Dataset Information

Interplay between IDO1 and iNOS in human retinal pigment epithelial cells.

ABSTRACT: 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-?), 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-? stimulation of hPRE cells induced the expression of IDO1, which inhibited the growth of T. gondii and S. aureus. A co-stimulation with IFN-?, 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-? together with tumor necrosis factor alpha and Interleukin-1 beta and prevents potential IDO1-dependent tissue damage.

SUBMITTER: Spekker-Bosker K

PROVIDER: S-EPMC6817751 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Interplay between IDO1 and iNOS in human retinal pigment epithelial cells.

Spekker-Bosker Katrin K Ufermann Christoph-Martin CM Oldenburg Maike M Däubener Walter W Eller Silvia Kathrin SK

Medical microbiology and immunology 20190702 6

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 respon ...[more]

PMID: 31267172

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Project description:BackgroundCurcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro.Methodology/principal findingsCellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM) and delayed apoptosis (above 1 µM). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein.ConclusionIt is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (?10 µM) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of cancer or in the treatment of eye diseases, retinal function should be monitored carefully.

Project description:BACKGROUND: Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. METHODOLOGY AND PRINCIPAL FINDINGS: Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca²? imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. CONCLUSIONS AND SIGNIFICANCE: Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic.

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Interplay between IDO1 and iNOS in human retinal pigment epithelial cells.

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Interplay between IDO1 and iNOS in human retinal pigment epithelial cells.

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