Morphological analysis of JAK1 intracellular pathway activation after pro-inflammatory psoriatic cytokines exposure: inside-out and outside-in the epidermis

For their normal growth, cells depend on a continuous flow of signals from the environment. The Janus kinases (JAK) 1 transducers signalling pathway is a pleiotropic cascade used to transduce a multitude of signals among cells. A variety of ligands including cytokines, hormones, growth factors, and their receptors stimulate the JAK1 pathway. Cytokines, a large and very heterogeneous family of small and generally soluble glycoproteins, both control multiple biological processes as haematopoiesis, inflammation, and immunity playing a central role in cell-cell communication. Their action is mediated by the binding to specific receptors on the cell surface, thus transducing biological information to target cells [1]. Pro-inflammatory cytokines play a pivotal role in several inflammatory illnesses including psoriasis. Among them, interleukin (IL)-17, IL-22, IL-23 and tumor necrosis factor (TNF)-alpha play a central role. In the formation and progression of the psoriatic lesion a typical marker is keratin (K) 17 which is correlated with psoriasis severity. The aims of this study were to evaluate the early, direct, and specific effects of pro-inflammatory psoriatic cytokines i) on the activation of the intracellular pathway JAK1 and ii) on the correlation with the induction of K17 expression in a three-dimensional model (3D) of human skin (n=7) by immunofluorescence. Biopsies were cultured overnight epidermal side-up in a Transwell system and exposed to 50 ng/ml IL-17, or 100 ng/ml IL-22, or 50 ng/ml IL-23 or 100 ng/ml TNF-alpha. Samples were harvested 24 (T24), 48 (T48), and 72 (T72) hours after cytokine incubation. In samples not exposed to cytokines, a JAK1 slight labelling was observed throughout the epidermis, decreasing at T72 in the lower layers. At T24, IL-17 and IL-22, but not IL-23 and TNF-alpha, induced an expression of JAK1 in the spinous layer. At T72, JAK1 immunostaining decreased in all samples, similarly to controls. K17 immunopositivity was induced and progressively increased with time in the suprabasal layers of epidermis in all experimental groups, with the exception of the TNF-alpha group. These results suggest that cytokines exert parallel effects on JAK1 pathway activation and K17 induction. In conclusion, 3D this model, reproducing some features of psoriatic microenvironment, represents a useful experimental approach to dissect the specific role of each cytokine in the different steps of psoriatic lesion formation

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 \ub5g/mL and T: 1 \ub5g/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 harvested 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 substances 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

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. The simultaneous inhibition of the effect of different cytokines - all with a defined role in the pathogenesis of psoriasis - could further improve psoriasis treatment

Ultrastructural features in organotypic cultures of normal human skin in an in vitro microenvironment mimicking atopic dermatitis

Atopic dermatitis (AD) is a common inflammatory skin disease charactided by chronic, systemic inflammation, early age of onset, persistent itch, marked redness, cracking, and dryness of the skin. Skin infections occur frequently in AD, contribute to the disordered immune activity and are probably related to disruption of skin barrier by inhibiting lipids, tight junctions, and antimicrobial peptide formation. Among the several cytokines involved in the AD pathogenesis interleukin (IL) -4, IL- 1 3 and IL22 play a key role. The present study is focused on the early effects of pro-inflammatory cytokines on ultrastructural changes using transmission electron microscopy (TEM) in a well standardized 3D model of normal human skin organotypic culture. Skin explants obtained from plastic surgery of healthy 20-40 yearold women (n = 5) after informed consent were cultured ovemight in Dulbecco's modified Eagle's medium and treated with 50 nglml IL-4, 50 nglml IL-13 and 100 nglml IL-22 alone or in combination (TRIS). Samples were then harvested 24 and 48 hours after the cytokine incubation. In all samples exposed to cytokines, desmosomes appeared well preserved, comparable to controls. TEM analysis revealed that, starting from24 hours of culture, the exposure toIL-4, but not to IL13, caused a marked enlargement of intercellular spaces and chromatin condensation. In TRIS treated samples, these alterations were less evident, however, we have observed condensation of cytoskeletal filaments. Altogether, this present study strongly suggest that this experimental approach useful for studying the early, direct, and specific effects of pro-inflammatory AD cytokines and may be useful for assessing new biological drugs directed against a specific cytokine

An innovative three-dimensional model of normal human skin to study the proinflammatory psoriatic effects of tumor necrosis factor-alpha and interleukin-17

Background: Among all cytokines involved in the pathogenesis and in the progression of psoriasis, Tumor Necrosis Factor (TNF)-alpha and interleukin (IL)-17 play a pivotal role. Objective: The aim of the present study was to mimic a psoriatic microenvironment and to investigate the early effects of TNF-alpha and IL-17 in a three-dimensional model of organotypic normal human skin. Methods: Human skin explants were obtained from plastic aesthetic surgery of healthy young women 20-40. years old ( n= 7). The study was approved by the Institutional Review Board and written informed consent was obtained from all subjects. Bioptic fragments were cultured at the air-liquid interface overnight in a Transwell system and further divided before adding either 50. ng/ml IL-17 or 100. ng/ml TNF-alpha or a combination of both cytokines. For each subject, a control sample was cultured without any cytokine. Samples were harvested 24 or 48. h after cytokine incubation. At both time points and for all cytokine treatments a bioptic fragment obtained from each patient was processed. Epidermal proliferation, expressions of terminal differentiation (keratin 10, K10, and 14, K14) and of intercellular adhesion (occludin for tight junctions and E-cadherin for adherens junctions) biomarkers were investigated by indirect immunofluorescence. Results: IL-17 and TNF-alpha induced an early and statistically significant inhibition of keratinocyte proliferation (more than 80% compared with their respective controls). At 24. h, the combination of both cytokines did not further reduce cell proliferation. Starting from 24. h of incubation, a non-continuous occludin expression in the granular layer was observed after both IL-17 and TNF-alpha exposure. Immunolabelling for E-cadherin in adherens junctions, for K10 in the suprabasal layers, and for K14 in the basal layer was similar in all experimental groups and unaffected after cytokine treatment. Conclusions: These results suggest that in this experimental model IL-17 and TNF-alpha induced an early alteration of the homeostasis of the inner proliferative layer and of the upper granular layer, as shown by cell proliferation inhibition and occludin expression.

Effects of cytokines involved in a microenvironment mimicking atopic dermatitis in a well standardized three-dimensional model of normal human skin

Atopic dermatitis (AD) is one of the most common chronic skin inflammatory disorders, driven by several pro-inflammatory cytokines among which interleukin (IL)-4, IL-13, and IL-22 are the most involved mediators. Tight junctions (TJs) are targeted in AD from both a morphological and a molecular level, leading an epidermal barrier disruption, which is one of the clinical manifestations of AD. TJs seal adjacent keratinocytes in the granular layer and are composed by transmembrane proteins such as occludin and claudins, in addition to cytosolic scaffold proteins. The present study was aimed at evaluating the early effects of IL-4 and IL-13 using a well-standardized three-dimensional model of normal human skin. Skin explants were obtained from plastic surgery of healthy women (n=5) 20-40 years old after informed consent, and cultured overnight in Dulbecco\u2019s modified Eagle\u2019s medium and treated with 50 ng/mL IL-4, 50 ng/mL IL-13. Secondly, the samples were harvested 24 hours after the cytokine incubation and in parallel processed for morphological and molecular biology analysis through both Western Blot. Compared to control samples, a decrease of the expression of occludin was found in samples incubated with either IL-4 or IL-13. TEM analysis revealed an increase of the intercellular spaces and chromatine condensation only in samples treated with IL-4, with well preserved desmosomes. Conversely, samples incubated with IL-13 showed similar features as control ones. All these results suggest the usefulness of this experimental model, which could be used in order to study and evaluate the early and direct effects of several pro-inflammatory cytokines involved in AD, providing a well and standardize method for pre-clinic dermatology research

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

Skin, the most superficial tissue of our body, is the first target of environmental 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 \u3bcM), 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