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1.  cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING 
Nature  2013;498(7454):380-384.
Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens1. Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions2, 3. The endoplasmic-reticulum-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin4, 5, 6, 7, 8. Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of cyclic dinucleotides, which function as second messenger molecules in bacteria9, 10, 11, 12, 13. DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING14, 15. We here show in vivo and in vitro that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides. Using a combinatorial approach based on mass spectrometry, enzymatic digestion, NMR analysis and chemical synthesis we demonstrate that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2′-5′ and a 3′-5′ phosphodiester linkage >Gp(2′-5′)Ap(3′-5′)>. We found that the presence of this 2′-5′ linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyses the synthesis of a linear 2′-5′-linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3′-5′ phosphodiester linkage. This 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2′-5′-linked antiviral biomolecules.
PMCID: PMC4143541  PMID: 23722158
2.  Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP 
Nature  2013;503(7477):530-534.
The innate immune defence of multicellular organisms against microbial pathogens requires cellular collaboration. Information exchange allowing immune cells to collaborate is generally attributed to soluble protein factors secreted by pathogen-sensing cells. Cytokines, such as type I interferons (IFNs), serve to alert non-infected cells to the possibility of pathogen challenge1. Moreover, in conjunction with chemokines they can instruct specialized immune cells to contain and eradicate microbial infection. Several receptors and signalling pathways exist that couple pathogen sensing to the induction of cytokines, whereas cytosolic recognition of nucleic acids seems to be exquisitely important for the activation of type I IFNs, master regulators of antiviral immunity2. Cytosolic DNA is sensed by the receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), which catalyses the synthesis of the second messenger cGAMP(2′-5′)3, 4, 5, 6, 7, 8. This molecule in turn activates the endoplasmic reticulum (ER)-resident receptor STING9, 10, 11, thereby inducing an antiviral state and the secretion of type I IFNs. Here we find in murine and human cells that cGAS-synthesized cGAMP(2′-5′) is transferred from producing cells to neighbouring cells through gap junctions, where it promotes STING activation and thus antiviral immunity independently of type I IFN signalling. In line with the limited cargo specificity of connexins, the proteins that assemble gap junction channels, most connexins tested were able to confer this bystander immunity, thus indicating a broad physiological relevance of this local immune collaboration. Collectively, these observations identify cGAS-triggered cGAMP(2′-5′) transfer as a novel host strategy that serves to rapidly convey antiviral immunity in a transcription-independent, horizontal manner.
PMCID: PMC4142317  PMID: 24077100
3.  A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes 
Nature biotechnology  2013;31(1):76-81.
Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly of >600 TALEN genes in a single day. Given its simplicity, throughput and fidelity, LIC assembly will permit the generation of TAL effector gene libraries for large-scale functional genomics studies.
PMCID: PMC4142318  PMID: 23242165
4.  iGLuc: a luciferase-based inflammasome and protease activity reporter 
Nature methods  2013;10(2):147-154.
Measurement of protease activity in living cells or organisms remains a challenging task. We here present a transgene-encoded biosensor that reports the proteolytic activity of caspase-1 in the course of inflammasome activation and that of other proteases in a highly sensitive and specific manner. This protease reporter is based on the biological activity of a pro-interleukin (IL)-1β-Gaussia luciferase (iGLuc) fusion construct, in which pro-IL-1β-dependent formation of protein aggregates renders GLuc enzyme inactive. Cleavage leads to monomerization of this biosensor protein, resulting in a strong gain in luciferase activity. Exchange of the canonical caspase-1 cleavage site in this reporter construct allows the generation of protease biosensors with additional specificities. The high sensitivity, signal-to-background ratio and specificity of the iGLuc system renders it a useful tool to study proteolytic events in mouse and human cells at high throughput and to monitor protease activity in mice in vivo.
PMCID: PMC4127477  PMID: 23291722
5.  DNA sensing unchained 
Cell Research  2013;23(5):585-587.
In two recent reports in Science, James Chen and colleagues provide compelling evidence that detection of cytosolic DNA triggers the production of a novel second messenger, cyclic GMP-AMP (cGAMP), which in turn activates a signaling pathway that induces type I interferons (IFNs) in a STING-dependent manner. They further unravel a key role for a so far uncharacterized murine protein E330016A19 (human homolog: C6ORF150), now termed cGAMP synthetase (cGAS), to act as the DNA sensor that generates cGAMP.
PMCID: PMC3641596  PMID: 23419517
6.  Critical functions of priming and lysosomal damage for NLRP3 activation 
European journal of immunology  2010;40(3):620-623.
Inflammasomes are cytosolic multi-protein complexes that form in response to infectious or injurious challenges. Inflammasomes control the activity of caspase-1, which is essential for the maturation and release of IL-1b family cytokines. The NLRP1, IPAF and AIM2 inflammasomes recognize specific substances, while the NLRP3 inflammasome responds to many structurally and chemically diverse triggers. Here, we discuss the critical roles of priming and lysosomal damage in NLRP3 inflammasome activation.
PMCID: PMC3893565  PMID: 20201015
Inflammasome; NLRP3; IL-1b
7.  RIG-I dependent sensing of poly(dA-dT) via the induction of an RNA polymerase III transcribed RNA intermediate 
Nature immunology  2009;10(10):10.1038/ni.1779.
Viral RNA is sensed by TLR 7 and 8 or by the RNA helicases LGP2, MDA5 and RIG-I to trigger antiviral responses. Much less is known about sensors for DNA. Here we identify a novel DNA sensing pathway involving RNA polymerase III and RIG-I. AT-rich dsDNA serve as a template for RNA polymerase III, which is transcribed into dsRNA harboring a 5′ triphosphate moiety which signals via RIG-I to activate type I IFN gene transcription and NF-κB. This pathway is also important in sensing Epstein-Barr virus encoded small RNAs, which are transcribed by RNA polymerase III and then trigger RIG-I activation. Thus, RNA Pol III and RIG-I play a pivotal role in coordinating anti-viral defenses in the innate immune response.
PMCID: PMC3878616  PMID: 19609254
8.  Structural mechanism of cytosolic DNA sensing by cGAS 
Nature  2013;498(7454):332-337.
Cytosolic DNA arising from intracellular bacteria or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host defense by the production of type I interferon and inflammatory cytokines. Recognition of cytosolic DNA by the recently discovered cyclic-GMP-AMP (cGA) synthase (cGAS) induces the production of cGA to activate the stimulator of interferon genes (STING). Here we report the crystal structure of cGAS alone and in complex with DNA, ATP and GTP along with functional studies. Our results explain cGAS’ broad specificity DNA sensing, show how cGAS catalyzes di-nucleotide formation and indicate activation by a DNA-induced structural switch. cGAS possesses a remarkable structural similarity to the antiviral cytosolic dsRNA sensor 2’-5’oligoadenylate synthase (OAS1), but contains a unique zinc-thumb that recognizes B-form dsDNA. Our results mechanistically unify dsRNA and dsDNA innate immune sensing by OAS1 and cGAS nucleotidyl transferases.
PMCID: PMC3768140  PMID: 23722159
9.  Molecular Mechanism for p202-mediated Specific Inhibition of AIM2 Inflammasome Activation 
Cell reports  2013;4(2):327-339.
Mouse p202 containing two HIN domains antagonizes AIM2 inflammasome signaling and potentially modifies lupus susceptibility. We found only HIN1 of p202 binds dsDNA, while HIN2 forms a homo-tetramer. Crystal structures of HIN1 revealed that dsDNA is bound on the opposite face to the site used in AIM2 and IFI16. The structure of HIN2 revealed a dimer of dimers, with the face analogous to the HIN1 dsDNA binding site being a dimerization interface. Electron microscopy imaging showed that HIN1 is flexibly linked to HIN2 in p202, and tetramerization provided enhanced avidity for dsDNA. Surprisingly, HIN2 of p202 interacts with AIM HIN domain. We propose this results in spatial separation of AIM2 pyrin domains, and indeed p202 prevented dsDNA-dependent clustering of ASC and AIM2 inflammasome activation. We hypothesize that while p202 was evolutionarily selected to limit AIM2-mediated inflammation in some mouse strains, the same mechanism contributes to increased interferon production and lupus susceptibility.
PMCID: PMC3760141  PMID: 23850291
10.  Of inflammasomes and pathogens – sensing of microbes by the inflammasome 
EMBO Molecular Medicine  2013;5(6):814-826.
Inflammasomes are signalling platforms that sense a diverse range of microbial products and also a number of stress and damage associated endogenous signals. Inflammasome complexes can be formed by members of the Nod-like receptor family or the PYHIN family member AIM2. Upon formation, inflammasomes trigger proteolysis of caspase-1, which subsequently leads to a potent inflammatory response through the maturation and secretion of IL-1 family cytokines, which can be accompanied by an inflammatory cell death termed pyroptosis. Here, we review the sensing mechanisms of the currently characterized inflammasome complexes and discuss how they are involved in the innate immune response against microbial pathogens. We especially highlight recent advances in the molecular understanding of how microbial patterns are detected and discriminated from endogenous compounds by inflammasome sensors. Further, we review how inflammasomes contribute to the anti microbial host defense by cytokine-dependent and cell autonomous mechanisms.
PMCID: PMC3779445  PMID: 23666718
caspase-1; inflammasome; NLRC4; NLRP3; pathogens
11.  Structures of The HIN Domain:DNA Complexes Reveal Ligand Binding and Activation Mechanisms of The AIM2 Inflammasome and IFI16 Receptor 
Immunity  2012;36(4):561-571.
Recognition of DNA by the innate immune system is central to anti-viral and anti-bacterial defenses, as well as an important contributor to autoimmune diseases involving self DNA. AIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16) have been identified as DNA receptors that induce inflammasome formation and interferon production, respectively. Here we present the crystal structures of their HIN domains in complex with double-stranded (ds) DNA. Non-sequence specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone. An intramolecular complex of the AIM2 Pyrin and HIN domains in an autoinhibited state is liberated by DNA binding, which may facilitate the assembly of inflammasomes along the DNA staircase. These findings provide novel mechanistic insights into dsDNA as the activation trigger and oligomerization platform for the assembly of large innate signaling complexes such as the inflammasomes.
PMCID: PMC3334467  PMID: 22483801
12.  Reactive oxygen species inhibitors block priming, but not activation of the NLRP3 inflammasome 
A common denominator among the multiple damage-inducing agents that ultimately lead to the activation of NLRP3 has not yet been identified. Recently, the production of reactive oxygen species (ROS) has been suggested to act as a common event upstream of the NLRP3 inflammasome machinery. Since de novo translation of NLRP3 is an essential step in the activation of NLRP3, we investigated the role of substances that either inhibit ROS production or its oxidative activity. While we observe that NLRP3 inflammasome activation is unique amongst other known inflammasomes due to its sensitivity to ROS inhibition, we have found that this phenomenon is attributable to the fact that NLRP3 strictly requires priming by a pro-inflammatory signal, a step that is blocked by ROS inhibitors. While these data do not exclude a general role of ROS production in the process of NLRP3-triggered inflammation, they put ROS upstream of NLRP3 induction, but not activation.
PMCID: PMC3131480  PMID: 21677136
13.  Cytosolic DNA Triggers Inflammasome Activation in Keratinocytes in Psoriatic Lesions 
Science translational medicine  2011;3(82):82ra38.
The proinflammatory cytokine interleukin-1β (IL-1β) plays a central role in the pathogenesis and the course of inflammatory skin diseases, including psoriasis. Posttranscriptional activation of IL-1β is mediated by inflammasomes; however, the mechanisms triggering IL-1β processing remain unknown. Recently, cytosolic DNA has been identified as a danger signal that activates inflammasomes containing the DNA sensor AIM2. In this study, we detected abundant cytosolic DNA and increased AIM2 expression in keratinocytes in psoriatic lesions but not in healthy skin. In cultured keratinocytes, interferon-γ induced AIM2, and cytosolic DNA triggered the release of IL-1β via the AIM2 inflammasome. Moreover, the antimicrobial cathelicidin peptide LL-37, which can interact with DNA in psoriatic skin, neutralized cytosolic DNA in keratinocytes and blocked AIM2 inflammasome activation. Together, these data suggest that cytosolic DNA is an important disease-associated molecular pattern that can trigger AIM2 inflammasome and IL-1β activation in psoriasis. Furthermore, cathelicidin LL-37 interfered with DNA-sensing inflammasomes, which thereby suggests an anti-inflammatory function for this peptide. Thus, our data reveal a link between the AIM2 inflammasome, cathelicidin LL-37, and autoinflammation in psoriasis, providing new potential targets for the treatment of this chronic skin disease.
PMCID: PMC3235683  PMID: 21562230
14.  Listeria monocytogenes is sensed by the NLRP3 and AIM2 Inflammasome 
European journal of immunology  2010;40(6):1545-1551.
The inflammasome pathway functions to regulate caspase-1 activation in response to a broad range of stimuli. Caspase-1 activation is required for the maturation of the pivotal pro-inflammatory cytokines of the pro-IL-1β family. In addition, caspase-1 activation leads to a certain type of cell death known as pyroptosis. Activation of the inflammasome has been shown to play a critical role in the recognition and containment of various microbial pathogens, including the intracellularly replicating Listeria monocytogenes; however, the inflammasome pathways activated during L. monocytogenes infection are only poorly defined. Here, we demonstrate that L. monocytogenes activates both the NLRP3 and the AIM2 inflammasome, with a predominant involvement of the AIM2 inflammasome. In addition, L. monocytogenes-triggered cell death was diminished in the absence of both AIM2 and NLRP3, and is concomitant with increased intracellular replication of L. monocytogenes. Altogether, these data establish a role for DNA sensing through the AIM2 inflammasome in the detection of intracellularly replicating bacteria.
PMCID: PMC3128919  PMID: 20333626
Listeria monocytogenes; Inflammasome; caspase-1; AIM2; NLRP3
15.  The NALP3 inflammasome is involved in the innate immune response to amyloid-β 
Nature immunology  2008;9(8):857-865.
The fibrillar peptide amyloid-β (Aβ) has a chief function in the pathogenesis of Alzheimer’s disease. Interleukin 1β (IL-1β) is a key cytokine in the inflammatory response to Aβ. Insoluble materials such as crystals activate the inflammasome formed by the cytoplasmic receptor NALP3, which results in the release of IL-1β. Here we identify the NALP3 inflammasome as a sensor of Aβ in a process involving the phagocytosis of Aβ and subsequent lysosomal damage and release of cathepsin B. Furthermore, the IL-1β pathway was essential for the microglial synthesis of proinflammatory and neurotoxic factors, and the inflammasome, caspase-1 and IL-1β were critical for the recruitment of microglia to exogenous Aβ in the brain. Our findings suggest that activation of the NALP3 inflammasome is important for inflammation and tissue damage in Alzheimer’s disease.
PMCID: PMC3101478  PMID: 18604209
16.  The AIM2 inflammasome is essential for host-defense against cytosolic bacteria and DNA viruses 
Nature immunology  2010;11(5):395-402.
Inflammasomes regulate the activity of capase-1 and maturation of interleukin-1β and interleukin-18. Recently, AIM2 was shown to bind DNA and engage ASC to form a caspase-1 activating inflammasome. Using Aim2-deficient mice, we reveal a central role for AIM2 in regulating caspase-1-dependent maturation of IL-1β and IL-18, as well as pyroptosis in response to synthetic dsDNA. AIM2 is essential for inflammasome activation in response to Fransicella tularensis, vaccinia virus, mouse cytomegalovirus and plays a partial role in sensing Listeria monocytogenes. Moreover, production of IL-18 and NK cell-dependent IFN-γ production, events critical in early control of virus replication were dependent on AIM2 during mCMV infection in vivo. Collectively, these observations reveal the importance of AIM2 in sensing both bacterial and viral pathogens and triggering innate immunity.
PMCID: PMC2887480  PMID: 20351692
18.  Silica crystals and aluminum salts mediate NALP-3 inflammasome activation via phagosomal destabilization 
Nature immunology  2008;9(8):847-856.
Inhalation of silica crystals causes inflammation in the alveolar space. Prolonged silica exposure can lead to the development of silicosis, an irreversible, fibrotic pulmonary disease. The mechanisms by which silica and other crystals activate immune cells are not well understood. Here, we demonstrate that silica and aluminum salt crystals activate the NALP3 inflammasome. NALP3 activation requires crystal phagocytosis and crystal uptake leads to lysosomal damage and rupture. Sterile lysosomal damage is also sufficient to induce NALP3 activation and inhibition of phagosomal acidification or cathepsin B impairs NALP3 activation. These results indicate that the NALP3 inflammasome can sense lysosomal damage induced by various means as an endogenous danger signal.
PMCID: PMC2834784  PMID: 18604214
19.  NF-kB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression 
The interleukin (IL)-1 family cytokines are regulated on transcriptional and posttranscriptional levels. Pattern recognition and cytokine receptors control pro-IL-1β transcription while inflammasomes regulate the proteolytic processing of pro-IL-1β. The NLRP3 inflammasome, however, assembles in response to extracellular ATP, poreforming toxins or crystals only in the presence of proinflammatory stimuli. How activation of gene transcription by signaling receptors enables the NLRP3 activation remains elusive and controversial. Here, we show that cell priming through multiple signaling receptors induce NLRP3 expression, which we identified to be a critical checkpoint for NLRP3 activation. Signals provided by NF-κB activators are necessary but not sufficient for NLRP3 activation and a second stimulus, such as ATP or crystal-induced damage is required for NLRP3 activation.
PMCID: PMC2824855  PMID: 19570822
20.  Recognition of 5′-triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative strand virus 
Immunity  2009;31(1):25-34.
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 which lack long double-stranded RNA but do contain panhandle blunt short double-stranded 5′triphosphate RNA in their single-stranded genome.
PMCID: PMC2824854  PMID: 19576794
5′-triphosphate RNA; immunorecognition of RNA virus; RIG-I
21.  AIM2 recognizes cytosolic dsDNA and forms a caspase-1 activating inflammasome with ASC 
Nature  2009;458(7237):514-518.
The innate immune system senses nucleic acids via germ-line encoded pattern recognition receptors. RNA is sensed via Toll-like receptor (TLR)−3, −7 and −8 or by the RNA helicases RIG-I and MDA-51. Little is known about sensors for cytoplasmic DNA which trigger antiviral and/or inflammatory responses2–6. The best characterized of these responses involves activation of the TANK-binding kinase (TBK1)-Interferon Regulatory Factor (IRF)-3 signaling axis to trigger transcriptional induction of IFN〈/® genes2,3. A second, less well-defined pathway leads to the activation of an ‘inflammasome’ which via caspase-1, controls the catalytic cleavage of the pro-forms of the cytokines IL-1β and IL-186,7. Here we identify the IFI20X/IFI16 (PYHIN) family member8, absent in melanoma 2 (AIM2), as a receptor for cytosolic DNA which regulates caspase-1. The HIN200 domain of AIM2 binds to DNA, while the PYD domain (but not that of the other PYHIN family members) associates with the adapter molecule ASC to activate both NF-κB and caspase-1. Knockdown of AIM2 abrogates caspase-1 activation in response to cytoplasmic dsDNA and the dsDNA virus, vaccinia. Collectively, these observations identify AIM2 as a novel receptor for cytoplasmic DNA, which forms an inflammasome with the ligand and ASC to activate caspase-1.
PMCID: PMC2726264  PMID: 19158675
22.  Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon–independent apoptosis in human melanoma cells 
The Journal of Clinical Investigation  2009;119(8):2399-2411.
The retinoic acid–inducible gene I (RIG-I) and melanoma differentiation–associated antigen 5 (MDA-5) helicases sense viral RNA in infected cells and initiate antiviral responses such as the production of type I IFNs. Here we have shown that RIG-I and MDA-5 also initiate a proapoptotic signaling pathway that is independent of type I IFNs. In human melanoma cells, this signaling pathway required the mitochondrial adapter Cardif (also known as IPS-1) and induced the proapoptotic BH3-only proteins Puma and Noxa. RIG-I– and MDA-5–initiated apoptosis required Noxa but was independent of the tumor suppressor p53. Triggering this pathway led to efficient activation of mitochondrial apoptosis, requiring caspase-9 and Apaf-1. Surprisingly, this proapoptotic signaling pathway was also active in nonmalignant cells, but these cells were much less sensitive to apoptosis than melanoma cells. Endogenous Bcl-xL rescued nonmalignant, but not melanoma, cells from RIG-I– and MDA-5–mediated apoptosis. In addition, we confirmed the results of the in vitro studies, demonstrating that RIG-I and MDA-5 ligands both reduced human tumor lung metastasis in immunodeficient NOD/SCID mice. These results identify an IFN-independent antiviral signaling pathway initiated by RIG-I and MDA-5 that activates proapoptotic signaling and, unless blocked by Bcl-xL, results in apoptosis. Due to their immunostimulatory and proapoptotic activity, RIG-I and MDA-5 ligands have therapeutic potential due to their ability to overcome the characteristic resistance of melanoma cells to apoptosis.
PMCID: PMC2719920  PMID: 19620789
23.  A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing 
Cell  2007;129(7):1401-1414.
MicroRNAs (miRNAs) are small non-coding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents, enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide novel computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the difference in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.
PMCID: PMC2681231  PMID: 17604727
24.  Superior Immunogenicity of Inactivated Whole Virus H5N1 Influenza Vaccine is Primarily Controlled by Toll-like Receptor Signalling 
PLoS Pathogens  2008;4(8):e1000138.
In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (SU) vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs) of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP) recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR). The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic.
Author Summary
The rise and spread of the highly pathogenic avian H5N1 influenza virus has seriously increased the risk of a new influenza pandemic. However, the number of vaccine doses that can be produced with today's production capacity will fall short of the demand in times of a pandemic. Use of inactivated whole virus (WIV) vaccines, which are more immunogenic than split virus or subunit vaccines in an unprimed population, could contribute to a dose-sparing strategy. Yet, the mechanisms underlying the superior immunogenicity of WIV vaccine formulations are unknown. Here, we demonstrate that the viral RNA present in inactivated virus particles is crucial for the improved immunogenic properties of WIV in mice. By triggering Toll-like receptor 7 (TLR7), the viral RNA activates innate immune mechanisms that augment and determine subsequent adaptive responses. Efficient TLR7 signalling is lost in split virus and subunit vaccines with the processing steps that lead to disruption of the integrity of the virus particle and exclusion of the RNA. Our results prove for the first time to our knowledge that the immune-potentiating mechanism of a classic vaccine is based on activation of the innate immune system by one of its structural components. These findings may reflect a general principle for viral vaccines and provide a rational basis for further improvement of influenza vaccines, which are urgently needed in the face of the current H5N1 pandemic threat.
PMCID: PMC2516931  PMID: 18769719
25.  Inhibition of Toll-Like Receptor 7- and 9-Mediated Alpha/Beta Interferon Production in Human Plasmacytoid Dendritic Cells by Respiratory Syncytial Virus and Measles Virus 
Journal of Virology  2005;79(9):5507-5515.
Human plasmacytoid dendritic cells (PDC) are key sentinels alerting both innate and adaptive immune responses through production of huge amounts of alpha/beta interferon (IFN). IFN induction in PDC is triggered by outside-in signal transduction pathways through Toll-like receptor 7 (TLR7) and TLR9 as well as by recognition of cytosolic virus-specific patterns. TLR7 and TLR9 ligands include single-stranded RNA and CpG-rich DNA, respectively, as well as synthetic derivatives thereof which are being evaluated as therapeutic immune modulators promoting Th1 immune responses. Here, we identify the first viruses able to block IFN production by PDC. Both TLR-dependent and -independent IFN responses are abolished in human PDC infected with clinical isolates of respiratory syncytial virus (RSV), RSV strain A2, and measles virus Schwarz, in contrast to RSV strain Long, which we previously identified as a potent IFN inducer in human PDC (Hornung et al., J. Immunol. 173:5935-5943, 2004). Notably, IFN synthesis of PDC activated by the TLR7 and TLR9 agonists resiquimod (R848) and CpG oligodeoxynucleotide 2216 is switched off by subsequent infection by RSV A2 and measles virus. The capacity of RSV and measles virus of human PDC to shut down IFN production should contribute to the characteristic features of these viruses, such as Th2-biased immune pathology, immune suppression, and superinfection.
PMCID: PMC1082779  PMID: 15827165

Results 1-25 (25)