Research Article
Interleukin 22 early affects keratinocyte differentiation, but not proliferation, in a three-dimensional model of normal human skin

https://doi.org/10.1016/j.yexcr.2016.05.004Get rights and content

Abstract

Interleukin (IL)-22 is a pro-inflammatory cytokine driving the progression of the psoriatic lesion with other cytokines, as Tumor Necrosis Factor (TNF)-alpha and IL-17. Our study was aimed at evaluating the early effect of IL-22 alone or in combination with TNF-alpha and IL-17 by immunofluorescence on i) keratinocyte (KC) proliferation, ii) terminal differentiation biomarkers as keratin (K) 10 and 17 expression, iii) intercellular junctions. Transmission electron microscopy (TEM) analysis was performed. A model of human skin culture reproducing a psoriatic microenvironment was used. Plastic surgery explants were obtained from healthy young women (n=7) after informed consent. Fragments were divided before adding IL-22 or a combination of the three cytokines, and harvested 24 (T24), 48 (T48), and 72 (T72) h later. From T24, in IL-22 samples we detected a progressive decrease in K10 immunostaining in the spinous layer paralleled by K17 induction. By TEM, after IL-22 incubation, keratin aggregates were evident in the perinuclear area. Occludin immunostaining was not homogeneously distributed. Conversely, KC proliferation was not inhibited by IL-22 alone, but only by the combination of cytokines. Our results suggest that IL-22 affects keratinocyte terminal differentiation, whereas, in order to induce a proliferation impairment, a more complex psoriatic-like microenvironment is needed.

Introduction

The interplay among pro-inflammatory cytokines such as Tumor Necrosis Factor (TNF)-alpha, interleukin (IL)-17 and IL-22 and the two main epidermal cytotypes, i.e. keratinocytes (KCs) and Langerhans cells (LCs), is a central issue in the pathogenesis of psoriasis. Many different stimuli can trigger the onset of psoriasis in genetically predisposed individuals [1]. Psoriasis has been recently defined as “a T-cell mediated inflammatory skin disease with T helper cell type 1 (Th1), type 17 (Th17) and IL-22-producing CD4+ T cells as principal mediators” [2]. On the other hand, cytokine-stimulated KCs may secrete a great variety of pro-inflammatory factors [3]. As a result, this microenvironment is able to promote KC activation and maintain a high rate of KC proliferation. In this milieu, epidermal terminal differentiation is profoundly affected as demonstrated by the expression switch from the typical keratin (K) isoforms K1/K10 towards K17 in the suprabasal layers. K17 is not expressed in normal skin [4], but it is detected in the basal cells of finger nails, hair follicles, sebaceous glands and nail bed epithelium [5]. K17 expression levels are positively associated with psoriasis severity [6] and a K17/T-cell/cytokine autoimmune loop was found, which strengthens the hypothesis of a correlation between K17 and the pathogenesis of psoriasis [7]. Pro-inflammatory cytokines IL-17 and IL-22, both produced by Th17 cells [8], are known to upregulate K17 expression in vitro [9], [10] and in psoriatic lesions [11]. Recent in vitro [12] and in vivo [13], [14] studies demonstrated the role of IL-22 in the process of KC terminal differentiation, but its effect on proliferation is still debated. IL-22 promoted proliferation and injury repair of hyperglycemic KCs in diabetic mice wounds [14], but it did non induce a proliferative increase in HaCat cells [12]. Considering that much of the data regarding IL-22 has been obtained from animal models and are thus difficult to extrapolate to the human pathology, human systems can greatly help in further exploring the role of IL-22 in psoriasis.

Up to now a specific activity of IL-17 and TNF-alpha was demonstrated on KC proliferation [15] and LC immunophenotype [16] in a model of human skin organotypic culture standardized in our laboratory.

As IL-17 and TNF-alpha enhance by powerful synergies the skin effects exerted by IL-22 [17], the present study was aimed at evaluating the epidermal response to IL-22 alone or in combination with IL-17 and TNF-alpha up to 72 h of stimulation in this experimental setting. Immunofluorescence was used to address i) KC proliferation, ii) terminal differentiation biomarkers as K10 and 17 expression and iii) the expression and localization of molecules of intercellular junctions (desmocollin 1 (Dsc 1), E-cadherin, and occludin). Transmission electron microscopy (TEM) was used to analyze ultrastructure.

Section snippets

Materials and methods

Normal human skin explants were obtained from abdomen or mammary gland after plastic surgery of healthy 20–40 year-old women (n=7) after informed consent. Procedures were in accordance with the ethical standards of the Institutional committee on human experimentation and with the Helsinki Declaration. The fragments (1 cm2) were placed in a Transwell system epidermis upwards at air-liquid interface and the dermis immersed in the culture medium (Costar, Corning, NY, USA) thus reproducing as close

Immunofluorescence of epidermal markers of terminal differentiation and intercellular adhesion

In control group the immunoreactivity for the terminal differentiation biomarker of suprabasal layers, K10, was homogeneously distributed throughout all the epidermis at all time points (Fig. 1A-C). Upon IL-22 incubation, clusters of K10-negative KCs were clearly detectable after 24 h and became progressively more evident in the lower stratum spinosum (Fig. 1D-F, asterisks). In samples exposed to the three cytokines, only scattered K10-negative KCs were present in the suprabasal layers (Fig. 1

Discussion

Although the extensive implication of pro-inflammatory cytokines in psoriasis pathogenesis has been widely ascertained, the intrinsic and synergistic effects of these cytokines in the early phases of the plaque formation still have not been fully investigated. Psoriasis used to be regarded as a Th1-driven disease with a focus on TNF-alpha [18], but, in recent years, other cytokines have emerged as major players in the pathogenesis of psoriasis [19], [20]. In particular, IL-17 and IL-22 are both

Acknowledgments

We would thank Mrs. Maria Gaman and Mr. Paolo Monti for their technical help in transmission electron microscopy.

References (29)

  • C. Bonifati et al.

    Cytokines in psoriasis

    Int. J. Dermatol.

    (1999)
  • Y.J. Lan et al.

    Immunolocalization of vimentin, keratin 17, Ki-67, involucrin, β-Catenin and E-Cadherin in cutaneous squamous cell carcinoma

    Pathol. Oncol. Res.

    (2014)
  • S.M. Troyanovsky et al.

    Patterns of expression of keratin 17 in human epithelia: dependency on cell position

    J. Cell Sci.

    (1989)
  • E.M. de Jong et al.

    Keratin 17: a useful marker in anti-psoriatic therapies

    Arch. Dermatol. Res.

    (1991)
  • Cited by (18)

    • Inside-out and outside-in organotypic normal human skin culture: JAK-STAT pathway is activated after pro-inflammatory psoriatic cytokine exposure

      2022, Tissue and Cell
      Citation Excerpt :

      In samples incubated with the Mix, K17 expression was evident starting from 24 h of culture and K17-positive cells were detectable also in the basal layer (Fig. 3g–i). Densitometric analysis of immunoreactive bands after Western blot confirmed the pattern expression previously investigated by immunofluorescence after IL-17, IL-22 and TNF-alpha administration (data not shown) (Donetti et al., 2016) and revealed a tendency to increased K17 induced by IL-23 at all considered time points, with a strong up-regulation induced by Mix after 72 h (Fig. 3j). Possible disturbances in JAK/STAT pathway were described in various inflammatory and autoimmune skin diseases, such as atopic dermatitis, alopecia areata as well as psoriasis (Gündüz, 2019).

    • Dupilumab progressively improves systemic and cutaneous abnormalities in patients with atopic dermatitis

      2019, Journal of Allergy and Clinical Immunology
      Citation Excerpt :

      In addition, our results suggest that IL-4/IL-13 signaling contributes to regulation of the IL-17 and IL-22 cytokine networks in patients with AD, given the significant modulation of these pathways that results from IL-4Rα inhibition. These data demonstrate that inhibition of IL-4Rα, which results in dual inhibition of IL-4/IL-13 signaling, can effectively suppress key pathogenic processes in patients with AD, thus supporting the notion that cytokines induce and perpetuate the epidermal alterations in patients with AD.2,3,68-70,73,120,129,132,133,136 In parallel with increased cytokine activation in skin lesions before treatment,8-11,49 patients with severe AD have robust systemic cytokine induction, as reflected by the broad abnormalities already observed at the nonlesional skin level.11,54

    • Epidermal barrier reaction to an in vitro psoriatic microenvironment

      2017, Experimental Cell Research
      Citation Excerpt :

      At present, no experimental evidences exist regarding the specific and early effect of psoriatic pro-inflammatory cytokines on this complex epidermal tableau. In the present study, we sought to dissect the effects of TNF-alpha and IL-17 on i) phenotype of LCs and ii) TLR and filaggrin expressions, i.e. three primary components of the healthy epidermal barrier, in the three-dimensional model of organotypic cultures standardized in our laboratory in the last decade [13–17]. By immunofluorescence we investigated, after cytokine exposure at different time points, the expression pattern of filaggrin, TLR2, 7, and 9, and the intracellular localization of NF-kB.

    • Regulation of Gastric Carcinogenesis by Inflammatory Cytokines

      2017, Cellular and Molecular Gastroenterology and Hepatology
      Citation Excerpt :

      Not all cytokines activate JAK-STAT signaling, and different signal transduction pathways can be used that ultimately activate transcription factors such as activator protein 1, mitogen-activated protein kinases (MAPKs), and nuclear factor kappa B (NF-κB).12–15 In addition to their ability to act on immune cells and regulate the type and degree of inflammation, cytokines also act on epithelial cells and other cell types to regulate secretion,9,16 proliferation,16,17 and differentiation.18–20 Because of their broad and pleiotropic effects on immune and epithelial cells, cytokines are an obvious candidate for analysis as gastric cancer risk factors.

    View all citing articles on Scopus
    View full text