Unmethylated CpG motifs in bacterial DNA, plasmid DNA and synthetic oligodeoxynucleotides (CpG ODN) activate dendritic cells (DC) and macrophages in a CD40-CD40 ligand-independent fashion. To understand the molecular mechanisms involved we focused on the cellular uptake of CpG ODN, the need for endosomal maturation and the role of the stress kinase pathway. Here we demonstrate that CpG-DNA induces phosphorylation of Jun N-terminal kinase kinase 1 (JNKK1/SEK/MKK4) and subsequent activation of the stress kinases JNK1/2 and p38 in murine macrophages and dendritic cells. This leads to activation of the transcription factor activating protein-1 (AP-1) via phosphorylation of its constituents c-Jun and ATF2. Moreover, stress kinase activation is essential for CpG-DNA-induced cytokine release of tumor necrosis factor α (TNFα) and interleukin-12 (IL-12), as inhibition of p38 results in severe impairment of this biological response. We further demonstrate that cellular uptake via endocytosis and subsequent endosomal maturation is essential for signalling, since competition by non-CpG-DNA or compounds blocking endosomal maturation such as chloroquine or bafilomycin A prevent all aspects of cellular activation. The data suggest that endosomal maturation is required for translation of intraendosomal CpG ODN sequences into signalling via the stress kinase pathway, where p38 kinase activation represents an essential step in CpG-ODN-triggered activation of antigen-presenting cells.

SummaryBecause mice are more resistant than humans to the pathogenic effects of bacterial toxins, we used D-Galactosamine-(D-Gal) sensitized mice as a modal system to evaluate potential toxic shock symptoms triggered by the superantigcn staphylococcal enterotoxin B (SEB). We show that similar to endotoxin (lipopolysaccharide) [LPS], the exotoxin SEB causes lethal shock within 8 h in D-Gal-sensitized mice, inducing 100% and about 50% lethality with 20 and 2 ~g SEB, respectively. The lethal shock triggered by the superantigcn SEB is mediated by T cells, a conclusion based on the observation that T cell repopulation of SCID mice conferred sensitivity to SEB. Since CSA also conferred protection, the role of T ceU-derived lymphokines in mediating lethal shock was evaluated. Within 30-60 min after SEB injection, serum tumor necrosis factor (TNF) levels peaked, followed immediately by interleukin-2 (IL-2). Serum-borne lymphokines were detected weU in advance of signs of T ceU activation, as assessed by IL-2 receptor expression of SEB-reactive VB8 + T ceUs. Passive immunization with anti-TNF-a/~-neutralizing monodonal antibody also conferred protection, indicating that it is TNF which is critical for initiating toxic shock symptoms. Taken together, this study defines basic differences between endotoxin (LPS)-and cxotoxin (SEB)-mediated lethal shock, in that the former is mediated by macrophages and the latter by T calls. Yet the pathogcnesis distal to the lymphokine/cytokine-producing cells appears surprisingly similar in that TNF represents a key mediator in inducing shock.

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG-oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as "nature's adjuvant" since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG-ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen-presenting cell (APC) function. Unfractionated murine bone marrow-derived DC and FACS-fractionated MHC class IIlow (termed immature DC) or MHC class IIhigh populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG-ODN. Similar to lipopolysaccharide, i.s. CpG-ODN caused up-regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL-12, IL-6 and TNF-alpha. CpG-ODN-activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B-driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG-ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.

Cell surface components of pathogens, such as lipopolysaccharide (LPS), are an important signal for receptor-mediated activation of immune cells. Here we demonstrate that DNA of gram-positive and gram-negative bacteria or certain synthetic oligonucleotides displaying unmethylated CpG-motifs can trigger macrophages in vitro to induce nuclear translocation of nuclear factor-kappa B, accumulate tumor necrosis factor (TNF)-alpha mRNA and release large amounts of TNF-alpha. In vivo these events culminate in acute cytokine-release syndrome which includes systemic but transient accumulation of TNF-alpha. D-Galactosamine (DGalN)-sensitized mice succumb to lethal toxic shock due to macrophage-derived TNF-alpha resulting in fulminant apoptosis of liver cells. LPS and a specific oligonucleotide synergized in vivo as measured by TNF-alpha-release, suggesting that macrophages integrate the respective signals. The ability of macrophages to discriminate and to respond to bacterial DNA with acute release of pro-inflammatory cytokines may point out an important and as yet unappreciated sensing mechanism for foreign DNA.

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Immunity to infection with intracellular pathogens is regulated by interleukin 12 (IL-12), which mediates protective T helper type 1 (TH1) responses, or IL-4, which induces TH2 cells and susceptibility. Paradoxically, we show here that when present during the initial activation of dendritic cells (DCs) by infectious agents, IL-4 instructed DCs to produce IL-12 and promote TH1 development. This TH1 response established resistance to Leishmania major in susceptible BALB/c mice. When present later, during the period of T cell priming, IL-4 induced TH2 differentiation and progressive leishmaniasis in resistant mice. Because immune responses developed via the consecutive activation of DCs and then T cells, the contrasting effects of IL-4 on DC development and T cell differentiation led to immune responses that had opposing functional phenotypes.

During infection, TLR agonists are released and trigger mature as well as differentiating innate immune cells. Early encounter with TLR agonists (R848; LPS) blocks conventional differentiation of CD14 1 monocytes into immature dendritic cells (iDCs) resulting in a deviated phenotype. We and others characterized these APCs (TLR-APC) by a retained expression of CD14 and a lack of CD1a. Here, we show in addition, expression of programmed death ligand-1 (PD-L1). TLR-APCs failed to induce T-cell proliferation and furthermore were able to induce CD25 1 Foxp3 1 T regulatory cells (Tregs). Since PD-L1 is described as a key negative regulator and inducer of tolerance, we further analyzed its regulation. PD-L1 expression was regulated in a MAPK/cytokine/STAT-3-dependent manner: high levels of IL-6 and IL-10 that signal via STAT-3 were produced by TLR-APCs. Blocking of STAT-3 activation prevented PD-L1 expression. Moreover, chromatin immunoprecipitation revealed direct binding of STAT-3 to the PD-L1 promoter. Those findings indicate a pivotal role of STAT-3 in regulating PD-L1 expression. MAPKs were indirectly engaged, as blocking of p38 and p44/42 MAPKs decreased IL-6 and IL-10 thus reducing STAT-3 activation and subsequent PD-L1 expression. Hence, during DC differentiation TLR agonists induce a STAT-3-mediated expression of PD-L1 and favor the development of tolerogenic APCs.Keywords: DC . PD-L1 . STAT-3 . Tolerance . TLR See accompanying Commentary by Sumpter and ThomsonSupporting Information available online IntroductionDC are initiators and modulators of the adaptive immune response [1]. They are able to induce T-cell activation as well as T-cell tolerance. During infection, DCs are confronted with pathogen-associated molecular patterns (PAMP), which in turn trigger effector functions in innate immune cells. For example, immature DCs (iDCs) generated from monocytes by in vitro culture with GM-CSF and IL-4 (G4) mature and become fully activated upon stimulation with TLR agonists. Mature DCs (mDCs) in turn activate most efficiently naïve T cells [2]. However, during infection induction of inhibitory immune pathways can also be observed [3,4]. Here, we investigate an alternative TLR-induced APC phenotype, which inhibits immune reactivity. It has been shown that encounter of monocytes with LPS during the very beginning of the differentiation process blocks conventional differentiation to iDCs. A phenotypically distinct APC type (TLR-APC) is generated, characterized by a [5][6][7]. Activation of p38 MAPK, the secretion of IL-10 and the inactivation of ERK and NF-kB [7] have been correlated with the generation of TLR-APCs. LPS-treated cells showed in addition an intense STAT-3 phosphorylation. Differentiation processes of DCs are plastic and can be influenced by various factors, e.g. cytokines. Many cytokines mediate their cellular response via the JAK/STAT signaling pathway thereby controlling the status of transcription and cellular differentiation. For instance, during the maturation of DCs, a switch occurs from constit...

Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/ signal transducer and activator of transcription (JAK/ STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-3 and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I interferon. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-3, or CIS on prototypical TLR target genes such as tumor necrosis factor-␣. However, we found that TLR-2, TLR-3, TLR-4, and TLR-9 stimulation induced interferon-␤ (IFN-␤), which is able to exert auto-and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-3 and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NFB signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NFB pathway.