Regulation of Suppressors of Cytokine Signaling as a Therapeutic Approach in Autoimmune Diseases, with an Emphasis on Multiple Sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating, presumably autoimmune disease of the central nervous system (CNS). Among the available MS therapies, interferon (IFN)β and the recently introduced statins have been reported to exert their immunomodulatory effects through the induction of SOCS1 and SOCS3 in various inflammatory cell subsets. The SOCS proteins negatively regulate cytokine and Toll-like receptors- (TLR-) induced signaling in the inflammatory cells. SOCS1 and SOCS3 have been reported to play an important role in the regulation of Th17-cell differentiation through their effects on the cells of the innate and adaptive immune systems. IFNβ and statins inhibit Th17-cell differentiation directly and indirectly via induction of SOCS1 and SOCS3 expression in monocytes, dendritic cells (DCs), and B-cells. Due to their rapid induction and degradation, and SOCS-mediated regulation of multiple cytokine-signaling pathways, they represent an attractive therapeutic target in the autoimmune diseases, and particularly relapsing remitting (RR) MS

T Cell LFA-1 Engagement Induces HuR-Dependent Cytokine mRNA Stabilization through a Vav-1, Rac1/2, p38MAPK and MKK3 Signaling Cascade

Engagement of the β2 integrin, lymphocyte function-associated antigen-1 (LFA-1), results in stabilization of T cell mRNA transcripts containing AU-rich elements (AREs) by inducing rapid nuclear-to-cytosolic translocation of the RNA-stabilizing protein, HuR. However, little is known regarding integrin-induced signaling cascades that affect mRNA catabolism. This study examines the role of the GTPases, Rac 1 and Rac 2, and their downstream effectors, in the LFA-1-induced effects on mRNA.Engagement of LFA-1 to its ligand, ICAM-1, in human peripheral T cells resulted in rapid activation of Rac1 and Rac2. siRNA-mediated knockdown of either Rac1 or Rac2 prevented LFA-1-stimulated stabilization of the labile transcripts encoding IFN-γ and TNF-α, and integrin mediated IFN-γ mRNA stabilization was absent in T cells obtained from Rac2 gene-deleted mice. LFA-1 engagement-induced translocation of HuR and stabilization of TNF- α mRNA was lost in Jurkat cells deficient in the Rac guanine nucleotide exchange factor Vav-1 (J.Vav1). The transfection of J.Vav1 cells with constitutively active Rac1 or Rac2 stabilized a labile β-globin reporter mRNA, in a HuR-dependent manner. Furthermore, LFA-1-mediated mRNA stabilization and HuR translocation in mouse splenic T cells was dependent on the phosphorylation of the mitogen-activated protein kinase kinase, MKK3, and its target MAP kinase p38MAPK, and lost in T cells obtained from MKK3 gene-deleted mice.Collectively, these results demonstrate that LFA-1-induced stabilization of ARE-containing mRNAs in T cells is dependent on HuR, and occurs through the Vav-1, Rac1/2, MKK3 and p38MAPK signaling cascade. This pathway constitutes a molecular switch that enhances immune and pro-inflammatory gene expression in T cells undergoing adhesion at sites of activation and effector function

Self-complementary AAV Virus (scAAV) Safe and Long-term Gene Transfer in the Trabecular Meshwork of Living Rats and Monkeys

AAV vectors produce stable transgene expression and elicit low immune response in many tissues. AAVs have been the vectors of choice for gene therapy for the eye, in particular the retina. scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. The goal was to investigate the ability of AAV vectors to induce long-term, safe delivery of transgenes to the trabecular meshwork of living animals

T cell LFA-1-induced proinflammatory mRNA stabilization is mediated by the p38 pathway kinase MK2 in a process regulated by hnRNPs C, H1 and K.

Activation of the β2 integrin lymphocyte function-associated antigen-1 (LFA-1) in T cells induces stabilization of proinflammatory AU-rich element (ARE)-bearing mRNAs, by triggering the nuclear-to-cytoplasmic translocation of the mRNA-binding and -stabilizing protein HuR. However, the mechanism by which LFA-1 engagement controls HuR localization is not known. Here, we identify and characterize four key regulators of LFA-1-induced changes in HuR activity: the p38 pathway kinase MK2 and the constitutive nuclear proteins hnRNPs C, H1 and K. LFA-1 engagement results in rapid, sequential activation of p38 and MK2. Post-LFA-1 activation, MK2 inducibly associates with both hnRNPC and HuR, resulting in the dissociation of HuR from hnRNPs C, H1 and K. Freed from the three hnRNPs, HuR translocates from the nucleus to the cytoplasm, and mediates the stabilization of labile cytokine transcripts. Our results suggest that the modulation of T cell cytokine mRNA half-life is an intricate process that is negatively regulated by hnRNPs C, H1 and K and requires MK2 as a critical activator

p38 and MK2 are activated upon LFA-1 engagement, and MK2 is required for LFA-1-induced HuR translocation.

<p>(<b>A</b>, <b>B</b>) LFA-1-induced activation of p38 and MK2. Human Jurkat T cells were adhered to pLL- or ICAM-1-coated plates for 5, 15, 30, or 60 min, then lysed. Lysates were subjected to phospho-p38 (<b>A</b>) or phospho-MK2 (<b>B</b>) immunoblotting. Data are representative of three independent experiments. (<b>C</b>) Requirement of MK2 for LFA-1-induced HuR translocation. WT or MK2^-/- primary murine T cells were adhered to pLL- or ICAM-1-coated coverslips for 45 min, and subjected to IF using an anti-HuR antibody and the nuclear marker DAPI. Quantification of HuR translocation is shown as a ratio of area within the periphery of HuR staining (green contours) divided by nuclear area (blue contours). Data represent three independent experiments with standard error. Scale bar, 10 μm.</p

Activated p38 and MK2 are functionally required for LFA-1-induced proinflammatory mRNA stabilization.

<p>(<b>A</b>, <b>B</b>) WT or MK2^-/- primary murine T cells were adhered to pLL- or ICAM-1-coated plates, treated with PMA for 3 h to maximize <i>TNF</i> and <i>IFNG</i> gene transcription, then treated with transcriptional inhibitor DRB and lysed at 0, 20, 40, or 60 min. Total RNA was isolated from the lysates, and TNF-α (<b>A</b>) and IFN-γ (<b>B</b>) levels at each timepoint, normalized to GAPDH, were determined using qRT-PCR relative to time 0 (set at 1.0) levels. Data represent three independent experiments. (<b>C, D</b>) Jurkat T cells were pretreated with p38 inhibitor (C), MK2 inhibitor (D), or DMSO vehicle for 15 min, adhered to pLL- or ICAM-1-coated plates for 30 min, then treated with DRB and lysed at indicated timepoints. Total RNA was isolated from the lysates, and TNF-α mRNA levels at each timepoint, normalized to GAPDH, were determined using qRT-PCR and compared to time 0 levels. Data represent three independent experiments. **, p < 0.01.</p