Federica Landoni

Effects of UV Rays and Thymol/Thymus vulgaris L. Extract in an ex vivo Human Skin Model: Morphological and Genotoxicological Assessment.

Ultraviolet (UV) radiation is the major environmental factor affecting functions of the skin. Compounds rich in polyphenols, such as Thymus vulgaris leaf extract and thymol, have been proposed for the prevention of UV-induced skin damage. We compared the acute effects induced by UVA and UVB rays on epidermal morphology and proliferation, cytotoxicity, and genotoxicity. Normal human skin explants were obtained from young healthy women (n = 7) after informed consent and cultured at the air-liquid interface overnight. After 24 h, the samples were divided in 2 groups: the former exposed to UVA (16 or 24 J/cm2) and the latter irradiated with UVB (0.24 or 0.72 J/cm2). One hour after the end of irradiation, supernatants were collected for evaluation of the lactate dehydrogenase activity. Twenty-four hours after UVB exposure, biopsies were processed for light and transmission electron microscopy analysis, proliferation, cytotoxicity, and genotoxicity. UVB and UVA rays induced early inhibition of cell proliferation and DNA damage compared to controls. In particular, UVB rays were always more cytotoxic and genotoxic than UVA ones. For this reason, we evaluated the effect of either T. vulgaris L. extract (1.82 µg/ml) or thymol (1 µg/ml) on all samples treated for 1 h before UVB irradiation. While Thymus had a protective action for all of the endpoints evaluated, the action of the extract was less pronounced on epidermal proliferation and morphological features. The results presented in this study could be the basis for investigating the mechanism of thymol and T. vulgaris L. extract against the damage induced by UV radiation.

Tumor necrosis factor-alpha and interleukin-17 differently affects Langerhans cell distribution and activation in an innovative three-dimensional model of normal human skin.

Among the several cytokines involved in the psoriasis pathogenesis, tumor necrosis factor (TNF)-alpha and interleukin (IL)-17 play a central role. Many biomolecular steps remain unknown due to difficulty to obtain psoriatic models. To investigate the effect of TNF-alpha and IL-17 on the ultrastructure, immunophenotype, and number of epidermal Langerhans cells (LCs), human skin explants (n=7) were cultured air-liquid interface in a Transwell system. Four different conditions were used: medium alone (control), medium added with 100 ng/ml TNF-alpha or 50 ng/ml IL-17 or a combination of both cytokines. Samples were harvested 24 and 48 h after cytokine addition and were frozen. Samples harvested at 24h were also processed for transmission electron microscopy (TEM). By immunofluorescence analysis with anti-human Langerin antibody (three experiments/sample) we calculated the percentage of LCs/mm(2) of living epidermis after 24 and 48 h of incubation (considering control as 100%). At 24h LC number was significantly higher in samples treated with both cytokines (216.71+15.10%; p<0.001) and in TNF-alpha (125.74+26.24%; p<0.05). No differences were observed in IL-17-treated samples (100.14+38.42%). After 48 h, the number of epidermal Langerin-positive cells in IL-17- and TNF-alpha treated samples slightly decreased (94.99+36.79% and 101.37+23% vs. their controls, respectively). With the combination of both cytokines epidermal LCs strongly decreased (120+13.36%). By TEM, upon TNF-alpha stimulus LCs appeared with few organelles, mostly mitochondria, lysosomes, and scattered peripherical BGs. Upon IL-17 stimulus, LCs showed a cytoplasm with many mitochondria and numerous BGs close to the perinuclear space and Golgi apparatus, but also at the periphery, at the beginning of the dendrites. The addition of both cytokines did not affect LC ultrastructure. Our study showed that IL-17 induced significant changes in LC ultrastructure, while the combination of both cytokines seems to have a strong chemo-attractant effect on epidermal LCs, supporting the relevance of investigating the interplay between LCs and pro-inflammatory cytokines in the ongoing of the disease.

Interleukin 22 early affects keratinocyte differentiation, but not proliferation, in a three-dimensional model of normal human skin

Interleukin (IL)-22 is a pro-inflammatory cytokine driving the progression of the psoriatic lesion with other cytokines, as Tumor Necrosis Factor (TNF)-alpha and IL-17. Our study was aimed at evaluating the early effect of IL-22 alone or in combination with TNF-alpha and IL-17 by immunofluorescence on i) keratinocyte (KC) proliferation, ii) terminal differentiation biomarkers as keratin (K) 10 and 17 expression, iii) intercellular junctions. Transmission electron microscopy (TEM) analysis was performed. A model of human skin culture reproducing a psoriatic microenvironment was used. Plastic surgery explants were obtained from healthy young women (n=7) after informed consent. Fragments were divided before adding IL-22 or a combination of the three cytokines, and harvested 24 (T24), 48 (T48), and 72 (T72)h later. From T24, in IL-22 samples we detected a progressive decrease in K10 immunostaining in the spinous layer paralleled by K17 induction. By TEM, after IL-22 incubation, keratin aggregates were evident in the perinuclear area. Occludin immunostaining was not homogeneously distributed. Conversely, KC proliferation was not inhibited by IL-22 alone, but only by the combination of cytokines. Our results suggest that IL-22 affects keratinocyte terminal differentiation, whereas, in order to induce a proliferation impairment, a more complex psoriatic-like microenvironment is needed.

Modulation of epidermal proliferation and terminal differentiation in a promising ex vivo human skin model mimicking a psoriatic microenvironment

Epidermal keratinocyte hyperproliferation is one of the key features involved in the formation/progression of psoriatic lesions and is driven by cytokines, among which TNF-α, IL-17, IL-22 and IL-23, secreted by both activated resident immune cells and keratinocytes (1). The network orchestrated by these cytokines is essential for the communication between resident cells and infiltrating cells and is due to redun- dancy, synergism and, sometimes, the reciprocal antagonism of cytokines. The aims of our study were to investigate whether the exposure of normal human skin to four main psoriatic cytokines, i.e. TNF-α, IL-17, IL-22, and IL-23 (cytokine mix) induced i) a modulation of epidermal proliferation and ii) a modification of keratinocyte terminal differentiation (TD). Human skin samples (n=5) obtained from healthy 20-40 years- old women after plastic surgery, were exposed to the cytokine mix in a Transwell sys- tem at air-liquid interface as previously described (2). For each patient a control (ctr) group was not exposed to cytokine mix. Samples were harvested 5 (T5), 24 (T24), 48 (T48) and 72 (T72) hours after cytokine stimulation, processed for paraffin embedding and immunofluorescence analysis for the quantitative analysis of epidermal proliferation and the expression of the TD biomarkers, keratin (K) 10 and 17. A decrease of cell proliferation was evident starting from T5 in samples exposed to cytokine mix and was progressively more marked at later time points (T5 ctr 47.44 ± 6.90 vs mix 23.07 ± 8.84; T24 ctr 41.12 ± 12.78 vs mix 12.16 ± 1.53; T48 ctr 25.88 ± 10.21 vs mix 2.19 ± 2.44; T72 ctr 10.49 ± 2.52 vs mix 0.65 ± 1.02) (p<0.05). K17 expression was evi- dent in samples exposed to the cytokine mix. Altogether the present results suggest that cell proliferation inhibition and K17 expression could be regarded as the basis for a later response to injury leading to psoriatic lesion formation/progression. In conclu- sion, this model allows to investigate the intimate interplay among different psoriatic cytokines and new insights may be of potential value for future clinical treatments.

Langerhans cells and Toll Like Receptors: how do they act and react in an in vitro psoriatic microenvironment?

Tumor Necrosis Factor (TNF)-α, interleukin (IL)-17, IL-22 and IL-23 are involved in the psoriasis pathogenesis and represent a strong proinflammatory stimulus. Both epidermal keratinocytes (KCs) and Langerhans cells (LCs) early respond promoting an early epidermal response [1, 2]. Human skin can count on the cellular response supported by LCs and on innate immunity through the expression of Toll-like Receptors (TLRs) [4]. We aimed at investigate whether the exposure of normal human skin to a combination of TNF-α, IL-17, IL-22, and IL-23 (cytokine mix) affected i) LCs immunophenotype, ii) expression of TLR2 and TLR9 and iii) KC proliferation. Human skin samples were obtained after plastic surgery (n = 5) and exposed to the cytokine mix in a Transwell system at air-liquid interface, with a parallel control group. Samples were harvested 24 and 48 hours after cytokine stimulation, processed in parallel for immunofluorescence or ultrastructural analysis. A decrease of cell proliferation was evident in samples exposed to cytokine mix for 24 hours and this phenomenon was more and more evident later. TLR2 immunopositivity progressively disappeared in the basal layer after cytokine mix exposure compared to the control group, while TLR9 expression was induced in scattered granular keratinocytes. By TEM, LCs showed an activated phenotype. In conclusion, these results suggest that, in a microenvironment mimicking the psoriatic plaque, epidermis early stimulates two important lines of defense, thus proposing that a therapeutic intervention in this direction can interfere with the formation/progression of the psoriatic plaque.

Effect of TNF-alpha and IL-17 on TLR expression and Langerhans cells phenotype in a three-dimensional model of normal human skin: a morphological study

Toll-like receptors (TLRs) are essential for innate immunity and contribute to create the skin barrier. Their abnormal stimulation is involved in the development of several dermatological diseases, among which psoriasis. Tumor Necrosis Factor (TNF)-alpha and interleukin (IL)-17 play a pivotal role in the pathogenesis of psoriatic plaques and their proinflammatory activity can affect Langerhans cell (LC) phenotype. In a well characterized three-dimensional model of organotypic cultures of normal human skin [1-3] we evaluated the effect of TNF-alpha and IL-17 on the expression of TLR2 and 9 by immunofluorescence, on the ultrastructural morphology of keratinocytes and LCs by transmission electron microscopy (TEM). Human skin explants (n=7) were cultured at the air-liquid interface overnight in a Transwell system and exposed to 50 ng/ml IL-17 or 100 ng/ml TNF-alpha or a combination of both cytokines. Samples were harvested 24 (T24) and 48h (T48) after cytokines incubation. After incubation with IL-17 and IL-17+TNF-alpha, TLR2 immunostaining was not detectable in the basal layer, differently from controls and TNF-alpha-treated samples. Conversely, TLR9 expression was progressively induced in granular keratinocytes in all cytokine-exposed groups. By TEM, enlargements of intercellular spaces were evident especially and, after IL-17 treatment, LCs showed an activated phenotype. At T24 LCs number increased indicating that TNF-alpha and IL-17+TNF-alpha exert a chemoattractant activity, while at T48 only IL-17+TNF-alpha maintained this effect on trapping LCs in epidermis. TNF-alpha and IL-17 differently affect LCs behaviour and TLR expression, with a specific contribution to the inflammatory loop underlying the lesion formation. These results suggest that the simultaneous inhibition of the effect of different cytokines with a defined role in the pathogenesis of psoriasis could improve psoriasis treatment.

Interleukin 17 affects early and late biomarkers of terminal differentiation in a three-dimensional model of normal human skin

Interleukin (IL)-17 expression has been correlated with the pathogenesis of mul- tiple autoimmune diseases, as rheumatoid arthritis, multiple sclerosis, and, more recently, psoriasis (1). During plaque formation, the interplay between immunocytes and keratinocytes is deregulated, resulting in the altered expression of keratin (K) 17, occludin, and filaggrin in psoriatic lesional epidermis (2, 3). The involvement of IL-17 in psoriasis pathogenesis has been identified (4), but the specific and intrinsic effects exerted by this cytokine have not been thoroughly investigated. The aim of the pre- sent work was to study by indirect immunofluorescence the expressions of K17, K10, filaggrin, and occludin in a three-dimensional model of normal human skin stand- ardized in our laboratory (5). Human skin samples (n = 5) obtained from healthy 20-40 years-old women after plastic surgery, were exposed to IL-17 in a Transwell system at air-liquid interface as previously described (5). Samples were harvested 24 (T24), 48 (T48), and 72 (T72) hours after IL-17 stimulation and processed for paraffin embedding and immunofluorescence analysis for expressions of K17, K10, filaggrin, and occludin. After IL-17 exposition, K17 immunostaining progressively increased with time in the upper stratum spinosum, while K10 expression resulted homogene- ously distributed in the suprabasal layers. In IL-17 treated samples occludin staining became irregular starting from 24 hours. On the other hand, filaggrin distribution was affected in T48 samples, where the immunolabelling was discontinuously punc- tuate. In conclusion, our results strongly support the use of this experimental setting for investigating the time-dependent early effects induced by IL-17 in normal human skin.

Effects of UV rays and natural compound repairs using an ex-vivo human skin model: morphological and genotoxicological analysis

Among the key factors in skin disorders such as wrinkling, dryness and photo-aging, the exposure to solar ultraviolet (UV) radiation plays a central role (1). Recently, compounds rich in polyphenols such as Thymus Vulgaris Leaf (TVL) extract and its major component Thymol (T) have been proposed in the prevention of UV-induced skin damages (2). Experiments were carried out in a human ex-vivo skin model, in which biopsies were obtained from aesthetic surgery of healthy 20-40 year-old women (n=6) after written informed consent (3). After 24 h, samples were pre-treated for 1 h with comparable concentrations of two compounds (TVL: 1.82 μg /mL and T: 1 μg /mL) before being irradiated with different UVB doses (0.24 J/ cm2 to 0.72 J/cm2) or UVA radiation (8 J/cm2 to 32 J/cm2). Samples were harvest- ed 24 h after irradiation and were processed both for light and transmission electron microscopy.Cell proliferation, Lactate Dehydrogenase assay, alkaline comet test, and histone H2AX phosphorylationwere evaluated. Both UVB and UVA induced an early inhibition of cell proliferation and DNA damage compared with respective controls. In particular, UVB rays were always more cytotoxic and genotoxic than UVA. The T-pretreatment showed a reduction of UVB-induced structural/ultrastructural and genotoxic damages. These results suggest that polyphenol fraction of tested substanc- es may be useful for skin photoprotection after UV radiation damage in an ex-vivo human skin model. The present study suggests that this experimental setting can be a reliable approach for safety evaluation of UV skin exposure.

Evaluation of protective effect of Thymol on UVBinduced damage in an ex-vivo human skin tissue model: morphological analysis and genotoxic evaluations

insult, among which is the most important solar ultraviolet (UV) radiation (Bernerd et al., 2001). For this reason, the use of human skin tissue obtained from plastic aesthetic surgery represents a simple but efficient experimental approach to reproduce a physiological condition to test the early effects of an exogenous stimulus as UV radiation and the possibility of preventing or reducing the early epidermal effects. Normal human skin explants were obtained from healthy young non smoking women 20-40 years old (n=5) after informed consent and cultured epidermal side up at the air-liquid interface overnight in a Transwell system before treatment (Donetti et al., 2005; Bedoni et al., 2007). They were further divided in two groups: the first was exposed to UVB doses ranging from 0.24 J/cm2 to 0.72 J/cm2 and the other one pretreated for 1 h with Thymol (natural monoterpene phenol, 6.6 μM), before the UVB irradiation. In each experiment a cultured sample was not UVB exposed and represented the internal control. Samples were harvested 24 hours after of UVB exposure. Lactate Dehydrogenase (LDH) assay and alkaline comet and micronucleus tests were used to assess cytotoxicity and genotoxicity, respectively. Bioptic fragments were processed both for transmission electron (TEM) and light (LM) microscopy. Epidermal proliferation was investigated by indirect immunofluorescence after incorporation of 5-Bromo- 2’-deoxyuridine (BrdU). UVB induced evident ultrastructural alterations in nucleus and cytoskeleton, while the pretreatment with Thymol showed a reduction of damage in UVB exposed samples both from the morphological point of view that genotoxic aspects. Cell proliferation was strongly inhibited by UVB exposure, while in Thylom pretreated samples was comparable to control. Furthermore these results strongly support the use of ex vivo human skin as a relevant method for safety evaluation of UV skin exposure.

Morphological analysis of the effects of tumor necrosis factor-alpha and interleukin-17 in a three-dimensional organotipic model of normal human skin

Psoriasis is an autoimmune chronic inflammatory disease in which epidermal keratinocytes and innate immunity effector cells play a pivotal role in the lesion formation in genetically predisposed subjects (Bonifati et al., 1999). Among the several cytokines involved in psoriasis pathogenesis, tumor necrosis factor (TNF)-alpha and interleukin (IL)-17 play a relevant role. TNF-alpha stimulates the production of many chemokines, induces cell proliferation, and is proapoptotic. IL-17 is involved in the recruitment/activation of neutrophils and induces keratin 17 (K17) expression in psoriatic lesions. The present study is focussed on the early effects of these proinflammatory cytokines on i) the molecular composition of intercellular junctions (desmocollin (DSC)1/desmoglein (DSG)1, E-cadherin, and occludin) ii) on K17 expression iii) on immunophenotype/number of epidermal Langerhans cells (LCs) after cytokines exposure. Ultrastructural analysis was performed in on all samples. Skin explants obtained from plastic surgery of healthy 20-40 year-old women (n = 7) after informed consent, were cultured overnight in Dulbecco’s modified Eagle’s medium and divided before adding 100 ng/ml TNF-alpha or 50 ng/ml IL-17 or a combination of both cytokines (Donetti et al., 2014). Samples were harvested 24, 48, and 72 hours after cytokine incubation. Occludin immunostaining was non homogeneous in cytokine treated samples, starting from 24 hours of culture. Interestingly, K17 expression was induced only in IL-17-treated samples only. No differences were observed in DSC1, DSG1 and E-cadherin expression by immunofluorescence. LC number was significantly higher in samples treated with both cytokines (216.71±15.10%) than in TNF-alpha (125.74±26.24%) or IL-17 (100.14±38.42%) alone. TEM analysis revealed that spaces were enlarged in the basal and spinous layer, especially upon TNF-alpha treatment, but desmosomes were uniformly distributed. Upon TNF-alpha stimulus LCs appeared with few organelles, mostly mitochondria, lysosomes, and scattered peripherical Birbeck granules. Upon IL-17 stimulus, LCs showed a cytoplasm with many mitochondria and numerous Birbeck granules close to the perinuclear space and Golgi apparatus, but also at the periphery, at the beginning of the dendrites. The addition of both cytokines did not modify LC ultrastructure. Altogether this study strongly suggests that this model is useful to study the early, direct, and specific effects of specific psoriatic cytokines on the different cell population.