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Sökning: WFRF:(Barragan Antonio)

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1.
  • Ajibola Omotesho, Quadri, et al. (författare)
  • Epigenetic targets to enhance antitumor immune response through the induction of tertiary lymphoid structures
  • 2024
  • Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 15
  • Forskningsöversikt (refereegranskat)abstract
    • Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates found in sites of chronic inflammation such as tumors and autoimmune diseases. The discovery that TLS formation at tumor sites correlated with good patient prognosis has triggered extensive research into various techniques to induce their formation at the tumor microenvironment (TME). One strategy is the exogenous induction of specific cytokines and chemokine expression in murine models. However, applying such systemic chemokine expression can result in significant toxicity and damage to healthy tissues. Also, the TLS formed from exogenous chemokine induction is heterogeneous and different from the ones associated with favorable prognosis. Therefore, there is a need to optimize additional approaches like immune cell engineering with lentiviral transduction to improve the TLS formation in vivo. Similarly, the genetic and epigenetic regulation of the different phases of TLS neogenesis are still unknown. Understanding these molecular regulations could help identify novel targets to induce tissue-specific TLS in the TME. This review offers a unique insight into the molecular checkpoints of the different stages and mechanisms involved in TLS formation. This review also highlights potential epigenetic targets to induce TLS neogenesis. The review further explores epigenetic therapies (epi-therapy) and ongoing clinical trials using epi-therapy in cancers. In addition, it builds upon the current knowledge of tools to generate TLS and TLS phenotyping biomarkers with predictive and prognostic clinical potential.
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2.
  • Barragan, Antonio (författare)
  • Carbohydrate-mediated adhesion in Plasmodium falciparum malaria
  • 2000
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Characterization of virulence factors in P. falciparum malaria is essential in order to identify new therapeutic and prophylactic targets. Rosette formation, the binding of uninfected red blood cells to parasite-infected red blood cells, is a P. falciparum virulence phenotype associated with severe clinical manifestations, e. g. cerebral malaria and severe anemia. Humoral responses to rosetting epitopes seem to confer protective immunity and we have shown in these studies that individuals living in malaria endemic areas build up age-related humoral responses to rosetting epitopes that parallel the development of clinical immunity. Furthermore, epitopes that mediate the rosetting phenotype appear to be diverse but conserved between distant regions in Africa. We have performed a detailed study of the cell-cell binding mechanisms that govern rosetting. Polysaccharides belonging to the glycosaminoglycan family, especially heparin and heparan sulfate, inhibit rosette formation and treatments of host cells indicate that heparan sulfate-like glycans support adhesion. In addition, the ABO blood group phenotype of the infected host modulates rosetting and blood group A and B antigens have been shown to function as co-receptors to other receptors in rosetting. The parasite-derived adhesion molecule that mediates rosetting has been identified as a Plasmodium falciparum erythrocyte membrane protein I (PfEMP1) variant, a product of the vast family of var genes. PfEMP1 mediates adhesion by interacting with host cell glycan receptors, such as heparan sulfate and blood group A antigen. We have characterized the binding of heparan sulfate and heparin to the rosetting domain of PfEMP1. Important molecular features of oligosaccharides required for optimal binding, such as molecular size (12-mer oligosaccharide chain or larger) and N-sulfation, have thus been identified. In field studies, the heparin-binding phenotype of P. falciparum was found to be more common among patients with severe malaria. Furthermore, the clinical isolates had the ability to adhere to multiple receptors. We have characterized the binding properties of PfEMP1. to multiple host receptors, which may explain the poly-adhesive P. falciparum phenotype associated with severe disease in these studies. The present investigation contributes to the molecular elucidation of virulent adhesive phenotypes in P. falciparum malaria. The identified molecules involved in the cell-cell adherence may serve as targets for prophylactic and therapeutic measures.
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3.
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4.
  • Barragan, Antonio, et al. (författare)
  • Erythrocyte Glycans as Plasmodium falciparum Rosetting Receptors : Molecular Background of Strain Specific Rosette Disruption by Glycosaminoglycans and Sulfated Glycoconjugates
  • 1999
  • Ingår i: Experimental parasitology. - : Elsevier BV. - 0014-4894 .- 1090-2449. ; 91:2, s. 133-143
  • Tidskriftsartikel (refereegranskat)abstract
    • Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, is a virulent parasite phenotype associated with the occurrence of severe malaria, e.g., cerebral malaria. Compounds with specific anti-rosetting activity are potential therapeutic agents. Glycosaminoglycans and sulfated glycoconjugates were found to disrupt rosettes in a strain- and isolate-specific manner. Rosette disruption was strongly connected to the presence of N-sulfate groups in heparin/heparan sulfate as demonstrated by modified heparin preparations. This finding was corroborated by the disruption of rosettes with mono- and disaccharides derived from heparin/heparan sulfate that contained N-sulfated glucosamine. Furthermore, heparinase III treatment of erythrocyte cultures infected by FCR3S1 (and to some extent TM 284) P. falciparum strains abolished rosetting. Heparinase III treatment of the uninfected erythrocytes prior to mixing with the infected culture impeded formation of rosettes, indicating that the rosetting receptors at least partially are of glycosaminoglycan nature.
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5.
  • Barragan, Antonio, et al. (författare)
  • GABAergic signalling in the immune system
  • 2015
  • Ingår i: Acta Physiologica. - : Wiley. - 1748-1708 .- 1748-1716. ; 213:4, s. 819-827
  • Forskningsöversikt (refereegranskat)abstract
    • 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.
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6.
  • Barragan, Carlos Eduardo, et al. (författare)
  • Isolation of Arsenic Resistant and Arsenopyrite Oxidizing Acidithiobacillus Species from pH Neutral Colombian Mine Effluents
  • 2020
  • Ingår i: Geomicrobiology Journal. - : Taylor & Francis Group. - 0149-0451 .- 1521-0529. ; 37:7, s. 682-689
  • Tidskriftsartikel (refereegranskat)abstract
    • Inactive mines provide a great source of bacterial diversity for studying acidophilic communities and their biotechnological applications, but prospecting of these anthropogenic environments in Colombia has been limited. Conventional microbiological methods were used to isolate acidophilic bacterial strains from effluents emanating from the Colombian gold mine 'El Zancudo' (Titiribi, Antioquia). Despite the drainage waters having circumneutral pH, all of the isolated strains were phylogenetically related to the extreme acidophile Acidithiobacillus genus. However, based upon 16S rRNA gene sequences the mesophilic sulfur-oxidizing indigenous strains could not be assigned to a species. Pure cultures were selected by screening in medium with soluble inorganic arsenic (III) and their mineral-oxidative activity was evaluated at 30 degrees C in Erlenmeyer flasks with arsenopyrite ore under rotary shaking conditions. The indigenous strains were able to catalyze arsenopyrite oxidation in a mixed culture with a pulp density of 10%, maintaining their growth in the presence of >80 mM leached arsenic. This research provides information regarding the isolation of arsenic resistant bacterial communities from neutral effluents from El Zancudo mine and the possibility of the isolated strains to be useful in the biooxidation pretreatment of refractory gold-bearing arsenopyrite ores and concentrates.
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7.
  • Barragan, Carlos Eduardo, et al. (författare)
  • RNA transcript response by an Acidithiobacillus spp. mixed culture reveals adaptations to growth on arsenopyrite
  • 2021
  • Ingår i: Extremophiles. - : Springer. - 1431-0651 .- 1433-4909. ; 25, s. 143-158
  • Tidskriftsartikel (refereegranskat)abstract
    • Biooxidation of gold-bearing refractory mineral ores such as arsenopyrite (FeAsS) in stirred tanks produces solutions containing highly toxic arsenic concentrations. In this study, ferrous iron and inorganic sulfur-oxidizing Acidithiobacillus strain IBUN Ppt12 most similar to Acidithiobacillus ferrianus and inorganic sulfur compound oxidizing Acidithiobacillus sp. IBUNS3 were grown in co-culture during biooxidation of refractory FeAsS. Total RNA was extracted and sequenced from the planktonic cells to reveal genes with different transcript counts involved in the response to FeAsS containing medium. The co-culture's response to arsenic release during biooxidation included the ars operon genes that were independently regulated according to the arsenopyrite concentration. Additionally, increased mRNA transcript counts were identified for transmembrane ion transport proteins, stress response mechanisms, accumulation of inorganic polyphosphates, urea catabolic processes, and tryptophan biosynthesis. Acidithiobacillus spp. RNA transcripts also included those encoding the Rus and PetI proteins involved in ferrous iron oxidation and gene clusters annotated as encoding inorganic sulfur compound metabolism enzymes. Finally, mRNA counts of genes related to DNA methylation, management of oxidative stress, chemotaxis, and motility during biooxidation were decreased compared to cells growing without mineral. The results provide insights into the adaptation of Acidithiobacillus spp. to growth during biooxidation of arsenic-bearing sulfides.
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8.
  • Bhandage, Amol, 1988-, et al. (författare)
  • A motogenic GABAergic system of mononuclear phagocytes facilitates dissemination of coccidian parasites
  • 2020
  • Ingår i: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 9
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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9.
  • Bhandage, Amol, 1988-, et al. (författare)
  • Calling in the CaValry-Toxoplasma gondii Hijacks GABAergic Signaling and Voltage-Dependent Calcium Channel Signaling for Trojan horse-Mediated Dissemination
  • 2019
  • Ingår i: Frontiers in Cellular and Infection Microbiology. - : Frontiers Media S.A.. - 2235-2988. ; 9
  • Tidskriftsartikel (refereegranskat)abstract
    • 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 γ-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 is mediated via the L-type voltage-dependent calcium channel (L-VDCC) subtype Cav1.3. These studies have revealed that DCs possess a GABA/L-VDCC/Cav1.3 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 and microglia. 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.
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10.
  • Bhandage, Amol, 1988-, et al. (författare)
  • GABAergic signaling by cells of the immune system : more the rule than the exception.
  • 2021
  • Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 78:15, s. 5667-5679
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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11.
  • Bhandage, Amol K., et al. (författare)
  • GABAergic signaling in human and murine NK cells upon challenge with Toxoplasma gondii
  • 2021
  • Ingår i: Journal of Leukocyte Biology. - : Oxford University Press (OUP). - 0741-5400 .- 1938-3673. ; 110:4, s. 617-628
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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12.
  • Bhandage, Amol K., et al. (författare)
  • Toxoplasma-Induced Hypermigration of Primary Cortical Microglia Implicates GABAergic Signaling
  • 2019
  • Ingår i: Frontiers in Cellular and Infection Microbiology. - : Frontiers Media SA. - 2235-2988. ; 9
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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13.
  • Fuks, Jonas M, et al. (författare)
  • GABAergic Signaling Is Linked to a Hypermigratory Phenotype in Dendritic Cells Infected by Toxoplasma gondii
  • 2012
  • Ingår i: PLoS pathogens. - : Public Library of Science (PLoS). - 1553-7374. ; 8:12, s. e1003051-
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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14.
  • García-Sanchez, Marta, et al. (författare)
  • Differential Responses of Bovine Monocyte-Derived Macrophages to Infection by Neospora caninum Isolates of High and Low Virulence
  • 2019
  • Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 10
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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15.
  • Guldevall, Karolin (författare)
  • Development of Microchip-based Assays to Study Immune Cell Interactions at the Single Cell Level
  • 2011
  • Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Immune cell populations are constantly divided into smaller and smaller subsets defined by newly emerging cellular markers. However, there is a growing awareness of the functional heterogeneities in between cells even within small populations, in addition to the heterogeneity over time. One may ask whether a population is correctly defined only by cellular markers or if the functionality should be regarded as well? Many of today’s techniques only measures at the population level, giving an average estimate of the behavior of that pool of cells, but failing to detect rare possibly important events. Thus, high-throughput experimental approaches to analyze single cells over time are required to address cellular heterogeneity. Progress in the fields of microfabrication, microscopy and computing have paved the way for increasingly efficient tools for studies on the single cell level, and a variety of devices have been described by others. However, few of them are suitable for long-term imaging of dynamic events such as cell-cell interactions or migration. In addition, for efficient recording of many individual events it is desirable to scale down the cells’ interaction volume; not only to shorten the time to interaction, but also to increase the number of individual events in a given area; thereby pushing a screening approach. To address these questions, a complete microwell array system for imaging of immune cell responses with single-cell resolution was designed. The platform consists of a range of silicon-glass microchips with arrays of miniature wells for incubation of cells and a custom made holder that fits conventional microscopes. The device has been designed to allow cells to be kept viable for several days in the wells, to be easy to use and to allow high-resolution imaging. Five different designs were fabricated; all with a specific type of assay in mind, and were evaluated regarding biocompatibility and functionality. One design is aimed towards screening applications, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated; this program is also described and evaluated. We here show that our silicon microwell platform allows long-term studies (up to several days), with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using time-lapse imaging we confirmed immune cell heterogeneity in NK cell populations regarding both cytotoxicity and migrational behavior. The automatic counting program was tested and showed similar results compared to both manual counting and FACS. In addition, the large numbers of wells that can be simultaneously imaged, provide new statistical information that will lead to a better understanding of the function and regulation of the immune system at the single cell level. Altogether, our technique enables novel types of cellular imaging assays allowing data collection at a level of resolution not previously obtained – this was shown to be important for performing basic cell biological studies, but may also prove valuable in the proposed future medical applications.
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16.
  • Hildebrandt, Franziska, 1994-, et al. (författare)
  • scDual-Seq of Toxoplasma gondii-infected mouse BMDCs reveals heterogeneity and differential infection dynamics
  • 2023
  • Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 14
  • Tidskriftsartikel (refereegranskat)abstract
    • 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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17.
  • Hildebrandt, Franziska, 1994-, et al. (författare)
  • Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the mouse liver
  • 2021
  • Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 12:1
  • Tidskriftsartikel (refereegranskat)abstract
    • 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. Global transcriptional differences across lobular units in the liver remain unknown. Here the authors perform spatial transcriptomics of liver tissue to delineate transcriptional differences in physical space, confirm lobular zonation along transcriptional gradients and suggest the presence of previously uncharacterized structures within liver tissue.
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18.
  • Idro, Richard, et al. (författare)
  • Neuroimmunology of Common Parasitic Infections in Africa
  • 2022
  • Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 13
  • Forskningsöversikt (refereegranskat)abstract
    • 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 falciparum, Toxoplasma gondii, Trypanosoma 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.
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19.
  • Kanatani, Sachie, 1982- (författare)
  • Host-parasite interactions in the dissemination of Toxoplasma gondii
  • 2017
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Toxoplasma gondii is an obligate intracellular parasite that infects virtually all warm-blooded organisms. Systemic dissemination of T. gondii in the organism can cause life-threatening infection that manifests as Toxoplasma encephalitis in immune-compromised patients. In addition, mounting evidence from epidemiological studies indicates a link between chronic Toxoplasma infection and mental disorders. To better understand the pathogenesis of toxoplasmosis, basic knowledge on the host-parasite interactions and the dissemination mechanisms are essential. Previous findings have established that, upon infection with T. gondii, dendritic cells (DCs) and microglia exhibit enhanced migration, which was termed the hypermigratory phenotype. As a result of this enhanced migration, DCs and microglia are used as vehicle cells for dissemination (‘Trojan horse’) which potentiates dissemination of T. gondii in mice. However, the precise mechanisms behind the hypermigratory phenotype remained unknown. In this thesis, we characterized host-parasite interactions upon infection with T. gondii and investigated the basic mechanisms behind the hypermigratory phenotype of T. gondii-infected DCs and microglia.In paper I, we observed that upon infection with T. gondii, DCs underwent rapid morphological changes such as loss of adhesiveness and podosomes, with integrin redistribution. These rapid morphological changes were linked to hypermotility and were induced by active invasion of T. gondii within minutes. T. gondii-infected DCs exhibited up-regulation of the C-C chemokine receptor CCR7 and chemotaxis towards the CCR7 chemotactic cue, CCL19.In paper II, we developed a 3-dimensional migration assay in a collagen matrix, which allowed us to characterize the hypermigratory phenotype in a more in vivo-like environment. The migration of T. gondii-infected DCs exhibited features consistent with integrin-independent amoeboid type of migration. T. gondii-induced hypermigration of DCs was further potentiated in the presence of CCL19 in a 3D migration assay.In paper III, we identified a parasite effector molecule, a Tg14-3-3 protein derived from parasite secretory organelles. Tg14-3-3 was sufficient to induce the hypermigratory phenotype. Transfection with Tg14-3-3-containing fractions or recombinant Tg14-3-3 protein induced the hypermigratory phenotype in primary DCs and in a microglial cell line. In addition, Tg14-3-3 localized in the parasitophorous vacuolar space and host 14-3-3 proteins were rapidly recruited around the parasitophorous vacuole.In paper IV, we found that mouse DCs dominantly express the L-type voltage-dependent calcium channel, Cav1.3. Cav1.3 was linked to the GABAergic signaling-induced hypermigratory phenotype. Pharmacological inhibition of Cav1.3 and knockdown of Cav1.3 abolished the hypermigratory phenotype in T. gondii infected DCs. Blockade of voltage-dependent calcium channels reduced the dissemination of T. gondii in a mouse model.In paper V, we showed that microglia, resident immune cells in the brain, also exhibited rapid morphological changes and hypermotility upon infection with T. gondii. However, an alternative GABA synthesis pathway was shown to be involved in the hypermigratory phenotype in microglia.In summary, this thesis describes novel host-parasite interactions, including host cell migratory responses and key molecular mechanisms that mediate the hypermigratory phenotype. The findings define a novel motility-related signaling axis in DCs. Thus, T. gondii employs GABAergic non-canonical pathways to hijack host cell migration and facilitate dissemination. We believe that these findings represent a significant step forward towards a better understanding of the pathogenesis of T. gondii infection.
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20.
  • Kanatani, Sachie, et al. (författare)
  • Infection by Toxoplasma gondii Induces Amoeboid-Like Migration of Dendritic Cells in a Three-Dimensional Collagen Matrix
  • 2015
  • Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:9
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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21.
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22.
  • Kanatani, Sachie, et al. (författare)
  • Voltage-dependent calcium channel signaling mediates GABA(A) receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii
  • 2017
  • Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 13:12
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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23.
  • Lundqvist, Jenny, 1975- (författare)
  • Malaria and relapsing fever Borrelia : interactions and potential therapy
  • 2009
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Infectious diseases such as malaria and relapsing fever borreliosis (RF), cause severe human mortality and morbidity in developing countries. Malaria, caused by Plasmodium spp. parasites, is estimated by the World Health Organization to cause 1.5-2.7 million deaths annually. RF, caused by Borrelia spirochetes, has the highest prevalence described for any bacterial disease in Africa, with infection outcomes ranging from asymptomatic to fatal. RF borreliosis manifests in humans as a recurring fever and with other symptoms very similar to those of malaria. RF borreliosis has been regarded as a transient infection of the blood. However, B. duttonii exploits the brain as an immunoprivileged site escaping the host immune response while spirochetes in the blood are cleared. To investigate whether residual bacteria are dormant or actively dividing, mice with residual brain infection were administered ceftriaxone, a β-lactam antibiotic interfering with cell wall synthesis. Hence, it only affects actively dividing bacteria. Ceftriaxone eradicated brain RF infection in all treated mice, demonstrating that the bacteria are actively multiplying rather than in a dormant state. The findings support the therapeutic use of ceftriaxone for RF neuroborreliosis since penetration into cerebrospinal fluid is greater for ceftriaxone than for the often recommended doxycycline. The clinical features of malaria and RF are similar and diagnosis is further complicated by the frequently occurring concomitant malaria-RF infections. Therefore, we established a mouse model to study the pathogenesis and immunological response to Plasmodium/Borrelia mixed infection. Interestingly, malaria was suppressed in the co-infected animals whereas spirochete numbers were elevated 21-fold. The immune response in the concomitantly infected mice was polarized towards malaria leaving the spirochetes unharmed. Mice with co-infections also exhibited severe anemia and internal damages, probably attributed to escalating spirochete numbers. A secondary malaria infection reactivated the residual brain RF infection in 60% of the mice. This highlights the importance of co-infections as diagnostic pitfalls as well as the need for novel treatment strategies. Currently there is no commercial malaria vaccine and increasing drug resistance presents an urgent need for new malaria chemotherapeutics. Blood-stage malaria parasites are rapidly growing with high metabolic and biosynthetic activity, making them highly sensitive to limitations in polyamine supply. Disrupting polyamine synthesis in vivo with trans-4-methylcyclohexylamine (4MCHA) eradicated the malaria infection gradually, resulting in protective immunity. This leads the way for further biochemical and pharmacological development of the polyamine inhibitor 4MCHA and similar compounds as antimalarial drugs
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24.
  • Mulas, Floriana, et al. (författare)
  • The deubiquitinase OTUB1 augments NF-κB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13
  • 2021
  • Ingår i: Cellular & Molecular Immunology. - : Springer Science and Business Media LLC. - 1672-7681 .- 2042-0226. ; 18, s. 1512-1527
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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25.
  • Ólafsson, Einar B., et al. (författare)
  • Convergent Met and voltage-gated Ca2+ channel signaling drives hypermigration of Toxoplasma-infected dendritic cells
  • 2021
  • Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 134:5
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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26.
  • Ólafsson, Einar B., 1988- (författare)
  • Signaling determinants in Trojan horse-mediated dissemination of Toxoplasma gondii
  • 2019
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Toxoplasma gondii is an obligate intracellular parasite that infects all warm-blooded vertebrates including one third of the global human population. While infection is typically asymptomatic in healthy human hosts, reactivated and acute infection in immunosuppressed or immunecompromised individuals can lead to lethal toxoplasmic encephalitis After ingestion, the parasite crosses the intestinal epithelium and rapidly achieves systemic dissemination, ultimately establishing chronic infection in the brain. Shortly after crossing the intestinal epithelium T. gondii encounters dendritic cells (DCs). Paradoxically, T. gondii tachyzoites exploit the inherent migratory ability of DCs for dissemination via a “Trojan horse” mechanism. Within minutes of active invasion by T. gondii tachyzoites, DCs adopt a hypermigratory phenotype that mediates rapid systemic dissemination of T. gondii in mice. Previous studies have demonstrated that the hypermigratory phenotype involves cytoskeletal rearrangement, redistribution of integrins and high-velocity in vitro cell migration (termed hypermotility), which is initiated by GABAergic signaling. However, the downstream effectors of GABAergic signaling in parasitized DCs remain enigmatic. Leukocyte migration often relies on adhesion and proteolysis of extracellular matrix (ECM). However, the role of ECM proteolysis in hypermigration has not been addressed. In this thesis, the migratory activation of T. gondii-infected DCs is characterized in terms of cell signaling and ECM proteolysis.In paper I we demonstrate that MMP-mediated proteolytic activity of DCs is abolished upon T. gondii infection. To investigate DC pericellular proteolysis at the single cell level, we developed a high-content imaging and automated image analysis method. With pharmacological inhibitors and gene silencing, we show that T. gondii-infected DCs lose the ability to degrade ECM through the upregulation of TIMP1 and the loss of podosome structures.In paper II we show that the hypermigratory phenotype induced by GABAergic signaling in T. gondii-infected DCs is dependent on L-type voltage dependent Ca2+ channel (L-VDCC) activation, predominantly CaV1.3. Pharmacological antagonism of CaV1.3 and gene silencing of cav1.3 blocked hypermotility. Further, inhibition of L-VDCCs with benidipine significantly reduced T. gondii dissemination in a mouse model.In paper III we address the impact of TIMP1 on the migratory activation of T. gondii-infected DCs. Using pharmacological antagonism and shRNA-mediated gene silencing, we demonstrate that secreted TIMP1 induces motility and migration in T. gondii-infected DCs by activating ITGB1-FAK signaling through interactions with CD63.In paper IV we report that the GTPase Ras functions as a molecular switch in the migratory activation of T. gondii-infected DCs. We identify that VDCC-CaM-CaMkII and Met signaling converge on Ras-mediated Erk phosphorylation leading to migratory activation of T. gondii-infected DCs.In summary, my thesis details novel host signaling pathways hijacked by the protozoan parasite T. gondii in Trojan horse DCs for dissemination. Through the investigation of host-parasite interactions, we shed new light on mechanisms that govern leukocyte migration and strategies employed by T. gondii to achieve pervasive dissemination. Gaining further insights into the biology that underlies T. gondii pathogenesis and persistence will help ameliorate toxoplasmosis in at-risk groups.
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27.
  • Ólafsson, Einar B., et al. (författare)
  • The unicellular eukaryotic parasite Toxoplasma gondii hijacks the migration machinery of mononuclear phagocytes to promote its dissemination
  • 2020
  • Ingår i: Biology of the Cell. - : Wiley. - 0248-4900 .- 1768-322X. ; 112:9, s. 239-250
  • Forskningsöversikt (refereegranskat)abstract
    • 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.
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28.
  • Ólafsson, Einar B., et al. (författare)
  • TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63-ITGB1-FAK signaling
  • 2019
  • Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 132:3
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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29.
  • Ólafsson, Einar B., et al. (författare)
  • Toxoplasma gondii infection shifts dendritic cells into an amoeboid rapid migration mode encompassing podosome dissolution, secretion of TIMP-1, and reduced proteolysis of extracellular matrix
  • 2018
  • Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 20:3
  • Tidskriftsartikel (refereegranskat)abstract
    • 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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30.
  • Olivera, Gabriela Carina, et al. (författare)
  • Blood-brain barrier-restricted translocation of Toxoplasma gondii from cortical capillaries
  • 2021
  • Ingår i: eLIFE. - 2050-084X. ; 10
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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31.
  • Pelosi, Benedetta (författare)
  • The labyrinth of protein classification: a pipeline forselection and classification of biological data
  • 2022
  • Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Recent progress in fundamental biological sciences and medicine has considerably increased the quantity ofdata that can be studied and processed. The main limitation now is not retrieving data, but rather extractinguseful biological insights from the large datasets accumulated. More recent advances have provided detailedhigh-density data regarding metabolism (metabolomics) and protein expression (proteomics). Clearly, no single analytic methods, can provide a comprehensive understanding. Rather, the ability to link available datatogether in a coherent manner is required to obtain a complete view. The improving application of MachineLearning (ML) techniques provides the means to make continuous progress in processing complex data sets.A brief discussion is offered on the advantages of ML, the state-of-the-art in Deep Learning (DL) for proteinpredictions and the importance of ML in biological data processing. Noise stemming from incorrect classification or arbitrary/ambiguous labelling of data may arise when ML techniques are applied to large datasets. Furthermore, the stochasticity of biological systems needs to be considered for correctly evaluating theoutputs. Here we show the potential of a workflow to respond biological questions taking into consideration aperturbation of the biological data.  For controlling the applicability of models and maximizing the predictivity, in silico filtering schemescan usefully be applied as an “Ockham’s razor” before using any ML technique. After reviewing differentDL approaches for protein prediction purposes, this work shows that a computational approach in filteringsteps is a valuable tool for proteins classification when biological features are not fully annotated or reviewed.The in silico approach has identified putative proline transporters in fungi and plants as well as carotenoidbiosynthetic gene products in the plant family Brassicaceae. The proposed method is suitable for extractingfeatures of classification and then maximizing the use of a DL approach. 
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32.
  • Ross, Emily C., et al. (författare)
  • Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers
  • 2019
  • Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 21:9
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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33.
  • Ross, Emily Charlotte, 1991-, et al. (författare)
  • Early passage of Toxoplasma gondii across the blood–brain barrier
  • 2022
  • Ingår i: Trends in Parasitology. - : Elsevier BV. - 1471-4922 .- 1471-5007. ; 38:6, s. 450-461
  • Forskningsöversikt (refereegranskat)abstract
    • 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.
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34.
  • Ross, Emily C., et al. (författare)
  • Integrin-dependent migratory switches regulate the translocation of Toxoplasma-infected dendritic cells across brain endothelial monolayers
  • 2021
  • Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 78, s. 5197-5212
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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35.
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36.
  • Ross, Emily Charlotte, 1991-, et al. (författare)
  • Toxoplasma effector-induced ICAM-1 expression by infected dendritic cells potentiates transmigration across polarised endothelium
  • 2022
  • Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 13
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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37.
  • Ross, Emily C., 1991- (författare)
  • Transmigration of Toxoplasma gondii across biological barriers
  • 2022
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Toxoplasma gondii is an obligate intracellular parasite that can likely infect all warm-blooded vertebrates, with estimates of up to 30% of the global human population being infected. Although infection with T. gondii is usually asymptomatic or mild, in immunocompromised individuals infection can lead to lethal toxoplasmic encephalitis. Infection acquired during pregnancy can also lead to serious ocular or neurological damage and even death of the foetus. Following ingestion, the parasite is able to cross the first biological barrier it encounters, the gut epithelium and convert to the rapidly replicating tachyzoite stage. It can then disseminate throughout the body of the host, eventually reaching sites such as the brain, after crossing the blood-brain barrier (BBB). Previous findings have shown that T. gondii can use leukocytes, such as dendritic cells (DCs), for dissemination via a “Trojan horse”-type mechanism, but how T. gondii then crosses restrictive barriers such as the BBB is still not fully understood. The overall objective of this work has been to investigate how T. gondii crosses biological barriers and how infection impacts host cell signalling.In paper I we demonstrate that T. gondii can cross polarised cell monolayers without significantly perturbing barrier integrity. Reduced phosphorylation of focal adhesion kinase (FAK) was observed in cell monolayers upon T. gondii challenge, and inhibition or gene silencing of FAK (Ptk2) facilitated transmigration of T. gondii across polarised cell monolayers. In paper II we found that upon T. gondii infection of DCs, secreted TIMP-1 induces hypermotility by activating β1 integrin-FAK signalling through interactions with CD63. In paper III we show that T. gondii can cross polarised endothelial cell monolayers inside DCs. We also report that parasitised DCs on endothelium do not display a hypermotile phenotype, switching to integrin-dependent motility. Blockade of β1 and β2 integrins or ICAM-1, and gene silencing of β1 (Itgb1) or talin (Tln1) restored infected-DC motility, and reduced the frequency of transmigration of T. gondii-challenged DCs across endothelium. In paper IV we demonstrate that, shortly after T. gondii inoculation in mice, parasites mainly localised to cortical capillaries of the brain. Early invasion to the brain parenchyma occurred in absence of a significant increase in BBB permeability, perivascular leukocyte cuffs or haemorrhage. Further, pharmacological inhibition or endothelial cell-specific knockout of FAK facilitated parasite transmigration to the brain parenchyma. In paper V we report that DCs challenged with type II T. gondii transmigrate across polarised endothelial cell monolayers at a higher frequency than type I T. gondii, while type I infected DCs exhibited increased migratory velocities on endothelium. We also show that T. gondii-induced upregulation of ICAM-1 in DCs is genotype-dependent, and requires the T. gondii secreted effector GRA15. Finally, gene silencing of leukocyte ICAM-1 (Icam-1) or deletion of T. gondii GRA15 reduced transmigration across endothelial cell monolayers.In summary, the work in this thesis provides novel insights into how T. gondii can potentially cross biological barriers on its journey to the brain. We find that T. gondii can cross polarised monolayers both as free parasites and using DCs as a “Trojan horse”, and identify new ways in which T. gondii can alter host cell dynamics to benefit its own dissemination.
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38.
  • Sangaré, Lamba Omar, et al. (författare)
  • In Vivo CRISPR Screen Identifies TgWIP as a Toxoplasma Modulator of Dendritic Cell Migration
  • 2019
  • Ingår i: Cell Host and Microbe. - : Elsevier BV. - 1931-3128 .- 1934-6069. ; 26:4, s. 478-492
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
39.
  • Schlüter, Dirk, et al. (författare)
  • Advances and Challenges in Understanding Cerebral Toxoplasmosis
  • 2019
  • Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 10
  • Forskningsöversikt (refereegranskat)abstract
    • 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.
  •  
40.
  • Shen, Xianli, et al. (författare)
  • Glioma-induced inhibition of caspase-3 in microglia promotes a tumor-supportive phenotype
  • 2016
  • Ingår i: Nature Immunology. - : Springer Science and Business Media LLC. - 1529-2908 .- 1529-2916. ; 17:11, s. 1282-1290
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
41.
  • Sultana, M. Afifa, et al. (författare)
  • Downmodulation of Effector Functions in NK Cells upon Toxoplasma gondii Infection
  • 2017
  • Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 85:10
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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42.
  • ten Hoeve, Arne L., MSc, 1989- (författare)
  • Modulation of mononuclear phagocyte function in the dissemination of Toxoplasma gondii
  • 2022
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Toxoplasma gondii is an obligate intracellular Apicomplexan parasite that could possibly infect all warm-blooded animals. Acute infections with T. gondii produce generally mild symptoms in healthy individuals, but infections of the fetus during pregnancy and infections in those that are immunocompromised can cause severe and life-threatening pathology. The parasite gains entry to the host by crossing the biological barriers of the intestine or placenta. Mononuclear phagocytes (MPs) and other leukocytes at these barriers become infected by T. gondii. Previous work has established that upon T. gondii infection, dendritic cells (DCs) undergo morphological and phenotypical changes and display enhanced migration. Components of this ‘hypermigratory phenotype’ have been confirmed in other MPs, such as monocytes, macrophages and microglia. The mechanisms underlying the hypermigratory phenotype are the subject of this thesis.In paper I, we describe a non-canonical extended upregulation of the transcription factor Egr1 in T. gondii-infected DCs. While the rapid and transient canonical induction of Egr1 depends on the ERK1/2-pathway, the extended upregulation was dependent on p38 MAPK and p38-activating parasite-derived effector GRA24. The hypermotility component of the hypermigratory phenotype did not depend on GRA24/p38, but on ERK1/2. We determined that EGR1 acts as an inhibitor of phenotypic maturation and that GRA24 stimulates Il2 and Il12p40 expression in T. gondii-infected DCs.In paper II, we characterize actors upstream of ERK1/2 in hypermotility of T. gondii-infected DCs. Two axes that output on the ERK1/2 pathway were found to be required for hypermotility. The first involves Ca2+ influx through voltage-gated calcium channel Cav1.3, resulting in activation of calcium/calmodulin-dependent protein kinase II (CaMKII) via Ca2+ sensor calmodulin (CaM). The other axis relies on hepatocyte growth factor (HGF), which is secreted by DCs, and its receptor Met. Both axes converge on the ERK1/2 pathway via the GTPase Ras.In paper III, we study the migratory behavior of T. gondii-infected DCs on and across endothelial cell monolayers. Particularly infected DCs transmigrated across endothelial cell monolayers, but were, unlike on 2D surfaces, not hypermotile on endothelial cells. We characterize the differential involvement of β1 and β2 integrins, cell adhesion molecules ICAM-1 and PECAM-1 and pan-integrin-cytoskeleton linker talin in transmigration across endothelial cells and in migration on endothelial cells and 2D surfaces.Finally, we report in paper IV that T. gondii imparts a DC-like transcriptional signature on infected macrophages. Infected macrophages upregulate chemokine receptor CCR7 and display chemotaxis to CCR7-ligand CCL19, like DCs. Concomitantly, these macrophages upregulate the expression of transcription factors associated with DCs and of immune activation-related genes and markers. T. gondii-infected macrophages thus display a remarkable transcriptional and functional plasticity. We identify GRA28 as the primary T. gondii-derived effector protein responsible for these phenotypes, with parasite-derived ROP16 having partially opposing effects.Altogether, my thesis identifies novel aspects of the hypermigratory phenotype in T. gondii-infected MPs and provides insights into the molecular components and signaling that underlie them.
  •  
43.
  • ten Hoeve, Arne L., et al. (författare)
  • Sustained Egr-1 Response via p38 MAP Kinase Signaling Modulates Early Immune Responses of Dendritic Cells Parasitized by Toxoplasma gondii
  • 2019
  • Ingår i: Frontiers in Cellular and Infection Microbiology. - : Frontiers Media SA. - 2235-2988. ; 9
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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44.
  •  
45.
  • ten Hoeve, Arne L., MSc, et al. (författare)
  • The Toxoplasma effector GRA28 promotes parasite dissemination by inducing dendritic cell-like migratory properties in infected macrophages
  • 2022
  • Ingår i: Cell Host and Microbe. - : Elsevier BV. - 1931-3128 .- 1934-6069. ; 30:11, s. 1570-1588.e7
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
46.
  • Theopold, Ulrich, et al. (författare)
  • TRP channels, the missing link for Ca2+ tuning by a unicellular eukaryotic parasite?
  • 2021
  • Ingår i: Cell Calcium. - : Elsevier BV. - 0143-4160 .- 1532-1991. ; 98
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
47.
  • Tjärnlund, Anna, 1975- (författare)
  • Does IgA play a role in protection against pulmonary tuberculosis?
  • 2005
  • Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • More than a century after the identification of the tubercle bacillus and the first attempts at vaccination, tuberculosis (TB) still remains one of the world’s most serious infectious diseases. TB is typically a disease of the lung, which serves both as port of entry and as the major site of disease manifestation. The currently used vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is administered parentally and induces a systemic immune response. However, it fails to protect against pulmonary TB, thereby raising the question whether vaccination targeting the mucosal immunity in the lungs could be favourable.The respiratory mucosal surfaces represent the first line of defence against a multitude of pathogens. Secretory IgA (sIgA) in mucosal secretions has an important function by blocking entrance of pathogenic organisms and preventing infections. Yet, another role for IgA in protection against intracellular pathogens has lately been appreciated, when sIgA was demonstrated to neutralize viruses intracellulary. We aimed to investigate the relevance of sIgA in protection against mycobacterial infections using mice deficient for IgA and the polymeric Ig receptor. Mice were immunized intranasally with a mycobacterial antigen which elicited, in wild-type mice, a strong IgA response in mucosal secretions in the respiratory tract. Gene-targeted mice failed to induce the same response and more importantly, were more susceptible to mycobacterial infections in the respiratory tract, as demonstrated by higher bacterial loads in the lungs than wild-type mice. Analysis of immune responses after infection revealed reduced production of proinflammatory, and protective, factors such as IFN-γ and TNF-α in the lungs of deficient mice, which was in concordance with the higher bacterial burden seen in the lungs of these mice. The mechanisms explaining the defective proinflammatory responses in the lungs of deficient mice are not clear but might involve impaired signalling through Fcα receptors, or homologous receptors, which could lead to inadequate activation of pulmonary macrophages. This could subsequently result in suboptimal induction and production of cytokines and chemokines important for attraction and migration of cells to sites of infection in the lungs.Our results demonstrate a role for IgA in protection against mycobacterial infection in the respiratory tract by blocking the entrance of the mycobacterium into the lungs, and/or by modulating the locally induced proinflammatory immune responses.
  •  
48.
  • Vogt, Anna M, et al. (författare)
  • Heparan sulfate on endothelial cells mediates the binding of Plasmodium falciparum-infected erythrocytes via the DBL1alpha domain of PfEMP1
  • 2003
  • Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 101:6, s. 2405-2411
  • Tidskriftsartikel (refereegranskat)abstract
    • Plasmodium falciparum may cause severe forms of malaria when excessive sequestration of infected and uninfected erythrocytes occurs in vital organs. The capacity of wild-type isolates of P falciparum-infected erythrocytes (parasitized red blood cells [pRBCs]) to bind glycosaminoglycans (GAGs) such as heparin has been identified as a marker for severe disease. Here we report that pRBCs of the parasite FCR3S1.2 and wild-type clinical isolates from Uganda adhere to heparan sulfate (HS) on endothelial cells. Binding to human umbilical vein endothelial cells (HUVECs) and to human lung endothelial cells (HLECs) was found to be inhibited by HS/heparin or enzymes that remove HS from cell surfaces. (35)S-labeled HS extracted from HUVECs bound directly to the pRBCs' membrane. Using recombinant proteins corresponding to the different domains of P falciparum erythrocyte membrane protein 1 (PfEMP1), we identified Duffy-binding-like domain-1alpha (DBL1alpha) as the ligand for HS. DBL1alpha bound in an HS-dependent way to endothelial cells and blocked the adherence of pRBCs in a dose-dependent manner. (35)S-labeled HS bound to DBL1alpha-columns and eluted as a distinct peak at 0.4 mM NaCl. (35)S-labeled chondroitin sulfate (CS) of HUVECs did not bind to PfEMP1 or to the pRBCs' membrane. Adhesion of pRBCs of FCR3S1.2 to platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, mediated by the cysteine-rich interdomain region 1alpha (CIDR1alpha), was found be operative with, but independent of, the binding to HS. HS and the previously identified HS-like GAG on uninfected erythrocytes may act as coreceptors in endothelial and erythrocyte binding of rosetting parasites, causing excessive sequestration of both pRBCs and RBCs.
  •  
49.
  • Weidner, Jessica M., et al. (författare)
  • Migratory activation of parasitized dendritic cells by the protozoan Toxoplasma gondii 14-3-3 protein
  • 2016
  • Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 18:11, s. 1537-1550
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
50.
  • Weidner, Jessica M., et al. (författare)
  • Rapid cytoskeleton remodelling in dendritic cells following invasion by Toxoplasma gondii coincides with the onset of a hypermigratory phenotype
  • 2013
  • Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 15:10, s. 1735-1752
  • Tidskriftsartikel (refereegranskat)abstract
    • Host cell manipulation is an important feature of the obligate intracellular parasite Toxoplasma gondii. Recent reports have shown that the tachyzoite stages subvert dendritic cells (DC) as a conduit for dissemination (Trojan horse) during acute infection. To examine the cellular basis of these processes, we performed a detailed analysis of the early events following tachyzoite invasion of human monocyte-derived DC. We demonstrate that within minutes after tachyzoite penetration, profound morphological changes take place in DC that coincide with a migratory activation. Active parasite invasion of DC led to cytoskeletal actin redistribution with loss of adhesive podosome structures and redistribution of integrins (CD18 and CD11c), that concurred with the onset of DC hypermotility in vitro. Inhibition of parasite rhoptry secretion and invasion, but not inhibition of parasite or host cell protein synthesis, abrogated the onset of morphological changes and hypermotility in DC dose-dependently. Also, infected DC, but not by-stander DC, exhibited upregulation of C-C chemokine receptor 7 (CCR7). Yet, the onset of parasite-induced DC hypermotility preceded chemotactic migratory responsesin vitro. Collectively, present data reveal that invasion of DC by T. gondii initiates a series of regulated events, including rapid cytoskeleton rearrangements, hypermotility and chemotaxis, that promote the migratory activation of DC.
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