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.

Some bacterial pathogens utilize cell-cell communication systems, such as quorum sensing (QS), to coordinate genetic programs during host colonization and infection. The human-restricted pathosymbiont Streptococcus pyogenes (group A streptococcus [GAS]) uses the Rgg2/Rgg3 QS system to modify the bacterial surface, enabling biofilm formation and lysozyme resistance. Here, we demonstrate that innate immune cell responses to GAS are substantially altered by the QS status of the bacteria. We found that macrophage activation, stimulated by multiple agonists and assessed by cytokine production and NF-κB activity, was substantially suppressed upon interaction with QS-active GAS but not QS-inactive bacteria. Neither macrophage viability nor bacterial adherence, internalization, or survival were altered by the QS activation status, yet tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interferon beta (IFN-β) levels and NF-κB reporter activity were drastically lower following infection with QS-active GAS. Suppression required contact between viable bacteria and macrophages. A QS-regulated biosynthetic gene cluster (BGC) in the GAS genome, encoding several putative enzymes, was also required for macrophage modulation. Our findings suggest a model wherein upon contact with macrophages, QS-active GAS produce a BGC-derived factor capable of suppressing inflammatory responses. The suppressive capability of QS-active GAS is abolished after treatment with a specific QS inhibitor. These observations suggest that interfering with the ability of bacteria to collaborate via QS can serve as a strategy to counteract microbial efforts to manipulate host defenses. IMPORTANCE Streptococcus pyogenes is restricted to human hosts and commonly causes superficial diseases such as pharyngitis; it can also cause severe and deadly manifestations including necrotizing skin disease or severe postinfectious sequelae like rheumatic heart disease. Understanding the complex mechanisms used by this pathogen to manipulate host defenses could aid in developing new therapeutics to treat infections. Here, we examine the impact of a bacterial cell-cell communication system, which is highly conserved across S. pyogenes, on host innate immune responses. We find that S. pyogenes uses this system to suppress macrophage proinflammatory cytokine responses in vitro. Interference with this communication system could serve as a strategy to disarm bacteria and maintain an effective immune response.

IL-27, a multifunctional cytokine produced by antigen-presenting cells, 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. Prostaglandin E2 (PGE2), a major immunomodulatory prostanoid, acts as a pro-inflammatory agent in several models of inflammatory/autoimmune diseases, promoting primarily Th17 development and function. In this study, we report on a novel mechanism which promotes the pro-inflammatory function of PGE2. We showed previously that PGE2 inhibits IL-27 production in murine bone marrow derived DCs. Here, we show that, in addition to BMDCs, 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 IRF1 expression and binding to the p28 ISRE site. The inhibitory effect of PGE2 on p28 and irf1 expression does not involve endogenous IFNβ, STAT1 or STAT2, and inhibition of IL-27 does not appear to be mediated through PKA, EPAC, 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 pro-inflammatory functions.

We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5 to 30 mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4–6 months to produce. To shorten the construction time, we replaced the muti-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2 months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ~5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines.