Interferon-beta induction through toll-like receptor 3 depends on double-stranded RNA structure

Type I interferons (IFN-alpha/beta) play an essential role in both innate and adaptive antiviral immune responses. IFN- beta is produced by fibroblasts and myeloid dendritic cells (DCs) upon viral infection or in response to doublestranded RNA (dsRNA). Several intracellular molecules having a dsRNA-binding motif such as dsRNA-dependent protein kinase recognize dsRNA in a sequence-independent manner and induce antiviral innate responses. Toll-like receptor (TLR) 3, a member of TLR family proteins, recognizes extracellular dsRNA and activates NF- kappaB and the IFN-beta promoter leading to the induction of IFN-beta production. Here we analyzed the dsRNA structure capable of inducing TLR3-mediated IFN-beta production using various synthetic RNA duplexes. In contrast to the recognition of dsRNA by intracellular molecules, TLR3 preferentially recognizes polyriboinocinic:polyribocytidylic acid (poly(I:C)) rather than synthetic virus-derived dsRNAs. 2'-O-methyl or 2'-fluoro modification of cytidylic acid abolished the IFN-beta-inducing ability of the poly(I:C) duplex, and these modified dsRNAs inhibited poly(I:C)-induced TLR3-mediated IFN-beta production by fibroblasts and DCs. In addition, poly(dI:dC), a non-IFN inducer, also blocked poly(I:C)-induced IFN-beta induction. Since TLR3 is localized in the intracellular compartment of DCs where signaling occurs, modified dsRNAs may compete with poly(I:C) for binding to the cell-surface receptor that transfers dsRNA into TLR3-enriched vesicles. Thus, TLR3 recognizes a unique dsRNA structure that largely differs from those recognized by other dsRNA-binding proteins.     

The Human Antimicrobial Peptide LL-37, but Not the Mouse Ortholog,mCRAMP, Can Stimulate Signaling by Poly(I:C) through a FPRL1-dependent Pathway

LL-37 is an antimicrobial peptide produced by human cells that can down-regulate the lipopolysaccharide-induced innate immune responses and up-regulate double-stranded (ds) RNA-induced innate responses through Toll-like receptor 3 (TLR3). The murine LL-37 ortholog, mCRAMP, also inhibited lipopolysaccharide-induced responses, but unlike LL-37, it inhibited viral-induced responses in mouse cells. A fluorescence polarization assay showed that LL-37 was able to bind dsRNA better than mCRAMP. In the human lung epithelial cell line BEAS-2B, LL-37, but not mCRAMP, colocalized with TLR3, and the colocalization was increased in the presence of dsRNA. The presence of poly(I:C) increased the accumulation of LL-37 in Rab5 endosomes. Signaling by cells induced with both LL-37 and poly(I:C) was sensitive to inhibitors that affect clathrin-independent trafficking, whereas signaling by poly(I:C) alone was not, suggesting that the LL-37-poly(I:C) complex trafficked to signaling endosomes by a different mechanism than poly(I:C) alone. siRNA knockdown of known LL-37 receptors identified that FPRL1 was responsible for TLR3 signaling induced by LL-37-poly(I:C). These results show that LL-37 and mCRAMP have different activities in TLR3 signaling and that LL-37 can redirect trafficking of poly(I:C) to effect signaling by TLR3 in early endosomes in a mechanism that involves FPRL1.     

Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling

A monoclonal antibody (mAb) against human Toll-like receptor (TLR) 3 was established and its effect on TLR3-mediated responses was tested using human fibroblast cell lines expressing TLR3 on the cell surface. Fibroblasts are known to produce IFN-beta upon viral infection or treatment with double-stranded RNA (dsRNA) through distinct signaling pathways. Here, we show the mAb to TLR3 suppressed poly(I):poly(C)-mediated IFN-beta production by human fibroblasts naturally expressing TLR3 on their surface. By reporter gene assay using HEK293 cells transfected with a human TLR3 expression vector, TLR3 recognized dsRNA to activate NF-kappaB and the IFN-beta promoter. TLR3 signaling was not elicited by either single-stranded RNA (ssRNA) or dsDNA. Thus, specific recognition of dsRNA by extracellular TLR3 is essential for induction of type I IFN: the interassociation between dsRNA and TLR3, regardless of direct or indirect binding, should be disrupted by mAb being attached to TLR3. The mAb against TLR3 reported herein may serve as a regulator for virus-mediated immune response via an alternative pathway involving the dsRNA-TLR3 recognition which might occur on host cells.     

The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

Recognition of viral dsRNA by Toll-like receptor 3 (TLR3) leads to induction of interferons (IFNs) and proinflammatory cytokines, and innate antiviral response. Here we identified the RNA-binding protein Mex3B as a positive regulator of TLR3-mediated signaling by expression cloning screens. Cells from Mex3b^−/− mice exhibited reduced production of IFN-β in response to the dsRNA analog poly(I:C) but not infection with RNA viruses. Mex3b^−/− mice injected with poly(I:C) was more resistant to poly(I:C)-induced death. Mex3B was associated with TLR3 in the endosomes. It bound to dsRNA and increased the dsRNA-binding activity of TLR3. Mex3B also promoted the proteolytic processing of TLR3, which is critical for its activation. Mutants of Mex3B lacking its RNA-binding activity inhibited TLR3-mediated IFN-β induction. These findings suggest that Mex3B acts as a coreceptor of TLR3 in innate antiviral response.     

TLR3-specific double-stranded RNA oligonucleotide adjuvants induce dendritic cell cross-presentation, CTL responses, and antiviral protection

Maturation of dendritic cells (DC) to competent APC is essential for the generation of acquired immunity and is a major function of adjuvants. dsRNA, a molecular signature of viral infection, drives DC maturation by activating TLR3, but the size of dsRNA required to activate DC and the expression patterns of TLR3 protein in DC subsets have not been established. In this article, we show that cross-priming CD8α(+) and CD103(+) DC subsets express much greater levels of TLR3 than other DC. In resting DC, TLR3 is located in early endosomes and other intracellular compartments but migrates to LAMP1(+) endosomes on stimulation with a TLR3 ligand. Using homogeneous dsRNA oligonucleotides (ONs) ranging in length from 25 to 540 bp, we observed that a minimum length of ～90 bp was sufficient to induce CD86, IL-12p40, IFN-β, TNF-α, and IL-6 expression, and to mature DC into APC that cross-presented exogenous Ags to CD8(+) T cells. TLR3 was essential for activation of DC by dsRNA ONs, and the potency of activation increased with dsRNA length and varied between DC subsets. In vivo, dsRNA ONs, in a size-dependent manner, served as adjuvants for the generation of Ag-specific CTL and for inducing protection against lethal challenge with influenza virus when given with influenza nucleoprotein as an immunogen. These results provide the basis for the development of TLR3-specific adjuvants capable of inducing immune responses tailored for viral pathogens.     

Insulin-dependent phosphatidylinositol 3-kinase/Akt and ERK signaling pathways inhibit TLR3-mediated human bronchial epithelial cell apoptosis

TLR3, one of the TLRs involved in the recognition of infectious pathogens for innate and adaptive immunity, primarily recognizes viral-associated dsRNA. Recognition of dsRNA byproducts released from apoptotic and necrotic cells is a recently proposed mechanism for the amplification of toxicity, suggesting a pivotal participation of TLR3 in viral infection, as well as in lung diseases where apoptosis plays a critical role, such as asthma and chronic obstructive pulmonary disease. In addition to metabolic control, insulin signaling was postulated to be protective by inhibiting apoptosis. Therefore, we explored the role of insulin signaling in protecting against TLR3-mediated apoptosis of human bronchial epithelial cells. Significant TLR3-mediated apoptosis was induced by polyinosinic-polycytidylic acid, a dsRNA analog, via caspase-8-dependent mechanisms. However, insulin efficiently inhibited TLR3/polyinosinic-polycytidylic acid-induced human bronchial epithelial cell apoptosis via PI3K/Akt and ERK pathways, at least in part, via upregulation of cellular FLIPs and through protein synthesis-independent mechanisms. These results indicate the significance of TLR3-mediated dsRNA-induced apoptosis in the pathogenesis of apoptosis-driven lung disease and provide evidence for a novel protective role of insulin.     

dsRNA induces apoptosis through an atypical death complex associating TLR3 to caspase-8

Toll-like receptor 3 (TLR3) is a pattern-recognition receptor known to initiate an innate immune response when stimulated by double-stranded RNA (dsRNA). Components of TLR3 signaling, including TIR domain-containing adapter inducing IFN-α (TRIF), have been demonstrated to contribute to dsRNA-induced cell death through caspase-8 and receptor interacting protein (RIP)1 in various human cancer cells. We provide here a detailed analysis of the caspase-8 activating machinery triggered in response to Poly(I:C) dsRNA. Engagement of TLR3 by dsRNA in both type I and type II lung cancer cells induces the formation of an atypical caspase-8-containing complex that is devoid of classical death receptors of the TNFR superfamily, but instead is physically associated to TLR3. The recruitment of caspase-8 to TLR3 requires RIP1, and is negatively modulated by cellular inhibitor of apoptosis protein (cIAP)2-TNF receptor-associated factor (TRAF)2-TNFR-associated death domain (TRADD) ubiquitin ligase complex, which regulates RIP1 ubiquitination. Intriguingly, unlike Fas- or TRAILR-dependent death signaling, caspase-8 recruitment and activation within the TLR3 death-signaling complex appears not to be stringently dependent on Fas-associated with death domain (FADD). Our findings uncover a novel aspect of the molecular mechanisms involved during apoptosis induced by the innate immune receptor TLR3 in cancer cells.     

Teleost TLR22 recognizes RNA duplex to induce IFN and protect cells from birnaviruses

TLR22 occurs exclusively in aquatic animals and its role is unknown. Herein we show that the fugu (Takifugu rubripes) (fg)TLR3 and fgTLR22 link the IFN-inducing pathway via the fg Toll-IL-1R homology domain-containing adaptor protein 1(fgTICAM-1, or TRIF) adaptor in fish cells. fgTLR3 resides in endoplasmic reticulum and recognizes relatively short-sized dsRNA, whereas fgTLR22 recognizes long-sized dsRNA on the cell surface. On poly(I:C)-stimulated fish cells, both recruit fgTICAM-1, which in turn moves from the TLR to a cytoplasmic signalosome region. Thus, fgTICAM-1 acts as a shuttling platform for IFN signaling. When fish cells expressing fgTLR22 are exposed to dsRNA or aquatic dsRNA viruses, cells induce IFN responses to acquire resistance to virus infection. Thus, fish have a novel TICAM-1-coupling TLR that is distinct from the mammalian TLR3 in cellular localization, ligand selection, and tissue distribution. TLR22 may be a functional substitute of human cell-surface TLR3 and serve as a surveillant for infection with dsRNA virus to alert the immune system for antiviral protection in fish.     

Double-stranded RNA induces shedding of the 34-kDa soluble TNFR1 from human airway epithelial cells via TLR3-TRIF-RIP1-dependent signaling: roles for dual oxidase 2- and caspase-dependent pathways

TNF, an important mediator of inflammatory and innate immune responses, can be regulated by binding to soluble TNF receptors. The 55-kDa type 1 TNFR (TNFR1), the key receptor for TNF signaling, is released to the extracellular space by two mechanisms, the inducible cleavage and shedding of 34-kDa soluble TNFR1 (sTNFR1) ectodomains and the constitutive release of full-length 55-kDa TNFR1 within exosome-like vesicles. The aim of this study was to identify and characterize TLR signaling pathways that mediate TNFR1 release to the extracellular space. To our knowledge, we demonstrate for the first time that polyinosinic-polycytidylic acid [poly (I:C)], a synthetic dsRNA analogue that signals via TLR3, induces sTNFR1 shedding from human airway epithelial (NCI-H292) cells, whereas ligands for other microbial pattern recognition receptors, including TLR4, TLR7, and nucleotide-binding oligomerization domain containing 2, do not. Furthermore, poly (I:C) selectively induces the cleavage of 34-kDa sTNFR1 ectodomains but does not enhance the release of full-length 55-kDa TNFR1 within exosome-like vesicles. RNA interference experiments demonstrated that poly (I:C)-induced sTNFR1 shedding is mediated via activation of TLR3-TRIF-RIP1 signaling, with subsequent activation of two downstream pathways. One pathway involves the dual oxidase 2-mediated generation of reactive oxygen species, and the other pathway is via the caspase-mediated activation of apoptosis. Thus, the ability of dsRNA to induce the cleavage and shedding of the 34-kDa sTNFR1 from human bronchial epithelial cells represents a novel mechanism by which innate immune responses to viral infections are modulated.     

West Nile virus envelope protein inhibits dsRNA-induced innate immune responses

The immune response against viral infection relies on the early production of cytokines that induce an antiviral state and trigger the activation of immune cells. This response is initiated by the recognition of virus-associated molecular patterns such as dsRNA, a viral replication intermediate recognized by TLR3 and certain RNA helicases. Infection with West Nile virus (WNV) can lead to lethal encephalitis in susceptible individuals and constitutes an emerging health threat. In this study, we report that WNV envelope protein (WNV-E) specifically blocks the production of antiviral and proinflammatory cytokines induced by dsRNA in murine macrophages. This immunosuppressive effect was not dependent on TLR3 or its adaptor molecule Trif. Instead, our experiments show that WNV-E acts at the level of receptor-interacting protein 1. Our results also indicate that WNV-E requires a certain glycosylation pattern, specifically that of dipteran cells, to inhibit dsRNA-induced cytokine production. In conclusion, these data show that the major structural protein of WNV impairs the innate immune response and suggest that WNV exploits differential vector/host E glycosylation profiles to evade antiviral mechanisms.     

LL-37 Peptide Enhancement of Signal Transduction by Toll-like Receptor 3 Is Regulated by pH: IDENTIFICATION OF A PEPTIDE ANTAGONIST OF LL-37

LL-37 is a peptide secreted by human epithelial cells that can lyse bacteria, suppress signaling by Toll-like receptor 4 (TLR4), and enhance signaling to double-stranded RNA (dsRNA) by TLR3. How LL-37 interacts with dsRNA to affect signal transduction by TLR3 is not completely understood. We determined that LL-37 binds dsRNA and traffics to endosomes and releases the dsRNA in a pH-dependent manner. Using dynamic light scattering spectroscopy and cell-based FRET experiments, LL-37 was found to form higher order complexes independent of dsRNA binding. Upon acidification LL-37 will dissociate from a larger complex. In cells, LL-37 has a half-live of ∼1 h. LL-37 half-life was increased by inhibiting endosome acidification or inhibiting cathepsins, which include proteases whose activity are activated by endosome acidification. Residues in LL-37 that contact poly(I:C) and facilitate oligomerization in vitro were mapped. Peptide LL-29, which contains the oligomerization region of LL-37, inhibited LL-37 enhancement of TLR3 signal transduction. LL-29 prevented LL-37·poly(I:C) co-localization to endosomes containing TLR3. These results shed light on the requirements for LL-37 enhancement of TLR3 signaling.