Skin morphological analysis upon treatment with anti-TNF-alpha agents in psoriatic patients

Psoriasis is the most common immune-mediated skin disease worldwide, with an estimated incidence of about 2%. It is characterized by erythematous scaly plaques and its pathogenesis is mostly due to an interplay among epidermal cells, immunocompetent cells, and pro-inflammatory cytokines. Previous studies on psoriatic skin demonstrated delayed terminal differentiation, keratinocyte hyperproliferation, and abnormal occludin expression in tight junctions. However, evidences about desmosomal cadherin distribution in psoriatic patients are not available so far, and only scattered studies have been carried out on adherens junctions. In psoriasis, Tumor Necrosis Factor- alpha (TNF-alpha) plays a central role, strongly supporting the treatment with anti-TNF-alpha agents, but the effects of these agents on epidermal intercellular adhesion, terminal differentiation, and proliferation have still to be elucidated. In this preliminary study, we investigated by immunofluorescence the expression of transmembrane proteins in tight junctions (occludin), adherens junctions (E-cadherin), and desmosomes (desmocollin-1 and desmoglein-1) in normal (N=5) and psoriatic skin before/after treatment with anti-TNF-alpha agents (N=5). Differentiation biomarkers (keratin-10, keratin-14, and involucrin) and epithelial proliferation were also evaluated. In psoriatic epidermis occludin, keratin-14, and involucrin were expressed also in the spinous layer, differently from controls. Desmoglein-1, desmocollin-1, E-cadherin, and keratin-10 localizations were comparable in psoriatic and healthy subjects. Moreover, in all considered patients the hyperproliferative condition was accompanied by an unusual suprabasal distribution of replicating keratinocytes. Interestingly, the distribution pattern of all considered biomarkers of intercellular adhesion and terminal differentiation was reverted to the physiological condition upon treatment with anti-TNF-alpha agents. What’s more, the proliferative rate registered in anti-TNF-alpha treated patients was reduced and similar to controls, even though scattered suprabasal dividing cells were still present. Our results highlight that anti-TNF-alpha biologicals, next to their proven inhibitory action on the cytokinic pathway, are effective in restoring an efficient junctional apparatus, a more differentiated phenotype, and the typical proliferation rate in the epidermis of psoriatic patients

Effect of a psoriatic microenvironment in a threedimensional model of normal human skin

Among the cytokines involved in the pathogenesis and in the progression of the disease, tumor necrosis factor (TNF)-alpha and interleukin (IL)-17 are the most relevant. A three dimensional model of organotypic human skin cultures is a valuable approach for exposing the whole skin to TNF-alpha and IL-17 as specific proinflammatory stimuli, thus mimicking a psoriatic microenvironment. Normal human skin explants were obtained from plastic surgery of healthy 20-40 year-old women (n = 7) after informed consent. Bioptic fragments were cultured overnight in a DMEM medium and further divided before adding either 100 ng/ml TNF-alpha or 50 ng/ml IL-17 or a combination of both cytokines. Samples were harvested 24 hours after cytokine incubation. Each patient was represented in all experimental groups. Epidermal proliferation together with the expression of terminal differentiation biomarkers (keratin 10, K10, and 14, K14) and of intercellular adhesion (occludin for tight junctions and E-cadherin for adherens junctions) were investigated by indirect immunofluorescence. Vibrational spectroscopy analysis by a confocal micro-Raman system (785nm laser) has been carried out in three skin samples to evaluate differences of the spectrum versus normal skin. Both cytokines induced a strong inhibition of keratinocyte proliferation (more than 80% compared with their respective controls). A non-continuous occludin expression in the granular layer was observed after the TNF-alpha and IL-17 exposure. Immunolabelings for E-cadherin in tight junctions, for K10 in the suprabasal layers, and for K14 in the basal layer were similar in all experimental groups. The preliminary Raman results highlighted some biomolecules modifications in TNF-alpha- and IL-17-treated skin samples related to ceramide and amide III (keratin proteins) peaks. These results suggest that in this experimental model we reproduced a psoriatic microenvironment in which TNF-alpha and IL-17 induce an early alteration of the homeostasis of the inner proliferative layer, the upper granular layer, and stratum corneum as shown by cell proliferation inhibition, occludin expression, and the biomolecules Raman bands

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

Efficacy and safety of adalimumab after failure of other anti-TNFα agents for plaque-type psoriasis: clinician behavior in real life clinical practice

Introduction: During treatment with biologic agents for psoriasis (Pso) in a number of patients a failure may occur and discontinuation with transitioning to another drug or an optimization strategy, consisting in a dose-adjustment or a co-medication with a traditional systemic agent, represent two possible alternatives. Objective: The SAFARI study objective was a retrospective observation of adalimumab efficacy and safety profile after switching from other anti-TNFα agents related to clinician behavior after the failure of the first-line agent. Results: The retrospective multicenter observation demonstrated that after a first-line anti-TNFα failure adalimumab efficacy was consistent at week-12 and 24 with a further significant improvement at week-48 with a proportion of patients achieving PASI75/PASI90/PASI100 of 83.3, 71.6, and 56.9.%, respectively. Clinician strategies to extend drug-survival after first-line anti-TNFα failure, such as co-medication or dose-adjustment, were irrelevant to future drug effectiveness. Conclusions: Adalimumab profile was excellent in this 5-year retrospective observation, showing the clinical validity of interclass transitioning among anti-TNFα options