Antiinflammatory Effects of Glucocorticoids in Brain Cells, Independent of NF-kB

Glucocorticoids are potent antiinflammatory drugs. They inhibit the expression of proinflammatory cytokines and adhesion molecules. It has recently been proposed that the underlying basis to such inhibition is the induction of the protein IkB, which inhibits the transcription factor NF-kB. The latter is a key activator of the genes encoding cytokines and adhesion molecules. The present study shows that the synthetic glucocorticoid, dexamethasone, inhibits the induction of the proinflammatory cytokine IL-8 and the adhesion molecules VCAM-1 and ICAM-1 in human 1321N1 astrocytoma and SK.N.SH neuroblastoma cells. However, dexamethasone failed to induce IkB or inhibit activation of NF-kB by IL-1 in the two cell types. EMSA confirmed the identity of the activated NF-kB by demonstrating that an oligonucleotide, containing the wild-type NF-kB-binding motif, inhibited formation of the NF-kB-DNA complexes whereas a mutated form of the NF-kB-binding motif was ineffective. In addition, supershift analysis showed that the protein subunits p50 and p65 were prevalent components in the activated NF-kB complexes. The lack of effect of dexamethasone on the capacity of IL-1 to activate NF-kB correlated with its inability to induce IkB and the ability of IL-1 to cause degradation of IkB, even in the presence of dexamethasone. The results presented in this paper strongly suggest that glucocorticoids may exert antiinflammatory effects in cells of neural origin by a mechanism(s) independent of NF-kB

Evidence for a role for the group I metabotropic glutamate receptor in the inhibitory effect of tumor necrosis factor-α on long-term potentiation

Pro-inflammatory cytokines are known to be elevated in several neuropathological states that are associated with learning and memory. We have previously demonstrated in our laboratory that the inhibition of long-term potentiation (LTP) in the dentate gyrus region of the rat hippocampus, by tumor necrosis factor (TNF)-α, represents a biphasic response, an early phase dependent on p38 mitogen activated protein kinase (MAPK) activation and a later phase, possible dependent on protein synthesis. Many of the factors involved in the early modulation of LTP by TNF-α have yet to be elucidated. This study investigated if metabotropic glutamate receptors (mGluRs) are functionally linked to the inhibitory effect of TNF-α on LTP in the rat dentate gyrus in vitro. We report that the impairment of early-LTP by TNF-α is significantly attenuated by prior application of the group I/II mGluR antagonist MCPG and more specifically the mGluR5 antagonist MPEP. Since TNF-α is now known to cause transient increases in intracellular Ca2+ levels from ryanodine-sensitive stores, we explored the possibility that disruption of intracellular Ca2+ homeostasis could be involved. Ryanodine was found to significantly reverse the inhibition of LTP by TNF-α. From these studies we propose that the TNF-α inhibition of LTP is dependent upon the activation of TNFR1 and mGlu5-receptors. Importantly this study provides the first proof of the involvement of ryanodine-sensitive intracellular Ca2+ stores in TNF-α mediated inhibition of LTP

Nitric oxide affects IL-6 expression in human peripheral blood mononuclear cells involving cGMP-dependent modulation of NF-jB activity

Interleukin 6 (IL-6) and nitric oxide (NO) are important mediators of the inflammatory response. We report that in human peripheral blood mononuclear cells (PBMCs), NO exerts a biphasic effect on the expression of IL-6. Using sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) as NO-donating compounds, we observed that both mRNA and protein levels of IL-6 increased at lower (610 lM) and decreased at higher (>100 lM) concentrations of NO donors. Changes in the expression of IL-6 correlated with changes in the activity of NF-jB, which increased at lower and decreased at higher concentrations of both NO donors as shown by the electrophoretic mobility shift assay (EMSA). The effects of NO on NF-jB activity were cGMP-dependent because they were reversed in the presence of ODQ, the inhibitor of soluble guanylyl cyclase (sGC), and KT5823, the inhibitor of cGMP-dependent protein kinase (PKG). Moreover, the membrane permeable analog of cGMP (8-Br-cGMP) mimicked the effect of the NO donors. These observations show that NO, depending on its concentration, may act in human PBMCs as a stimulator of IL-6 expression involving the sGC/cGMP/PKG pathway

Characterisation of expression patterns and functional role of Cactin in early zebrafish development

The immune system of teleost zebrafish (Danio rerio) shows high similarity to mammalian counterparts sharing many innate immune components including Toll-Like Receptors (TLRs), cytokines, chemokines and complement molecules. As in mammals, zebrafish also contains the transcription factor NF-jB that plays dualist roles in innate immunity and early development. Indeed NF-jB members are expressed in different temporal patterns during the early stages of zebrafish embryogenesis indicating that each molecule is involved in specific developmental events. In the present study we employ zebrafish as a model to characterise the expression pattern and role of a novel NF-jB regulator, termed Cactin, in early development. Cactin was first characterised in Drosophila as a new member of the Rel pathway that could affect the generation of dorsal–ventral polarity. To explore the potential developmental role of Cactin in zebrafish, we initially investigated its expression pattern and functional role during early embryonic developmental stages. We detect Cactin expression at all stages of early development and knockdown of Cactin by specific morpholino antisense oligonucleotides causes developmental abnormalities manifested by an overall dysmorphic cellular organisation. These results indicate that Cactin has been highly conserved during evolution and plays a key role in early embryonic development

Characterisation of expression patterns and functional role of Cactin in early zebrafish development

The immune system of teleost zebrafish (Danio rerio) shows high similarity to mammalian counterparts sharing many innate immune components including Toll-Like Receptors (TLRs), cytokines, chemokines and complement molecules. As in mammals, zebrafish also contains the transcription factor NF-jB that plays dualist roles in innate immunity and early development. Indeed NF-jB members are expressed in different temporal patterns during the early stages of zebrafish embryogenesis indicating that each molecule is involved in specific developmental events. In the present study we employ zebrafish as a model to characterise the expression pattern and role of a novel NF-jB regulator, termed Cactin, in early development. Cactin was first characterised in Drosophila as a new member of the Rel pathway that could affect the generation of dorsal–ventral polarity. To explore the potential developmental role of Cactin in zebrafish, we initially investigated its expression pattern and functional role during early embryonic developmental stages. We detect Cactin expression at all stages of early development and knockdown of Cactin by specific morpholino antisense oligonucleotides causes developmental abnormalities manifested by an overall dysmorphic cellular organisation. These results indicate that Cactin has been highly conserved during evolution and plays a key role in early embryonic development

Mal Mediates TLR-Induced Activation of CREB and Expression of IL-10

TLRs initiate immune responses by direct detection of molecular motifs that distinguish invading microbes from host cells. Five intracellular adaptor proteins, each containing a Toll/IL-1R (TIR) domain, are used by TLRs and play key roles in dictating gene expression patterns that are tailored to the invader. Such gene expression is mediated by transcription factors, and although TIR adaptor-induced activation of NF-kB and the IFN regulatory factors have been intensively studied, there is a dearth of information on the role of TIR adaptors in regulating CREB. In this paper, we describe a role for the TIR adaptor Mal in enhancing activation of CREB. Mal-deficient murine bone marrow-derived macrophages show a loss in responsiveness to TLR2 and TLR4 ligands with respect to activation of CREB. Mal-deficient cells also fail to express the CREB-responsive genes IL-10 and cyclooxygenase 2 in response to Pam2Cys-Ser-(Lys)4 and LPS. We reveal that Mal-mediated activation of CREB is dependent on Pellino3 and TNFR-associated factor 6, because CREB activation is greatly diminished in Pellino3 knockdown cells and TNFRassociated factor 6-deficient cells. We also demonstrate the importance of p38 MAPK in this pathway with the p38 inhibitor SB203580 abolishing activation of CREB in murine macrophages. MAPK-activated protein kinase 2 (MK2), a substrate for p38 MAPK, is the likely downstream mediator of p38 MAPK in this pathway, because Mal is shown to activate MK2 and inhibition of MK2 decreases TLR4-induced activation of CREB. Overall, these studies demonstrate a new role for Mal as a key upstream regulator of CREB and as a contributor to the expression of both pro- and anti-inflammatory gen

Mal Mediates TLR-Induced Activation of CREB and Expression of IL-10

TLRs initiate immune responses by direct detection of molecular motifs that distinguish invading microbes from host cells. Five intracellular adaptor proteins, each containing a Toll/IL-1R (TIR) domain, are used by TLRs and play key roles in dictating gene expression patterns that are tailored to the invader. Such gene expression is mediated by transcription factors, and although TIR adaptor-induced activation of NF-kB and the IFN regulatory factors have been intensively studied, there is a dearth of information on the role of TIR adaptors in regulating CREB. In this paper, we describe a role for the TIR adaptor Mal in enhancing activation of CREB. Mal-deficient murine bone marrow-derived macrophages show a loss in responsiveness to TLR2 and TLR4 ligands with respect to activation of CREB. Mal-deficient cells also fail to express the CREB-responsive genes IL-10 and cyclooxygenase 2 in response to Pam2Cys-Ser-(Lys)4 and LPS. We reveal that Mal-mediated activation of CREB is dependent on Pellino3 and TNFR-associated factor 6, because CREB activation is greatly diminished in Pellino3 knockdown cells and TNFRassociated factor 6-deficient cells. We also demonstrate the importance of p38 MAPK in this pathway with the p38 inhibitor SB203580 abolishing activation of CREB in murine macrophages. MAPK-activated protein kinase 2 (MK2), a substrate for p38 MAPK, is the likely downstream mediator of p38 MAPK in this pathway, because Mal is shown to activate MK2 and inhibition of MK2 decreases TLR4-induced activation of CREB. Overall, these studies demonstrate a new role for Mal as a key upstream regulator of CREB and as a contributor to the expression of both pro- and anti-inflammatory gen

Nuclear Factor κB Subunits RelB and cRel Negatively Regulate Toll-like Receptor 3-mediated β-Interferon Production via Induction of Transcriptional Repressor Protein YY1

The induction of β-interferon (IFN-β) is a key anti-viral response to infection by RNA viruses. Virus-induced expression of IFN-β requires the co-operative action of the transcription factors IRF-3/7, NF-κB, and ATF-2/c-Jun on the IFN-β promoter leading to the orderly recruitment of chromatin remodeling complexes. Although viruses strongly activate NF-κB and promote its binding to the IFN-β promoter, recent studies have indicated that NF-κB is not essential for virus-induced expression of IFN-β. Herein, we examined the role of NF-κB in regulating IFN-β expression in response to the viral-sensing Toll-like receptor 3 (TLR3). Intriguingly pharmacological inhibition of the NF-κB pathway augments late phase expression of IFN-β expression in response to TLR3 stimulation. We show that the negative effect of NF-κB on IFN-β expression is dependent on the induction of the transcriptional repressor protein YinYang1. We demonstrate that the TLR3 ligand polyriboinosinic:polyribocytidylic acid (poly(I:C)) induces expression and nuclear translocation of YinYang1 where it interacts with the IFN-β promoter and inhibits the binding of IRF7 to the latter. Evidence is also presented showing that the NF-κB subunits c-Rel and RelB are the likely key drivers of these negative effects on IFN-β expression. These findings thus highlight for the first time a novel self-regulatory mechanism that is employed by TLR3 to limit the level and duration of IFN-β expression

Nuclear Factor κB Subunits RelB and cRel Negatively Regulate Toll-like Receptor 3-mediated β-Interferon Production via Induction of Transcriptional Repressor Protein YY1

The induction of β-interferon (IFN-β) is a key anti-viral response to infection by RNA viruses. Virus-induced expression of IFN-β requires the co-operative action of the transcription factors IRF-3/7, NF-κB, and ATF-2/c-Jun on the IFN-β promoter leading to the orderly recruitment of chromatin remodeling complexes. Although viruses strongly activate NF-κB and promote its binding to the IFN-β promoter, recent studies have indicated that NF-κB is not essential for virus-induced expression of IFN-β. Herein, we examined the role of NF-κB in regulating IFN-β expression in response to the viral-sensing Toll-like receptor 3 (TLR3). Intriguingly pharmacological inhibition of the NF-κB pathway augments late phase expression of IFN-β expression in response to TLR3 stimulation. We show that the negative effect of NF-κB on IFN-β expression is dependent on the induction of the transcriptional repressor protein YinYang1. We demonstrate that the TLR3 ligand polyriboinosinic:polyribocytidylic acid (poly(I:C)) induces expression and nuclear translocation of YinYang1 where it interacts with the IFN-β promoter and inhibits the binding of IRF7 to the latter. Evidence is also presented showing that the NF-κB subunits c-Rel and RelB are the likely key drivers of these negative effects on IFN-β expression. These findings thus highlight for the first time a novel self-regulatory mechanism that is employed by TLR3 to limit the level and duration of IFN-β expression

Identification of the Synthetic Cannabinoid R()WIN55,212-2 as a Novel Regulator of IFN Regulatory Factor 3 Activation and IFN- Expression

Beta Interferons (IFN-βs) represent one of the first line treatments for relapsing remitting multiple sclerosis (RRMS), slowing disease progression whilst reducing the frequency of relapses. Despite this, more effective, well tolerated therapeutic strategies are needed. Cannabinoids palliate experimental autoimmune encephalomyelitis (EAE) symptoms and have therapeutic potential in MS patients although the precise molecular mechanism for these effects is not understood. Toll-like receptor (TLR) signaling controls innate immune responses and TLRs are implicated in MS. Here we demonstrate that the synthetic cannabinoid R(+)WIN55,212-2 is a novel regulator of TLR3 and TLR4 signaling by inhibiting the pro-inflammatory signaling axis triggered by TLR3 and TLR4 whilst selectively augmenting TLR3-induced activation of IFN regulatory factor 3 (IRF3) and expression of IFN-β. We present evidence that R(+)WIN55,212-2 strongly promotes the nuclear localization of IRF3. The potentiation of IFN-β expression by R(+)WIN55,212-2 is critical for manifesting its protective effects in the murine MS model EAE as evidenced by its reduced therapeutic efficacy in the presence of an anti-IFN-β antibody. R(+)WIN55,212-2 also induces IFN-β expression in MS patient peripheral blood mononuclear cells (PBMCs), whilst downregulating inflammatory signaling in these cells. These findings identify R(+)WIN55,212-2 as a novel regulator of TLR3 signaling to IRF3 activation and IFN-β expression and highlights a new mechanism that may be open to exploitation in the development of new therapeutics for the treatment of MS