PDE8 controls CD4(+) T cell motility through the PDE8A-Raf-1 kinase signaling complex

The levels of cAMP are regulated by phosphodiesterase enzymes (PDEs), which are targets for the treatment of inflammatory disorders. We have previously shown that PDE8 regulates T cell motility. Here, for the first time, we report that PDE8A exerts part of its control of T cell function through the V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase signaling pathway. To examine T cell motility under physiologic conditions, we analyzed T cell interactions with endothelial cells and ligands in flow assays. The highly PDE8-selective enzymatic inhibitor PF-04957325 suppresses adhesion of in vivo myelin oligodendrocyte glycoprotein (MOG) activated inflammatory CD4(+) T effector (Teff) cells to brain endothelial cells under shear stress. Recently, PDE8A was shown to associate with Raf-1 creating a compartment of low cAMP levels around Raf-1 thereby protecting it from protein kinase A (PKA) mediated inhibitory phosphorylation. To test the function of this complex in Teff cells, we used a cell permeable peptide that selectively disrupts the PDE8A-Raf-1 interaction. The disruptor peptide inhibits the Teff-endothelial cell interaction more potently than the enzymatic inhibitor. Furthermore, the LFA-1/ICAM-1 interaction was identified as a target of disruptor peptide mediated reduction of adhesion, spreading and locomotion of Teff cells under flow. Mechanistically, we observed that disruption of the PDE8A-Raf-1 complex profoundly alters Raf-1 signaling in Teff cells. Collectively, our studies demonstrate that PDE8A inhibition by enzymatic inhibitors or PDE8A-Raf-1 kinase complex disruptors decreases Teff cell adhesion and migration under flow, and represents a novel approach to target T cells in inflammation

Treatment of Experimental Autoimmune Encephalomyelitis with an Inhibitor of Phosphodiesterase-8 (PDE8)

After decades of development, inhibitors targeting cyclic nucleotide phosphodiesterases (PDEs) expressed in leukocytes have entered clinical practice for the treatment of inflammatory disorders, with three PDE4 inhibitors being in clinical use as therapeutics for psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease and atopic dermatitis. In contrast, the PDE8 family that is upregulated in pro-inflammatory T cells is a largely unexplored therapeutic target. We have previously demonstrated a role for the PDE8A-Raf-1 kinase complex in the regulation of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) activated CD4+ effector T cell adhesion and locomotion by a mechanism that differs from PDE4 activity. In this study, we explored the in vivo treatment of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS) induced in mice immunized with MOG using the PDE8-selective inhibitor PF-04957325. For treatment in vivo, mice with EAE were either subcutaneously (s.c.) injected three times daily (10 mg/kg/dose), or were implanted subcutaneously with Alzet mini-osmotic pumps to deliver the PDE8 inhibitor (15.5 mg/kg/day). The mice were scored daily for clinical signs of paresis and paralysis which were characteristic of EAE. We observed the suppression of the clinical signs of EAE and a reduction of inflammatory lesion formation in the CNS by histopathological analysis through the determination of the numbers of mononuclear cells isolated from the spinal cord of mice with EAE. The PDE8 inhibitor treatment reduces the accumulation of both encephalitogenic Th1 and Th17 T cells in the CNS. Our study demonstrates the efficacy of targeting PDE8 as a treatment of autoimmune inflammation in vivo by reducing the inflammatory lesion load

Use Of An Alpha-smooth Muscle Actin (SMAA) GFP Reporter To Identify An Osteoprogenitor Population

Identification of a reliable marker of skeletal precursor cells within calcified and soft tissues remains a major challenge for the field. To address this, we used a transgenic model in which osteoblasts can be eliminated by pharmacological treatment. Following osteoblast ablation a dramatic increase in a population of α-smooth muscle actin (α-SMA) positive cells was observed. During early recovery phase from ablation we have detected cells with the simultaneous expression of SMAA and a preosteoblastic 3.6GFP marker, indicating the potential for transition of α-SMA+ cells towards osteoprogenitor lineage. Utilizing α-SMAGFP transgene, α-SMAGFP+ positive cells were detected in the microvasculature and in the osteoprogenitor population within bone marrow stromal cells. Osteogenic and adipogenic induction stimulated expression of bone and fat markers in the α-SMAGFP+ population derived from bone marrow or adipose tissue. In adipose tissue, α-SMA+ cells were localized within the smooth muscle cell layer and in pericytes. After in vitro expansion, α-SMA+/CD45−/Sca1+ progenitors were highly enriched. Following cell sorting and transplantation of expanded pericyte/myofibroblast populations, donor-derived differentiated osteoblasts and new bone formation was detected. Our results show that cells with a pericyte/myofibroblast phenotype have the potential to differentiate into functional osteoblasts

Virion Assembly Factories in the Nucleus of Polyomavirus-Infected Cells

<div><p>Most DNA viruses replicate in the cell nucleus, although the specific sites of virion assembly are as yet poorly defined. Electron microscopy on freeze-substituted, plastic-embedded sections of murine polyomavirus (PyV)-infected 3T3 mouse fibroblasts or mouse embryonic fibroblasts (MEFs) revealed tubular structures in the nucleus adjacent to clusters of assembled virions, with virions apparently “shed” or “budding” from their ends. Promyelocytic leukemia nuclear bodies (PML-NBs) have been suggested as possible sites for viral replication of polyomaviruses (BKV and SV40), herpes simplex virus (HSV), and adenovirus (Ad). Immunohistochemistry and FISH demonstrated co-localization of the viral T-antigen (Tag), PyV DNA, and the host DNA repair protein MRE11, adjacent to the PML-NBs. In PML<b>^−/−</b> MEFs the co-localization of MRE11, Tag, and PyV DNA remained unchanged, suggesting that the PML protein itself was not responsible for their association. Furthermore, PyV-infected PML<b>^−/−</b> MEFs and PML<b>^−/−</b> mice replicated wild-type levels of infectious virus. Therefore, although the PML protein may identify sites of PyV replication, neither the observed “virus factories” nor virus assembly were dependent on PML. The ultrastructure of the tubes suggests a new model for the encapsidation of small DNA viruses.</p> </div

PyV DNA and T-antigen localization in PyV-infected PML^−/− MEFs.

<p>PML^−/− MEFs were infected with PyV at an MOI of 30–40 pfu/cell. At 22 or 24 hpi cells were fixed, permeabilized, and co-stained with either anti-Tag and/or anti-MRE11a antibodies followed by AlexaFluor-conjugated secondary antibodies, a fluorescently-labeled PyV DNA FISH probe, and DAPI staining of nuclei. <b>A</b>) FISH for PyV DNA at 24 hpi followed by antibody staining for Tag. <b>B</b>) Infected cells were stained by FISH for PyV DNA at 22 hpi followed by antibody staining for MRE11 or co-stained for MRE11 and Tag. All images represent a 0.1 mm z-stack slice.</p

Tubular structures contain VP1.

<p>PyV-infected 3T3 cells or PML^−/− MEFs (MOI of 10–20 pfu/cell) at 32 hpi were frozen by high pressure and processed by cryo-substitution for immunoelectron microscopy. Thin sections (45 nm or 70 nm) of Lowicryl-embedded samples were stained with either anti-PML or anti-VP1 antibodies followed by a secondary antibody conjugated to 10 or 15 nm colloidal gold. Top panels: 45 nm sections stained for the PML protein; white arrows, anti-PML staining, black arrow, tubular structures. Bottom panels: 70 nm sections stained for VP1; white arrows, anti-VP1 staining, black arrows, tubular structures.</p

Temporal progression of PyV nuclear assembly.

<p>Images from dual-axis tomograms of high pressure frozen, Epon-embedded, 300 nm thick sections of 3T3 cells infected with PyV (MOI of 10–20 pfu/cell) and harvested at 32 hpi. <b>A</b>) Tubular structures (black arrowhead) are present in the periphery of the condensed chromatin (Chr) adjacent to occasional virions (white arrowhead). <b>B</b>) A 1 nm section extracted from a 2×2 montage over six serial sections (1.8 µm thick) of a 3T3 nucleus in which the interchromatin space is partially filled with virion clusters and each cluster is associated with tubular structures. As infection proceeds, the number of virus clusters (white arrowhead) increases while the tubular structures (black arrowhead) are less prominent. <b>C</b>) Late in infection virions fill the entire interchromatin space and tubular structures are not seen. <b>D</b>) 3-D model of the 2×2 montage over six serial sections (each 300 nm thick) of a PyV-infected 3T3 nucleus. The model represents 1.8 µm thick section of the nucleus showing the connections between virion clusters and tubular structures. An image extracted from the tomogram is shown in (B); a video of the tomograms of each section and the model can be found as supporting information <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002630#ppat.1002630.s002" target="_blank">Video S1</a>. Pink spheres, full virions; yellow cylinders, tubular structures. Chr, host condensed chromatin; Cyt, cytoplasm.</p

Tomographic reconstruction of a virus factory.

<p>3T3 cells were infected with PyV as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002630#ppat-1002630-g001" target="_blank">Figure 1</a>. <b>A</b>) 70 nm thin section of Epon-embedded cells showing tubular structures adjacent to a virus cluster. (Black arrowhead, “full” tubular structure; white arrowhead, full virion; black arrow, “empty” tubular structure; white arrow, empty virion) <b>B</b>) A cluster of virions packed in a highly ordered array at the periphery of a bundle of tubular structures. One of the tubular structures appears in the plane (black arrowhead). Empty (white arrow) and full (white arrowhead) virions are identified in the virus clusters. <b>C</b>) 3-D model of (B) showing ≈2000 full assembled virions (pink spheres) and ≈2% empty virions (red spheres) in a 300 nm thick section. The tubular structures are either filled with electron-dense material (yellow cylinders) or appear empty (red cylinders). (See also Supporting information <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002630#ppat.1002630.s003" target="_blank">Video S2</a>).</p

Tubule ultrastructure and association with virions.

<p>PyV-infected 3T3 cells (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002630#ppat-1002630-g001" target="_blank">Figure 1</a>) were processed by high pressure freezing and cryo-substitution for electron microscopy and a series of images was extracted from dual-axis tomograms of Epon-embedded, 300 nm thick sections. <b>A</b>) Two distinct virions show a lighter and well-arranged density at their periphery (white arrowhead) corresponding to capsid protein density as well as a dense core suggesting DNA. The tubular structure exhibited a similar organization with a lighter and well-arranged density at its periphery (white arrowhead) as well as a patched and electron-dense core (black arrowhead). Occasionally, “bulges” or opaque regions (thick black arrows, also see inset) are seen along the length of the tube. <b>B</b>) A tubular structure (black arrowhead) exhibiting bulges forming at both ends (thick black arrows). The bulges are defined by an inner layer of dense material surrounded by well-arranged lighter densities. <b>C</b>) A mature virion is partially attached to the tubular structure (black arrowhead). A constriction point is seen where the virion may be shed from the tubular structure (thin black arrow). (See also Supporting information <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002630#ppat.1002630.s001" target="_blank">Figure S1</a>).</p

PyV DNA and T-antigen are localized near PML-NBs.

<p>C57 MEFs were infected at an MOI of 30–40 pfu/cell. At 24 or 28 hpi cells were fixed, permeabilized, and co-stained with either anti-PML, anti-Tag and/or anti-MRE11 antibodies followed by AlexaFluor-conjugated secondary antibodies, a fluorescently-labeled PyV DNA FISH probe, and DAPI staining of nuclei. <b>A</b>) PyV-infected C57 MEFs at 24 hpi stained for either PML (top) or Tag (bottom) followed by fluorescent <i>in situ</i> hybridization (FISH) for PyV DNA. <b>B</b>) PyV-infected C57 MEFs at 28 hpi co-stained for PML and Tag. <b>C</b>) C57 MEFs at 24 hpi co-stained for either MRE11 and PyV DNA (by FISH) or MRE11 and Tag. All images represent a 0.1 mm z-stack slice. Insets show enlarged regions from image to illustrate the localization of proteins in relation to each other (B) or PyV DNA (C).</p