Double-stranded RNA upregulates the expression of inflammatory mediators in human aortic valve cells through the TLR3-TRIF-noncanonical NF-κB pathway.
Journal: 2017/June - American Journal of Physiology - Cell Physiology
ISSN: 1522-1563
Abstract:
Calcific aortic valve disease is a chronic inflammatory condition, and the inflammatory responses of aortic valve interstitial cells (AVICs) play a critical role in the disease progression. Double-stranded RNA (dsRNA) released from damaged or stressed cells is proinflammatory and may contribute to the mechanism of chronic inflammation observed in diseased aortic valves. The objective of this study is to determine the effect of dsRNA on AVIC inflammatory responses and the underlying mechanism. AVICs from normal human aortic valves were stimulated with polyinosinic-polycytidylic acid [poly(I:C)], a mimic of dsRNA. Poly(I:C) increased the production of IL-6, IL-8, monocyte chemoattractant protein-1, and ICAM-1. Poly(I:C) also induced robust activation of ERK1/2 and NF-κB. Knockdown of Toll-like receptor 3 (TLR3) or Toll-IL-1 receptor domain-containing adapter-inducing IFN-β (TRIF) suppressed ERK1/2 and NF-κB p65 phosphorylation and reduced inflammatory mediator production induced by poly(I:C). Inhibition of NF-κB, not ERK1/2, reduced inflammatory mediator production in AVICs exposed to poly(I:C). Interestingly, inhibition of NF-κB by prevention of p50 migration failed to suppress inflammatory mediator production. NF-κB p65 intranuclear translocation induced by the TLR4 agonist was reduced by inhibition of p50 migration; however, poly(I:C)-induced p65 translocation was not, although the p65/p50 heterodimer is present in AVICs. Poly(I:C) upregulates the production of multiple inflammatory mediators through the TLR3-TRIF-NF-κB pathway in human AVICs. The NF-κB activated by dsRNA appears not to be the canonical p65/p50 heterodimers.
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Am J Physiol Cell Physiol 312(4): C407-C417

Double-stranded RNA upregulates the expression of inflammatory mediators in human aortic valve cells through the TLR3-TRIF-noncanonical NF-κB pathway

MATERIALS AND METHODS

Materials.

Antibodies against ICAM-1, TLR3, TRIF, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX). Antibodies against phosphorylated NF-κB p65, total NF-κB p65, total NF-κB p50, phosphorylated ERK1/2, and total ERK1/2 were purchased from Cell Signaling Technology (Beverly, MA). Specific small interfering RNA (siRNA) for human TLR3 and TRIF and scrambled siRNA were purchased from Thermo Fisher Scientific (Waltham, MA). HiPerFect Transfection Reagents were purchased from Qiagen (Germantown, MD), and other transfection-related reagents were purchased from GE Dharmacon (Lafayette, CO). Medium 199 was purchased from Lonza (Walkersville, MD). ELISA kits for IL-6, IL-8, and MCP-1 were purchased from R&D Systems (Minneapolis, MN). Poly(I:C), molecular size 1.5–8 kb, was purchased from InvivoGen (San Diego, CA). NF-κB p50 migration inhibitor (SN50) and control (SN50M) were purchased from Enzo Life Sciences (Farmingdale, NY). MAPK kinase 1 (MEK1) inhibitors (PD98059 and 328000ERK) were purchased from EMD Millipore (Billerica, MA). IKK inhibitor (Bay11-7082), LPS, and all other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Cell isolation and culture.

This study was approved by the Colorado Multiple Institutional Review Board of the University of Colorado Denver. Normal aortic valve leaflets were collected from the explanted hearts of six patients (4 males and 2 females; mean age 59 ± 8.1 yr), due to advanced cardiomyopathy, undergoing heart transplantation at the University of Colorado Hospital. All patients gave informed consent for the use of their aortic valves for this study.

AVICs were isolated and cultured using a previously described method (25) with modifications (24). Briefly, valve leaflets were subjected to sequential digestions with collagenase, and cells were collected by centrifugation. Cells isolated from each donor valve were used as a cell line. Cells were cultured in M199 growth medium containing penicillin G, streptomycin, amphotericin B, and 10% FBS. Cells of Passages 3–5 were used for this study, subcultured on plates, and treated when they reached 80–90% confluence.

Varied concentrations of poly(I:C) (0.5–10 μg/ml) were applied to examine the dose response. To determine the effect of poly(I:C) on the production of ICAM-1, IL-6, IL-8, and MCP-1, cells were treated with the optimal dose of poly(I:C) for 24 h. Cells treated with poly(I:C) for 30 min–8 h were analyzed for the phosphorylation of NF-κB p65 and ERK1/2.

To determine the role of TLR3 and TRIF in NF-κB and ERK1/2 phosphorylation, as well as inflammatory mediator production, TLR3 and TRIF knockdown, with specific siRNAs, was performed. To determine the role of the NF-κB and ERK1/2 pathways in inflammatory mediator production, IKKα inhibitor Bay11-7082 (2.5 μmol/l), NF-κB p50 migration inhibitor SN50 (100 μg/ml), and MEK1 inhibitors (PD98059, 25 μmol/l; 328000ERK, 40 µmol/l) were added to cultured cells, 1 h before poly(I:C) stimulation.

Immunoblotting.

Immunoblotting was applied to analyze the levels of ICAM-1, TRIF, TLR3, NF-κB p50, phosphorylated and total NF-κB p65, and phosphorylated and total ERK1/2 with β-actin as a loading control. Cells were lysed in a sample buffer (100 mmol/l Tris-HCl, 2% SDS, 0.02% bromophenol blue, and 10% glycerol, pH 6.8). Protein samples were separated on gradient (4–20%) mini-gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% skim milk solution for 1 h at room temperature. The blocked membranes were incubated with a primary antibody. After washing with PBS containing 0.05% Tween 20, the membranes were incubated with a peroxidase-linked secondary antibody specific to the primary antibody. After further washes, membranes were treated with enhanced chemiluminescence reagents. The membrane was then exposed on X-ray film. Band density was analyzed using ImageJ software (Wayne Rasband, U.S. National Institutes of Health, Bethesda, MD).

Gene knockdown.

Gene knockdown was performed as described previously (35). Briefly, cells (60–70% confluence) in 24-well plates were incubated with a mixture of TLR3 siRNA (150 nmol/l) or TRIF siRNA (100 nmol/l) and transfection reagent (6.0 μl/ml medium) for 72 h to knock down TLR3 or TRIF. Control cells were treated with scrambled siRNA (150 nmol/l).

ELISA assay.

Levels of IL-6, IL-8, and MCP-1 in cell culture supernatants were analyzed using ELISA kits following the manufacturer’s protocols.

Immunofluorescent staining.

Immunofluorescent staining was performed, as described previously, to localize NF-κB p65 and p50 subunits (44). After permeabilization with a methanol/acetone mixture, cells were fixed in 4% paraformaldehyde and incubated with the primary antibody (rabbit MAb against human NF-κB p65 and mouse MAb against human NF-κB p50) overnight at 4°C. After washing with PBS, cells were incubated with Cy3-tagged secondary antibody (imaged on the red channel). Nuclei were stained with bis-benzimide [4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI), imaged on the blue channel]. Glycoproteins on cell surfaces were stained with Alexa 488-tagged wheat-germ agglutinin (imaged on the green channel). Microscopy was performed with a Leica DM RXA digital microscope (Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany).

Coimmunoprecipitation.

AVICs (80–90% confluence), in six-well plates, were rinsed three times with PBS. Cells were lysed in Pierce cell lysis buffer (25 mmol/l Tris-HCl, 150 mmol/l NaCl, 1 mmol/l EDTA, 1% Tergitol-type Nonidet P-40, and 5% glycerol, pH 7.4; Thermo Fisher Scientific), and the lysates were centrifuged at 13,000 g for 20 min at 4°C. Clarified supernatants were incubated with a rabbit MAb against human NF-κB p50 (4 µg/sample) or nonimmune rabbit IgG-agarose, conjugated (4 µg/sample) overnight at 4°C with rocking. Precipitation was performed at 4°C for 2 h using 20 μl protein A/G Plus agarose per sample. The immune complexes, collected by centrifugation at 10,000 g for 1 min, were gently washed five times with 0.5 ml ice-cold PBS and solubilized by 50 μl, 2× SDS sample buffer (100 mmol/l Tris-HCl, 2% SDS, 0.02% bromophenol blue, and 10% glycerol, pH 6.8). NF-κB p65 and p50 were analyzed by immunoblotting with mouse MAb.

Statistical analysis.

Data are presented as means ± SE. Statistical analysis was performed using StatView software (Abacus Concepts, Berkeley, CA). Student’s t-test was used for comparison between two groups. ANOVA with the post hoc Bonferroni/Dunn test was used to analyze differences among multiple groups. Statistical significance was defined as P ≤ 0.05. Nonparametric Mann-Whitney U-test was performed to confirm the difference of the two-group comparison. For multiple-group comparisons, nonparametric Kruskal-Wallis test was performed to confirm the differences.

Materials.

Antibodies against ICAM-1, TLR3, TRIF, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX). Antibodies against phosphorylated NF-κB p65, total NF-κB p65, total NF-κB p50, phosphorylated ERK1/2, and total ERK1/2 were purchased from Cell Signaling Technology (Beverly, MA). Specific small interfering RNA (siRNA) for human TLR3 and TRIF and scrambled siRNA were purchased from Thermo Fisher Scientific (Waltham, MA). HiPerFect Transfection Reagents were purchased from Qiagen (Germantown, MD), and other transfection-related reagents were purchased from GE Dharmacon (Lafayette, CO). Medium 199 was purchased from Lonza (Walkersville, MD). ELISA kits for IL-6, IL-8, and MCP-1 were purchased from R&D Systems (Minneapolis, MN). Poly(I:C), molecular size 1.5–8 kb, was purchased from InvivoGen (San Diego, CA). NF-κB p50 migration inhibitor (SN50) and control (SN50M) were purchased from Enzo Life Sciences (Farmingdale, NY). MAPK kinase 1 (MEK1) inhibitors (PD98059 and 328000ERK) were purchased from EMD Millipore (Billerica, MA). IKK inhibitor (Bay11-7082), LPS, and all other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Cell isolation and culture.

This study was approved by the Colorado Multiple Institutional Review Board of the University of Colorado Denver. Normal aortic valve leaflets were collected from the explanted hearts of six patients (4 males and 2 females; mean age 59 ± 8.1 yr), due to advanced cardiomyopathy, undergoing heart transplantation at the University of Colorado Hospital. All patients gave informed consent for the use of their aortic valves for this study.

AVICs were isolated and cultured using a previously described method (25) with modifications (24). Briefly, valve leaflets were subjected to sequential digestions with collagenase, and cells were collected by centrifugation. Cells isolated from each donor valve were used as a cell line. Cells were cultured in M199 growth medium containing penicillin G, streptomycin, amphotericin B, and 10% FBS. Cells of Passages 3–5 were used for this study, subcultured on plates, and treated when they reached 80–90% confluence.

Varied concentrations of poly(I:C) (0.5–10 μg/ml) were applied to examine the dose response. To determine the effect of poly(I:C) on the production of ICAM-1, IL-6, IL-8, and MCP-1, cells were treated with the optimal dose of poly(I:C) for 24 h. Cells treated with poly(I:C) for 30 min–8 h were analyzed for the phosphorylation of NF-κB p65 and ERK1/2.

To determine the role of TLR3 and TRIF in NF-κB and ERK1/2 phosphorylation, as well as inflammatory mediator production, TLR3 and TRIF knockdown, with specific siRNAs, was performed. To determine the role of the NF-κB and ERK1/2 pathways in inflammatory mediator production, IKKα inhibitor Bay11-7082 (2.5 μmol/l), NF-κB p50 migration inhibitor SN50 (100 μg/ml), and MEK1 inhibitors (PD98059, 25 μmol/l; 328000ERK, 40 µmol/l) were added to cultured cells, 1 h before poly(I:C) stimulation.

Immunoblotting.

Immunoblotting was applied to analyze the levels of ICAM-1, TRIF, TLR3, NF-κB p50, phosphorylated and total NF-κB p65, and phosphorylated and total ERK1/2 with β-actin as a loading control. Cells were lysed in a sample buffer (100 mmol/l Tris-HCl, 2% SDS, 0.02% bromophenol blue, and 10% glycerol, pH 6.8). Protein samples were separated on gradient (4–20%) mini-gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% skim milk solution for 1 h at room temperature. The blocked membranes were incubated with a primary antibody. After washing with PBS containing 0.05% Tween 20, the membranes were incubated with a peroxidase-linked secondary antibody specific to the primary antibody. After further washes, membranes were treated with enhanced chemiluminescence reagents. The membrane was then exposed on X-ray film. Band density was analyzed using ImageJ software (Wayne Rasband, U.S. National Institutes of Health, Bethesda, MD).

Gene knockdown.

Gene knockdown was performed as described previously (35). Briefly, cells (60–70% confluence) in 24-well plates were incubated with a mixture of TLR3 siRNA (150 nmol/l) or TRIF siRNA (100 nmol/l) and transfection reagent (6.0 μl/ml medium) for 72 h to knock down TLR3 or TRIF. Control cells were treated with scrambled siRNA (150 nmol/l).

ELISA assay.

Levels of IL-6, IL-8, and MCP-1 in cell culture supernatants were analyzed using ELISA kits following the manufacturer’s protocols.

Immunofluorescent staining.

Immunofluorescent staining was performed, as described previously, to localize NF-κB p65 and p50 subunits (44). After permeabilization with a methanol/acetone mixture, cells were fixed in 4% paraformaldehyde and incubated with the primary antibody (rabbit MAb against human NF-κB p65 and mouse MAb against human NF-κB p50) overnight at 4°C. After washing with PBS, cells were incubated with Cy3-tagged secondary antibody (imaged on the red channel). Nuclei were stained with bis-benzimide [4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI), imaged on the blue channel]. Glycoproteins on cell surfaces were stained with Alexa 488-tagged wheat-germ agglutinin (imaged on the green channel). Microscopy was performed with a Leica DM RXA digital microscope (Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany).

Coimmunoprecipitation.

AVICs (80–90% confluence), in six-well plates, were rinsed three times with PBS. Cells were lysed in Pierce cell lysis buffer (25 mmol/l Tris-HCl, 150 mmol/l NaCl, 1 mmol/l EDTA, 1% Tergitol-type Nonidet P-40, and 5% glycerol, pH 7.4; Thermo Fisher Scientific), and the lysates were centrifuged at 13,000 g for 20 min at 4°C. Clarified supernatants were incubated with a rabbit MAb against human NF-κB p50 (4 µg/sample) or nonimmune rabbit IgG-agarose, conjugated (4 µg/sample) overnight at 4°C with rocking. Precipitation was performed at 4°C for 2 h using 20 μl protein A/G Plus agarose per sample. The immune complexes, collected by centrifugation at 10,000 g for 1 min, were gently washed five times with 0.5 ml ice-cold PBS and solubilized by 50 μl, 2× SDS sample buffer (100 mmol/l Tris-HCl, 2% SDS, 0.02% bromophenol blue, and 10% glycerol, pH 6.8). NF-κB p65 and p50 were analyzed by immunoblotting with mouse MAb.

Statistical analysis.

Data are presented as means ± SE. Statistical analysis was performed using StatView software (Abacus Concepts, Berkeley, CA). Student’s t-test was used for comparison between two groups. ANOVA with the post hoc Bonferroni/Dunn test was used to analyze differences among multiple groups. Statistical significance was defined as P ≤ 0.05. Nonparametric Mann-Whitney U-test was performed to confirm the difference of the two-group comparison. For multiple-group comparisons, nonparametric Kruskal-Wallis test was performed to confirm the differences.

Materials.

Antibodies against ICAM-1, TLR3, TRIF, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX). Antibodies against phosphorylated NF-κB p65, total NF-κB p65, total NF-κB p50, phosphorylated ERK1/2, and total ERK1/2 were purchased from Cell Signaling Technology (Beverly, MA). Specific small interfering RNA (siRNA) for human TLR3 and TRIF and scrambled siRNA were purchased from Thermo Fisher Scientific (Waltham, MA). HiPerFect Transfection Reagents were purchased from Qiagen (Germantown, MD), and other transfection-related reagents were purchased from GE Dharmacon (Lafayette, CO). Medium 199 was purchased from Lonza (Walkersville, MD). ELISA kits for IL-6, IL-8, and MCP-1 were purchased from R&D Systems (Minneapolis, MN). Poly(I:C), molecular size 1.5–8 kb, was purchased from InvivoGen (San Diego, CA). NF-κB p50 migration inhibitor (SN50) and control (SN50M) were purchased from Enzo Life Sciences (Farmingdale, NY). MAPK kinase 1 (MEK1) inhibitors (PD98059 and 328000ERK) were purchased from EMD Millipore (Billerica, MA). IKK inhibitor (Bay11-7082), LPS, and all other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Cell isolation and culture.

This study was approved by the Colorado Multiple Institutional Review Board of the University of Colorado Denver. Normal aortic valve leaflets were collected from the explanted hearts of six patients (4 males and 2 females; mean age 59 ± 8.1 yr), due to advanced cardiomyopathy, undergoing heart transplantation at the University of Colorado Hospital. All patients gave informed consent for the use of their aortic valves for this study.

AVICs were isolated and cultured using a previously described method (25) with modifications (24). Briefly, valve leaflets were subjected to sequential digestions with collagenase, and cells were collected by centrifugation. Cells isolated from each donor valve were used as a cell line. Cells were cultured in M199 growth medium containing penicillin G, streptomycin, amphotericin B, and 10% FBS. Cells of Passages 3–5 were used for this study, subcultured on plates, and treated when they reached 80–90% confluence.

Varied concentrations of poly(I:C) (0.5–10 μg/ml) were applied to examine the dose response. To determine the effect of poly(I:C) on the production of ICAM-1, IL-6, IL-8, and MCP-1, cells were treated with the optimal dose of poly(I:C) for 24 h. Cells treated with poly(I:C) for 30 min–8 h were analyzed for the phosphorylation of NF-κB p65 and ERK1/2.

To determine the role of TLR3 and TRIF in NF-κB and ERK1/2 phosphorylation, as well as inflammatory mediator production, TLR3 and TRIF knockdown, with specific siRNAs, was performed. To determine the role of the NF-κB and ERK1/2 pathways in inflammatory mediator production, IKKα inhibitor Bay11-7082 (2.5 μmol/l), NF-κB p50 migration inhibitor SN50 (100 μg/ml), and MEK1 inhibitors (PD98059, 25 μmol/l; 328000ERK, 40 µmol/l) were added to cultured cells, 1 h before poly(I:C) stimulation.

Immunoblotting.

Immunoblotting was applied to analyze the levels of ICAM-1, TRIF, TLR3, NF-κB p50, phosphorylated and total NF-κB p65, and phosphorylated and total ERK1/2 with β-actin as a loading control. Cells were lysed in a sample buffer (100 mmol/l Tris-HCl, 2% SDS, 0.02% bromophenol blue, and 10% glycerol, pH 6.8). Protein samples were separated on gradient (4–20%) mini-gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% skim milk solution for 1 h at room temperature. The blocked membranes were incubated with a primary antibody. After washing with PBS containing 0.05% Tween 20, the membranes were incubated with a peroxidase-linked secondary antibody specific to the primary antibody. After further washes, membranes were treated with enhanced chemiluminescence reagents. The membrane was then exposed on X-ray film. Band density was analyzed using ImageJ software (Wayne Rasband, U.S. National Institutes of Health, Bethesda, MD).

Gene knockdown.

Gene knockdown was performed as described previously (35). Briefly, cells (60–70% confluence) in 24-well plates were incubated with a mixture of TLR3 siRNA (150 nmol/l) or TRIF siRNA (100 nmol/l) and transfection reagent (6.0 μl/ml medium) for 72 h to knock down TLR3 or TRIF. Control cells were treated with scrambled siRNA (150 nmol/l).

ELISA assay.

Levels of IL-6, IL-8, and MCP-1 in cell culture supernatants were analyzed using ELISA kits following the manufacturer’s protocols.

Immunofluorescent staining.

Immunofluorescent staining was performed, as described previously, to localize NF-κB p65 and p50 subunits (44). After permeabilization with a methanol/acetone mixture, cells were fixed in 4% paraformaldehyde and incubated with the primary antibody (rabbit MAb against human NF-κB p65 and mouse MAb against human NF-κB p50) overnight at 4°C. After washing with PBS, cells were incubated with Cy3-tagged secondary antibody (imaged on the red channel). Nuclei were stained with bis-benzimide [4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI), imaged on the blue channel]. Glycoproteins on cell surfaces were stained with Alexa 488-tagged wheat-germ agglutinin (imaged on the green channel). Microscopy was performed with a Leica DM RXA digital microscope (Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany).

Coimmunoprecipitation.

AVICs (80–90% confluence), in six-well plates, were rinsed three times with PBS. Cells were lysed in Pierce cell lysis buffer (25 mmol/l Tris-HCl, 150 mmol/l NaCl, 1 mmol/l EDTA, 1% Tergitol-type Nonidet P-40, and 5% glycerol, pH 7.4; Thermo Fisher Scientific), and the lysates were centrifuged at 13,000 g for 20 min at 4°C. Clarified supernatants were incubated with a rabbit MAb against human NF-κB p50 (4 µg/sample) or nonimmune rabbit IgG-agarose, conjugated (4 µg/sample) overnight at 4°C with rocking. Precipitation was performed at 4°C for 2 h using 20 μl protein A/G Plus agarose per sample. The immune complexes, collected by centrifugation at 10,000 g for 1 min, were gently washed five times with 0.5 ml ice-cold PBS and solubilized by 50 μl, 2× SDS sample buffer (100 mmol/l Tris-HCl, 2% SDS, 0.02% bromophenol blue, and 10% glycerol, pH 6.8). NF-κB p65 and p50 were analyzed by immunoblotting with mouse MAb.

Statistical analysis.

Data are presented as means ± SE. Statistical analysis was performed using StatView software (Abacus Concepts, Berkeley, CA). Student’s t-test was used for comparison between two groups. ANOVA with the post hoc Bonferroni/Dunn test was used to analyze differences among multiple groups. Statistical significance was defined as P ≤ 0.05. Nonparametric Mann-Whitney U-test was performed to confirm the difference of the two-group comparison. For multiple-group comparisons, nonparametric Kruskal-Wallis test was performed to confirm the differences.

RESULTS

dsRNA promotes the production of inflammatory mediators in human AVICs.

The result in Fig. 1A shows that treatment of human AVICs with 0.5–10 μg/ml poly(I:C) resulted in a dose-dependent increase in ICAM-1 protein levels. It appears that 2.5 μg/ml poly(I:C) induces a marked increase in ICAM-1 levels, although lower concentrations of poly(I:C) also have effects.

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Poly(I:C) upregulates the production of IL-6, IL-8, MCP-1, and ICAM-1 in human AVICs. A: AVICs isolated from normal human aortic valves were treated with varied concentrations of poly(I:C) for 24 h. Representative immunoblots and densitometric data show that poly(I:C) increases ICAM-1 levels in a dose-dependent fashion. B: human AVICs were treated with 2.5 μg/ml poly(I:C) for 24 h. ELISA data show that poly(I:C) upregulates the production of IL-6, IL-8, and MCP-1. Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control.

We analyzed the levels of IL-6, IL-8, and MCP-1 in culture supernatants of cells exposed to 2.5 μg/ml poly(I:C). As shown in Fig. 1B, poly(I:C) markedly elevated the levels of IL-6 (712 ± 67.2 pg/ml vs. 140 ± 22.4 pg/ml in untreated control; P < 0.05), IL-8 (1,485 ± 366 pg/ml vs. 11.1 ± 7.45 pg/ml in untreated control; P < 0.05), and MCP-1 (829 ± 135 pg/ml vs. 272 ± 66.2 pg/ml in untreated control; P < 0.05). Together, these results show that dsRNA is potent to promote the production of multiple inflammation mediators in human AVICs.

TLR3 and TRIF mediate the proinflammatory effect of dsRNA.

dsRNA can activate multiple signaling pathways, including TLR3, in a variety of cell types (1, 12, 13). TLR3 recruits the adaptor molecule TRIF to activate proinflammatory signaling (37). To test the hypothesis that poly(I:C) induces the inflammatory responses in human AVICs, mainly through the TLR3-TRIF pathway, we applied specific TLR3 siRNA (150 μmol/l) and TRIF siRNA (100 μmol/l) to knock down TLR3 and TRIF expression. Treatment with TLR3 siRNA reduced the levels of TLR3 by 60% and suppressed the effect of poly(I:C) on the production of the inflammatory mediators examined (Fig. 2, A and B). Similarly, treatment with TRIF siRNA reduced TRIF protein to extremely low levels and essentially abrogated poly(I:C)-induced production of ICAM-1, IL-6, IL-8, and MCP-1 (Fig. 2, A and C). These results demonstrate that the TLR3-TRIF signaling pathway mediates poly(I:C)-induced proinflammatory responses in human AVICs.

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TLR3 and TRIF mediate the upregulation of inflammatory mediator production by poly(I:C). AVICs were treated with TLR3 siRNA (150 nmol/l) or TRIF siRNA (100 nmol/l) for 72 h to knock down these molecules before stimulation with 2.5 μg/ml poly(I:C) for 24 h. Control cells were treated with scrambled siRNA. A: representative immunoblots show that the protein levels of TLR3 and TRIF are markedly lower in cells treated with specific siRNA. B and C: representative immunoblots with densitometric assessment and ELISA data show that knockdown of either TLR3 or TRIF suppresses the production of ICAM-1, IL-6, IL-8, and MCP-1 induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control; #P < 0.05 vs. poly(I:C) + scrambled siRNA.

dsRNA activates NF-κB and ERK1/2 in a TLR3- and TRIF-dependent manner.

dsRNA activates proinflammatory signaling pathways, including the ERK1/2, p38 MAPK, NF-κB, and IFN regulatory factor 3 pathways (20). We analyzed the phosphorylation of NF-κB p65 and ERK1/2 after treatment with poly(I:C) and observed that poly(I:C) induced rapid and sustained phosphorylation of both NF-κB and ERK1/2 (Fig. 3A). The levels of phosphorylated NF-κB p65 increased at 1 h and were still detectable at 8 h. The level of phosphorylated ERK1/2 increased at 30 min and remained higher at 8 h.

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Poly(I:C) induces the phosphorylation of NF-κB p65 and ERK1/2 via the TLR3-TRIF pathway. A: AVICs were treated with 2.5 μg/ml poly(I:C) for 30 min–8 h. Representative immunoblots and densitometric data show that poly(I:C) increases the levels of phosphorylated NF-κB p65 and phosphorylated ERK1/2, with peaks at 2 h. B and C: AVICs were treated with TLR3 siRNA (150 nmol/l) or TRIF siRNA (100 nmol/l) for 72 h before poly(I:C) stimulation for 2 or 4 h. Representative immunoblots and densitometric data show that knockdown of either TLR3 or TRIF suppresses the phosphorylation of NF-κB p65 and ERK1/2 induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control (Ctrl); #P < 0.05 vs. poly(I:C) + scrambled siRNA. p, phosphorylated; t, total.

We analyzed NF-κB p65 and ERK1/2 phosphorylation after knockdown of TLR3 or TRIF. We stimulated cells with poly(I:C) for 2 or 4 h after treatment with TLR3 siRNA or TRIF siRNA. Knockdown of either TLR3 or TRIF markedly reduced phosphorylated NF-κB p65 and phosphorylated ERK1/2 levels following stimulation with poly(I:C) (Fig. 3, B and C). Thus poly(I:C) activates NF-κB and ERK1/2 in human AVICs through the TLR3-TRIF signaling pathway.

NF-κB mediates the upregulation of inflammatory mediator production by dsRNA.

To determine the relative role of NF-κB and ERK1/2 in poly(I:C)-induced inflammatory mediator production in human AVICs, we applied specific inhibitors of NF-κB and ERK1/2. Interestingly, inhibition of ERK1/2 with MEK1 inhibitor PD98059 failed to reduce ICAM-1 production (Fig. 4A). Whereas MEK1 inhibitor 328000ERK abrogated poly(I:C)-induced ERK1/2 phosphorylation, it also failed to reduce the production of inflammatory mediators (Fig. 4B). In contrast, inhibition of NF-κB with IKK inhibitor Bay11-7082 essentially abrogated the effect of poly(I:C) on inflammatory mediator production (Fig. 4C). These results show that NF-κB, rather than ERK1/2, is the critical signaling molecule that mediates the inflammatory mediator production induced by dsRNA in human AVICs.

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NF-κB, not ERK1/2, is critical for the upregulation of inflammatory mediator production by poly(I:C). A: AVICs were treated with MEK1 inhibitor PD98059 (25 μmol/l), 1 h before poly(I:C) stimulation for 24 h. Representative immunoblot of 3 separate experiments using cell isolates from different donor valves shows that inhibition of ERK1/2 with PD98059 fails to suppress ICAM-1 production induced by poly(I:C). B: AVICs were treated with MEK1 inhibitor 328000ERK (40 μmol/l), 1 h before poly(I:C) stimulation. Representative immunoblot of 3 separate experiments using cell isolates from different donor valves shows that 328000ERK inhibits ERK1/2 phosphorylation. Quantitative data show that inhibition of ERK1/2 with 328000ERK fails to suppress ICAM-1 and cytokine production induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves. C: AVICs were treated with an IKKα inhibitor (Bay11-7082, 2.5 μmol/l), 1 h before poly(I:C) stimulation for 24 h. Representative immunoblots with densitometric analysis and ELISA data show that inhibition of NF-κB essentially abrogates inflammatory mediator production induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control; #P < 0.05 vs. poly(I:C) + DMSO.

NF-κB activated by dsRNA appears not to be the canonical p65/p50 heterodimer.

A previous study found that the specific p50 migration inhibitor SN50 (50 μg/ml) fails to inhibit the poly(I:C)-induced expression of COX-2, PGE2, and ICAM-1 in AVICs (22). To determine the effect of SN50 on cytokine production in human AVICs, we treated cells with a higher concentration of SN50 (100 μg/ml) before poly(I:C) stimulation. As shown in Fig. 5A, treatment with SN50 had no effect on ICAM-1 or cytokine levels. Surprisingly, the levels of ICAM-1 and MCP-1 were increased in cells treated with SN50 and poly(I:C). To confirm that SN50 has an effect on human AVICs, we applied the same concentration of SN50 before treating cells with LPS. As shown in Fig. 5B, SN50 markedly reduced the production of ICAM-1 induced by LPS in human AVICs.

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NF-κB p50 migration inhibitor fails to suppress the upregulation of inflammatory mediator production by poly(I:C). Cells were treated with a NF-κB p50 migration inhibitor (SN50, 100 μg/ml), 1 h before poly(I:C) or LPS (0.20 µg/ml) stimulation for 24 h. A: representative immunoblots with densitometric analysis and ELISA data show that SN50 has no effect on the production of IL-6, IL-8, and MCP-1 in cells treated with poly(I:C) but increases MCP-1 and ICAM-1 levels. Experiments (n = 6) using cell isolates from different donor valves;*P < 0.05 vs. untreated control; #P < 0.05 vs. poly(I:C) alone or poly(I:C) + SN50M. B: representative immunoblots with densitometric analysis show that SN50 markedly reduces ICAM-1 production induced by LPS. Experiments (n = 4) using cell isolates from different donor valves; *P < 0.05 vs. untreated control; #P < 0.05 vs. LPS alone or LPS + SN50M.

To understand why the p50 migration inhibitor has no effect on the proinflammatory effect of poly(I:C), we examined whether the NF-κB p50 subunit and p65/p50 heterodimer are present in human AVICs. As shown in Fig. 6A, NF-κB p50 was immunoprecipitated from human AVICs, and p65 and p105 were coprecipitated with p50. Thus the NF-κB p50 subunit and p65/p50 heterodimers are present in human AVICs. It should be noted that neither poly(I:C) nor LPS affected the association of p65 with p50.

An external file that holds a picture, illustration, etc.
Object name is zh00031780920006.jpg

Inhibition of NF-κB with SN50 prevents TLR4-mediated p65 intranuclear translocation but has a minimal influence on TLR3-mediated p65 intranuclear translocation. A: AVICs were treated with LPS or poly(I:C) for 1 or 2 h. Coimmunoprecipitation was performed to examine heterodimers containing p50. Representative immunoblots of 2 separate experiments show that NF-κB p65 is coprecipitated with p50 in untreated cells and those treated with LPS or poly(I:C). IP, immunoprecipitation; IB, immunoblotting. B: AVICs were treated with poly(I:C) for 1 or 2 h in the presence of SN50 (100 μg/ml). Immunofluorescence staining was applied to localize NF-κB p65 (red). Nuclei were counterstained with bis-benzimide (DAPI; blue), and cells were outlined with Alexa 488-tagged wheat germ agglutinin (green). Representative images of 3 separate experiments show robust intranuclear localization of NF-κB p65 following a treatment with poly(I:C) for 2 h. SN50 has a minimal effect on that induced by poly(I:C). C: AVICs were treated with LPS for 1 or 2 h in the presence of SN50 (100 μg/ml). Immunofluorescence staining was applied to localize NF-κB p65 and p50 (red). Counterstaining was performed as described above. Representative images of 3 separate experiments show that SN50 markedly reduces NF-κB p65 intranuclear translocation induced by LPS. It also prevents NF-κB p50 translocation; ×40 objective.

To understand whether the p65/p50 heterodimer is involved in the proinflammatory responses in human AVICs, we examined NF-κB p65 intranuclear translocation in the presence or absence of SN50. As shown in Fig. 6, B and C, NF-κB p65 intranuclear translocation was observed in cells exposed to poly(I:C) or LPS. Whereas SN50 had a minimal effect on NF-κB p65 intranuclear localization induced by poly(I:C), it greatly reduced NF-κB p65 intranuclear localization induced by LPS (Fig. 6C). In addition, SN50 abrogated NF-κB p50 intranuclear translocation (Fig. 6C). These results suggest that the NF-κB complex, activated through TLR4, is primarily the p65/p50 heterodimer. However, the NF-κB complex, activated by TLR3, is not. This observation may explain why inhibition of p50 migration fails to inhibit the inflammatory responses to TLR3 stimulation.

dsRNA promotes the production of inflammatory mediators in human AVICs.

The result in Fig. 1A shows that treatment of human AVICs with 0.5–10 μg/ml poly(I:C) resulted in a dose-dependent increase in ICAM-1 protein levels. It appears that 2.5 μg/ml poly(I:C) induces a marked increase in ICAM-1 levels, although lower concentrations of poly(I:C) also have effects.

An external file that holds a picture, illustration, etc.
Object name is zh00031780920001.jpg

Poly(I:C) upregulates the production of IL-6, IL-8, MCP-1, and ICAM-1 in human AVICs. A: AVICs isolated from normal human aortic valves were treated with varied concentrations of poly(I:C) for 24 h. Representative immunoblots and densitometric data show that poly(I:C) increases ICAM-1 levels in a dose-dependent fashion. B: human AVICs were treated with 2.5 μg/ml poly(I:C) for 24 h. ELISA data show that poly(I:C) upregulates the production of IL-6, IL-8, and MCP-1. Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control.

We analyzed the levels of IL-6, IL-8, and MCP-1 in culture supernatants of cells exposed to 2.5 μg/ml poly(I:C). As shown in Fig. 1B, poly(I:C) markedly elevated the levels of IL-6 (712 ± 67.2 pg/ml vs. 140 ± 22.4 pg/ml in untreated control; P < 0.05), IL-8 (1,485 ± 366 pg/ml vs. 11.1 ± 7.45 pg/ml in untreated control; P < 0.05), and MCP-1 (829 ± 135 pg/ml vs. 272 ± 66.2 pg/ml in untreated control; P < 0.05). Together, these results show that dsRNA is potent to promote the production of multiple inflammation mediators in human AVICs.

TLR3 and TRIF mediate the proinflammatory effect of dsRNA.

dsRNA can activate multiple signaling pathways, including TLR3, in a variety of cell types (1, 12, 13). TLR3 recruits the adaptor molecule TRIF to activate proinflammatory signaling (37). To test the hypothesis that poly(I:C) induces the inflammatory responses in human AVICs, mainly through the TLR3-TRIF pathway, we applied specific TLR3 siRNA (150 μmol/l) and TRIF siRNA (100 μmol/l) to knock down TLR3 and TRIF expression. Treatment with TLR3 siRNA reduced the levels of TLR3 by 60% and suppressed the effect of poly(I:C) on the production of the inflammatory mediators examined (Fig. 2, A and B). Similarly, treatment with TRIF siRNA reduced TRIF protein to extremely low levels and essentially abrogated poly(I:C)-induced production of ICAM-1, IL-6, IL-8, and MCP-1 (Fig. 2, A and C). These results demonstrate that the TLR3-TRIF signaling pathway mediates poly(I:C)-induced proinflammatory responses in human AVICs.

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TLR3 and TRIF mediate the upregulation of inflammatory mediator production by poly(I:C). AVICs were treated with TLR3 siRNA (150 nmol/l) or TRIF siRNA (100 nmol/l) for 72 h to knock down these molecules before stimulation with 2.5 μg/ml poly(I:C) for 24 h. Control cells were treated with scrambled siRNA. A: representative immunoblots show that the protein levels of TLR3 and TRIF are markedly lower in cells treated with specific siRNA. B and C: representative immunoblots with densitometric assessment and ELISA data show that knockdown of either TLR3 or TRIF suppresses the production of ICAM-1, IL-6, IL-8, and MCP-1 induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control; #P < 0.05 vs. poly(I:C) + scrambled siRNA.

dsRNA activates NF-κB and ERK1/2 in a TLR3- and TRIF-dependent manner.

dsRNA activates proinflammatory signaling pathways, including the ERK1/2, p38 MAPK, NF-κB, and IFN regulatory factor 3 pathways (20). We analyzed the phosphorylation of NF-κB p65 and ERK1/2 after treatment with poly(I:C) and observed that poly(I:C) induced rapid and sustained phosphorylation of both NF-κB and ERK1/2 (Fig. 3A). The levels of phosphorylated NF-κB p65 increased at 1 h and were still detectable at 8 h. The level of phosphorylated ERK1/2 increased at 30 min and remained higher at 8 h.

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Poly(I:C) induces the phosphorylation of NF-κB p65 and ERK1/2 via the TLR3-TRIF pathway. A: AVICs were treated with 2.5 μg/ml poly(I:C) for 30 min–8 h. Representative immunoblots and densitometric data show that poly(I:C) increases the levels of phosphorylated NF-κB p65 and phosphorylated ERK1/2, with peaks at 2 h. B and C: AVICs were treated with TLR3 siRNA (150 nmol/l) or TRIF siRNA (100 nmol/l) for 72 h before poly(I:C) stimulation for 2 or 4 h. Representative immunoblots and densitometric data show that knockdown of either TLR3 or TRIF suppresses the phosphorylation of NF-κB p65 and ERK1/2 induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control (Ctrl); #P < 0.05 vs. poly(I:C) + scrambled siRNA. p, phosphorylated; t, total.

We analyzed NF-κB p65 and ERK1/2 phosphorylation after knockdown of TLR3 or TRIF. We stimulated cells with poly(I:C) for 2 or 4 h after treatment with TLR3 siRNA or TRIF siRNA. Knockdown of either TLR3 or TRIF markedly reduced phosphorylated NF-κB p65 and phosphorylated ERK1/2 levels following stimulation with poly(I:C) (Fig. 3, B and C). Thus poly(I:C) activates NF-κB and ERK1/2 in human AVICs through the TLR3-TRIF signaling pathway.

NF-κB mediates the upregulation of inflammatory mediator production by dsRNA.

To determine the relative role of NF-κB and ERK1/2 in poly(I:C)-induced inflammatory mediator production in human AVICs, we applied specific inhibitors of NF-κB and ERK1/2. Interestingly, inhibition of ERK1/2 with MEK1 inhibitor PD98059 failed to reduce ICAM-1 production (Fig. 4A). Whereas MEK1 inhibitor 328000ERK abrogated poly(I:C)-induced ERK1/2 phosphorylation, it also failed to reduce the production of inflammatory mediators (Fig. 4B). In contrast, inhibition of NF-κB with IKK inhibitor Bay11-7082 essentially abrogated the effect of poly(I:C) on inflammatory mediator production (Fig. 4C). These results show that NF-κB, rather than ERK1/2, is the critical signaling molecule that mediates the inflammatory mediator production induced by dsRNA in human AVICs.

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NF-κB, not ERK1/2, is critical for the upregulation of inflammatory mediator production by poly(I:C). A: AVICs were treated with MEK1 inhibitor PD98059 (25 μmol/l), 1 h before poly(I:C) stimulation for 24 h. Representative immunoblot of 3 separate experiments using cell isolates from different donor valves shows that inhibition of ERK1/2 with PD98059 fails to suppress ICAM-1 production induced by poly(I:C). B: AVICs were treated with MEK1 inhibitor 328000ERK (40 μmol/l), 1 h before poly(I:C) stimulation. Representative immunoblot of 3 separate experiments using cell isolates from different donor valves shows that 328000ERK inhibits ERK1/2 phosphorylation. Quantitative data show that inhibition of ERK1/2 with 328000ERK fails to suppress ICAM-1 and cytokine production induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves. C: AVICs were treated with an IKKα inhibitor (Bay11-7082, 2.5 μmol/l), 1 h before poly(I:C) stimulation for 24 h. Representative immunoblots with densitometric analysis and ELISA data show that inhibition of NF-κB essentially abrogates inflammatory mediator production induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control; #P < 0.05 vs. poly(I:C) + DMSO.

NF-κB activated by dsRNA appears not to be the canonical p65/p50 heterodimer.

A previous study found that the specific p50 migration inhibitor SN50 (50 μg/ml) fails to inhibit the poly(I:C)-induced expression of COX-2, PGE2, and ICAM-1 in AVICs (22). To determine the effect of SN50 on cytokine production in human AVICs, we treated cells with a higher concentration of SN50 (100 μg/ml) before poly(I:C) stimulation. As shown in Fig. 5A, treatment with SN50 had no effect on ICAM-1 or cytokine levels. Surprisingly, the levels of ICAM-1 and MCP-1 were increased in cells treated with SN50 and poly(I:C). To confirm that SN50 has an effect on human AVICs, we applied the same concentration of SN50 before treating cells with LPS. As shown in Fig. 5B, SN50 markedly reduced the production of ICAM-1 induced by LPS in human AVICs.

An external file that holds a picture, illustration, etc.
Object name is zh00031780920005.jpg

NF-κB p50 migration inhibitor fails to suppress the upregulation of inflammatory mediator production by poly(I:C). Cells were treated with a NF-κB p50 migration inhibitor (SN50, 100 μg/ml), 1 h before poly(I:C) or LPS (0.20 µg/ml) stimulation for 24 h. A: representative immunoblots with densitometric analysis and ELISA data show that SN50 has no effect on the production of IL-6, IL-8, and MCP-1 in cells treated with poly(I:C) but increases MCP-1 and ICAM-1 levels. Experiments (n = 6) using cell isolates from different donor valves;*P < 0.05 vs. untreated control; #P < 0.05 vs. poly(I:C) alone or poly(I:C) + SN50M. B: representative immunoblots with densitometric analysis show that SN50 markedly reduces ICAM-1 production induced by LPS. Experiments (n = 4) using cell isolates from different donor valves; *P < 0.05 vs. untreated control; #P < 0.05 vs. LPS alone or LPS + SN50M.

To understand why the p50 migration inhibitor has no effect on the proinflammatory effect of poly(I:C), we examined whether the NF-κB p50 subunit and p65/p50 heterodimer are present in human AVICs. As shown in Fig. 6A, NF-κB p50 was immunoprecipitated from human AVICs, and p65 and p105 were coprecipitated with p50. Thus the NF-κB p50 subunit and p65/p50 heterodimers are present in human AVICs. It should be noted that neither poly(I:C) nor LPS affected the association of p65 with p50.

An external file that holds a picture, illustration, etc.
Object name is zh00031780920006.jpg

Inhibition of NF-κB with SN50 prevents TLR4-mediated p65 intranuclear translocation but has a minimal influence on TLR3-mediated p65 intranuclear translocation. A: AVICs were treated with LPS or poly(I:C) for 1 or 2 h. Coimmunoprecipitation was performed to examine heterodimers containing p50. Representative immunoblots of 2 separate experiments show that NF-κB p65 is coprecipitated with p50 in untreated cells and those treated with LPS or poly(I:C). IP, immunoprecipitation; IB, immunoblotting. B: AVICs were treated with poly(I:C) for 1 or 2 h in the presence of SN50 (100 μg/ml). Immunofluorescence staining was applied to localize NF-κB p65 (red). Nuclei were counterstained with bis-benzimide (DAPI; blue), and cells were outlined with Alexa 488-tagged wheat germ agglutinin (green). Representative images of 3 separate experiments show robust intranuclear localization of NF-κB p65 following a treatment with poly(I:C) for 2 h. SN50 has a minimal effect on that induced by poly(I:C). C: AVICs were treated with LPS for 1 or 2 h in the presence of SN50 (100 μg/ml). Immunofluorescence staining was applied to localize NF-κB p65 and p50 (red). Counterstaining was performed as described above. Representative images of 3 separate experiments show that SN50 markedly reduces NF-κB p65 intranuclear translocation induced by LPS. It also prevents NF-κB p50 translocation; ×40 objective.

To understand whether the p65/p50 heterodimer is involved in the proinflammatory responses in human AVICs, we examined NF-κB p65 intranuclear translocation in the presence or absence of SN50. As shown in Fig. 6, B and C, NF-κB p65 intranuclear translocation was observed in cells exposed to poly(I:C) or LPS. Whereas SN50 had a minimal effect on NF-κB p65 intranuclear localization induced by poly(I:C), it greatly reduced NF-κB p65 intranuclear localization induced by LPS (Fig. 6C). In addition, SN50 abrogated NF-κB p50 intranuclear translocation (Fig. 6C). These results suggest that the NF-κB complex, activated through TLR4, is primarily the p65/p50 heterodimer. However, the NF-κB complex, activated by TLR3, is not. This observation may explain why inhibition of p50 migration fails to inhibit the inflammatory responses to TLR3 stimulation.

dsRNA promotes the production of inflammatory mediators in human AVICs.

The result in Fig. 1A shows that treatment of human AVICs with 0.5–10 μg/ml poly(I:C) resulted in a dose-dependent increase in ICAM-1 protein levels. It appears that 2.5 μg/ml poly(I:C) induces a marked increase in ICAM-1 levels, although lower concentrations of poly(I:C) also have effects.

An external file that holds a picture, illustration, etc.
Object name is zh00031780920001.jpg

Poly(I:C) upregulates the production of IL-6, IL-8, MCP-1, and ICAM-1 in human AVICs. A: AVICs isolated from normal human aortic valves were treated with varied concentrations of poly(I:C) for 24 h. Representative immunoblots and densitometric data show that poly(I:C) increases ICAM-1 levels in a dose-dependent fashion. B: human AVICs were treated with 2.5 μg/ml poly(I:C) for 24 h. ELISA data show that poly(I:C) upregulates the production of IL-6, IL-8, and MCP-1. Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control.

We analyzed the levels of IL-6, IL-8, and MCP-1 in culture supernatants of cells exposed to 2.5 μg/ml poly(I:C). As shown in Fig. 1B, poly(I:C) markedly elevated the levels of IL-6 (712 ± 67.2 pg/ml vs. 140 ± 22.4 pg/ml in untreated control; P < 0.05), IL-8 (1,485 ± 366 pg/ml vs. 11.1 ± 7.45 pg/ml in untreated control; P < 0.05), and MCP-1 (829 ± 135 pg/ml vs. 272 ± 66.2 pg/ml in untreated control; P < 0.05). Together, these results show that dsRNA is potent to promote the production of multiple inflammation mediators in human AVICs.

TLR3 and TRIF mediate the proinflammatory effect of dsRNA.

dsRNA can activate multiple signaling pathways, including TLR3, in a variety of cell types (1, 12, 13). TLR3 recruits the adaptor molecule TRIF to activate proinflammatory signaling (37). To test the hypothesis that poly(I:C) induces the inflammatory responses in human AVICs, mainly through the TLR3-TRIF pathway, we applied specific TLR3 siRNA (150 μmol/l) and TRIF siRNA (100 μmol/l) to knock down TLR3 and TRIF expression. Treatment with TLR3 siRNA reduced the levels of TLR3 by 60% and suppressed the effect of poly(I:C) on the production of the inflammatory mediators examined (Fig. 2, A and B). Similarly, treatment with TRIF siRNA reduced TRIF protein to extremely low levels and essentially abrogated poly(I:C)-induced production of ICAM-1, IL-6, IL-8, and MCP-1 (Fig. 2, A and C). These results demonstrate that the TLR3-TRIF signaling pathway mediates poly(I:C)-induced proinflammatory responses in human AVICs.

An external file that holds a picture, illustration, etc.
Object name is zh00031780920002.jpg

TLR3 and TRIF mediate the upregulation of inflammatory mediator production by poly(I:C). AVICs were treated with TLR3 siRNA (150 nmol/l) or TRIF siRNA (100 nmol/l) for 72 h to knock down these molecules before stimulation with 2.5 μg/ml poly(I:C) for 24 h. Control cells were treated with scrambled siRNA. A: representative immunoblots show that the protein levels of TLR3 and TRIF are markedly lower in cells treated with specific siRNA. B and C: representative immunoblots with densitometric assessment and ELISA data show that knockdown of either TLR3 or TRIF suppresses the production of ICAM-1, IL-6, IL-8, and MCP-1 induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control; #P < 0.05 vs. poly(I:C) + scrambled siRNA.

dsRNA activates NF-κB and ERK1/2 in a TLR3- and TRIF-dependent manner.

dsRNA activates proinflammatory signaling pathways, including the ERK1/2, p38 MAPK, NF-κB, and IFN regulatory factor 3 pathways (20). We analyzed the phosphorylation of NF-κB p65 and ERK1/2 after treatment with poly(I:C) and observed that poly(I:C) induced rapid and sustained phosphorylation of both NF-κB and ERK1/2 (Fig. 3A). The levels of phosphorylated NF-κB p65 increased at 1 h and were still detectable at 8 h. The level of phosphorylated ERK1/2 increased at 30 min and remained higher at 8 h.

An external file that holds a picture, illustration, etc.
Object name is zh00031780920003.jpg

Poly(I:C) induces the phosphorylation of NF-κB p65 and ERK1/2 via the TLR3-TRIF pathway. A: AVICs were treated with 2.5 μg/ml poly(I:C) for 30 min–8 h. Representative immunoblots and densitometric data show that poly(I:C) increases the levels of phosphorylated NF-κB p65 and phosphorylated ERK1/2, with peaks at 2 h. B and C: AVICs were treated with TLR3 siRNA (150 nmol/l) or TRIF siRNA (100 nmol/l) for 72 h before poly(I:C) stimulation for 2 or 4 h. Representative immunoblots and densitometric data show that knockdown of either TLR3 or TRIF suppresses the phosphorylation of NF-κB p65 and ERK1/2 induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control (Ctrl); #P < 0.05 vs. poly(I:C) + scrambled siRNA. p, phosphorylated; t, total.

We analyzed NF-κB p65 and ERK1/2 phosphorylation after knockdown of TLR3 or TRIF. We stimulated cells with poly(I:C) for 2 or 4 h after treatment with TLR3 siRNA or TRIF siRNA. Knockdown of either TLR3 or TRIF markedly reduced phosphorylated NF-κB p65 and phosphorylated ERK1/2 levels following stimulation with poly(I:C) (Fig. 3, B and C). Thus poly(I:C) activates NF-κB and ERK1/2 in human AVICs through the TLR3-TRIF signaling pathway.

NF-κB mediates the upregulation of inflammatory mediator production by dsRNA.

To determine the relative role of NF-κB and ERK1/2 in poly(I:C)-induced inflammatory mediator production in human AVICs, we applied specific inhibitors of NF-κB and ERK1/2. Interestingly, inhibition of ERK1/2 with MEK1 inhibitor PD98059 failed to reduce ICAM-1 production (Fig. 4A). Whereas MEK1 inhibitor 328000ERK abrogated poly(I:C)-induced ERK1/2 phosphorylation, it also failed to reduce the production of inflammatory mediators (Fig. 4B). In contrast, inhibition of NF-κB with IKK inhibitor Bay11-7082 essentially abrogated the effect of poly(I:C) on inflammatory mediator production (Fig. 4C). These results show that NF-κB, rather than ERK1/2, is the critical signaling molecule that mediates the inflammatory mediator production induced by dsRNA in human AVICs.

An external file that holds a picture, illustration, etc.
Object name is zh00031780920004.jpg

NF-κB, not ERK1/2, is critical for the upregulation of inflammatory mediator production by poly(I:C). A: AVICs were treated with MEK1 inhibitor PD98059 (25 μmol/l), 1 h before poly(I:C) stimulation for 24 h. Representative immunoblot of 3 separate experiments using cell isolates from different donor valves shows that inhibition of ERK1/2 with PD98059 fails to suppress ICAM-1 production induced by poly(I:C). B: AVICs were treated with MEK1 inhibitor 328000ERK (40 μmol/l), 1 h before poly(I:C) stimulation. Representative immunoblot of 3 separate experiments using cell isolates from different donor valves shows that 328000ERK inhibits ERK1/2 phosphorylation. Quantitative data show that inhibition of ERK1/2 with 328000ERK fails to suppress ICAM-1 and cytokine production induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves. C: AVICs were treated with an IKKα inhibitor (Bay11-7082, 2.5 μmol/l), 1 h before poly(I:C) stimulation for 24 h. Representative immunoblots with densitometric analysis and ELISA data show that inhibition of NF-κB essentially abrogates inflammatory mediator production induced by poly(I:C). Experiments (n = 6) using cell isolates from different donor valves; *P < 0.05 vs. untreated control; #P < 0.05 vs. poly(I:C) + DMSO.

NF-κB activated by dsRNA appears not to be the canonical p65/p50 heterodimer.

A previous study found that the specific p50 migration inhibitor SN50 (50 μg/ml) fails to inhibit the poly(I:C)-induced expression of COX-2, PGE2, and ICAM-1 in AVICs (22). To determine the effect of SN50 on cytokine production in human AVICs, we treated cells with a higher concentration of SN50 (100 μg/ml) before poly(I:C) stimulation. As shown in Fig. 5A, treatment with SN50 had no effect on ICAM-1 or cytokine levels. Surprisingly, the levels of ICAM-1 and MCP-1 were increased in cells treated with SN50 and poly(I:C). To confirm that SN50 has an effect on human AVICs, we applied the same concentration of SN50 before treating cells with LPS. As shown in Fig. 5B, SN50 markedly reduced the production of ICAM-1 induced by LPS in human AVICs.

An external file that holds a picture, illustration, etc.
Object name is zh00031780920005.jpg

NF-κB p50 migration inhibitor fails to suppress the upregulation of inflammatory mediator production by poly(I:C). Cells were treated with a NF-κB p50 migration inhibitor (SN50, 100 μg/ml), 1 h before poly(I:C) or LPS (0.20 µg/ml) stimulation for 24 h. A: representative immunoblots with densitometric analysis and ELISA data show that SN50 has no effect on the production of IL-6, IL-8, and MCP-1 in cells treated with poly(I:C) but increases MCP-1 and ICAM-1 levels. Experiments (n = 6) using cell isolates from different donor valves;*P < 0.05 vs. untreated control; #P < 0.05 vs. poly(I:C) alone or poly(I:C) + SN50M. B: representative immunoblots with densitometric analysis show that SN50 markedly reduces ICAM-1 production induced by LPS. Experiments (n = 4) using cell isolates from different donor valves; *P < 0.05 vs. untreated control; #P < 0.05 vs. LPS alone or LPS + SN50M.

To understand why the p50 migration inhibitor has no effect on the proinflammatory effect of poly(I:C), we examined whether the NF-κB p50 subunit and p65/p50 heterodimer are present in human AVICs. As shown in Fig. 6A, NF-κB p50 was immunoprecipitated from human AVICs, and p65 and p105 were coprecipitated with p50. Thus the NF-κB p50 subunit and p65/p50 heterodimers are present in human AVICs. It should be noted that neither poly(I:C) nor LPS affected the association of p65 with p50.

An external file that holds a picture, illustration, etc.
Object name is zh00031780920006.jpg

Inhibition of NF-κB with SN50 prevents TLR4-mediated p65 intranuclear translocation but has a minimal influence on TLR3-mediated p65 intranuclear translocation. A: AVICs were treated with LPS or poly(I:C) for 1 or 2 h. Coimmunoprecipitation was performed to examine heterodimers containing p50. Representative immunoblots of 2 separate experiments show that NF-κB p65 is coprecipitated with p50 in untreated cells and those treated with LPS or poly(I:C). IP, immunoprecipitation; IB, immunoblotting. B: AVICs were treated with poly(I:C) for 1 or 2 h in the presence of SN50 (100 μg/ml). Immunofluorescence staining was applied to localize NF-κB p65 (red). Nuclei were counterstained with bis-benzimide (DAPI; blue), and cells were outlined with Alexa 488-tagged wheat germ agglutinin (green). Representative images of 3 separate experiments show robust intranuclear localization of NF-κB p65 following a treatment with poly(I:C) for 2 h. SN50 has a minimal effect on that induced by poly(I:C). C: AVICs were treated with LPS for 1 or 2 h in the presence of SN50 (100 μg/ml). Immunofluorescence staining was applied to localize NF-κB p65 and p50 (red). Counterstaining was performed as described above. Representative images of 3 separate experiments show that SN50 markedly reduces NF-κB p65 intranuclear translocation induced by LPS. It also prevents NF-κB p50 translocation; ×40 objective.

To understand whether the p65/p50 heterodimer is involved in the proinflammatory responses in human AVICs, we examined NF-κB p65 intranuclear translocation in the presence or absence of SN50. As shown in Fig. 6, B and C, NF-κB p65 intranuclear translocation was observed in cells exposed to poly(I:C) or LPS. Whereas SN50 had a minimal effect on NF-κB p65 intranuclear localization induced by poly(I:C), it greatly reduced NF-κB p65 intranuclear localization induced by LPS (Fig. 6C). In addition, SN50 abrogated NF-κB p50 intranuclear translocation (Fig. 6C). These results suggest that the NF-κB complex, activated through TLR4, is primarily the p65/p50 heterodimer. However, the NF-κB complex, activated by TLR3, is not. This observation may explain why inhibition of p50 migration fails to inhibit the inflammatory responses to TLR3 stimulation.

DISCUSSION

CAVD is a leading cardiovascular disease in the elderly. Chronic inflammation and calcification processes eventually result in valvular failure. Currently, no pharmacological intervention is available for prevention of CAVD progression. Inflammatory responses of AVICs to noxious stimuli are known to elicit the osteogenic responses in human AVICs, leading to exaggerated valvular calcification (6, 11, 30). Whereas AVIC inflammatory responses are important in CAVD progression, the molecular mechanism underlying the responses remains incompletely understood. In this study, we found that poly(I:C), a synthetic analog of dsRNA, is capable of upregulating the production of multiple inflammatory mediators in human AVICs through activation of the TLR3-TRIF-noncanonical NF-κB pathway.

dsRNA induces the inflammatory responses in human AVICs through TLR3 and TRIF.

dsRNA derived from viruses or endogenous origin is a potent modulator of cell functions and is involved in antiviral defense (2, 34). Extracellular nucleic acids are detected in aortic valve lesions and atherosclerotic lesions and are able to evoke inflammatory responses in cultured vascular cells (32). Inflammatory cell infiltration, resident cell damage, and extracellular matrix remodeling occur with the development of aortic valve lesions associated with CAVD (21).

The dsRNA mimic, poly(I:C), has been reported to induce the production of ICAM-1 in AVICs (22), but the effect of poly(I:C) on proinflammatory cytokine production in human AVICs remains unclear. In the present study, we observed that poly(I:C) is capable of upregulating the production of MCP-1, IL-8, IL-6, and ICAM-1 in human AVICs. MCP-1, IL-8, and ICAM-1 are critical mediators for the recruitment of leukocytes. IL-6 can induce osteogenic response in vascular and valvular cells (6, 28). As poly(I:C) is potent in elevating the production of all of these inflammatory mediators in human AVICs, it is possible that extracellular dsRNA in aortic valve tissues with cell damage causes the production and release of these inflammatory mediators that recruit inflammatory cells to exaggerate the inflammatory changes associated with the development and progression of CAVD.

Our results show that TLR3 and TRIF play a critical role in mediating the proinflammation effect of poly(I:C). Knockdown of either TLR3 or TRIF markedly suppressed the effect of poly(I:C) on the production of IL-6, IL-8, MCP-1, and ICAM-1. Together, these results demonstrate that poly(I:C) exerts its proinflammatory effect on human AVICs through the TLR3-TRIF signaling pathway.

TLR3-TRIF-dependent NF-κB activation is required for the inflammatory responses to dsRNA in human AVICs.

Upon activation of TLR3 by dsRNA, TRIF interacts with TLR3 and acts as an adaptor to recruit downstream signaling molecules. TLR3 activates NF-κB in 293T human embryonic kidney cells (26). TLR3 signaling also activates the MAP3 kinase tumor progression locus 2 to mediate ERK-dependent signaling in bone marrow-derived macrophages (17). Our previous study has shown that stimulation of TLR4 upregulates IL-8 and ICAM-1 production in human AVICs through NF-κB and ERK1/2 (44). We tested the hypothesis that NF-κB and ERK1/2 play a role in the dsRNA-induced inflammatory response in human AVICs. The results of the present study show that poly(I:C) induces rapid and sustained phosphorylation of NF-κB p65 and ERK1/2. Phosphorylation of these two signaling molecules was markedly reduced by knockdown of either TLR3 or TRIF. These results indicate that dsRNA activates NF-κB and ERK1/2 in human AVICs through the TLR3-TRIF signaling pathway.

To confirm the role of NF-κB and ERK1/2 in the upregulation of IL-6, IL-8, MCP-1, and ICAM-1 production, we applied the specific inhibitor Bay11-7082 to inhibit NF-κB and 328000ERK to inhibit ERK1/2. Inhibition of NF-κB with Bay11-7082 abrogated the effect of poly(I:C) on the production of inflammatory mediators. However, inhibition of ERK1/2 with either PD98059 or 328000ERK had no effect on ICAM-1 production induced by poly(I:C). In addition, 328000ERK had no effect on cytokine levels in cells exposed to poly(I:C), although it abolished ERK1/2 phosphorylation. Thus NF-κB plays a critical role in poly(I:C)-induced production of IL-6, IL-8, MCP-1, and ICAM-1 in human AVICs. Previous studies have shown that poly(I:C) induces ERK-dependent TNF-α and IL-1 production in macrophages (17, 36). The results of the present study show that poly(I:C) upregulates the production of IL-6, IL-8, MCP-1, and ICAM-1, independent of the ERK1/2 pathway, although poly(I:C) induces ERK1/2 phosphorylation. This difference in the role of ERK1/2 may be due to distinct cell types examined.

Noncanonical NF-κB complex mediates the inflammatory responses to dsRNA in human AVICs.

The family of NF-κB consists of five members: NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA (p65), c-Rel, and RelB. RelA, c-Rel, and RelB can form homodimers or heterodimers contributing to the variety and selectivity of NF-κB response (8, 33). Studies have reported that the canonical activation of NF-κB by TLR4 involves mainly the p65/p50 heterodimer (7, 9), which is predominant in most mammalian cell types (33).

SN50 prevents p50 translocation into the nucleus. In contrast to the IKK inhibitor, NF-κB p50 migration inhibitor SN50 fails to suppress the upregulation of inflammatory mediator production by poly(I:C). The results from the experiments using SN50 are consistent with the study by López et al. (22) that suggests that NF-κB is not involved in the upregulation of COX-2, PGE2, and ICAM-1 by a high concentration of poly(I:C) in AVICs. However, inhibition of NF-κB p50 translocation suppressed the inflammatory responses induced by the TLR4 agonist LPS. It appears that the NF-κB dimer activated by TLR4 includes p65/p50, but that activated by TLR3 does not.

We confirmed the existence of p50 and p65/p50 heterodimers in human AVICs. NF-κB p65 translocates into the nucleus in response to TLR3 stimulation by poly(I:C) or TLR4 stimulation by LPS. Inhibition of p50 migration with SN50 cannot prevent p65 intranuclear translocation induced by poly(I:C), whereas p65 intranuclear translocation induced by LPS is essentially abolished by SN50. Taken together, stimulation of TLR4 activates NF-κB p65/p50 heterodimers in human AVICs, whereas the NF-κB complex activated by stimulation of TLR3 is not the canonical p65/p50 heterodimer. Moreover, the fact that NF-κB-dependent inflammatory responses to TLR3 are not suppressed by SN50 also supports this notion. It appears that TLR3 activates NF-κB dimers containing p65 without p50 in human AVICs. Five family members have been identified in mammals—RelA (p65), c-Rel, RelB, and the precursor proteins NF-κB1 (p105) and NF-κB2 (p100)—that are processed into p52 and p50, respectively. Studies have reported that dsRNA activates p52, c-Rel, and RelB in immune cells (5, 31). It is possible that dimers of p65 with one of these subunits play a role in mediating the inflammatory responses to TLR3 stimulation in human AVICs. Further study is needed to identify the noncanonical NF-κB complex involved.

Interestingly, we observed that the levels of ICAM-1 and MCP-1 are higher in AVICs exposed to SN50 plus poly(I:C) than cells exposed to poly(I:C) alone. It appears that inhibition of p50 translocation enhances the inflammatory responses to TLR3 stimulation. Studies have reported that the p50/p50 homodimer acts as an anti-inflammatory regulator (18, 19). Thus in human AVICs, stimulation of TLR3 may also lead to the activation of the NF-κB p50/p50 homodimer to balance the pro- and anti-inflammatory responses.

Conclusion.

Poly(I:C), a mimic of dsRNA, upregulates the production of IL-6, IL-8, MCP-1, and ICAM-1 in human AVICs through the TLR3-TRIF-NF-κB signaling pathway. The NF-κB dimer complex, which mediates the inflammatory responses to TLR3 stimulation by dsRNA, is not the p65/p50 heterodimer. These findings provide insights into the mechanism underlying AVIC inflammatory responses to noxious stimuli.

dsRNA induces the inflammatory responses in human AVICs through TLR3 and TRIF.

dsRNA derived from viruses or endogenous origin is a potent modulator of cell functions and is involved in antiviral defense (2, 34). Extracellular nucleic acids are detected in aortic valve lesions and atherosclerotic lesions and are able to evoke inflammatory responses in cultured vascular cells (32). Inflammatory cell infiltration, resident cell damage, and extracellular matrix remodeling occur with the development of aortic valve lesions associated with CAVD (21).

The dsRNA mimic, poly(I:C), has been reported to induce the production of ICAM-1 in AVICs (22), but the effect of poly(I:C) on proinflammatory cytokine production in human AVICs remains unclear. In the present study, we observed that poly(I:C) is capable of upregulating the production of MCP-1, IL-8, IL-6, and ICAM-1 in human AVICs. MCP-1, IL-8, and ICAM-1 are critical mediators for the recruitment of leukocytes. IL-6 can induce osteogenic response in vascular and valvular cells (6, 28). As poly(I:C) is potent in elevating the production of all of these inflammatory mediators in human AVICs, it is possible that extracellular dsRNA in aortic valve tissues with cell damage causes the production and release of these inflammatory mediators that recruit inflammatory cells to exaggerate the inflammatory changes associated with the development and progression of CAVD.

Our results show that TLR3 and TRIF play a critical role in mediating the proinflammation effect of poly(I:C). Knockdown of either TLR3 or TRIF markedly suppressed the effect of poly(I:C) on the production of IL-6, IL-8, MCP-1, and ICAM-1. Together, these results demonstrate that poly(I:C) exerts its proinflammatory effect on human AVICs through the TLR3-TRIF signaling pathway.

TLR3-TRIF-dependent NF-κB activation is required for the inflammatory responses to dsRNA in human AVICs.

Upon activation of TLR3 by dsRNA, TRIF interacts with TLR3 and acts as an adaptor to recruit downstream signaling molecules. TLR3 activates NF-κB in 293T human embryonic kidney cells (26). TLR3 signaling also activates the MAP3 kinase tumor progression locus 2 to mediate ERK-dependent signaling in bone marrow-derived macrophages (17). Our previous study has shown that stimulation of TLR4 upregulates IL-8 and ICAM-1 production in human AVICs through NF-κB and ERK1/2 (44). We tested the hypothesis that NF-κB and ERK1/2 play a role in the dsRNA-induced inflammatory response in human AVICs. The results of the present study show that poly(I:C) induces rapid and sustained phosphorylation of NF-κB p65 and ERK1/2. Phosphorylation of these two signaling molecules was markedly reduced by knockdown of either TLR3 or TRIF. These results indicate that dsRNA activates NF-κB and ERK1/2 in human AVICs through the TLR3-TRIF signaling pathway.

To confirm the role of NF-κB and ERK1/2 in the upregulation of IL-6, IL-8, MCP-1, and ICAM-1 production, we applied the specific inhibitor Bay11-7082 to inhibit NF-κB and 328000ERK to inhibit ERK1/2. Inhibition of NF-κB with Bay11-7082 abrogated the effect of poly(I:C) on the production of inflammatory mediators. However, inhibition of ERK1/2 with either PD98059 or 328000ERK had no effect on ICAM-1 production induced by poly(I:C). In addition, 328000ERK had no effect on cytokine levels in cells exposed to poly(I:C), although it abolished ERK1/2 phosphorylation. Thus NF-κB plays a critical role in poly(I:C)-induced production of IL-6, IL-8, MCP-1, and ICAM-1 in human AVICs. Previous studies have shown that poly(I:C) induces ERK-dependent TNF-α and IL-1 production in macrophages (17, 36). The results of the present study show that poly(I:C) upregulates the production of IL-6, IL-8, MCP-1, and ICAM-1, independent of the ERK1/2 pathway, although poly(I:C) induces ERK1/2 phosphorylation. This difference in the role of ERK1/2 may be due to distinct cell types examined.

Noncanonical NF-κB complex mediates the inflammatory responses to dsRNA in human AVICs.

The family of NF-κB consists of five members: NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA (p65), c-Rel, and RelB. RelA, c-Rel, and RelB can form homodimers or heterodimers contributing to the variety and selectivity of NF-κB response (8, 33). Studies have reported that the canonical activation of NF-κB by TLR4 involves mainly the p65/p50 heterodimer (7, 9), which is predominant in most mammalian cell types (33).

SN50 prevents p50 translocation into the nucleus. In contrast to the IKK inhibitor, NF-κB p50 migration inhibitor SN50 fails to suppress the upregulation of inflammatory mediator production by poly(I:C). The results from the experiments using SN50 are consistent with the study by López et al. (22) that suggests that NF-κB is not involved in the upregulation of COX-2, PGE2, and ICAM-1 by a high concentration of poly(I:C) in AVICs. However, inhibition of NF-κB p50 translocation suppressed the inflammatory responses induced by the TLR4 agonist LPS. It appears that the NF-κB dimer activated by TLR4 includes p65/p50, but that activated by TLR3 does not.

We confirmed the existence of p50 and p65/p50 heterodimers in human AVICs. NF-κB p65 translocates into the nucleus in response to TLR3 stimulation by poly(I:C) or TLR4 stimulation by LPS. Inhibition of p50 migration with SN50 cannot prevent p65 intranuclear translocation induced by poly(I:C), whereas p65 intranuclear translocation induced by LPS is essentially abolished by SN50. Taken together, stimulation of TLR4 activates NF-κB p65/p50 heterodimers in human AVICs, whereas the NF-κB complex activated by stimulation of TLR3 is not the canonical p65/p50 heterodimer. Moreover, the fact that NF-κB-dependent inflammatory responses to TLR3 are not suppressed by SN50 also supports this notion. It appears that TLR3 activates NF-κB dimers containing p65 without p50 in human AVICs. Five family members have been identified in mammals—RelA (p65), c-Rel, RelB, and the precursor proteins NF-κB1 (p105) and NF-κB2 (p100)—that are processed into p52 and p50, respectively. Studies have reported that dsRNA activates p52, c-Rel, and RelB in immune cells (5, 31). It is possible that dimers of p65 with one of these subunits play a role in mediating the inflammatory responses to TLR3 stimulation in human AVICs. Further study is needed to identify the noncanonical NF-κB complex involved.

Interestingly, we observed that the levels of ICAM-1 and MCP-1 are higher in AVICs exposed to SN50 plus poly(I:C) than cells exposed to poly(I:C) alone. It appears that inhibition of p50 translocation enhances the inflammatory responses to TLR3 stimulation. Studies have reported that the p50/p50 homodimer acts as an anti-inflammatory regulator (18, 19). Thus in human AVICs, stimulation of TLR3 may also lead to the activation of the NF-κB p50/p50 homodimer to balance the pro- and anti-inflammatory responses.

Conclusion.

Poly(I:C), a mimic of dsRNA, upregulates the production of IL-6, IL-8, MCP-1, and ICAM-1 in human AVICs through the TLR3-TRIF-NF-κB signaling pathway. The NF-κB dimer complex, which mediates the inflammatory responses to TLR3 stimulation by dsRNA, is not the p65/p50 heterodimer. These findings provide insights into the mechanism underlying AVIC inflammatory responses to noxious stimuli.

dsRNA induces the inflammatory responses in human AVICs through TLR3 and TRIF.

dsRNA derived from viruses or endogenous origin is a potent modulator of cell functions and is involved in antiviral defense (2, 34). Extracellular nucleic acids are detected in aortic valve lesions and atherosclerotic lesions and are able to evoke inflammatory responses in cultured vascular cells (32). Inflammatory cell infiltration, resident cell damage, and extracellular matrix remodeling occur with the development of aortic valve lesions associated with CAVD (21).

The dsRNA mimic, poly(I:C), has been reported to induce the production of ICAM-1 in AVICs (22), but the effect of poly(I:C) on proinflammatory cytokine production in human AVICs remains unclear. In the present study, we observed that poly(I:C) is capable of upregulating the production of MCP-1, IL-8, IL-6, and ICAM-1 in human AVICs. MCP-1, IL-8, and ICAM-1 are critical mediators for the recruitment of leukocytes. IL-6 can induce osteogenic response in vascular and valvular cells (6, 28). As poly(I:C) is potent in elevating the production of all of these inflammatory mediators in human AVICs, it is possible that extracellular dsRNA in aortic valve tissues with cell damage causes the production and release of these inflammatory mediators that recruit inflammatory cells to exaggerate the inflammatory changes associated with the development and progression of CAVD.

Our results show that TLR3 and TRIF play a critical role in mediating the proinflammation effect of poly(I:C). Knockdown of either TLR3 or TRIF markedly suppressed the effect of poly(I:C) on the production of IL-6, IL-8, MCP-1, and ICAM-1. Together, these results demonstrate that poly(I:C) exerts its proinflammatory effect on human AVICs through the TLR3-TRIF signaling pathway.

TLR3-TRIF-dependent NF-κB activation is required for the inflammatory responses to dsRNA in human AVICs.

Upon activation of TLR3 by dsRNA, TRIF interacts with TLR3 and acts as an adaptor to recruit downstream signaling molecules. TLR3 activates NF-κB in 293T human embryonic kidney cells (26). TLR3 signaling also activates the MAP3 kinase tumor progression locus 2 to mediate ERK-dependent signaling in bone marrow-derived macrophages (17). Our previous study has shown that stimulation of TLR4 upregulates IL-8 and ICAM-1 production in human AVICs through NF-κB and ERK1/2 (44). We tested the hypothesis that NF-κB and ERK1/2 play a role in the dsRNA-induced inflammatory response in human AVICs. The results of the present study show that poly(I:C) induces rapid and sustained phosphorylation of NF-κB p65 and ERK1/2. Phosphorylation of these two signaling molecules was markedly reduced by knockdown of either TLR3 or TRIF. These results indicate that dsRNA activates NF-κB and ERK1/2 in human AVICs through the TLR3-TRIF signaling pathway.

To confirm the role of NF-κB and ERK1/2 in the upregulation of IL-6, IL-8, MCP-1, and ICAM-1 production, we applied the specific inhibitor Bay11-7082 to inhibit NF-κB and 328000ERK to inhibit ERK1/2. Inhibition of NF-κB with Bay11-7082 abrogated the effect of poly(I:C) on the production of inflammatory mediators. However, inhibition of ERK1/2 with either PD98059 or 328000ERK had no effect on ICAM-1 production induced by poly(I:C). In addition, 328000ERK had no effect on cytokine levels in cells exposed to poly(I:C), although it abolished ERK1/2 phosphorylation. Thus NF-κB plays a critical role in poly(I:C)-induced production of IL-6, IL-8, MCP-1, and ICAM-1 in human AVICs. Previous studies have shown that poly(I:C) induces ERK-dependent TNF-α and IL-1 production in macrophages (17, 36). The results of the present study show that poly(I:C) upregulates the production of IL-6, IL-8, MCP-1, and ICAM-1, independent of the ERK1/2 pathway, although poly(I:C) induces ERK1/2 phosphorylation. This difference in the role of ERK1/2 may be due to distinct cell types examined.

Noncanonical NF-κB complex mediates the inflammatory responses to dsRNA in human AVICs.

The family of NF-κB consists of five members: NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA (p65), c-Rel, and RelB. RelA, c-Rel, and RelB can form homodimers or heterodimers contributing to the variety and selectivity of NF-κB response (8, 33). Studies have reported that the canonical activation of NF-κB by TLR4 involves mainly the p65/p50 heterodimer (7, 9), which is predominant in most mammalian cell types (33).

SN50 prevents p50 translocation into the nucleus. In contrast to the IKK inhibitor, NF-κB p50 migration inhibitor SN50 fails to suppress the upregulation of inflammatory mediator production by poly(I:C). The results from the experiments using SN50 are consistent with the study by López et al. (22) that suggests that NF-κB is not involved in the upregulation of COX-2, PGE2, and ICAM-1 by a high concentration of poly(I:C) in AVICs. However, inhibition of NF-κB p50 translocation suppressed the inflammatory responses induced by the TLR4 agonist LPS. It appears that the NF-κB dimer activated by TLR4 includes p65/p50, but that activated by TLR3 does not.

We confirmed the existence of p50 and p65/p50 heterodimers in human AVICs. NF-κB p65 translocates into the nucleus in response to TLR3 stimulation by poly(I:C) or TLR4 stimulation by LPS. Inhibition of p50 migration with SN50 cannot prevent p65 intranuclear translocation induced by poly(I:C), whereas p65 intranuclear translocation induced by LPS is essentially abolished by SN50. Taken together, stimulation of TLR4 activates NF-κB p65/p50 heterodimers in human AVICs, whereas the NF-κB complex activated by stimulation of TLR3 is not the canonical p65/p50 heterodimer. Moreover, the fact that NF-κB-dependent inflammatory responses to TLR3 are not suppressed by SN50 also supports this notion. It appears that TLR3 activates NF-κB dimers containing p65 without p50 in human AVICs. Five family members have been identified in mammals—RelA (p65), c-Rel, RelB, and the precursor proteins NF-κB1 (p105) and NF-κB2 (p100)—that are processed into p52 and p50, respectively. Studies have reported that dsRNA activates p52, c-Rel, and RelB in immune cells (5, 31). It is possible that dimers of p65 with one of these subunits play a role in mediating the inflammatory responses to TLR3 stimulation in human AVICs. Further study is needed to identify the noncanonical NF-κB complex involved.

Interestingly, we observed that the levels of ICAM-1 and MCP-1 are higher in AVICs exposed to SN50 plus poly(I:C) than cells exposed to poly(I:C) alone. It appears that inhibition of p50 translocation enhances the inflammatory responses to TLR3 stimulation. Studies have reported that the p50/p50 homodimer acts as an anti-inflammatory regulator (18, 19). Thus in human AVICs, stimulation of TLR3 may also lead to the activation of the NF-κB p50/p50 homodimer to balance the pro- and anti-inflammatory responses.

Conclusion.

Poly(I:C), a mimic of dsRNA, upregulates the production of IL-6, IL-8, MCP-1, and ICAM-1 in human AVICs through the TLR3-TRIF-NF-κB signaling pathway. The NF-κB dimer complex, which mediates the inflammatory responses to TLR3 stimulation by dsRNA, is not the p65/p50 heterodimer. These findings provide insights into the mechanism underlying AVIC inflammatory responses to noxious stimuli.

GRANTS

Support for this study was provided, in part, by the National Heart, Lung, and Blood Institute (Grants HL106582 and {"type":"entrez-nucleotide","attrs":{"text":"HL121776","term_id":"1051700249","term_text":"HL121776"}}HL121776).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Q. Zhan, R.S., and L.A. performed experiments; Q. Zhan and Q. Zeng analyzed data; X.M. interpreted results of experiments; Q. Zhan and F.L. prepared figures; Q. Zhan and F.L. drafted manuscript; Q. Zeng, D.X., D.A.F., and X.M. edited and revised manuscript; X.M. approved final version of manuscript.

Department of Surgery, University of Colorado Denver, Aurora, Colorado; and
Department of Cardiology, Nanfang Hospital, Southern Medical University, Guangzhou, China
Corresponding author.
Address for reprint requests and other correspondence: X. Meng, Dept. of Surgery, University of Colorado, 12700 E. 19th Ave., Aurora, CO 80045 (e-mail: ude.revnedcu@gnem.gnohznaix).
Received 2016 Aug 10; Revised 2016 Dec 8; Accepted 2017 Jan 3.

Abstract

Calcific aortic valve disease is a chronic inflammatory condition, and the inflammatory responses of aortic valve interstitial cells (AVICs) play a critical role in the disease progression. Double-stranded RNA (dsRNA) released from damaged or stressed cells is proinflammatory and may contribute to the mechanism of chronic inflammation observed in diseased aortic valves. The objective of this study is to determine the effect of dsRNA on AVIC inflammatory responses and the underlying mechanism. AVICs from normal human aortic valves were stimulated with polyinosinic-polycytidylic acid [poly(I:C)], a mimic of dsRNA. Poly(I:C) increased the production of IL-6, IL-8, monocyte chemoattractant protein-1, and ICAM-1. Poly(I:C) also induced robust activation of ERK1/2 and NF-κB. Knockdown of Toll-like receptor 3 (TLR3) or Toll-IL-1 receptor domain-containing adapter-inducing IFN-β (TRIF) suppressed ERK1/2 and NF-κB p65 phosphorylation and reduced inflammatory mediator production induced by poly(I:C). Inhibition of NF-κB, not ERK1/2, reduced inflammatory mediator production in AVICs exposed to poly(I:C). Interestingly, inhibition of NF-κB by prevention of p50 migration failed to suppress inflammatory mediator production. NF-κB p65 intranuclear translocation induced by the TLR4 agonist was reduced by inhibition of p50 migration; however, poly(I:C)-induced p65 translocation was not, although the p65/p50 heterodimer is present in AVICs. Poly(I:C) upregulates the production of multiple inflammatory mediators through the TLR3-TRIF-NF-κB pathway in human AVICs. The NF-κB activated by dsRNA appears not to be the canonical p65/p50 heterodimers.

Keywords: TLR3, TRIF, NF-κB, inflammation, aortic valve interstitial cells
Abstract

calcificaorticvalvedisease (CAVD) is the most commonly acquired valvular heart disease in the western society. It occurs in 3% of people who are 65 yr or older in the United States (38). The prevalence of CAVD is increasing with the prolongation of people’s lifespan. However, there are no pharmacological interventions capable of delaying or halting CAVD progression. Thus it is important to understand the mechanism responsible for CAVD progression.

Histological analysis revealed that the early pathological change in CAVD is characterized by chronic inflammation with leukocyte infiltration, including macrophages, T lymphocytes, and mast cells (28, 29). The presence of inflammatory infiltrates is associated with osteogenic metaplastic changes in the aortic valve tissue (4). In this regard, proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, are believed to play a role in exaggeration of inflammation-related valvular calcification (40).

Aortic valve interstitial cells (AVICs), the main cellular component of the aortic valve, play an important role in the pathogenesis of CAVD (27). Our previous studies have demonstrated that AVICs can produce ICAM-1, IL-8, and monocyte chemoattractant protein-1 (MCP-1) when stimulated with agonists to Toll-like receptor 2 (TLR2) and TLR4 (24, 44). More importantly, AVICs differentiate into “osteoblast-like” cells characterized by increased alkaline phosphatase activity and greater levels of Runt-related transcription factor 2 when they are stimulated by cytokines and TLR2/4 agonists (24, 41, 42). Thus the proinflammatory mechanisms contribute to aortic valve calcification through upregulation of AVIC pro-osteogenic activity.

Cells produce and secrete nucleotides, including double-stranded RNA (dsRNA), when they are damaged or under stress. Extracellular nucleic acids, including dsRNA, act as damage-associated molecular patterns and play a role in the pathophysiology of a number of diseases and conditions (14, 15). TLRs recognize pathogen- and damage-associated molecular patterns. In addition to playing a key role in host defense against infection and damage, TLRs have been linked to the pathogenesis of many chronic inflammatory conditions and autoimmune diseases.

dsRNA can be recognized by TLR3 and several other receptors, including retinoic acid-inducible gene I-like receptors, the nucleotide-binding oligomerization domain-like receptor family, and protein kinase RNA activated (10, 12, 13). TLR3 is a sensor for dsRNA derived from cells with viral infection or sterile damage (39, 43). TLR3 is located on the cell surface or in intracellular endosomes (23, 37). It recruits adaptor protein Toll-IL-1 receptor domain-containing adapter-inducing IFN-β (TRIF) to activate NF-κB, MAPKs, and IFN regulatory factor 3 pathways that regulate the inflammatory responses. Cellular RNA released from damaged cells and polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of dsRNA, are capable of inducing the cytokine response in several cell types through a TLR3-dependent mechanism (3, 16). In addition, a high concentration of poly(I:C) has been shown to increase the expression of cyclooxygenase-2 (COX-2), PGE2, and ICAM-1 in the AVIC culture (22). Whereas proinflammatory cytokines are capable of modulating aortic valve osteogenic activity, it remains unclear whether dsRNA induces the production of proinflammatory cytokines in human AVICs and which signaling pathway mediates AVIC inflammatory responses to dsRNA. The present study was to determine the proinflammatory effect of dsRNA on human AVICs and the signaling mechanism involved.

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