The identification of the specific cellular events underlying and/or triggering a diffuse chronic inflammatory skin disorder as atopic dermatitis (AD) is more and more challenging. In particular, the peculiar role played by interleukin (IL)-4 and IL-13 in promoting and supporting AD etiopathogenesis is still unclear. The aim of the present study is to dissect the early, specific, and direct effects of these pro-inflammatory cytokines in a 3D model of organotypic cultures of normal human skin standardized in our lab, strictly mimicking the physiological condition. Bioptic skin fragments obtained after aesthetic surgery of healthy women (n=7) were cultured in a Transwell system with either IL-4 (50 ng/ml) or IL-13 (50 ng/ml) and harvested after 24 and 48 hours, with parallel control groups. All samples were processed for light microscopy (LM), immunofluorescence (IF), and transmission electron microscopy (TEM) analysis. As biomarkers of terminal differentiation, keratin (K)14 and K10, respectively expressed in the basal and the suprabasal layers, and filaggrin, typically found in the granular layer, were analysed. K16 and K17, absent in normal skin, were assessed as skin alarmins. The molecular composition of tight junction (TJs) encompassed claudin-1 and zonula occludens (ZO)-1 expression. Quantitative analysis was performed for i) Langerhans cells (LCs) on blue toluidine-stained semithin sections, ii) keratin immunostaining after indirect IF, and iii) intercellular spaces by TEM observations. Both cytokines induced a time-dependent increase of LCs number, a K14 immunostaining weakening, and K17 expression in scattered suprabasal keratinocytes. At 24 hours, IL-13 elicited the dilation of intercellular spaces throughout the entire epidermal compartment and the restriction of K10 immunoreactivity from the medium spinous layer upwards. K16 induction and discontinuity of filaggrin immunostaining were always more evident in IL-13 than in IL-4 groups. Regarding TJs, the two cytokines inhibited similarly claudin-1 expression at both time points. Conversely, ZO-1 distribution was more affected by IL-13 than by IL-4. In our experimental conditions, IL-13 initially triggers the modulation of keratin expression and the molecular composition of TJs. Later, IL-4 sustains the existing pro-inflammatory milieu, with weaker effects than IL-13. Synergic or additional actions between IL-4 and IL-13 in AD progression will be evaluated.

Dissecting the role of IL-4 and IL-13 using a 3D model of normal human skin: an innovative experimental approach / S.L. Indino, G. Lombardo, E. Rosi, F. Baruffaldi Preis, E.B. Donetti, F. Prignano. ((Intervento presentato al 93. convegno Congresso della Società Italiana di Biologia Sperimentale-SIBS tenutosi a online-Palermo nel 2021.

Dissecting the role of IL-4 and IL-13 using a 3D model of normal human skin: an innovative experimental approach

S.L. Indino
Investigation
;
G. Lombardo
Investigation
;
E.B. Donetti
Project Administration
;
2021-04-24

Abstract

The identification of the specific cellular events underlying and/or triggering a diffuse chronic inflammatory skin disorder as atopic dermatitis (AD) is more and more challenging. In particular, the peculiar role played by interleukin (IL)-4 and IL-13 in promoting and supporting AD etiopathogenesis is still unclear. The aim of the present study is to dissect the early, specific, and direct effects of these pro-inflammatory cytokines in a 3D model of organotypic cultures of normal human skin standardized in our lab, strictly mimicking the physiological condition. Bioptic skin fragments obtained after aesthetic surgery of healthy women (n=7) were cultured in a Transwell system with either IL-4 (50 ng/ml) or IL-13 (50 ng/ml) and harvested after 24 and 48 hours, with parallel control groups. All samples were processed for light microscopy (LM), immunofluorescence (IF), and transmission electron microscopy (TEM) analysis. As biomarkers of terminal differentiation, keratin (K)14 and K10, respectively expressed in the basal and the suprabasal layers, and filaggrin, typically found in the granular layer, were analysed. K16 and K17, absent in normal skin, were assessed as skin alarmins. The molecular composition of tight junction (TJs) encompassed claudin-1 and zonula occludens (ZO)-1 expression. Quantitative analysis was performed for i) Langerhans cells (LCs) on blue toluidine-stained semithin sections, ii) keratin immunostaining after indirect IF, and iii) intercellular spaces by TEM observations. Both cytokines induced a time-dependent increase of LCs number, a K14 immunostaining weakening, and K17 expression in scattered suprabasal keratinocytes. At 24 hours, IL-13 elicited the dilation of intercellular spaces throughout the entire epidermal compartment and the restriction of K10 immunoreactivity from the medium spinous layer upwards. K16 induction and discontinuity of filaggrin immunostaining were always more evident in IL-13 than in IL-4 groups. Regarding TJs, the two cytokines inhibited similarly claudin-1 expression at both time points. Conversely, ZO-1 distribution was more affected by IL-13 than by IL-4. In our experimental conditions, IL-13 initially triggers the modulation of keratin expression and the molecular composition of TJs. Later, IL-4 sustains the existing pro-inflammatory milieu, with weaker effects than IL-13. Synergic or additional actions between IL-4 and IL-13 in AD progression will be evaluated.
Settore BIO/16 - Anatomia Umana
Società Italiana di Biologia Sperimentale -SIBS
Dissecting the role of IL-4 and IL-13 using a 3D model of normal human skin: an innovative experimental approach / S.L. Indino, G. Lombardo, E. Rosi, F. Baruffaldi Preis, E.B. Donetti, F. Prignano. ((Intervento presentato al 93. convegno Congresso della Società Italiana di Biologia Sperimentale-SIBS tenutosi a online-Palermo nel 2021.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/839817
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