**1. Introduction**

Psoriasis is an autoimmune skin disease affecting 3% of the population worldwide and for which no cure currently exists [1]. Clinical manifestation of psoriasis is defined by the apparition of red plaques with white scales, which have detrimental consequences on patient's quality of life [2,3]. Psoriasis patches can range from a few spots of dandruff-like scaling to major eruptions that cover large areas of the body. The histological hallmarks of psoriasis are a marked thickening of the epidermis, due to keratinocyte hyperproliferation, abnormal epidermal differentiation, and immune keratinocytes activation accompanied with immune cell infiltrate [4]. Consequently, the granular layer of the epidermis is reduced in thickness and the horny layer contains some undifferentiated keratinocytes, which still contain cell nuclei [5]. Furthermore, altered keratinocyte differentiation in psoriasis results at a molecular level in the deregulation of the epidermal differentiation marker proteins, such as

involucrin (up-regulated), filaggrin (down-regulated), and keratin 10 (down-regulated) [6–8]. It is now well documented that the complex etiology of psoriasis involves interactions between environmental factors and complex genetic background [9]. However, the exact cause of psoriasis is still unknown, making it di fficult to develop an e ffective treatment for the pathology [1].

Since the development of the first tissue-engineered skin model in 1978, by Green H. and colleagues, grea<sup>t</sup> deal of research has been conducted in this field, leading to the generation of highly reproducible and sophisticated skin models. In the last decade, the limits of tissue engineering have been constantly pushed back by the development of new emerging techniques, such as bio-printing [10,11]. However, the reconstruction of pathological skin models, such as psoriatic skin substitutes, remains challenging, especially since the exact causes of the pathology are still unknown. However, many psoriatic skin models were reconstructed in vitro using di fferent tissue-engineered techniques [12]. According to Niehues et al., the self-assembly method is the one which leads to the reconstruction of substitutes faithfully mimicking psoriatic characteristics and which, consequently, o ffers an e ffective tool for the screening of new molecules [12–14]. Indeed, reconstructed psoriatic skin substitutes produced using cells from patients with psoriasis were shown to closely mimic the pathology, as they displayed enhanced epidermal thickness, hyperproliferative keratinocytes and disturbed epidermal di fferentiation. The morphology of reconstructed skin models is directly a ffected by culture conditions. While developing the first healthy tissue engineered model of skin in 1978, Green H. reported the importance of adding cAMP stimulator, such as cholera toxin (CT) or isoproterenol (ISO), to induce the proliferation of cells in culture [15]. Therefore, skin models are produced using culture media supplemented with a stimulator of the AC system, which is crucial for stimulating the colony growth of human keratinocytes and thus essential to obtain a fully di fferentiated epidermis [15–20].

The cAMP signaling pathway is composed of the first messenger, the G protein-coupled receptor (GPCR), the adenylate cyclase enzyme (AC) and the cAMP-degrading enzyme (Figure 1a). The epidermis contains four independent receptor AC systems: the β-adrenergic, prostaglandin E, adenosine, and histamine receptors [21–23]. The cAMP mainly acts as a second messenger, by stimulating di fferent proteins, such as protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC). PKA and EPAC are then able to modify cell activity by phosphorylating diverse proteins [18]. The action of cAMP ends upon its hydrolysis by phosphodiesterases [24–26]. CT and ISO induce the production of cAMP following a di fferent pathway. ISO is an analogue to epinephrine and binds exclusively to β-adrenergic receptors, which then stimulates cAMP production following the classical pathway (Figure 1a) [27,28]. CT binds to ganglioside GM1 receptors on epithelial cells, which triggers endocytosis, thus transporting the receptors to the endoplasmic reticulum (ER). From the lumen of the ER, the A1 peptide of the CT is transported into the cytoplasm, where it prevents the G protein from cleaving GTP to GDP, leading to a tremendous increase in cAMP levels (Figure 1b) [16,29,30].

**Figure 1.** The cAMP signaling pathways. (**a**) 1: The first messenger (red) binds to the G protein-coupled receptor (GPCR). The receptor then changes conformation, leading to the replacement of GDP by GTP on the α subunit of the G protein and inducing the subsequent release of the α subunit from the β and γ subunits of the G protein [21,31]. 2: The α subunit binds to the catalytic domain of adenylate cyclase (AC). The α subunit can have inhibitory (αi) or stimulating (αs) properties, which will lead to the inhibition or stimulation of AC [16,32]. The activated AC will convert ATP into cAMP. Isoproterenol induces the conversion of ATP to cAMP following the classical pathway (**b**) The A1 peptide of cholera toxin (CT) (purple) binds to the complex composed of the GTP-α subunit of the G protein and AC and prevents the G protein from cleaving GTP to GDP, leading to a tremendous increase in cAMP levels [30].

The impact of different cAMP inducers (CT and ISO) on the morphology of psoriatic skin models has not ye<sup>t</sup> been investigated, whereas it should be chosen with much consideration, especially since controversial results have been found regarding the involvement of cAMP in psoriasis. Indeed, Voorhes et al. suggested in the seventies that an alteration in cAMP levels could be involved in psoriasis [33]. Moreover, a few studies have reported lower levels of cAMP in psoriatic skin, suggesting that low levels of cAMP were linked with enhanced cell proliferation and thus contrasting with finding of Green H [33–37]. The aim of the present study was to therefore compare the use of both cAMP enhancers (CT or ISO) in the reconstruction of psoriatic skin substitutes to establish which one would lead to the better psoriatic phenotype, with traits, such as cell hyperproliferation and disturbed cell differentiation. The cAMP signaling pathway was then investigated in detail to demystify cAMP levels, as well as the activity of various agents of the AC system, in the pathology of psoriasis. Therefore, the current study brings new insights to the long-standing debate as to whether cAMP is increased or decreased in psoriasis.
