Prostaglandin E2 Inhibition of IL-27 Production in Murine Dendritic Cells: A Novel Mechanism That Involves IRF1.

IL-27, a multifunctional cytokine produced by APCs, antagonizes inflammation by affecting conventional dendritic cells (cDC), inducing IL-10, and promoting development of regulatory Tr1 cells. Although the mechanisms involved in IL-27 induction are well studied, much less is known about the factors that negatively impact IL-27 expression. PGE2, a major immunomodulatory prostanoid, acts as a proinflammatory agent in several models of inflammatory/autoimmune disease, promoting primarily Th17 development and function. In this study, we report on a novel mechanism that promotes the proinflammatory function of PGE2 We showed previously that PGE2 inhibits IL-27 production in murine bone marrow-derived DCs. In this study, we show that, in addition to bone marrow-derived DCs, PGE2 inhibits IL-27 production in macrophages and in splenic cDC, and we identify a novel pathway consisting of signaling through EP2/EP4→induction of cAMP→downregulation of IFN regulatory factor 1 expression and binding to the p28 IFN-stimulated response element site. The inhibitory effect of PGE2 on p28 and irf1 expression does not involve endogenous IFN-ß, STAT1, or STAT2, and inhibition of IL-27 does not appear to be mediated through PKA, exchange protein activated by cAMP, PI3K, or MAPKs. We observed similar inhibition of il27p28 expression in vivo in splenic DC following administration of dimethyl PGE2 in conjunction with LPS. Based on the anti-inflammatory role of IL-27 in cDC and through the generation of Tr1 cells, we propose that the PGE2-induced inhibition of IL-27 in activated cDC represents an important additional mechanism for its in vivo proinflammatory functions.

The natural dual cyclooxygenase and 5-lipoxygenase inhibitor flavocoxid is protective in EAE through effects on Th1/Th17 differentiation and macrophage/microglia activation.

Prostaglandins and leukotrienes, bioactive mediators generated by cyclooxygenases (COX) and 5-lipoxygenase (5-LO) from arachidonic acid, play an essential role in neuroinflammation. High levels of LTB4 and PGE2 and increased expression of COX and 5-LO, as well as high expression of PGE2 receptors were reported in multiple sclerosis (MS) patients and in experimental autoimmune encephalomyelitis (EAE). Prostaglandins and leukotrienes have an interdependent and compensatory role in EAE, which led to the concept of therapy using dual COX/5-LO inhibitors. The plant derived flavocoxid, a dual COX/5-LO inhibitor with anti-inflammatory and antioxidant properties, manufactured as a prescription pharmaconutrient, was reported to be neuroprotective in models of transient ischemic stroke and brain injury. The present study is the first report on prophylactic and therapeutic effects of flavocoxid in EAE. The beneficial effects correlate with reduced expression of proinflammatory cytokines and of COX2 and 5-LO in spinal cords and spleens of EAE mice. The protective mechanisms include: 1. reduction in expression of MHCII/costimulatory molecules and production of proinflammatory cytokines; 2. promotion of the M2 phenotype including IL-10 expression and release by macrophages and microglia; 3. inhibition of Th1 and Th17 differentiation through direct effects on T cells. The direct inhibitory effect on Th1/Th17 differentiation, and promoting the development of M2 macrophages and microglia, represent novel mechanisms for the flavocoxid anti-inflammatory activity. As a dual COX/5-LO inhibitor with antioxidant properties, flavocoxid might be useful as a potential therapeutic medical food agent in MS patients.

Glioblastoma exosomes and IGF-1R/AS-ODN are immunogenic stimuli in a translational research immunotherapy paradigm.

Glioblastomas are primary intracranial tumors for which there is no cure. Patients receiving standard of care, chemotherapy and irradiation, survive approximately 15 months prompting studies of alternative therapies including vaccination. In a pilot study, a vaccine consisting of Lucite diffusion chambers containing irradiated autologous tumor cells pre-treated with an antisense oligodeoxynucleotide (AS-ODN) directed against the insulin-like growth factor type 1 receptor was found to elicit positive clinical responses in 8/12 patients when implanted in the rectus sheath for 24 h. Our preliminary observations supported an immune response, and we have since reopened a second Phase 1 trial to assess this possibility among other exploratory objectives. The current study makes use of a murine glioma model and samples from glioblastoma patients in this second Phase 1 trial to investigate this novel therapeutic intervention more thoroughly. Implantation of the chamber-based vaccine protected mice from tumor challenge, and we posit this occurred through the release of immunostimulatory AS-ODN and antigen-bearing exosomes. Exosomes secreted by glioblastoma cultures are immunogenic, eliciting and binding antibodies present in the sera of immunized mice. Similarly, exosomes released by human glioblastoma cells bear antigens recognized by the sera of 6/12 patients with recurrent glioblastomas. These results suggest that the release of AS-ODN together with selective release of exosomes from glioblastoma cells implanted in chambers may drive the therapeutic effect seen in the pilot vaccine trial.

Enhancement of glioma-specific immunity in mice by "NOBEL", an insulin-like growth factor 1 receptor antisense oligodeoxynucleotide.

Autologous glioblastoma multiforme tumor cells treated with an antisense oligodeoxynucleotide (AS-ODN) targeting insulin-like growth factor receptor-1 (IGF-1R) are the basis of a vaccine with therapeutic effects on tumor recurrence in a pilot clinical trial. As a preface to continued clinical investigation of this vaccination strategy, we have studied the contribution of an optimized IGF-1R AS-ODN, designated "NOBEL", to the induction of immunity to mouse GL261 glioma cells. The impact of NOBEL on mechanisms contributing to the development of GL261 immunity was first examined in the periphery. GL261 cells are naturally immunogenic when implanted into the flanks of congenic C57BL/6 mice, immunizing rather than forming tumors in around 50 % of these animals but causing tumors in the majority of mice lacking T and B lymphocytes. Overnight treatment with NOBEL in vitro reduces IGF-1R expression by GL261 cells but has minimal effect on cell viability and does not reduce the capacity of the cells to form tumors upon implantation. In contrast, tumors are extremely rare when GL261 cells are mixed with NOBEL at inoculation into the flanks of C57BL/6, and the recipient mice become immune to subcutaneous and intracranial challenge with untreated GL261. Adaptive immune mechanisms contribute to this effect, as immunocompromised mice fail to either fully control tumor formation or develop immunity following flank administration of the GL261/NOBEL mix. NOBEL's structure has known immunostimulatory motifs that likely contribute to the immunogenicity of the mix, but its specificity for IGF-1R mRNA is also important as a similarly structured sense molecule is not effective.

The cytokine network type I IFN-IL-27-IL-10 is augmented in murine and human lupus.

IL-10 is elevated in the autoimmune disease systemic lupus erythematosus (SLE). Here, we show that conventional dendritic cells (cDCs) from predisease lupus-prone B6.NZM Sle1/Sle2/Sle3 triple congenic (TCSle) mice produce more IL-10 than wild-type congenic cDCs upon TLR stimulation, and this overproduction is prevented by blocking the type I IFN receptor (IFNAR) with specific Abs. Priming wild-type cDCs with type I IFN mimics the IL-10 overproduction of TCSle cDCs. The MAPK ERK is more phosphorylated in lupus cDCs, partially contributing to IL-10 overproduction. Moreover, we found that TCSle cDCs express higher levels of IL-27 upon TLR7/TLR9 stimulation, and IFNAR blockade reduced IL-27 levels in TCSle cDCs. These results suggest that dysregulated type I IFNs in cDCs contribute to the increased IL-10 and IL-27 in SLE. Since IL-27 neutralization did not inhibit TLR-induced IL-10 production, we propose that type I IFNs enhanced IL-10 in TCSle cDCs independently from IL-27. Moreover, RNA sequencing analysis of a cohort of SLE patients reveals higher gene expression of these cytokines in SLE patients expressing a high IFN signature. Since IL-27 and IL-10 have both pro- and anti-inflammatory effects, our results also suggest that these cytokines can be modulated by the therapeutic IFN blockade in trials in SLE patients and have complex effects on the autoimmune response.

Differential effects of IFN-ß 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-ß, 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-ß. 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-ß on DC expression of cytokines involved in the control of Th1/Th17 differentiation and expansion. Administration of IFN-ß 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-ß 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-ß 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-ß induces IRF-7 and, to a lesser degree, IRF-1. However, neither IRF mediated the effects of IFN-ß 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.

IL-1R1 signaling facilitates Munro's microabscess formation in psoriasiform imiquimod-induced skin inflammation.

Munro's microabscesses contain polymorphonuclear leukocytes and form specifically in the epidermis of psoriasis patients. The mechanism whereby the neutrophils are recruited into the epidermis is poorly understood. Using a combination of human and mouse primary keratinocyte cell cultures and the imiquimod-induced psoriasis-like mouse model of skin inflammation, we explored the role of IL-1 signaling in microabscess formation. In vitro imiquimod stimulated production of IL-1α and neutrophil recruiting chemokines. Imiquimod-activated chemokine expression was dependent upon adenosine signaling and independent of IL-1α and IL-1 receptor type 1 (IL-1R1); nevertheless, IL-1α could enhance chemokine expression initiated by imiquimod. Topical application of imiquimod in vivo led to epidermal microabscess formation, acanthosis, and increased IL-1α and chemokine expression in the skin of wild-type mice. However, in IL-1R1-deficient mice these responses were either absent or dramatically reduced. These results demonstrate that IL-1α and IL-1R1 signaling is essential for microabscess formation, neutrophil recruiting chemokine expression, and acanthosis in psoriasis-like skin inflammation induced by imiquimod.