Toll様受容体を介して誘導された気道のIL-17Cは、自己分泌的に上皮の宿主防御を増強する

浜松医科大学学位論文　医博第690号（平成27年3月16日

Idiopathic pleuroparenchymal fibroelastosis: consideration of a clinicopathological entity in a series of Japanese patients

Abstract Background Idiopathic pleuroparenchymal fibroelastosis (IPPFE) is a recently reported group of disorders characterized by fibrotic thickening of the pleural and subpleural parenchyma predominantly in the upper lobes. We report five Japanese cases fulfilling the criteria of IPPFE and address whether it should be considered a separate clinicopathologic entity. And this study was an attempt to identify features in common between IPPFE and previously described idiopathic upper lobe fibrosis (IPUF), allowing IPPFE to be considered as a distinct entity in our Japanese series. Methods Five consecutive cases of idiopathic interstitial lung disease confirmed as IPPFE by surgical lung biopsy were studied. Results There were four males and one female, aged 70±2.76 yr. No associated disorder or presumed cause was found in any case. Lung function tests found a restrictive ventilatory defect (4/5) and/or impairment of DLco (4/5). Chest X-ray showed marked apical pleural thickening in all cases. Computed tomography of the chest in all cases mainly showed intense pleural thickening and volume loss associated with evidence of fibrosis, predominantly in the upper lobes. In all cases in this study, markedly thickened visceral pleura and prominent subpleural fibrosis characterized by both elastic tissue and dense collagen were clearly shown. All cases were alive at the last follow-up, 17.6±13.59 months after diagnosis; however, all had deteriorated both clinically and radiologically. Conclusions IPPFE deserves to be defined as a separate, original clinicopathologic entity owing to its uniformity and IPPFE has some features in common with previously described idiopathic upper lobe fibrosis (IPUF). Our limited experience with a cohort of 5 subjects suggests that IPPFE can be rapidly progressive.</p

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

<div><p>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 <i>de novo</i> 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 <i>in vitro</i>.</p></div

Blocking of TNF receptor signaling did not affect IL-17A/polyI:C-induced proinflammatory cytokine gene expression.

<p>BEAS-2B cells were transfected with TNFR1 siRNA (solid bars) or a random oligomer negative control (shadowed bars) for 24 hours. At 72 hours after transfection, transfected cells were harvested for real-time quantitative PCR analysis following 24 hours of stimulation with IL-17A and/or polyI:C. Treatment with TNFR1 siRNAs reduced endogenous TNFR1 mRNA level about 80% as compared with the negative control (A). Expression of G-CSF and IL-8 mRNAs that were induced by polyI:C or co-treatment with IL-17A/polyI:C was not attenuated by knockdown of TNFR1 (B, C). * p < 0.001, TNFR1 siRNA vs. negative control.</p

Time course analysis of mRNA expression and protein induction in NHBE cells.

<p>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.</p

IL-17A/polyI:C-provoked synergistic induction of proinflammatory cytokines and the contribution of the posttranscriptional process.

<p>Submerged BEAS-2B cells were stimulated with IL-17A and/or polyI:C for 24 hours. G-CSF (A) and IL-8 (B) mRNA induction was evaluated by real-time RT-qPCR and normalized to β-actin levels. Co-treatment with IL-17A and polyI:C synergistically upregulated mRNA expression of these genes. Next, BEAS-2B cells were treated for 24 hours with IL-17A, polyI:C, and cycloheximide (CHX) (5 μg/ml). The mRNA levels of G-CSF (C) and IL-8 (D) were evaluated using real-time RT-qPCR and normalized to β-actin levels. CHX had no effect on the IL-17A/polyI:C-provoked synergistic induction. After overnight stimulation with polyI:C (50 μg/ml) in submerged cultures, BEAS-2B cells were incubated with actinomycin D (Act D, 1μg/ml) together with IL-17A and/or polyI:C. Total RNA was harvested at different time points (0, 0.5, 2 and 6 hours) from Act D, IL-17A and/or polyI:C treated cells. Next, G-CSF (E) and IL-8 (F) mRNA levels were evaluated by RT-qPCR and normalized to GAPDH levels. There was no significant difference between each treatment in two-way ANOVA analyses. Gray dashed lines with circles, Act D only; gray solid lines with squares, Act D + IL-17A; black dashed lines with triangles, Act D + polyI:C; and black solid lines with diamonds, Act D + IL-17A + polyI:C. Results are shown as the mean with S.E. of three independent experiments. * p < 0.01, † p < 0.05 vs. co-treatment with IL-17A and polyI:C.</p

Activation of NF-κB and IRF3 signaling pathways after treatment with IL-17A and/or polyI:C.

<p>BEAS-2B cells were stimulated with IL-17A and/or polyI:C in submerged cultures. Whole cell lysates were obtained at different time points (30, 60, 120 and 240 minutes) after treatment. Phosphorylation of IκBα was assessed by western blotting. β-actin was used as a loading control. Band images from the representative experiment (A) and semi-quantification of phosphorylated IκBα using densitometry (B) are shown. IκB-α phosphorylation was strongly induced at 30–240 minutes in cells treated with polyI:C alone or co-treated with IL-17A and polyI:C. The relative amount of IκB-αphosphorylation was significantly higher in co-treatment conditions with IL-17A and polyI:C as compared to polyI:C treatment alone at 120 and 240 minutes (p = 0.036 and 0.044, respectively). Phosphorylation of IRF3 was also assessed by western blotting. The representative experiment (C) and densitometric analysis (D) are shown. IRF3 phosphorylation was enhanced by polyI:C treatment or co-treatment with IL-17A and polyI:C, and no difference in the relative amount of IRF3 phosphorylation was observed between polyI:C treatment alone and co-treatment with IL-17A and polyI:C. Results of densitometoric analysis represent the mean with S.E. from three independent experiments. C, control. * p < 0.05, polyI:C vs. co-treatment with IL17-A and polyI:C.</p