PRMT5-Selective Inhibitors Suppress Inflammatory T Cell Responses and Experimental Autoimmune Encephalomyelitis

In the autoimmune disease multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), expansion of pathogenic, myelin-specific Th1 cell populations drives active disease; selectively targeting this process may be the basis for a new therapeutic approach. Previous studies have hinted at a role for protein arginine methylation in immune responses, including T cell–mediated autoimmunity and EAE. However, a conclusive role for the protein arginine methyltransferase (PRMT) enzymes that catalyze these reactions has been lacking. PRMT5 is the main PRMT responsible for symmetric dimethylation of arginine residues of histones and other proteins. PRMT5 drives embryonic development and cancer, but its role in T cells, if any, has not been investigated. In this article, we show that PRMT5 is an important modulator of CD4+ T cell expansion. PRMT5 was transiently upregulated during maximal proliferation of mouse and human memory Th cells. PRMT5 expression was regulated upstream by the NF-κB pathway, and it promoted IL-2 production and proliferation. Blocking PRMT5 with novel, highly selective small molecule PRMT5 inhibitors severely blunted memory Th expansion, with preferential suppression of Th1 cells over Th2 cells. In vivo, PRMT5 blockade efficiently suppressed recall T cell responses and reduced inflammation in delayed-type hypersensitivity and clinical disease in EAE mouse models. These data implicate PRMT5 in the regulation of adaptive memory Th cell responses and suggest that PRMT5 inhibitors may be a novel therapeutic approach for T cell–mediated inflammatory disease

NF-κB/mTOR/MYC Axis Drives PRMT5 Protein Induction After T Cell Activation via Transcriptional and Non-transcriptional Mechanisms

Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) mediated by CD4+ T cells and modeled via experimental autoimmune encephalomyelitis (EAE). Inhibition of PRMT5, the major Type II arginine methyltransferase, suppresses pathogenic T cell responses and EAE. PRMT5 is transiently induced in proliferating memory inflammatory Th1 cells and during EAE. However, the mechanisms driving PRMT5 protein induction and repression as T cells expand and return to resting is currently unknown. Here, we used naive mouse and memory mouse and human Th1/Th2 cells as models to identify mechanisms controlling PRMT5 protein expression in initial and recall T cell activation. Initial activation of naive mouse T cells resulted in NF-κB-dependent transient Prmt5 transcription and NF-κB, mTOR and MYC-dependent PRMT5 protein induction. In murine memory Th cells, transcription and miRNA loss supported PRMT5 induction to a lesser extent than in naive T cells. In contrast, NF-κB/MYC/mTOR-dependent non-transcriptional PRMT5 induction played a major role. These results highlight the importance of the NF-κB/mTOR/MYC axis in PRMT5-driven pathogenic T cell expansion and may guide targeted therapeutic strategies for MS

Modulation of Epithelial Morphology, Monolayer Permeability, and Cell Migration by Growth Arrest Specific 3/Peripheral Myelin Protein 22

Peripheral myelin protein 22 (PMP22) is associated with a subset of hereditary peripheral neuropathies. Although predominantly recognized as a transmembrane constituent of peripheral nerve myelin, PMP22 is localized to epithelial and endothelial cell-cell junctions, where its function remains unknown. In this report, we investigated the role of PMP22 in epithelial biology. Expression of human PMP22 (hPMP22) slows cell growth and induces a flattened morphology in Madin-Darby canine kidney (MDCK) cells. The transepithelial electrical resistance (TER) and paracellular flux of MDCK monolayers are elevated by hPMP22 expression. After calcium switch, peptides corresponding to the second, but not the first, extracellular loop of PMP22 perturb the recovery of TER and paracellular flux. Finally, subsequent to wounding, epithelial monolayers expressing hPMP22 fail to migrate normally. These results indicate that PMP22 is capable of modulating several aspects of epithelial cell biology, including junctional permeability and wound closure

Control of the Inflammatory Macrophage Transcriptional Signature by miR-155

<div><p>Inflammatory M1 spectrum macrophages protect from infection but can cause inflammatory disease and tissue damage, whereas alternatively activated/M2 spectrum macrophages reduce inflammation and promote tissue repair. Modulation of macrophage phenotype may be therapeutically beneficial and requires further understanding of the molecular programs that control macrophage differentiation. A potential mechanism by which macrophages differentiate may be through microRNA (miRNA), which bind to messenger RNA and post-transcriptionally modify gene expression, cell phenotype and function. We hypothesized that the inflammation-associated miRNA, miR-155, would be required for typical development of macrophage inflammatory state. miR-155 was rapidly up-regulated over 100-fold in inflammatory M1(LPS + IFN-γ), but not M2(IL-4), macrophages. Inflammatory genes <i>Inos</i>, <i>Il1b</i> and <i>Tnfa</i> and their corresponding protein or enzymatic products were reduced up to 72% in miR-155 knockout mouse M1(LPS + IFN-γ) macrophages, but miR-155 deficiency did not affect expression of the M2-associated gene <i>Arg1</i> in M2(IL-4) macrophages. Additionally, a miR-155 oligonucleotide inhibitor efficiently suppressed <i>Inos</i> and <i>Tnfa</i> gene expression in wild-type M1(LPS + IFN-γ) macrophages. Comparative transcriptional profiling of unstimulated and M1(LPS + IFN-γ) macrophages derived from wild-type (WT) and miR-155 knockout (KO) mice revealed that half (approximately 650 genes) of the signature we previously identified in WT M1(LPS + IFN-γ) macrophages was dependent on miR-155. Real-Time PCR of independent datasets confirmed that miR-155 contributed to suppression of its validated mRNA targets <i>Inpp5d</i>, <i>Tspan14</i>, <i>Ptprj</i> and <i>Mafb</i> and induction of <i>Inos</i>, <i>Il1b</i>, <i>Tnfa</i>, <i>Il6</i> and <i>Il12</i>. Overall, these data indicate that miR-155 plays an essential role in driving the inflammatory phenotype of M1(LPS+ IFN-γ) macrophages.</p></div

miR-155 is associated with the classically activated macrophage phenotype.

<p>Expression of miRNAs was determined by Taqman Real-Time PCR and expressed as mean relative expression (+ SEM) in <b>(A)</b> macrophages stimulated <i>in vitro</i> for 24 hours in M0, M1, and M2 (n = 3) conditions; expression relative to M0 condition; Post-hoc ANOVA, **p<0.005. <b>(B)</b> M1 and M2 macrophages activated <i>in vitro</i> over a 48 hour period; expression relative to 0 hour pre-stimulation time-point. Unpaired t-test, ***p<0.001. Results reproduced in two independent experiments.</p

Classically activated macrophage signature in wild-type and miR-155 knockout macrophages.

<p>Fold Change (FC) vs. Mean Expression Value (MEV) plot of microarray data highlighting 2 FC or higher up-regulated genes (red, p≤0.05) or down-regulated genes (blue, p≤0.05) in <b>(A)</b> knockout <b>(</b>KO) M1 to M0 macrophages comparison (n = 3). (<b>B, C</b>) FC vs. MEV plot with previously described classical M1 markers highlighted (in red) and the top 15 M1-exclusive genes (identified in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159724#pone.0159724.ref017" target="_blank">17</a>]) most decreased in KO M1 macrophages (in green) in the WT M1 vs. M0 comparison (<b>B</b>) and the KO M1 vs. M0 comparison (<b>C</b>). Red lines represent a +/- 2FC cut-off.</p

Novel Markers to Delineate Murine M1 and M2 Macrophages.

Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages

Reduced classically activated M1 marker expression in miR-155 knock-out (KO) macrophages.

<p><b>(A)</b> Inducible nitric oxide synthase (<i>Nos2</i>), <b>(B)</b> <i>IL1b</i>, (<b>C</b>) Tumor Necrosis Factor-α (<i>Tnfa</i>) <b>(H</b>) and Arginase-1 (<i>Arg1</i>) expression was determined by Real-Time PCR in wild-type (WT, n = 8–11) and miR-155 knockout (KO n = 8–12) bone marrow-derived macrophages <i>in vitro</i> activated in M1 or M2 conditions for 24 hours in three independent experiments. Gene expression is expressed as a percentage +/- SEM of the WT M1 condition. Unpaired t-test, *p<0.05, **p<0.005. Relative concentration of <b>(D)</b> nitric oxide (NO), <b>(E)</b> IL-1β protein and <b>(F)</b> TNF-α protein was determined using Griess assay (for NO) or Bio-Plex Suspension Array (For IL-1β and TNF-α) in cell lysates from WT (n = 5) and miR-155 KO (n = 5) bone marrow-derived macrophages <i>in vitro</i> activated in M1 or M2 conditions for 24 hours. Individual protein concentrations expressed as fraction of total protein concentration in either M0 or M1 condition. (<b>G</b>) <i>IL1b</i> expression was determined by Real-Time PCR in WT and KO bone marrow-derived macrophages transfected with a scrambled miR control (n = 5) or a miR-155 oligonucleotide mimic (n = 5) and activated in M0 (untreated), M1 (LPS+IFN-γ) or M2(IL-4) conditions for 24 hours. Gene expression is expressed as a percentage +/- SEM of the scrambled M1 condition. Unpaired t-test, ***p<0.0005, ****p<0.00005. (A-G) Data from 2–3 independent experiments.</p

miR-155 targets up-regulated more than 2 fold-change in KOM1(LPS + IFN-γ) vs. WTM1(LPS + IFN-γ) macrophages.

<p>miR-155 targets up-regulated more than 2 fold-change in KOM1(LPS + IFN-γ) vs. WTM1(LPS + IFN-γ) macrophages.</p