TLR3 activation efficiency by high or low molecular mass poly I:C

Abstract

Toll-like receptor 3 (TLR3) plays a critical role in initiating type I IFN-mediated innate immunity against viral infections. TLR3 recognizes various forms of double stranded (ds) RNA, including viral dsRNA and a synthetic mimic of dsRNA, poly I:C, which has been used extensively as a TLR3 ligand to induce antiviral immunity. The activation efficiency of TLR3 by poly I:C is influenced by various factors, including size of the ligands, delivery methods and cell types. In this study, we examined the stimulatory effect of two commercially-available poly I:Cs [high molecular mass (HMM) and low molecular mass (LMM)] on TLR3 activation in various human cell types by determining the induction of type I and type III IFNs, as well as the antiviral effect. We demonstrated that the direct addition of both HMM- and LMM-poly I:C to the cultures of primary macrophages or a neuroplastoma cell line could activate TLR3. However, the transfection of poly I:C was necessary to induce TLR3 activation in other cell types studied. In all the cell lines tested, the efficiency of TLR3 activation by HMM-poly I:C was significantly higher than that by LMM-poly I:C. These observations indicate the importance and necessity of developing effective TLR3 ligands for antiviral therapy.

Keywords

TLR3 poly I:C type I IFN type III IFN LyoVec

Introduction

Innate immunity is important for the control of many viral infections including human immunodeficiency virus (HIV).^1,2 Induction of innate antiviral responses depends largely on a number of innate immune receptors, including the TLR-family. TLRs are a family of pattern recognition receptors (PRRs) that are expressed on many cell types and recognize non-specifically pathogen-associated molecular patterns.² Engagement of TLRs by the corresponding ligands activates complex signaling cascades that culminate in the inflammatory and immune defense responses.³ Among TLRs that have been identified, TLR3 plays an important role in the innate responses to viral infections, as it recognizes double stranded (ds) RNA, a common intermediate of viral replication.⁴ In response to dsRNA, TLR3 is activated, initiating the expression of antiviral genes, including type I and type III IFNs.^5–10

TLR3 recognizes various forms of dsRNA, including viral dsRNA and a synthetic mimic of dsRNA, poly I:C.^4,11,12 Poly I:C has been used extensively as a TLR3 ligand to induce antiviral immunity.^13–17 However, TLR3 activation by its ligand can be greatly influenced by a number of factors, including the molecular structure and size of the ligands. It has been reported that longer strands of dsRNA are more capable of inducing IFN responses than shorter ones in target cells.^18,19 A recent study demonstrated that RNA-induced innate immune response in human monocyte-derived dendritic cells is largely dependent on RNA molecular size.²⁰ In the present study, we examined the effectiveness of two commercially-available poly I:Cs [low molecular mass (LMM) (0.2–1 kb) and high molecular mass (HMM) (1.5–8 kb) poly I:C] on TLR3 activation in various human cell types. We demonstrated that the activation efficiency of TLR3 is affected by the molecular size of poly I:C, as well as the cell types.

Materials and methods

Reagents and Antibodies

The synthetic dsRNA poly I:Cs (LMM, HMM, rhodamine conjugated-HMM) and Lyovec transfection reagent were purchased from InvivoGen (San Diego, CA, USA). Avian myeloblastosis virus (AMV) transcriptase and RNasin were from Promega (Madison, WI, USA). Goat anti-human TLR3 Ab was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and FITC-conjugated donkey anti-goat IgG1 was purchased from Southern Biotechnology Associates (Birmingham, AL, USA).

Cell culture

Human hepatic cell line (Huh7) was kindly provided by Dr Charles M. Rice (Laboratory of Virology and Infectious Diseases, the Rockefeller University, New York). Cells were maintained in conditioned DMEM supplemented with 10% FCS, 10 mM Hepes, 100 units/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine at 5% CO₂.

The brain microvascular endothelial cell line hCMEC/D3 was generously provided by Dr Yuri Perdsisky (Department of Pathology and Laboratory Medicine, Temple University, PA, USA). hCMEC/D3 cells were maintained in complete EGM-2 medium (Lonza, Walkersville, MD, USA). Purified human monocytes obtained from Human Immunology Core at the University of Pennsylvania were plated in 96-well plates in complete DMEM with 10% FCS and differentiated into macrophages (MDM) after 5–7 d of culture. Human neuroblastoma cell line CHP212 was obtained from ATCC (Manassas, VA, USA). Cells were cultured in ATCC-formulated Eagle’s minimum essential medium with 10% FBS.

Poly I:C treatment

Macrophages, CHP212, hCMEC/D3 and Huh7 cells were seeded in 48-well plates with a density of 5 × 10⁵ or 1 × 10⁵ cells/well depending on cell type. Cells were then treated with poly-I:C (LMM, HMM) at different concentrations (1 µg/ml, 10 µg/ml) for 16 h. Untreated cells were included as a negative control (N.C.). In some experiments, poly I:C was transfected by a cationic lipid-based transfection reagent, LyoVec^TM from InvivoGen (San Diego, CA, USA) according to the manufacturer’s manual. Briefly, poly I:C was mixed gently with 20 μl LyoVec and then incubated at 18–22°C (room temperature) for 15 min to allow the formation of lipid–RNA complex. The complex was added into cell cultures at a 1:20 volume ratio for further incubation. For the antiviral experiments, Huh7 cells or macrophages were activated by poly I:C for 6 h prior to hepatitis C virus (HCV) or HIV infection as described below. HMM-Poly I:C-rhodamine treated or transfected cells were visualized for rhodamine-positive cells under microscopy.

Viral infection

Production of infectious HCV Japanese fulminant hepatitis virus 1 (JFH1) and infection of Huh7 cells with JFH1 [multiplicity of infection (MOI) of 0.01] were carried out as previously described.²¹ Real time RT-PCR was used to detect the levels of HCV RNA.²² For HIV infection of macrophages, cells were infected with an equal amount (p24 protein content) of cell-free HIV Bal strains for 2 h at 37°C. The cells were washed three times with DMEM to remove excessive uninfected viruses and fresh medium was added to the cultures. The cells were incubated for 8 d and then subjected to the real-time PCR assay for HIV gag gene.

Real-time RT-PCR

Total cellular RNA was extracted from cells using Tri-Reagent (Molecular Research Center, Cincinnati, OH, USA) as described previously. Total RNA was subjected to the reverse transcription using reagents obtained from Promega (Madison, WI, USA). RT-PCR for the quantification of HCV or HIV RNA, as well as messenger RNA for IFN-β, IFN-λ1, TLR3 and GAPDH, etc. were performed with the iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) as described previously. The levels of GAPDH mRNA were used as an endogenous reference to normalize the quantities of target mRNA. The special oligonucleotide primers used in this study are listed as follows: HCV: 5′-RAYCACTCCCCTGTGAGGAAC-3′(sense) and 5′-TGR TGCACGGTCTACGAGACCTC-3′ (anti-sense); TLR3: 5′-AGCCACCTGAAGTT GACTCAGG-3′ (sense) and 5′-CAGTCAAATTCGTGCAGAAGGC-3′ (anti-sense); Retinoic acid inducible gene I (RIG-I): 5’-CTTGGCATGTTACACAGCTGAC-3′ (sense) and 5’-GCTTGGGATGTGG TCTTACTCA-3′ (anti-sense); Melanoma differentiation-associated gene 5 (MDA5): 5′-ACATAACAGCAACATGGGCAGTG-3’ (sense) and 5′-TTTGGTAAGGCCTGAGCTGGAG-3′ (anti-sense); IFN-β: 5′-GCCGCATTGACCATCTATGAGA-3′ (sense) and 5′-GAGATC TTCAGTTT CGGAGGTAAC-3′(anti-sense); IFN-λ1: 5′-CTTCCAAGCCCACCCC AACT-3′ (sense) and 5′-GGCCTCCAGGACCTTCAGC-3′ (anti-sense); GAPDH: 5′-GGTGGTCTCCTCTGACTTCAACA-3′ (sense) and 5′-GTTGCTGTAGCCAAATTC GTTGT-3′ (anti-sense). The oligonucleotide primers were synthesized by Integrated DNA Technologies Inc. (Coralville, IA, USA).

Immunofluorescence assay

hCMEC/D3, Huh7, CHP212 and macrophages were cultured on glass cover slips at a density of 2 × 10⁵ or 5 × 10⁵ cells/well in 24-well plates. For measuring TLR3 protein expression, cells were fixed with PBS containing 4% paraformaldehyde and 4% sucrose for 20 min at 48°C, and then permeabilized in pre-cold 100% methanol for an additional 10 min, followed by 0.2% Triton X-100 for another 10 min. Cells were blocked by blocking solution for 1 h at room temperature. The coverslips were then incubated with goat anti-TLR3 (1:50) Ab in blocking solution at room temperature for 60 min and were subsequently incubated with the secondary Abs (FITC-conjugated donkey anti-goat IgG1) (1:100) for 1 h. For monitoring delivery of poly I:C, the cells were treated or transfected with rhodamine-conjugated poly I:C (10 µg/ml) for 24 h and then fixed. The coverslips were washed six times with PBS, mounted in Vectorshield (Vector Laboratories, Burlingame, CA, USA) and viewed with a fluorescence microscope (Olympus, Tokyo, Japan).

Statistical analysis

Where appropriate, data are expressed as mean ± SD of triplicate cultures. For comparison of the mean of two groups (treated vs untreated), statistical significance was assessed by Student’s t-test. If there were more than two groups, one-way repeated measures of analysis of variance were used. Statistical analyses were performed with Graphpad Instat Statistical Software (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was defined as P < 0.05.

Results

Electrophoresis and delivery efficiency of LMM- and HMM-poly I:C

To determine the quality and size of LMM- and HMM-poly I:C used in this study, we first performed the electrophoresis of both LMM- and HMM-poly I:C. As shown in Figure 1, the size of HMM-poly I:C is substantially larger than that of LMM-poly I:C. We then investigated the delivery efficiency of poly I:C in several types of human cells (hCMEC/D3, Huh7, CHP212 and macrophages) either by treatment (direct addition to cell cultures) or by LyoVec transfection using rhodamine-labeled HMM-poly I:C. We found that treatment resulted in efficient delivery of HMM-poly I:C in macrophages (Figure 2H) and CHP212 cells (Figure 2K), but not in hCMEC/D3 (Figure 2B) and Huh7 (Figure 2E). In contrast, transfection successfully delivered HMM-poly I:C in all four cell types (Figure 2C, F, I and L). These findings were confirmed quantitatively by counting the percentage of rhodamine-positive cells (Figure 2M).

Figure 1.

Electrophoresis of LMM- and HMM-poly I:Cs. Low (middle) and high molecular mass (right) poly I:Cs (purchased from InvivoGen) were evaluated for size and quality by loading them on 1% agarose gel containing ethidium bromide. A 1-kb ladder was included (left). After electrophoresis, the gel was placed on a UV transilluminator for nucleic acid visualization and analysis.

Figure 2.

Delivery efficiency of HMM-poly I:C with or without transfection in different cell types. Four different cell types indicated were either treated (B, E, H and K) or transfected (C, F, I and L) with rhodamine-labeled HMM-poly I:C (1 µg/ml) for 24 h. Non-treated cells are included as a control for corresponding cell types (A, D, G and J). The fluorescence of rhodamine-labeled HMM-poly I:C was visualized under fluorescence microscopy (magnification × 100). The percentage of rhodamine-positive cells was counted under a microscope and the results shown are the mean ± SD of positive cells in triplicate wells (M) (**P < 0.01).

Transfection of poly I:C is necessary to induce IFNs

We next determined poly I:C-mediated TLR3 functional activation in different cell types by treatment (direct addition to cell cultures) or transfection delivery. We were interested in TLR3 activation-mediated IFN expression, as IFNs (IFN-α/β and λ₁ are potent innate antiviral cytokines regulated by TLR3 signaling.^8,23 We found that transfection with either HMM- or LMM-poly I:C efficiently induced the expression of IFN-β and IFN-λ1 in hCMEC/D3 cells (Figure 3A, B). In Huh7 cells, transfection of HMM-poly I:C, but not LMM-poly I:C, induced substantially high levels of both IFN-β and IFN-λ1 (Figure 3C, D). In contrast, direct addition of either HMM- or LMM-poly I:C to these cell cultures could not effectively induce IFN expression (Figure 3).

Figure 3.

Effect of LMM- or HMM-poly I:C on IFN induction. hCMEC/D3 (A, B) or Huh7 cells (C, D) were treated or transfected with HMM- or LMM-poly I:C (10 µg/ml) for 16 h. Total RNA was then subjected to real-time RT-PCR for quantifying mRNA expression of IFN-β (A, C) and IFN-λ1 (B, D), as well as GAPDH. The results for GAPDH-normalized expression of mRNA are expressed as fold change of target gene expression relative to the control (without poly I:C treatment, which is defined as 1). The results shown are the mean ± SD of triplicate wells, representing three independent experiments (**P < 0.01).

HMM-poly I:C is more effective in inducing IFNs than LMM-poly I:C

To further determine the effect of HMM-poly I:C or LMM-poly I:C on TLR3 activation, we compared the expression of IFN-β and IFN-λ1 mRNA in hCMEC/D3, Huh7, macrophages and CHP212 cells transfected with either LMM- or HMM-poly I:C. We showed that HMM-poly I:C induced higher levels of both IFN-β (Figure 4A, C, E and G) and IFN-λ1 (Figure 4B, D, F and H) than LMM-poly I:C in these cells. In Huh7 cells, the differences in TLR3 activation by HMM- than LMM-poly I:C were as high as 15-fold (IFN-β) to 60-fold (IFN-λ1) (Figure 4C, 4D).

Figure 4.

Effect of LMM- or HMM-poly I:C on IFNs in different cell types. (A–D) hCMEC/D3 (A, B) and Huh7 (C, D) were transfected with LMM- or HMM-poly I:C at two concentrations (1 µg/ml, 10 µg/ml). LyoVec vector alone without poly I:C was included as control. (E–H) Macrophages (E, F) and CHP212 (G, H) were directly treated by LMM- or HMM-poly I:C (no transfection) at indicated concentrations. Non-treated cells were included as a control. Cells were collected and subjected to RNA extraction 16 h after poly I:C stimulation. Total RNA was subjected to real-time RT-PCR for the mRNA levels of IFN-β (A, C, E and G), and IFN-λ1 (B, D, F and H) and GAPDH. The data are expressed as IFN-β and IFN-λ1 mRNA levels relative (fold change) to the control (without poly I:C treatment, which is defined as 1). The results shown are the mean ± SD of triplicate wells, representing three independent experiments (*P < 0.05, **P < 0.01).

HMM-poly I:C is more effective in inhibiting viral replication than LMM-poly I:C

Our previous studies demonstrated that TLR3 activation could result in the inhibition of viral replication in macrophages and neuronal cells.^9,10 We thus examined the antiviral efficiency of HMM-poly I:C or LMM-poly I:C in macrophages and Huh7 cells. As shown in Figure 5, LMM-poly I:C had little effect on HCV JFH1 expression in Huh7 cells, whereas HMM-poly I:C inhibited HCV replication by more than 90% (Figure 5A). Although LMM-poly I:C treatment of macrophages could inhibit HIV replication (Figure 5B), HMM-poly I:C was significantly more effective than LMM-poly I:C in diminishing HIV replication in macrophages (Figure 5B).

Figure 5.

Comparison of antiviral activity induced by LMM- and HMM-poly I:C. (A) Huh7 cells were transfected with HMM- or LMM-poly I:C (10 µg/ml) for 6 h and then infected with HCV JFH1 for 72 h. Total cellular RNA was subjected to real-time RT-PCR for the RNA levels of HCV JFH1 and GAPDH. The data are expressed as HCV JFH1 RNA levels relative (fold) to the control (without poly I:C treatment, which is defined as 1). (B) Monocyte-derived macrophages were treated with LMM- or HMM-poly I:C for 6 h. Cells were then infected with HIV Bal for 24 h. Cells collected at d 8 after HIV infection were subjected to real-time RT-PCR for the RNA levels of the HIV-gag gene and GAPDH. The data are expressed as HIV-gag RNA levels relative (fold change) to the control (without poly I:C treatment, which is defined as 1). The results shown are the mean ± SD of triplicate wells, representing three independent experiments (*P < 0.05, **P < 0.01).

HMM-poly I:C is more effective in inducing TLR3, RIG-I and MDA5 than LMM-poly I:C

It has been shown that cellular expression of innate immune receptors, such as TLR3, and cytoplasmic RNA sensors RIG-I and MDA5 could be regulated in response to various signals, such as viral infections and poly I:C.^24–27 We then determined whether the expression of TLR3, RIG-I and MDA5 is differentially regulated by HMM- or LMM-poly I:C in Huh7 cells. Compared with LMM-poly I:C, HMM-poly I:C induced significantly higher levels of TLR3 expression at both mRNA (Figure 6A) and protein levels (Figure 6B–E). Similarly, HMM-poly I:C was more effective in inducing the expression of RIG-I and MDA5 than LMM poly I:C (Figure 6A).

Figure 6.

Effects of LMM- or HMM-poly I:C on TLR3, RIG-I and MDA5 in Huh7 cells. (A) Huh7 cells were transfected with or without LMM- or HMM-poly I:C (10 µg/ml) by lyoVec transfection. Non-transfected cells were included as control. Total cellular RNA was extracted 16 h post-transfection with poly I:C and then subjected to real-time RT-PCR for the mRNA levels of TLR3, RIG-I, MDA5 and GAPDH. The data are expressed as mRNA levels relative (fold change) to the control (without transfection, which is defined as 1). The results shown are the mean ± SD of triplicate wells, representing three independent experiments (*P < 0.05, **P < 0.01). (B–D) Fluorescent staining and visualization of TLR3 protein in Huh7 cells after poly I:C transfection. Huh7 cells seeded on chamber slides were transfected with 10 µg/ml LMM- (C) or HMM-poly I:C (D) for 12 h and then stained with Ab to TLR3. Cells transfected with LyoVec vector were included as control (B). The positive staining of TLR3 (arrow) was visualized under a fluorescence microscopy (magnification × 200). (E) Fluorescence intensity of positive cells was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Discussion

Among the family members of TLRs, TLR3 plays a uniquely important role in the generation of innate responses to viral infections, which is because of its ability to sense dsRNA, a common intermediate of viral replication, initiating potent antiviral pathways.^4,7,18 Various forms of dsRNA, including viral dsRNA, as well as a synthetic mimic of dsRNA, poly I:C, can be recognized by TLR3.^4,12 Studies from our group and others showed that the activation of TLR3 by poly I:C potently inhibits several viruses, including HSV¹⁰ and HIV.⁹ In addition to type I IFNs, TLR3 activation also induces type III IFN expression.^5,6 Because of the importance of TLR3 in host innate immune responses to viral infections, great attention is now given to the use of effective TLR3 ligands for the treatment of viral diseases, including HCV and HIV.²⁸ In this study, we compared the effectiveness of two different sizes of poly I:C in activating TLR3 in several cell systems, showing that HMM-poly I:C is more efficient in activating TLR3 than LMM-poly I:C in terms of IFN induction and antiviral activities.

There are two major ways to introduce foreign dsRNA into cells: viral infection and therapeutic dsRNA, such as RNA interfering (RNAi). RNAi studies have shown that while short interfering RNA (siRNA) duplexes, ranging in size between 17 and 31 nt, are usually well tolerated in cells where no significant innate responses were detected, the introduction of long dsRNA induced much stronger type I IFN and other defense responses in target cells.^18,19 These findings suggest that compared with short dsRNA, long dsRNA is more immunogenic, inducing stronger activation of RNA sensors. In line with these important observations, we showed that HMM-poly I:C with sizes of 1.5–8 kb induced stronger TLR3 activation than LMM-poly I:C with sizes of 0.2–1 kb, which was evidenced by higher levels of IFN expression and the inhibition of HIV or HCV. Although the mechanisms for the effectiveness of poly I:C on TLR3 remains to be determined, it is likely that the activation efficiency of poly I:C could be influenced by the size of its molecule and duplex structure that contributes to the binding affinity of poly I:C to TLR3. However, one recent study by Jiang et al. showed that compared with longer dsRNA (with sizes ranging between 308 and 1808 bp), the short dsRNA (58–108 bp) induced stronger type I/III IFN and pro-inflammatory responses (TNF-α, IL-6 and IL-1β) in human dendritic cells.²⁰ These discrepancies could be owing to the cell types used in the study by Jiang et al. Monocyte-derived dendritic cells may have a different profile of innate receptors and RNA sensors. In addition, the synthetic bacteriophage and viral dsRNA used in the study by Jiang et al. may have different characteristics to the poly I:C used in this study in terms of the RNA duplex and secondary structure. This assumption is supported by reports that TLR3 activation is highly dependent on the RNA duplex rather than 5’-triphosphate.^4,12,29 Finally, there is no information about whether dsRNA larger than 3 kb could induce type I/III IFNs in the study by Jiang et al.

In addition to TLR3, cytoplasmic RNA sensors (RIG-I and MDA5) also play a coordinating role in the innate immune response to viral infections.²⁷ We observed that poly I:C not only induced TLR3 expressions, but also up-regulated the expression of RIG-I and MDA5 in Huh7 cells (Figure 6). These data suggested the possible involvement of RIG-I or MDA5 in poly I:C-induced IFN activation. To explore the potential role of RIG-I in poly I:C-mediated IFN induction, we performed the experiments with Huh7.5.1 cells that had a mutationally-inactivated RIG-I.³⁰ We found that poly I:C could activate TLR3 and induce IFNs expression in Huh7.5.1 cells, indicating that RIG-I is not involved in the poly I:C-induced IFN activation in Huh7.5.1 cells. To determine the contribution of MDA5 to the poly I:C action on IFN-β we used specific siRNA to knock down MDA5 expression (data not shown) and found that the knock down of MDA5 had little effect on poly I:C-mediated induction of IFN-β in Huh7.5.1 cells (data not shown). These findings indicate that TLR3 is a major receptor for poly I:C in Huh7.5.1 cells. The role of TLR3 as the major sensor of poly I:C has also been confirmed by Wang et al.,³¹ showing that the expression of TLR3 in Huh7 cells was necessary for poly I:C-mediated activation of IFN-β promoter. In addition, they demonstrated that TLR3 acted independently of RIG-I in inducing an antiviral state in Huh7 cells.³¹

Because of its ability to induce innate antiviral immunity,^13–16 poly I:C has been suggested to be used as an adjuvant for vaccine therapy. Efforts in applying TLR3 ligand to enhance vaccine therapy have been made in mouse tumor implant model, as well as in a mouse viral infection model.^13–15,32 Using TLR3 ligands as an adjuvant could induce selective expression of TLR3 in mouse dendritic cells and the subsequent immune responses, resulting in the expression of type I IFNs, dendritic cells maturation and cytotoxic T-lymphocyte (CTL) response, which were associated with increased protective immunity.¹⁵ Several studies also reported that poly I:C₁₂ U induces IFN-β production and exhibits protective antiviral effects in mice.^29,33,34 We demonstrated that TLR3 activation by poly I:C induced innate antiviral activity against HIV in macrophages⁹ and HSV-1 in neuronal cells.¹⁰ These observations justify clinical studies on TLR3 ligand-based antiviral therapy. Therefore, to understand and determine the optimal molecular pattern and size of the TLR3 ligand is important for the development of effective TLR3 ligand-based antiviral therapy.

Footnotes

Funding

The study was supported by grants NIDA012815, NIDA027550 and NIDA022177 (to W.Z. Ho) from the National Institutes of Health.

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