Jami Dwyer

Defining the CREB Regulon: A Genome-Wide Analysis of Transcription Factor Regulatory Regions

The CREB transcription factor regulates differentiation, survival, and synaptic plasticity. The complement of CREB targets responsible for these responses has not been identified, however. We developed a novel approach to identify CREB targets, termed serial analysis of chromatin occupancy (SACO), by combining chromatin immunoprecipitation (ChIP) with a modification of SAGE. Using a SACO library derived from rat PC12 cells, we identified approximately 41,000 genomic signature tags (GSTs) that mapped to unique genomic loci. CREB binding was confirmed for all loci supported by multiple GSTs. Of the 6302 loci identified by multiple GSTs, 40% were within 2 kb of the transcriptional start of an annotated gene, 49% were within 1 kb of a CpG island, and 72% were within 1 kb of a putative cAMP-response element (CRE). A large fraction of the SACO loci delineated bidirectional promoters and novel antisense transcripts. This study represents the most comprehensive definition of transcription factor binding sites in a metazoan species.

Mitogen- and Stress-Activated Protein Kinase 1 Mediates cAMP Response Element-Binding Protein Phosphorylation and Activation by Neurotrophins

Activation of the transcription factor cAMP response element-binding protein (CREB) by neurotrophins is believed to regulate the survival, differentiation, and maturation of neurons in the CNS and PNS. Although phosphorylation of Ser133 is critical for the expression of CREB-regulated genes, the identity of neurotrophin-regulated Ser133 kinases has remained controversial. We show here that neurotrophin-induced CREB phosphorylation in CNS neurons depends exclusively on the extracellular signal-regulated kinase 1/2-activated kinase mitogen- and stress-activated protein kinase 1 (MSK1). Small interfering RNA directed against ribosomal S6 kinase 1 (RSK1) and RSK2 reduced phosphorylation of a RSK substrate but did not effect CREB-dependent transcription. However, expression of a selective inhibitory MSK1 mutant markedly attenuated BDNF-stimulated CREB phosphorylation and CREB-mediated transcription. Moreover, the ability of neurotrophins to stimulate CREB phosphorylation was abolished in CNS neurons from MSK1 knock-out mice. Consistent with a role for MSK1 in Ser133 phosphorylation, neurotrophin-induced expression of CREB-regulated genes was attenuated in MSK-deficient neurons. These results indicate that MSK1 is the major neurotrophin-activated Ser133 kinase in CNS neurons.

Estrogen inhibition of EAE involves effects on dendritic cell function

Estrogen has been found to have suppressive effects on the induction of experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. We have investigated the effects of 17β‐estradiol (E2) treatment on dendritic cells (DCs) in two different mouse models of EAE. The frequency of CD11b+/CD11c+ DCs was significantly decreased in the brain of mice protected from EAE induction by E2 treatment. In addition, the frequency of CD11c+/CD8α+ DCs producing tumor necrosis factor (TNF)α and interferon (IFN)γ in the spleen of E2‐treated mice was dramatically decreased compared to that in control mice with EAE, demonstrating an effect of E2 on DC function. In order to examine E2 effects on DCs in more detail, splenic DCs were cultured in the presence of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4to promote maturation. E2 pretreatment was found to suppress the ability of cultured DCs bearing a mature phenotype to present Ag to myelin basic protein (MBP)‐specific T cells. Analysis of cytokine production demonstrated that E2 decreased TNFα, IFNγ and IL‐12 production in mature DCs. In addition, MBP‐specific T cells cocultured with E2‐pretreated mature DCs in the presence of antigen demonstrated a shift towards production of Th2 cytokines IL‐4 and IL‐10 and a concomitant decrease in the production of Th1 cytokines TNFα and IFNγ. Thus, E2 treatment appears to have multiple effects on the DC population, which may contribute to a down‐regulation or block in the activation of Th1 cells involved in the induction of EAE.

Evaluation of the effects of 17β-estradiol (17β-E2) on gene expression in experimental autoimmune encephalomyelitis using DNA microarray

The aim of this study was to identify immune-related genes affected by treatment with 17-estradiol (17-E2) that contribute to protection of T cell antigen receptor double transgenic mice from experimental autoimmune encephalomyelitis (EAE). The Affymetrix microarray system was used to screen more than 12,000 genes from E2-treated mice protected from EAE vs. control mice with severe EAE. In general, E2 treatment affected about 10% of the genes tested, but only 18 cytokine, chemokine/receptor, adhesion molecule, or activation genes were up- or down-regulated more than 2.4-fold by E2 treatment. Down-regulated genes included TNF (an important proinflammatory cytokine in EAE); peptidoglycan recognition proteins (Pgrp); regulated on activation, normal T cell expressed and secreted (RANTES); and neural cell adhesion molecule (MCP-1). Up-regulated genes included cytotoxic T lymphocyte antigen-4 (CTLA-4; known to inhibit T cell activation), TGF3, IL-18, and two interferon--induced genes, the chemokines: monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1), vascular cell adhesion molecule (VCAM), and disintegrin metalloprotease (thought to regulate TNF production). These results implicate a limited set of known and previously unsuspected E2-sensitive genes that may be crucial for inhibition of EAE and potentially the human disease, multiple sclerosis. (Endocrinology 143: 313–319, 2002)

Estrogen treatment induces a novel population of regulatory cells, which suppresses experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a debilitating neurological disease characterized by a progressive loss of motor and sensory function, eventually leading to paralysis and death. The primary cause of neurological impairment is demyelination of the central nervous system (CNS) caused by an inflammatory autoimmune response. Previous studies have shown that the severity of MS is reduced during pregnancy, suggesting that the increased level of sex hormones may reduce the autoimmune response. Recently, we have shown that estrogen treatment confers protection from experimental autoimmune encephalomyelitis (EAE), which is an animal model for MS. However, the cellular basis of estrogens action remains unknown. In the current study, we demonstrate that estrogen treatment led to the induction of a novel subpopulation of regulatory cells in spleen and CNS, which also occurs naturally in pregnant mice. These previously uncharacterized cells display a low level expression of CD45 (CD45dim) and no detectable expression of many cell surface markers related to TCR signaling, including CD3 and TCR. However, these cells retained expression of VLA‐4, an extracellular protein involved in cellular migration. Several lines of evidence suggest that these novel cells, defined as CD45dimVLA‐4+ cells, may play a role in the protective effects of estrogen in EAE. Injection of purified CD45dimVLA‐4+ cells conferred protection from spontaneous EAE (Sp‐EAE). In contrast, injection of CD45highVLA‐4+ cells exacerbated the disease course. CD45dimVLA‐4+ cells also suppressed antigen‐specific proliferation of primed lymphocytes in coculture. A better understanding of how CD45dimVLA‐4+ cells suppress the harmful immune response of EAE may help in explaining the induction of immune tolerance during pregnancy and lead to novel therapeutic approaches to combat MS and other autoimmune diseases.

Transfer of Severe Experimental Autoimmune Encephalomyelitis by IL-12- and IL-18-Potentiated T Cells Is Estrogen Sensitive

The aim of this study was to evaluate the roles of IL-18 and IL-12 in potentiating the encephalitogenic activity of T cell lines specific for myelin oligodendrocyte glycoprotein (MOG35–55). MOG-specific T cells stimulated with anti-CD3 and anti-CD28 in the presence of IL-12 or IL-18 alone transferred only mild experimental autoimmune encephalomyelitis (EAE) into a low percentage of recipients. However, T cells cocultured with both cytokines transferred aggressive clinical and histological EAE into all recipients. Coculture of T cells with IL-12 enhanced the secretion of IFN-γ, but not TNF-α, whereas coculture with IL-18 enhanced the secretion of TNF-α, but not INF-γ. However, coculture with both IL-18 and IL-12 induced high levels of both TNF-α and IFN-γ. Additionally, IL-12 selectively enhanced mRNA expression of CCR5, whereas IL-18 selectively enhanced the expression of CCR4 and CCR7, and CCR4 and CCR5 were coexpressed on the surface of T cells cocultured with IL-12 and IL-18. Finally, estrogen treatment, previously found to inhibit both TNF-α and IFN-γ production, completely abrogated all signs of passive EAE. These data demonstrate that optimal potentiation of encephalitogenic activity can be achieved by conditioning MOG-specific T cells with the combination of IL-12 and IL-18, which, respectively, induce the secretion of IFN-γ/CCR5 and TNF-α/CCR4/CCR7, and that estrogen treatment, which is known to inhibit both proinflammatory cytokines, can completely ablate this aggressive form of passive EAE.

Effects of cytokine deficiency on chemokine expression in CNS of mice with EAE

Although both cytokines and chemokines have been implicated in the pathogenesis of clinical and histological EAE, their interactions in vivo have not yet been clearly established. To address this issue, we evaluated expression of chemokines and receptors in the CNS of wild‐type control and cytokine deficient mice at the peak of EAE induced with MOG‐35–55 peptide in CFA. Our results demonstrate that: 1) expression of most chemokines/receptors was drastically inhibited in TNF‐α deficient mice, and was reflective of delayed onset and reduced severity of EAE; 2) distinct patterns of chemokine expression occurred in various other cytokine knockout mice that did not significantly affect expression of clinical EAE; 3) there was a strong association between expression of MIP‐1α, MIP‐2 and MCP‐1 in CNS and overall severity of EAE in wild‐type and cytokine knockout mice; and 4) among CNS infiltrating cells at the peak of EAE, macrophages and CD8+ T cells were the primary cellular source of most of the chemokines. Of note, we present evidence that TNF‐α may be involved in regulating RANTES and MIP‐1α, and that IL‐4 may be involved in regulating MCP‐1. Our results not only identify the cellular source of chemokines in CNS, but also implicate MIP‐1α, MIP‐2, and MCP‐1 in controlling CNS inflammation and severity of EAE.

CNS gene expression pattern associated with spontaneous experimental autoimmune encephalomyelitis.

Transgenic mice with T‐cell receptor (TCR) specific for myelin basic protein (MBP)‐Ac1‐11 peptide and homozygous for the RAG‐1 mutation (T/R− mice) spontaneously develop acute progressive experimental autoimmune encephalomyelitis (Sp‐EAE) mediated by CD4+ T cells. Microarray analysis of spinal cord tissue obtained from symptomatic versus non‐symptomatic T/R− mice revealed strongly upregulated transcripts for genes involved in antigen presentation and processing, signal transduction, transcription regulation, metabolism, development, cell cycle, and many other processes involved in the induction of clinical and pathological signs of Sp‐EAE. Several highly expressed genes were related directly to inflammation, including cytokines/receptors, chemokines/receptors, acute phase, complement molecules, and others. Many CNS‐specific genes were also upregulated in sick mice. Abundance of message for the Tg TCR BV8S2 gene as well as several monocyte/macrophage‐associated genes would suggest that both components play a crucial role in the pathogenesis of Sp‐EAE. The profile of transcriptional changes found during the development of Sp‐EAE provides the first description of the encephalitogenic process in the absence of purposeful immunization with myelin peptides and immune‐enhancing adjuvants. This unique approach is the first to implicate molecules and pathways that contribute naturally to onset of paralysis and demyelination, and thus may provide unique insights and novel treatment strategies for human diseases such as multiple sclerosis.

Endogenous CD4+BV8S2- T cells from TG BV8S2+ donors confer complete protection against spontaneous experimental encephalomyelitis (Sp-EAE) in TCR transgenic, RAG-/- mice

To investigate regulatory mechanisms which naturally prevent autoimmune diseases, we adopted the genetically restricted immunodeficient (RAG‐1−/−) myelin basic protein (MBP)‐specific T cell receptor (TCR) double transgenic (T/R−) mouse model of spontaneous experimental autoimmune encephalomyelitis (Sp‐EAE). Sp‐EAE can be prevented after transfer of CD4+splenocytes from naïve immunocompetent mice. RAG‐1+ double transgenic (T/R+) mice do not develop Sp‐EAE due to the presence of a very small population (about 2%) of non‐Tg TCR specificities. In this study, CD4+BV8S2+ T cells that predominate in T/R+ mice, and three additional populations, CD4+BV8S2−, CD4−CD8−BV8S2+, and CD4−CD8+BV8S2+ T cells that expanded in T/R+ mice after immunization with MBP‐Ac1‐11 peptide, were studied for their ability to prevent Sp‐EAE in T/R− mice. Only the CD4+BV8S2− T cell population conferred complete protection against Sp‐EAE, similar to unfractionated splenocytes from non‐Tg donors, whereas CD4−CD8−BV8S2+ and CD4+BV8S2+ T cells conferred partial protection. In contrast, CD4−CD8+BV8S2+ T cells had no significant protective effects. The highly protective CD4+BV8S2− subpopulation was CD25+, contained non‐clonotypic T cells, and uniquely expressed the CCR4 chemokine receptor. Protected recipient T/R− mice had marked increases in CD4+CD25+ Treg‐like cells, retention of the pathogenic T cell phenotype in the spleen, and markedly reduced inflammation in CNS tissue. Partially protective CD4+BV8S2+ and CD4− CD8−BV8S2+ subpopulations appeared to be mainly clonotypic T cells with altered functional properties. These three Sp‐EAE protective T cell subpopulations possessed distinctive properties and induced a variety of effects in T/R− recipients, thus implicating differing mechanisms of protection.