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Toll-like receptor 3 activation is required for normal skin barrier repair following UV damage.

Borkowski AW, Kuo IH, Bernard JJ, Yoshida T, Williams MR, Hung NJ, Yu BD, Beck LA, Gallo RL - J. Invest. Dermatol. (2014)

Bottom Line: UV damage to the skin leads to the release of noncoding RNA (ncRNA) from necrotic keratinocytes that activates Toll-like receptor 3 (TLR3).This release of ncRNA triggers inflammation in the skin following UV damage.Combined, these data further validate the conclusion that recognition of endogenous RNA by TLR3 is an important step in the program of skin barrier repair.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, Department of Medicine, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
UV damage to the skin leads to the release of noncoding RNA (ncRNA) from necrotic keratinocytes that activates Toll-like receptor 3 (TLR3). This release of ncRNA triggers inflammation in the skin following UV damage. Recently, TLR3 activation was also shown to aid wound repair and increase the expression of genes associated with permeability barrier repair. Here, we sought to test whether skin barrier repair after UVB damage is dependent on the activation of TLR3. We observed that multiple ncRNAs induced expression of skin barrier repair genes, that the TLR3 ligand Poly (I:C) also induced expression and function of tight junctions, and that the ncRNA U1 acts in a TLR3-dependent manner to induce expression of skin barrier repair genes. These observations were shown to have functional relevance as Tlr3-/- mice displayed a delay in skin barrier repair following UVB damage. Combined, these data further validate the conclusion that recognition of endogenous RNA by TLR3 is an important step in the program of skin barrier repair.

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Tlr3−/− mice exhibit delayed barrier repair following UV-treatment(a) TEWL values were measured daily for 5 days in WT and Tlr3−/− mice exposed to 5 kJ/m2 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 ANOVA. (b) Barrier recovery between day 3 and 4. One-tailed t-test. Skin was harvested from mice 24 hours after treatment with 5 kJ/m2 UVB. (c) Toluidine blue stained ultrathin sections. Scale bar = 20 μm (d) Transmission electron microscopy images of UVB-treated skin of WT and Tlr3−/− mice. Scale bar = 1 μm. (e+f) Mice were lethally irradiated and subsequently reconstituted with bone marrow 7 weeks prior to UVB irradiation and TEWL measurements. Data are mean +/− SEM, n = 6–8, and are representative of at least two independent experiments. * = P < 0.05, ** = P < 0.01 Two-way ANOVA.
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Figure 5: Tlr3−/− mice exhibit delayed barrier repair following UV-treatment(a) TEWL values were measured daily for 5 days in WT and Tlr3−/− mice exposed to 5 kJ/m2 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 ANOVA. (b) Barrier recovery between day 3 and 4. One-tailed t-test. Skin was harvested from mice 24 hours after treatment with 5 kJ/m2 UVB. (c) Toluidine blue stained ultrathin sections. Scale bar = 20 μm (d) Transmission electron microscopy images of UVB-treated skin of WT and Tlr3−/− mice. Scale bar = 1 μm. (e+f) Mice were lethally irradiated and subsequently reconstituted with bone marrow 7 weeks prior to UVB irradiation and TEWL measurements. Data are mean +/− SEM, n = 6–8, and are representative of at least two independent experiments. * = P < 0.05, ** = P < 0.01 Two-way ANOVA.

Mentions: The capacity of UVB irradiated NHEK and dsRNAs to alter the expression of genes involved in barrier repair and increase NHEK tight junction function prompted us to directly test whether TLR3 influences permeability barrier function after UVB injury. Tlr3−/− mice and WT controls were exposed to a single 5 kJ/m2 dose of UVB as previously described (Bernard et al. 2012), and transepidermal water loss (TEWL) was examined to evaluate the kinetics of permeability barrier disruption and repair. This high dose of UVB has been reported to cause apoptosis and necrosis in cell culture and cause permeability barrier disruption in mice (Haratake et al. 1997; Lai et al. 2009; Uchida et al. 2010). Although levels of TEWL in WT and Tlr3−/− mice were similar over the first 3 days following UVB light exposure, Tlr3−/− mice displayed elevated and prolonged high levels of TEWL with significantly higher TEWL at Day 4 (Figure 5a). WT mice exhibited a 3.3-fold faster recovery between days 3 and 4 (p = 0.055) than Tlr3−/− mice (Figure 5b). Interestingly, permeability barrier disruption caused by a chemical depilatory reagent or by tape stripping did not have a significantly different effect on barrier disruption or repair in Tlr3−/− or Trif−/− mice, respectively, when compared to WT mice (Supplementary Figure 1). 24 hours after UVB exposure, no gross morphological differences were detected between WT and Tlr3−/− mice in semi-thin Toluidine blue stained sections (Figure 5c). However, transmission electron microscopy images demonstrated that Tlr3−/− mice display more abundant vacuolization of cells subjacent to the first layer of the stratum granulosum in comparison to WT mice (Figure 5d). Interestingly, 80% of Tlr3−/− mice exhibited chronic non-healing wounds at 8 and 16 weeks following a single acute 5 kJ/m2 dose of UVB. Photographs of these mice reveal that 4 of 5 Tlr3−/− mice failed to completely reepithelialize at both 8 and 16 weeks after UVB exposure (Supplementary Figure 2).


Toll-like receptor 3 activation is required for normal skin barrier repair following UV damage.

Borkowski AW, Kuo IH, Bernard JJ, Yoshida T, Williams MR, Hung NJ, Yu BD, Beck LA, Gallo RL - J. Invest. Dermatol. (2014)

Tlr3−/− mice exhibit delayed barrier repair following UV-treatment(a) TEWL values were measured daily for 5 days in WT and Tlr3−/− mice exposed to 5 kJ/m2 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 ANOVA. (b) Barrier recovery between day 3 and 4. One-tailed t-test. Skin was harvested from mice 24 hours after treatment with 5 kJ/m2 UVB. (c) Toluidine blue stained ultrathin sections. Scale bar = 20 μm (d) Transmission electron microscopy images of UVB-treated skin of WT and Tlr3−/− mice. Scale bar = 1 μm. (e+f) Mice were lethally irradiated and subsequently reconstituted with bone marrow 7 weeks prior to UVB irradiation and TEWL measurements. Data are mean +/− SEM, n = 6–8, and are representative of at least two independent experiments. * = P < 0.05, ** = P < 0.01 Two-way ANOVA.
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Figure 5: Tlr3−/− mice exhibit delayed barrier repair following UV-treatment(a) TEWL values were measured daily for 5 days in WT and Tlr3−/− mice exposed to 5 kJ/m2 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 ANOVA. (b) Barrier recovery between day 3 and 4. One-tailed t-test. Skin was harvested from mice 24 hours after treatment with 5 kJ/m2 UVB. (c) Toluidine blue stained ultrathin sections. Scale bar = 20 μm (d) Transmission electron microscopy images of UVB-treated skin of WT and Tlr3−/− mice. Scale bar = 1 μm. (e+f) Mice were lethally irradiated and subsequently reconstituted with bone marrow 7 weeks prior to UVB irradiation and TEWL measurements. Data are mean +/− SEM, n = 6–8, and are representative of at least two independent experiments. * = P < 0.05, ** = P < 0.01 Two-way ANOVA.
Mentions: The capacity of UVB irradiated NHEK and dsRNAs to alter the expression of genes involved in barrier repair and increase NHEK tight junction function prompted us to directly test whether TLR3 influences permeability barrier function after UVB injury. Tlr3−/− mice and WT controls were exposed to a single 5 kJ/m2 dose of UVB as previously described (Bernard et al. 2012), and transepidermal water loss (TEWL) was examined to evaluate the kinetics of permeability barrier disruption and repair. This high dose of UVB has been reported to cause apoptosis and necrosis in cell culture and cause permeability barrier disruption in mice (Haratake et al. 1997; Lai et al. 2009; Uchida et al. 2010). Although levels of TEWL in WT and Tlr3−/− mice were similar over the first 3 days following UVB light exposure, Tlr3−/− mice displayed elevated and prolonged high levels of TEWL with significantly higher TEWL at Day 4 (Figure 5a). WT mice exhibited a 3.3-fold faster recovery between days 3 and 4 (p = 0.055) than Tlr3−/− mice (Figure 5b). Interestingly, permeability barrier disruption caused by a chemical depilatory reagent or by tape stripping did not have a significantly different effect on barrier disruption or repair in Tlr3−/− or Trif−/− mice, respectively, when compared to WT mice (Supplementary Figure 1). 24 hours after UVB exposure, no gross morphological differences were detected between WT and Tlr3−/− mice in semi-thin Toluidine blue stained sections (Figure 5c). However, transmission electron microscopy images demonstrated that Tlr3−/− mice display more abundant vacuolization of cells subjacent to the first layer of the stratum granulosum in comparison to WT mice (Figure 5d). Interestingly, 80% of Tlr3−/− mice exhibited chronic non-healing wounds at 8 and 16 weeks following a single acute 5 kJ/m2 dose of UVB. Photographs of these mice reveal that 4 of 5 Tlr3−/− mice failed to completely reepithelialize at both 8 and 16 weeks after UVB exposure (Supplementary Figure 2).

Bottom Line: UV damage to the skin leads to the release of noncoding RNA (ncRNA) from necrotic keratinocytes that activates Toll-like receptor 3 (TLR3).This release of ncRNA triggers inflammation in the skin following UV damage.Combined, these data further validate the conclusion that recognition of endogenous RNA by TLR3 is an important step in the program of skin barrier repair.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, Department of Medicine, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
UV damage to the skin leads to the release of noncoding RNA (ncRNA) from necrotic keratinocytes that activates Toll-like receptor 3 (TLR3). This release of ncRNA triggers inflammation in the skin following UV damage. Recently, TLR3 activation was also shown to aid wound repair and increase the expression of genes associated with permeability barrier repair. Here, we sought to test whether skin barrier repair after UVB damage is dependent on the activation of TLR3. We observed that multiple ncRNAs induced expression of skin barrier repair genes, that the TLR3 ligand Poly (I:C) also induced expression and function of tight junctions, and that the ncRNA U1 acts in a TLR3-dependent manner to induce expression of skin barrier repair genes. These observations were shown to have functional relevance as Tlr3-/- mice displayed a delay in skin barrier repair following UVB damage. Combined, these data further validate the conclusion that recognition of endogenous RNA by TLR3 is an important step in the program of skin barrier repair.

Show MeSH
Related in: MedlinePlus