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Errata: Corrections Volume 56, Issue 1, 397, Article first published online: 3 July 2012
Potential conflict of interest: Nothing to report.
This work was supported by National Institutes of Health grants AI069285 (to K.L.) and CA133322 (to L.M.P.).
Chemokines and inflammatory cytokines are key regulators of immunity and inflammation during viral infections. Hepatitis C virus (HCV) is a hepatotropic RNA virus frequently associated with chronic liver inflammation and hepatocellular carcinoma. Intrahepatic levels of chemokines and cytokines are elevated in chronic HCV infections, but the underlying mechanisms remain unclear. We found that Toll-like receptor-3 (TLR3) senses HCV infection in cultured hepatoma cells, leading to nuclear factor kappa B (NF-κB) activation and the production of numerous chemokines and inflammatory cytokines, such as regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, MIP-1β, IP-10, and interleukin-6. The chemokine/cytokine induction occurred late in HCV infection and was abrogated when HCV was ultraviolet-inactivated before infection, indicating a dependence on the cellular recognition of HCV replication products. Gel-shift and chromatin immunoprecipitation assays revealed that NF-κB plays a pivotal role in HCV-induced chemokine/cytokine transcription. Mutations specifically disrupting the double-stranded RNA (dsRNA)-binding activity of TLR3 ablated the chemokine/cytokine response to HCV infection, indicating that HCV dsRNA was the pathogen-associated molecular pattern triggering TLR3 signaling. In vitro synthesized HCV dsRNAs, with a minimal length of ∼80-100 base pairs, activated TLR3-dependent chemokine expression, regardless of the genome position from which they derived. In contrast, HCV single-stranded RNAs, including those derived from the structured 3'nontranslated region highly potent for RIG-I activation, failed to do so. Moreover, robust production of chemokines and inflammatory cytokines was also observed in primary human hepatocytes after stimulation with extracellular poly-I:C, a TLR3 ligand. Conclusion: Our data suggest that TLR3-mediated chemokine and inflammatory cytokine responses may play an important role in host immune responses to HCV and the pathogenesis of HCV-associated liver diseases. (HEPATOLOGY 2011)
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Infections with the hepatitis C virus (HCV) affect approximately 130 million people worldwide and pose a major threat to human health. HCV is a positive-sense, single-stranded RNA (ssRNA) virus that has a restricted tropism for hepatocytes. Remarkably, HCV persists in ∼70% of infected individuals, causing chronic intrahepatic inflammation and putting patients at risk of developing cirrhosis and hepatocellular carcinoma.1 Clearance of HCV infection depends on the development of vigorous, broad cluster of differentiation (CD)4 and CD8 T-cell responses, which, however, often fail and are replaced with an intermediate cytotoxic T-cell response unable to eliminate the infection, but strong enough to cause hepatocyte destruction.2 Central to T-cell homing to the liver is the induction of a family of small chemotactic cytokines, called chemokines, that regulate the migration of leukocytes and their recruitment to inflammation sites. As such, chemokines play crucial roles in inflammatory processes and the transition from innate to adaptive immunity.3, 4 Intrahepatic chemokines, such as the CCR5 ligands, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1β and MIP-1α, and the CXCR3 ligand, IP-10, are elevated in HCV patients, and the levels of some of them are reported to correlate with the severity of liver inflammation.3-5 However, the cellular source and underlying mechanism of chemokine induction during HCV infection remain elusive.2
Toll-like receptor-3 (TLR3) and the retinoic inducible gene I (RIG-I)-like receptors (RLRs; RIG-I and melanoma differentiation-associated gene 5; MDA5) constitute two parallel classes of cellular sensors that recognize viral pathogen-associated molecular patterns (PAMPs) and initiate innate immune responses. Despite operating via distinct adaptors and mechanisms, both pathways culminate in the activation of interferon regulatory factor-3 (IRF3)-dependent interferon (IFN) antiviral response and the production of nuclear factor kappa B (NF-κB)-dependent proinflammatory mediators. TLR3 and RLRs sense viral double-stranded RNA (dsRNA), a major viral PAMP, in endosomal and cytoplasmic compartments, respectively. RIG-I also recognizes viral RNAs bearing 5'-triphosphates.6 The importance of RLR and TLR3 pathways in innate immunity to HCV is suggested by the fact that HCV encodes a serine protease, nonstructural protein (NS)3/4A, which inactivates both pathways by cleaving two adaptor proteins, mitochondrial antiviral signaling protein (MAVS) and Toll-interleukin (IL)-1 receptor homology domain containing adaptor-inducing interferon β (TRIF).7-10 Though much has been learned concerning how TLR3 and RIG-I pathways act to control HCV replication through activating IRF3 and antiviral interferon-stimulated gene (ISG) expression,8, 11-13 little is known about the mechanisms governing the chemokine and proinflammatory cytokine response to HCV infection. A better understanding of the latter is crucial to broadening our knowledge on immune response to, and pathogenesis of, HCV and to design new effective immunotherapies for HCV. Here, we demonstrate that TLR3 senses HCV infection in cultured hepatocytes, leading to NF-κB activation and production of proinflammatory chemokines/cytokines previously reported in hepatitis C patients. Our study also defines the molecular features of HCV dsRNA that serves as the PAMP for TLR3.
See Supporting Materials for a detailed description.
Plasmids, HCV ssRNAs, and dsRNAs.
Conventional polymerase chain reaction (PCR) and recombinant DNA techniques were used to generate the HCV complementary DNA (cDNA) plasmids (all from HCV-N strain) for in vitro transcription of HCV ssRNAs and dsRNAs. pBSII-core and pBSII-E1-p7 contained cDNA inserts encoding the full-length HCV core and E1/E2/p7, respectively, in pBluscript-II backbone (Stratagene, La Jolla, CA). pBSII-HCV-NS-3'NTR (nontranslated region) contained a 6.6-kb (kilobase pair) insert spanning partial NS2 through NS5B and the entire 3'NTR sequences. Plasmid cytomegalovirus (pCMV)-NS5A contained the full-length NS5A-coding sequence in pCMV-Tag4a (Stratagene). HCV ssRNAs were synthesized from linearized vectors using T7 (for +strand RNAs of core, E1-p7, and NS-3'NTR origins and –strand of NS5A RNA) and T3 (for –strand RNAs of core, E1-p7, and NS-3'NTR origins and +strand of NS5A RNA) MegaScript kits (Ambion, Inc., Austin, TX). The cDNA templates for synthesizing HCV dsRNAs with varying lengths (ranging from 50 to 250 bp [base pair]) were amplified by PCR using pBSII-E1-P7 as the template and the indicated oligonucleotide primers (sequences available online in Supporting Table 1) with introduced T7 promoter sequence at 5' termini. In vitro transcriptions were performed using the MEGAshortScript T7 kit (Ambion). To form dsRNA duplexes, equal molar amounts of +ssRNAs and –ssRNAs were combined in a microcentrifuge tube and incubated at 100°C for 5 minutes, followed by a slow cool-down to room temperature.
Bio-Plex Multiplex Cytokine Assay.
Culture supernatants of cells after the indicated treatments were assessed for cytokines and chemokines by the Bio-Plex Pro human cytokine 27-plex immunoassay (Bio-Rad, Hercules, CA), as per the manufacturer's suggestions.
Total RNA was isolated from cells after the indicated treatments, using Trizol (Invitrogen, Carlsbad, CA), and subsequently was programmed for the synthesis of cDNA. The abundance of RANTES, MIP-1β and 28S (internal control) transcripts, and HCV RNA were analyzed by quantitative PCR (qPCR) using gene-specific primers (sequences available online in Supporting Table 2) and GoTaq qPCR Master Mix (Promega, Madison, WI) on an iCycler IQ5 real-time PCR system (Bio-Rad).
Reporter Gene Assay.
The promoter activities of RANTES and PRDII in transfected cells mock infected or infected with HCV for the indicated periods were determined using the luciferase reporter assay, as previously described.12, 14
Detailed methods have been presented elsewhere.7, 12, 15 The following polyclonal (pAb) and monoclonal (mAb) antibodies were used: anti-IκBα (C-21) and anti-p65 (C-20) pAbs and anti-Lamin A/C (636) mAb (Santa Cruz Biotechonology, Santa Cruz, CA); anti-HCV NS5A (9E10) mAb (a gift from Charles Rice, Center for the Study of Hepatitis C, The Rockefeller University); anti-actin mAb (Sigma-Aldrich, St. Louis, MO); anti-ISG15 (a gift from Dr. Arthur Haas, Louisiana State University), and anti-ISG56 pAbs12; and peroxidase-conjugated secondary antirabbit and antimouse pAbs (Southern Biotech, Birmingham, AL). The protein bands were visualized by enhanced chemiluminescence (Millipore, Billerica, MA), followed by exposure to X-ray films.
Chromatin Immunoprecipitation Assay.
7.5-TLR3 cells (∼5 × 106) were mock infected or infected with HCV (multiplicity of infection [MOI] = 0.5) for 48 hours, then were processed for chromatin immunoprecipitation (ChIP) assay, as described previously.14 The antibodies used for ChIP were from Santa Cruz (anti-p65) and Active Motif (control immunoglobulin G). The ChIP-enriched samples were subjected to qPCR quantification of various chemokine and Trefoil family factor 1 (TFF1) (negative control) promoter sequences using specific primers presented online in Supporting Table 3.
HCV Induces Production of Proinflammatory Cytokines and Chemokines in Huh7.5 cells Stably Reconstituted With Functional TLR3 Signaling.
To investigate whether TLR3 plays a role in hepatoceullar proinflammatory response to HCV infection, we determined the production of cytokines/chemokines in HCV-infected 7.5-TLR3 cells, which were derived from Huh7.5 cells by stably reconstituting the expression of human TLR3. As controls, we studied Huh7.5 cells expressing the control vector (Vect) and mutant TLR3s defective for signaling as a result of incapability of dsRNA binding (H539E and N541A). All these cells are RIG-I defective, and the effects observed were the result of activation of the TLR3 pathway in the absence of RIG-I. We have previously utilized these cell lines to demonstrate that TLR3 senses HCV infection and triggers a weak ISG response, thereby moderately reducing HCV propagation when cells were infected at low MOIs.12 We infected cells with JFH1 virus at an MOI of 0.2 for 72 hours, then harvested the culture supernatants to measure cytokine/chemokine production by a Bio-Plex Multiplex Cytokine Assay. At this MOI, HCV did not replicate differentially among these Huh7.5 derivatives,12 and all the cells were infected at 72 hours, as determined by immunostaining of NS5A (data not shown). Compared to mock-infected cells, six chemokines/cytokines were strongly induced by HCV infection in 7.5-TLR3 cells, including RANTES (37-fold), MIP-1β (25-fold), MIP-1α (17-fold), IL-6 (13-fold), IP-10 (10-fold), and tumor necrosis factor alpha (TNF-α) (10-fold) (Fig. 1). In addition, HCV weakly induced the production of IL-17 (5-fold), granulocyte colony-stimulating factor (G-CSF) (4-fold), IL-1β (3-fold), granulocyte macrophage colony-stimulating factor (2-fold), and eotaxin (2-fold) (data not shown). Importantly, none of these cytokines/chemokines were induced by HCV in the 7.5-Vect cells or 7.5-H593E and 7.5-N541A cells expressing mutant TLR3 (Fig. 1 and data not shown). These data reveal that TLR3 signaling, but not TLR3 expression per se, confers Huh7.5 cells the ability to produce proinflammatory mediators in response to HCV infection.
TLR3-Mediated Up-regulation of Chemokine Transcription After HCV infection Depends on Viral Replication.
To characterize the mechanism of TLR3-mediated chemokine/cytokine induction in HCV-infected cells, we studied the temporal kinetics of messenger RNA (mRNA) expression by qPCR for RANTES, one of the most up-regulated chemokines. In 7.5-TLR3 cells, RANTES mRNA levels did not increase at 8 and 24 hours postinfection, but were elevated by 477- and 1,326-fold at 48 and 72 hours, respectively. In contrast, RANTES mRNA abundance was relatively unaffected in HCV-infected Huh7.5 cells. As a positive control, poly-I:C strongly stimulated RANTES mRNA expression in 7.5-TLR3 cells (by 365- and 3,034-fold at 4 and 24 hours post-treatment, respectively), but had little effect in Huh7.5 cells (Fig. 2A, left panel). We obtained similar results when examining MIP-1β mRNA expression (right panel). The delayed kinetics of chemokine induction by HCV in infected 7.5-TLR3 cells implies that viral replication is needed to generate the HCV PAMP to activate TLR3 signaling, whereas HCV entry and uncoating are insufficient to do so. Consistent with this point, ultraviolet (UV)-inactivated HCV virions completely lost the chemokine-inducing capacity (Fig. 2B). We next determined whether RANTES induction by HCV was regulated at the transcriptional level. To this end, 7.5-TLR3 and Huh7.5 cells were transfected with a luciferase reporter construct under the control of the RANTES promoter, followed by infection with HCV (MOI = 1). Consistent with the mRNA quantitation data (Fig. 2A), activation of the RANTES promoter was observed only in 7.5-TLR3 cells at 48 hours post-HCV infection, but not at 24 hours (Fig. 2C). Collectively, these data suggest that HCV replication product(s) induces chemokine expression by triggering TLR3-dependent activation of chemokine transcription.
NF-κB Is Activated in HCV-Infected 7.5-TLR3 Cells in a Manner That Parallels the Kinetics of Chemokine Induction.
Because NF-κB plays a pivotal role in regulating the expression of proinflammatory cytokines and chemokines, we assessed NF-κB activity in HCV-infected cells. Reporter gene assays demonstrated that the NF-κB-dependent positive regulatory domain (PRD)II promoter was activated by 2- and 9-fold, respectively, in 7.5-TLR3 cells at 48 and 72 hours post-HCV infection, but not at earlier times (Fig. 3A). In contrast, the PRDII promoter activity did not change significantly in HCV-infected Huh7.5 cells. The kinetics of TLR3-dependent activation of the PRDII promoter by HCV infection thus closely mirrored that of the chemokine up-regulation (Fig. 2). The signal-induced degradation of inhibitor of kappa B (IκB) resulted in the liberation of the p65/p50 heterodimer complexes of NF-κB, followed by their nuclear translocation and binding to target promoters. In HCV-infected 7.5-TLR3 cells, degradation of IκBα was evident at 48 hours and further enhanced at 72 hours, concomitant with the increase in HCV replication, as visualized by the accumulation of viral NS5A protein (Fig. 3B). Consistent with this, substantial nuclear accumulation of p65 NF-κB subunit was observed at 48 hours postinfection in 7.5-TLR3 cells (Fig. 3C). Furthermore, at 48 hours, the nuclear p65/p50 complex was induced by HCV infection, as revealed by electrophoretic mobility shift assay (EMSA), with an NF-κB-specific DNA probe (Fig. 3D). This effect resulted specifically from TLR3 signaling, because substantially less p65/p50 complex was formed in HCV-infected Huh7.5 cells and 7.5-N541A cells expressing a TLR3 mutant defective for dsRNA binding, than was in 7.5-TLR3 cells (Supporting Fig. 1). Taken together, these data demonstrate that HCV replication induces NF-κB activity in a TLR3-dependent fashion with kinetics similar to that of chemokine/cytokine induction, indicating a regulatory role for NF-κB in the latter process.
We next performed ChIP experiments to determine whether NF-κB would bind to chemokine promoters in HCV-infected 7.5-TLR3 cells. We found that HCV infection substantially augmented p65 NF-κB subunit binding to three chemokine promoters—RANTES, MIP-1β, and IP-10—but not to the control TFF1 promoter devoid of the NF-κB recognition site (Fig. 4A), indicating that the p65/p50 NF-κB complexes formed in the nucleus of HCV-infected 7.5-TLR3 cells (Fig. 3D) directly controlled the transcription from these chemokine promoters. Confirming this, treatment with caffeic acid phenethyl ester (CAPE), a potent NF-κB inhibitor,16 abrogated HCV-induced up-regulation of transcripts for RANTES (Fig. 4B) and MIP-1β (data not shown). Collectively, these data confirm that NF-κB governs TLR3-mediated chemokine/cytokine induction by HCV infection.
HCV dsRNA Duplex, but not Structured Viral RNA, Is the Ligand for Activating TLR3.
HCV RNA is highly structured and contains ds regions in various portions of the HCV genome, especially in the 5'- and 3'-NTRs and the core- and NS5B-coding regions.17 Because TLR3 binds dsRNA, the activation of TLR3 signaling during HCV infection may involve TLR3 recognition of structured HCV RNA (of either positive or negative strand) or HCV dsRNA intermediates generated during viral RNA replication, or both. To distinguish between these possibilities, we synthesized in vitro a 6.6-kb subgenomic HCV RNA of both positive (+ss) and negative sense (−ss), encompassing a partial NS2 sequence, the entire NS3 to NS5B coding region, and the intact 3'NTR (here referred to as NS-3'NTR) (Fig. 5A, lanes 11 and 12). To resemble HCV dsRNA replicative intermediates, the +ss and −ss NS-3'NTR RNAs were annealed to form dsRNA duplexes (lane 13). When added to culture medium of 7.5-TLR3 cells, NS-3'NTR dsRNA duplexes, but not +ss or –ss NS-3'NTR RNA, up-regulated RANTES transcripts (by 17-fold) (Fig. 5B). As expected, Huh7.5 cells had no response to either ds or ss NS-3'NTR RNA. When the various NS-3'NTR RNA ligands were complexed with Lipofectamine (Invitrogen) to enhance their intracellular delivery, again, only NS-3'NTR dsRNA stimulated RANTES expression. RANTES was up-regulated by ∼1,600-fold in transfected 7.5-TLR3 cells, but was only induced by 70-fold in Huh7.5 cells. The latter phenotype most likely resulted from MDA5 signaling, which is diminished, yet weakly detectable, in Huh7.5 cells (our unpublished observations). Collectively, these data suggest that HCV dsRNAs generated during viral replication are the HCV PAMP that activates TLR3, whereas structured HCV RNAs of either strand are not. We further confirmed these findings in a more physiologic setting using PH5CH8 cells, which are T-antigen-immortalized human hepatocytes expressing TLR3 endogenously and containing a robust TLR3-signaling pathway.15 NS-3'NTR dsRNA, but not +ss or –ss NS-3'NTR RNA, potently up-regulated RANTES transcripts (by 1,975-fold) when added to culture medium (to specifically engage TLR3 in these cells) (Fig. 5B, right panel). When transfected into PH5CH8 cells, both +ss and –ss NS-3'NTR RNA potently activated RANTES transcription, as did NS-3'NTR dsRNA. This was not unexpected, because PH5CH8 cells contain an intact RIG-I pathway15 (as opposed to Huh7.5 cells) that recognizes 3'NTR HCV RNA, which is a potent ligand for RIG-I.11
dsRNAs are readily detectable in cells replicating HCV genomes.18 We confirmed this observation in HCV-infected cells using a mAb specifically reacting to dsRNA. More importantly, we observed that HCV dsRNAs substantially colocalized with TLR3 as discrete cytoplasmic foci (Supporting Fig. 2). This provides further support for the notion that HCV dsRNA replicative intermediates are sensed by TLR3 intracellularly.
Activation of TLR3-Dependent Chemokine Induction by HCV dsRNAs Is Independent of the Genome-Position From Which HCV dsRNAs Derive.
RIG-I preferentially senses a segment of HCV RNA derived from 3'NTR rich in poly-U/UC motifs.11 To determine whether the activation of TLR3 is influenced by genome position and/or nucleotide composition of HCV dsRNA, we compared the chemokine-inducing abilities of various HCV dsRNAs derived from different regions of the HCV genome with varying lengths, including core (0.6 kb), E1 to p7 (1.9 kb), NS5A (1.4 kb), and the NS-3'NTR (6.6 kb) (Fig. 5A). Regardless of their length and nucleotide composition, these different HCV dsRNAs stimulated RANTES mRNA expression with comparable efficiency in both 7.5-TLR3 and PH5CH8 cells (Fig. 5C). Therefore, the TLR3 sensing of HCV dsRNA solely relies on the dsRNA structure, but not on the genome position or nucleotide composition of HCV dsRNA. Additionally, the data reveal that the ability to stimulate TLR3-dependent chemokine production reaches a plateau when HCV dsRNA is longer than 0.6 kb.
The Activation of TLR3 Signaling Requires HCV dsRNAs With a Minimal Length of Approximately 80-100 bp.
A minimal length of 40-48 bp is required for dsRNA activation of NF-κB in HEK293 cells stably expressing TLR3.19 To determine the length requirement for HCV dsRNA to activate TLR3 in hepatocytes, we synthesized in vitro a number of HCV dsRNAs derived from the E2/p7-coding region of increasing length (i.e., 50, 100, and 250 bp) (Fig. 6A) and compared them to the 1.9-kb E1-p7 dsRNA for the capacity to induce RANTES in 7.5-TLR3 cells (Fig. 6B). We found that HCV dsRNAs, with a length of ≥100 bp, all reproducibly up-regulated RANTES transcripts when added to culture medium or introduced into cells by transfection. In contrast, there was no reproducible effect on RANTES induction by the two 50-bp HCV dsRNAs, irrespective of the delivery route. Additional refined length-mapping experiments revealed that whereas a 79-bp HCV dsRNA weakly activated RANTES expression, robust activation of TLR3 signaling was achieved when HCV dsRNAs were ≥89 bp (Fig. 6C). These data suggest that the efficient activation of TLR3 in hepatocytes requires HCV dsRNA with a minimal length of approximately 80-100 bp.
Robust Production of Cytokines and Chemokines in Primary Human Hepatocytes After Engagement of TLR3.
We previously demonstrated that human hepatocytes express TLR3 in situ, and that isolated primary human hepatocytes (PHHs) mount a strong ISG response to extracellular poly-I:C stimulation in vitro.12 To determine whether TLR3 signaling in PHHs would lead to the production of proinflammatory chemokines/cytokines, as we observed in HCV-infected 7.5-TLR3 cells, we stimulated PHHs with poly-I:C for 18 hours and measured various cytokine/chemokine levels in culture supernatants. It was found that all the cytokines/chemokines induced by HCV in 7.5-TLR3 cells (Fig. 1) were secreted in large quantities from poly-I:C-treated PHHs (Fig. 7). Specifically, the production of RANTES, MIP-1α, MIP-1β, IP-10, and IL-6 was up-regulated, by at least 100-fold, by poly-I:C, a phenomenon also observed in Sendai virus (SeV)-infected PHHs. Interestingly, TNF-α was more efficiently up-regulated by poly-I:C (11-fold) than by SeV (4-fold), as was G-CSF (229-fold by poly-I:C versus 3-fold by SeV; data not shown), indicating that these two cytokines are preferentially induced via the TLR3 pathway over RIG-I in PHHs. When PHHs were treated with the Toll-like receptor-7 (TLR7)/8 ligand, R-848, there was weak up-regulation (4- to 10-fold) of MIP-1α, MIP-1β, IP-10, and IL-6, but no induction of RANTES, TNF-α (Fig. 7), and G-CSF (data not shown), suggesting that although the engagement of TLR7/8 could moderately induce certain cytokines/chemokines, this pathway plays a minor role in sensing viral infections to produce inflammatory mediators in hepatocytes, as compared with the TLR3 and RIG-I pathways. Taken together, the experiments in PHHs demonstrate that TLR3 is a prominent innate immune pathway in human hepatocytes responsible for the induction of proinflammatory response to viral infections.
Chemokines and cytokines are critical regulators of liver inflammation, and innate and adaptive immunity to HCV, the complex orchestration of which is suggested to determine the outcome of HCV infection.3, 4 The recruitment of T cells to the liver is central in host immune response to HCV infection2 and is believed to depend mainly on two chemokine receptors expressed on activated T cells: CXCR3 and CCR5.3, 4 Intrahepatic expression of the ligands for CXCR3 (IP-10, I-TAC, and Mig) and CCR5 (RANTES, MIP-1β, and MIP-1α) is elevated in HCV patients, and levels of IP-10 and RANTES have been linked to degree of liver inflammation.3-5 However, the cellular source and mechanism of induction for these chemokines were unclear. We demonstrate, in this study, that upon infection by HCV, cultured hepatoma cells secrete proinflammatory mediators, including RANTES, MIP-1β, MIP-1α, and IP-10, via the TLR3-mediated recognition of HCV dsRNA and activation of NF-κB. Importantly, these observations were not limited to hepatoma Huh7.5 cells reconstituted for TLR3 expression, and we have shown the same repertoire of chemokines and cytokines to be highly up-regulated after stimulation by poly-I:C in PHHs (Fig. 7), which contain a robust TLR3-signaling pathway.12 Therefore, not only does the TLR3 pathway mediate the establishment of an antiviral state against HCV infection,12 but it also plays an important role in initiating proinflammatory responses to HCV in hepatocytes and in bridging innate and adaptive immunity.
The induction of chemokines/cytokines via the TLR3 pathway showed delayed kinetics after HCV infection and did not commence until robust viral replication took place (Fig. 2), implying that HCV replication is needed to produce the PAMP for the engagement of TLR3. Consistent with this, UV-inactivated HCV virions were unable to up-regulate chemokines (Fig. 2B). The latter result also indicates that HCV-entry and virion-uncoating processes do not trigger TLR3 activation and neither do the HCV genomic RNAs released upon virion disassembly early after infection. Our finding that HCV dsRNA duplexes, but not structured HCV ssRNAs highly potent for RIG-I activation, are capable of stimulating chemokine expression in 7.5-TLR3 cells (Fig. 5 and see discussion below) explains why TLR3 activation depends on HCV replication, because the latter process yields viral dsRNAs (Supporting Fig. 2),18 the HCV ligands for TLR3. Our results suggest a model in which TLR3 mediates the late-phase hepatocellular response to HCV infection by sensing viral dsRNA replicative intermediates, secondary to RIG-I-mediated early response built upon sensing genomic HCV RNA.8, 11, 20 Additionally, TLR3-mediated IFN12 and cytokine responses may provide a positive feedback to that via RIG-I, whose expression is inducible by IFNs and certain cytokines, such as TNF-α.21
The mechanism of TLR3-mediated chemokine/cytokine induction in HCV-infected hepatoma cells revealed in the current study mainly involves the activation of NF-κB-dependent gene transcription, at least for several of the most up-regulated chemokines, such as RANTES and MIP-1β (Figs. 2-4). Induction of IFNs is not involved in this process, because neither mRNAs for IFN-β and IFN-λs nor secreted antiviral activity were detected at 48 hours post-HCV infection (data not shown), when active NF-κB complex and chemokine transcription were first observed (Figs. 2 and 3). Apart from being transcriptionally controlled by NF-κB, a subset of cytokines/chemokines contains AU-rich elements (AREs) in the 3'NTR of their mRNAs that regulate their expression post-transcritionally by controlling mRNA stability and/or translation. Among these cytokines/chemokines are IL-6, TNF-α, and MIP-1α,22 and so on, which were found to be induced in HCV-infected 7.5-TLR3 cells (Fig. 1). Although the TLR3-TRIF pathway is less capable of promoting the stabilization of ARE-containing mRNAs than are the myeloid differentiation primary response gene (88)–-dependent pathways,23 further studies are needed to investigate whether ARE-mediated enhancement of mRNA stability/translation also contributes to TLR3-dependent up-regulation of specific proinflammatory mediators during HCV infection in hepatocytes.
Our study defines the molecular features of HCV PAMP required for TLR3 activation as HCV dsRNA duplexes ≥80-100-bp, regardless of genome location or nucleotide composition. Although it is established that TLR3 senses dsRNA,24 whether TLR3 recognizes HCV RNA duplexes or the secondary structures present in HCV RNA (such as the stem loops in the core- and NS5B-coding and 3'NTR regions) has not been determined previously. Our data demonstrate that HCV dsRNA duplexes generated during viral RNA replication are the ligands for TLR3 (Fig. 5), whereas the secondary structures present in either +ss or –ss HCV RNAs are not, as evidenced by the finding that neither strand of the NS-3'NTR RNA containing complex stem-loop structures in NS5B and 3'NTR sequences activated RANTES expression (Fig. 5B). Further supporting this, the +ss and –ss HCV RNAs derived from the HCV core-coding region, which contains two stem-loops,17 also failed to activate TLR3, unless the two RNA strands were annealed to form dsRNA duplexes (data not shown). We speculate that the highly structured HCV ssRNAs may not present the perfect dsRNA structure required for binding to and/or form a stable complex with TLR3, as opposed to HCV dsRNA duplexes.
RIG-I preferentially recognizes a segment of HCV 3'NTR RNA rich in poly-U/UC nucleotides.11 This is not the case for TLR3, because HCV dsRNA duplexes derived from core, E-p7, NS5A, and NS-3'NTR regions all activated TLR3 with similar potency (Fig. 5C), regardless of their nucleotide compositions. This suggests that the dsRNA duplex structure is the sole determinant for the HCV dsRNA recognition by TLR3, as long as the dsRNA meets the minimal length requirement (see below). We found that HCV dsRNA has to be at least 80-100-bp to reproducibly activate TLR3 and trigger chemokine induction (Fig. 6). This is consistent with the intracellular localization of TLR3 expressed in reconstituted 7.5-TLR3 cells and of that expressed endogenously in human hepatocytes in situ.12 Although 40-48-bp-long dsRNAs bind to TLR3 in vitro and are able to activate TLR3 expressed on the surface of HEK293 cells, only dsRNAs longer than 90 bp can activate TLR3 that is exclusively intracellular.19 A longer stretch of dsRNA may be required to form stable dsRNA-TLR3 receptor dimer complexes in the endosomes,19 where TLR3 appears to be expressed in hepatocytes (Supporting Fig. 2).12
How dsRNAs generated during HCV RNA replication are delivered to TLR3 in the luminal compartment of endolysosomes is unknown. Possibly, this process is facilitated by autophagy, as demonstrated for the recognition of vesicular stomatitis virus infection in plasmacytoid dendritic cells by TLR7,25 a viral RNA-sensing TLR also residing in endosomes. Though this hypothesis requires further investigation, preliminary evidence suggests that inhibition of autophagy by 3-methyladenine disrupts poly-I:C-induced TLR3 signaling to RANTES and ISG56 induction in both 7.5-TLR3 and PH5CH8 cells (Supporting Fig. 3). Furthermore, bafilomycin A1, which specifically inhibits acidification of the endolysosome (a terminal step in autophagy), blocked poly-I:C-induced ISG expression and NF-κB activation in these hepatocyte cell lines (Supporting Fig. 4). The potential dependence on autophagy for HCV activation of TLR3 signaling would contrast with that reported for RIG-I, which is negatively regulated by autophagy.26
The identification of TLR3 as an active player in mediating proinflammatory cytokine/chemokine responses to HCV in hepatocytes provides novel insights into host immune response to HCV and the pathogenesis of HCV-associated liver injury. It remains to be investigated in vivo how much TLR3-mediated signaling contributes to antiviral defense and protective immune responses that culminate in HCV clearance and how much it is involved in chronic liver inflammation and the progression to fibrosis and, ultimately, hepatocellular carcinoma. Answers to these questions would yield valuable information toward developing novel immunotherapies for hepatitis C.