Limits...
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

Effects of IRF3 siRNA on inflammatory cytokines gene expression.BEAS-2B cells were transfected with IRF3 siRNA (solid bars) or a random oligomer negative control (shadowed bars). After 24 hours of transfection, the siRNA transfection mix was replaced with a fresh LHC-9 medium. At 2 days after siRNA treatment, transfected cells were incubated for 24 hours with IL-17A and/or polyI:C and mRNA expression was evaluated by real-time RT-qPCR. The IRF3 siRNA reduced endogenous IRF3 mRNA expression (A). Synergistic mRNA induction of G-CSF (B) and IL-8 (C) was significantly suppressed by the siRNA knockdown of IRF3. All measurements were averaged from duplicate wells, and the experiment was repeated three times. * p < 0.001, IRF3 siRNA vs. negative control. † p < 0.01, IRF3 siRNA vs. negative control in polyI:C treatment. ‡ p < 0.01, IRF3 siRNA vs. negative control in co-treatment with IL-17A and polyI:C.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4587973&req=5

pone.0139491.g006: Effects of IRF3 siRNA on inflammatory cytokines gene expression.BEAS-2B cells were transfected with IRF3 siRNA (solid bars) or a random oligomer negative control (shadowed bars). After 24 hours of transfection, the siRNA transfection mix was replaced with a fresh LHC-9 medium. At 2 days after siRNA treatment, transfected cells were incubated for 24 hours with IL-17A and/or polyI:C and mRNA expression was evaluated by real-time RT-qPCR. The IRF3 siRNA reduced endogenous IRF3 mRNA expression (A). Synergistic mRNA induction of G-CSF (B) and IL-8 (C) was significantly suppressed by the siRNA knockdown of IRF3. All measurements were averaged from duplicate wells, and the experiment was repeated three times. * p < 0.001, IRF3 siRNA vs. negative control. † p < 0.01, IRF3 siRNA vs. negative control in polyI:C treatment. ‡ p < 0.01, IRF3 siRNA vs. negative control in co-treatment with IL-17A and polyI:C.

Mentions: To further confirm the involvement of IRF3 in the IL-17A/polyI:C-induced synergistic proinflammatory response, we used an siRNA approach to knock down IRF3. As shown in Fig 6A, IRF3 siRNA was effective in reducing IRF3 expression 80% of its original level. The IRF3 siRNA treatment also suppressed the IL-17A/polyI:C-provoked induction of G-CSF and IL-8 (Fig 6B and 6C). The IRF3 siRNA decreased the amplification ratio (co-treatment/polyI:C) of IL-8 mRNA expression (mean, 9.94 to 5.26, S2 Table), however, there was no reduction observed in the amplification ratio of G-CSF mRNA expression (mean, 6.78 to 11.0, S2 Table), which suggests that the IRF3 pathway was involved in the synergy of IL-8 mRNA expression but was not in the synergy of G-CSF mRNA expression. The detailed mechanism of IL-17A/polyI:C-provoked synergistic G-CSF expression and that of IL-17A/polyI:C-provoked synergistic IL-8 expression may be slightly different. These results indicate that the IRF3 pathway is partially involved in the IL-17A/polyI:C-induced synergistic proinflammatory response in airway epithelial cells, on the other hand, the NF-κB pathway is more important for the synergism than the IRF3 pathway.


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)

Effects of IRF3 siRNA on inflammatory cytokines gene expression.BEAS-2B cells were transfected with IRF3 siRNA (solid bars) or a random oligomer negative control (shadowed bars). After 24 hours of transfection, the siRNA transfection mix was replaced with a fresh LHC-9 medium. At 2 days after siRNA treatment, transfected cells were incubated for 24 hours with IL-17A and/or polyI:C and mRNA expression was evaluated by real-time RT-qPCR. The IRF3 siRNA reduced endogenous IRF3 mRNA expression (A). Synergistic mRNA induction of G-CSF (B) and IL-8 (C) was significantly suppressed by the siRNA knockdown of IRF3. All measurements were averaged from duplicate wells, and the experiment was repeated three times. * p < 0.001, IRF3 siRNA vs. negative control. † p < 0.01, IRF3 siRNA vs. negative control in polyI:C treatment. ‡ p < 0.01, IRF3 siRNA vs. negative control in co-treatment with IL-17A and polyI:C.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139491.g006: Effects of IRF3 siRNA on inflammatory cytokines gene expression.BEAS-2B cells were transfected with IRF3 siRNA (solid bars) or a random oligomer negative control (shadowed bars). After 24 hours of transfection, the siRNA transfection mix was replaced with a fresh LHC-9 medium. At 2 days after siRNA treatment, transfected cells were incubated for 24 hours with IL-17A and/or polyI:C and mRNA expression was evaluated by real-time RT-qPCR. The IRF3 siRNA reduced endogenous IRF3 mRNA expression (A). Synergistic mRNA induction of G-CSF (B) and IL-8 (C) was significantly suppressed by the siRNA knockdown of IRF3. All measurements were averaged from duplicate wells, and the experiment was repeated three times. * p < 0.001, IRF3 siRNA vs. negative control. † p < 0.01, IRF3 siRNA vs. negative control in polyI:C treatment. ‡ p < 0.01, IRF3 siRNA vs. negative control in co-treatment with IL-17A and polyI:C.
Mentions: To further confirm the involvement of IRF3 in the IL-17A/polyI:C-induced synergistic proinflammatory response, we used an siRNA approach to knock down IRF3. As shown in Fig 6A, IRF3 siRNA was effective in reducing IRF3 expression 80% of its original level. The IRF3 siRNA treatment also suppressed the IL-17A/polyI:C-provoked induction of G-CSF and IL-8 (Fig 6B and 6C). The IRF3 siRNA decreased the amplification ratio (co-treatment/polyI:C) of IL-8 mRNA expression (mean, 9.94 to 5.26, S2 Table), however, there was no reduction observed in the amplification ratio of G-CSF mRNA expression (mean, 6.78 to 11.0, S2 Table), which suggests that the IRF3 pathway was involved in the synergy of IL-8 mRNA expression but was not in the synergy of G-CSF mRNA expression. The detailed mechanism of IL-17A/polyI:C-provoked synergistic G-CSF expression and that of IL-17A/polyI:C-provoked synergistic IL-8 expression may be slightly different. These results indicate that the IRF3 pathway is partially involved in the IL-17A/polyI:C-induced synergistic proinflammatory response in airway epithelial cells, on the other hand, the NF-κB pathway is more important for the synergism than the IRF3 pathway.

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