Type I IFN production in response to the DNA virus herpes simplex virus type-1 (HSV-1) is essential in controlling viral replication. We investigated whether plasmacytoid dendritic cells (pDC) were the major tissue source of IFN-␣, and whether the production of IFN-␣ in response to HSV-1 depended on Toll-like receptor 9 (TLR9). Total spleen cells or bone marrow (BM) cells, or fractions thereof, including highly purified pDC, from WT, TLR9, and MyD88 knockout mice were stimulated with known ligands for TLR9 or active HSV-1. pDC freshly isolated from both spleen and BM were the major source of IFN-␣ in response to oligodeoxynucleotides containing CpG motifs, but in response to HSV-1 the majority of IFN-␣ was produced by other cell types. Moreover, IFN-␣ production by non-pDC was independent of TLR9. The tissue source determined whether pDC responded to HSV-1 in a strictly TLR9-dependent fashion. Freshly isolated BM pDC or pDC derived from culture of BM precursors with FMS-like tyrosine kinase-3 ligand, produced IFN-␣ in the absence of functional TLR9, whereas spleen pDC did not. Heat treatment of HSV-1 abolished maturation and IFN-␣ production from all TLR9-deficient DC but not WT DC. Thus pDC and non-pDC produce IFN-␣ in response to HSV-1 via both TLR9-independent and -dependent pathways.

CpG motifs within phosphorothioate (PS)-modified DNA drive Toll-like receptor 9 (TLR9) activation, but the rules governing recognition of natural phosphodiester (PD) DNA are less understood. Here, we showed that the sugar backbone determined DNA recognition by TLR9. Homopolymeric, base-free PD 2' deoxyribose acted as a basal TLR9 agonist as it bound to and activated TLR9. This effect was enhanced by DNA bases, even short of CpG motifs. In contrast, PS-modified 2' deoxyribose homopolymers acted as TLR9 and TLR7 antagonists. They displayed high affinity to both TLRs and did not activate on their own, but they competitively inhibited ligand-TLR interaction and activation. Although addition of random DNA bases to the PS 2' deoxyribose backbone did not alter these effects, CpG motifs transformed TLR9-inhibitory to robust TLR9-stimulatory activity. Our results identified the PD 2' deoxyribose backbone as an important determinant of TLR9 activation by natural DNA, restrict CpG-motif dependency of TLR9 activation to synthetic PS-modified ligands, and define PS-modified 2' deoxyribose as a prime effector of TLR9 and TLR7 inhibition.

TLRs discriminate foreign from self via their specificity for pathogen-derived invariant ligands, an example being TLR9 recognizing bacterial unmethylated CpG motifs. In this study we report that endosomal translocation of CpG DNA via the natural endocytotic pathway is inefficient and highly saturable, whereas endosomal translocation of DNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) is not. Interestingly, DOTAP-mediated enhanced endosomal translocation of otherwise nonstimulatory vertebrate DNA or of certain noncanonical CpG motifs triggers robust dendritic cell activation in terms of both up-regulation of CD40/CD69 and cytokine production, such as type I IFN and IL-6. We report that the stimulatory activity of phosphorothioated noncanonical CpG oligodeoxynucleotides is TLR9 dependent, whereas phosphodiester DNA, such as vertebrate DNA, in addition trigger TLR9-independent pathways. We propose that the inefficiency of the natural route for DNA internalization hinders low affinity TLR9 ligands in endosomes to reach threshold concentrations required for TLR9 activation. Endosomal compartmentalization of TLR9 may thus reflect an evolutionary strategy to avoid TLR9 activation by self-DNA.

The mammalian target of rapamycin (mTOR) can be viewed as cellular master complex scoring cellular vitality and stress. Whether mTOR controls also innate immune-defenses is currently unknown. Here we demonstrate that TLR activate mTOR via phosphoinositide 3-kinase/Akt. mTOR physically associates with the MyD88 scaffold protein to allow activation of interferon regulatory factor-5 and interferon regulatory factor-7, known as master transcription factors for pro-inflammatory cytokine-and type I IFN-genes. Unexpectedly, inactivation of mTOR did not prevent but increased lethality of endotoxinmediated shock, which correlated with increased levels of IL-1b. Mechanistically, mTOR suppresses caspase-1 activation, thus inhibits release of bioactive IL-1b. We have identified mTOR as indispensable component of PRR signal pathways, which orchestrates the defense program of innate immune cells.Key words: Caspase-1 . IRF . mTOR . TLR Supporting Information available online IntroductionThe phosphoinositide 3-kinase (PI3K) represents a signaling gateway for the activation of various cellular effector functions including cell growth, proliferation, survival and vesicular transport [1,2]. Activated PI3K catalyzes the phosphorylation of membrane-anchored phosphoinositides (PI) and binding of PI-3,4,5-tri-phosphate to both Akt and PI-dependent protein kinase 1, which then drives PI-dependent protein kinase 1 to activate Akt via Thr308 phosphorylation [1]. It is known that inhibition of PI3K interferes with functions of innate immune cells [3,4], yet the molecular basis for this is still unclear.Upon inhibition of Akt, TLR-activated macrophages and DC mimic the phenotype of TLR-stimulated PI3K deficient cells [5]. Therefore, we reasoned that PI3K executes its regulatory function along the Akt pathway. One of the major targets of Akt is the mammalian target of rapamycin (mTOR), known to influence multiple cellular functions including cell cycle control, cellular growth, apoptosis, transcription and translational efficacy [6,7]. Whether and how mTOR signaling becomes integrated into TLR signaling pathways is unknown. Eur. J. Immunol. 2008. 38: 2981-2992 DOI 10.1002 HIGHLIGHTS 2981 FrontlineHere we describe that mTOR signaling is indispensable for the signal pathways of various PRR. First we show that membrane bound TLR directly activate mTOR via the PI3K/Akt axis. Activated mTOR subsequently transcriptionally controls in innate immune cells cytokine and type I IFN production. Essential steps in this transcriptional process include recruitment of activated mTOR to the MyD88 scaffold protein, the site at which interferon regulatory factor (IRF)-5 and IRF-7 become activated in an mTOR-dependent fashion. In addition, mTOR negatively regulates bioactive IL-1b production by inhibiting caspase-1 activation. These data characterize mTOR as transcriptional regulator and controller of acute innate immune reactions. Results TLR activate mTORTo analyze whether TLR signaling drives mTOR activation, we asked whether TLR-mediated activation of bon...