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Individual liver plasmacytoid dendritic cells are capable of producing IFN? and multiple additional cytokines during chronic HCV infection.

ABSTRACT: Plasmacytoid dendritic cells (pDCs) are "natural" interferon ? (IFN?)-producing cells. Despite their importance to antiviral defense, autoimmunity, and ischemic liver graft injury, because DC subsets are rare and heterogeneous, basic questions about liver pDC function and capacity to make cytokines remain unanswered. Previous investigations failed to consistently detect IFN? mRNA in HCV-infected livers, suggesting that pDCs may be incapable of producing IFN?. We used a combination of molecular, biochemical, cytometric, and high-dimensional techniques to analyze DC frequencies/functions in liver and peripheral blood mononuclear cells (PBMCs) of hepatitis C virus (HCV)-infected patients, to examine correlations between DC function and gene expression of matched whole liver tissue and liver mononuclear cells (LMCs), and to determine if pDCs can produce multiple cytokines. T cells often produce multiple cytokines/chemokines but until recently technical limitations have precluded tests of polyfunctionality in individual pDCs. Mass cytometry (CyTOF) revealed that liver pDCs are the only LMC that produces detectable amounts of IFN? in response TLR-7/8 stimulation. Liver pDCs secreted large quantities of IFN? (~2 million molecules of IFN?/cell/hour) and produced more IFN? than PBMCs after stimulation, p = 0.0001. LMCs secreted >14-fold more IFN? than IFN? in 4 hours. Liver pDC frequency positively correlated with whole liver expression of "IFN?-response" pathway (R2 = 0.58, p = 0.007) and "monocyte surface" signature (R2 = 0.54, p = 0.01). Mass cytometry revealed that IFN?-producing pDCs were highly polyfunctional; >90% also made 2-4 additional cytokines/chemokines of our test set of 10. Liver BDCA1 DCs, but not BDCA3 DCs, were similarly polyfunctional. pDCs from a healthy liver were also polyfunctional. Our data show that liver pDCs retain the ability to make abundant IFN? during chronic HCV infection and produce many other immune modulators. Polyfunctional liver pDCs are likely to be key drivers of inflammation and immune activation during chronic HCV infection.

SUBMITTER: Doyle EH

PROVIDER: S-EPMC6687199 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Individual liver plasmacytoid dendritic cells are capable of producing IFNα and multiple additional cytokines during chronic HCV infection.

Doyle Erin Heather EH Rahman Adeeb A Aloman Costica C Klepper Arielle L AL El-Shamy Ahmed A Eng Francis F Rocha Chiara C Kim Sang S Haydel Brandy B Florman Sander S SS Fiel M Isabel MI Schiano Thomas T Branch Andrea D AD

PLoS pathogens 20190729 7

Plasmacytoid dendritic cells (pDCs) are "natural" interferon α (IFNα)-producing cells. Despite their importance to antiviral defense, autoimmunity, and ischemic liver graft injury, because DC subsets are rare and heterogeneous, basic questions about liver pDC function and capacity to make cytokines remain unanswered. Previous investigations failed to consistently detect IFNα mRNA in HCV-infected livers, suggesting that pDCs may be incapable of producing IFNα. We used a combination of molecular, ...[more]

PMID: 31356648

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Project description:Plasmacytoid dendritic cells (pDCs) are ÒnaturalÓ interferon _ (IFN_)-producing cells. Despite their importance to antiviral defense, autoimmunity, and ischemic liver graft injury, because DC subsets are rare and heterogeneous, basic questions about liver pDC function and capacity to make cytokines remain unanswered. Previous investigations failed to consistently detect IFN_ mRNA in HCV-infected livers, suggesting that pDCs may be incapable of producing IFN_. We used a combination of molecular, biochemical, cytometric, and high-dimensional techniques to analyze DC frequencies/functions in liver and peripheral blood mononuclear cells (PBMCs) of hepatitis C virus (HCV)-infected patients, to examine correlations between DC function and gene expression of matched whole liver tissue and liver mononuclear cells (LMCs), and to determine if pDCs can produce multiple cytokines. T cells often produce multiple cytokines/chemokines but until recently technical limitations have precluded tests of polyfunctionality in individual pDCs. Mass cytometry (CyTOF) revealed that liver pDCs are the only LMC that produces detectable amounts of IFN_ in response TLR-7/8 stimulation. Liver pDCs secreted large quantities of IFN_ (~2 million molecules of IFN_/cell/hour) and produced more IFN_ than PBMCs after stimulation, p=0.0001. LMCs secreted >14-fold more IFN_ than IFN_ in 4 hours. Liver pDC frequency positively correlated with whole liver expression of ÒIFN_-responseÓ pathway (R2=0.58, p=0.007) and Òmonocyte surfaceÓ signature (R2=0.54, p=0.01). Mass cytometry revealed that IFN_-producing pDCs were highly polyfunctional; >90% also made 2-4 additional cytokines/chemokines of our test set of 10. Liver BDCA1 DCs, but not BDCA3 DCs, were similarly polyfunctional. pDCs from a healthy liver were also polyfunctional. Our data show that liver pDCs retain the ability to make abundant IFN_ during chronic HCV infection and produce many other immune modulators. Polyfunctional liver pDCs are likely to be key drivers of inflammation and immune activation during chronic HCV infection.

Project description:BACKGROUND:The murine discs large homolog 2 (DLG2; post synaptic density 93 (PSD-93); Chapsyn-110) is a member of the membrane-associated guanylate kinase (MAGUK) protein family involved in receptor assembly and associated with signaling enzymes on cell membranes. In neurons, DLG2 protein isoforms derived from alternatively spliced transcripts have been described to bind to NMDA (N-methyl-aspartate) receptors and K channels and to mediate clustering of these channels in the postsynaptic membrane. In myeloid cells of the immune system, such as dendritic cells (DCs), a lack of data exists on the expression or function of DLG2. In cDNA microarray transcriptome analyses, we found Dlg2 highly expressed in a subpopulation of plasmacytoid DCs (pDCs) stimulated to produce type I interferons (IFNs) such as IFN?. RESULTS:Using RACE- and RT-PCR as well as immunoprecipitation followed by Western blotting we characterised the differential expression of the Dlg2 splice variants in IFN?-producing pDCs. Besides Dlg2? this cell population expressed a novel short Dlg2? transcript we termed Dlg2?3. Our expression data were integrated into information from genome databases to obtain a novel and comprehensive overview of the mouse Dlg2 gene architecture. To elucidate the intracellular localisation pattern of protein isoforms, ectopical expression analysis of fluorescently tagged DLG2 splice variants was performed. Here we found an enrichment of the larger isoform DLG2?1 at the plasma membrane while the newly identified shorter (DLG2?) isoform as well as DLG2? were equally distributed throughout the cytoplasm. Additionally, DLG2? was also found in the nucleus. Analysis of Dlg2-knockout mice previously generated by deleting exon 9 surprisingly revealed that the protein for the novel DLG2? isoform was still expressed in the brain and in bone marrow-derived pDCs from mice carrying the homozygous deletion (Dlg2 ?E9/?E9 ). CONCLUSION:We describe a novel splice variant of the mouse Dlg2 gene termed Dlg2? and define the differential expression pattern of DLG2 isoforms in IFN?-producing pDCs. The presence of DLG2? protein in the CNS of Dlg2 ?E9/?E9 mice might influence the phenotype of these mice and has to be taken into account in the interpretation of results regarding the functional role of DLG2 in neuronal postsynaptic membranes.

Dataset Information

Individual liver plasmacytoid dendritic cells are capable of producing IFN? and multiple additional cytokines during chronic HCV infection.

Publications

Individual liver plasmacytoid dendritic cells are capable of producing IFNα and multiple additional cytokines during chronic HCV infection.

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