Journal of Investigative Dermatology , 18 September 2014 2014.12.01 ゼミ M1 中山千華 Toll-Like Receptor 3 Activation Is Required for Normal Skin Barrier Repair Following UV Damage UVダメージからの皮膚バリア機能回復にはToll-Like Receptor3の活性化が必要である [背景と目的] TLR3は微生物由来の核酸を認識して自然免疫を活性化するシグナル伝達レセプターである。さらに微生物の他にも、 UVBダメージによってNHEKから放出されたncRNAがTLR3を活性化され、続いて炎症がされることが分かっている。また、 最近の報告ではこうした免疫応答だけでなく、TLR3のアゴニストであるPoly (I:C)がTLR3の活性化を介して創傷治癒を促 進することが報告されている。そこで筆者達は、 UVBダメージ後の、皮膚バリア機能の回復にはTLR3の活性化が必要で あることを確かめた。 ［略語］ ncRNA, noncoding RNA TLR3, Toll-like receptor 3 snRNA, small nuclear RNA TJ, tight junction NHEK, normal human epidermal keratinocyte TEER, transepithelial electrical resistance Fig1. UVBダメージを受けたケラチノサイトは皮膚バリアに重要な遺伝子 を刺激する Normal human keratinocytes were treated with 1 μg ml−1 Poly (I:C), sonicated keratinocytes, or UVB-treated keratinocytes for 24 hours. Real-time PCR was used to quantify mRNA levels, and fold-change values are calculated relative and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression for (a) lipid transport (ATP-binding cassette subfamily A member 12 (ABCA12)), lipid metabolism (glucocerebrosidase (GBA) and acid sphingomyelinase (SMPD1)), transglutaminase-1 (TGM1), and (b) desmosome (CDSN) and tight junction (occludin (OCLN), TJ protein 1 (TJP1), and claudin 1 (CLDN1)) transcripts. Data are mean±SEM, n=3 and are representative of at least three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 compared with control. τP<0.05, ττP<0.01, and τττP<0.001 comparing sonicated with UVB-treated normal human epidermal keratinocyte (NHEK) treatments. One-way analysis of variance (ANOVA) with the Bonferroni post-test. Fig2. Poly(I:C)はタイトジャンクション機能を増強する Transepithelial electrical resistance (TEER) was measured in confluent differentiated primary human keratinocytes grown in transwell inserts that were treated with various concentrations of Poly (I:C) for 24 hours (a) and 48 hours (b). (c) Time-course data of TEER values. (d) Paracellular flux was measured 30 minutes after addition of fluorescein sodium to differentiated keratinocytes that were treated with various concentrations of Poly (I:C) for 48 hours. Data are mean±SEM, n=3–8, and are representative of at least three independent experiments. *P<0.05, **P<0.01, and ***P<0.001. One-tailed t-test. Fig3. TLR3の活性化はU1 RNA起因の皮膚バリア遺伝子の発現変化に必要である . TLR3 was silenced in normal human epidermal keratinocytes (NHEKs) for 48 hours before treatment with 1 μg ml−1 U1 RNA or 1 μg ml−1 Poly (I:C) for 24 hours. Real-time PCR was used to quantify (a) ATP-binding cassette subfamily A member 12 (ABCA12), (b) glucocerebrosidase (GBA), (c) acid sphingomyelinase (SMPD1), and (d) tumor necrosis factor-α (TNFα) mRNA levels, and fold-change values are calculated relative to and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Data are mean±SEM, n=3, and are representative of at least three independent experiments. *P<0.05, **P<0.01, and ***P<0.001. Two-tailed t-test. Ctrl, control. Fig4.バリア機能関連・炎症性サイトカイン遺伝子発現に与える様々なsnRNA. (a) Structures of snRNA species generated using RNAfold and VARNA applet. (b) Normal human epidermal keratinocytes (NHEKs) were treated with 1 μg ml−1 in vitro transcribed snRNAs for 24 hours in the presence of Dharmafect 1. Real-time PCR was used to quantify mRNA levels, and fold-change values are calculated relative to and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and then to NHEKs that have been treated with a control in vitro transcribed RNA. Data are mean±SEM, n=3, and are representative of at least three independent experiments. *P<0.05, **P<0.01, and ***P<0.001. Two-tailed t-test. ABCA12, ATP-binding cassette subfamily A member 12; GBA, glucocerebrosidase; SMPD1, acid sphingomyelinase; TGM1, transglutaminase 1; TNF, tumor necrosis factor. Fig5 Tlr3-/-マウスではUVBによって崩れたバリア機能からの回復が遅れる Tlr3−/− mice exhibit delayed barrier repair following UV treatment. (a) Transepidermal water loss (TEWL) values were measured daily for 5 days in wild-type (WT) and Tlr3−/− mice exposed to 5 kJ m−2 UVB. Data are mean±SEM, n=3 WT, n=5 Tlr3−/−, and are representative of at least two independent experiments. *P<0.05, two-way analysis of variance (ANOVA). (b) Barrier recovery between days 3 and 4. One-tailed t-test. Skin was harvested from mice 24 hours after treatment with 5 kJ m−2 UVB. (c) Toluidine blue-stained ultrathin sections. Bar=20 μm. (d) Transmission electron microscopy images of UVB-treated skin of WT and Tlr3−/− mice. Bar=1 μm. (e, f) Mice were lethally irradiated and subsequently reconstituted with bone marrow 7 weeks before UVB irradiation and TEWL measurements. Data are mean±SEM, n=6–8, and are representative of at least two independent experiments. *P<0.05 and **P<0.01. Two-way analysis of variance (ANOVA). TLR, Toll-like receptor 3. (e, f) Mice were lethally irradiated and subsequently reconstituted with bone marrow 7 weeks before UVB irradiation and TEWL measurements. Data are mean±SEM, n=6–8, and are representative of at least two independent experiments. *P<0.05 and **P<0.01. Two-way analysis of variance (ANOVA). TLR, Toll-like receptor 3. まとめ ●UVBダメージによって、NHEKはバリア機能関連遺伝子の発現を促進する ●UVBダメージを受けたNHEKから放出される物質がTLR3を介してバリア機能の回復の影響を与える ● snRNAとPoly(I:C)はNHEKにおいて同様の応答をする ●TLR3欠損マウスはバリア機能の回復が遅れる ➣紫外線によってダメージを受けた皮膚のバリア機能回復にはTLR3の活性化が必要である
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