IFN- Negatively Regulates CpG-Induced IL-10 in Bone Marrow-Derived Dendritic Cells 1 Rafael R. Flores, Kelly A. Diggs, Lauren M. Tait, and Penelope A. Morel 2 Dendritic cells (DCs) are important players in the regulation of Th1- and Th2-dominated immune responses. In these studies we showed that IFN-, the key mediator of Th1 immunity, actively suppressed the production of IL-10 in murine DCs when activated with LPS or CpG. Our analysis revealed that both LPS and CpG induced IL-10 and IL-12 production but that the presence of IFN-, in a dose-dependent manner, suppressed the production of IL-10 while enhancing that of IL-12. The observed inhibition of IL-10 production was independent of IL-12. Experiments performed with STAT-1 knockout mice demonstrated that the primary production of IL-12 induced by CpG was STAT-1 dependent, whereas the production of IL-10 was not. This finding was confirmed by the observation that CpG-induced IL-12 production could be inhibited by anti-IFN- Abs, whereas CpG-induced IL-10 production could not be inhibited. These data also demonstrated that the inhibitory effect of IFN- on IL-10 expression was STAT-1 dependent and transcriptionally regulated. Thus, DCs respond to CpG by producing proinflammatory and anti- inflammatory cytokines such as IL-12 and IL-10, respectively, and IFN- acts to not only enhance IL-12 but also to inhibit IL-10 production. The current data demonstrate a novel pathway for IFN--mediated immunoregulation and suggest that IFN-- dependent suppression of IL-10 production by DCs may be involved in the antagonism between Th1 and Th2 patterns of immune reactivity. The Journal of Immunology, 2007, 178: 211–218. D endritic cells (DCs) 3 are APCs that provide a link be- tween the innate and adaptive immune systems. Imma- ture DCs reside in peripheral tissue and sample the en- vironment for pathogens. The uptake of pathogens and the secretion of inflammatory cytokines induce DC maturation and migration to draining lymph nodes, where DCs encounter Ag-spe- cific T cells (1). During this encounter, DCs present Ag, in the form of MHC-peptide complexes, and also provide costimulatory signals that induce T cell activation. Cytokines secreted by DCs have a profound impact on subse- quent Th cell differentiation (2, 3). IL-12 and/or IL-18 contribute significantly to the development of Th1 cells, whereas IL-6 and IL-10 stimulate the development of Th2 or Tr1 cells (2–5). In addition, the CD40-CD40L interaction plays an important role in stimulating IL-12 production (6), which is augmented in the pres- ence of IFN- (7, 8). Recent work by Mailliard et al. (9, 10) has shown that IFN-, produced by activated NK cells or CD8 + T cells, can skew the development of DCs toward a type I phenotype, characterized by their ability to secrete high levels of IL-12p70 following CD40 ligation and to stimulate Th1 differentiation. The innate response by DCs is mediated by receptors that rec- ognize conserved molecular structures on pathogens known as pathogen-associated molecular patterns. The recognition of these patterns is performed by pattern recognition receptors that include lectins (DC-SIGN, DEC-205, and the mannose receptor) (11, 12) and TLRs of which there are 11 human and 10 murine (13). TLR2, TLR4, and TLR6 form multisubunit complexes and preferentially respond to bacterial structures. Other TLRs such as TLR3, TLR7, and TLR9 respond to and detect intracellular pathogens. TLR9 is found in endosomal vesicles within the cell, and recent studies have shown that activated TLR9 signals from these vesicles (14, 15). TLR9 binds unmethylated DNA structures containing a C-G dinucleotide motif, known as CpG. Activation of TLR9 induces DC maturation, characterized by increases in the expression of costimulatory markers and MHC class II molecules (16), as well as the secretion of cytokines such as TNF-, IL-1, IL-6, IL-10, IL-12, or IFN-, IP-10, and RANTES, depending on which DC subset is activated (17). Due to its ability to stimulate DC activation, CpG has been considered for use as an adjuvant in immunotherapy for various conditions such as allergies, infections, or tumors (18, 19). In a murine model of atopic asthma, mice sensitized with methacho- line, along with CpG, had reduced eosinophilic infiltration of the lung, decreased serum levels of IgE, and decreased airway hyper- reactivity (20). Atopic mice treated with CpG experienced an in- crease in IL-12 and IFN- detected in the bronchoalveolar lavage fluid. Similar effects of CpG have been shown in infectious models such as Leishmania major (19, 21). In another study, mice injected with a tumor cell line were treated with CpG resulting in the in- filtration of the tumor by DCs expressing/producing high levels of costimulatory molecules and IL-12. This stimulated a Th1 re- sponse complete with tumor-specific cytolytic activity (22–24). These results have led to the use of CpG in a phase I clinical trial testing a cancer vaccine (25). In the present study, we analyzed the impact of IFN-, a key mediator of Th1-dominated immunity, upon the ability of different TLR ligands to induce the production of DC cytokines, IL-12 and Department of Immunology and Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 Received for publication November 23, 2005. Accepted for publication October 18, 2006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant CA73743 (to P.A.M.) and Department of Defense Training Grant DAMD17-99-1-9352 (to R.R.F.). K.A.D. and L.M.T. were supported by a National Science Foundation Research Ex- periences for Undergraduates Award 0243735. 2 Address correspondence and reprint requests to Dr. Penelope A. Morel, Department of Immunology, University of Pittsburgh, Biomedical Science Tower E-1048, 200 Lothrop Street, Pittsburgh, PA 15261. E-mail address: morel@pitt.edu 3 Abbreviations used in this paper: DC, dendritic cell; BMDC, bone marrow-derived DC; KO, knockout; WT, wild type; IRF, IFN regulatory factor. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$2.00 The Journal of Immunology www.jimmunol.org