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Synergistic Proinflammatory Responses by IL-17A and Toll-Like Receptor 3 in Human Airway Epithelial Cells.

Mori K, Fujisawa T, Kusagaya H, Yamanaka K, Hashimoto D, Enomoto N, Inui N, Nakamura Y, Maekawa M, Suda T - PLoS ONE (2015)

Bottom Line: In this study, we demonstrated that IL-17A and polyI:C, the ligand of TLR3, synergistically induced the expression of proinflammatory cytokines and chemokines (G-CSF, IL-8, CXCL1, CXCL5, IL-1F9), but not type I interferon (IFN-α1, -β) in primary culture of normal human bronchial epithelial cells.Inhibition of the TLR3, TLR/TIR-domain-containing adaptor-inducing interferon β (TRIF), NF-κB, and IRF3 pathways decreased the polyI:C- and IL-17A/polyI:C-induced G-CSF and IL-8 mRNA expression.In western blotting analysis, activation of both NF-κB and IRF3 was observed in treatment with polyI:C and co-treatment with IL-17A/polyI:C; moreover, co-treatment with IL-17A/polyI:C augmented IκB-α phosphorylation as compared to polyI:C treatment alone.

View Article: PubMed Central - PubMed

Affiliation: Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama Higashi-ku, Hamamatsu 431-3192, Japan.

ABSTRACT
Viral respiratory infections activate the innate immune response in the airway epithelium through Toll-like receptors (TLRs) and induce airway inflammation, which causes acute exacerbation of asthma. Although increases in IL-17A expression were observed in the airway of severe asthma patients, the interaction between IL-17A and TLR activation in airway epithelium remains poorly understood. In this study, we demonstrated that IL-17A and polyI:C, the ligand of TLR3, synergistically induced the expression of proinflammatory cytokines and chemokines (G-CSF, IL-8, CXCL1, CXCL5, IL-1F9), but not type I interferon (IFN-α1, -β) in primary culture of normal human bronchial epithelial cells. Synergistic induction after co-stimulation with IL-17A and polyI:C was observed from 2 to 24 hours after stimulation. Treatment with cycloheximide or actinomycin D had no effect, suggesting that the synergistic induction occurred without de novo protein synthesis or mRNA stabilization. Inhibition of the TLR3, TLR/TIR-domain-containing adaptor-inducing interferon β (TRIF), NF-κB, and IRF3 pathways decreased the polyI:C- and IL-17A/polyI:C-induced G-CSF and IL-8 mRNA expression. Comparing the levels of mRNA induction between co-treatment with IL-17A/polyI:C and treatment with polyI:C alone, blocking the of NF-κB pathway significantly attenuated the observed synergism. In western blotting analysis, activation of both NF-κB and IRF3 was observed in treatment with polyI:C and co-treatment with IL-17A/polyI:C; moreover, co-treatment with IL-17A/polyI:C augmented IκB-α phosphorylation as compared to polyI:C treatment alone. Collectively, these findings indicate that IL-17A and TLR3 activation cooperate to induce proinflammatory responses in the airway epithelium via TLR3/TRIF-mediated NF-κB/IRF3 activation, and that enhanced activation of the NF-κB pathway plays an essential role in synergistic induction after co-treatment with IL-17A and polyI:C in vitro.

No MeSH data available.


Related in: MedlinePlus

Time course analysis of mRNA expression and protein induction in NHBE cells.NHBE cells in submerged cultures were stimulated with IL-17A and/or polyI:C. Total RNA was extracted from cells at different time points (0, 2, 6, 12, and 24 hours) after treatment. G-CSF (A), IL-8 (B), IFN-α1 (C), and IFN-β (D) levels were evaluated by real-time RT-qPCR and normalized to β-actin levels. Inductions of G-CSF and IL-8 mRNA expression increased over time and was significantly higher in co-treatments with IL-17A and polyI:C than in controls or other treatments. IFN-β mRNA was significantly upregulated by polyI:C or co-treatment with IL-17A and polyI:C at 2, and 6 hours. However, IFN mRNA expression was not different between polyI:C-treatment and co-treatment with IL-17A/polyI:C. Gray dashed lines with circles, unstimulated control; gray solid lines with squares, IL-17A; black dashed lines with triangles, polyI:C; black solid lines with diamonds, co-treatment with IL-17A and polyI:C. The concentration of G-CSF (E) and IL-8 (F) proteins in the conditioned medium of submerged cultures treated with IL-17A and/or polyI:C for 24 hours were detected by ELISA. Synergistic increases in G-CSF and IL-8 in protein levels were observed after co-treatment with IL-17A and polyI:C. Results are shown as the mean with S.E. of three independent experiments. * p < 0.01 versus co-treatment with IL-17A and polyI:C.
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pone.0139491.g002: Time course analysis of mRNA expression and protein induction in NHBE cells.NHBE cells in submerged cultures were stimulated with IL-17A and/or polyI:C. Total RNA was extracted from cells at different time points (0, 2, 6, 12, and 24 hours) after treatment. G-CSF (A), IL-8 (B), IFN-α1 (C), and IFN-β (D) levels were evaluated by real-time RT-qPCR and normalized to β-actin levels. Inductions of G-CSF and IL-8 mRNA expression increased over time and was significantly higher in co-treatments with IL-17A and polyI:C than in controls or other treatments. IFN-β mRNA was significantly upregulated by polyI:C or co-treatment with IL-17A and polyI:C at 2, and 6 hours. However, IFN mRNA expression was not different between polyI:C-treatment and co-treatment with IL-17A/polyI:C. Gray dashed lines with circles, unstimulated control; gray solid lines with squares, IL-17A; black dashed lines with triangles, polyI:C; black solid lines with diamonds, co-treatment with IL-17A and polyI:C. The concentration of G-CSF (E) and IL-8 (F) proteins in the conditioned medium of submerged cultures treated with IL-17A and/or polyI:C for 24 hours were detected by ELISA. Synergistic increases in G-CSF and IL-8 in protein levels were observed after co-treatment with IL-17A and polyI:C. Results are shown as the mean with S.E. of three independent experiments. * p < 0.01 versus co-treatment with IL-17A and polyI:C.

Mentions: Time course analyses indicated that the mRNA expression of G-CSF and IL-8 increased after stimulation with polyI:C (Fig 2A and 2B). Notably, co-treatment with IL-17A and polyI:C synergistically increased G-CSF and IL-8 mRNA expression from 2 to 24 hours after stimulation (Fig 2A and 2B). Although the IFN-β mRNA levels were induced at 2 hours after polyI:C stimulation, there was no synergistic increase in IFN-α1 or –β mRNA levels by co-treatment with IL-17A/polyI:C throughout the time course (Fig 2C and 2D). ELISAs demonstrated a similar trend for the synergistic induction of G-CSF or IL-8 proteins in NHBE cells after 24 hour of treatments (Fig 2E and 2F).


Synergistic Proinflammatory Responses by IL-17A and Toll-Like Receptor 3 in Human Airway Epithelial Cells.

Mori K, Fujisawa T, Kusagaya H, Yamanaka K, Hashimoto D, Enomoto N, Inui N, Nakamura Y, Maekawa M, Suda T - PLoS ONE (2015)

Time course analysis of mRNA expression and protein induction in NHBE cells.NHBE cells in submerged cultures were stimulated with IL-17A and/or polyI:C. Total RNA was extracted from cells at different time points (0, 2, 6, 12, and 24 hours) after treatment. G-CSF (A), IL-8 (B), IFN-α1 (C), and IFN-β (D) levels were evaluated by real-time RT-qPCR and normalized to β-actin levels. Inductions of G-CSF and IL-8 mRNA expression increased over time and was significantly higher in co-treatments with IL-17A and polyI:C than in controls or other treatments. IFN-β mRNA was significantly upregulated by polyI:C or co-treatment with IL-17A and polyI:C at 2, and 6 hours. However, IFN mRNA expression was not different between polyI:C-treatment and co-treatment with IL-17A/polyI:C. Gray dashed lines with circles, unstimulated control; gray solid lines with squares, IL-17A; black dashed lines with triangles, polyI:C; black solid lines with diamonds, co-treatment with IL-17A and polyI:C. The concentration of G-CSF (E) and IL-8 (F) proteins in the conditioned medium of submerged cultures treated with IL-17A and/or polyI:C for 24 hours were detected by ELISA. Synergistic increases in G-CSF and IL-8 in protein levels were observed after co-treatment with IL-17A and polyI:C. Results are shown as the mean with S.E. of three independent experiments. * p < 0.01 versus co-treatment with IL-17A and polyI:C.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4587973&req=5

pone.0139491.g002: Time course analysis of mRNA expression and protein induction in NHBE cells.NHBE cells in submerged cultures were stimulated with IL-17A and/or polyI:C. Total RNA was extracted from cells at different time points (0, 2, 6, 12, and 24 hours) after treatment. G-CSF (A), IL-8 (B), IFN-α1 (C), and IFN-β (D) levels were evaluated by real-time RT-qPCR and normalized to β-actin levels. Inductions of G-CSF and IL-8 mRNA expression increased over time and was significantly higher in co-treatments with IL-17A and polyI:C than in controls or other treatments. IFN-β mRNA was significantly upregulated by polyI:C or co-treatment with IL-17A and polyI:C at 2, and 6 hours. However, IFN mRNA expression was not different between polyI:C-treatment and co-treatment with IL-17A/polyI:C. Gray dashed lines with circles, unstimulated control; gray solid lines with squares, IL-17A; black dashed lines with triangles, polyI:C; black solid lines with diamonds, co-treatment with IL-17A and polyI:C. The concentration of G-CSF (E) and IL-8 (F) proteins in the conditioned medium of submerged cultures treated with IL-17A and/or polyI:C for 24 hours were detected by ELISA. Synergistic increases in G-CSF and IL-8 in protein levels were observed after co-treatment with IL-17A and polyI:C. Results are shown as the mean with S.E. of three independent experiments. * p < 0.01 versus co-treatment with IL-17A and polyI:C.
Mentions: Time course analyses indicated that the mRNA expression of G-CSF and IL-8 increased after stimulation with polyI:C (Fig 2A and 2B). Notably, co-treatment with IL-17A and polyI:C synergistically increased G-CSF and IL-8 mRNA expression from 2 to 24 hours after stimulation (Fig 2A and 2B). Although the IFN-β mRNA levels were induced at 2 hours after polyI:C stimulation, there was no synergistic increase in IFN-α1 or –β mRNA levels by co-treatment with IL-17A/polyI:C throughout the time course (Fig 2C and 2D). ELISAs demonstrated a similar trend for the synergistic induction of G-CSF or IL-8 proteins in NHBE cells after 24 hour of treatments (Fig 2E and 2F).

Bottom Line: In this study, we demonstrated that IL-17A and polyI:C, the ligand of TLR3, synergistically induced the expression of proinflammatory cytokines and chemokines (G-CSF, IL-8, CXCL1, CXCL5, IL-1F9), but not type I interferon (IFN-α1, -β) in primary culture of normal human bronchial epithelial cells.Inhibition of the TLR3, TLR/TIR-domain-containing adaptor-inducing interferon β (TRIF), NF-κB, and IRF3 pathways decreased the polyI:C- and IL-17A/polyI:C-induced G-CSF and IL-8 mRNA expression.In western blotting analysis, activation of both NF-κB and IRF3 was observed in treatment with polyI:C and co-treatment with IL-17A/polyI:C; moreover, co-treatment with IL-17A/polyI:C augmented IκB-α phosphorylation as compared to polyI:C treatment alone.

View Article: PubMed Central - PubMed

Affiliation: Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama Higashi-ku, Hamamatsu 431-3192, Japan.

ABSTRACT
Viral respiratory infections activate the innate immune response in the airway epithelium through Toll-like receptors (TLRs) and induce airway inflammation, which causes acute exacerbation of asthma. Although increases in IL-17A expression were observed in the airway of severe asthma patients, the interaction between IL-17A and TLR activation in airway epithelium remains poorly understood. In this study, we demonstrated that IL-17A and polyI:C, the ligand of TLR3, synergistically induced the expression of proinflammatory cytokines and chemokines (G-CSF, IL-8, CXCL1, CXCL5, IL-1F9), but not type I interferon (IFN-α1, -β) in primary culture of normal human bronchial epithelial cells. Synergistic induction after co-stimulation with IL-17A and polyI:C was observed from 2 to 24 hours after stimulation. Treatment with cycloheximide or actinomycin D had no effect, suggesting that the synergistic induction occurred without de novo protein synthesis or mRNA stabilization. Inhibition of the TLR3, TLR/TIR-domain-containing adaptor-inducing interferon β (TRIF), NF-κB, and IRF3 pathways decreased the polyI:C- and IL-17A/polyI:C-induced G-CSF and IL-8 mRNA expression. Comparing the levels of mRNA induction between co-treatment with IL-17A/polyI:C and treatment with polyI:C alone, blocking the of NF-κB pathway significantly attenuated the observed synergism. In western blotting analysis, activation of both NF-κB and IRF3 was observed in treatment with polyI:C and co-treatment with IL-17A/polyI:C; moreover, co-treatment with IL-17A/polyI:C augmented IκB-α phosphorylation as compared to polyI:C treatment alone. Collectively, these findings indicate that IL-17A and TLR3 activation cooperate to induce proinflammatory responses in the airway epithelium via TLR3/TRIF-mediated NF-κB/IRF3 activation, and that enhanced activation of the NF-κB pathway plays an essential role in synergistic induction after co-treatment with IL-17A and polyI:C in vitro.

No MeSH data available.


Related in: MedlinePlus