapedesis, the findings provide novel insights in how an intracellular pathogen impacts the migratory plasticity of leukocytes in response to the cellular environment, to promote infection-related dissemination.

  • 18.
    Ross, Emily Charlotte
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Olivera, Gabriela C.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Early passage of Toxoplasma gondii across the blood–brain barrier2022In: Trends in Parasitology, ISSN 1471-4922, E-ISSN 1471-5007, Vol. 38, no 6, p. 450-461Article, review/survey (Refereed)
    Abstract [en]

    The blood–brain barrier (BBB) efficiently protects the central nervous system (CNS) from infectious insults. Yet, the apicomplexan parasite Toxoplasma gondii has a remarkable capability to establish latent cerebral infection in humans and other vertebrates. In addition to the proposed mechanisms for access to the brain parenchyma, recent findings highlight a paramount role played by the BBB in restricting parasite passage and minimizing parasite loads in the brain. Consistent with clinically asymptomatic primary infections in humans, mounting evidence indicates that the original colonization of the brain by T. gondii encompasses previously unappreciated, nondisruptive translocation processes that precede the onset of parasite-limiting immune responses.

  • 19.
    Ross, Emily Charlotte
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    ten Hoeve, Arne L.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Saeij, Jeroen P. J.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Toxoplasma effector-induced ICAM-1 expression by infected dendritic cells potentiates transmigration across polarised endothelium2022In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 13, article id 950914Article in journal (Refereed)
    Abstract [en]

    The obligate intracellular parasite Toxoplasma gondii makes use of infected leukocytes for systemic dissemination. Yet, how infection impacts the processes of leukocyte diapedesis has remained unresolved. Here, we addressed the effects of T. gondii infection on the trans-endothelial migration (TEM) of dendritic cells (DCs) across polarised brain endothelial monolayers. We report that upregulated expression of leukocyte ICAM-1 is a feature of the enhanced TEM of parasitised DCs. The secreted parasite effector GRA15 induced an elevated expression of ICAM-1 in infected DCs that was associated with enhanced cell adhesion and TEM. Consequently, gene silencing of Icam-1 in primary DCs or deletion of parasite GRA15 reduced TEM. Further, the parasite effector TgWIP, which impacts the regulation of host actin dynamics, facilitated TEM across polarised endothelium. The data highlight that the concerted action of the secreted effectors GRA15 and TgWIP modulate the leukocyte-endothelial interactions of TEM in a parasite genotype-related fashion to promote dissemination. In addition to the canonical roles of endothelial ICAM-1, this study identifies a previously unappreciated role for leukocyte ICAM-1 in infection-related TEM.

  • 20.
    Ross, Emily Charlotte
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    ten Hoeve, Arne L.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Saeij, Jeroen P.J.
    Department of Pathology, Microbiology, and Immunology, University of California Davis.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Toxoplasma effector-induced ICAM-1 expression by infected dendritic cells potentiates transmigration across polarised endotheliumManuscript (preprint) (Other academic)
  • 21. Sangaré, Lamba Omar
    et al.
    Ólafsson, Einar B.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wang, Yifan
    Yang, Ninghan
    Julien, Lindsay
    Camejo, Ana
    Pesavento, Patricia
    Sidik, Saima M.
    Lourido, Sebastian
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Saeij, Jeroen P. J.
    In Vivo CRISPR Screen Identifies TgWIP as a Toxoplasma Modulator of Dendritic Cell Migration2019In: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 26, no 4, p. 478-492Article in journal (Refereed)
    Abstract [en]

    Toxoplasma can reach distant organs, especially the brain, leading to a lifelong chronic phase. However, genes involved in related in vivo processes are currently unknown. Here, we use focused CRISPR libraries to identify Toxoplasma genes that affect in vivo fitness. We focus on TgWIP, whose deletion affects Toxoplasmadissemination to distant organs. We show that TgWIP is secreted into the host cell upon invasion and interacts with the host WAVE regulatory complex and SHP2 phosphatase, both of which regulate actin dynamics. TgWIP affects the morphology of dendritic cells and mediates the dissolution of podosomes, which dendritic cells use to adhere to extracellular matrix. TgWIP enhances the motility and transmigration of parasitized dendritic cells, likely explaining its effect on in vivofitness. Our results provide a framework for systemic identification of Toxoplasmagenes with in vivo effects at the site of infection or on dissemination to distant organs, including the brain.

  • 22. Schlüter, Dirk
    et al.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Advances and Challenges in Understanding Cerebral Toxoplasmosis2019In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 10, article id 242Article, review/survey (Refereed)
    Abstract [en]

    Toxoplasma gondii is a widespread parasitic pathogen that infects over one third of the global human population. The parasite invades and chronically persists in the central nervous system (CNS) of the infected host. Parasite spread and persistence is intimately linked to an ensuing immune response, which does not only limit parasite-induced damage but also may facilitate dissemination and induce parasite-associated immunopathology. Here, we discuss various aspects of toxoplasmosis where knowledge is scarce or controversial and, the recent advances in the understanding of the delicate interplay of T. gondii with the immune system in experimental and clinical settings. This includes mechanisms for parasite passage from the circulation into the brain parenchyma across the blood-brain barrier during primary acute infection. Later, as chronic latent infection sets in with control of the parasite in the brain parenchyma, the roles of the inflammatory response and of immune cell responses in this phase of the disease are discussed. Additionally, the function of brain resident cell populations is delineated, i.e., how neurons, astrocytes and microglia serve both as target cells for the parasite but also actively contribute to the immune response. As the infection can reactivate in the CNS of immune-compromised individuals, we bring up the immunopathogenesis of reactivated toxoplasmosis, including the special case of congenital CNS manifestations. The relevance, advantages and limitations of rodent infection models for the understanding of human cerebral toxoplasmosis are discussed. Finally, this review pinpoints questions that may represent challenges to experimental and clinical science with respect to improved diagnostics, pharmacological treatments and immunotherapies.

  • 23. Shen, Xianli
    et al.
    Burguillos, Miguel A.
    Osman, Ahmed M.
    Frijhoff, Jeroen
    Carrillo-Jimenez, Alejandro
    Kanatani, Sachie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Augsten, Martin
    Saidi, Dalel
    Rodhe, Johanna
    Kavanagh, Edel
    Rongvaux, Anthony
    Rraklli, Vilma
    Nyman, Ulrika
    Holmberg, Johan
    Östman, Arne
    Flavell, Richard A.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Venero, Jose Luis
    Blomgren, Klas
    Joseph, Bertrand
    Glioma-induced inhibition of caspase-3 in microglia promotes a tumor-supportive phenotype2016In: Nature Immunology, ISSN 1529-2908, E-ISSN 1529-2916, Vol. 17, no 11, p. 1282-1290Article in journal (Refereed)
    Abstract [en]

    Glioma cells recruit and exploit microglia (the resident immune cells of the brain) for their proliferation and invasion ability. The underlying molecular mechanism used by glioma cells to transform microglia into a tumor-supporting phenotype has remained elusive. We found that glioma-induced microglia conversion was coupled to a reduction in the basal activity of microglial caspase-3 and increased S-nitrosylation of mitochondria-associated caspase-3 through inhibition of thioredoxin-2 activity, and that inhibition of caspase-3 regulated microglial tumor-supporting function. Furthermore, we identified the activity of nitric oxide synthase 2 (NOS2, also known as iNOS) originating from the glioma cells as a driving stimulus in the control of microglial caspase-3 activity. Repression of glioma NOS2 expression in vivo led to a reduction in both microglia recruitment and tumor expansion, whereas depletion of microglial caspase-3 gene promoted tumor growth. Our results provide evidence that inhibition of the denitrosylation of S-nitrosylated procaspase-3 mediated by the redox protein Trx2 is a part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells.

  • 24. Sultana, M. Afifa
    et al.
    Du, Ann
    Carow, Berit
    Angbjär, Catrine M.
    Weidner, Jessica M.
    Kanatani, Sachie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Fuks, Jonas M.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Muliaditan, Tamara
    James, Jaime
    Mansfield, Imogen O.
    Campbell, Tessa M.
    Liu, Lifeng
    Kadri, Nadir
    Lambert, Henrik
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Karolinska University Hospital, Sweden.
    Chambers, Benedict J.
    Downmodulation of Effector Functions in NK Cells upon Toxoplasma gondii Infection2017In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 85, no 10, article id e00069-17Article in journal (Refereed)
    Abstract [en]

    The obligate intracellular parasite Toxoplasma gondii can actively infect any nucleated cell type, including cells from the immune system. The rapid transfer of T. gondii from infected dendritic cells to effector natural killer (NK) cells may contribute to the parasite's sequestration and shielding from immune recognition shortly after infection. However, subversion of NK cell functions, such as cytotoxicity or production of proinflammatory cytokines, such as gamma interferon (IFN-γ), upon parasite infection might also be beneficial to the parasite. In the present study, we investigated the effects of T. gondii infection on NK cells. In vitro, infected NK cells were found to be poor at killing target cells and had reduced levels of IFN-γ production. This could be attributed in part to the inability of infected cells to form conjugates with their target cells. However, even upon NK1.1 cross-linking of NK cells, the infected NK cells also exhibited poor degranulation and IFN-γ production. Similarly, NK cells infected in vivo were also poor at killing target cells and producing IFN-γ. Increased levels of transforming growth factor β production, as well as increased levels of expression of SHP-1 in the cytosol of infected NK cells upon infection, were observed in infected NK cells. However, the phosphorylation of STAT4 was not altered in infected NK cells, suggesting that transcriptional regulation mediates the reduced IFN-γ production, which was confirmed by quantitative PCR. These data suggest that infection of NK cells by T. gondii impairs NK cell recognition of target cells and cytokine release, two mechanisms that independently could enhance T. gondii survival.

  • 25.
    ten Hoeve, Arne L.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Braun, L.
    Rodriguez, Matias E.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Olivera, Gabriela C.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Bougdour, A.
    Belmudes, L.
    Couté, Y.
    Saeij, J. P. J.
    Hakimi, M.-A.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The Toxoplasma effector GRA28 promotes parasite dissemination by inducing dendritic cell-like migratory properties in infected macrophages2022In: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 30, no 11, p. 1570-1588.e7Article in journal (Refereed)
    Abstract [en]

    Upon pathogen detection, macrophages normally stay sessile in tissues while dendritic cells (DCs) migrate to secondary lymphoid tissues. The obligate intracellular protozoan Toxoplasma gondii exploits the trafficking of mononuclear phagocytes for dissemination via unclear mechanisms. We report that, upon T. gondii infection, macrophages initiate the expression of transcription factors normally attributed to DCs, upregulate CCR7 expression with a chemotactic response, and perform systemic migration when adoptively transferred into mice. We show that parasite effector GRA28, released by the MYR1 secretory pathway, cooperates with host chromatin remodelers in the host cell nucleus to drive the chemotactic migration of parasitized macrophages. During in vivo challenge studies, bone marrow-derived macrophages infected with wild-type T. gondii outcompeted those challenged with MYR1- or GRA28-deficient strains in migrating and reaching secondary organs. This work reveals how an intracellular parasite hijacks chemotaxis in phagocytes and highlights a remarkable migratory plasticity in differentiated cells of the mononuclear phagocyte system.

  • 26.
    ten Hoeve, Arne L.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hakimi, Mohamed-Ali
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sustained Egr-1 Response via p38 MAP Kinase Signaling Modulates Early Immune Responses of Dendritic Cells Parasitized by Toxoplasma gondii2019In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 9, article id 349Article in journal (Refereed)
    Abstract [en]

    As a response to a diverse array of external stimuli, early growth response protein 1 (Egr-1) plays important roles in the transcriptional regulation of inflammation and the cellular immune response. However, a number of intracellular pathogens colonize immune cells and the implication of Egr-1 in the host-pathogen interplay has remained elusive. Here, we have characterized the Egr-1 responses of primary murine and human dendritic cells (DCs) upon challenge with the obligate intracellular parasite Toxoplasma gondii. We report that live intracellular parasites induce a sustained high expression of Egr-1 in DCs, different from the immediate-early Egr-1 response to parasite lysates, inactivated parasites or LPS. Moreover, a distinct nuclear localization of elevated amounts of Egr-1 protein was detected in infected DCs, but not in by-stander DCs. The ERK1/2 MAPK signaling pathway mediated the canonical immediate-early Egr-1 response to soluble antigens in a MyD88/TLR-dependent fashion. In contrast, a non-canonical extended Egr-1 response that relied primarily on p38 MAPK signaling was induced by intracellular parasites and was exhibited similarly by MyD88-deficient and wildtype DCs. The extended phase Egr-1 response was dramatically reduced upon challenge of DCs with T. gondii parasites deficient in GRA24, a secreted p38-interacting protein. Further, Egr-1-silenced primary DCs maintained their migratory responses upon T. gondii challenge. Importantly, Egr-1 silencing led to elevated expression of co-stimulatory molecules (CD40, CD80) in Toxoplasma-infected DCs and in LPS-challenged immature DCs, indicating that Egr-1 responses suppressed maturation of DCs. Moreover, the IL-12 and IL-2 responses of Toxoplasma-challenged DCs were modulated in a GRA24-dependent fashion. Jointly, the data show that the Egr-1 responses of DCs to microbial external stimuli and intracellular stimuli can be selectively mediated by ERK1/2 or p38 MAPK signaling, and that Egr-1 can act as an intrinsic negative modulator of maturation in primary DCs.

  • 27.
    ten Hoeve, Arne L.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Olivera, Gabriela C.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Saeij, Jeroen P.J.
    Department of Pathology, Microbiology, and Immunology, University of California Davis.
    Hakimi, Mohamed-Ali
    Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309, Université Grenoble Alpes.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The Toxoplasma effector GRA28 promotes parasite dissemination by inducing dendritic cell-like migratory properties in infected macrophagesManuscript (preprint) (Other academic)
  • 28.
    Theopold, Ulrich
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    TRP channels, the missing link for Ca2+ tuning by a unicellular eukaryotic parasite?2021In: Cell Calcium, ISSN 0143-4160, E-ISSN 1532-1991, Vol. 98, article id 102449Article in journal (Refereed)
    Abstract [en]

    Sensing and responding to changes in the cellular environments are essential for the diverse family of Apicomplexan parasites, which undergo complex life cycles comprised of both extracellular and obligate intracellular stages. Despite evidence of paramount roles for Ca2+, the molecular players behind how parasites sense Ca2+ and initiate Ca2+ signaling cascades have remained enigmatic. In a recent publication, Marquez-Nogueras et al., identify a transient receptor potential (TRP)-like channel in Toxoplasma gondii and show its implication in the crucial processes of parasite invasion and egress from host cells.

  • 29.
    Weidner, Jessica M.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Tightly regulated migratory subversion of immune cells promotes the dissemination of Toxoplasma gondii2014In: International Journal of Parasitology, ISSN 0020-7519, E-ISSN 1879-0135, Vol. 44, no 2, p. 85-90Article, review/survey (Refereed)
    Abstract [en]

    While the spread of Toxoplasma gondii within the infected human or animal host is associated with pathology, the pathways of dissemination have remained enigmatic. From the time point of entry into the gut, to the quiescent chronic infection in the central nervous system, Toxoplasma is detected and surveyed by immune cells that populate the tissues, for example dendritic cells. Paradoxically, this protective migratory function of leukocytes appears to be targeted by Toxoplasma to mediate its dissemination in the organism. Recent findings show that tightly regulated events take place shortly after host cell invasion that promote the migratory activation of infected dendritic cells. Here, we review the emerging knowledge on how this obligate intracellular protozoan orchestrates the subversion of leukocytes to achieve systemic dissemination and reach peripheral organs where pathology manifests.

  • 30.
    Weidner, Jessica M.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Kanatani, Sachie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Uchtenhagen, Hannes
    Varas-Godoy, Manuel
    Schulte, Tim
    Engelberg, Klemens
    Gubbels, Marc-Jan
    Sun, He Song
    Harrison, Rene E.
    Achour, Adnane
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Migratory activation of parasitized dendritic cells by the protozoan Toxoplasma gondii 14-3-3 protein2016In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 18, no 11, p. 1537-1550Article in journal (Refereed)
    Abstract [en]

    The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon infection, parasitized dendritic cells (DCs) and microglia exhibit a hypermigratory phenotype in vitro that has been associated with enhancing parasite dissemination in vivo in mice. One unresolved question is how parasites commandeer parasitized cells to achieve systemic dissemination by a Trojan-horse' mechanism. By chromatography and mass spectrometry analyses, we identified an orthologue of the 14-3-3 protein family, T. gondii 14-3-3 (Tg14-3-3), as mediator of DC hypermotility. We demonstrate that parasite-derived polypeptide fractions enriched for Tg14-3-3 or recombinant Tg14-3-3 are sufficient to induce the hypermotile phenotype when introduced by protein transfection into murine DCs, human DCs or microglia. Further, gene transfer of Tg14-3-3 by lentiviral transduction induced hypermotility in primary human DCs. In parasites expressing Tg14-3-3 in a ligand-regulatable fashion, overexpression of Tg14-3-3 was correlated with induction of hypermotility in parasitized DCs. Localization studies in infected DCs identified Tg14-3-3 within the parasitophorous vacuolar space and a rapid recruitment of host cell 14-3-3 to the parasitophorous vacuole membrane. The present work identifies a determinant role for Tg14-3-3 in the induction of the migratory activation of immune cells by T. gondii. Collectively, the findings reveal Tg14-3-3 as a novel target for an intracellular pathogen that acts by hijacking the host cell's migratory properties to disseminate.

  • 31.
    Ólafsson, Einar B.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The unicellular eukaryotic parasite Toxoplasma gondii hijacks the migration machinery of mononuclear phagocytes to promote its dissemination2020In: Biology of the Cell, ISSN 0248-4900, E-ISSN 1768-322X, Vol. 112, no 9, p. 239-250Article, review/survey (Refereed)
    Abstract [en]

    Toxoplasma gondii is an obligate intracellular protozoan with the ability to infect virtually any type of nucleated cell in warm-blooded vertebrates including humans. Toxoplasma gondii invades immune cells, which the parasite employs as shuttles for dissemination by a Trojan horse mechanism. Recent findings are starting to unveil how this parasite orchestrates the subversion of the migratory functions of parasitised mononuclear phagocytes, especially dendritic cells (DCs) and monocytes. Here, we focus on how T. gondii impacts host cell signalling that regulates leukocyte motility and systemic migration in tissues. Shortly after active parasite invasion, DCs undergo mesenchymal-to-amoeboid transition and adopt a high-speed amoeboid mode of motility. To trigger migratory activation - termed hypermigratory phenotype - T. gondii induces GABAergic signalling, which results in calcium fluxes mediated by voltage-gated calcium channels in parasitised DCs and brain microglia. Additionally, a TIMP-1-CD63-ITGB1-FAK signalling axis and signalling via the receptor tyrosine kinase MET promotes sustained hypermigration of parasitised DCs. Recent reports show that the activated signalling pathways converge on the small GTPase Ras to activate the MAPK Erk signalling cascade, a central regulator of cell motility. To date, three T. gondii-derived putative effector molecules have been linked to hypermigration: Tg14-3-3, TgWIP and ROP17. Here, we discuss their impact on the hypermigratory phenotype of phagocytes. Altogether, the emerging concept suggests that T. gondii induces metastasis-like migratory properties in parasitised mononuclear phagocytes to promote infection-related dissemination.

  • 32.
    Ólafsson, Einar B.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ross, Emily C.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Varas-Godoy, Manuel
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63-ITGB1-FAK signaling2019In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 132, no 3, article id UNSP jcs225193Article in journal (Refereed)
    Abstract [en]

    Tissue inhibitor of metalloproteinases-1 (TIMP-1) exerts pleiotropic effects on cells including conferring metastatic properties to cancer cells. As for metastatic cells, recent paradigms of leukocyte migration attribute important roles to the amoeboid migration mode of dendritic cells (DCs) for rapid locomotion in tissues. However, the role of TIMP-1 in immune cell migration and in the context of infection has not been addressed. We report that, upon challenge with the obligate intracellular parasite Toxoplasma gondii, primary DCs secrete TIMP-1 with implications for their migratory properties. Using a short hairpin RNA (shRNA) gene silencing approach, we demonstrate that secreted TIMP-1 and its ligand CD63 are required for the onset of hypermotility in DCs challenged with T. gondii. Further, gene silencing and antibody blockade of the beta 1-integrin CD29 (ITGB1) inhibited DC hypermotility, indicating that signal transduction occurred via ITGB1. Finally, gene silencing of the ITGB1-associated focal adhesion kinase (FAK, also known as PTK2), as well as pharmacological antagonism of FAK and associated kinases SRC and PI3K, abrogated hypermotility. The present study identifies a TIMP-1-CD63-ITGB1-FAK signaling axis in primary DCs, which T. gondii hijacks to drive high-speed amoeboid migration of the vehicle cells that facilitate its systemic dissemination.

  • 33.
    Ólafsson, Einar B.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    ten Hoeve, Arne L.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Li-Wang, Xiaoze
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Westermark, Linda
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Varas-Godoy, Manuel
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Convergent Met and voltage-gated Ca2+ channel signaling drives hypermigration of Toxoplasma-infected dendritic cells2021In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 134, no 5, article id jcs241752Article in journal (Refereed)
    Abstract [en]

    Ras–Erk MAPK signaling controls many of the principal pathways involved in metazoan cell motility, drives metastasis of multiple cancer types and is targeted in chemotherapy. However, its putative roles in immune cell functions or in infections have remained elusive. Here, using primary dendritic cells (DCs) in an infection model with the protozoan Toxoplasma gondii, we show that two pathways activated by infection converge on Ras–Erk MAPK signaling to promote migration of parasitized DCs. We report that signaling through the receptor tyrosine kinase Met (also known as HGF receptor) contributes to T. gondii-induced DC hypermotility. Furthermore, voltage-gated Ca2+ channel (VGCC, subtype CaV1.3) signaling impacted the migratory activation of DCs via calmodulin–calmodulin kinase II. We show that convergent VGCC signaling and Met signaling activate the GTPase Ras to drive Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively) phosphorylation and hypermotility of T. gondii-infected DCs. The data provide a molecular basis for the hypermigratory mesenchymal-to-amoeboid transition (MAT) of parasitized DCs. This emerging concept suggests that parasitized DCs acquire metastasis-like migratory properties that promote infection-related dissemination.

  • 34.
    Ólafsson, Einar B.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Varas-Godoy, Manuel
    Barragan, Antonio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Toxoplasma gondii infection shifts dendritic cells into an amoeboid rapid migration mode encompassing podosome dissolution, secretion of TIMP-1, and reduced proteolysis of extracellular matrix2018In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 20, no 3, article id e12808Article in journal (Refereed)
    Abstract [en]

    Dendritic cells (DCs) infected by Toxoplasma gondii rapidly acquire a hypermigratory phenotype that promotes systemic parasite dissemination by a Trojan horse mechanism in mice. Recent paradigms of leukocyte migration have identified the amoeboid migration mode of DCs as particularly suited for rapid locomotion in extracellular matrix and tissues. Here, we have developed a microscopy-based high-throughput approach to assess motility and matrix degradation by Toxoplasma-challenged murine and human DCs. DCs challenged with T.gondii exhibited dependency on metalloproteinase activity for hypermotility and transmigration but, strikingly, also dramatically reduced pericellular proteolysis. Toxoplasma-challenged DCs up-regulated expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) and their supernatants impaired matrix degradation by naive DCs and by-stander DCs dose dependently. Gene silencing of TIMP-1 by short hairpin RNA restored matrix degradation activity in Toxoplasma-infected DCs. Additionally, dissolution of podosome structures in parasitised DCs coincided with abrogated matrix degradation. Toxoplasma lysates inhibited pericellular proteolysis in a MyD88-dependent fashion whereas abrogated proteolysis persevered in Toxoplasma-infected MyD88-deficient DCs. This indicated that both TLR/MyD88-dependent and TLR/MyD88-independent signalling pathways mediated podosome dissolution and the abrogated matrix degradation. We report that increased TIMP-1 secretion and cytoskeletal rearrangements encompassing podosome dissolution are features of Toxoplasma-induced hypermigration of DCs with an impact on matrix degradation. Jointly, the data highlight how an obligate intracellular parasite orchestrates key regulatory cellular processes consistent with non-proteolytic amoeboid migration of the vehicle cells that facilitate its dissemination.

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