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Administration of cannabinoid receptor 2 (CB2R) agonists in inflammatory and autoimmune disease and CNS injury models results in significant attenuation of clinical disease, and reduction of inflammatory mediators. Previous studies reported that CB2R signaling also reduces leukocyte migration. Migration of dendritic cells (DCs) to various sites is required for their activation and for the initiation of adaptive immune responses. Here, we report for the first time that CB2R signaling affects DC migration in vitro and in vivo, primarily through the inhibition of matrix metalloproteinase 9 (MMP-9) expression. Reduced MMP-9 production by DCs results in decreased migration to draining lymph nodes in vivo and in vitro in the matrigel migration assay. The effect on Mmp-9 expression is mediated through CB2R, resulting in reduction in cAMP levels, subsequent decrease in ERK activation, and reduced binding of c-Fos and c-Jun to Mmp-9 promoter activator protein 1 sites. We postulate that, by dampening production of MMP-9 and subsequent MMP-9–dependent DC migration, cannabinoids contribute to resolve acute inflammation and to reestablish homeostasis. Selective CB2R agonists might be valuable future therapeutic agents for the treatment of chronic inflammatory conditions by targeting activated immune cells, including DCs.

We reported previously that prostaglandin E2 (PGE2) up-regulates IL-23 in vitro in bone marrow-derived dendritic cells and in vivo in models of collagen-induced arthritis and inflammatory bowel disease, leading to preferential Th17 development and activity. There is very little information on the molecular mechanisms involved in the PGE2-induced up-regulation of Il23a gene expression. In this study we investigated the signaling pathways and transcription factors involved in the stimulatory effect of PGE2. Although PGE2 does not induce IL-23p19 expression by itself, it synergizes with both extra- and intracellular Toll-like receptor ligands and with inflammatory cytokines such as TNFα. We established that the effect of PGE2 in conjunction with either LPS or TNFα is mediated through the EP4 receptor and the cAMP-dependent activation of both protein kinase A (PKA) and exchange protein activated by cAMP (EPAC). Using the EP4 agonist PGE(1)OH in conjunction with TNFα, we found that PKA-induced phosphorylation of cAMP-response element-binding protein ((P)CREB) and EPAC-induced phosphorylation of C/AATT enhancer-binding protein β ((P)C/EBPβ) mediate the stimulatory effect of PGE2 on IL-23p19 expression. This is the first report of CREB and C/EBPβ involvement in Il23a promoter activation. Mutation within the putative CREB and C/EBP sites combined with in vivo DNA binding (ChIP) assays identified the distal CREB site (-1125) and the two proximal C/EBP sites (-274 and -232) as essential for PKA-activated CREB and EPAC-activated C/EBPβ-induced IL-23p19 expression.

Dendritic Cells (DCs) play an important role in the initiation of the immune response by migrating to regional lymph nodes and presenting antigen processed at the inflammatory site to antigen-specific naïve T cells. Prostaglandin E2 (PGE2) has been reported to play an essential role in DC migration. We reported previously that PGE2 induces matrix metalloproteinase 9 (MMP-9) expression in DCs and that PGE2-induced MMP-9 is required for DC migration in vivo and in vitro. In this study, we investigated the signaling mechanisms involved in PGE2-induced MMP-9 expression in DCs. We show that PGE2-induced MMP-9 expression is mediated primarily through the EP2/EP4 → cAMP → protein kinase A (PKA)/PI3K → ERK signaling pathway, leading to c-Fos expression, and through JNK-mediated activation of c-Jun in a PKA/PI3K/ERK-independent manner. EP2 and EP4 receptor agonists, as well as cAMP analogs, mimic the up-regulation of MMP-9 by PGE2. PKA, PI3K, and ERK inhibitors abolished PGE2- and cAMP-induced c-Fos and MMP-9 up-regulation, and ERK activation was required for the binding of activator protein 1 (AP-1) transcription factor to the MMP-9 promoter. Our results describe a new molecular mechanism for the effect of PGE2 on MMP-9 production in DCs that could lead to future therapeutic approaches using ERK inhibitors to regulate DC migration.

Neuroinflammation characterizes various neurological disorders. Peripheral immune cells and CNS-resident glia contribute to neuroinflammation and impact CNS degeneration, recovery and regeneration. Recently, the role of dendritic cells in neuroinflammation received special attention. The function of infiltrating immune cells and resident glia is affected by various factors, including lipid mediators. Polyunsaturated fatty acids, especially n-6 arachidonic acid and n-3 docosahexaenoic acid (DHA), the most abundant in the CNS, play an important role in neuroinflammation. The major arachidonic acid bioactive derivative in immune cells, PGE2, and DHA have been reported to have opposite effects on dendritic cells in terms of cytokine production and activation/differentiation of CD4(+) T cells. Here we review the existing information on PGE2 and DHA modulation of dendritic cell function and the potential impact of these lipid mediators of dendritic cells in CNS inflammatory disorders.

Summary: This study analyzed the antigen-specific (Gag, Nef, Rev, and Tat) IgM, IgG, and IgA humoral responses during the first 200 days of SIVmac251 infection in cynomolgus macaques. These responses were tested for correlation with the CD4+ T-cell-related hematologic parameters and viral load throughout the course of the study (acute and chronic infection, during and after antiretroviral therapy). Strong inverse correlations were observed between the percentage of CD4+ T cells at almost every timepoint of the study and the levels of IgM (but not IgG and IgA) against Gag, Nef, and Rev (but not Tat) measured after, but not during, the primary peak of IgM response. Significant levels of persistent antigen-specific IgMs may reflect the prevalence of mature plasma cells that have not undergone immunoglobulin class switching, possibly due to defects in helper T-cell function. Strong correlations were observed between the preinfection CD4+ T-cell count or CD4/CD8 ratio and the same parameters measured throughout the study, suggesting the importance of preinfection immune status as a determinant of disease progression. The negative correlations between the post-acute-phase IgM levels and the percentage of CD4+ T cells at later times during the study suggest the potential prognostic value of this measurement.

In murine models of multiple sclerosis (experimental autoimmune encephalomyelitis or EAE), cannabinoid receptor 2 (CB2) agonists have been shown to be beneficial, reducing the number of CNS infiltrating leukocytes. Adhesion molecules, chemokines, and matrix metalloproteinases (MMPs), particularly MMP-9, play important roles in migration to the CNS. Antigen presenting cells such as dendritic cells (DC) and macrophages, as well as activated microglia are major producers of MMP-9. In this study, we investigated the effects of CB2 receptor agonists on DC migration and MMP-9 production utilizing both in vitro and in vivo migration assays. DC activated with cytokine cocktail (TNFα+IL-1β+IL-6+PGE2) in the presence or absence of CB2R agonist exhibited similar migratory capacity in transwell migration assays. However, the CB2R agonist treated cells migrated less in matrigel migration assays as compared to untreated cells. We found that this effect was due to reduced MMP-9 production in CB2R agonist treated cells. Also, in the in vivo migration assay using cytokine cocktail-activated DCs injected into mice footpads, we found significantly fewer CB2R agonist-treated DCs in the draining lymph nodes. Our data indicate that one of the mechanisms by which CB2R agonists attenuate inflammation is the inhibition of MMP-9 release which results in decreased migration of activated dendritic cells.

Dendritic Cells (DCs) play an important role in the initiation of the immune response. Immature DCs take up antigen, mature and migrate to the regional lymph nodes where they activate antigen-specific naïve T cells. Prostaglandins such as PGE2 are essential for the migration of DCs to lymph nodes. Matrix metalloproteinases (MMPs) play an essential role in DC migration, through the degradation of extracellular matrix (ECM) and basement membranes. MMP2, and particularly MMP9, are essential for dendritic cell migration in vitro, as well as in vivo to the draining lymph nodes. In this study, we show that PGE2-induced MMP9 expression is mediated through the EP2/EP4-cAMP-PKA/PI3K-ERK signaling pathway. EP2 and EP4 receptors as well as cAMP analog mimic the effect of PGE2 to upregulate MMP-9. In the presence of PKA, PI3K or ERK inhibitors, PGE2-induced MMP-9 production is abolished. Furthermore, PGE2-induced c-Fos expression mediated through ERK activation is required for the binding of AP-1 transcription factor to the MMP-9 promoter. In the presence of ERK inhibitors, PGE2-induced AP-1 binding is abrogated. Our results describe a new molecular mechanism for the effect of PGE on MMP-9 production in DCs, which could lead to a new therapeutic approach by using ERK inhibitors to regulate DC migration.

We reported previously that PGE2 promotes IL-23p19 expression in dendritic cells (DC) treated with LPS. The effect is mediated through EP4 through the induction of cAMP. This study is focused on the signaling pathways involved in PGE1OH (an EP4 agonist)-induced p19 expression. EP4 activates adenylate cyclase and increases intracellular cAMP which leads to activation of PKA and EPAC. We found that adenylate cyclase, cAMP, and EPAC play a role in IL-23p19 induction. Using chemical inhibitors of PKA, PI3K, MAPK, and GSK3, we did not see a decrease in IL-23p19 expression. Previous studies reported the involvement of NFkB/c-Rel, ATF-2, SMAD-3 and AP-1 in p19 expression. Since two CRE-binding sites have been identified in the p19 promoter, we focused on CREB as a potential transcription factor activated by PGE1OH. Luciferase experiments including vectors containing mutated CRE sites and expressing dominant negative versions of CREB demonstrated the essential role CREB plays in IL-23p19 expression. In a transcription factor assay we found increased CREB binding activity in nuclear extracts from cells treated with PGE1OH. In addition, a ChIP assay confirmed CREB binding to the p19-promoter in the presence of PGE1OH. Overall, we identified CREB as a new transcription factor required for PGE1OH induction of IL-23p19 expression.

Previously, we have shown that PGE2 inhibits IL-12p35 expression & promotes IL-23p19 expression in dendritic cells (DC) treated with LPS or Poly I:C. The effect is mediated through EP4 and to a lesser degree EP2 receptors and induction of cAMP. This study is focused on essential transcriptional factors and signaling pathways involved in PGE1-OH (EP4 agonist) induced p19 expression. Previous studies reported involvement of NFkB (c-Rel), ATF-2, SMAD-3 & AP-1. CREB is a target of cAMP-activated PKA and two CREB binding sites have been identified in the p19 promoter. Therefore, CREB is a potential transcription factor activated by PGE. Experiments with p19-luciferase constructs containing wt & CREB-mutated sites indicate the importance of CREB sites in p19 transcriptional activity. PGE2/PGE1-OH also induced STAT-3 tyrosine phosphorylation in DC and in the DC cell line. Blocking STAT-3 phosphorylation with the chemical inhibitor S3I-201 and the specific peptide inhibitor PpYLKTK-mts reduced p19 expression in DC. Although no STAT-3 binding site was found in the p19 promoter, STAT-3 was proposed to act as a co-activator for c-Rel to induce p19 expression. These data show for the first time that CREB and STAT-3 are important regulators of PGE2-induced p19 expression in DC. These observations show the versatility that prostaglandins have to differentially regulate expression patterns of cytokines that orchestrate an immune response.