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Short Wavelength Visible Light Suppresses Innate Immunity-Related Responses by Modulating Protein S-Nitrosylation in Keratinocytes

Open ArchivePublished:December 10, 2015DOI:https://doi.org/10.1016/j.jid.2015.12.004

      Abbreviations:

      AMPs (antimicrobial peptides), HBD (human beta-defensins), NHEKs (normal human epithelial keratinocytes), poly I:C (polyinosinic-polycytidylic acid), SNO (S-nitrosylated), TLRs (Toll-like receptors), UVA (ultraviolet A)
      To the Editor
      The solar radiation spectrum reaching the earth’s surface consists mostly of ultraviolet A (UVA), visible, and infrared light. UVA irradiation has clinical applications for patients requiring local immune-suppression therapy (
      • Weatherhead S.C.
      • Farr P.M.
      • Reynolds N.J.
      Spectral effects of UV on psoriasis.
      ). However, UV radiation is carcinogenic and is not recommended for long-term treatment (
      • Kunisada M.
      • Kumimoto H.
      • Ishizaki K.
      • Sakumi K.
      • Nakabeppu Y.
      • Nishigori C.
      Narrow-band UVB induces more carcinogenic skin tumors than broad-band UVB through the formation of cyclobutane pyrimidine dimer.
      ). Thus, visible light-based therapies with less harmful effects on human skin are desirable. For example, blue light (400–450 nm) irradiation is used therapeutically to treat severe atopic dermatitis (
      • Becker D.
      • Langer E.
      • Seemann M.
      • Seemann G.
      • Fell I.
      • Saloga J.
      • et al.
      Clinical efficacy of blue light full body irradiation as treatment option for severe atopic dermatitis.
      ), 632.8-nm light enhances cell proliferation, and red light (550–670 nm) accelerates epidermal permeability barrier recovery after disruption (
      • Denda M.
      • Fuziwara S.
      Visible radiation affects epidermal permeability barrier recovery: selective effects of red and blue light.
      ,
      • Hu W.P.
      • Wang J.J.
      • Yu C.L.
      • Lan C.C.E.
      • Chen G.S.
      • Yu H.S.
      Helium-neon laser irradiation stimulates cell proliferation through photostimulatory effects in mitochondria.
      ). However, the mechanisms underlying various effects of visible light are not clear.
      Human skin exhibits innate immune responses, such as epithelial defense via antimicrobial peptides (AMPs), and the release of proinflammatory cytokines involved in the recognition of microbes via toll-like receptors (TLRs) (
      • Gallo R.L.
      • Nakatsuji T.
      Microbial symbiosis with the innate immune defense system of the skin.
      ,
      • Meyer T.
      • Stockfleth E.
      • Christophers E.
      Immune response profiles in human skin.
      ). Thus, we investigated the effects of visible light on innate immunity. The survival rate of normal human epithelial keratinocytes (NHEKs) was not affected by visible light irradiation (Supplementary Figure S1 online). Violet or blue light downregulated the mRNA expression levels of AMPs after one or three exposures (Figure 1a, b and Supplementary Figure S2a online). By contrast, UV irradiation upregulated AMPs, except for LL-37 (Supplementary Figure S2b). The expression levels of AMPs (human beta-defensins (HBD-1, -3)) and proinflammatory cytokines (RANTES, MCP-1, and IL-8) decreased after violet light irradiation in 3D skin (Supplementary Figure S2c). This violet light-induced downregulation of AMPs affected bacterial survival. Bacteria grew better in a violet light-irradiated NHEK-conditioned medium than in a control NHEK-conditioned medium, irrespective of polyinosinic-polycytidylic acid (poly I:C), which amplifies innate immune responses (Supplementary Figure S3 online).
      Figure 1
      Figure 1Short wavelength visible light suppressed innate immunity responses in normal human epithelial keratinocytes (NHEKs). Real-time Q-RT-PCR was performed for NHEKs irradiated with each visible light (a) one or (b) three times, and (c) NHEKs were incubated with Pam3CSK4, poly I:C, lipopolysaccharide (LPS), LPS (ultrapure), flagellin, or CpG for 24 hours after violet light irradiation. The graphs depict means ± SD of three independent experiments. *P < 0.05, using one-way ANOVA followed by (a, b) Dunnett’s tests or (c) t-tests. Poly I:C, flagellin, or CpG was incubated with NHEKs for 30 minutes after violet light irradiation or no irradiation. And then, (d) immunoblotting or (e) immunofluorescence was conducted. The arrow designated NF-κB nuclear translocation. Scale bar = 50 μm. Each image was taken under a fluorescence microscope. These data were representative of three independent experiments. ANOVA, analysis of variance; poly I:C; polyinosinic-polycytidylic acid; Q-RT-PCR, quantitative real-time reverse transcriptase-PCR.
      We examined whether violet light influences TLR ligand-induced responses. Poly I:C, but not Pam3, lipopolysaccharide, or CpG, increased HBD-1, -2, and -3 simultaneously, which was significantly decreased by violet light. Flagellin-induced increases in HBD-2 and S100A7 were reduced by violet light (Figure 1c). Poly I:C-induced increases in proinflammatory cytokines were decreased by violet light in NHEKs and 3D models (Supplementary Figure S4a online). Unlike violet light, red light did not affect the poly I:C-induced augmentation of AMPs and proinflammatory cytokines (Supplementary Figure S4b).
      When TLRs are activated via ligands, the NF-κB signaling cascade is activated and proinflammatory cytokines are produced (
      • Goldminz A.M.
      • Au S.C.
      • Kim N.
      • Gottlieb A.B.
      • Lizzul P.F.
      NF-κB: an essential transcription factor in psoriasis.
      ). Interestingly, violet light effectively blocked TLR3- or TLR5-induced NF-κB phosphorylation and IκB degradation, but not UVA (Figure 1d and Supplementary Figure S5 online). Although the long-wavelength range of UVA could not be completely excluded from violet light, we assume that the observed effects were not induced by UVA, based on the distinct effects of UVA and violet light (Supplementary Figures S1b, 2, and 5). p-NF-κB nuclear translocation via poly I:C or flagellin was also inhibited by violet light (Figure 1e).
      Wavelengths of 420–453 nm induce nonenzymatic nitric oxide (NO) generation (
      • Opländer C.
      • Deck A.
      • Volkmar C.M.
      • Kirsch M.
      • Liebmann J.
      • Born M.
      • et al.
      Mechanism and biological relevance of bluelight (420-453 nm)-induced nonenzymatic nitric oxide generation from photolabile nitric oxide derivates in human skin in vitro and in vivo.
      ). Violet light increased nitrite levels, an indirect index of NO production, in the cytosol (Supplementary Figure S6a online). The nitroso donor S-nitroso-cysteine efficiently blocked poly I:C-induced increases in HBD-1, -2, proinflammatory cytokines, and COX-2, but not HBD-3 (Supplementary Figure S6b). HBDs are differentially expressed depending on stimulatory factors and cell type (
      • Yin L.
      • Chino T.
      • Horst O.V.
      • Hacker B.M.
      • Clark E.A.
      • Dale B.A.
      • et al.
      Differential and coordinated expression of defensins and cytokines by gingival epithelial cells and dendritic cells in response to oral bacteria.
      ).
      Nitro-l-arginine, an nitric oxide synthase inhibitor, did not antagonize the effects of violet light (Supplementary Figure S7a online). 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO), an NO scavenger, dramatically blocked the effects of violet light in poly I:C-treated NHEKs, whereas N-acetyl-l-cysteine (NAC), a reactive oxygen species scavenger, did not (Figure 2a and Supplementary Table S1 online). These results suggested that violet light effects are mediated by NO, but not by reactive oxygen species. The violet light-induced abnormal differentiation morphology of the upper spinous and granular layers and downregulation of innate immunity-related proteins were partially recovered by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide in 3D skin (Supplementary Figure S8 online). Although the soluble guanylyl cyclase protein kinase G pathway is involved in NO signaling, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, did not block the effects of violet light (
      • Sawa T.
      • Ihara H.
      • Ida T.
      • Fujii S.
      • Nishida M.
      • Akaike T.
      Formation, signaling functions, and metabolisms of nitrated cyclic nucleotide.
      ) (Supplementary Figure S9 online).
      Figure 2
      Figure 2Violet light-induced alterations in the S-nitrosylation status of proteins. After violet light irradiation, NHEKs were incubated with poly I:C and treated with N-acetyl-l-cysteine (NAC) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO). (a) After 12 hours, real-time Q-RT-PCR was performed. The graphs depict the means ± SD of three independent experiments. *P < 0.05 using one-way ANOVA followed by Bonferroni’s post hoc tests. After violet light irradiation, SNO proteins were visualized on a silver staining gel. The arrowheads indicate the representative bands of SNO proteins showing (b) dose-dependent decreases or (c) fluctuations over time. (d) After violet or red light irradiation, HSP60, FAS, Annexin A2, HSP A8, myosin-9, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), clathrin, or pyruvate kinase was detected. (e) TRIF, Myd88, NF-κB (p50), or NF-κB (p65) was also detected. These data were representative of three independent experiments. ANOVA, analysis of variance; c-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide; NAC, N-acetyl-l-cysteine; NHEKs, normal human epithelial keratinocytes; poly I:C, polyinosinic-polycytidylic acid; Q-RT-PCR, quantitative real-time reverse transcriptase-PCR; SD, standard deviation; SNO, S-nitrosylated.
      A major NO-mediated signaling pathway is S-nitrosylation, a reversible redox modification of cysteine residues (
      • Nakamura T.
      • Tu S.
      • Akhtar M.W.
      • Sunico C.R.
      • Okamoto S.I.
      • Lipton S.A.
      Aberrant protein s-nitrosylation in neurodegenerative diseases.
      ). To examine whether violet light affects S-nitrosylation, we performed a biotin-switch assay to detect S-nitrosylated (SNO) proteins. Violet light, but not UV, decreased the S-nitrosylation status of most proteins compared with control treatments (Figure 2b and Supplementary Figure S10a online). After violet light irradiation, the S-nitrosylation status of proteins changed over time (Figure 2c). Violet light induced more extensive changes in protein S-nitrosylation than green or red light (Supplementary Figure S10b).
      Using liquid chromatography-tandem mass spectrometry, we identified 21 SNO proteins affected by violet light, including proteins involved in inflammation (Supplementary Figure S11 and Table S2 online). Among them, we verified 8 SNO proteins by immunoblotting. S-Nitrosylation of HSP60 and FAS increased, whereas that of Annexin A2, HSPA8, and myosin-9 decreased in response to violet light exposure. S-Nitrosylation of glyceraldehyde-3-phosphate dehydrogenase, clathrin, and pyruvate kinase fluctuated (Figure 2d and Supplementary Figure S12a online). Myd88 and NF-κB (p65/p50) undergo S-nitrosylation, resulting in a loss of activity (
      • Into T.
      • Inomata M.
      • Nakashima M.
      • Shibata K.I.
      • Häcker H.
      • Matsushita K.
      Regulation of MyD88-dependent signaling events by S nitrosylation retards toll-like receptor signal transduction and initiation of acutephase immune responses.
      ,
      • Marshall H.E.
      • Stamler J.S.
      Inhibition of NF-kappa B by S-nitrosylation.
      ). In addition, S-nitrosylation inhibits IKKβ and prevents NF-κB activation (
      • Reynaert N.L.
      • Ckless K.
      • Korn S.H.
      • Vos N.
      • Guala A.S.
      • Wouters E.F.M.
      • et al.
      Nitric oxide represses inhibitory kappaB kinase through S-nitrosylation.
      ). Consistent with these previous reports, we observed S-nitrosylation of TIR-domain-containing adapter-inducing interferon-β (TRIF), Myd88, and NF-κB after violet light irradiation (Figure 2e and Supplementary Figure S12b), implying that this phenomenon is a potential mechanism by which violet light induces anti-inflammatory effects.
      Alteration of S-nitrosylation by violet light irradiation may be implicated in NO transfer between SNO proteins that function as NO acceptors or donors. This redistribution of NO by violet light irradiation may influence SNO protein activity. In addition, cPTIO- attenuation of S-nitrosylation fluctuation stimulated by violet light also strongly supports the possible involvement of NO transfer (Supplementary Figure S13 online).
      Our results suggest that S-nitrosylation is deeply involved in violet light-induced anti-inflammatory effects. In the future, it is necessary to identify SNO proteins that are important in this process; these studies will enable the development of violet light-based phototherapy.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We thank Hee Hwan Kim and Dong Kwon Lee for technical support in operating the LED irradiation system.

      Supplementary Material

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