Sabina Adhikary

Docosahexaenoic acid prevents dendritic cell maturation and in vitro and in vivo expression of the IL-12 cytokine family

BackgroundAcute and chronic inflammation play essential roles in inflammatory/autoimmune conditions. Protective anti-inflammatory effects of the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were reported in animal models of colitis, sepsis, and stroke. Since dendritic cells (DC) represent the essential cellular link between innate and adaptive immunity and have a prominent role in tolerance for self-antigens, we sought to investigate the impact of DHA on DC maturation and proinflammatory cytokine production.MethodsMurine bone marrow-derived DC were treated with DHA and stimulated with various toll-like receptor (TLR) ligands. Flow cytometry was used to determine the levels of surface maturation markers and endocytic activity. Cytokine expression and secretion were measured by real-time RT-PCR and ELISA assays. PPARγ and NFκB activity in nuclear extracts were determined by binding to specific oligonucleotide sequences using ELISA-based assays. In vivo effects of DHA were assessed in splenic DC from LPS-inoculated mice maintained on a DHA-enriched diet.ResultsDHA maintained the immature phenotype in bone marrow-derived DC by preventing the upregulation of MHCII and costimulatory molecules (CD40, CD80 and CD86) and maintaining high levels of endocytic activity. DHA inhibited the production of pro-inflammatory cytokines, including the IL-12 cytokine family (IL-12p70, IL-23, and IL-27), from DC stimulated with TLR2, 3, 4, and 9 ligands. DHA inhibition of IL-12 expression was mediated through activation of PPARγ and inhibition of NFκBp65 nuclear translocation. DHA exerted a similar inhibitory effect on IL-12 and IL-23 expression in vivo in LPS-inoculated mice maintained on a DHA-enriched diet.ConclusionsExposure of bone marrow-derived DC to DHA resulted in the maintenance of an immature phenotype and drastic reduction in proinflammatory cytokine release. DHA inhibited the expression and secretion of the IL-12 cytokine family members (IL-12p70, IL-23 and IL-27), which play essential roles in the differentiation of the proinflammatory Th1/Th17 effector cells. The effect of DHA on IL-12 expression was mediated through activation of PPARγ and inhibition of NFκB. Inhibition of IL-12 and IL-23 expression was also evident in splenic DC from mice fed a DHA-enriched diet, suggesting that dietary DHA acts as an anti-inflammatory agent in vivo.

Modulation of inflammatory responses by a cannabinoid-2-selective agonist after spinal cord injury.

The goal of the current investigation was to evaluate the mechanisms through which administration of a selective cannabinoid-2 (CB2) agonist (O-1966) modifies inflammatory responses and helps to improve function following spinal cord injury. A comparison of motor function, autonomic function, and inflammatory responses was made between animals treated with O-1966 (5 mg/kg IP) and animals treated with vehicle 1 h and 24 h following contusion injury to the spinal cord. Motor function was significantly improved in the treated animals at each time point during the 14 days of evaluation. The percentage of animals able to spontaneously void their bladder was also greater over the entire study period in the group treated with the selective CB2 agonist. Seven days following injury there was a significant reduction in both hematopoietic and myeloid cell invasion of the spinal cord, and a reduction in the number of immunoreactive microglia. The results of the evaluation of chemokine/cytokine expression and inflammatory cell invasion also demonstrated a significant effect of treatment on inflammatory reactions following injury. Two days after injury, animals treated with O-1966 had significant reductions in CXCL-9 and CXCL-11, and dramatic reductions in IL-23p19 expression and its receptor IL-23r. Treatment with O-1966 also caused inhibition of toll-like receptor expression (TLR1, TLR4, TLR6 and TLR7) following injury. These results demonstrate that the improvement in motor and autonomic function resulting from treatment with a selective CB2 agonist is associated with a significant effect on inflammatory responses in the spinal cord following injury.

Prostaglandin E2-induced IL-23p19 Subunit Is Regulated by cAMP-responsive Element-binding Protein and C/AATT Enhancer-binding Protein β in Bone Marrow-derived Dendritic Cells

Background: The molecular mechanisms involved in IL-23 up-regulation by PGE2 are not elucidated. Results: PGE2 induces IL-23p19 through the EP4 cAMP-PKA/EPAC-CREB/C/EBPβ signaling pathway. Conclusion: PGE2 synergizes with TLR ligands and with proinflammatory cytokines such as TNFα to up-regulate Il23a gene expression. Significance: Understanding is gained of one of the major functions of PGE2 leading to activation of pathogenic Th17 cells. 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 PGE1OH in conjunction with TNFα, we found that PKA-induced phosphorylation of cAMP-response element-binding protein (PCREB) and EPAC-induced phosphorylation of C/AATT enhancer-binding protein β (PC/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.