USP 18 lack in microglia causes destructive interferonopathy of the mouse brain
2015; Springer Nature; Volume: 34; Issue: 12 Linguagem: Inglês
10.15252/embj.201490791
ISSN1460-2075
AutoresTobias Goldmann, Nicolas Zeller, Jenni Raasch, Katrin Kierdorf, Kathrin Frenzel, Lars Ketscher, Anja Basters, Ori Staszewski, Stefanie M. Brendecke, Alena Spieß, Tuan Leng Tay, Clemens Kreutz, Jens Timmer, Grazia M.S. Mancini, Thomas Blank, G. Fritz, Knut Biber, Roland Lang, Danielle Malo, Doron Merkler, Mathias Heikenwälder, Klaus‐Peter Knobeloch, Marco Prinz,
Tópico(s)Immune Response and Inflammation
ResumoArticle20 April 2015free access USP18 lack in microglia causes destructive interferonopathy of the mouse brain Tobias Goldmann Tobias Goldmann Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Nicolas Zeller Nicolas Zeller Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Jenni Raasch Jenni Raasch Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Katrin Kierdorf Katrin Kierdorf Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Kathrin Frenzel Kathrin Frenzel Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Lars Ketscher Lars Ketscher Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Anja Basters Anja Basters Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Ori Staszewski Ori Staszewski Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Stefanie M Brendecke Stefanie M Brendecke Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Alena Spiess Alena Spiess Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Tuan Leng Tay Tuan Leng Tay Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Clemens Kreutz Clemens Kreutz Institute of Physics & Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Search for more papers by this author Jens Timmer Jens Timmer Institute of Physics & Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Grazia MS Mancini Grazia MS Mancini Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Thomas Blank Thomas Blank Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Günter Fritz Günter Fritz Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Knut Biber Knut Biber Department of Psychiatry, University of Freiburg, Freiburg, Germany Department of Neuroscience, University Medical Center Groningen, Groningen, The Netherlands Search for more papers by this author Roland Lang Roland Lang Institute of Clinical Microbiology, Immunology and Hygiene, University Hospital Erlangen, Erlangen, Germany Search for more papers by this author Danielle Malo Danielle Malo Department of Human Genetics, McGill University, Montreal, QC, Canada Search for more papers by this author Doron Merkler Doron Merkler Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland Search for more papers by this author Mathias Heikenwälder Mathias Heikenwälder Institute of Virology, Technische Universität München/Helmholtz-Zentrum Munich, München, Germany Search for more papers by this author Klaus-Peter Knobeloch Klaus-Peter Knobeloch Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Marco Prinz Corresponding Author Marco Prinz Institute of Neuropathology, University of Freiburg, Freiburg, Germany BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Tobias Goldmann Tobias Goldmann Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Nicolas Zeller Nicolas Zeller Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Jenni Raasch Jenni Raasch Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Katrin Kierdorf Katrin Kierdorf Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Kathrin Frenzel Kathrin Frenzel Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Lars Ketscher Lars Ketscher Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Anja Basters Anja Basters Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Ori Staszewski Ori Staszewski Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Stefanie M Brendecke Stefanie M Brendecke Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Alena Spiess Alena Spiess Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Tuan Leng Tay Tuan Leng Tay Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Clemens Kreutz Clemens Kreutz Institute of Physics & Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Search for more papers by this author Jens Timmer Jens Timmer Institute of Physics & Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Grazia MS Mancini Grazia MS Mancini Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Thomas Blank Thomas Blank Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Günter Fritz Günter Fritz Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Knut Biber Knut Biber Department of Psychiatry, University of Freiburg, Freiburg, Germany Department of Neuroscience, University Medical Center Groningen, Groningen, The Netherlands Search for more papers by this author Roland Lang Roland Lang Institute of Clinical Microbiology, Immunology and Hygiene, University Hospital Erlangen, Erlangen, Germany Search for more papers by this author Danielle Malo Danielle Malo Department of Human Genetics, McGill University, Montreal, QC, Canada Search for more papers by this author Doron Merkler Doron Merkler Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland Search for more papers by this author Mathias Heikenwälder Mathias Heikenwälder Institute of Virology, Technische Universität München/Helmholtz-Zentrum Munich, München, Germany Search for more papers by this author Klaus-Peter Knobeloch Klaus-Peter Knobeloch Institute of Neuropathology, University of Freiburg, Freiburg, Germany Search for more papers by this author Marco Prinz Corresponding Author Marco Prinz Institute of Neuropathology, University of Freiburg, Freiburg, Germany BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Author Information Tobias Goldmann1, Nicolas Zeller1, Jenni Raasch1, Katrin Kierdorf1, Kathrin Frenzel1, Lars Ketscher1, Anja Basters1, Ori Staszewski1, Stefanie M Brendecke1, Alena Spiess1, Tuan Leng Tay1, Clemens Kreutz2, Jens Timmer2,3, Grazia MS Mancini4, Thomas Blank1, Günter Fritz1, Knut Biber5,6, Roland Lang7, Danielle Malo8, Doron Merkler9, Mathias Heikenwälder10, Klaus-Peter Knobeloch1,‡ and Marco Prinz 1,3,‡ 1Institute of Neuropathology, University of Freiburg, Freiburg, Germany 2Institute of Physics & Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany 3BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany 4Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands 5Department of Psychiatry, University of Freiburg, Freiburg, Germany 6Department of Neuroscience, University Medical Center Groningen, Groningen, The Netherlands 7Institute of Clinical Microbiology, Immunology and Hygiene, University Hospital Erlangen, Erlangen, Germany 8Department of Human Genetics, McGill University, Montreal, QC, Canada 9Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland 10Institute of Virology, Technische Universität München/Helmholtz-Zentrum Munich, München, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 761 270 51050; Fax: +49 761 270 50500; E-mail: [email protected] The EMBO Journal (2015)34:1612-1629https://doi.org/10.15252/embj.201490791 See also: K Takata & F Ginhoux (June 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Microglia are tissue macrophages of the central nervous system (CNS) that control tissue homeostasis. Microglia dysregulation is thought to be causal for a group of neuropsychiatric, neurodegenerative and neuroinflammatory diseases, called “microgliopathies”. However, how the intracellular stimulation machinery in microglia is controlled is poorly understood. Here, we identified the ubiquitin-specific protease (Usp) 18 in white matter microglia that essentially contributes to microglial quiescence. We further found that microglial Usp18 negatively regulates the activation of Stat1 and concomitant induction of interferon-induced genes, thereby terminating IFN signaling. The Usp18-mediated control was independent from its catalytic activity but instead required the interaction with Ifnar2. Additionally, the absence of Ifnar1 restored microglial activation, indicating a tonic IFN signal which needs to be negatively controlled by Usp18 under non-diseased conditions. These results identify Usp18 as a critical negative regulator of microglia activation and demonstrate a protective role of Usp18 for microglia function by regulating the Ifnar pathway. The findings establish Usp18 as a new molecule preventing destructive microgliopathy. Synopsis This study identifies Usp18 as a new critical negative regulator of microglia activation and demonstrates a protective role of Usp18 for microglia function by regulating IFN signaling. The protease USP18 is expressed in white matter microglia. Loss of USP18 induces white matter microglia activation and leads to microgliopathy. Microglia activation in the absence of USP18 is due to prolonged STAT1 phosphorylation. Constitutive IFN type I signaling in microglia during steady state. The Ifnar2 interaction domain rather then the protease function of USP19 controls microglia activation. Introduction Microglia are the tissue macrophages of the brain, crucially involved in the scavenging of dying cells, pathogens and molecules through phagocytosis/endocytosis and the use of pathogen-associated molecular pattern (PAMPs) receptors (Hanisch & Kettenmann, 2007; Ransohoff & Perry, 2009). Moreover, dysregulation of microglia activation is nowadays considered the pathogenetic basis for a group of neurodegenerative and neuroinflammatory conditions, called “microgliopathies” (Prinz & Priller, 2014). These include roles for several microglia molecules such as Csf1r in hereditary diffuse leukoencephalopathy with spheroids (Rademakers et al, 2012), CD33 in Alzheimer's disease (Hollingworth et al, 2011; Naj et al, 2011), Trem2 in frontotemporal dementia (Guerreiro et al, 2013), and Tnfrsf1a and Irf8 in multiple sclerosis (De Jager et al, 2009). In general, engagement of recognition receptors initiates a complex machinery of various signaling pathways that lead to the induction of inflammatory cytokines and type I interferons such as interferon-α (IFN-α) and IFN-β, which are critical for inhibiting early viral replication in the host (Gonzalez-Navajas et al, 2012). The induction of such inflammatory mediators is controlled in a multifaceted fashion at the transcriptional level (Gonzalez-Navajas et al, 2012). These activation mechanisms have to be tightly regulated to prevent harmful tissue damage caused by hyperinflammatory reactions. Type I interferons signal through a common heterodimeric receptor known as the IFN-α/β receptor (Ifnar), which is expressed by nearly all cell types (Gonzalez-Navajas et al, 2012). This receptor consists of two subunits—Ifnar1 and Ifnar2—that are associated with Janus kinase 1 (Jak1) (Honda et al, 2006). Upon Jak1 activation, several signal transducer and activator of transcription (Stat) family members, such as Stat1, are activated that finally induce the induction of a plethora of interferon-induced genes (ISGs) such as Isg15, 2′5′Oas, Mx1 and many more (Honda et al, 2006). Recent data have also uncovered potentially harmful sides of type I IFNs, including roles in inflammatory diseases such as autoimmunity and diabetes (Gonzalez-Navajas et al, 2012; Gough et al, 2012). For example, mutations in the human 3′ repair exonuclease 1 (Trex1) gene cause Aicardi-Goutières syndrome (AGS), an IFN-associated inflammatory disorder found in the brains of infants that suffer from epileptic seizures, intracerebral calcifications and leukodystrophy (Gall et al, 2012; Prinz & Knobeloch, 2012; Crow, 2015). On the other side, constitutive type I IFN levels are important for the maintenance, maturation and mobilization of the innate immune system in the body (Gough et al, 2012). Taking into account these highly divergent effects of type I IFNs, their tight regulation is imperative for ensuring immune homeostasis. However, it is not known yet how microglia under homeostatic conditions are kept in a quiescent state, but intracellular proteases are potential candidates for such regulatory functions. Among them, ubiquitin-specific proteases (Usps) form the largest family of deubiquitinating enzymes (Dubs) that have key functions in immune responses and many other biological processes (Hershko & Ciechanover, 1998; Liu et al, 2005). Cylindromatosis (Cyld) has been extensively studied and shown to regulate various immune functions (Sun, 2008). It is now clear that Usps like Cyld target multiple signaling molecules, such as members of the Traf (tumor necrosis factor (Tnf) receptor (Tnfr)-associated factor) family, the Ikk (inhibitor of the NF-κB (ΙκB) kinase) regulatory subunit Ikkγ (also known as Nemo) (Brummelkamp et al, 2003; Kovalenko et al, 2003; Trompouki et al, 2003), the Src protein tyrosine kinase Lck (Reiley et al, 2006), the transforming growth factor-β (Tgfβ)-activated kinase 1 (Tak1) (Reiley et al, 2007) and many more. Not surprisingly, Usps regulate diverse biological functions, including host defense against infections, immune-cell development, activation and inflammation, cell survival, cell proliferation and tumorigenesis, microtubule assembly and cell migration, mitotic cell entry, calcium-channel function, spermatogenesis, osteoclastogenesis and many more (Sun, 2008). Furthermore, individual Dubs such as A20 or Uspl1 were shown to possess additional functions besides their protease activity further extending potential functions of this class of proteins (Schulz et al, 2012; De et al, 2014). Although the role of some Usps for the peripheral immune system is starting to emerge, the Usp family members that shape the innate immune system in the CNS, especially in microglia, have been less well studied. Here, we identified Usp18 as a novel microglia protein that is essential to prevent aberrant activation and that is required for the termination of the Ifnar2 activation signal mediated by the Ifnar1 subunit of the Ifnar heterodimer complex upon stimulation. Our data further indicate that Usp18-mediated control of the type I IFN system is critical for microglia quiescence preventing uncontrolled tissue damage. Our data additionally suggest that microglia heterogeneity in cortical and subcortical regions is determined by diverse endogenous functional programs. Results Usp18 silences white matter microglia under homeostatic conditions It is not yet known how microglia are kept in a quiescent state under homeostatic conditions, but intracellular proteases are potential candidates for regulatory functions. Among them, ubiquitin-specific proteases (Usps) form the largest family of deubiquitinating enzymes (Dubs) that have key functions in immune responses and other biological processes (Hershko & Ciechanover, 1998; Liu et al, 2005). We first performed whole-genome gene expression analysis of CD11b+CD45lo microglia from the white and gray matter and examined the expression of ubiquitin-specific proteases (Usps) as important regulators of the immune response (Sun, 2008). We found several Usps expressed in both white and gray matter microglia with only few differently expressed proteases in the gray and white matter, most prominently Usp18 (Fig 1A and B). Usp18 transcripts were found to be highly expressed in unstimulated microglia with only background expression levels in other CNS cells (Fig 1C). We next confirmed microglia specificity of this protease in the white matter of Usp18LacZ/LacZ (designated Usp18−/−) mice by X-gal staining (Fig 1D). Figure 1. Usp18 is a distinct feature of white matter microglia and essentially regulates microglia quiescence A. Spatial distribution of ubiquitin-specific protease (USP) transcripts based on FACS-sorted adult microglia isolated from the white or gray matter that were subsequently examined by MouseRef-8 v2.0 Expression Bead Chip (Illumina) array analysis (Olah et al, 2012). Each USP shown exceeds a median expression value of two resulting from five mice compared to the mean expression value of the same gene in the other brain region. B. Quantitative RT–PCR of indicated genes in FACS-isolated adult microglia. Bars represent means ± s.e.m. with three mice in each group (*P < 0.05). Significant differences are determined by an unpaired t-test. C. Expression of Usp18 mRNA measured by qRT–PCR in primary microglia (micro.), astrocytes (astro.), neurons (neu.) and oligodendrocytes (oli.). Bars represent means ± s.e.m with at least three samples in each group normalized to the mean expression value of Usp18 transcripts in the whole brain. D. Cell-specific expression of Usp18. Light microscopic analysis of X-gal-stained (blue) white matter brain tissue of adult Usp18LacZ/LacZ mice. Iba-1 staining (brown) reveals microglia. Inserts show microglia from the cortex. Scale bar, 20 μm. E–G. Histology of different brain areas in the cerebrum of adult Usp18+/+ and Usp18LacZ/LacZ (Usp18−/−) mice. Cortex (Co), hippocampus (Hc), thalamus (Th) and hypothalamus (Hypo) represent areas of the gray matter, whereas corpus callosum (CC) and fimbria (Fi) are defined as white matter. Scale bar, 10 μm. H–J. Histological pictures of different cerebellar regions of adult Usp18+/+ and Usp18LacZ/LacZ (Usp18−/−) mice. Molecular layer (ML) and granular layer (GL) represent areas of the gray matter, whereas arbor vitae (Arb) is part of the white matter. K, L. Quantification of Iba-1+ microglia in the different areas of Usp18LacZ/LacZ (Usp18−/−) mice. Microglia numbers are normalized to that found in Usp18+/+ littermates and are displayed as % of control. At least five mice per genotype were counted. Significant differences are determined by an unpaired t-test or Mann–Whitney U-test and marked with asterisks (*P < 0.05, **P < 0.01). Bars represent means ± s.e.m. M. Immunofluorescence of white matter and gray matter microglia (Iba-1, red) in adult Usp18−/− animals (green, scale bar, 10 μm). Three animals per genotype were examined. One characteristic picture is shown. N. Transmission electron microscopy of myelin-phagocytosing microglia in adult Usp18LacZ/LacZ (Usp18−/−) mice. Scale bars, 1 μm (overview) and 250 nm (zoom). Download figure Download PowerPoint To further investigate the physiological role of Usp18 for the CNS, we performed a thorough histopathological analysis of different brain areas of adult Usp18LacZ/LacZ mice. While there were no obvious histopathological abnormalities in the gray matter, a significant increase of Iba-1+ microglia numbers was detectable in several white matter regions in Usp18-deficient mice (Fig 1E–L). Furthermore, only subcortical white matter microglia but not cortical microglia exhibited strong expression of the activation marker MHC class II and of MAC-3 (LAMP2, CD107b, Fig 1M). Transmission electron microscopy was used to confirm that myelin debris was visible in phagocytotically active microglia (Fig 1N). Importantly, Usp18−/− brains did not show any infiltrating lymphocytes or monocytes (Supplementary Fig S1), indicating a sole microglia activation phenotype that we defined as “white matter microglia activation” (WMMA). We were next interested at what developmental stage the WMMA is first detectable. Hardly any MAC-3+ microglia were visible on postnatal day P0 in both genotypes (Fig 2A). On postnatal day P4, when microglia expansion occurs in foci within the periventricular subcortical white matter called “fountains of microglia” (Hristova et al, 2010), we observed no changes in the number of MAC-3+ microglia in mice lacking Usp18 (Fig 2B and E). However, from P10 onwards until adulthood, Usp18-deficient mice exhibited accumulations of MAC-3+ microglia in the white matter that expressed the activation marker inducible nitric oxide synthase (iNOS) and S100 calcium binding protein a9 (S100a9) (Fig 2C–E, Supplementary Fig S2A). Notably, in vitro oligodendrocyte differentiation as well as overall oligodendrocyte numbers was found to be independent of the presence of Usp18 (Supplementary Figs S3 and S4). In order to examine the underlying mechanisms of WMMA, we analyzed brain homogenates on P4 and P7 when Usp18−/− mice were still histologically undistinguishable from their Usp18+/+ littermates (Fig 2F, Supplementary Fig S2B). We found a robust induction of the myeloattracting and myeloactivating chemokines Ccl2, Ccl3, Ccl5 and Cxcl10, and a slight induction of the cytokines Tnf-α and Il-6, which was also present in adulthood. In situ hybridizations of P4 brain sections revealed Ccl2 mRNA expression in white matter Iba-1+ microglia (Fig 2G). Interestingly, adult Usp18−/− mice occasionally displayed intracerebral calcifications reminiscent to human brains suffering from interferonopathies (Fig 2H) (Crow, 2015). Figure 2. WMMA starts at early postnatal stages A–D. Histology of brain sections of newborn (P0), 4-day-old (P4), 10-day-old (P10) and adult Usp18+/+ and Usp18LacZ/LacZ (Usp18−/−) mice revealing an early white matter microglia activation (WMMA). Hematoxylin and eosin (H&E), MAC-3 for activated microglia. Scale bars, 100 μm (overviews in H&E and MAC-3) and 50 μm (insert in MAC-3). Data are representative of two experiments with two mice each. E. Quantification of MAC-3-labeled microglia of P4 and adult Usp18+/+ and Usp18LacZ/LacZ (Usp18−/−) mice. At least three animals per genotype were examined. Bars represent means ± s.e.m. Significant differences are determined by an unpaired t-test and marked with an asterisk (*P < 0.05). F. Gene expression levels of Ccl5 and Cxcl10 mRNA in the brains of P4, P7 and adult Usp18+/+ (white bars) and Usp18LacZ/LacZ (Usp18−/−, black bars) mice. Data are expressed as the ratio of induced factors normalized to endogenous Gapdh compared to Usp18+/+ mice and expressed as mean ± s.e.m. At least three mice were used in two independent experiments. Significant differences are determined by an unpaired t-test and marked with asterisks (*P < 0.05, **P < 0.01). G. In situ hybridization of Ccl2 mRNA displays co-labeling in Iba-1+ white matter microglia of Usp18LacZ/LacZ (Usp18−/−) mice. Scale bar, 20 μm. Two mice were used in two independent experiments. H. Occurrence of cerebral calcifications in adult Usp18LacZ/LacZ (Usp18−/−) mice. Scale bars, 200 μm (overview) and 50 μm (zoom). Download figure Download PowerPoint To test whether microglia activation found in the WMMA of Usp18-deficient mice is induced by cell autonomous or non-autonomous mechanisms, we targeted the Usp18 locus and generated Usp18 floxed animals (Usp18fl/fl, Fig 3A and B). We next crossed these newly generated Usp18fl/fl mice with a transgenic line expressing the Cre recombinase under the control of the CX3CR1 promoter that drives recombination in myeloid cells in general (Yona et al, 2013) including microglia in the brain (Goldmann et al, 2013; Wieghofer et al, 2015). Resulting Cx3cr1Cre:Usp18fl/fl mice showed an Usp18 deletion in microglia but not in neuroectodermal cells of the CNS (Fig 3C). Strikingly, the brains of adult Cx3cr1Cre:Usp18fl/fl mice exhibited marked microgliosis with activated amoboid Iba-1+Mac3+ microglia that precisely mirrored the WMMA pathology observed in adult Usp18−/− mice (Fig 3D–J). Figure 3. WMMA due to Usp18 deletion occurs in a cell-autonomous manner A. Targeting strategy for the conditional mutagenesis of the Usp18 gene. A targeting vector was used to modify the Usp18 gene locus. Upon homologous recombination and elimination of the frt-flanked selection marker (neo), the third exon of the gene was flanked by loxP sites allowing Cre-mediated deletion of Usp18. B. Homologous recombination in ES cells was detected by genomic Southern blot analysis. As depicted in (A), probe A detects a diagnostic 3.5-kb band upon KpnI restriction digest diagnostic for the mutated Usp18 allele. C. PCR analysis of the Usp18 deletion in primary microglia, astrocytes, oligodendrocytes or neurons of Usp18fl/fl, Cx3cr1Cre:Usp18fl/fl and wild-type mice. Recombination is only taking place in microglia but not in astrocytes, oligodendrocytes or neurons. One representative experiment out of two performed is shown. D, E. Histology of different brain areas in the cerebrum of adult Usp18fl/fl and Cx3cr1Cre:Usp18fl/fl mice. Cortex (Co), hippocampus (Hc), thalamus (Th) and hypothalamus (Hypo) represent areas of the gray matter, whereas corpus callosum (CC) and fimbria (Fi) are parts of the white matter. Scale bar = 10 μm. F, G. Histological pictures of different cerebellar regions of adult Usp18fl/fl and Cx3cr1Cre:Usp18fl/fl mice. Molecular layer (ML) and granular layer (GL) represent areas of the gray matter, whereas arbor vitae (Arb) is part of white matter. H, I. Quantification of Iba-1+ cells in Cx3cr1Cre:Usp18fl/fl mice. Microglia numbers are normalized to that found in Usp18+/+ littermates and are displayed as % of control. At least five mice per genotype were counted. Significant differences are determined by an unpaired t-test or Mann–Whitney U-test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.0001). Bars represent means ± s.e.m. J. Immunofluorescence of white and gray matter microglia (Iba-1, red) in adult Usp18−/− animals (green, scale bar, 10 μm). Three animals per genotype were examined. One characteristic sample is shown. Download figure Download PowerPoint Microglia activation in the absence of Usp18 is due to prolonged STAT1 phosphorylation We subsequently isolated microglia, and whole-genome gene expression was determined. Investigation of the gene ontology enrichment network revealed that mostly type I IFN-regulated molecules were affected in Usp18−/− microglia (Fig 4A). IFN-β treatment induced Usp18 expression in a time-dependent and dose-independent fashion in primary microglia (Fig 4B). Constitutive type I IFN levels have been shown for some tissues (Gough et al, 2012) but have never been reported in the healthy murine brain. We used Mx1Cre mice intercrossed with the R26-confetti reporter line to examine the constitutive expression of the classical type I IFN-induced gene Mx1 and found Iba-1+Mx1+ microglia in white matter under SPF conditions indicating the presence of constitutive IFN signaling in these cells. Figure 4. Lack of Usp18 enhances type I IFN gene expression in microglia due to prolonged Stat1 phosphorylation Gene ontology enrichment network on differentially expressed genes in microglia from unstimulated Usp18+/+ and Usp18LacZ/LacZ (Usp18−/−) microglia on the basis of an Affymetrix DNA microarray analysis. Diagram depicts results of GO clustering through GORilla. Only very highly significantly overrepresented GO terms are included with P-values ranging from P < 10−9 (yellow) to P < 10−24 (red). Quantitative RT–PCR for Usp18 transcripts in primary microglia stimulated for the designated time points with 100 U/ml of IFN-β (left panel) or with 10, 100 or 1,000 U/ml of IFN-β and measured after 4 h (right panel). Bars represent means ± s.e.m with three to four samples in each group. Data are representative of two independently performed experiments. Fluorescence microscopy of the white matter (corpus callosum) of Mx1Cre:R26-confetti mice raised under specific pathogen-free conditions. Recombination of GFP, RFP, CFP or YFP (combined into one channel to XFP and displayed in red) was found in Iba-1+ microglia of the white matter. Scale bars: 20 μm (overview) and 10 μm (zoom). Flow cytometric quantification of Stat1 phosphorylation in BV-2 microglial cells transfected with control siRNA (siRNA co) or siRNA against Usp18. Representative dot blots of IFN-β-treated cells at indicated time points are shown that were obtained from two independent experiments. FSC: forward scatter. Immunoblot analysis of type I IFN signaling in microglia lack
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