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Insight into the redox status of inflammatory skin equivalents as determined by EPR spectroscopy
Chemico-Biological Interactions 310 (2019) 108752
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Insight into the redox status of inflammatory skin equivalents as determined by EPR spectroscopy
T
Anja Elpelta,b,1, Stephanie Albrechta,1, Christian Teutloffc, Martina Hügingd, Siavash Saeidpourc, Silke B. Lohana, Sarah Hedtrichb,e, Martina C. Meinkea,* a
Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Department of Dermatology, Venerology and Allergology, Center of Experimental and Applied Cutaneous Physiology, Charitéplatz 1, 10117, Berlin, Germany b Institute of Pharmacy, Department of Pharmacology, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195, Berlin, Germany c Department of Physics, Institute of Experimental Physics, Freie Universität Berlin, Arnimallee 14, 14195, Berlin, Germany d Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Department of Pediatric Surgery, Augustenburger Platz 1, 13353, Berlin, Germany e University of British Columbia, Faculty of Pharmaceutical Sciences, 2405 Wesbrook Mall, V6T1Z3, Vancouver, Canada
ARTICLE INFO
ABSTRACT
Keywords: Atopic dermatitis Skin equivalents Redox status Irradiation Electron paramagnetic resonance spectroscopy
Atopic dermatitis (AD) is a chronic inflammatory skin disease whose pathogenesis is still not fully understood. Since inflammatory processes correlate with oxidative stress, the redox status may play a key role in AD. In this study, electron paramagnetic resonance (EPR) spectroscopy was mainly used to investigate the redox status in normal and inflammatory skin equivalents mimicking characteristics of AD in vitro using EPR spin probes (TEMPO, PCA) and a spin trap (DMPO). The total antioxidant status in the hydrophilic and lipophilic compartments of skin (microenvironment) showed no differences between the skin equivalents. In the inflammatory skin equivalents, a decreased glutathione concentration in the epidermis and an increased metabolic radical production could be observed compared to normal skin equivalents. The induction of external stress by simulated solar irradiation (UVB-NIR) resulted in the same amount and type of radicals in normal and inflammatory skin equivalents. For the first time, the antioxidant and oxidant status of inflammatory in vitro skin equivalents was analyzed by EPR to elucidate their redox status using different methods which focus on various microenvironments. Our investigations suggested that the redox status in atopic skin could be different, but this should be investigated more comprehensively, because the results can vary depending on the used methods and where the investigations take place.
1. Introduction Atopic dermatitis (AD) is a chronic inflammatory skin disease representing a common health problem worldwide. The disease affects 15–20% of children and 1–3% of adults. The main symptoms are skin eczema characterized by itching, dryness and crusting, which can impair substantially the quality of life of AD patients. AD is a multifactorial disease combining genetic, immunological and environmental aspects [1,2]. So far, it is known that up to 50% of AD patients have one loss-of-function mutation in the filaggrin gene (FLG), which is an important skin differentiation marker [3]. Furthermore, a changed lipid composition and a lower lipid order in the stratum corneum (outermost skin layer) of atopic skin likely correlates with a disturbed barrier function [4,5]. It is assumed that the disturbed skin barrier increases
the penetration of allergens and pathogens into the skin, which can activate the immune system. A dysfunctional immune response characterized by a Th2-dominated reaction in the acute phase causes the release of proinflammatory cytokines, supporting the development of inflammations. In addition, inflammation facilitates an impaired skin barrier, leading to an increased transepidermal water loss (TEWL), which is responsible for dry skin [1,6]. AD patients also have a higher risk of developing further allergic diseases like allergic rhinitis, asthma and food allergy, which is known as the atopic march [7]. To prevent the development of AD and to reduce symptoms for improving the quality of life of AD patients, further fundamental studies are necessary to better understand the pathogenesis of AD. Oxidative stress, describing an imbalance of oxidants and antioxidants, represents an aspect to be associated with the development of
*
Corresponding author. E-mail address: [email protected] (M.C. Meinke). 1 Both researchers contributed equally to authoring this manuscript. https://doi.org/10.1016/j.cbi.2019.108752 Received 24 May 2019; Received in revised form 8 July 2019; Accepted 15 July 2019 Available online 19 July 2019 0009-2797/ © 2019 Published by Elsevier B.V.
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List of abbreviations AD ⋅CCR DMPO EPR Exp FLG GEE GSH
IR MED NIR PCA PCR Sim TEMPO UV VIS
atopic dermatitis carbon-centered radicals 5,5-Dimethyl-1-pyrroline-N-oxide electron paramagnetic resonance experimental filaggrin generalized estimating equations glutathione
infrared minimal erythema dose near infrared 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy polymerase chain reaction simulated 2,2,6,6-Tetramethyl-1-piperidinyloxy ultraviolet visible
4/IL-13) skin equivalents was performed according to previously published research [28]. Briefly, primary human keratinocytes and fibroblasts were isolated from juvenile foreskin following circumcisions. The procedure was approved by the Ethics Committee of Charité-Universitätsmedizin Berlin (EA1/081/13) and conducted according to the principles of the Declaration of Helsinki. 3.0 × 105 primary human fibroblasts were embedded in a matrix consisting of fetal calf serum and bovine collagen I (PureCol®, Advanced BioMatrix, San Diego, USA) and brought to a neutral pH value. After 4 h, 4.2 × 106 primary human keratinocytes were seeded on top of the collagen matrix. The skin equivalents were cultivated in 3D cell culture well inserts with a growth area of 4.2 cm2 (BD Biosciences, Heidelberg, Germany). After 24 h, the skin equivalents were lifted to the air-liquid interface. For inflammatory skin equivalents, the filaggrin gene (UniGene 2723828 - Hs.654510) was knocked down into keratinocytes by transfection with a specific siRNA (Stealth RNAi™, siRNA ID: HSS177192, sequence: CAGCUCCA GACAAUCAGGCACUCAU; LifeTechnologies, Darmstadt, Germany) and the transfection reagent HiPerFect® (Qiagen, Hilden, Germany). Starting at day 10, the culture medium of the inflammatory skin equivalents was supplemented with 15 ng/ml of the proinflammatory cytokines IL-4 and IL-13 (Milteny Biotec, Bergisch Gladbach, Germany). The efficiency of the filaggrin knock down was checked at day 6 by quantitative PCR. After 14 days of cultivation, the skin equivalents were used for further investigations.
inflammatory processes. Therefore, oxidative stress could also play a role in the pathogenesis of AD, as in other inflammatory skin diseases, e.g. psoriasis [8,9]. It is known that oxidative stress promotes tissue inflammation by an upregulation of proinflammatory cytokines. In turn, a signal cascade is triggered and inflammatory cells release endogenous free radicals which can lead to oxidative stress [10]. In vivo studies investigating the blood and/or urine of AD patients showed clearly that oxidative markers are upregulated and antioxidative markers downregulated in AD patients compared to control group [11–13]. Oxidative stress is characterized by an excessive radical accumulation caused by either increased radical production or an impaired antioxidative protection system [14]. This may result in an increased oxidation of cell components such as proteins, lipids in cell membranes and nucleic acids leading to cell and tissue damages [15]. An increase in the radical concentration can be caused by either exogenous factors or physiological processes, e.g. mitochondrial electron transport [16]. Therefore, especially the skin as the interface between the organism and the environment is a major target for oxidative stress, because it is permanently exposed to a prooxidative environment, like solar irradiation and pollutants [17]. To control these radical cascades, the skin possesses a complex endogenous antioxidative protection system including non-enzymatic, e.g. glutathione (GSH), and enzymatic, e.g. superoxide dismutase, components. Exogenous antioxidants like vitamin C and vitamin E are not produced physiologically, but they are taken up by food. Such antioxidants are chemical compounds that can either capture or reduce radicals and/or support other antioxidants in their effects (synergistic effects). Since oxidized antioxidants are constantly regenerating, they prevent undesirable oxidation processes of cellular components. The correlation of oxidants and antioxidants is described as redox status, and in the normal state oxidants and antioxidants are in balance [8,18,19]. Previous in vivo study results about the redox status of atopic skin are rare and controversial [20,21]. Since the role of oxidative stress is not clarified in atopic skin, in this study the redox status of inflammatory skin was investigated mainly by EPR spectroscopy to see possible differences in the antioxidant and oxidant status. The redox status in vivo is influenced by a various number of environmental factors like nutrition including exogenous antioxidants and stress [22,23]. To exclude these factors, normal and inflammatory skin equivalents mimicking characteristics of AD in vitro were used in this study to analyze exclusively the endogenous redox status. For this purpose, the spin probe TEMPO was used to monitor the antioxidant status in different microenvironments [24,25], the spin probe PCA to investigate the metabolic and irradiation-induced radical production [26] and the spin trap DMPO to characterize the radicals produced in the skin equivalents [27].
2.2. EPR spectroscopy 2.2.1. EPR spin probes and trap 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO, 98%), 3-(carboxy)2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-Carboxy-PROXYL, PCA, 98%) were purchased from Sigma-Aldrich (Steinheim, Germany). 5,5Dimethyl-1-pyrroline-N-oxide (DMPO, ≥99%) was purchased from Dojindo Molecular Technologies (Japan). All chemicals were of analytical grade. 2.2.2. Sample preparation For EPR measurements, the normal and inflammatory skin equivalents were prepared as follows: First, a skin punch (Ø 8 mm) was taken from skin and incubated with the spin probe TEMPO, PCA or spin trap DMPO using filter paper disc (SmartPractice®, Phoenix, USA), respectively, for 10 min at 32 °C. Afterwards, a smaller skin punch (Ø 4.5 mm) was taken from the incubated skin sample. The skin samples were placed in an EPR tissue sample cell (GZ 170–5.0 × 0.5, Magnettech GmbH, Berlin, Germany for TEMPO and ER 162 TC-HS for HS and SHQCavities, Bruker BioSpin GmbH, Karlsruhe, Germany for PCA and DMPO) and measured over a certain time period by EPR spectroscopy. The detailed sample preparation for PCA and DMPO is described in Albrecht et al. [27].
2. Material and methods 2.1. Skin equivalents
2.2.3. TEMPO decay To investigate the antioxidant status, a filter paper disc (Ø 6 mm) was placed on the top of the skin punch which were incubated with 15 μl of the EPR spin probe TEMPO (0.25 mM in PBS). After 10 min
For all investigations, reconstructed human skin equivalents were used. The reconstruction of normal (FLG+) and inflammatory (FLG-/IL2
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5.7 mW/cm2 UVB-NIR (economical handheld laser power meter 843R, Newport Corporation, California, USA), 0.026 mW/cm2 UVB, 0.31 mW/ cm2 UVA (radiometer photometer ILT1400, International Light Technologies, Waldbronn, Germany). The maximal dose of UVB irradiation was 24 mJ/cm2 which corresponds to 0.8 minimal erythema dose (MED). The initial measurement at time point 0 was performed without irradiation. Afterwards, the samples were irradiated over the whole time period of 15.4 min. For evaluation, the PCA amount of irradiated samples was determined by the Bruker software using spin fit analysis (Xepr 2.6b143, Bruker BioSpin GmbH, Karlsruhe, Germany) according to the non-irradiated samples. The PCA decay with irradiation was also normalized and plotted over time. The cumulative radical production was also calculated for the irradiated samples. To exclude metabolic effects, the cumulative radical production of non-irradiated skin was subtracted from cumulative radical production of irradiated skin. The relative radical production of each donor induced by irradiation was analyzed and was plotted over time according to non-irradiated skin. The detailed description for EPR measurements and data analysis for the EPR spin probe PCA is described in Albrecht et al. [27].
incubation, the EPR measurements were performed using an X-band EPR spectrometer (9.4 GHz) of Magnettech GmbH (MiniScope MS400, Berlin, Germany) and Magnettech GmbH by Freiberg Instruments (MiniScope MS5000, Freiberg, Germany) with the following instrument parameters: center field: 335.46 mT (MS400)/ 337.32 mT (MS5000), sweep: 6.86 mT, sweep time: 30 s, modulation amplitude: 0.2 mT, modulation frequency: 100 kHz, microwave power: 10 mW, time constant: 0.1 s. All EPR measurements were performed at 32 °C using a biotemperator device (BTC01, Magnettech GmbH, Berlin, Germany) to ensure constant temperature and similar conditions as in the stratum corneum. Automatic repetition measurements were performed over a time interval of 23.3 min using the software “MiniScopeCtrl” (Magnettech GmbH, Berlin, Germany) and “ESR Studio” (Magnettech GmbH by Freiberg Instruments, Freiberg, Germany). For evaluation, the TEMPO spectra of selected time points were simulated using the toolbox EasySpin version 5.2.11 [29], a MATLAB® package (The MathWorks GmbH, Ismaning, Germany) to deconvolute the total TEMPO spectrum in hydrophilic and lipophilic TEMPO spectra. In particular, the function “chili” was used for the spectra simulations. The magnetic parameters for simulation are presented in the supplemental material (Table S1). The integral of the simulated EPR spectrum of total TEMPO spectrum was determined by EasySpin toolbox and MATLAB®. The integral of hydrophilic and lipophilic TEMPO spectrum was obtained by multiplication of integral of total TEMPO spectrum and the appropriate percentage of TEMPO spectra of the hydrophilic or lipophilic microenvironment. For the determination of the signal decay rate (rate constant k) of the hydrophilic and lipophilic TEMPO decay, the slope of semi-logarithmic intensity plots was used. Due to signal to noise limitations of the lipophilic fraction, the initial 12 min of TEMPO decay were used.
2.2.6. Characterization of irradiation-induced radical production For the characterization of produced radicals, the skin samples were incubated with the EPR spin trap DMPO from top and bottom (1.2 mM in distilled water/PBS) according to the skin samples incubated with spin probe PCA. The EPR measurements were performed using the Xband EPR system “Elexsys E 500” (9.6 GHz) with a TMHS resonator (E2044500TMHS, Bruker BioSpin GmbH, Karlsruhe, Germany) with the following parameters: center field: 348.0 mT, sweep: 14.0 mT, sweep time: 45 s, modulation amplitude: 0.12 mT, modulation frequency: 100 kHz, microwave power: 10.02 mW. The initial measurement at time point 0 min was performed without irradiation. Afterwards, the following UVB-NIR irradiation was used for inducing radicals: 49 mW/ cm2 UVB-NIR (economical handheld laser power meter 843R, Newport Corporation, California, USA), 0.29 mW/cm2 UVB, 2.5 mW/cm2 UVA (radiometer photometer ILT1400, International Light Technologies, Waldbronn, Germany). The maximal dose of UVB irradiation over the time period of 2.3 min was 40.3 mJ/cm2 which corresponds to 1.3 MED. For evaluation, the EPR spectra of defined time points and all donors were averaged by MATLAB®. Subsequently, the obtained EPR spectra were simulated by WinSim, a public electron paramagnetic resonance software tool of NIEHS [30]. The total intensity of the EPR spectra was calculated from the integral and the ratio of reactive oxygen species (⋅OH radicals) and lipid oxygen species (carbon centered radicals, ⋅CCR) determined by WinSim. The detailed description for EPR measurements and data analysis for the EPR spin trap DMPO is described in Albrecht et al. [27].
2.2.4. Metabolic PCA decay For the quantification of radical production, the skin samples were incubated from both top and bottom with 38.4 μl of the EPR spin probe PCA using a Ø 8 mm filter paper (1.5 mM in distilled water/PBS). The EPR measurements were performed using the X-band EPR system “Elexsys E 500” (9.6 GHz) with a TMHS resonator (E2044500TMHS, Bruker BioSpin GmbH, Karlsruhe, Germany) using the following instrument parameters: center field: 348.0 mT, sweep: 14.0 mT, sweep time: 45 s, modulation amplitude: 0.15 mT, modulation frequency: 100 kHz, microwave power: 1.26 mW. All EPR measurements were performed at ambient temperature and measured in single-scan operation mode over a total time of 15.4 min. For evaluation, the PCA amount of each spectrum was determined by the Bruker software using spin fit analysis (Xepr 2.6b143, Bruker BioSpin GmbH, Karlsruhe, Germany). On the one site, the PCA amount was normalized and plotted over the measurement time resulting in PCA decay. On the other site, the cumulative radical production at each time point was calculated by subtraction of the PCA amount at a certain time point from the initial PCA amount at time point 0 min. To exclude differences between the different donors, the highest radical production of each donor (normal, inflammatory) was set to 1 and the radical production at each time point was related to this value. The relative cumulative radical production of normal and inflammatory skin equivalents was plotted over time.
2.3. MTT assay After the EPR measurements the cell viability of the skin samples was measured by MTT assay. The skin punches (Ø 4.5 mm) were placed on filter paper discs soaked with 0.5 mg/ml MTT solution (SigmaAldrich, Steinheim, Germany) dissolved in PBS and incubated for 4 h at 37 °C and 5% CO2. Afterwards, the skin samples were transferred to 400 μl isopropanol (Merck KGaA, Darmstadt, Germany) to extract the formazan salt overnight in the dark. The absorption of formazan salt was measured at 560 nm by multimode plate reader (PerkinElmer, Waltham, USA). The viability of skin samples was calculated to untreated control, which was set to 100%. Skin punches incubated with 20% SDS solution for 1 h served as positive control.
2.2.5. PCA decay induced by irradiation To induce radical production by an exogenous stress factor, the skin samples were irradiated during the EPR measurements additionally using a sun simulator (LOT-QuantumDesign GmbH, Darmstadt, Germany, classified to IEC 60904–9, class A; Xe-lamp, 150 W) which was directly coupled into the EPR resonator by an optical UV-VIS fiber (LOT-QuantumDesign GmbH, Darmstadt, Germany). The skin samples were exposed to the whole simulated solar spectrum; ultraviolet B (UVB) to near infrared (NIR) region (305–2200 nm) which mimics a solar spectrum by air mas 0. The following irradiances were used:
2.4. Determination of GSH concentration For determining the concentration of the reduced form of GSH, the epidermis of all skin equivalents, normal and inflammatory, was separated from the dermis by tweezers. Epidermis and dermis were then 3
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homogenized in isotonic PBS using the TissueLyserII from Qiagen GmbH (Hilden, Germany) for 1 min at 30 Hz, followed by sonification for 90 s (amplitude: 90, interval: 0.5) (Sonorex Super RK102H, BANDELIN electronic GmbH & Co. KG, Berlin, Germany). Afterwards, the skin homogenates were centrifuged at 12,000×g for 10 min. The supernatant was collected and part of it was used to determine the protein concentration using Pierce Protein Reagent Assay BCA Kit according to the manufacturers' instructions (Thermo Fisher Scientific, Waltham, USA). The remaining sample was incubated with cold 5% sulfosalicylic acid (Sigma Aldrich, Steinheim, Germany) for 10 min at 4 °C to eliminate interfering proteins. The GSH concentration was determined by means of a Glutathione Fluorescent Detection Kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturers' instructions.
skin equivalents (Fig. 1). Due to its amphiphilicity (logP = 1.8) [31,32], TEMPO is able to partition between hydrophilic and lipophilic microenvironments [25,33]. The total TEMPO spectra were simulated using the magnetic parameters shown in Table S1. The partitioning of TEMPO in normal and inflammatory skin equivalents is significantly different (p ≤ 0.05). In normal skin equivalents 33 ± 2% of TEMPO partitions in the lipophilic microenvironment compared to 25 ± 2% in inflammatory skin equivalents. However, in both skin equivalents most TEMPO is located in the hydrophilic microenvironment. 3.2. Antioxidant status in hydrophilic and lipophilic microenvironment To determine the antioxidant status in skin equivalents, the signal decay of TEMPO over time was investigated. TEMPO is a nitroxide radical including a six-membered piperidine ring which can cross cell membranes very easily and consequently spread in intra- and extracellular compartments of the whole skin equivalent [14]. The spin probe TEMPO can react potentially with oxidants and antioxidants in the skin, but it reacts predominantly with antioxidants as described in the literature [14,23]. TEMPO is reduced to the corresponding (diamagnetic) hydroxylamine, which is EPR-silent, mainly by ascorbate and thiol-dependent antioxidant activities like GSH in the skin [24]. Consequently, the decay of TEMPO over time indicates the radical scavenging capacity of skin. In Fig. 2A the total signal decay of TEMPO in both skin equivalents is illustrated which can be described as a biexponential function I (t ) = I0 (e khydro t + e klipo t ). Therefore, the TEMPO decay in both individual microenvironments was analyzed by evaluating the signal decay rate. The TEMPO decay of a single component, hydrophilic and lipophilic microenvironment, was assumed to be a monoexponential decay I (t ) = I0 e kt , which gave reasonable fits. The semi-logarithmic plots of the TEMPO signal intensity over the time for hydrophilic and lipophilic microenvironment are presented in the supplemental material (Fig. S2). Our investigations showed that there are no significant differences between normal and inflammatory skin equivalents in hydrophilic and lipophilic TEMPO decay. Although the signal decay rate is significantly different between hydrophilic and lipophilic microenvironments in both skin equivalents, the differences are very small (Fig. 2B). Notably, the viability of normal and inflammatory skin equivalents was not affected during the EPR measurements using TEMPO (Fig. S3).
2.5. Statistical analysis All data are presented as mean ± standard error of the mean (SEM). The statistical analysis was performed using IBM® Statistical Package for the Social Sciences (SPSS®) Statistics version 23 (IBM Corporation, Armonk, USA). For experiments over a certain time period, the method of generalized estimating equations (GEE) was used. Normal and inflammatory skin equivalents were compared by Wilcoxon test. A p-value ≤ 0.05 was considered to be statistically significant. 3. Results 3.1. TEMPO as indicator for different microenvironments within the skin equivalents To investigate the redox status in normal and inflammatory skin equivalents, several EPR markers were employed. First, the spin probe TEMPO was applied to the skin equivalents. Representative TEMPO spectra of normal and inflammatory skin equivalents are depicted in Fig. 1. The total TEMPO spectrum of both skin equivalents showed a splitting of the high field peak into two individual peaks in an X-band EPR spectrometer, whereas the low and middle field peak are only broadened compared to a TEMPO spectrum in PBS shown in supplemental material (Fig. S1). This can be interpreted, that the total TEMPO spectrum consists of two individual spectra, which represent TEMPO in the hydrophilic and lipophilic compartment (microenvironment) of the
Fig. 1. Partitioning of TEMPO in skin equivalents. Representative X-band EPR spectra of TEMPO of normal (A) and inflammatory (B) skin equivalents. Illustration of experimental (exp) and simulated (sim) total TEMPO spectrum, simulated hydrophilic and simulated lipophilic TEMPO spectrum including percentage of TEMPO in each microenvironment; mean ± SEM (n = 6). 4
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Fig. 2. Signal decay of TEMPO in skin equivalents. Illustration of biexponential fit of total TEMPO decay (A) and the resulting signal decay rates (B) of normal and inflammatory skin equivalents; mean ± SEM (n = 5). * statistically significant difference as indicated at p ≤ 0.05 (*).
3.3. Lower GSH levels in epidermis of inflammatory skin equivalents
metabolic PCA decay over the measured time period compared to normal skin equivalents (Fig. 4A). Expressing the PCA decay in quantitative radical formation, in normal skin equivalents 3.8 ± 1.5 × 1013 radicals/mm3 and in inflammatory skin equivalents 9.4 ± 3.5 × 1013 radicals/mm3 were detected after 15.4 min. Evaluating each donor of normal and inflammatory skin equivalents separately, the difference in relative radical production was highly significant (p ≤ 0.001) shown in Fig. 4B.
To get a deeper insight into the antioxidant status of hydrophilic microenvironment, the concentration of the hydrophilic antioxidant GSH was determined in epidermis and dermis homogenates of both normal and inflammatory skin equivalents. GSH represents a major regulator predominately in the intracellular redox status, because it is involved in the enzyme-mediated antioxidant system and the detoxification of hydrogen peroxide [16,34]. Epidermal homogenates of inflammatory skin equivalents showed a significantly lower GSH concentration compared to normal skin equivalents (Fig. 3A). In contrast, no significant differences in the GSH concentration was observed in the dermis homogenates between both skin equivalents (Fig. 3B).
3.5. UVB-NIR irradiation induced no differences in amount and type of radicals between normal and inflammatory skin equivalents Another possibility to monitor the redox status is the application of a stress response test. Irradiation is a common exogenous stress factor of the environment. Not only UVA and UVB, but also visible (VIS), near infrared (NIR) and infrared (IR) radiation are responsible for the radical production during solar exposure in skin [37–39]. In the experimental set up the whole simulated solar spectrum (UVB-NIR) was used to induce radicals within the skin equivalents (normal, inflammatory) to see the stress response and thus the antioxidant capacity to irradiation. The application of irradiation promoted a faster PCA decay in both skin equivalents compared to the absence of irradiation (Fig. 5A), but resulted in a similar irradiation-induced radical production for both skin equivalents (Fig. 5B). After the irradiation time, in normal skin equivalents 2.1 ± 0.4 × 1014 radicals/mm3 (1.7 ± 0.4 × 1014 induced radicals/mm3) and in inflammatory skin equivalents 2.8 ± 0.2 × 1014 radicals/mm3 (1.8 ± 0.4 × 1014 induced radicals/ mm3) were measured. Consequently, irradiation leads to a 3–5.5 fold higher radical production as without irradiation.
3.4. Higher metabolic radical production in inflammatory skin equivalents In addition to the antioxidant status, the oxidant status was investigated in normal and inflammatory skin equivalents using the spin probe PCA. Since PCA mainly reacts with free radicals in the skin resulting in EPR-silent products [26], the PCA decay was analyzed to quantify metabolic and irradiation-induced radicals by EPR spectroscopy. In contrast to TEMPO, PCA includes a five-membered pyrrolidine ring and is a hydrophilic nitroxide radical (logP = −1.7) and thus it mainly detects free radicals in hydrophilic microenvironment [26,35,36]. Its spectrum is comparable to the one of TEMPO in aqueous solution (Fig. S1). PCA penetrates slower into cells than TEMPO due to its charged carboxyl group at pH 7.4, but it distributes in intracellular and extracellular compartments [36] and detect the radicals present there. The inflammatory skin equivalents showed a significantly faster
Fig. 3. Glutathione (GSH) concentration of tissue homogenates. GSH concentration of epidermis homogenates (A) and dermis homogenates (B) of normal and inflammatory skin equivalents determined by fluorescence-based GSH assay and adjusted to the total protein amount of the skin homogenate; mean ± SEM (n = 4); * statistically significant difference as indicated at p ≤ 0.05 (*).
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Fig. 4. Metabolic radical production of skin equivalents using PCA (without UVB-NIR irradiation). (A) Normalized signal decay of PCA over time. (B) Relative cumulative radical production over time was calculated by subtraction of the PCA amount at a certain time point from the initial PCA amount at time point 0 min. To exclude differences between the different donors, the highest radical production of each donor (normal, inflammatory) was set to 1 and the radical production at each time point was adjusted to this value; mean ± SEM (n = 5); * statistically significant difference as indicated at p ≤ 0.05 (*) and p ≤ 0.001 (***).
To assess the type of radicals produced during irradiation, normal and inflammatory skin equivalents were investigated by using the EPR spin trap DMPO. It can enter cells very easily and reacts intracellularly and extracellularly with different free radicals locating in the hydrophilic microenvironment of skin (logP = −1.0) [40]. It forms DMPOradical-adducts (spin trapping) which are stable and measurable [41,42]. By that, DMPO can give information about the occurrence of different types of free radicals as well as reflect relative changes. However, the absolute quantification and the detection of low radical concentrations are challenging [34]. Therefore, in the experimental setup radicals were induced by UVB-NIR irradiation. The DMPO spectra of the analyzed skin equivalents include six peaks characterizing ⋅CCR, four peaks characterizing ⋅OH radicals and three peaks characterizing the nitroxide (Fig. 6A) [41]. The DMPO spectra were simulated using the magnetic parameters shown in Table S2. For both, normal and inflammatory skin equivalents, ⋅OH radicals and ⋅CCR were produced at similar ratio during irradiation (Fig. 6B). ⋅OH radicals were produced to 89% and ⋅CCR to 11% on average. The skin samples measured with PCA and DMPO showed a viability of at least 80% and thus they were still viable after the EPR measurements (Fig. S3).
4. Discussion Since the redox status in atopic skin is not known yet, the antioxidant and oxidant status in normal and inflammatory skin equivalents mimicking characteristics of AD in vitro were investigated mainly by EPR spectroscopy to get an insight into the role of redox status in atopic skin. Nitroxide radicals, like TEMPO and PCA are often used for skin investigations using EPR spectroscopy, because they can offer different information about the redox status and the measured microenvironment due to their various structural and physicochemical properties [34,43]. The spin probe TEMPO is able to partition between hydrophilic and lipophilic microenvironment in skin and thus its decay can be monitored in different microenvironments (Fig. 1) [25,33]. TEMPO in the hydrophilic microenvironment mainly corresponds to the viable epidermis and dermis and the lipophilic microenvironment corresponds to the stratum corneum (unpublished data). The main part of TEMPO is distributed in the hydrophilic microenvironment, because the viable epidermis and dermis representing the hydrophilic microenvironment are relatively larger compared to the stratum corneum representing the lipophilic microenvironment in the investigated skin sample. Consequently, the higher percentage of TEMPO in the lipophilic microenvironment in normal skin equivalents can be explained by a thicker stratum corneum in normal skin compared to atopic skin [44]. Although
Fig. 5. Irradiation-induced radical production of skin equivalents using PCA (with UVB-NIR irradiation). (A) Normalized signal decay of PCA over irradiation time. (B) Relative cumulative radical production induced by UVB-NIR irradiation (metabolic radical production was substracted). To exclude differences between the different donors, the highest radical production of each donor (normal, inflammatory) was set to 1 and the radical production at each time point was adjusted to this value. UVB-NIR irradiation (305–2200 nm) was used: 5.7 mW/cm2 UVB-NIR, 0.026 mW/cm2 UVB, 0.31 mW/cm2 UVA. The maximal dose of UVB irradiation was 24 mJ/cm2, which corresponds to 0.8 minimal erythema dose; mean ± SEM (n = 5). 6
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Fig. 6. Characterization of irradiation-induced radical production in skin equivalents using DMPO. (A) Representative X-band EPR spectrum of DMPO of nonirradiated and irradiated normal and inflammatory skin equivalents including labeled peaks for DMPO-CCR (carbon-centered radicals), DMPO-OH and nitroxide radicals. (B) Ratio between DMPO-CCR and DMPO-OH radicals. UVB-NIR irradiation (305–2200 nm) was used: 49 mW/cm2 UVB-NIR, 0.29 mW/cm2 UVB, 2.5 mW/ cm2 UVA. The maximal dose of UVB irradiation was 40.3 mJ/cm2, which corresponds to 1.3 minimal erythema dose; mean ± SEM (n = 4).
normal and inflammatory skin equivalents showed different partitioning of TEMPO in hydrophilic and lipophilic microenvironments, the TEMPO decay and thus the antioxidant status in the microenvironments were similar. Comparing the TEMPO decay in the hydrophilic and lipophilic microenvironments, a not relevant lower signal decay rate was detected in the lipophilic microenvironment of both skin equivalents, which can be due to lower amount of antioxidants in the stratum corneum [45]. To the best of our knowledge, such analysis of the TEMPO decay into hydrophilic and lipophilic microenvironments in skin equivalents was shown for the first time. Although the TEMPO decay showed no differences in the total antioxidant status between normal and inflammatory skin equivalents, we found a lower concentration of the hydrophilic antioxidant GSH in the epidermis of inflammatory skin equivalent compared to normal skin equivalent. In contrast, this difference was not present in the dermis. Consequently, it is illustrated that the results of redox status can differ between different skin layers and depending on whether the total antioxidant status in the skin or only one specific intracellular antioxidant is analyzed. In addition, EPR investigations using PCA showed a significantly higher metabolic radical formation in the inflammatory skin equivalents compared to normal skin equivalents (Fig. 4B). This could result from inflammation processes that are connected with oxidative stress [46,47]. To induce an inflammatory response in the skin equivalents, the proinflammatory cytokines IL-4 and IL-13 were supplemented to the culture medium. It has already been shown that these cytokines increase the free radical production [48]. The increased metabolic radical production in the inflammatory skin equivalents can lead, in turn, to a lower antioxidant status for protection, which was shown by a lower GSH concentration in the epidermis (Fig. 3A). Consequently, our data support the results of Sapuntsova et al. who detected a decompensated accumulation of oxidants and inhibition of antioxidative systems in skin biopsies of AD patients [21]. In contrast, Antille et al. published a lower oxidative status in the stratum corneum of AD patients [20]. These differences might have been caused by the fact that the authors investigated non-lesional skin areas of AD patients in contrast to our skin equivalents which mimic characteristics of lesional atopic skin. Since the inflammatory skin equivalents showed a significantly higher radical production without irradiation, we expected also a faster PCA decay and thus a higher radical production by irradiation. Surprisingly, the UVB-NIR irradiation induced the same PCA decay and thus the same amount of irradiation-induced radicals in normal and inflammatory skin equivalents (Fig. 5B). Consequently, the skin
equivalents showed the same resistance to irradiation which is in accordance to the similar antioxidant status determined by TEMPO decay (Fig. 2). It is known that AD patients show no modifications or even improvement of eczema during sun exposure, which can explain the missing negative effect of solar sun irradiation [49]. In addition to the similar irradiation-induced radical amount in normal and inflammatory skin equivalents a similar ratio between ⋅OH radicals and ⋅CCR were detected in the skin equivalents by the spin trap DMPO. The DMPO data of normal skin equivalents are consistent with published data [27]. Since radicals are already induced in the DMPO solution by UVB irradiation, DMPO measurements were not used for quantitative analysis, but to characterize the radical type and determine possible differences in the ratio of radical production (⋅OH radicals to ⋅CCR) between normal and inflammatory skin equivalents. Since normal and inflammatory skin equivalents were measured under the same conditions, the produced radicals by DMPO solution will be similar for both. Albrecht et al., already showed differences in the ratio between ⋅OH radicals and ⋅CCR between different skin models (human, porcine, skin equivalents) by using DMPO, whereby a comparable tendency in semiquantitative data to PCA was shown [27]. The spin marker PCA was used for radical quantification, because no radicals can be detected in PCA solution during irradiation. However, the ratio between ⋅OH radicals and ⋅CCR showed no differences in normal and inflammatory skin equivalents, which is in accordance to the irradiationinduced radical production quantified by PCA (Fig. 5B). ⋅OH radicals represent the main radicals produced in the skin equivalents after irradiation, which are formed in the first steps of oxidative stress development. In the next steps ⋅OH radicals interact with lipid and membrane structures, promoting the formation of ⋅CCR, like lipid radicals [50]. The produced radicals are in agreement with former investigations on ex vivo human and porcine skin [27,39]. The differences in redox status investigations may result from the applied methods and the part of skin, on which the focus is set. As shown in our investigations the GSH concentration in epidermis and metabolic radical formation differ between the skin equivalents but the total antioxidant status, GSH in dermis and the stress response to irradiation did not show differences between normal and inflammatory skin equivalents. We have to admit, our results cover one part of the redox status in inflammatory skin equivalents, because only a limited range of nitroxides were used and not all aspects of redox status were considered, e.g. the extracellular redox status. Therefore, atopic skin has to be studied further to confirm these results. The skin equivalents 7
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offer the possibility and advantage to investigate the endogenous antioxidant system produced exclusively by the metabolism of cells. These results showed that a supply of antioxidants could be beneficial for atopic skin, in particular hydrophilic ones could be more effective than lipophilic. Exogenous antioxidants like vitamin C, vitamin E and carotenoids are not contained in skin equivalents, however they have an essential influence on the redox status leading to a changed TEMPO decay and thus antioxidant system as already shown in vivo after supplementation of different exogenous antioxidants [23,51].
[12] [13]
[14]
5. Conclusions
[15]
In conclusion, we could gain a first insight into the endogenous redox status of inflammatory skin equivalents by using three different EPR spin markers. Our investigations showed differences in the redox status of inflammatory skin equivalents concerning epidermal GSH and metabolic radical production compared to normal skin equivalents. Consequently, our results suggest that the redox status could play a role in developing AD and that antioxidants could be beneficial for atopic skin. To investigate the redox status comprehensively, the applied methods (total status or individual component) as well as the focused skin compartment should be considered. Nevertheless, the results presented here only cover parts of the redox status (e.g. no exogenous antioxidants) in inflammatory skin diseases and do not offer a complete overview. Therefore, further detailed investigations of the redox status in atopic skin are indispensable.
[16] [17] [18] [19]
[20] [21]
Funding
[22]
This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Germany via the Collaborative Research Center 1112 (project B01 and C02).
[23]
Conflicts of interest
[24]
The authors declare no conflict of interest.
[25]
Appendix A. Supplementary data
[26]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbi.2019.108752.
[27]
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Insight into the redox status of inflammatory skin equivalents as determined by EPR spectroscopy
T
Anja Elpelta,b,1, Stephanie Albrechta,1, Christian Teutloffc, Martina Hügingd, Siavash Saeidpourc, Silke B. Lohana, Sarah Hedtrichb,e, Martina C. Meinkea,* a
Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Department of Dermatology, Venerology and Allergology, Center of Experimental and Applied Cutaneous Physiology, Charitéplatz 1, 10117, Berlin, Germany b Institute of Pharmacy, Department of Pharmacology, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195, Berlin, Germany c Department of Physics, Institute of Experimental Physics, Freie Universität Berlin, Arnimallee 14, 14195, Berlin, Germany d Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Department of Pediatric Surgery, Augustenburger Platz 1, 13353, Berlin, Germany e University of British Columbia, Faculty of Pharmaceutical Sciences, 2405 Wesbrook Mall, V6T1Z3, Vancouver, Canada
ARTICLE INFO
ABSTRACT
Keywords: Atopic dermatitis Skin equivalents Redox status Irradiation Electron paramagnetic resonance spectroscopy
Atopic dermatitis (AD) is a chronic inflammatory skin disease whose pathogenesis is still not fully understood. Since inflammatory processes correlate with oxidative stress, the redox status may play a key role in AD. In this study, electron paramagnetic resonance (EPR) spectroscopy was mainly used to investigate the redox status in normal and inflammatory skin equivalents mimicking characteristics of AD in vitro using EPR spin probes (TEMPO, PCA) and a spin trap (DMPO). The total antioxidant status in the hydrophilic and lipophilic compartments of skin (microenvironment) showed no differences between the skin equivalents. In the inflammatory skin equivalents, a decreased glutathione concentration in the epidermis and an increased metabolic radical production could be observed compared to normal skin equivalents. The induction of external stress by simulated solar irradiation (UVB-NIR) resulted in the same amount and type of radicals in normal and inflammatory skin equivalents. For the first time, the antioxidant and oxidant status of inflammatory in vitro skin equivalents was analyzed by EPR to elucidate their redox status using different methods which focus on various microenvironments. Our investigations suggested that the redox status in atopic skin could be different, but this should be investigated more comprehensively, because the results can vary depending on the used methods and where the investigations take place.
1. Introduction Atopic dermatitis (AD) is a chronic inflammatory skin disease representing a common health problem worldwide. The disease affects 15–20% of children and 1–3% of adults. The main symptoms are skin eczema characterized by itching, dryness and crusting, which can impair substantially the quality of life of AD patients. AD is a multifactorial disease combining genetic, immunological and environmental aspects [1,2]. So far, it is known that up to 50% of AD patients have one loss-of-function mutation in the filaggrin gene (FLG), which is an important skin differentiation marker [3]. Furthermore, a changed lipid composition and a lower lipid order in the stratum corneum (outermost skin layer) of atopic skin likely correlates with a disturbed barrier function [4,5]. It is assumed that the disturbed skin barrier increases
the penetration of allergens and pathogens into the skin, which can activate the immune system. A dysfunctional immune response characterized by a Th2-dominated reaction in the acute phase causes the release of proinflammatory cytokines, supporting the development of inflammations. In addition, inflammation facilitates an impaired skin barrier, leading to an increased transepidermal water loss (TEWL), which is responsible for dry skin [1,6]. AD patients also have a higher risk of developing further allergic diseases like allergic rhinitis, asthma and food allergy, which is known as the atopic march [7]. To prevent the development of AD and to reduce symptoms for improving the quality of life of AD patients, further fundamental studies are necessary to better understand the pathogenesis of AD. Oxidative stress, describing an imbalance of oxidants and antioxidants, represents an aspect to be associated with the development of
*
Corresponding author. E-mail address: [email protected] (M.C. Meinke). 1 Both researchers contributed equally to authoring this manuscript. https://doi.org/10.1016/j.cbi.2019.108752 Received 24 May 2019; Received in revised form 8 July 2019; Accepted 15 July 2019 Available online 19 July 2019 0009-2797/ © 2019 Published by Elsevier B.V.
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List of abbreviations AD ⋅CCR DMPO EPR Exp FLG GEE GSH
IR MED NIR PCA PCR Sim TEMPO UV VIS
atopic dermatitis carbon-centered radicals 5,5-Dimethyl-1-pyrroline-N-oxide electron paramagnetic resonance experimental filaggrin generalized estimating equations glutathione
infrared minimal erythema dose near infrared 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy polymerase chain reaction simulated 2,2,6,6-Tetramethyl-1-piperidinyloxy ultraviolet visible
4/IL-13) skin equivalents was performed according to previously published research [28]. Briefly, primary human keratinocytes and fibroblasts were isolated from juvenile foreskin following circumcisions. The procedure was approved by the Ethics Committee of Charité-Universitätsmedizin Berlin (EA1/081/13) and conducted according to the principles of the Declaration of Helsinki. 3.0 × 105 primary human fibroblasts were embedded in a matrix consisting of fetal calf serum and bovine collagen I (PureCol®, Advanced BioMatrix, San Diego, USA) and brought to a neutral pH value. After 4 h, 4.2 × 106 primary human keratinocytes were seeded on top of the collagen matrix. The skin equivalents were cultivated in 3D cell culture well inserts with a growth area of 4.2 cm2 (BD Biosciences, Heidelberg, Germany). After 24 h, the skin equivalents were lifted to the air-liquid interface. For inflammatory skin equivalents, the filaggrin gene (UniGene 2723828 - Hs.654510) was knocked down into keratinocytes by transfection with a specific siRNA (Stealth RNAi™, siRNA ID: HSS177192, sequence: CAGCUCCA GACAAUCAGGCACUCAU; LifeTechnologies, Darmstadt, Germany) and the transfection reagent HiPerFect® (Qiagen, Hilden, Germany). Starting at day 10, the culture medium of the inflammatory skin equivalents was supplemented with 15 ng/ml of the proinflammatory cytokines IL-4 and IL-13 (Milteny Biotec, Bergisch Gladbach, Germany). The efficiency of the filaggrin knock down was checked at day 6 by quantitative PCR. After 14 days of cultivation, the skin equivalents were used for further investigations.
inflammatory processes. Therefore, oxidative stress could also play a role in the pathogenesis of AD, as in other inflammatory skin diseases, e.g. psoriasis [8,9]. It is known that oxidative stress promotes tissue inflammation by an upregulation of proinflammatory cytokines. In turn, a signal cascade is triggered and inflammatory cells release endogenous free radicals which can lead to oxidative stress [10]. In vivo studies investigating the blood and/or urine of AD patients showed clearly that oxidative markers are upregulated and antioxidative markers downregulated in AD patients compared to control group [11–13]. Oxidative stress is characterized by an excessive radical accumulation caused by either increased radical production or an impaired antioxidative protection system [14]. This may result in an increased oxidation of cell components such as proteins, lipids in cell membranes and nucleic acids leading to cell and tissue damages [15]. An increase in the radical concentration can be caused by either exogenous factors or physiological processes, e.g. mitochondrial electron transport [16]. Therefore, especially the skin as the interface between the organism and the environment is a major target for oxidative stress, because it is permanently exposed to a prooxidative environment, like solar irradiation and pollutants [17]. To control these radical cascades, the skin possesses a complex endogenous antioxidative protection system including non-enzymatic, e.g. glutathione (GSH), and enzymatic, e.g. superoxide dismutase, components. Exogenous antioxidants like vitamin C and vitamin E are not produced physiologically, but they are taken up by food. Such antioxidants are chemical compounds that can either capture or reduce radicals and/or support other antioxidants in their effects (synergistic effects). Since oxidized antioxidants are constantly regenerating, they prevent undesirable oxidation processes of cellular components. The correlation of oxidants and antioxidants is described as redox status, and in the normal state oxidants and antioxidants are in balance [8,18,19]. Previous in vivo study results about the redox status of atopic skin are rare and controversial [20,21]. Since the role of oxidative stress is not clarified in atopic skin, in this study the redox status of inflammatory skin was investigated mainly by EPR spectroscopy to see possible differences in the antioxidant and oxidant status. The redox status in vivo is influenced by a various number of environmental factors like nutrition including exogenous antioxidants and stress [22,23]. To exclude these factors, normal and inflammatory skin equivalents mimicking characteristics of AD in vitro were used in this study to analyze exclusively the endogenous redox status. For this purpose, the spin probe TEMPO was used to monitor the antioxidant status in different microenvironments [24,25], the spin probe PCA to investigate the metabolic and irradiation-induced radical production [26] and the spin trap DMPO to characterize the radicals produced in the skin equivalents [27].
2.2. EPR spectroscopy 2.2.1. EPR spin probes and trap 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO, 98%), 3-(carboxy)2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-Carboxy-PROXYL, PCA, 98%) were purchased from Sigma-Aldrich (Steinheim, Germany). 5,5Dimethyl-1-pyrroline-N-oxide (DMPO, ≥99%) was purchased from Dojindo Molecular Technologies (Japan). All chemicals were of analytical grade. 2.2.2. Sample preparation For EPR measurements, the normal and inflammatory skin equivalents were prepared as follows: First, a skin punch (Ø 8 mm) was taken from skin and incubated with the spin probe TEMPO, PCA or spin trap DMPO using filter paper disc (SmartPractice®, Phoenix, USA), respectively, for 10 min at 32 °C. Afterwards, a smaller skin punch (Ø 4.5 mm) was taken from the incubated skin sample. The skin samples were placed in an EPR tissue sample cell (GZ 170–5.0 × 0.5, Magnettech GmbH, Berlin, Germany for TEMPO and ER 162 TC-HS for HS and SHQCavities, Bruker BioSpin GmbH, Karlsruhe, Germany for PCA and DMPO) and measured over a certain time period by EPR spectroscopy. The detailed sample preparation for PCA and DMPO is described in Albrecht et al. [27].
2. Material and methods 2.1. Skin equivalents
2.2.3. TEMPO decay To investigate the antioxidant status, a filter paper disc (Ø 6 mm) was placed on the top of the skin punch which were incubated with 15 μl of the EPR spin probe TEMPO (0.25 mM in PBS). After 10 min
For all investigations, reconstructed human skin equivalents were used. The reconstruction of normal (FLG+) and inflammatory (FLG-/IL2
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5.7 mW/cm2 UVB-NIR (economical handheld laser power meter 843R, Newport Corporation, California, USA), 0.026 mW/cm2 UVB, 0.31 mW/ cm2 UVA (radiometer photometer ILT1400, International Light Technologies, Waldbronn, Germany). The maximal dose of UVB irradiation was 24 mJ/cm2 which corresponds to 0.8 minimal erythema dose (MED). The initial measurement at time point 0 was performed without irradiation. Afterwards, the samples were irradiated over the whole time period of 15.4 min. For evaluation, the PCA amount of irradiated samples was determined by the Bruker software using spin fit analysis (Xepr 2.6b143, Bruker BioSpin GmbH, Karlsruhe, Germany) according to the non-irradiated samples. The PCA decay with irradiation was also normalized and plotted over time. The cumulative radical production was also calculated for the irradiated samples. To exclude metabolic effects, the cumulative radical production of non-irradiated skin was subtracted from cumulative radical production of irradiated skin. The relative radical production of each donor induced by irradiation was analyzed and was plotted over time according to non-irradiated skin. The detailed description for EPR measurements and data analysis for the EPR spin probe PCA is described in Albrecht et al. [27].
incubation, the EPR measurements were performed using an X-band EPR spectrometer (9.4 GHz) of Magnettech GmbH (MiniScope MS400, Berlin, Germany) and Magnettech GmbH by Freiberg Instruments (MiniScope MS5000, Freiberg, Germany) with the following instrument parameters: center field: 335.46 mT (MS400)/ 337.32 mT (MS5000), sweep: 6.86 mT, sweep time: 30 s, modulation amplitude: 0.2 mT, modulation frequency: 100 kHz, microwave power: 10 mW, time constant: 0.1 s. All EPR measurements were performed at 32 °C using a biotemperator device (BTC01, Magnettech GmbH, Berlin, Germany) to ensure constant temperature and similar conditions as in the stratum corneum. Automatic repetition measurements were performed over a time interval of 23.3 min using the software “MiniScopeCtrl” (Magnettech GmbH, Berlin, Germany) and “ESR Studio” (Magnettech GmbH by Freiberg Instruments, Freiberg, Germany). For evaluation, the TEMPO spectra of selected time points were simulated using the toolbox EasySpin version 5.2.11 [29], a MATLAB® package (The MathWorks GmbH, Ismaning, Germany) to deconvolute the total TEMPO spectrum in hydrophilic and lipophilic TEMPO spectra. In particular, the function “chili” was used for the spectra simulations. The magnetic parameters for simulation are presented in the supplemental material (Table S1). The integral of the simulated EPR spectrum of total TEMPO spectrum was determined by EasySpin toolbox and MATLAB®. The integral of hydrophilic and lipophilic TEMPO spectrum was obtained by multiplication of integral of total TEMPO spectrum and the appropriate percentage of TEMPO spectra of the hydrophilic or lipophilic microenvironment. For the determination of the signal decay rate (rate constant k) of the hydrophilic and lipophilic TEMPO decay, the slope of semi-logarithmic intensity plots was used. Due to signal to noise limitations of the lipophilic fraction, the initial 12 min of TEMPO decay were used.
2.2.6. Characterization of irradiation-induced radical production For the characterization of produced radicals, the skin samples were incubated with the EPR spin trap DMPO from top and bottom (1.2 mM in distilled water/PBS) according to the skin samples incubated with spin probe PCA. The EPR measurements were performed using the Xband EPR system “Elexsys E 500” (9.6 GHz) with a TMHS resonator (E2044500TMHS, Bruker BioSpin GmbH, Karlsruhe, Germany) with the following parameters: center field: 348.0 mT, sweep: 14.0 mT, sweep time: 45 s, modulation amplitude: 0.12 mT, modulation frequency: 100 kHz, microwave power: 10.02 mW. The initial measurement at time point 0 min was performed without irradiation. Afterwards, the following UVB-NIR irradiation was used for inducing radicals: 49 mW/ cm2 UVB-NIR (economical handheld laser power meter 843R, Newport Corporation, California, USA), 0.29 mW/cm2 UVB, 2.5 mW/cm2 UVA (radiometer photometer ILT1400, International Light Technologies, Waldbronn, Germany). The maximal dose of UVB irradiation over the time period of 2.3 min was 40.3 mJ/cm2 which corresponds to 1.3 MED. For evaluation, the EPR spectra of defined time points and all donors were averaged by MATLAB®. Subsequently, the obtained EPR spectra were simulated by WinSim, a public electron paramagnetic resonance software tool of NIEHS [30]. The total intensity of the EPR spectra was calculated from the integral and the ratio of reactive oxygen species (⋅OH radicals) and lipid oxygen species (carbon centered radicals, ⋅CCR) determined by WinSim. The detailed description for EPR measurements and data analysis for the EPR spin trap DMPO is described in Albrecht et al. [27].
2.2.4. Metabolic PCA decay For the quantification of radical production, the skin samples were incubated from both top and bottom with 38.4 μl of the EPR spin probe PCA using a Ø 8 mm filter paper (1.5 mM in distilled water/PBS). The EPR measurements were performed using the X-band EPR system “Elexsys E 500” (9.6 GHz) with a TMHS resonator (E2044500TMHS, Bruker BioSpin GmbH, Karlsruhe, Germany) using the following instrument parameters: center field: 348.0 mT, sweep: 14.0 mT, sweep time: 45 s, modulation amplitude: 0.15 mT, modulation frequency: 100 kHz, microwave power: 1.26 mW. All EPR measurements were performed at ambient temperature and measured in single-scan operation mode over a total time of 15.4 min. For evaluation, the PCA amount of each spectrum was determined by the Bruker software using spin fit analysis (Xepr 2.6b143, Bruker BioSpin GmbH, Karlsruhe, Germany). On the one site, the PCA amount was normalized and plotted over the measurement time resulting in PCA decay. On the other site, the cumulative radical production at each time point was calculated by subtraction of the PCA amount at a certain time point from the initial PCA amount at time point 0 min. To exclude differences between the different donors, the highest radical production of each donor (normal, inflammatory) was set to 1 and the radical production at each time point was related to this value. The relative cumulative radical production of normal and inflammatory skin equivalents was plotted over time.
2.3. MTT assay After the EPR measurements the cell viability of the skin samples was measured by MTT assay. The skin punches (Ø 4.5 mm) were placed on filter paper discs soaked with 0.5 mg/ml MTT solution (SigmaAldrich, Steinheim, Germany) dissolved in PBS and incubated for 4 h at 37 °C and 5% CO2. Afterwards, the skin samples were transferred to 400 μl isopropanol (Merck KGaA, Darmstadt, Germany) to extract the formazan salt overnight in the dark. The absorption of formazan salt was measured at 560 nm by multimode plate reader (PerkinElmer, Waltham, USA). The viability of skin samples was calculated to untreated control, which was set to 100%. Skin punches incubated with 20% SDS solution for 1 h served as positive control.
2.2.5. PCA decay induced by irradiation To induce radical production by an exogenous stress factor, the skin samples were irradiated during the EPR measurements additionally using a sun simulator (LOT-QuantumDesign GmbH, Darmstadt, Germany, classified to IEC 60904–9, class A; Xe-lamp, 150 W) which was directly coupled into the EPR resonator by an optical UV-VIS fiber (LOT-QuantumDesign GmbH, Darmstadt, Germany). The skin samples were exposed to the whole simulated solar spectrum; ultraviolet B (UVB) to near infrared (NIR) region (305–2200 nm) which mimics a solar spectrum by air mas 0. The following irradiances were used:
2.4. Determination of GSH concentration For determining the concentration of the reduced form of GSH, the epidermis of all skin equivalents, normal and inflammatory, was separated from the dermis by tweezers. Epidermis and dermis were then 3
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homogenized in isotonic PBS using the TissueLyserII from Qiagen GmbH (Hilden, Germany) for 1 min at 30 Hz, followed by sonification for 90 s (amplitude: 90, interval: 0.5) (Sonorex Super RK102H, BANDELIN electronic GmbH & Co. KG, Berlin, Germany). Afterwards, the skin homogenates were centrifuged at 12,000×g for 10 min. The supernatant was collected and part of it was used to determine the protein concentration using Pierce Protein Reagent Assay BCA Kit according to the manufacturers' instructions (Thermo Fisher Scientific, Waltham, USA). The remaining sample was incubated with cold 5% sulfosalicylic acid (Sigma Aldrich, Steinheim, Germany) for 10 min at 4 °C to eliminate interfering proteins. The GSH concentration was determined by means of a Glutathione Fluorescent Detection Kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturers' instructions.
skin equivalents (Fig. 1). Due to its amphiphilicity (logP = 1.8) [31,32], TEMPO is able to partition between hydrophilic and lipophilic microenvironments [25,33]. The total TEMPO spectra were simulated using the magnetic parameters shown in Table S1. The partitioning of TEMPO in normal and inflammatory skin equivalents is significantly different (p ≤ 0.05). In normal skin equivalents 33 ± 2% of TEMPO partitions in the lipophilic microenvironment compared to 25 ± 2% in inflammatory skin equivalents. However, in both skin equivalents most TEMPO is located in the hydrophilic microenvironment. 3.2. Antioxidant status in hydrophilic and lipophilic microenvironment To determine the antioxidant status in skin equivalents, the signal decay of TEMPO over time was investigated. TEMPO is a nitroxide radical including a six-membered piperidine ring which can cross cell membranes very easily and consequently spread in intra- and extracellular compartments of the whole skin equivalent [14]. The spin probe TEMPO can react potentially with oxidants and antioxidants in the skin, but it reacts predominantly with antioxidants as described in the literature [14,23]. TEMPO is reduced to the corresponding (diamagnetic) hydroxylamine, which is EPR-silent, mainly by ascorbate and thiol-dependent antioxidant activities like GSH in the skin [24]. Consequently, the decay of TEMPO over time indicates the radical scavenging capacity of skin. In Fig. 2A the total signal decay of TEMPO in both skin equivalents is illustrated which can be described as a biexponential function I (t ) = I0 (e khydro t + e klipo t ). Therefore, the TEMPO decay in both individual microenvironments was analyzed by evaluating the signal decay rate. The TEMPO decay of a single component, hydrophilic and lipophilic microenvironment, was assumed to be a monoexponential decay I (t ) = I0 e kt , which gave reasonable fits. The semi-logarithmic plots of the TEMPO signal intensity over the time for hydrophilic and lipophilic microenvironment are presented in the supplemental material (Fig. S2). Our investigations showed that there are no significant differences between normal and inflammatory skin equivalents in hydrophilic and lipophilic TEMPO decay. Although the signal decay rate is significantly different between hydrophilic and lipophilic microenvironments in both skin equivalents, the differences are very small (Fig. 2B). Notably, the viability of normal and inflammatory skin equivalents was not affected during the EPR measurements using TEMPO (Fig. S3).
2.5. Statistical analysis All data are presented as mean ± standard error of the mean (SEM). The statistical analysis was performed using IBM® Statistical Package for the Social Sciences (SPSS®) Statistics version 23 (IBM Corporation, Armonk, USA). For experiments over a certain time period, the method of generalized estimating equations (GEE) was used. Normal and inflammatory skin equivalents were compared by Wilcoxon test. A p-value ≤ 0.05 was considered to be statistically significant. 3. Results 3.1. TEMPO as indicator for different microenvironments within the skin equivalents To investigate the redox status in normal and inflammatory skin equivalents, several EPR markers were employed. First, the spin probe TEMPO was applied to the skin equivalents. Representative TEMPO spectra of normal and inflammatory skin equivalents are depicted in Fig. 1. The total TEMPO spectrum of both skin equivalents showed a splitting of the high field peak into two individual peaks in an X-band EPR spectrometer, whereas the low and middle field peak are only broadened compared to a TEMPO spectrum in PBS shown in supplemental material (Fig. S1). This can be interpreted, that the total TEMPO spectrum consists of two individual spectra, which represent TEMPO in the hydrophilic and lipophilic compartment (microenvironment) of the
Fig. 1. Partitioning of TEMPO in skin equivalents. Representative X-band EPR spectra of TEMPO of normal (A) and inflammatory (B) skin equivalents. Illustration of experimental (exp) and simulated (sim) total TEMPO spectrum, simulated hydrophilic and simulated lipophilic TEMPO spectrum including percentage of TEMPO in each microenvironment; mean ± SEM (n = 6). 4
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Fig. 2. Signal decay of TEMPO in skin equivalents. Illustration of biexponential fit of total TEMPO decay (A) and the resulting signal decay rates (B) of normal and inflammatory skin equivalents; mean ± SEM (n = 5). * statistically significant difference as indicated at p ≤ 0.05 (*).
3.3. Lower GSH levels in epidermis of inflammatory skin equivalents
metabolic PCA decay over the measured time period compared to normal skin equivalents (Fig. 4A). Expressing the PCA decay in quantitative radical formation, in normal skin equivalents 3.8 ± 1.5 × 1013 radicals/mm3 and in inflammatory skin equivalents 9.4 ± 3.5 × 1013 radicals/mm3 were detected after 15.4 min. Evaluating each donor of normal and inflammatory skin equivalents separately, the difference in relative radical production was highly significant (p ≤ 0.001) shown in Fig. 4B.
To get a deeper insight into the antioxidant status of hydrophilic microenvironment, the concentration of the hydrophilic antioxidant GSH was determined in epidermis and dermis homogenates of both normal and inflammatory skin equivalents. GSH represents a major regulator predominately in the intracellular redox status, because it is involved in the enzyme-mediated antioxidant system and the detoxification of hydrogen peroxide [16,34]. Epidermal homogenates of inflammatory skin equivalents showed a significantly lower GSH concentration compared to normal skin equivalents (Fig. 3A). In contrast, no significant differences in the GSH concentration was observed in the dermis homogenates between both skin equivalents (Fig. 3B).
3.5. UVB-NIR irradiation induced no differences in amount and type of radicals between normal and inflammatory skin equivalents Another possibility to monitor the redox status is the application of a stress response test. Irradiation is a common exogenous stress factor of the environment. Not only UVA and UVB, but also visible (VIS), near infrared (NIR) and infrared (IR) radiation are responsible for the radical production during solar exposure in skin [37–39]. In the experimental set up the whole simulated solar spectrum (UVB-NIR) was used to induce radicals within the skin equivalents (normal, inflammatory) to see the stress response and thus the antioxidant capacity to irradiation. The application of irradiation promoted a faster PCA decay in both skin equivalents compared to the absence of irradiation (Fig. 5A), but resulted in a similar irradiation-induced radical production for both skin equivalents (Fig. 5B). After the irradiation time, in normal skin equivalents 2.1 ± 0.4 × 1014 radicals/mm3 (1.7 ± 0.4 × 1014 induced radicals/mm3) and in inflammatory skin equivalents 2.8 ± 0.2 × 1014 radicals/mm3 (1.8 ± 0.4 × 1014 induced radicals/ mm3) were measured. Consequently, irradiation leads to a 3–5.5 fold higher radical production as without irradiation.
3.4. Higher metabolic radical production in inflammatory skin equivalents In addition to the antioxidant status, the oxidant status was investigated in normal and inflammatory skin equivalents using the spin probe PCA. Since PCA mainly reacts with free radicals in the skin resulting in EPR-silent products [26], the PCA decay was analyzed to quantify metabolic and irradiation-induced radicals by EPR spectroscopy. In contrast to TEMPO, PCA includes a five-membered pyrrolidine ring and is a hydrophilic nitroxide radical (logP = −1.7) and thus it mainly detects free radicals in hydrophilic microenvironment [26,35,36]. Its spectrum is comparable to the one of TEMPO in aqueous solution (Fig. S1). PCA penetrates slower into cells than TEMPO due to its charged carboxyl group at pH 7.4, but it distributes in intracellular and extracellular compartments [36] and detect the radicals present there. The inflammatory skin equivalents showed a significantly faster
Fig. 3. Glutathione (GSH) concentration of tissue homogenates. GSH concentration of epidermis homogenates (A) and dermis homogenates (B) of normal and inflammatory skin equivalents determined by fluorescence-based GSH assay and adjusted to the total protein amount of the skin homogenate; mean ± SEM (n = 4); * statistically significant difference as indicated at p ≤ 0.05 (*).
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Fig. 4. Metabolic radical production of skin equivalents using PCA (without UVB-NIR irradiation). (A) Normalized signal decay of PCA over time. (B) Relative cumulative radical production over time was calculated by subtraction of the PCA amount at a certain time point from the initial PCA amount at time point 0 min. To exclude differences between the different donors, the highest radical production of each donor (normal, inflammatory) was set to 1 and the radical production at each time point was adjusted to this value; mean ± SEM (n = 5); * statistically significant difference as indicated at p ≤ 0.05 (*) and p ≤ 0.001 (***).
To assess the type of radicals produced during irradiation, normal and inflammatory skin equivalents were investigated by using the EPR spin trap DMPO. It can enter cells very easily and reacts intracellularly and extracellularly with different free radicals locating in the hydrophilic microenvironment of skin (logP = −1.0) [40]. It forms DMPOradical-adducts (spin trapping) which are stable and measurable [41,42]. By that, DMPO can give information about the occurrence of different types of free radicals as well as reflect relative changes. However, the absolute quantification and the detection of low radical concentrations are challenging [34]. Therefore, in the experimental setup radicals were induced by UVB-NIR irradiation. The DMPO spectra of the analyzed skin equivalents include six peaks characterizing ⋅CCR, four peaks characterizing ⋅OH radicals and three peaks characterizing the nitroxide (Fig. 6A) [41]. The DMPO spectra were simulated using the magnetic parameters shown in Table S2. For both, normal and inflammatory skin equivalents, ⋅OH radicals and ⋅CCR were produced at similar ratio during irradiation (Fig. 6B). ⋅OH radicals were produced to 89% and ⋅CCR to 11% on average. The skin samples measured with PCA and DMPO showed a viability of at least 80% and thus they were still viable after the EPR measurements (Fig. S3).
4. Discussion Since the redox status in atopic skin is not known yet, the antioxidant and oxidant status in normal and inflammatory skin equivalents mimicking characteristics of AD in vitro were investigated mainly by EPR spectroscopy to get an insight into the role of redox status in atopic skin. Nitroxide radicals, like TEMPO and PCA are often used for skin investigations using EPR spectroscopy, because they can offer different information about the redox status and the measured microenvironment due to their various structural and physicochemical properties [34,43]. The spin probe TEMPO is able to partition between hydrophilic and lipophilic microenvironment in skin and thus its decay can be monitored in different microenvironments (Fig. 1) [25,33]. TEMPO in the hydrophilic microenvironment mainly corresponds to the viable epidermis and dermis and the lipophilic microenvironment corresponds to the stratum corneum (unpublished data). The main part of TEMPO is distributed in the hydrophilic microenvironment, because the viable epidermis and dermis representing the hydrophilic microenvironment are relatively larger compared to the stratum corneum representing the lipophilic microenvironment in the investigated skin sample. Consequently, the higher percentage of TEMPO in the lipophilic microenvironment in normal skin equivalents can be explained by a thicker stratum corneum in normal skin compared to atopic skin [44]. Although
Fig. 5. Irradiation-induced radical production of skin equivalents using PCA (with UVB-NIR irradiation). (A) Normalized signal decay of PCA over irradiation time. (B) Relative cumulative radical production induced by UVB-NIR irradiation (metabolic radical production was substracted). To exclude differences between the different donors, the highest radical production of each donor (normal, inflammatory) was set to 1 and the radical production at each time point was adjusted to this value. UVB-NIR irradiation (305–2200 nm) was used: 5.7 mW/cm2 UVB-NIR, 0.026 mW/cm2 UVB, 0.31 mW/cm2 UVA. The maximal dose of UVB irradiation was 24 mJ/cm2, which corresponds to 0.8 minimal erythema dose; mean ± SEM (n = 5). 6
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Fig. 6. Characterization of irradiation-induced radical production in skin equivalents using DMPO. (A) Representative X-band EPR spectrum of DMPO of nonirradiated and irradiated normal and inflammatory skin equivalents including labeled peaks for DMPO-CCR (carbon-centered radicals), DMPO-OH and nitroxide radicals. (B) Ratio between DMPO-CCR and DMPO-OH radicals. UVB-NIR irradiation (305–2200 nm) was used: 49 mW/cm2 UVB-NIR, 0.29 mW/cm2 UVB, 2.5 mW/ cm2 UVA. The maximal dose of UVB irradiation was 40.3 mJ/cm2, which corresponds to 1.3 minimal erythema dose; mean ± SEM (n = 4).
normal and inflammatory skin equivalents showed different partitioning of TEMPO in hydrophilic and lipophilic microenvironments, the TEMPO decay and thus the antioxidant status in the microenvironments were similar. Comparing the TEMPO decay in the hydrophilic and lipophilic microenvironments, a not relevant lower signal decay rate was detected in the lipophilic microenvironment of both skin equivalents, which can be due to lower amount of antioxidants in the stratum corneum [45]. To the best of our knowledge, such analysis of the TEMPO decay into hydrophilic and lipophilic microenvironments in skin equivalents was shown for the first time. Although the TEMPO decay showed no differences in the total antioxidant status between normal and inflammatory skin equivalents, we found a lower concentration of the hydrophilic antioxidant GSH in the epidermis of inflammatory skin equivalent compared to normal skin equivalent. In contrast, this difference was not present in the dermis. Consequently, it is illustrated that the results of redox status can differ between different skin layers and depending on whether the total antioxidant status in the skin or only one specific intracellular antioxidant is analyzed. In addition, EPR investigations using PCA showed a significantly higher metabolic radical formation in the inflammatory skin equivalents compared to normal skin equivalents (Fig. 4B). This could result from inflammation processes that are connected with oxidative stress [46,47]. To induce an inflammatory response in the skin equivalents, the proinflammatory cytokines IL-4 and IL-13 were supplemented to the culture medium. It has already been shown that these cytokines increase the free radical production [48]. The increased metabolic radical production in the inflammatory skin equivalents can lead, in turn, to a lower antioxidant status for protection, which was shown by a lower GSH concentration in the epidermis (Fig. 3A). Consequently, our data support the results of Sapuntsova et al. who detected a decompensated accumulation of oxidants and inhibition of antioxidative systems in skin biopsies of AD patients [21]. In contrast, Antille et al. published a lower oxidative status in the stratum corneum of AD patients [20]. These differences might have been caused by the fact that the authors investigated non-lesional skin areas of AD patients in contrast to our skin equivalents which mimic characteristics of lesional atopic skin. Since the inflammatory skin equivalents showed a significantly higher radical production without irradiation, we expected also a faster PCA decay and thus a higher radical production by irradiation. Surprisingly, the UVB-NIR irradiation induced the same PCA decay and thus the same amount of irradiation-induced radicals in normal and inflammatory skin equivalents (Fig. 5B). Consequently, the skin
equivalents showed the same resistance to irradiation which is in accordance to the similar antioxidant status determined by TEMPO decay (Fig. 2). It is known that AD patients show no modifications or even improvement of eczema during sun exposure, which can explain the missing negative effect of solar sun irradiation [49]. In addition to the similar irradiation-induced radical amount in normal and inflammatory skin equivalents a similar ratio between ⋅OH radicals and ⋅CCR were detected in the skin equivalents by the spin trap DMPO. The DMPO data of normal skin equivalents are consistent with published data [27]. Since radicals are already induced in the DMPO solution by UVB irradiation, DMPO measurements were not used for quantitative analysis, but to characterize the radical type and determine possible differences in the ratio of radical production (⋅OH radicals to ⋅CCR) between normal and inflammatory skin equivalents. Since normal and inflammatory skin equivalents were measured under the same conditions, the produced radicals by DMPO solution will be similar for both. Albrecht et al., already showed differences in the ratio between ⋅OH radicals and ⋅CCR between different skin models (human, porcine, skin equivalents) by using DMPO, whereby a comparable tendency in semiquantitative data to PCA was shown [27]. The spin marker PCA was used for radical quantification, because no radicals can be detected in PCA solution during irradiation. However, the ratio between ⋅OH radicals and ⋅CCR showed no differences in normal and inflammatory skin equivalents, which is in accordance to the irradiationinduced radical production quantified by PCA (Fig. 5B). ⋅OH radicals represent the main radicals produced in the skin equivalents after irradiation, which are formed in the first steps of oxidative stress development. In the next steps ⋅OH radicals interact with lipid and membrane structures, promoting the formation of ⋅CCR, like lipid radicals [50]. The produced radicals are in agreement with former investigations on ex vivo human and porcine skin [27,39]. The differences in redox status investigations may result from the applied methods and the part of skin, on which the focus is set. As shown in our investigations the GSH concentration in epidermis and metabolic radical formation differ between the skin equivalents but the total antioxidant status, GSH in dermis and the stress response to irradiation did not show differences between normal and inflammatory skin equivalents. We have to admit, our results cover one part of the redox status in inflammatory skin equivalents, because only a limited range of nitroxides were used and not all aspects of redox status were considered, e.g. the extracellular redox status. Therefore, atopic skin has to be studied further to confirm these results. The skin equivalents 7
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offer the possibility and advantage to investigate the endogenous antioxidant system produced exclusively by the metabolism of cells. These results showed that a supply of antioxidants could be beneficial for atopic skin, in particular hydrophilic ones could be more effective than lipophilic. Exogenous antioxidants like vitamin C, vitamin E and carotenoids are not contained in skin equivalents, however they have an essential influence on the redox status leading to a changed TEMPO decay and thus antioxidant system as already shown in vivo after supplementation of different exogenous antioxidants [23,51].
[12] [13]
[14]
5. Conclusions
[15]
In conclusion, we could gain a first insight into the endogenous redox status of inflammatory skin equivalents by using three different EPR spin markers. Our investigations showed differences in the redox status of inflammatory skin equivalents concerning epidermal GSH and metabolic radical production compared to normal skin equivalents. Consequently, our results suggest that the redox status could play a role in developing AD and that antioxidants could be beneficial for atopic skin. To investigate the redox status comprehensively, the applied methods (total status or individual component) as well as the focused skin compartment should be considered. Nevertheless, the results presented here only cover parts of the redox status (e.g. no exogenous antioxidants) in inflammatory skin diseases and do not offer a complete overview. Therefore, further detailed investigations of the redox status in atopic skin are indispensable.
[16] [17] [18] [19]
[20] [21]
Funding
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This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Germany via the Collaborative Research Center 1112 (project B01 and C02).
[23]
Conflicts of interest
[24]
The authors declare no conflict of interest.
[25]
Appendix A. Supplementary data
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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbi.2019.108752.
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