ISG15 Regulates Peritoneal Macrophages Functionality against Viral Infection

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG152/2 macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogensThis work was supported by grants from the Spanish Ministry of Health FIS2011-00127, Comunidad de Madrid UAM-CM-CCG10-4911 and UAM-Banco de Santander to SG. This work was also partly supported by NIAID grant U19AI083025 and by CRIP (Center for Research on Influenza Pathogenesis, HHSN266200700010C), a NIAID Center of Excellence for Influenza Research and Surveillance (CEIRS) to AGS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Long-term STED imaging of amyloid fibers with exchangeable Thioflavin T

We report the use of the amyloid probe Thioflavin T (ThT) as a specific and exchangeable fluorophore for stimulated emission depletion (STED) super-resolution imaging of amyloid fibers. This method achieves a spatial resolution in the range of 60–70 nm, low image background and increased photostability that enables long-term STED imaging. These results expand the widespread uses of ThT and can be potentially extended to other common amyloid fluorescent probes, providing new tools for the study of amyloid diseases.This research was supported by the Ministerio de Ciencia, Innovación y Universidades through projects PCI2018-093064 and PGC2018-094802-B-I00 (C. F.), SAF2017-87305-R (B. S.) and SEV-2016-0686 (IMDEA Nanociencia), and the Comunidad Autónoma de Madrid project S2017/BMD-3867 (C.F.)

R-RAS2 overexpression in tumors of the human central nervous system

Malignant tumors of the central nervous system (CNS) are the 10th most frequent cause of cancer mortality. Despite the strong malignancy of some such tumors, oncogenic mutations are rarely found in classic members of the RAS family of small GTPases. This raises the question as to whether other RAS family members may be affected in CNS tumors, excessively activating RAS pathways. The RAS-related subfamily of GTPases is that which is most closely related to classical Ras and it currently contains 3 members: RRAS, RRAS2 and RRAS3. While R-RAS and R-RAS2 are expressed ubiquitously, R-RAS3 expression is restricted to the CNS. Significantly, both wild type and mutated RRAS2 (also known as TC21) are overexpressed in human carcinomas of the oral cavity, esophagus, stomach, skin and breast, as well as in lymphomas. Hence, we analyzed the expression of R-RAS2 mRNA and protein in a wide variety of human CNS tumors and we found the R-RAS2 protein to be overexpressed in all of the 90 CNS cancer samples studied, including glioblastomas, astrocytomas and oligodendrogliomas. However, R-Ras2 was more strongly expressed in low grade (World Health Organization grades I-II) rather than high grade (grades III-IV) tumors, suggesting that R-RAS2 is overexpressed in the early stages of malignancy. Indeed, R-RAS2 overexpression was evident in pre-malignant hyperplasias, both at the mRNA and protein levels. Nevertheless, such dramatic changes in expression were not evident for the other two subfamily members, which implies that RRAS2 is the main factor triggering neural transformation.This work was supported by grants SAF2012-31279 from the Comisión Interministerial de Ciencia y Tecnología and the Ramón y Cajal program (RYC-2010-06251, to B.C.). We also thank the Fundación Ramón Areces for its institutional support of the Centro de Biología Molecular Severo OchoaPeer Reviewe

Harnessing DNA for nanothermometry

16 p.-4 fig.-1 graph. abst.Temperature measurement at the nano-scale has brought insight to a wide arrayof research interests in modern chemis-try, physics, and biology. These measure-ments have been enabled by the adventof nanothermometers, which relay nano-scale temperature information throughthe analysis of their intrinsic photo-physical behavior. In the past decade, several nanothermometers have beendeveloped including dyes, nanodiamonds, fluorescent proteins, nucleotides,and nanoparticles. However, temperature measurement using intact DNA hasnot yet been achieved. Here, we present a method to study the temperaturesensitivity of the DNA molecule within a physiologic temperature range whencomplexed with fluorescent dye. We theoretically and experimentally reportthe temperature sensitivity of the DNA-Hoechst 33342 complex in differentsizes of double-stranded oligonucleotides and plasmids, showing its potentialuse as a nanothermometer. These findings allow for extending the thermalstudy of DNA to several research fields including DNA nanotechnology, opti-cal tweezers, and DNA nanoparticles.This work was supported by RETOS RTI2018-101050-J-I00 (ST), National Institute of General Medical Sciences of the National Institutes of Health under award numberR01GM124133 (AA), Ramon y Cajal Grant (RYC-2013-13546) and by SAF2015-67538-R (MCIU/AEI/FEDER, UE) (AE)Peer reviewe

ISG15 Regulates Peritoneal Macrophages Functionality against Viral Infection

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG152/2 macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogens

Phagocytosis of GFP-latex beads by ISG15 +/+ or ISG15 −/− macrophages.

<p><b>A–C.</b> Peritoneal macrophages isolated for ISG15+/+ or ISG15<b>−/−</b> mice were seeded in 8-well tissue culture plates and treated with type I IFN (100units/ml) for 16 hours (<b>A</b>) or not (<b>C</b>). After that, the cells were incubated with 1<b>-</b>µm-diameter latex beads conjugated to GFP in a ratio of 10 latex beads per cell. Phagocytized beads and cells were visualized with the time by fluorescent and phase contrast microscopy. <b>B.</b> Quantification of phagocytic cells in type I IFN alpha treated or not treated macrophages. Cells were quantified by IF in three independent experiments. Black bars represent ISG15+/+ macrophages, white bars represents ISG15<b>−/−</b> macrophages. Results represent the mean ± the standard deviation of three independent experiments. P values from a two-tailed t-test assuming non-equal variance were determined. In all the cases, P<0.05. <b>D.</b> Phagocytosis of GFP-latex beads by ISG15+/+ and ISG15<b>−/−</b> macrophages detected by immunofluorescence. Peritoneal macrophages were cultured on coverslips and incubated for 1 h with GFP-latex beads (20 beads per cell) and then washed three times with phosphate-buffered saline (PBS) and incubated with Dulbecco's medium (DMEM) medium for an additional hour. Cells were fixed with 4% PFA and processed for immunofluorescence analysis. Phagocytized beads and cells were visualized by confocal fluorescent microscopy. Representative fields are shown at a magnification of 40× (left panels) and 100× (right panels).</p

Effect of ISG15 on cytotoxicity and infection progression in ISG15+/+ or ISG15−/− MEFs or macrophages infected with influenza virus (FluV).

<p><b>A–B. </b><i>De novo</i> protein synthesis in FluV- infected ISG15+/+ or ISG15−/− MEFs (<b>A</b>) or peritoneal macrophages (<b>B</b>). ISG15+/+or ISG15<b>−/−</b> cells were infected (10⁶ cells/time post-infection; 3 PFU/cell) with FluV (A/<i>WSN</i>/1933 strain) and labeled with ³⁵S-methionine (50 µCi/ml, 30 min) at the times indicated. Cellular lysates were analyzed by 12% SDS-PAGE, transferred to nitrocellulose membranes and visualized by autoradiography. Protein standards are indicated. Uninfected cells (Mock) served as control. In the same membranes, the expression of ISG15 or tubuline (protein loading control) was detected by Western blot using specific antibodies. <b>C.</b> ISG15+/+ or ISG15<b>−/−</b> peritoneal macrophages were infected with FluV (A/<i>WSN</i>/1933 strain, 5 PFU/cell) and CPE was visualized by phase-contrast microscopy at indicated times post-infection. A selected cell is indicated with an arrow.</p

ISG15 control the VACV entry.

<p>Peritoneal macrophages isolated from ISG15+/+ or ISG15<b>−/−</b> mice were seeded in 8-well tissue culture plates and treated with type I interferon (IFN) (100units/ml) for 16 hours. After that, the cells infected with VACV-YFP (60 PFU/cell). Infected-cells were visualized with the time by fluorescent and phase contrast microscopy. Representative fields are shown at a magnification of 40× (left panels).</p

ISG15 regulates the phagocytosis and the clearance of VACV-infected MEFS regulating AKT phosphorylation.

<p><b>A.</b> ISG15−/− MEFS were infected with VACV-YFP at 1 PFU/cell for 8 hours, and added to the ISG15+/+ or ISG15−/− macrophages culture with a ratio of one MEF cells to four macrophages. The mixed culture was monitored for 2,5 hours at 37°C by time-lapse microscopy and representative images were illustrated. <b>B.</b> Inhibition of VACV growth in the presence of ISG15+/+ macrophages. (A) ISG15−/− MEFS were infected with VACV-YFP at 1 PFU/cell for 8 hours and further cultured with DMEM (as negative control) or macrophages (ISG15+/+ or ISG15<b>−/−</b>) with a ratio of 1∶2 using 10⁵ macrophages. Infected cells were harvested at different times postinfection and virus yields were determined by plaque assay. Results represent the mean ± the standard deviation of three independent experiments. P values from a two-tailed t test assuming non-equal variance were determined. In all the cases, P<0.05. <b>C-D-E.</b> Viral protein expression (<b>C</b>) ISG15 (<b>D</b>) and AKT phosphorylation levels (<b>E</b>) were measured in the above described co-cultures. Total proteins from cells described above were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-ISG15 or anti-phosphoSer473<b>-</b>AKT. The expression of AKT was evaluated using a specific antibody. Actin was measured as protein loading control. Protein standards are indicated. <b>F–G</b> Quantification of phagocytic cell, expressed as relative amount of the total number of macrophages, and the viral titer was determined in the absence (<b>F</b>) or presence of 100 nM wortmannin (<b>G</b>). Means and standard deviations of a typical example from three independent experiments are presented. P values from a two-tailed t test assuming non-equal variance were determined. In all the cases, P<0.05.</p

Wortmannin treatment inhibits phagocytosis of GFP-latex beads by ISG15+/+ macrophages.

<p>Peritoneal macrophages isolated from ISG15+/+ mice were seeded in 8-well tissue culture plates and treated with type I IFN (100 units/ml) for 16 hours. After that, were untreated or treated with wortmannin (100 nM) or rapamycin (100 nM) for 1 hour and subsequently incubated with 1<b>-</b>µm-diameter latex beads conjugated to GFP in a ratio of 20 latex beads per cell. Phagocytized beads and cells were visualized with the time by fluorescent and phase contrast microscopy. Representative fields are shown at a magnification of 40× (left panels) and 100× (right panels).</p