EFFECTS OF A PSORIATIC INFLAMMATORY MICROENVIRONMENT ON KERATINOCYTE MORPHOLOGY IN 3D AND 2D BIOLOGICAL EXPERIMENTAL MODELS OF NORMAL HUMAN SKIN

The interplay between proinflammatory circulating cytokines and keratinocytes is a crucial event in the development and progression of psoriatic lesions. However, the early phases of the pathogenesis of psoriasis are still to be elucidated, in particular regarding the epidermal barrier. A pivotal role is played by tight junctions (TJs), i.e. claudin-mediated intercellular attachment structures, localized in the upper granular layer. A barrier impairment associated to an alteration in TJ proteins is described in psoriasis as a response to a proinflammatory microenvironment. We investigated by immunofluorescence analysis the modulation of the expression of claudin 1 (CLDN-1), a transmembrane integral TJ protein, and of Zonula Occludens 1 (ZO-1), a scaffold plaque protein, after the incubation with MIX, a combination of interleukin (IL)-17, IL-22, IL-23, tumor necrosis factor (TNF)-alpha, for 24 (T24) and 48 (T48) hours. We considered as experimental models the standardized 3D organotypic cultures of normal human skin (n=7) and in vitro cultures of primary normal human keratinocytes (n=3) in basal or differentiating cell growth conditions. On skin bioptic samples, ultrastructural analysis by transmission electron microscopy (TEM) was performed. In control skin samples, CLDN-1 immunopositivity increased from the basal layer upwards, but its expression was early reduced in the basal and suprabasal layers starting from T24 in MIX-incubated group. At this time point, ZO-1 expression in control samples increased gradually, starting from the basal layers towards the epidermal surface and the incubation with MIX induced its immunopositivity in the basal and suprabasal layers. At T24, CLDN-1 expression was unaffected by MIX in undifferentiated and also calcium- differentiated keratinocytes. Unexpectedly, undifferentiated cells relocated ZO-1 at cell-cell contact points after the incubation with MIX, and in calcium-differentiated keratinocytes, ZO-1 synthesis was stimulated, too. By TEM, after MIX incubation, the overall architecture of the epidermal compartment was maintained, but apoptosis and enlargement of intercellular spaces were evident. The present results strongly suggest that the i) broadening of ZO-1 expression and ii) the downregulation of CLDN-1, typical features of psoriasis, can be induced as early as 24 hours in both models, suggesting that they represent a valid experimental approach. To complete this study, the effect of this microenvironment on keratinocyte proliferation and differentiation will be investigated, obtaining further insights into the early processes leading to the formation/progression of psoriatic plaques. Donetti E, Cornaghi L, Gualerzi A, Baruffaldi Preis FW, Prignano F. An innovative three-dimensional model of normal human skin to study the proinflammatory psoriatic effects of tumor necrosis factor-alpha and interleukin-17. Cytokine; 2014; 68:1-8. Donetti E, Cornaghi L, Arnaboldi F, Ricceri F, Pescitelli L, Maiocchi M, Carriero F, Baruffaldi Preis FW, Prignano F. Epidermal barrier reaction to an in vitro psoriatic microenvironment. Exp Cell Res; 2017; 15:180-188. Donetti E, Lombardo G, Baruffaldi Preis FW, Cornaghi L, Pescitelli L, Prignano F. 3D skin model to investigate the early epidermal morphological psoriatic features. J Transl Sci; 2019; doi: 10.15761/JTS.1000361 Riva F, Casasco A, Nespoli E, Cornaglia AI, Casasco M, Faga A, Scevola S, Mazzini G, Calligaro A Generation of human epidermal constructs on a collagen layer alone. Tissue Eng; 2007; 13: 2769-79. Riva F, Casasco A, Casasco M, Calligaro A, Cornaglia AI. Growth and stratification of epithelial cells in minimal culture conditions. Methods Mol Biol; 2010; 585:25-43.