high-throughput-quantitative-real-time-rt-pcr-assay-for-determining

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Expression of chemokines at the endothelial surface depends on their rate of synthesis, the capacity of the endothelium to bind chemokines and the rate of clearance from the surface. The aim of this study was to establish how these factors depend on the chemokine and the tissue of origin of the endothelium. Human lung and dermal microvascular endothelium, saphenous and umbilical vein endothelium, and a bone marrow endothelial line were assayed in vitro. Chemokine expression, localization and transport was measured by immunoassay and confocal microscopy. All endothelia bound CCL3 (MIP-1alpha), CCL5 (RANTES) and CXCL10 (IP-10). CCL3 and CCL5 bound at high levels, and CXCL10 bound less strongly. However, the profile of chemokine expression varied between endothelia, and different chemokines were shown to bind to the endothelial surface by distinct mechanisms. The half-life of CCL3 and CCL5 at the cell surface was approximately 30 min and chemokines were cleared primarily by endocytosis into caveolae. Endothelia from different tissues synthesize distinctive sets of chemokines, but the profile of surface-expressed chemokines also depends on the distinctive characteristics of each endothelia. These two mechanisms may contribute to the differential recruitment of leucocyte subsets to different tissues.

Expression of chemokines on the surface of different human endothelia.

Leukocyte migration across vascular endothelium is mediated by chemokines that are either synthesized by the endothelium or transferred across the endothelium from the tissue. The mechanism of transfer of two chemokines, CXCL10 (interferon gamma-inducible protein [IP]-10) and CCL2 (macrophage chemotactic protein [MCP]-1), was compared across dermal and lung microvessel endothelium and saphenous vein endothelium. The rate of transfer depended on both the type of endothelium and the chemokine. The permeability coefficient (Pe) for CCL2 movement across saphenous vein was twice the value for dermal endothelium and four times that for lung endothelium. In contrast, the Pe value for CXCL10 was lower for saphenous vein endothelium than the other endothelia. The differences in transfer rate between endothelia was not related to variation in paracellular permeability using a paracellular tracer, inulin, and immunoelectron microscopy showed that CXCL10 was transferred from the basal membrane in a vesicular compartment, before distribution to the apical membrane. Although all three endothelia expressed high levels of the receptor for CXCL10 (CXCR3), the transfer was not readily saturable and did not appear to be receptor dependent. After 30 min, the chemokine started to be reinternalized from the apical membrane in clathrin-coated vesicles. The data suggest a model for chemokine transcytosis, with a separate pathway for clearance of the apical surface.

Chemokine transport across human vascular endothelial cells.

Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.

Resting CD4+ effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function.

To standardize the amount of biological material between samples (e.g., number of cells or amount of tissue) for quantitative real-time reverse transcriptase PCR (qRT-PCR), the cycle of the target gene at which expression is detected (the cycle threshold, or Ct) is divided by the Ct of a gene either thought to be unaffected by experimental conditions or similarly expressed among donors. Genes that maintain cellular structure or homeostasis, referred to as housekeeping genes, or 18S ribosomal RNA are often used for this purpose. Although unstable or inconsistent housekeeping gene expression will misrepresent experimental effects on target gene expression, housekeeping genes are often chosen arbitrarily rather than systematically. We designed a simple and systematic approach towards selection of housekeeping genes based on Ct variance (as reflected by the standard deviation) and normality of distribution. We validated this approach by comparing stability and consistency of expression of 11 housekeeping genes across different types of cells, experimental treatments, and human donors. Finally, we demonstrated the consequences of inconsistent housekeeping gene expression on the calculation of target gene expression, and conclude that validation of stability of housekeeping gene expression by considering both distribution normality and standard deviation is straightforward and critical for proper experimental design.

Systematic method for determining an ideal housekeeping gene for real-time PCR analysis.

IL-23 is a pro-inflammatory cytokine proposed to be central to the development of autoimmune disease. We investigated whether IL-23, together with the downstream mediator IL-17A, was present and functional in RA in humans.

Investigating the role of the interleukin-23/-17A axis in rheumatoid arthritis.

Human respiratory syncytial virus (HRSV), and to a lesser extent human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), re-infect symptomatically throughout life without antigenic change, suggestive of incomplete immunity. One causative factor is thought to be viral interference with dendritic cell (DC)-mediated stimulation of CD4+ T cells.

Effects of human respiratory syncytial virus, metapneumovirus, parainfluenza virus 3 and influenza virus on CD4+ T cell activation by dendritic cells.

We recently demonstrated that the respiratory syncytial virus (RSV) NS1 protein, an antagonist of host type I interferon (IFN-I) production and signaling, has a suppressive effect on the maturation of human dendritic cells (DC) that was only partly dependent on released IFN-I. Here we investigated whether NS1 affects the ability of DC to activate CD8+ and CD4+ T cells. Human DC were infected with RSV deletion mutants lacking the NS1 and/or NS2 genes and assayed for the ability to activate autologous T cells in vitro, which were analyzed by multi-color flow cytometry. Deletion of the NS1, but not NS2, protein resulted in three major effects: (i) an increased activation and proliferation of CD8+ T cells that express CD103, a tissue homing integrin that directs CD8+ T cells to mucosal epithelial cells of the respiratory tract and triggers cytolytic activity; (ii) an increased activation and proliferation of Th17 cells, which have recently been shown to have anti-viral effects and also indirectly attract neutrophils; and (iii) decreased activation of IL-4-producing CD4+ T cells--which are associated with enhanced RSV disease--and reduced proliferation of total CD4+ T cells. Except for total CD4+ T cell proliferation, none of the T cell effects appeared to be due to increased IFN-I signaling. In the infected DC, deletion of the NS1 and NS2 genes strongly up-regulated the expression of cytokines and other molecules involved in DC maturation. This was partly IFN-I-independent, and thus might account for the T cell effects. Taken together, these data demonstrate that the NS1 protein suppresses proliferation and activation of two of the protective cell populations (CD103+ CD8+ T cells and Th17 cells), and promotes proliferation and activation of Th2 cells that can enhance RSV disease.

Respiratory syncytial virus interferon antagonist NS1 protein suppresses and skews the human T lymphocyte response.

Human respiratory syncytial virus (HRSV) and, to a lesser extent, human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), can re-infect symptomatically throughout life without significant antigenic change, suggestive of incomplete or short-lived immunity. In contrast, re-infection by influenza A virus (IAV) largely depends on antigenic change, suggestive of more complete immunity. Antigen presentation by dendritic cells (DC) is critical in initiating the adaptive immune response. Antigen uptake by DC induces maturational changes that include decreased expression of the chemokine receptors CCR1, CCR2, and CCR5 that maintain DC residence in peripheral tissues, and increased expression of CCR7 that mediates the migration of antigen-bearing DC to lymphatic tissue. We stimulated human monocyte-derived DC (MDDC) with virus and found that, in contrast to HPIV3 and IAV, HMPV and HRSV did not efficiently decrease CCR1, 2, and 5 expression, and did not efficiently increase CCR7 expression. Consistent with the differences in CCR7 mRNA and protein expression, MDDC stimulated with HRSV or HMPV migrated less efficiently to the CCR7 ligand CCL19 than did IAV-stimulated MDDC. Using GFP-expressing recombinant virus, we showed that the subpopulation of MDDC that was robustly infected with HRSV was particularly inefficient in chemokine receptor modulation. HMPV- or HRSV-stimulated MDDC responded to secondary stimulation with bacterial lipopolysaccharide or with a cocktail of proinflammatory cytokines by increasing CCR7 and decreasing CCR1, 2 and 5 expression, and by more efficient migration to CCL19, suggesting that HMPV and HRSV suboptimally stimulate rather than irreversibly inhibit MDDC migration. This also suggests that the low concentration of proinflammatory cytokines released from HRSV- and HMPV-stimulated MDDC is partly responsible for the low CCR7-mediated migration. We propose that inefficient migration of HRSV- and HMPV-stimulated DC to lymphatic tissue contributes to reduced adaptive responses to these viruses.

Low CCR7-mediated migration of human monocyte derived dendritic cells in response to human respiratory syncytial virus and human metapneumovirus.

Recent genome-wide association studies suggest distinct roles for 12 human interferon-alpha (IFN-Î±) and 3 IFN-Î» subtypes that may be elucidated by defining the expression patterns of these sets of genes. To overcome the impediment of high homology among each of the sets, we designed a quantitative real-time PCR assay that incorporates the use of molecular beacon and locked nucleic acid (LNA) probes, and in some instances, LNA oligonucleotide inhibitors. We then measured IFN subtype expression by human peripheral blood mononuclear cells and by purified monocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), and monocyte-derived macrophages (MDM), and -dendritic cells (MDDC) in response to poly I:C, lipopolysaccharide (LPS), imiquimod and CpG oligonucleotides. We found that in response to poly I:C and LPS, monocytes, MDM and MDDC express a subtype pattern restricted primarily to IFN-Î² and IFN-Î»1. In addition, while CpG elicited expression of all type I IFN subtypes by pDC, imiquimod did not. Furthermore, MDM and mDC highly express IFN-Î», and the subtypes of IFN-Î» are expressed hierarchically in the order IFN-Î»1 followed by IFN-Î»2, and then IFN-Î»3. These data support a model of coordinated cell- and ligand-specific expression of types I and III IFN. Defining IFN subtype expression profiles in a variety of contexts may elucidate specific roles for IFN subtypes as protective, therapeutic or pathogenic mediators.

Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent.

Neuere genomweiten Verband-Studien legen nahe, unterschiedliche Rollen f&uuml;r 12 menschlichen Interferon-Alpha (IFN-&alpha;) und 3 IFN-&lambda; Subtypen, die durch die Definition der Ausdrucksmuster dieser Gruppen von Genen aufgekl&auml;rt werden kann. Um die Behinderung durch hohe Homologie zwischen den einzelnen Sets zu &uuml;berwinden, haben wir eine quantitative Echtzeit-PCR-Assay, das beinhaltet die Verwendung von molekularen Leuchtfeuer und verschlossene Nukleins&auml;ure (LNA) Sonden, und in einigen F&auml;llen LNA-Oligonukleotid-Hemmer entwickelt. Wir messen dann IFN Untertyp Ausdruck von Menschenblut peripheren mononukle&auml;ren Zellen und gereinigten Monozyten, myeloide dendritische Zellen (mDC), Plasmacytoid von dendritischen Zellen (pDC), und Monozyte abgeleiteten Makrophagen (MDM) und -dendritische Zellen (MDDC) als Reaktion auf Poly ich: C, Lipopolysaccharid (LPS), Imiquimod und CpG-Oligonukleotiden. Wir fanden, dass als Reaktion auf Poly ich: C und LPS, Monozyten, MDM und MDDC Express eine Subtyp-Muster in erster Linie auf IFN-&beta; und IFN-&lambda;1 beschr&auml;nkt. Dar&uuml;ber hinaus w&auml;hrend der CpG entlockt Ausdruck aller Typ I IFN-Subtypen von pDC, Imiquimod nicht. Dar&uuml;ber hinaus MDM und mDC sehr schnelles IFN-&lambda; und die Untertypen von IFN-&lambda; werden in der Reihenfolge, die IFN-&lambda;1 gefolgt von IFN-&lambda;2 und IFN-&lambda;3 hierarchisch ausgedr&uuml;ckt. Diese Daten unterst&uuml;tzen ein Modell f&uuml;r die koordinierte und Ligand-zellspezifische Ausdruck der Typen I und III IFN. Definieren von IFN Untertyp Ausdruck Profilen in einer Vielzahl von Kontexten kann bestimmte Rollen f&uuml;r IFN-Subtypen als Schutz-, therapeutische oder pathogenen Vermittler erhellen.

Ausdruck profile menschlichen Interferon-Alpha und Interferon-Lambda-Untertypen sind Ligand und Zelle-abh&auml;ngige.

Human type I interferons (IFNs) include IFN-β and 12 subtypes of IFN-α. During viral infection, infiltrating memory CD4(+) T cells are exposed to IFNs, but their impact on memory T-cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry. IFN-α8 and -α10 most potently enhanced expression of IFN-γ, IL-2, and IL-4. Potency among the subtypes differed most at doses between 10 and 100 U/mL. While enhancement of IL-2 and IL-4 correlated with the time of preincubation with type I IFN, IFN-γ production was enhanced best when IFN-α was added immediately preceding or simultaneously with T-cell stimulation. Comparison of T-cell responses to multiple doses of Staphylococcus aureus enterotoxin B and to peptide libraries from RSV or CMV demonstrated that IFN-α best enhanced cytokine expression when CD4(+) T cells were suboptimally stimulated. We conclude that type I IFNs enhance Th1 and Th2 function with dose dependency and subtype specificity, and best when T-cell stimulation is suboptimal. While type I IFNs may beneficially enhance CD4(+) T-cell memory responses to vaccines or viral pathogens, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation.

Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4(+) T cells.

Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We used gene-deletion viruses to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on virus uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro and on subsequent MDDC maturation and activation of autologous T cells. HMPV with deletion of G and SH (ΔSHG) exhibited increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ΔSHG induced increased proliferation of autologous Th1-polarized CD4(+) T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4(+) T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4(+) T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to that of ΔSHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether, our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4(+) T cells. This study describes a previously unknown mechanism of virus immune evasion.

Article	Year
Expression of chemokines on the surface of different human endothelia. Immunology and cell biology\| PubMed ID: 16033532	2005
Chemokine transport across human vascular endothelial cells. Endothelium : journal of endothelial cell research\| PubMed ID: 17364892	2007
Resting CD4+ effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function. Arthritis research & therapy\| PubMed ID: 18353171	2008
Systematic method for determining an ideal housekeeping gene for real-time PCR analysis. Journal of biomolecular techniques : JBT\| PubMed ID: 19183798	2008
Investigating the role of the interleukin-23/-17A axis in rheumatoid arthritis. Rheumatology (Oxford, England)\| PubMed ID: 19815670	2009
Effects of human respiratory syncytial virus, metapneumovirus, parainfluenza virus 3 and influenza virus on CD4+ T cell activation by dendritic cells. PloS one\| PubMed ID: 21124776	2010
Respiratory syncytial virus interferon antagonist NS1 protein suppresses and skews the human T lymphocyte response. PLoS pathogens\| PubMed ID: 21533073	2011
Low CCR7-mediated migration of human monocyte derived dendritic cells in response to human respiratory syncytial virus and human metapneumovirus. PLoS pathogens\| PubMed ID: 21731495	2011
Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent. Immunology and cell biology\| PubMed ID: 22249201	2012
Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4(+) T cells. European journal of immunology\| PubMed ID: 24030809	2013
Human metapneumovirus SH and G glycoproteins inhibit macropinocytosis-mediated entry into human dendritic cells and reduce CD4+ T cell activation. Journal of virology\| PubMed ID: 24672038	2014

Philippa Hillyer

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