Victoria Barygina

SIRT1 regulates MAPK pathways in vitiligo skin: insight into the molecular pathways of cell survival

Vitiligo is an acquired and progressive hypomelanotic disease that manifests as circumscribed depigmented patches on the skin. The aetiology of vitiligo remains unclear, but recent experimental data underline the interactions between melanocytes and other typical skin cells, particularly keratinocytes. Our previous results indicate that keratinocytes from perilesional skin show the features of damaged cells. Sirtuins (silent mating type information regulation 2 homolog) 1, well‐known modulators of lifespan in many species, have a role in gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection and healthy ageing. In the literature there is no evidence for SIRT1 signalling in vitiligo and its possible involvement in disease progression. Here, biopsies were taken from the perilesional skin of 16 patients suffering from non‐segmental vitiligo and SIRT1 signalling was investigated in these cells. For the first time, a new SIRT1/Akt, also known as Protein Kinase B (PKB)/mitogen‐activated protein kinase (MAPK) signalling has been revealed in vitiligo. SIRT1 regulates MAPK pathway via Akt‐apoptosis signal‐regulating kinase‐1 and down‐regulates pro‐apoptotic molecules, leading to decreased oxidative stress and apoptotic cell death in perilesional vitiligo keratinocytes. We therefore propose SIRT1 activation as a novel way of protecting perilesional vitiligo keratinocytes from damage.

Low dose cytokines reduce oxidative stress in primary lesional fibroblasts obtained from psoriatic patients

Psoriasis vulgaris is an inflammatory systemic disease with unclear pathogenesis characterized by the appearance of scaly erythematous plaques on the skin. These plaques are the sites of keratinocytes hyper-proliferation and misdifferentiation and inflammatory infiltration by T cells skewed toward Th1/Th17 with respect to Th2/TREG. Such disequilibrium between T cells population features psoriasis as an autoimmune disease [1]. A prominent attention is given to biologic drugs aimed to restore the balance between T cells populations in psoriasis: the use of antibodies against the overproduced cytokines or the use of cytokines-antagonists represent two successful strategies that have been shown to ameliorate Psoriasis Area Severity Index (PASI). Although targeting specific parts of immune system, such treatments require the use of high dose biologics and are frequently associated with adverse effects [2]. There are evidences that low doses of cytokines prepared by sequential-kinetic-activation (SKA) are effective and reduce adverse effects in psoriasis treatment with respect to standard dose of recombinant cytokines. Thus, in a clinical trial involving 48 patients affected by psoriasis vulgaris, the oral administration of SKA low-dose IL-4, IL-10 and IL-11 led to a significant reduction in PASI index with no adverse events [3]. Previously, signs of oxidative stress were found in the blood and skin of psoriatic patients [4,5] and according to our data, anti-TNFa drug Infliximab reduces oxidative stress in Peripheral Blood Mononuclear cells (PBMC) from psoriatic patients [4]. Moreover, we recently showed that the treatment with low dose SKA (femtograms per ml) IL-4, IL10, basic fibroblasts growth factor (bFGF) and b-endorphin (b-End) decreases oxidative stress in perilesional keratinocytes obtained from vitiligo skin [6]. In the present study the effect of low dose SKA cytokines in fibroblasts from lesional skin of psoriatic patients was investigated. We focused our attention on dermal fibroblasts because these cells provide a crucial microenvironment for epidermal keratinocyte function and, together with infiltrating polymorphonuclear leukocytes were shown to extensively produce superoxide and H2O2 impairing the redox balance in psoriatic derma and participating, at least partially, in abnormal keratinocytes growth in psoriasis [7].

A Biochemical Approach to Detect Oxidative Stress in Infertile Women Undergoing Assisted Reproductive Technology Procedures

Oxidative stress plays a major role in critical biological processes in human reproduction. However, a reliable and biologically accurate indicator of this condition does not yet exist. On these bases, the aim of this study was to assess and compare the blood and follicular fluid (FF) redox status of 45 infertile subjects (and 45 age-matched controls) undergoing in vitro fertilization (IVF), and explore possible relationships between the assessed redox parameters and IVF outcomes. Reactive Oxygen Species (ROS) production, assessed by flow cytometry analysis in blood leukocytes and granulosa cells, significantly increased (p < 0.05) in infertile patients. Also, oxidative stress markers—ThioBarbituric Acid-Reactive Substances (TBARS) as an index of lipid peroxidation, and Oxygen Radical Absorbance Capacity (ORAC) to account for total antioxidant capacity, both assayed by fluorometric procedures—in blood and FF were significantly (p < 0.001) modified in infertile patients compared to the control group. Moreover, a significant correlation between blood redox markers and FF redox markers was evident. An ORAC/TBARS ratio, defined as the redox index (RI), was obtained in the plasma and FF of the patients and controls. In the patients, the plasma RI was about 3.4-fold (p < 0.0001) lower than the control, and the FF RI was about six-fold (p < 0.0001) lower than the control. Interestingly, both the plasma RI and FF RI results were significantly correlated (p < 0.05) to the considered outcome parameters (metaphase II, fertilization rate, and ongoing pregnancies). Given the reported findings, a strict monitoring of redox parameters in assisted reproductive techniques and infertility management is recommended.

SIRT1 activity is decreased in lesional psoriatic skin.

Psoriasis is a chronic immune-mediated hyperproliferative inflammatory skin disease of unknown etiology characterized by the appearance of sore patches of thick, red skin with silvery scales. It affects about up to 3 % of the world population and it is equally prevalent in both genders, although results from a recent study have shown that on average, men have more severe forms of the disease. Psoriasis treatment often requires a varied team of clinicians with a range of expertise due to the fact that high prevalence, chronicity, disfiguration, disability, and associated comorbidity characterize the disease. The deep understanding of the role of immune function in psoriasis permits the management of this complex disease, which dramatically affects patients far beyond the skin. Hence, psoriasis is associated with impairment in health related quality of life, and, recently, it has emerged as a systemic disease, which affects not only cutaneous and articular sites, but it can also result in metabolic impairments and adverse cardiovascular outcomes [1]. Cytokines and growth factors released by activated T cells have been shown to display a prominent role in keratinocyte hyperproliferation; however, we focused our attention on dermal fibroblasts, which are also directly involved in the developing psoriatic lesions accelerating keratinocyte proliferation [1]. Emerging research suggests that reactive oxygen species (ROS) play a central role in the pathogenesis of psoriasis. Indeed, a number of studies reveal an altered blood redox status in psoriatic patients as shown by significantly increased levels of oxidative stress markers and decreased activity of the main antioxidant enzymes [2]. SIRT1, a mammalian ortholog of Silent information regulator 2 (Sir2) family, is significantly upregulated in response to oxidative stress [3]. Sirtuins are a family of NAD-dependent protein deacetylases that are evolutionally conserved from yeast to human. SIRT1, the most extensively studied member of the sirtuin family, targets a number of substrates—histones, transcription factors, DNA repair proteins, autophagy factors, and others—to modulate metabolism, stress responses, and many other cellular processes [3]. We have previously demonstrated that SIRT1 activation regulates mitogen-activated protein kinases (MAPK) pathway and down-regulates pro-apoptotic molecules, decreasing oxidative stress and reducing apoptotic cell death in keratinocytes from vitiligo skin. In the literature, there is neither evidence for SIRT1 expression and activity in lesional psoriatic fibroblasts, nor for its possible involvement in oxidative stress-related pathways. Here, for the first time, we investigated SIRT1 expression and activity in a fibroblast primary culture from lesional psoriatic skin, and we hypothesize its involvement in oxidative-mediated alterations. The study was approved by the Local Ethics Committee, and carried out according to the Helsinki Declaration. Analyses were carried out in eight patients (four females and four males) affected by moderate psoriasis (PASI = 12.5 ± 0.5) with a mean age of 40.1 ± 8.7 years and with a mean duration of disease of 15.8 years (from 8 M. Becatti and V. Barygina are equally contributed.

Use of Curcumin in Psoriasis

Curcumin is a polyphenol derived from the golden spice turmeric, which is widely used for different purposes, such as culinary spice and alimentary addictive, make - up and, finally, as a natural product for the treatment of different diseases, especially for the chronic inflammatory ones. Recently, curcumin has been proposed as a valid and safe therapeutic option for psoriasis.

Evaluation of the oxidative stress of psoriatic fibroblasts based on spectral two-photon fluorescence lifetime imaging

Psoriasis is an autoimmune disease of the skin characterized by hyperkeratosis, hyperproliferation of the epidermis, inflammatory cell accumulation and increased dilatation of dermal papillary blood vessels. Metabolic activity is increased in the epidermis and the dermis. Oxidative stress is high mainly due to reactive oxygen species (ROS) originating from the skin environment and cellular metabolism. We employed a custom multiphoton microscope coupled with a FLIM setup to image primary culture fibroblast cells from perilesional and lesional psoriatic skin in-vitro. Twophoton excited fluorescence images revealed the morphological differences between healthy and psoriatic fibroblasts. Based on the spectral analysis of the NADH and FAD components the oxidative stress was assessed and found to be higher in psoriatic cells. Furthermore the fluorescence lifetime properties were investigated with a TCSPC FLIM module. Mean fluorescence lifetime was found to be longer in psoriatic lesional cells. Analysis of the fast (τ1) and slow (τ2) decay lifetimes revealed a decrease of the ratio of the contribution of the fast (α1) parameter to the contribution of the slow (α2) parameter. The fluorescence in the examined part of the spectrum is attributed mainly to NADH. The decrease of the ratio (α1)/ (α2) is believed to correlate strongly with the anti-oxidant properties of NADH which can lead to the variation of its population in high ROS environment. This methodology could serve as an index of the oxidative status in cells and furthermore could be used to probe the oxidative stress of tissues in-vivo.

ROS-challenged keratinocytes as a new model for oxidative stress-mediated skin diseases: BARYGINA et al.

In the current study, the effects of the reactive oxygen species (ROS) generator 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH) on extracellular and intracellular ROS production in human keratinocytes (HACAT) were studied. AAPH is a water‐soluble compound able to generate ROS at known and constant rates at 37°C. The short treatment (2 h) with AAPH brought a significant dose‐dependent increase in NADPH oxidase activity in intact keratinocytes. The long‐term treatment (24 h) with AAPH led to a persistent increase in NADPH oxidase activity for up to 48 hour following the AAPH removal from cell incubation medium. ROS and nitric oxide levels, lipoperoxidation, intracellular calcium, mitochondrial superoxide production, and membrane potential were significantly modified in AAPH‐treated HACAT. Superoxide dismutase (SOD) and/or catalase addition to HACAT revealed that untreated keratinocytes produce mostly superoxide anion (O 2 −), while AAPH‐treated keratinocytes overproduce hydrogen peroxide (H 2O 2) in extracellular medium. H 2O 2 is particularly stable and plays important roles in several cell signaling pathways. Taken together, our findings suggest a cost‐effective and easily reproducible in vitro model of stressed human keratinocytes releasing significantly elevated ROS amounts in extracellular medium with respect to control keratinocytes. The possible application of the proposed model for keratinocytes‐melanocytes cross‐talk studies is also suggested. The model of AAPH‐stressed human keratinocytes described here can represent a useful tool for redox cross‐talk studies between keratinocytes and other skin cell types, and applied for researches regarding skin pathologies associated with oxidative stress.

Sirt1 Protects against Oxidative Stress-Induced Apoptosis in Fibroblasts from Psoriatic Patients: A New Insight into the Pathogenetic Mechanisms of Psoriasis

Psoriasis, a multisystem chronic disease characterized by abnormal keratinocyte proliferation, has an unclear pathogenesis where systemic inflammation and oxidative stress play mutual roles. Dermal fibroblasts, which are known to provide a crucial microenvironment for epidermal keratinocyte function, represented the selected experimental model in our study which aimed to clarify the potential role of SIRT1 in the pathogenetic mechanisms of the disease. We firstly detected the presence of oxidative stress (lipid peroxidation and total antioxidant capacity), significantly reduced SIRT1 expression level and activity, mitochondrial damage and apoptosis (caspase-3, -8 and -9 activities) in psoriatic fibroblasts. Upon SIRT1 activation, redox balance was re-established, mitochondrial function was restored and apoptosis was no longer evident. Furthermore, we examined p38, ERK and JNK activation, which was strongly altered in psoriatic fibroblasts, in response to SIRT1 activation and we measured caspase-3 activity in the presence of specific MAPK inhibitors demonstrating the key role of the SIRT1 pathway against apoptotic cell death via MAPK modulation. Our results clearly demonstrate the involvement of SIRT1 in the protective mechanisms related to fibroblast injury in psoriasis. SIRT1 activation exerts an active role in restoring both mitochondrial function and redox balance via modulation of MAPK signaling. Hence, SIRT1 can be proposed as a specific tool for the treatment of psoriasis.

Functional nutrition as integrated approach in vitiligo management

Vitiligo is a common disease of unknown cause that produces disfiguring white patches of depigmentation that can be treated using various new and experimental therapies, such as narrow‐band ultraviolet B (NB‐UVB) microphototherapy, NB‐UVB excimer laser, and monochromatic excimer light. Medical treatments include topical corticosteroids and other topical treatments, such as antioxidants, tacrolimus and pimecrolimus, prostaglandin E, and vitamin D derivatives (Lotti, Berti, & Moretti, 2009). The goal of treating vitiligo is to make it less noticeable either by restoring lost pigment or by eliminating remaining pigment. Functional foods and healthy diet, with nutrients, form a variety of sources, could be considered an integral part, as well as helpful, of vitiligos medical therapy.

Secukinumab reduces plasma oxidative stress in psoriasis: A case-based experience

Dear Editor, Psoriasis (Pso) is a chronic immune-mediated skin disease, with variable incidence and prevalence in the world population. It is usually considered a condition associated with increased metabolic and cardiovascular disorders (Danielsen et al., 2015). Reactive oxygen species (ROS) production has a prominent role in premature atherothrombosis not only in cardiovascular diseases (Becatti et al., 2013, 2014) but also in several immune-mediated disorders (Becatti et al., 2016; Emmi et al., 2015). ROS production is also involved in Pso pathogenesis, and we have previously demonstrated that biological agents, such as anti-TNFα, display a redox-balancing effect in psoriatic patients (Barygina et al., 2013). A new treatment targeting the IL-17 pathway (namely secukinumab; SEC) has been recently approved for the treatment of Pso (Langley et al., 2014); SEC has proved to be highly effective in Pso and in spondyloarthropathies (Smith, 2016), but at the present time, no data about its effect on redox status have been reported. In order to estimate a possible redox balancing effect of SEC, we evaluated, before and after treatment, plasma redox status (lipid peroxidation markers and total antioxidant capacity [TAC]) and blood cell ROS production in a psoriatic patient resistant to conventional treatments, successfully treated with SEC. This study was approved by our institutional review board. The reported patient was a 42-year-old Caucasian woman affected by severe plaque Pso of the trunk, lower and upper limbs since the age of 35 years. She was already treated with phototherapy (or Psoralen and UVA therapy), methotrexate, and cyclosporine, with unsatisfactory results. Due to the extent and severity of the cutaneous involvement, a deep impairment in physical and psychological quality of life was also present (DLQI 20). On June 2016, due to the presence of active severe Pso (PASI: 18.6) especially on the back of the hands and lower limb and the absence of contraindications, the treatment with SEC was started. After only 48 hr, there was a significant reduction in the cutaneous lesions and, 1 month after the beginning of the treatment, a complete resolution of skin manifestations was evident (Figure 1a) (PASI: 0.2). After 6 months of treatment, the patient maintained a good clinical response (PASI: 0.3, DLQI 3). Throughout treatment, SEC displayed a favorable safety profile and a good tolerability. Concomitantly, we evaluated the patient blood redox status before and after 1 month of SEC therapy. In particular, plasma lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS), was significantly reduced in SEC-treated patients with respect to pretreatment (0.11 0.07 vs. 0.43 0.09 nmol/ml) (Figure 1b), while TAC significantly increased (15.23 1.62 vs. 12.09 0.71 μmol Trolox equivalent/ml) (Figure 1b). Similarly, significantly lower levels of leukocyte-derived ROS were detected in SEC-treated patients with respect to pretreatment (Figure 1c). In the last years, several new anticytokine treatments have been developed and, recently, the anti-IL-17 drug SEC has been approved for Pso therapy (Burkett & Kuchroo, 2016; Langley et al., 2014). In psoriatic patients, IL-17 is a pivotal modulator of immune responses being produced by cells belonging to both the adaptive (T lymphocytes) and innate (neutrophils and mast cells) immunities (Keijsers, Joosten, van Erp, Koenen, & van de Kerkhof, 2014). Interestingly, IL-17, produced by innate immune response cells, is also considered to play a pivotal role in atherosclerosis and cardiovascular events (Keijsers et al., 2014). In conclusion, T lymphocyte/neutrophil-derived IL-17 seems to contribute not only to the inflammatory cutaneous alterations but also to the cardiovascular and metabolic complications in Pso patients. Here, for the first time, we report that SEC treatment is able to reduce overall blood oxidative stress in Pso patients. These data need to be confirmed in a wider population of patients with Pso; nevertheless, our case-based experience suggests that targeting the IL-17 pathway could be also effective in reducing Pso comorbidities, thus improving the overall outcomes of these patients via a blood redox balancing effect.