Publications by authors named "Martin Schlee"

44 Publications

Immune Sensing of Synthetic, Bacterial, and Protozoan RNA by Toll-like Receptor 8 Requires Coordinated Processing by RNase T2 and RNase 2.

Immunity 2020 04;52(4):591-605.e6

Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany. Electronic address:

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2 or RNASET2 but not RNASE2RNASET2 cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.
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http://dx.doi.org/10.1016/j.immuni.2020.03.009DOI Listing
April 2020

Human TLR8 Senses RNA From -Infected Red Blood Cells Which Is Uniquely Required for the IFN-γ Response in NK Cells.

Front Immunol 2019 27;10:371. Epub 2019 Mar 27.

Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany.

During blood-stage malaria, the innate immune system initiates the production of pro-inflammatory cytokines, including IFN-γ, that are critical to host defense and responsible for severe disease. Nonetheless, the innate immune pathways activated during this process in human malaria remain poorly understood. Here, we identify TLR8 as an essential sensor of -infected red blood cells (iRBC). In human immune cells, iRBC and RNA purified from iRBC were detected by TLR8 but not TLR7 leading to IFN-γ induction in NK cells. While TLR7 and 9 have been shown to lead to IFN-γ in mice, our data demonstrate that TLR8 was the only TLR capable of inducing IFN-γ release in human immune cells. This unique capacity was mediated by the release of IL-12p70 and bioactive IL-18 from monocytes, the latter via a hitherto undescribed pathway. Altogether, our data are the first reported activation of TLR8 by protozoan RNA and demonstrate both the critical role of TLR8 in human blood-stage malaria and its unique functionality in the human immune system. Moreover, our study offers important evidence that mouse models alone may not be sufficient to describe the human innate immune response to malaria.
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http://dx.doi.org/10.3389/fimmu.2019.00371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445952PMC
July 2020

SOCS1 and SOCS3 Target IRF7 Degradation To Suppress TLR7-Mediated Type I IFN Production of Human Plasmacytoid Dendritic Cells.

J Immunol 2018 06 30;200(12):4024-4035. Epub 2018 Apr 30.

Department of Dermatology and Allergy, University of Bonn, 53127 Bonn, Germany;

Type I IFN production of plasmacytoid dendritic cells (pDCs) triggered by TLR-signaling is an essential part of antiviral responses and autoimmune reactions. Although it was well-documented that members of the cytokine signaling (SOCS) family regulate TLR-signaling, the mechanism of how SOCS proteins regulate TLR7-mediated type I IFN production has not been elucidated yet. In this article, we show that TLR7 activation in human pDCs induced the expression of SOCS1 and SOCS3. SOCS1 and SOCS3 strongly suppressed TLR7-mediated type I IFN production. Furthermore, we demonstrated that SOCS1- and SOCS3-bound IFN regulatory factor 7, a pivotal transcription factor of the TLR7 pathway, through the SH2 domain to promote its proteasomal degradation by lysine 48-linked polyubiquitination. Together, our results demonstrate that SOCS1/3-mediated degradation of IFN regulatory factor 7 directly regulates TLR7 signaling and type I IFN production in pDCs. This mechanism might be targeted by therapeutic approaches to either enhance type I IFN production in antiviral treatment or decrease type I IFN production in the treatment of autoimmune diseases.
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http://dx.doi.org/10.4049/jimmunol.1700510DOI Listing
June 2018

RIG-I Activation Protects and Rescues from Lethal Influenza Virus Infection and Bacterial Superinfection.

Mol Ther 2017 09 8;25(9):2093-2103. Epub 2017 Jul 8.

Department of Otolaryngology, University Hospital Bonn, 53127 Bonn, Germany.

Influenza A virus infection causes substantial morbidity and mortality in seasonal epidemic outbreaks, and more efficient treatments are urgently needed. Innate immune sensing of viral nucleic acids stimulates antiviral immunity, including cell-autonomous antiviral defense mechanisms that restrict viral replication. RNA oligonucleotide ligands that potently activate the cytoplasmic helicase retinoic-acid-inducible gene I (RIG-I) are promising candidates for the development of new antiviral therapies. Here, we demonstrate in an Mx1-expressing mouse model of influenza A virus infection that a single intravenous injection of low-dose RIG-I ligand 5'-triphosphate RNA (3pRNA) completely protected mice from a lethal challenge with influenza A virus for at least 7 days. Furthermore, systemic administration of 3pRNA rescued mice with pre-established fulminant influenza infection and prevented the fatal effects of a streptococcal superinfection. Type I interferon, but not interferon-λ, was required for the therapeutic effect. Our results suggest that the use of RIG-I activating oligonucleotide ligands has the clinical potential to confine influenza epidemics when a strain-specific vaccine is not yet available and to reduce lethality of influenza in severely infected patients.
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http://dx.doi.org/10.1016/j.ymthe.2017.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589155PMC
September 2017

RIG-I Resists Hypoxia-Induced Immunosuppression and Dedifferentiation.

Cancer Immunol Res 2017 06 3;5(6):455-467. Epub 2017 May 3.

Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany.

A hypoxic tumor microenvironment is linked to poor prognosis. It promotes tumor cell dedifferentiation and metastasis and desensitizes tumor cells to type-I IFN, chemotherapy, and irradiation. The cytoplasmic immunoreceptor retinoic acid-inducible gene-I (RIG-I) is ubiquitously expressed in tumor cells and upon activation by 5'-triphosphate RNA (3pRNA) drives the induction of type I IFN and immunogenic cell death. Here, we analyzed the impact of hypoxia on the expression of RIG-I in various human and murine tumor and nonmalignant cell types and further investigated its function in hypoxic murine melanoma. 3pRNA-inducible RIG-I-expression was reduced in hypoxic melanoma cells compared with normoxic controls, a phenomenon that depended on the hypoxia-associated transcription factor HIF1α. Still, RIG-I functionality was conserved in hypoxic melanoma cells, whereas responsiveness to recombinant type-I IFN was abolished, due to hypoxia-induced loss of type I IFN receptor expression. Likewise, RIG-I activation in hypoxic melanoma cells, but not exposure to recombinant IFNα, provoked melanocyte antigen-specific CD8 T-cell and NK-cell attack. Scavenging of hypoxia-induced reactive oxygen species by vitamin C restored the inducible expression of RIG-I under hypoxia , boosted anti-melanoma NK- and CD8 T-cell attack, and augmented 3pRNA antitumor efficacy These results demonstrate that RIG-I remains operational under hypoxia and that RIG-I function is largely insensitive to lower cell surface expression of the IFNα receptor. RIG-I function could be fortified under hypoxia by the combined use of 3pRNA with antioxidants. .
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http://dx.doi.org/10.1158/2326-6066.CIR-16-0129-TDOI Listing
June 2017

G-rich DNA-induced stress response blocks type-I-IFN but not CXCL10 secretion in monocytes.

Sci Rep 2016 12 12;6:38405. Epub 2016 Dec 12.

Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany.

Excessive inflammation can cause damage to host cells and tissues. Thus, the secretion of inflammatory cytokines is tightly regulated at transcriptional, post-transcriptional and post-translational levels and influenced by cellular stress responses, such as endoplasmic reticulum (ER) stress or apoptosis. Here, we describe a novel type of post-transcriptional regulation of the type-I-IFN response that was induced in monocytes by cytosolic transfection of a short immunomodulatory DNA (imDNA), a G-tetrad forming CpG-free derivative of the TLR9 agonist ODN2216. When co-transfected with cytosolic nucleic acid stimuli (DNA or 3P-dsRNA), imDNA induced caspase-3 activation, translational shutdown and upregulation of stress-induced genes. This stress response inhibited the type-I-IFN induction at the translational level. By contrast, the induction of most type-I-IFN-associated chemokines, including Chemokine (C-X-C Motif) Ligand (CXCL)10 was not affected, suggesting a differential translational regulation of chemokines and type-I-IFN. Pan-caspase inhibitors could restore IFN-β secretion, yet, strikingly, caspase inhibition did not restore global translation but instead induced a compensatory increase in the transcription of IFN-β but not CXCL10. Altogether, our data provide evidence for a differential regulation of cytokine release at both transcriptional and post-transcriptional levels which suppresses type-I-IFN induction yet allows for CXCL10 secretion during imDNA-induced cellular stress.
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http://dx.doi.org/10.1038/srep38405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5150577PMC
December 2016

RIG-I activation induces the release of extracellular vesicles with antitumor activity.

Oncoimmunology 2016;5(10):e1219827. Epub 2016 Aug 19.

Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn , Bonn, Germany.

Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5'-triphosphate-RNA (3pRNA) triggers antitumor immunity predominantly via NK cell activation and direct apoptosis induction in tumor cells. However, how NK cells are mobilized to attack the tumor cells remains elusive. Here, we show that RIG-I activation induced the secretion of extracellular vesicles (EVs) from melanoma cells, which by themselves revealed antitumor activity and . RIG-I-induced EVs from melanoma cells exhibited an increased expression of the NKp30-ligand (BAG6, BAT3) on their surface triggering NK cell-mediated lysis of melanoma cells via activation of the cytotoxicity NK cell-receptor NKp30. Moreover, systemic administration of RIG-I-induced melanoma-EVs showed a potent antitumor activity in a melanoma mouse model . In conclusion, our data establish a new RIG-I-dependent pathway leading to NK cell-mediated tumor cell killing.
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http://dx.doi.org/10.1080/2162402X.2016.1219827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087302PMC
August 2016

Discriminating self from non-self in nucleic acid sensing.

Nat Rev Immunol 2016 09 25;16(9):566-80. Epub 2016 Jul 25.

Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Sigmund-Freud-Strasse 25, D-53127 Bonn, Germany.

Innate immunity against pathogens relies on an array of immune receptors to detect molecular patterns that are characteristic of the pathogens, including receptors that are specialized in the detection of foreign nucleic acids. In vertebrates, nucleic acid sensing is the dominant antiviral defence pathway. Stimulation of nucleic acid receptors results in antiviral immune responses with the production of type I interferon (IFN), as well as the expression of IFN-stimulated genes, which encode molecules such as cell-autonomous antiviral effector proteins. This Review summarizes the tremendous progress that has been made in understanding how this sophisticated immune sensory system discriminates self from non-self nucleic acids in order to reliably detect pathogenic viruses.
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http://dx.doi.org/10.1038/nri.2016.78DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7097691PMC
September 2016

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.

Nat Immunol 2015 Oct 7;16(10):1025-33. Epub 2015 Sep 7.

Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany.

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.
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http://dx.doi.org/10.1038/ni.3267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669199PMC
October 2015

A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N1-2'O-Methylated Self RNA.

Immunity 2015 Jul 14;43(1):41-51. Epub 2015 Jul 14.

Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, 53105 Bonn, Germany. Electronic address:

The cytosolic helicase retinoic acid-inducible gene-I (RIG-I) initiates immune responses to most RNA viruses by detecting viral 5'-triphosphorylated RNA (pppRNA). Although endogenous mRNA is also 5'-triphosphorylated, backbone modifications and the 5'-ppp-linked methylguanosine ((m7)G) cap prevent immunorecognition. Here we show that the methylation status of endogenous capped mRNA at the 5'-terminal nucleotide (N1) was crucial to prevent RIG-I activation. Moreover, we identified a single conserved amino acid (H830) in the RIG-I RNA binding pocket as the mediator of steric exclusion of N1-2'O-methylated RNA. H830A alteration (RIG-I(H830A)) restored binding of N1-2'O-methylated pppRNA. Consequently, endogenous mRNA activated the RIG-I(H830A) mutant but not wild-type RIG-I. Similarly, knockdown of the endogenous N1-2'O-methyltransferase led to considerable RIG-I stimulation in the absence of exogenous stimuli. Studies involving yellow-fever-virus-encoded 2'O-methyltransferase and RIG-I(H830A) revealed that viruses exploit this mechanism to escape RIG-I. Our data reveal a new role for cap N1-2'O-methylation in RIG-I tolerance of self-RNA.
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http://dx.doi.org/10.1016/j.immuni.2015.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128463PMC
July 2015

Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5'-diphosphates.

Nature 2014 10 10;514(7522):372-375. Epub 2014 Aug 10.

Immunobiology Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.

Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.
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http://dx.doi.org/10.1038/nature13590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201573PMC
October 2014

Enzymatic synthesis and purification of a defined RIG-I ligand.

Methods Mol Biol 2014 ;1169:15-25

Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Sigmund-Freud-Strasse 25, 53127, Bonn, Germany.

Receptor-based nucleic acid sensing constitutes one of the most fundamental mechanisms of our innate immune system to sense viral infection. RIG-I is a cytosolic RNA helicase that senses the presence of 5' triphosphate RNA species, a common feature of many negative strand RNA viruses. We here describe a protocol to enzymatically synthesize and to purify a defined RIG-I ligand that can be used to study RIG-I activation in vitro and in vivo.
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http://dx.doi.org/10.1007/978-1-4939-0882-0_2DOI Listing
February 2015

A human in vitro whole blood assay to predict the systemic cytokine response to therapeutic oligonucleotides including siRNA.

PLoS One 2013 5;8(8):e71057. Epub 2013 Aug 5.

Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, University of Bonn, Bonn, Germany.

Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR) or RIG-I like helicases (RLH) are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA) that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4) was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC) based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand) in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071057PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733725PMC
March 2014

Master sensors of pathogenic RNA - RIG-I like receptors.

Authors:
Martin Schlee

Immunobiology 2013 Nov 1;218(11):1322-35. Epub 2013 Jul 1.

Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, 53105 Bonn, Germany. Electronic address:

Initiating the immune response to invading pathogens, the innate immune system is constituted of immune receptors (pattern recognition receptors, PRR) that sense microbe-associated molecular patterns (MAMPs). Detection of pathogens triggers intracellular defense mechanisms, such as the secretion of cytokines or chemokines to alarm neighboring cells and attract or activate immune cells. The innate immune response to viruses is mostly based on PRRs that detect the unusual structure, modification or location of viral nucleic acids. Most of the highly pathogenic and emerging viruses are RNA genome-based viruses, which can give rise to zoonotic and epidemic diseases or cause viral hemorrhagic fever. As viral RNA is located in the same compartment as host RNA, PRRs in the cytosol have to discriminate between viral and endogenous RNA by virtue of their structure or modification. This challenging task is taken on by the homologous cytosolic DExD/H-box family helicases RIG-I and MDA5, which control the innate immune response to most RNA viruses. This review focuses on the molecular basis for RIG-I like receptor (RLR) activation by synthetic and natural ligands and will discuss controversial ligand definitions.
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http://dx.doi.org/10.1016/j.imbio.2013.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114584PMC
November 2013

RIG-I detects triphosphorylated RNA of Listeria monocytogenes during infection in non-immune cells.

PLoS One 2013 30;8(4):e62872. Epub 2013 Apr 30.

Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, University of Bonn, Bonn, Germany.

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062872PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639904PMC
December 2013

Targeting the cytosolic innate immune receptors RIG-I and MDA5 effectively counteracts cancer cell heterogeneity in glioblastoma.

Stem Cells 2013 Jun;31(6):1064-74

Stem Cell Pathologies, University of Bonn Medical Center, Bonn, Germany. [email protected]

Cellular heterogeneity, for example, the intratumoral coexistence of cancer cells with and without stem cell characteristics, represents a potential root of therapeutic resistance and a significant challenge for modern drug development in glioblastoma (GBM). We propose here that activation of the innate immune system by stimulation of innate immune receptors involved in antiviral and antitumor responses can similarly target different malignant populations of glioma cells. We used short-term expanded patient-specific primary human GBM cells to study the stimulation of the cytosolic nucleic acid receptors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). Specifically, we analyzed cells from the tumor core versus "residual GBM cells" derived from the tumor resection margin as well as stem cell-enriched primary cultures versus specimens without stem cell properties. A portfolio of human, nontumor neural cells was used as a control for these studies. The expression of RIG-I and MDA5 could be induced in all of these cells. Receptor stimulation with their respective ligands, p(I:C) and 3pRNA, led to in vitro evidence for an effective activation of the innate immune system. Most intriguingly, all investigated cancer cell populations additionally responded with a pronounced induction of apoptotic signaling cascades revealing a second, direct mechanism of antitumor activity. By contrast, p(I:C) and 3pRNA induced only little toxicity in human nonmalignant neural cells. Granted that the challenge of effective central nervous system (CNS) delivery can be overcome, targeting of RIG-I and MDA5 could thus become a quintessential strategy to encounter heterogeneous cancers in the sophisticated environments of the brain.
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http://dx.doi.org/10.1002/stem.1350DOI Listing
June 2013

RIG-I detects infection with live Listeria by sensing secreted bacterial nucleic acids.

EMBO J 2012 Nov 12;31(21):4153-64. Epub 2012 Oct 12.

Institutes of Molecular Medicine and Experimental Immunology, Friedrich-Wilhelms-Universität Bonn, Bonn, Germany.

Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill-defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG-I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG-I-dependent IL-1β-production and inflammasome activation. The signalling molecule CARD9 contributed to IL-1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG-I provides a mechanistic explanation for efficient induction of immunity by live bacteria.
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http://dx.doi.org/10.1038/emboj.2012.274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492734PMC
November 2012

Exosomes as nucleic acid nanocarriers.

Adv Drug Deliv Rev 2013 Mar 28;65(3):331-5. Epub 2012 Jun 28.

Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany.

Exosomes are nano-sized vesicles produced naturally by many cell types. They are specifically loaded with nucleic acid cargo, dependent on the exosome-producing cell and its homeostatic state. As natural intercellular shuttles of miRNA, exosomes influence an array of developmental, physiological and pathological processes in the recipient cell or tissue to which they can be selectively targeted by their tetraspanin surface-domains. By a review of current research, we illustrate here why exosomes are ideal nanocarriers for use in the targeted in vivo delivery of nucleic acids.
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http://dx.doi.org/10.1016/j.addr.2012.06.011DOI Listing
March 2013

Delivery with polycations extends the immunostimulant Ribomunyl into a potent antiviral Toll-like receptor 7/8 agonist.

Antivir Ther 2011 ;16(5):751-8

Department of Clinical Chemistry and Clinical Pharmacology, University Hospital of Bonn, Bonn, Germany.

Background: Upper respiratory tract infection is a frequent cause of morbidity worldwide. Although the course of infection is usually mild, it is responsible for enormous social and economic costs. Immunostimulation with bacterial extracts consisting of ribosomal RNA and proteoglycans, such as Ribomunyl, was introduced into the clinic in the 1980s as a new treatment concept, but did not achieve widespread application. Ribomunyl has been proposed to activate innate immunity, but the contribution of its RNA content as well as its antiviral potential has not been studied.

Methods: Peripheral blood mononuclear cells from healthy donors and immune cells from adenoids were incubated with Ribomunyl either by itself or formulated in a complex with cationic polypeptides such as poly-l-arginine or protamine, and induction of cytokines was quantified by ELISA.

Results: Ribomunyl in complex with either poly-l-arginine or protamine, but not on its own, was able to strongly induce interferon-α (P<0.01) and interleukin-12 (P<0.01) in peripheral blood mononuclear cells, whereas induced tumour necrosis factor-α and interleukin-6 levels were independent of the formulation. Comparable results were obtained in immune cells from adenoids, suggesting efficacy also in virus-affected tissue. Cell sorting, RNase digests and selective receptor expression show that the RNA in Ribomunyl acts as an agonist of Toll-like receptor (TLR)7 and TLR8.

Conclusions: Ribomunyl is, in principle, able to potently induce antiviral interferon-α and interleukin-12 via TLR7 and TLR8, respectively, but only when formulated in a complex with cationic polypeptides.
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http://dx.doi.org/10.3851/IMP1822DOI Listing
February 2012

Monocyte-mediated inhibition of TLR9-dependent IFN-α induction in plasmacytoid dendritic cells questions bacterial DNA as the active ingredient of bacterial lysates.

J Immunol 2010 Dec 5;185(12):7367-73. Epub 2010 Nov 5.

Institute of Clinical Chemistry and Clinical Pharmacology, Rheinische Friedrich-Wilhelms University Bonn, Bonn, Germany.

Bacterial DNA contains unmethylated CpG dinucleotides and is a potent ligand for TLR9. Bacterial DNA has been claimed the active ingredient in bacterial lysates used for immunotherapy. Whereas the detection of viral DNA by TLR9 expressed in plasmacytoid dendritic cells (PDCs) with subsequent IFN-α production is well defined, the role of bacterial DNA during microbial infection is less clear. In fact, IFN-α is not a hallmark of antibacterial immune responses. Unlike in mice, TLR9 expression in humans is restricted to PDCs and B cells; thus, conclusions from murine models of infection have limitations. In this study, we demonstrate that lysates of heat-killed Escherichia coli containing bacterial DNA induced IFN-α in isolated PDCs but not in the mixed cell populations of human PBMCs. Depletion of monocytes restored IFN-α secretion by PDCs within PBMCs. We found that monocyte-derived IL-10 and PGs contribute to monocyte-mediated inhibition of IFN-α release in PDCs. We conclude that human PDCs can be stimulated by bacterial DNA via TLR9; however, in the physiological context of mixed-cell populations, PDC activation is blocked by factors released from monocytes stimulated in parallel by other components of bacterial lysates such as LPS. This functional repression of PDCs by concomitantly stimulated monocytes avoids production of antiviral IFN-α during bacterial infection and thus explains how the innate immune system is enabled to distinguish bacterial from viral CpG DNA and thus to elicit the appropriate responses despite the presence of CpG DNA in both types of infection.
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http://dx.doi.org/10.4049/jimmunol.1001798DOI Listing
December 2010

Structural and functional insights into 5'-ppp RNA pattern recognition by the innate immune receptor RIG-I.

Nat Struct Mol Biol 2010 Jul 27;17(7):781-7. Epub 2010 Jun 27.

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

RIG-I is a cytosolic helicase that senses 5'-ppp RNA contained in negative-strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies established that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5'-ppp RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD bound to both blunt ends of a self-complementary 5'-ppp dsRNA 12-mer, with interactions involving 5'-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD sequesters the observable 5'-pp of the bound RNA, with a stacked phenylalanine capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5'-ppp dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2'-OCH(3) RNA modifications on the interferon response.
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http://dx.doi.org/10.1038/nsmb.1863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3744876PMC
July 2010

The chase for the RIG-I ligand--recent advances.

Mol Ther 2010 Jul 11;18(7):1254-62. Epub 2010 May 11.

Institute of Clinical Chemistry and Pharmacology, University Hospital Bonn, Bonn, Germany.

Multicellular organisms evolved efficient host-defense mechanisms to sense viruses and to block their replication and spread. Invertebrates and plants mainly rely on RNA interference (RNAi) for antiviral defense. In mammals, the initiation of antiviral defense mechanisms is largely based on the detection of viral nucleic acids by innate receptors: retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) and Toll-like receptors (TLRs). RLHs play a pivotal role in sensing viral RNA and DNA in the cytoplasm of cells. RLHs, like Dicer of the RNAi pathway, belong to the phylogenetically conserved DExD/H-box family of helicases. Unlike TLRs, RLHs are functional in all somatic cells. Activation of RIG-I triggers antiviral responses including type I interferon (IFN), inflammasome activation and proapoptotic signaling. Here, we provide a comprehensive overview of the current literature on the ligand structures detected by RIG-I, and conclude with the molecular definition of the RIG-I ligand: short double-stranded blunt-end 5'-triphosphate RNA. The recent information on the RIG-I ligand now allows the design of short double-stranded RNA (dsRNA) oligonucleotides that are ideally suited alone or in combination with small-interfering RNA (siRNA) for the treatment of viral infection and cancer.
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http://dx.doi.org/10.1038/mt.2010.90DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911265PMC
July 2010

Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.

Nat Immunol 2010 Jan 15;11(1):63-9. Epub 2009 Nov 15.

III. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.
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http://dx.doi.org/10.1038/ni.1824DOI Listing
January 2010

Higher activation of TLR9 in plasmacytoid dendritic cells by microbial DNA compared with self-DNA based on CpG-specific recognition of phosphodiester DNA.

J Leukoc Biol 2009 Sep 20;86(3):663-70. Epub 2009 Jul 20.

Institute of Clinical Chemistry and Pharmacology, University Hospital of Bonn, D-53127 Bonn, Germany.

TLR9 detects DNA in endolysosomal compartments of human B cells and PDC. Recently, the concept of the CpG motif specificity of TLR9-mediated detection, specifically of natural phosphodiester DNA, has been challenged. Unlike in human B cells, CpG specificity of natural phosphodiester DNA recognition in human PDC has not been analyzed in the literature. Here, we found that the induction of IFN-alpha and TNF-alpha in human PDC by phosphodiester ODNs containing one or two CG dinucleotides was reduced to a lower level when the CG dinucleotides were methylated and was abolished if the CGs were switched to GCs. Consistent with a high frequency of unmethylated CG dinucleotides, bacterial DNA induced high levels of IFN-alpha in PDC; IFN-alpha was reduced but not abolished upon methylation of bacterial DNA. Mammalian DNA containing low numbers of CG dinucleotides, which are frequently methylated, induced IFN-alpha in PDC consistently but on a much lower level than bacterial DNA. For activation of PDC, phosphodiester ODNs and genomic DNA strictly required complexation with cationic molecules such as the keratinocyte-derived antimicrobial peptide LL37 or a scrambled derivative. In conclusion, we demonstrate that self-DNA complexed to cationic molecules activate PDC and thus, indeed, may function as DAMPs; nevertheless, the preference of PDC for CpG containing DNA provides the basis for the discrimination of microbial from self-DNA even if DNA is presented in the condensed form of a complex.
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http://dx.doi.org/10.1189/jlb.0509314DOI Listing
September 2009

Recognition of 5' triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus.

Immunity 2009 Jul 2;31(1):25-34. Epub 2009 Jul 2.

Institute of Clinical Chemistry and Pharmacology, University Hospital, University of Bonn, D-53127 Bonn, Germany.

Antiviral immunity is triggered by immunorecognition of viral nucleic acids. The cytosolic helicase RIG-I is a key sensor of viral infections and is activated by RNA containing a triphosphate at the 5' end. The exact structure of RNA activating RIG-I remains controversial. Here, we established a chemical approach for 5' triphosphate oligoribonucleotide synthesis and found that synthetic single-stranded 5' triphosphate oligoribonucleotides were unable to bind and activate RIG-I. Conversely, the addition of the synthetic complementary strand resulted in optimal binding and activation of RIG-I. Short double-strand conformation with base pairing of the nucleoside carrying the 5' triphosphate was required. RIG-I activation was impaired by a 3' overhang at the 5' triphosphate end. These results define the structure of RNA for full RIG-I activation and explain how RIG-I detects negative-strand RNA viruses that lack long double-stranded RNA but do contain blunt short double-stranded 5' triphosphate RNA in the panhandle region of their single-stranded genome.
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http://dx.doi.org/10.1016/j.immuni.2009.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824854PMC
July 2009

Selection of molecular structure and delivery of RNA oligonucleotides to activate TLR7 versus TLR8 and to induce high amounts of IL-12p70 in primary human monocytes.

J Immunol 2009 Jun;182(11):6824-33

Institute of Clinical Chemistry and Pharmacology, University Hospital, University of Bonn, Bonn, Germany.

Detection of non-self RNA by TLRs within endosomes and by retinoic acid-inducible gene I (RIG-I)-like helicases in the cytosol is central to mammalian antiviral immunity. In this study, we used pathway-specific agonists and targeted delivery to address RNA immunorecognition in primary human immune cells. Within PBMC, plasmacytoid dendritic cells (pDC) and monocytes were found to be responsible for IFN-alpha production upon immunorecognition of RNA. The mechanisms of RNA recognition in pDC and monocytes were distinct. In pDC, recognition of ssRNA and dsRNA oligonucleotides was TLR7-dependent, whereas a 5' triphosphate moiety (RIG-I ligand activity) had no major contribution to IFN-alpha production. In monocytes, the response to RNA oligonucleotides was mediated by either TLR8 or RIG-I. TLR8 was responsible for IL-12 induction upon endosomal delivery of ssRNA oligonucleotides and RIG-I was responsible for IFN-alpha production upon delivery of 5' triphosphate RNA into the cytosol. In conclusion, the dissection of these pathways by selecting the appropriate structure and delivery of RNA reveals pDC as major producer of IFN-alpha upon TLR-mediated stimulation and monocytes as major producer of IFN-alpha upon RIG-I-mediated stimulation. Furthermore, our results uncover the potential of monocytes to function as major producers of IL-12p70, a key Th1 cytokine classically ascribed to myeloid dendritic cells that cannot be induced by CpG oligonucleotides in the human system.
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http://dx.doi.org/10.4049/jimmunol.0803001DOI Listing
June 2009

Selective and direct activation of human neutrophils but not eosinophils by Toll-like receptor 8.

J Allergy Clin Immunol 2009 May 10;123(5):1026-33. Epub 2009 Apr 10.

Institute of Clinical Chemistry and Pharmacology, University Hospital, University of Bonn, Bonn, Germany.

Background: Granulocytes represent the largest fraction of immune cells in peripheral blood and are directly exposed to circulating Toll-like receptor (TLR) ligands. Although highly relevant for TLR-based therapies, because of the technical challenge, activation of the granulocyte subsets of neutrophils and eosinophils by TLR ligands is less well studied than activation of other immune cell subsets.

Objective: The aim of this work was to study direct versus indirect neutrophil and eosinophil activation by TLR7 and TLR8 ligands.

Methods: We used a new whole-blood assay, single cell-based cytokine detection, and highly purified primary human neutrophils and eosinophils to separate direct and indirect effects on these blood cell subsets.

Results: We found indirect but not direct activation of neutrophils but not eosinophils in whole blood by using unmodified immunostimulatory RNA (isRNA; TLR7/8 ligand). In contrast, direct activation and stimulation of the respiratory burst and degranulation was seen with nuclease-stable isRNA and with the small-molecule TLR8 agonist 3M002 but not 3M001 (TLR7). Neutrophils expressed TLR8 but none of the other 2 RNA-detecting TLRs (TLR3 and TLR7).

Conclusions: Together, these results demonstrate that neutrophils are directly and fully activated through TLR8 but not TLR7. Furthermore, the results predict that the clinical utility of small-molecule TLR8 ligands or nuclease-stable RNA ligands for TLR8 might be limited because of neutrophil-mediated toxicity and that no such limitation applies for unmodified isRNA, which is known to induce desired T(H)1 activities in other immune cell subsets.
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http://dx.doi.org/10.1016/j.jaci.2009.02.015DOI Listing
May 2009

c-Myc and Rel/NF-kappaB are the two master transcriptional systems activated in the latency III program of Epstein-Barr virus-immortalized B cells.

J Virol 2009 May 4;83(10):5014-27. Epub 2009 Mar 4.

Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6101, Centre Hospitalier Universitaire Dupuytren, Université de Limoges, Laboratoire d'Hématologie, 87025 Limoges, France.

The Epstein-Barr virus (EBV) latency III program imposed by EBNA2 and LMP1 is directly responsible for immortalization of B cells in vitro and is thought to mediate most immunodeficiency-related posttransplant lymphoproliferative diseases in vivo. To answer the question whether and how this proliferation program is related to c-Myc, we have established the transcriptome of both c-Myc and EBV latency III proliferation programs using a Lymphochip specialized microarray. In addition to EBV-positive latency I Burkitt lymphoma lines and lymphoblastoid cell lines (LCLs), we used an LCL expressing an estrogen-regulatable EBNA2 fusion protein (EREB2-5) and derivative B-cell lines expressing a constitutively active or tetracycline-regulatable c-myc gene. A total of 897 genes were found to be fourfold or more up- or downregulated in either one or both proliferation programs compared to the expression profile of resting EREB2-5 cells. A total of 661 (74%) of these were regulated similarly in both programs. Numerous repressed genes were known targets of STAT1, and most induced genes were known to be upregulated by c-Myc and to be involved in cell proliferation. In keeping with the gene expression patterns, inactivation of c-Myc by a chemical inhibitor or by conditional expression of dominant-negative c-Myc and Max mutants led to proliferation arrest of LCLs. Most genes differently regulated in both proliferation programs corresponded to genes induced by NF-kappaB in LCLs, and many of them coded for immunoregulatory and/or antiapoptotic molecules. Thus, c-Myc and NF-kappaB are the two main transcription factors responsible for the phenotype, growth pattern, and biological properties of cells driven into proliferation by EBV.
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http://dx.doi.org/10.1128/JVI.02264-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682111PMC
May 2009

Approaching the RNA ligand for RIG-I?

Immunol Rev 2009 Jan;227(1):66-74

Institute of Clinical Chemistry and Pharmacology, University Hospital Bonn, Bonn, Germany.

Innate and antigen-specific antiviral immunity are triggered by immunorecognition of viral nucleic acids. The helicase retinoic acid-inducible gene I (RIG-I) (also known as DDX58) is the key sensor of negative strand RNA viruses in the cytosol of cells. RNA containing a triphosphate at the 5'-end was shown to activate RIG-I, but the exact structure of RNA supporting 5'-triphosphate recognition, the requirement of a 5'-triphosphate group, as well as the existence of RNA structures detected by RIG-I in the absence of 5'-triphosphate remain controversial. Here, we revisit the literature on RIG-I and RIG-I ligands. The literature proposes at least six different RIG-I ligands: (i) single strand with a 5'-triphosphate, (ii) double-stranded RNA with a 5'-triphosphate, (iii) 5'-triphosphate single-stranded RNA with A- and U-rich 3'-sequences, (iv) double-stranded RNA of intermediate length (>300 and <2000 bp) without 5'-triphosphate, (v) blunt-end short double-stranded RNA (23-30 bp) without 5'-triphosphate, and (vi) short double-stranded RNA (23-30 bp) with 5'-monophosphate. RIG-I thus seems promiscuous for a variety of different RNA molecules, very similar to the Toll-like receptors, of which 10 family members are sufficient for the safe detection of the microbial cosmos. In the light of these outstanding publications, it seems an unlikely possibility that there is a fundamental shortcoming in the design of all studies. Looking closely, the only issue that comes to mind is the in vitro transcription technique used by all investigators without confirming the identity of RNA products. This technique, together with the different biological systems used, the lack of dose responses and of proper comparison of different published ligands and controls leave us with more questions than answers as to what the exact RIG-I ligand is, if in fact it exists.
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http://dx.doi.org/10.1111/j.1600-065X.2008.00724.xDOI Listing
January 2009