Frances A. Emig

The Proinflammatory Effect of Prostaglandin E2 in Experimental Inflammatory Bowel Disease Is Mediated through the IL-23→IL-17 Axis

Although Crohn’s disease has been traditionally considered to be Th1-mediated, the newly identified Th17 cells emerged recently as crucial participants. Th1/Th17 differentiation is controlled primarily by the IL-12 family of cytokines secreted by activated dendritic cells (DCs) and macrophages. IL-23 and IL-12/IL-27 have opposite effects, supporting the Th17 and Th1 phenotypes, respectively. We found that PGE2, a major lipid mediator released in inflammatory conditions, shifts the IL-12/IL-23 balance in DCs in favor of IL-23, and propose that high levels of PGE2 exacerbate the inflammatory process in inflammatory bowel disease through the IL-23→IL-17 axis. We assessed the effects of PGE2 on IL-12, IL-27, and IL-23 and found that PGE2 promotes IL-23, inhibits IL-12 and IL-27 expression and release from stimulated DCs, and subsequently induces IL-17 production in activated T cells. The effects of PGE2 are mediated through the EP2/EP4 receptors on DCs. In vivo, we assessed the effects of PGE analogs in an experimental model for inflammatory bowel disease and found that the exacerbation of clinical symptoms and histopathology correlated with an increase in IL-23 and IL-17, a decrease in IL-12p35 expression in colon and mesenteric lymph nodes, and a substantial increase in the number of infiltrating neutrophils and of CD4+IL-17+ T cells in the colonic tissue. These studies suggest that high levels of PGE2 exacerbate the inflammatory process through the preferential expression and release of DC-derived IL-23 and the subsequent support of the autoreactive/inflammatory Th17 phenotype.

Human Arginase II: Crystal Structure and Physiological Role in Male and Female Sexual Arousal†,‡

Arginase is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to form l-ornithine and urea. The X-ray crystal structure of a fully active, truncated form of human arginase II complexed with a boronic acid transition state analogue inhibitor has been determined at 2.7 A resolution. This structure is consistent with the hydrolysis of l-arginine through a metal-activated hydroxide mechanism. Given that human arginase II appears to play a role in regulating l-arginine bioavailability to NO synthase in human penile corpus cavernosum smooth muscle, the inhibition of human arginase II is a potential new strategy for the treatment of erectile dysfunction [Kim, N. N., Cox, J. D., Baggio, R. F., Emig, F. A., Mistry, S., Harper, S. L., Speicher, D. W., Morris, S. M., Ash, D. E., Traish, A. M., and Christianson, D. W. (2001) Biochemistry 40, 2678-2688]. Since NO synthase is found in human clitoral corpus cavernosum and vagina, we hypothesized that human arginase II is similarly present in these tissues and functions to regulate l-arginine bioavailability to NO synthase. Accordingly, hemodynamic studies conducted with a boronic acid arginase inhibitor in vivo are summarized, suggesting that the extrahepatic arginase plays a role in both male and female sexual arousal. Therefore, arginase II is a potential target for the treatment of male and female sexual arousal disorders.

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.

Differential effects of IFN-β on IL-12, IL-23, and IL-10 expression in TLR-stimulated dendritic cells

MS is an autoimmune disease characterized by immune cell infiltration in the CNS, leading to cumulative disability. IFN‐β, used clinically in RR‐MS reduces lesion formation and rates of relapse. Although the molecular mechanisms are not entirely elucidated, myeloid cells appear to be a major target for the therapeutic effects of IFN‐β. DCs have a critical role in experimental models of MS through their effect on encephalitogenic Th1/Th17 cell differentiation and expansion. Here we focused on the effects of IFN‐β on DC expression of cytokines involved in the control of Th1/Th17 differentiation and expansion. Administration of IFN‐β to mice immunized with MOG35–55 inhibited IL‐12 and IL‐23 expression in splenic DC and reduced in vivo differentiation of Th1/Th17 cells. IFN‐β affected cytokine expression in TLR‐stimulated DC in a similar manner in vitro, inhibiting IL‐12 and IL‐23 and stimulating IL‐10 at both mRNA and protein levels, by signaling through IFNAR. We investigated the role of the signaling molecules STAT1/STAT2, IRF‐1 and IRF‐7, and of the PI3K→GSK3 pathway. IFN‐β inhibition of the IL‐12 subunits p40 and p35 was mediated through STAT1/STAT2, whereas inhibition of IL‐23 was STAT1 dependent, and the stimulatory effect on IL‐10 expression was mediated through STAT2. IFN‐β induces IRF‐7 and, to a lesser degree, IRF‐1. However, neither IRF mediated the effects of IFN‐β on IL‐12, IL‐23, or IL‐10. We found that the PI3K pathway mediated IL‐12 inhibition but did not interfere with the inhibition of IL‐23 or stimulation of IL‐10.

48. Neuropeptides as cell gene therapy in experimental autoimmune encephalomyelitis

Dendritic cells (DC) initiate immune responses as well as antigen-specific tolerance. We showed previously that the neuropeptide vasoactive intestinal peptide (VIP) suppresses innate immune responses, generates tolerogenic dendritic cells, and has therapeutic effects in models of autoimmune/inflammatory disorders. To generate tolerogenic DC that also provide VIP locally, we engineered VIP-expressing DC using lentiviral vectors. LentiVIP-DC secrete VIP, switch from a proinflammatory to an anti-inflammatory cytokine profile and lose the capacity to activate T cells. A single inoculation of LentiVIP-DC in models of experimental autoimmune encephalomyelitis (EAE) led to a reduction in clinical symptoms, associated with reduced expression of proinflammatory cytokines and increased levels of the anti-inflammatory cytokine IL-10 in the spinal cord of treated mice. Induction of antigen-specific regulatory T cells and local delivery of VIP by LentiVIP-DC may represent a promising therapeutic tool for the treatment of autoimmune diseases and inflammatory disorders.