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

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

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

<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

MiR-30a-3p Negatively Regulates BAFF Synthesis in Systemic Sclerosis and Rheumatoid Arthritis Fibroblasts

<div><p>We evaluated micro (mi) RNA-mediated regulation of BAFF expression in fibroblasts using two concomitant models: (i) synovial fibroblasts (FLS) isolated from healthy controls (N) or Rheumatoid Arthritis (RA) patients; (ii) human dermal fibroblasts (HDF) isolated from healthy controls (N) or Systemic Sclerosis (SSc) patients. Using RT-qPCR and ELISA, we first showed that SScHDF synthesized and released BAFF in response to Poly(I:C) or IFN-γ treatment, as previously observed in RAFLS, whereas NHDF released BAFF preferentially in response to IFN-γ. Next, we demonstrated that miR-30a-3p expression was down regulated in RAFLS and SScHDF stimulated with Poly(I:C) or IFN-γ. Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model. Interestingly, FLS and HDF isolated from healthy subjects express higher levels of miR-30a-3p and lower levels of BAFF than RAFLS and SScHDF. Transfection of miR-30a-3p antisense in Poly(I:C)- and IFN-γ-activated NFLS and NHDF upregulated BAFF secretion, confirming that this microRNA is a basal repressors of BAFF expression in cells from healthy donors. Our data suggest a critical role of miR-30a-3p in the regulation of BAFF expression, which could have a major impact in the regulation of the autoimmune responses occurring in RA and SSc.</p></div

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

Increased protein expression of putative miRNA-targeted genes in <i>Dicer^d/d</i> mutants.

<p>A. CXCL10 was quantified in the serum of naïve controls (black dots) and Dicer mutant mice (white dots). B. CXCL10 (expressed in picograms for 1×10⁶ cells) quantification in the supernatant of macrophages harvested from wild-type (+/+) and <i>Dicer^d/d</i> mutants. C. STAT1 expression visualized by Western blot performed on spleen extracts from two control animals and two <i>Dicer^d/d</i> mutant mice. GAPDH serves as loading control. D. Quantification of STAT1 signals in spleen protein extracts normalized to GADPH was done for 3 controls and 4 mutants. E. Sequence alignment of murine miR-21 and its target sequence in the Cxcl10 3’UTR. The mutated nucleotides disrupting miR-21 binding in the mutated Cxcl10 3’UTR are shown in red. F. miR-21 expression is reduced in Dicer d/d splenocytes compared to wild-types. qRT-PCR was used to quantify miR-21 expression in wild-type and <i>Dicer^d/d</i> splenocytes. G. Normalized Luciferase expression (RLU) in HeLa cells. Control or anti-miR-21 (<a href="mailto:@miR-21" target="_blank">@miR-21</a>) tiny LNAs were incubated with HeLa cells for 24 h. Afterward, cells were transfected with empty vector (psiCHECK-2), or vectors containing a perfect match for miR-21 (PM21), the wild-type Cxcl10 3’UTR, or a mutated Cxcl10 3’UTR downstream of the f-luc gene.</p