- Email: [email protected]
MicroRNA profiles of CD46-stimulated T cells
Abstracts / Molecular Immunology 48 (2011) 1666–1733
P76 MicroRNA profiles of CD46-stimulated T cells B.C.R. King a,∗ , J. Esguerra a , L. Eliasson a , J. Cardone b , C. Kemper b , A.M. Blom a a b
Lund University, Malmö, Sweden King’s College London, London, United Kingdom
Introduction: CD46 co-stimulation of T cells results in heightened IL10 secretion and could be an important in vivo mechanism of immune regulation, as defects in CD46-mediated IL10 production can be demonstrated in patients with autoimmune disease. microRNAs (miRs) are small RNAs which bind specific target mRNAs, causing degradation or inhibition of translation. miRs are therefore a post-transcriptional mechanism for regulation of protein expression. Many functional roles of miRs have been identified in the development or differentiation of leukocytes. We are therefore investigating the possible roles of miRs in CD46-induced IL10 production from CD4+ T cells Methods: T cells were purified from peripheral blood of donors and stimulated with anti-CD3 and anti-CD46 in the presence of IL2 for 36 h. Cells were then stained for IL10 or IFNg secretion and sorted by flow cytometry depending on cytokine secretion profile. CD46 or CD28 co-stimulated cells were also taken at a 2 h post-stimulation timepoint to study early activation events. Whole RNA was then purified from the separate populations and run on Exiqon microRNA microarrays. Significant analysis of microarray (SAM) was used to identify significantly altered miRs in different populations, which were then validated using RT-qPCR. Results and conclusions: A number of miRs were found to be differentially expressed in the different T cell populations. Differential expression of miRs in different cytokine-expressing cell populations suggests that miRs could have a functional role to play in the phenotype of these cells, and in CD46-induced immune regulation. doi:10.1016/j.molimm.2011.06.296 P77 Challenging pattern recognition cross-talk: A gene profiling perspective of human Gram-negative bacteriaemia C.L. Lau a,∗ , S. Nygård b , H. Fure a , K.T. Lappegård c , O.L. Brekke c , E. Hovig b , T.E. Mollnes c a
Nordland Hospital Bodø, Bodø, Norway Department of informatics, University of Oslo, Oslo, Norway c Nordland Hospital Bodø, University of Tromsø, Nordland, Norway b
Introduction: Pattern recognition systems constitute a first line of defense against intruding pathogens and endogenous damage signals. The cross-talk between these systems, like complement and Toll-like receptors (TLR), has been recognized as a powerful regulatory mechanism. We have shown that combined inhibition of complement and CD14, a key molecule in TLR function, effectively attenuates a number of inflammatory responses,likecytokine productionand granulocyte oxidative burstin a whole blood model of human Gram-negative sepsis. We hypothesized this system to have potential as a general approach to treat inflammatory over-activation Methods: To evaluate this hypothesis, we performed a comprehensive DNA microarray analysis of Escherichia coli-induced transcriptional changes in human whole blood from healthy donors, and a C5-deficient patient, incubated with the pathogen in the presence or absence of inhibitors of C3 and/or CD14. A gene expression profile was generated from total RNA (Affymetrix Human Gene ST1.0 array), and analyzed using appropriate statistical tests and gene-annotation enrichment databases.
1691
Results: The expression of 2335 transcripts was affected in response to E. coli. Up to 70% of all responses were reversed mainly by combined inhibition or inhibition of CD14 alone, underlining the important mediating role for CD14. Consistent with an inverse effect on gene expression upon inhibition, complement emerged as repressor of inflammatory responses for about 20% of the genes. Importantly, this effect was partially counteracted by CD14 inhibition. Conclusion: Overall, the broad range of transcriptional eventsof the host defense reveal distinct and synergistic roles for complement and CD14 in controlling those. doi:10.1016/j.molimm.2011.06.297 P78 C5a receptor is critical for Flt3L-driven generation of functional plasmacytoid dendritic cells in vitro Laumonnier ∗ , Vollbrandt, Schmudde, Köhl Institute for Systemic Inflammatory Research, Lübeck, Germany Previous data suggest that C5a regulates the development of maladaptive Th2 immunity in experimental allergic asthma through modulation of plasmacytoid dendritic cell (pDC) function. To study C5a-mediated regulation of pDC function, we differentiated pDCs from bone marrow (BM) cells of wild type (wt) and C5aR-deficient (C5aR−/−) mice using Flt3-ligand (Flt3L). After in vitro differentiation of wt cells, we found three distinct pDC populations. Among those, only mPDCAint B220+ but not mPDCAhi B200− or mPDCAint B200hi cells produced large amounts of IFNa and IL12p40 following CPG oligodeoxynucleotide-mediated TLR9 stimulation or high amounts of IL-12p40 in response to TLR7 activation. Surprisingly, PDCAint B220+ cells from C5aR−/− mice neither produced IFN-a nor IL-12p40 in response to CpG stimulation but retained their ability to produce IL-12p40 upon TLR7 stimulation. TLR7 and TLR9 expression in pDCs from WT or C5aR−/− mice were similar, suggesting that C5aR does not regulate TLR expression. Transcription factors from the Interferon Responsive Factor (IRF) family, in particular IRF7, are critical for the production of IFN-a by pDCs. In C5aR−/− pDCs, IRF5 and IRF8 mRNA levels were markedly increased whereas IRF7 transcripts were significantly decreased as compared with the levels of WT pDCs. Our findings suggest that C5aR signaling is critically involved in the differentiation of BM cells towards fully functional pDCs in response to Flt3L by a strong impact on the expression of IRF transcription factors. This effect may explain the impaired pDC function after C5aR targeting in experimental allergic asthma. doi:10.1016/j.molimm.2011.06.298 P79 C5aR but not C3aR signaling in mDCs differentially regulates maladaptive immunity and inflammation in house dust mitemediated experimental allergic asthma Engelke a , Laumonnier b,∗ , Schmudde b , Köhl b a b
Lübeck, Germany Institute for Systemic Inflammation Research, Lübeck, Germany
Previous studies have highlighted opposing roles of the anaphylatoxin receptors C3aR and C5aR in the sensitization and the effector phases of experimental allergic asthma. Myeloid dendritic cells (mDCs) play critical roles in allergen uptake and Th cell differentiation towards Th2, Th17 and Th1 effector cells. The aim of this study was to delineate the roles of C3aR and C5aR signaling in mDCs for the development of maladaptive Th2, Th1 and Th17
P76 MicroRNA profiles of CD46-stimulated T cells B.C.R. King a,∗ , J. Esguerra a , L. Eliasson a , J. Cardone b , C. Kemper b , A.M. Blom a a b
Lund University, Malmö, Sweden King’s College London, London, United Kingdom
Introduction: CD46 co-stimulation of T cells results in heightened IL10 secretion and could be an important in vivo mechanism of immune regulation, as defects in CD46-mediated IL10 production can be demonstrated in patients with autoimmune disease. microRNAs (miRs) are small RNAs which bind specific target mRNAs, causing degradation or inhibition of translation. miRs are therefore a post-transcriptional mechanism for regulation of protein expression. Many functional roles of miRs have been identified in the development or differentiation of leukocytes. We are therefore investigating the possible roles of miRs in CD46-induced IL10 production from CD4+ T cells Methods: T cells were purified from peripheral blood of donors and stimulated with anti-CD3 and anti-CD46 in the presence of IL2 for 36 h. Cells were then stained for IL10 or IFNg secretion and sorted by flow cytometry depending on cytokine secretion profile. CD46 or CD28 co-stimulated cells were also taken at a 2 h post-stimulation timepoint to study early activation events. Whole RNA was then purified from the separate populations and run on Exiqon microRNA microarrays. Significant analysis of microarray (SAM) was used to identify significantly altered miRs in different populations, which were then validated using RT-qPCR. Results and conclusions: A number of miRs were found to be differentially expressed in the different T cell populations. Differential expression of miRs in different cytokine-expressing cell populations suggests that miRs could have a functional role to play in the phenotype of these cells, and in CD46-induced immune regulation. doi:10.1016/j.molimm.2011.06.296 P77 Challenging pattern recognition cross-talk: A gene profiling perspective of human Gram-negative bacteriaemia C.L. Lau a,∗ , S. Nygård b , H. Fure a , K.T. Lappegård c , O.L. Brekke c , E. Hovig b , T.E. Mollnes c a
Nordland Hospital Bodø, Bodø, Norway Department of informatics, University of Oslo, Oslo, Norway c Nordland Hospital Bodø, University of Tromsø, Nordland, Norway b
Introduction: Pattern recognition systems constitute a first line of defense against intruding pathogens and endogenous damage signals. The cross-talk between these systems, like complement and Toll-like receptors (TLR), has been recognized as a powerful regulatory mechanism. We have shown that combined inhibition of complement and CD14, a key molecule in TLR function, effectively attenuates a number of inflammatory responses,likecytokine productionand granulocyte oxidative burstin a whole blood model of human Gram-negative sepsis. We hypothesized this system to have potential as a general approach to treat inflammatory over-activation Methods: To evaluate this hypothesis, we performed a comprehensive DNA microarray analysis of Escherichia coli-induced transcriptional changes in human whole blood from healthy donors, and a C5-deficient patient, incubated with the pathogen in the presence or absence of inhibitors of C3 and/or CD14. A gene expression profile was generated from total RNA (Affymetrix Human Gene ST1.0 array), and analyzed using appropriate statistical tests and gene-annotation enrichment databases.
1691
Results: The expression of 2335 transcripts was affected in response to E. coli. Up to 70% of all responses were reversed mainly by combined inhibition or inhibition of CD14 alone, underlining the important mediating role for CD14. Consistent with an inverse effect on gene expression upon inhibition, complement emerged as repressor of inflammatory responses for about 20% of the genes. Importantly, this effect was partially counteracted by CD14 inhibition. Conclusion: Overall, the broad range of transcriptional eventsof the host defense reveal distinct and synergistic roles for complement and CD14 in controlling those. doi:10.1016/j.molimm.2011.06.297 P78 C5a receptor is critical for Flt3L-driven generation of functional plasmacytoid dendritic cells in vitro Laumonnier ∗ , Vollbrandt, Schmudde, Köhl Institute for Systemic Inflammatory Research, Lübeck, Germany Previous data suggest that C5a regulates the development of maladaptive Th2 immunity in experimental allergic asthma through modulation of plasmacytoid dendritic cell (pDC) function. To study C5a-mediated regulation of pDC function, we differentiated pDCs from bone marrow (BM) cells of wild type (wt) and C5aR-deficient (C5aR−/−) mice using Flt3-ligand (Flt3L). After in vitro differentiation of wt cells, we found three distinct pDC populations. Among those, only mPDCAint B220+ but not mPDCAhi B200− or mPDCAint B200hi cells produced large amounts of IFNa and IL12p40 following CPG oligodeoxynucleotide-mediated TLR9 stimulation or high amounts of IL-12p40 in response to TLR7 activation. Surprisingly, PDCAint B220+ cells from C5aR−/− mice neither produced IFN-a nor IL-12p40 in response to CpG stimulation but retained their ability to produce IL-12p40 upon TLR7 stimulation. TLR7 and TLR9 expression in pDCs from WT or C5aR−/− mice were similar, suggesting that C5aR does not regulate TLR expression. Transcription factors from the Interferon Responsive Factor (IRF) family, in particular IRF7, are critical for the production of IFN-a by pDCs. In C5aR−/− pDCs, IRF5 and IRF8 mRNA levels were markedly increased whereas IRF7 transcripts were significantly decreased as compared with the levels of WT pDCs. Our findings suggest that C5aR signaling is critically involved in the differentiation of BM cells towards fully functional pDCs in response to Flt3L by a strong impact on the expression of IRF transcription factors. This effect may explain the impaired pDC function after C5aR targeting in experimental allergic asthma. doi:10.1016/j.molimm.2011.06.298 P79 C5aR but not C3aR signaling in mDCs differentially regulates maladaptive immunity and inflammation in house dust mitemediated experimental allergic asthma Engelke a , Laumonnier b,∗ , Schmudde b , Köhl b a b
Lübeck, Germany Institute for Systemic Inflammation Research, Lübeck, Germany
Previous studies have highlighted opposing roles of the anaphylatoxin receptors C3aR and C5aR in the sensitization and the effector phases of experimental allergic asthma. Myeloid dendritic cells (mDCs) play critical roles in allergen uptake and Th cell differentiation towards Th2, Th17 and Th1 effector cells. The aim of this study was to delineate the roles of C3aR and C5aR signaling in mDCs for the development of maladaptive Th2, Th1 and Th17