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      The lectin-specific activity of Toxoplasma gondii microneme proteins 1 and 4 binds Toll-like receptor 2 and 4 N-glycans to regulate innate immune priming

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          Abstract

          Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-γ in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis.

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          Toxoplasmosis is caused by the protozoan Toxoplasma gondii, belonging to the Apicomplexa phylum. This phylum comprises important parasites able to infect a broad diversity of animals, including humans. A particularity of T. gondii is its ability to invade virtually any nucleated cell of all warm-blooded animals through an active process, which depends on the secretion of adhesin proteins. These proteins are discharged by specialized organelles localized in the parasite apical region, and termed micronemes and rhoptries. We show in this study that two microneme proteins from T. gondii utilize their adhesion activity to stimulate innate immunity. These microneme proteins, denoted MIC1 and MIC4, recognize specific sugars on receptors expressed on the surface of mammalian immune cells. This binding activates these innate immune cells to secrete cytokines, which promotes efficient host defense mechanisms against the parasite and regulate their pathogenesis. This activity promotes a chronic infection by controlling parasite replication during acute infection.

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          Innate immunity to Toxoplasma gondii infection.

          Toxoplasma gondii is a protozoan parasite of global importance. In the laboratory setting, T. gondii is frequently used as a model pathogen to study mechanisms of T helper 1 (TH1) cell-mediated immunity to intracellular infections. However, recent discoveries have shown that innate type 1 immune responses that involve interferon-γ (IFNγ)-producing natural killer (NK) cells and neutrophils, rather than IFNγ-producing T cells, predetermine host resistance to T. gondii. This Review summarizes the Toll-like receptor (TLR)-dependent mechanisms that are responsible for parasite recognition and for the induction of IFNγ production by NK cells, as well as the emerging data about the TLR-independent mechanisms that lead to the IFNγ-mediated elimination of T. gondii.
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            In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-gamma and TNF-alpha.

            To examine the function of IL-10 synthesis during early infection with the intracellular protozoan Toxoplasma gondii, IL-10 knockout (KO) mice were inoculated with an avirulent parasite strain (ME-49). In contrast to control littermates that displayed 100% survival, the IL-10-deficient animals succumbed within the first 2 wk of the infection, with no evidence of enhanced parasite proliferation. The mortality in the IL-10 KO mice was associated with enhanced liver pathology characterized by increased cellular infiltration and intense necrosis. Levels of IL-12 and IFN-gamma in sera of infected IL-10-deficient animals were four- to sixfold higher than those in sera from control mice, as were mRNA levels for IFN-gamma, IL-1 beta, TNF-alpha, and IL-12 in lung tissue. Similarly, macrophages from IL-10 KO mice activated in vitro or in vivo with T. gondii produced higher levels of TNF-alpha and IL-12 than macrophages from control animals. Moreover, spleen cells from IL-10 KO mice infected with T. gondii secreted more IFN-gamma than splenocytes from nondeficient animals. In vitro depletion experiments indicated that CD4+ lymphocytes are the major source of the latter cytokine in the spleen cell populations, and in vivo depletion with anti-CD4 Abs protected the IL-10 KO mice from parasite-induced mortality. Together the data suggest that endogenous IL-10 synthesis plays an important role in vivo in down-regulating monokine and IFN-gamma responses to acute intracellular infection, thereby preventing host immunopathology.
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              Conventional T-bet+Foxp3− Th1 cells are the major source of host-protective regulatory IL-10 during intracellular protozoan infection

              Although interferon γ (IFN-γ) secretion is essential for control of most intracellular pathogens, host survival often also depends on the expression of interleukin 10 (IL-10), a cytokine known to counteract IFN-γ effector functions. We analyzed the source of regulatory IL-10 in mice infected with the protozoan parasite Toxoplasma gondii. Unexpectedly, IFN-γ–secreting T-bet+Foxp3− T helper type 1 (Th1) cells were found to be the major producers of IL-10 in these animals. Further analysis revealed that the same IL-10+IFN-γγ population displayed potent effector function against the parasite while, paradoxically, also inducing profound suppression of IL-12 production by antigen-presenting cells. Although at any given time point only a fraction of the cells appeared to simultaneously produce IL-10 and IFN-γ, IL-10 production could be stimulated in IL-10−IFN-γ+ cells by further activation in vitro. In addition, experiments with T. gondii–specific IL-10+IFN-γ+ CD4 clones revealed that although IFN-γ expression is imprinted and triggered with similar kinetics regardless of the state of Th1 cell activation, IL-10 secretion is induced more rapidly from recently activated than from resting cells. These findings indicate that IL-10 production by CD4+ T lymphocytes need not involve a distinct regulatory Th cell subset but can be generated in Th1 cells as part of the effector response to intracellular pathogens.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Formal analysisRole: MethodologyRole: Visualization
                Role: MethodologyRole: Resources
                Role: Methodology
                Role: Methodology
                Role: Resources
                Role: ResourcesRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: MethodologyRole: ResourcesRole: Writing – review & editing
                Role: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                21 June 2019
                June 2019
                : 15
                : 6
                : e1007871
                Affiliations
                [1 ] Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeirao Preto Medical School, University of Sao Paulo- USP (FMRP/USP), Ribeirao Preto, Sao Paulo, Brazil
                [2 ] Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States of America
                [3 ] Department of Microbiology and Molecular Medicine, CMU, University of Geneva, Switzerland
                [4 ] Department of Biochemistry, Cambridge University, United Kingdom
                University of New Mexico, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-9440-2814
                http://orcid.org/0000-0002-2782-7169
                http://orcid.org/0000-0001-7053-2895
                http://orcid.org/0000-0001-7360-1809
                http://orcid.org/0000-0003-3465-3782
                http://orcid.org/0000-0001-7425-0908
                Article
                PPATHOGENS-D-17-00181
                10.1371/journal.ppat.1007871
                6608980
                31226171
                58a81847-fece-4a6b-b2b6-85049e58db37

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 1 February 2017
                : 25 May 2019
                Page count
                Figures: 7, Tables: 0, Pages: 24
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001807, Fundação de Amparo à Pesquisa do Estado de São Paulo;
                Award ID: 2012/12950-0
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001807, Fundação de Amparo à Pesquisa do Estado de São Paulo;
                Award ID: 2014/13324-1
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001807, Fundação de Amparo à Pesquisa do Estado de São Paulo;
                Award ID: 2013/04088-0
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award ID: 475357/2013-2
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award ID: 306298/2013-9
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100008353, Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo;
                Award Recipient :
                Funded by: National Institute of Allergy and Infectious Diseases, National Institutes of Health
                Award Recipient :
                We wish to acknowledge the grants from Consortium for Functional Glycomics (#GM62116), for doing the glycoarray assays; UK Medical Research Council (#G1000133 to NJG), and Wellcome Investigator Award (#WT100321/z/12/Z to NJG). This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grants #2012/12950-0 to ASS, #2014/13324-1 to FCMN and #2013/04088-0 to MCRB), National Institute of Allergy and Infectious Diseases, National Institutes of Health (ASS and MEG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grants #475357/2013-2 and #306298/2013-9 to MCRB), and Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto USP (FAEPA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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                Custom metadata
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                2019-07-03
                All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology
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