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.

Conditioning therapies before transplantation induce the release of uric acid, which triggers the NLRP3 inflammasome and IL-1β production contributing to graft-versus-host disease.

The Linear Ubiquitin chain Assembly Complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR11. Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, due to deregulation of TNFR1-mediated cell death2–8. In humans, deficiency in the third LUBAC component, HOIL-1, causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9–11. By creating HOIL-1-deficient mice, we here show that HOIL-1 is, however, as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is LUBAC’s catalytically active component, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1-signalling complex (TNFR1-SC), thereby preventing aberrant cell death. Both, HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of Caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- embryos, yet only combined loss of Caspase-8 with MLKL results in viable HOIL-1-deficient mice. Interestingly, Ripk3-/-Caspase-8-/-Hoil-1-/- embryos die at late-gestation due to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both, HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they unveil that, when LUBAC and Caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in foetal haematopoiesis.

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...

Activation of interferon regulatory factor (IRF)-3 and/or IRF-7 drives the expression of antiviral genes and the production of a/b IFN, a hallmark of antiviral responses triggered by Toll-like receptors (TLR). Here we describe a novel antiviral signaling pathway operating in myeloid (m) dendritic cells (DC) and macrophages that does not require IRF-3 and/or IRF-7 but is driven by IRF-1. IRF-1 together with myeloid differentiation factor 88 (MyD88) or IL-1 receptor-associated kinase (IRAK)-1 triggered IFN-b promoter activation. IRF-1 physically interacted with MyD88 and activation of mDC via TLR-9 induced IRF-1-dependent IFN-b production paralleled by rapid transcriptional activation of IFN-stimulated genes. The NF-jB-dependent production of pro-inflammatory cytokines, however, was not influenced by IRF-1. TLR-9 signaling through this pathway conferred cellular antiviral resistance while IRF-1-deficient mice displayed enhanced susceptibility to viral infection. These results demonstrate that TLR-9 activation of mDC and macrophages contributes to antiviral immunity via IRF-1. See accompanying commentary: http://dx

The molecular pathways that regulate the tissue repair function of type I interferon (IFN-I) during acute tissue damage are poorly understood. We describe a protective role for IFN-I and the RIG-I/MAVS signaling pathway during acute tissue damage in mice. Mice lacking mitochondrial antiviral-signaling protein (MAVS) were more sensitive to total body irradiation– and chemotherapy-induced intestinal barrier damage. These mice developed worse graft-versus-host disease (GVHD) in a preclinical model of allogeneic hematopoietic stem cell transplantation (allo-HSCT) than did wild-type mice. This phenotype was not associated with changes in the intestinal microbiota but was associated with reduced gut epithelial integrity. Conversely, targeted activation of the RIG-I pathway during tissue injury promoted gut barrier integrity and reduced GVHD. Recombinant IFN-I or IFN-I expression induced by RIG-I promoted growth of intestinal organoids in vitro and production of the antimicrobial peptide regenerating islet–derived protein 3 γ (RegIIIγ). Our findings were not confined to RIG-I/MAVS signaling because targeted engagement of the STING (stimulator of interferon genes) pathway also protected gut barrier function and reduced GVHD. Consistent with this, STING-deficient mice suffered worse GVHD after allo-HSCT than did wild-type mice. Overall, our data suggest that activation of either RIG-I/MAVS or STING pathways during acute intestinal tissue injury in mice resulted in IFN-I signaling that maintained gut epithelial barrier integrity and reduced GVHD severity. Targeting these pathways may help to prevent acute intestinal injury and GVHD during allogeneic transplantation.

The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in HoipE-KO and Hoil-1E-KO mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone.

Achieving durable clinical responses to immune checkpoint inhibitors remains a challenge. Here, we demonstrate that immunotherapy with anti–CTLA-4 and its combination with anti–PD-1 rely on tumor cell–intrinsic activation of the cytosolic RNA receptor RIG-I. Mechanistically, tumor cell–intrinsic RIG-I signaling induced caspase-3–mediated tumor cell death, cross-presentation of tumor-associated antigen by CD103+ dendritic cells, subsequent expansion of tumor antigen–specific CD8+ T cells, and their accumulation within the tumor tissue. Consistently, therapeutic targeting of RIG-I with 5′– triphosphorylated RNA in both tumor and nonmalignant host cells potently augmented the efficacy of CTLA-4 checkpoint blockade in several preclinical cancer models. In humans, transcriptome analysis of primary melanoma samples revealed a strong association between high expression of DDX58 (the gene encoding RIG-I), T cell receptor and antigen presentation pathway activity, and prolonged overall survival. Moreover, in patients with melanoma treated with anti–CTLA-4 checkpoint blockade, high DDX58 RIG-I transcriptional activity significantly associated with durable clinical responses. Our data thus identify activation of RIG-I signaling in tumors and their microenvironment as a crucial component for checkpoint inhibitor–mediated immunotherapy of cancer.