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1.
Med Microbiol Immunol ; 208(6): 811-824, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31267172

RESUMO

Human retinal pigment epithelial (hRPE) cells form a selectively permeable monolayer between the neural retina and the highly permeable choroidal vessels. Thus, hRPE cells bear important regulatory functions and are potential targets of pathogens in vivo. Endogenous bacterial endophthalmitis (EBE) is frequently caused by infections with the Gram-positive bacterium Staphylococcus aureus (S. aureus). Upon microbial infection, interferon gamma (IFN-γ), a major cytokine of the adaptive immune response, induces a broad spectrum of effector molecules, such as the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase-1 (IDO1). We stimulated human RPE (hRPE) cells in vitro with proinflammatory cytokines and analyzed the expression levels and enzymatic activities of IDO1 and inducible nitric oxide synthase (iNOS), another antimicrobial effector molecule. The antimicrobial capacity was analyzed in infection experiments using S. aureus and Toxoplasma gondii (T. gondii). Our aim was to characterize the particular importance of IDO1 and iNOS during EBE. We found that an IFN-γ stimulation of hPRE cells induced the expression of IDO1, which inhibited the growth of T. gondii and S. aureus. A co-stimulation with IFN-γ, interleukin-1 beta, and tumor necrosis factor alpha induced a strong expression of iNOS. The iNOS-derived nitric oxide production was dependent on cell-culture conditions; however, it could not cause antimicrobial effects. iNOS did not act synergistically with IDO1. Instead, iNOS activity inhibited IDO1-mediated tryptophan degradation and bacteriostasis. This effect was reversible by the addition of the iNOS inhibitor NG-monomethyl-L-arginine. In conclusion, iNOS mediates anti-inflammatory effects in hRPE cells stimulated with high amounts of IFN-γ together with tumor necrosis factor alpha and Interleukin-1 beta and prevents potential IDO1-dependent tissue damage.


Assuntos
Células Epiteliais/enzimologia , Células Epiteliais/imunologia , Fatores Imunológicos/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Linhagem Celular , Endoftalmite/imunologia , Humanos , Modelos Teóricos , Epitélio Pigmentado da Retina/enzimologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/imunologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/imunologia
2.
Artigo em Inglês | MEDLINE | ID: mdl-31231617

RESUMO

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite and belongs to the phylum Apicomplexa. T. gondii is of medical and veterinary importance, because T. gondii causes the parasitic disease toxoplasmosis. In human cells, the interferon-gamma inducible indoleamine 2,3-dioxygenase 1 (IDO1) is an antimicrobial effector mechanism that degrades tryptophan to kynurenine and thus limits pathogen proliferation in vitro. Furthermore, IDO is described to have immunosuppressive properties, e.g., regulatory T cell differentiation and T cell suppression in humans and mice. However, there is only little known about the role of IDO1 in mice during acute toxoplasmosis. To shed further light on the role of mIDO1 in vivo, we have used a specifically adjusted experimental model. Therein, we infected mIDO1-deficient (IDO-/-) C57BL/6 mice and appropriate wild-type (WT) control mice with a high dose of T. gondii ME49 tachyozoites (type II strain) via the intraperitoneal route and compared the phenotype of IDO-/- and WT mice during acute toxoplasmosis. During murine T. gondii infection, we found mIDO1 mRNA and mIDO1 protein, as well as mIDO1-mediated tryptophan degradation in lungs of WT mice. IDO-/- mice show no tryptophan degradation in the lung during infection. Even though T. gondii is tryptophan auxotroph and rapidly replicates during acute infection, the parasite load was similar in IDO-/- mice compared to WT mice 7 days post-infection. IDO1 is described to have immunosuppressive properties, and since T cell suppression is observed during acute toxoplasmosis, we analyzed the possible involvement of mIDO1. Here, we did not find differences in the intensity of ex vivo mitogen stimulated T cell proliferation between WT and IDO-/- mice. Concomitant nitric oxide synthase inhibition and interleukin-2 supplementation increased the T cell proliferation from both genotypes drastically, but not completely. In sum, we analyzed the involvement of mIDO1 during acute murine toxoplasmosis in our specifically adjusted experimental model and found a definite mIDO1 induction. Nevertheless, mIDO1 seems to be functional redundant as an antiparasitic defense mechanism during acute toxoplasmosis in mice. Furthermore, we suggest that the systemic T cell suppression observed during acute toxoplasmosis is influenced by nitric oxide activity and IL-2 deprivation.


Assuntos
Proliferação de Células/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/farmacologia , Linfócitos T/efeitos dos fármacos , Toxoplasma/efeitos dos fármacos , Toxoplasmose/tratamento farmacológico , Animais , Modelos Animais de Doenças , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interleucina-2/metabolismo , Cinurenina/farmacologia , Linfonodos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico , Óxido Nítrico Sintase/metabolismo , Células RAW 264.7 , RNA Mensageiro/metabolismo , Baço , Toxoplasmose/parasitologia , Transcriptoma , Triptofano/farmacologia
3.
Int J Mol Sci ; 20(4)2019 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-30781494

RESUMO

Human retinal pigment epithelial (hRPE) cells are important for the establishment and maintenance of the immune privilege of the eye. They function as target cells for human cytomegalovirus (hCMV), but are able to restrict viral replication. hCMV causes opportunistic posterior uveitis such as retinitis and chorioretinitis. Both mainly occur in severely immunocompromised patients and rarely manifest in immunocompetent individuals. In this study, hRPE cells were infected with hCMV in vitro and activated with proinflammatory cytokines. The enzymatic activities of indoleamine 2,3-dioxygenase-1 (IDO1) and inducible nitric oxide synthase (iNOS) were determined. The antimicrobial capacity of both molecules was analyzed in co-infection experiments using Staphylococcus aureus (S. aureus) and Toxoplasma gondii (T. gondii), causing uveitis in patients. We show that an hCMV infection of hRPE cells blocks IDO1 and iNOS mediated antimicrobial defense mechanisms necessary for the control of S. aureus and T. gondii. hCMV also inhibits immune suppressive effector mechanisms in hRPE. The interferon gamma-induced IDO1 dependent immune regulation was severely blocked, as detected by the loss of T cell inhibition. We conclude that an active hCMV infection in the eye might favor the replication of pathogens causing co-infections in immunosuppressed individuals. An hCMV caused blockade of IDO1 might weaken the eye's immune privilege and favor the development of post-infectious autoimmune uveitis.


Assuntos
Olho/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Epitélio Pigmentado da Retina/imunologia , Uveíte/imunologia , Proliferação de Células/genética , Coinfecção/imunologia , Coinfecção/microbiologia , Coinfecção/virologia , Citomegalovirus/genética , Citomegalovirus/imunologia , Olho/microbiologia , Olho/virologia , Citometria de Fluxo , Humanos , Privilégio Imunológico/genética , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Interferon gama/imunologia , Óxido Nítrico Sintase Tipo II/genética , Epitélio Pigmentado da Retina/microbiologia , Epitélio Pigmentado da Retina/virologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade , Linfócitos T/imunologia , Linfócitos T/microbiologia , Linfócitos T/virologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/patogenicidade , Uveíte/microbiologia , Uveíte/virologia
4.
Biomed Res Int ; 2017: 6746437, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29259983

RESUMO

The obligate intracellular apicomplexan parasite Neospora caninum (N. caninum) is closely related to Toxoplasma gondii (T. gondii). The dense granules, which are present in all apicomplexan parasites, are important secretory organelles. Dense granule (GRA) proteins are released into the parasitophorous vacuole (PV) following host cell invasion and are known to play important roles in the maintenance of the host-parasite relationship and in the acquisition of nutrients. Here, we provide a detailed characterization of the N. caninum dense granule protein NcGRA9. The in silico genomic organization and key protein characteristics are described. Immunofluorescence-based localization studies revealed that NcGRA9 is located in the dense granules and is released into the interior of the PV following host cell invasion. Immunogold-electron microscopy confirmed the dense granule localization and showed that NcGRA9 is associated with the intravacuolar network. In addition, NcGRA9 is found in the "excreted secreted antigen" (ESA) fraction of N. caninum. Furthermore, by analysing the distribution of truncated versions of NcGRA9, we provide evidence that the C-terminal region of this protein is essential for the targeting of NcGRA9 into the dense granules of N. caninum, and the truncated proteins show reduced secretion.


Assuntos
Interações Hospedeiro-Parasita/genética , Neospora/química , Proteínas de Protozoários/genética , Sequência de Aminoácidos/genética , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Antígenos de Protozoários/isolamento & purificação , Vesículas Citoplasmáticas/genética , Vesículas Citoplasmáticas/metabolismo , Neospora/genética , Neospora/patogenicidade , Proteínas de Protozoários/química , Toxoplasma/genética , Toxoplasma/patogenicidade
5.
Mediators Inflamm ; 2017: 3260289, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28883687

RESUMO

Porcine infections are currently not the state-of-the-art model to study human diseases. Nevertheless, the course of human and porcine toxoplasmosis is much more comparable than that of human and murine toxoplasmosis. For example, severity of infection, transplacental transmission, and interferon-gamma-induced antiparasitic effector mechanisms are similar in pigs and humans. In addition, the severe immunosuppression during acute infection described in mice does not occur in the experimentally infected ones. Thus, we hypothesise that porcine Toxoplasma gondii infection data are more representative for human toxoplasmosis. We therefore suggest that the animal model chosen must be critically evaluated for its assignability to human diseases.


Assuntos
Toxoplasma/patogenicidade , Toxoplasmose/parasitologia , Animais , Western Blotting , Modelos Animais de Doenças , Humanos , Interferon gama/metabolismo , Suínos
6.
Parasitol Res ; 113(4): 1473-80, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24532010

RESUMO

Toxoplasma gondii is a parasite which can be transmitted to humans via the consumption of contaminated meat products derived from different animal species, e.g., poultry. In Europe, the consumption rate of poultry meat is high and may pose a risk for humans. However, little is known about the prevalence and immune response against T. gondii in these animals. Based on these circumstances, we experimentally infected 18 turkeys and 16 chickens with the parasite. Turkeys were infected either with tachyzoites on different routes or with various amounts of oocysts. In contrast, chickens were only infected with different doses of oocysts. The immunoglobulin (Ig) Y humoral immune responses of these animals were investigated in a lineblot assay against the recombinant T. gondii antigens rGRA1, rGRA6, rGRA9, rSAG1, and rSUB1. By using the recombinant antigens rGRA6, rGRA9, and rSUB1 in the lineblot assay, we found a correlation between the humoral immune response and the parasite stage in turkeys. Thereby, an infection with oocysts induced a stronger, permanent long-lasting antibody response compared to tachyzoite-infected animals. Only a minor relation between the oocyst infection dose and the manifestation of the immune response in chickens was found 7 days post infection (dpi) by using rGRA1 and rGRA9. However, an inconstant detection of antigen-specific IgY antibodies in the lineblot assay seems not to be a sufficient method for the identification of a Toxoplasma infection in chickens. In contrast, the detection of anti-rGRA6, anti-rGRA9, and anti-rSUB1 IgY antibodies showed potential for the identification of an infection in turkeys.


Assuntos
Galinhas/imunologia , Imunidade Humoral , Toxoplasmose Animal/imunologia , Perus/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Imunoglobulinas/sangue , Imunoglobulinas/imunologia , Oocistos/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/parasitologia , Proteínas Recombinantes/imunologia , Toxoplasma
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