**2. Results**

#### *2.1. Differential Impact of CT and ISO on the Psoriatic Skin Substitute Phenotype: Epidermal Hyperproliferation*

According to the macroscopic aspect of the skin substitutes, the epidermis of psoriatic substitutes reconstructed using culture media supplemented with CT (PS+CT) and the epidermis of psoriatic substitutes reconstructed using ISO (PS+ISO) were more disorganized since they displayed a less opaque and uniform surface than those of healthy substitutes (HS+CT) and (HS+ISO) (Figure 2c,d vs. Figure 2a,b). Moreover, both PS+CT and PS+ISO had a significantly thicker epidermis than their respective counterparts, showing higher proliferation of the keratinocytes (Figure 2g,h vs. Figure 2e,f). Hematoxylin and eosin staining of the skin substitutes are presented in Figure S1. This enhanced proliferation was confirmed with Ki67 immunofluorescence showing more basal keratinocytes in cellular division in PS than in HS (Figure 2k,l vs. Figure 2i,j). However, keratinocytes from PS+ISO

were not as hyperproliferative as those in PS+CT. In fact, the epidermis of PS+ISO was not as thick as that of PS+CT (Figure 2m). The Ki67 staining confirmed these results (Figure 2n). Taken together, these results showed that both PS+CT and PS+ISO displayed higher levels of epidermal proliferation than their healthy counterparts HS+CT and HS+ISO, respectively. Moreover, the hyperproliferation was greater in the epidermis of PS+CT than PS+ISO.

**Figure 2.** Morphology and epidermal proliferation of healthy substitutes (HS) and psoriatic substitutes (PS) produced with cholera toxin (+CT) or isoproterenol (+ISO). (**<sup>a</sup>**–**d**) Skin substitute macroscopic aspects; (**<sup>e</sup>**–**h**) Masson's trichrome staining of skin substitute histological sections (D: Dermis, E: Epidermis living layers, SC: *Stratum corneum*); (**i**–**l**) Ki67 immunofluorescence (green) detecting keratinocytes in proliferation. White arrows indicate Ki67-positive cells. Nuclei were stained with Hoechst (blue); (**m**) quantification of the thickness of the epidermal living layer (designated as E in panels (**<sup>e</sup>**–**h**) measured from Masson's trichrome staining using the ImageJ software; (**n**) ratio of Ki67 positive cells to the number of total keratinocytes in the basal layer. Scale bars: (**<sup>a</sup>**–**d**) 5 mm; (**<sup>e</sup>**–**h**) 100 μm. Data presented are the means +SD (*N* = 2 donors per condition, *n* = 3 skin substitutes per donor). The statistical significance was determined using one-way ANOVA followed by a Tukey's post-hoc test. (\* *p*-value < 0.05; \*\* *p*-value < 0.01; \*\*\* *p*-value < 0.001; \*\*\*\* *p*-value < 0.0001).

#### *2.2. Di*ff*erential Impact of CT and ISO on the Psoriatic Skin Substitute Phenotype: Disturbed Epidermal Di*ff*erentiation*

The expression of differentiation markers was strongly altered in PS+CT compared with HS+CT, thus showing a psoriatic phenotype such as expected. Indeed, the late differentiation markers filaggrin and keratin 10 were both down-regulated while the expression of the early differentiation marker involucrin was up-regulated (Figure 3). On the other hand, the effects of ISO on the expression of the differentiation markers were not as conclusive as for CT. In fact, the expression of filaggrin and keratin 10 for PS+ISO was down-regulated but not as much as for PS+CT compared with their respective counterparts. The involucrin staining was even less conclusive, since its expression in PS+ISO appeared less intense than the other conditions. According to these results, PS produced with ISO displayed a complete epidermal differentiation similar to what was observed for HS, since late differentiation markers are expressed. Therefore, epidermal differentiation in PS+ISO did not properly mimic the disturbed epidermal differentiation characteristics of psoriasis, which further entail that ISO is not a suitable cAMP stimulator to produce psoriatic skin substitutes.

**Figure 3.** Expression of epidermal differentiation markers (green) in healthy substitutes (HS) and psoriatic substitutes (PS) produced with cholera toxin (+CT) or isoproterenol (+ISO) (*N* = 2 donors per condition, *n* = 3 skin substitutes per donor). The nuclei were stained with Hoechst (blue). The dotted line indicates the dermo-epidermal junction. Scale bar: 100 μm.

#### *2.3. Levels of cAMP in the Epidermis of Psoriatic Substitutes Produced with either CT or ISO*

The levels of cAMP in the epidermis of HS+CT and HS+ISO were approximately the same (Figure 4.) The two cAMP enhancers, therefore, have similar effects on the AC activity of HS. The level of cAMP for PS+CT was significantly higher than for the other three conditions, revealing a greater capacity of CT to stimulate the production of cAMP.

**Figure 4.** cAMP levels in the epidermis of healthy substitutes (HS) and psoriatic substitutes (PS) produced with either cholera toxin (+CT) or isoproterenol (+ISO). Data presented are the means +/− SD (*N* = 2 donors per condition, *n* = 2 skin substitutes per donor). The statistical significance was determined using one-way ANOVA followed by a Tukey's post-hoc test. (\* *p*-value < 0.05).

#### *2.4. Identification of Isoforms of cAMP-Related Protein Found in the Skin*

Gene profiling on microarray was exploited to examine which AC were expressed in the skin substitutes (Table 1). Among the 10 AC isoform genes (*ADCY1-10*), only *ADCY3*, *ADCY7*, and *ADCY9* displayed a linear signal over 100 and were therefore identified as expressed genes in the HS+CT and PS+CT. Interestingly, the levels of expression of the three *ADCY* genes were higher (2-fold) in PS+CT than in HS+CT. Furthermore, expression of the *ADRB2* gene encoding for the beta-2 adrenergic

receptor was also detected in both HS+CT and PS+CT. The expression of *ADRB2* was decreased (0.5-fold) in PS+CT.


**Table 1.** Linear signals and fold change for *ADCY1-10* and *ADRB2* genes between healthy and psoriatic substitutes produced with CT.

*2.5. Levels of cAMP-Related Proteins in the Epidermis of Psoriatic Substitutes Produced with Either CT or ISO*

Immunofluorescence staining was performed to validate the epidermal levels of AC9 and beta-2 adrenergic receptor, which are encoded by *ADCY3, ADCY7*, *ADCY9*, and *ADRB2* genes, respectively, as well as to compare the impact of CT and ISO on those protein levels (Figure 5a). Based on the immunofluorescence staining, the AC9 was found in the cells of both the dermis and the epidermis of the skin substitutes. Moreover, AC9 levels seemed up-regulated in psoriatic substitutes (PS+CT and PS+ISO) as compared with healthy ones. On the other hand, the β2-adrenergic receptor was found predominantly in the epidermis. Interestingly, high levels of the β2-adrenergic receptor were detected in HS+CT, while low levels were found in HS+ISO and it was not detected in PS+ISO. These results therefore support the conclusion that the levels of β2-adrenergic receptors are decreased in psoriatic skin.

**Figure 5.** (**a**) Expression of adenylate cyclase 9 (AC9) (green) and β2-adrenergic receptor (red) in healthy substitutes (HS) and psoriatic substitutes (PS) produced with cholera toxin (+CT) or isoproterenol (+ISO). The nuclei were stained with Hoechst (blue). The dotted line indicates the dermo-epidermal junction. Scale bar: 100 μm. (**b**) Ten micrograms of total protein from skin substitute epidermis were analyzed by immunoblot for the presence of adenylate cyclase 9 (AC9). β-actin was used to control equal loading. ( *N* = 2 donors per condition; *n* = 2 skin substitutes per donor). One representative immunoblot is shown. (**c**) Densitometric analyses of the immunoblot from panel (**b**) ( *N* = 2 donors per condition; *n* = 2 skin substitutes per donor). Data from healthy or psoriatic substitutes were combined irrespective of treatment regime. The statistical significance was determined using an unpaired *t*-test (\* *p*-value < 0.05).

Western blot analyses were next conducted to validate the levels of AC9 in the epidermis of the skin substitutes, since immunofluorescence staining of these proteins was used for qualitative purposes. AC9 was easily detected in the epidermis of all conditions, with a higher prevalence in PS+CT and PS+ISO (Figure 5b,c). AC3 and AC7 were not detected in the epidermis of either healthy nor psoriatic skin substitutes under our experimental conditions.
