Dicer-deficient mice exhibit constitutive up-regulation of IFN-stimulated genes in splenocytes.

<p>A. Scatterplot illustrating macroarray data performed on mRNA from non-infected (NI) spleens harvested from control mice (+/+) or <i>Dicer^d/d</i> mutants. Red dots indicate up-regulated genes in mutants, whereas green dots represent down-modulated genes. B. Repartition of up-regulated genes (red), down-regulated (green), or whose expression remains similar in <i>Dicer^d/d</i> splenocytes and macrophages compared to controls (blue). C–F. qRT-PCR experiments on selected IFN-dependant genes performed on mRNA extracted from control (+/+) or mutant (<i>Dicer^d/d</i>) spleens.</p

IFN-stimulated genes induction is impaired in MCMV-infected <i>Dicer^d/d</i> mice.

<p>A. Scatterplot shows multiple IFN-dependent genes up-regulation (red dots) in MCMV-infected control mice (+/+). B. Scatterplot analysis comparing MCMV-infected controls (+/+) and <i>Dicer^d/d</i> mice. C–E. qRT-PCR data on selected MCMV-inducible genes (<i>Irf7, Ifit1</i> and <i>Oas1a</i>). The graphs show relative gene expression in controls (black spots; +/+) and <i>Dicer^d/d</i> (white spots) spleen extracts. F. CXCL10 was measured in the serum of controls (white dots, +/+) and <i>Dicer^d/d</i> mice (black dots) 36 hours p.i. G. IRF7 expression was analyzed by Western blot performed on protein extracts from MCMV-infected wild-type (+/+) and mutant mice. GAPDH expression serves a loading control.</p

<i>Ex vivo</i> infected macrophages harvested from Dicer-deficient mice exhibit antiviral defects.

<p>A. Absolute quantification by qPCR of the MCMV genome in control (+/+, black bars) macrophages and cells isolated from <i>Dicer^d/d</i> mice (white bars) at different time points post infection (p.i). B. Northern blot experiments showing, miR-16 and MCMV miR-M23-2 expression in peritoneal macrophages isolated from <i>Dicer^flox/flox</i> mice 7 days and 13 days upon MCMV-Cre infection. Numbers below miR-16 panel indicate relative expression. Controls (Ctrl) are mock-infected macrophages. U6 expression serves as loading control. C. Quantification of Dicer excision efficacy upon MCMV-Cre infection of Dicer-floxed macrophages. Intensity of bands corresponding to excised and non-excised alleles was measured from agarose gels. Ratio is plotted for days 8, 12 and 16. D. Increased viral replication in Dicer-floxed macrophages upon MCMV-Cre infection. MCMV titer was measured by plaque assay in the supernatant of MCMV-Cre infected macrophages harvested from controls (+/+; dark spots) and <i>Dicer^flox/flox</i> mice (white spots).</p

Significantly induced genes in <i>Dicer^d/d</i> splenocytes.

<p>Significantly induced genes in <i>Dicer^d/d</i> splenocytes.</p

Deregulation of Type I IFN-Dependent Genes Correlates with Increased Susceptibility to Cytomegalovirus Acute Infection of Dicer Mutant Mice

<div><p>Regulation of gene expression by microRNAs (miRNAs) is now considered as an essential mechanism for cell development and homeostasis. Indeed, numerous studies have reported that modulating their expression, maturation, or activity can affect cell survival, identity or activation. In particular, miRNAs are key players in the tight regulation of signaling cascades, and as such, they appear as perfectly suited immunomodulators. Several immune-related processes, including inflammation, have recently been demonstrated to require specific miRNAs. In addition, the discovery of herpesvirus-encoded miRNAs has reinforced this assumption. To decipher the potential roles of miRNAs in innate antiviral immune response, we developed an <em>in vivo</em> model based on the inoculation of mouse cytomegalovirus (MCMV) in mice. Furthermore, we exploited a mouse line carrying a hypomorphic mutation in the <em>Dicer</em> gene to visualize the impact of impaired miRNA biogenesis upon the anti-MCMV response. Our data indicate that miRNAs are important actors in mounting an efficient response against herpesviruses. We suggest that a rapid and transient interferon response following viral infection requires miRNA-dependent repressor release. In addition, our <em>in vivo</em> efforts identified several miRNA targets, thus providing a conceptual framework for future analyzes on the regulation of specific actors involved in the Type I interferon pathway.</p> </div

Model for miR-dependent control of IFN-regulated genes.

<p>A. Schematic representation of our macroarray data. Interferon-stimulated genes (ISG) expression is indicated for controls (+/+, dark grey) and <i>Dicer^d/d</i> (light grey) cells in naïve or MCMV-stimulated conditions. B. In naïve control (+/+) cells, repression of ISGs is secured by miRNA-mediated repression exerted on transcriptional activators (in blue); on the contrary, rapid induction of ISGs upon virus (depicted in green) infection is permitted by MCMV-induced miRNA-mediated release of repressors (in red) along with virally-induced transcriptional induction of activators.</p

List of miRNAs strongly affected by the mutation in <i>Dicer^d/d</i> splenocytes.

<p>List of miRNAs strongly affected by the mutation in <i>Dicer^d/d</i> splenocytes.</p

Impaired maturation of a subset of miRNAs in <i>Dicer^d/d</i> mutant splenocytes and macrophages.

<p>A. Scatterplot showing relative expression of miRNAs by macroarray. In red, are shown miRNAs up-regulated in naïve (NI) splenocytes from <i>Dicer^d/d</i> mutants and green depicts down-regulated miRNAs. B. Scatterplot obtained upon macroarray analysis of mRNA extracted from control (+/+) and <i>Dicer^d/d</i> non-infected macrophages. C. Repartition of miRNAs down-regulated (green) or with similar expression (blue) in mutant splenocytes and macrophages. D. qRT-PCR experiments on selected miRNAs were performed on miRNA isolated from control (+/+, dark bars) and <i>Dicer^d/d</i> mutant (white bars) spleens.</p

<i>Dicer^d/d</i> mice are hypersusceptible to acute MCMV infection.

<p>A. Survival curve of control mice (+/+, black spots), <i>Dicer^d/d</i> (white spots) and BALB/c animals (black squares) upon acute MCMV infection (5×10⁵ pfu/mouse). B. Absolute quantification of the MCMV genome by qPCR in infected wild-type mice (+/+) and <i>Dicer^d/d</i> mutants. C. IFN-β quantification by ELISA in the serum of controls (+/+) and mutants 36 hours post-infection (h.p.i). D. Quantification of activated NK cells (NKp46+/IFN-γ+) in the spleen of non-infected (NI) and infected controls (+/+) and Dicer d/d mice 72 h.p.i. 6 animals/group were used. E. Snapshot images showing MCMV-derived luminescence 1 (D1) and 4 days (D4) upon MCMV-Luc infection of controls (+/+) and <i>Dicer^d/d</i> mice. F. Time-course analysis of the luminescence quantification in MCMV-Luc infected controls (n = 6 +/+; black spots) and mutants (n = 5; dotted line and white spots).</p

<i>Dicer^d/d</i> mutation affects the expression of a subset of MCMV-encoded miRNAs in macrophages.

<p>Virally-derived miRNAs were quantified by qRT-PCR in MCMV-infected macrophages harvested from control (dark bars, +/+) or <i>Dicer^d/d</i> mutant mice (light grey bars).</p