Development and validation of human psoriatic skin equivalents

Abstract

Psoriasis is an inflammatory skin disease driven by aberrant interactions between the epithelium and the immune system. Anti-psoriatic drugs can therefore target either the keratinocytes or the immunocytes. Here we sought to develop an in vitro reconstructed skin model that would display the molecular characteristics of psoriatic epidermis in a controlled manner, allowing the screening of anti-psoriatic drugs and providing a model in which to study the biology of this disease. Human skin equivalents generated from normal human adult keratinocytes after air exposure and stimulation by keratinocyte growth factor and epidermal growth factor displayed the correct morphological and molecular characteristics of normal human epidermis whereas the psoriasis-associated proteins, hBD-2, SKALP/elafin, and CK16, were absent. Skin equivalents generated from foreskin keratinocytes were clearly abnormal both morphologically and with respect to gene expression. When normal skin equivalents derived from adult keratinocytes were stimulated with psoriasis-associated cytokines [tumor necrosis factor-alpha, interleukin (IL)-1alpha, IL-6, and IL-22] or combinations thereof, strong expression of hBD-2, SKALP/elafin, CK16, IL-8, and tumor necrosis factor-alpha was induced as shown by quantitative polymerase chain reaction and immunohistochemistry. Retinoic acid but not cyclosporin A was found to inhibit cytokine-induced gene expression at both the mRNA and protein levels. These results illustrate the potential of this disease model to study the molecular pathology and pharmacological intervention in vitro.

Figure 1

Development of normal human skin equivalents. Adult human keratinocytes were seeded on DED, and cultured for 4 days submerged, followed by 0 (A), 3 (B), 7 (C), or 10 (D) days of culturing at the air-liquid interface. Skin equivalents were stained for H&E.

Figure 2

Comparison of adult and neonatal keratinocytes in the development of normal human skin equivalents. Abdominal (A--E) or foreskin (F--J) human keratinocytes were seeded on DED, and cultured for 4 days submerged, followed by 10 days of culture at the air-liquid interface. Morphology of the skin constructs was studied by H&E staining (A and F), whereas protein expression of CK10 (B and G), CK16 (C and H), hBD-2 (D and I), and SKALP/elafin (E and J) was determined by immunohistochemistry. Note the expression of SKALP/elafin and the presence of parakeratosis in the constructs derived from neonatal foreskin keratinocytes.

Figure 3

Effect of cytokine stimulation on SKALP/elafin and hBD-2 gene expression in human skin equivalents. Skin equivalents were stimulated the last 4 days of the air-liquid interface culture with 10 ng/ml of IL-1α, 10 ng/ml of IL-1α and 5 ng/ml of TNF–α, or 10 ng/ml of IL-1α, 5 ng/ml of TNF–α, and 5 ng/ml of IL-6. SKALP/elafin (A) and hBD-2 (B) gene expressions were determined by qPCR. All stimuli induced a significant induction of SKALP/elafin (P < 0.02) and hBD-2 (P < 0.001) gene expression (analysis of variance, repeated design). The cytokine mix of IL-1α, TNF-α, and IL-6 induced a significantly higher expression of hBD-2 than the other stimuli (P < 0.05, Duncan’s multiple range test).

Figure 4

Effect of cytokine stimulation on SKALP/elafin and hBD-2 protein expression in skin equivalents. Human skin equivalents were stimulated (B–D and F–H) or not (A and E) for 4 days with 10 ng/ml of IL-1α (B and F); 10 ng/ml of IL-1α and 5 ng/ml of TNF-α (C and G); or 10 ng/ml of IL-1α, 5 ng/ml of TNF-α, and 5 ng/ml of IL-6 (D and H). SKALP/elafin (A–D) and hBD-2 (E–H) protein expression was determined by immunohistochemistry.

Figure 5

Effect of cytokine stimulation on CK10 and CK16 protein expression in skin equivalents. Human skin equivalents were stimulated (B–D and F–H) or not (A and E) for 4 days with 10 ng/ml of IL-1α (B and F); 10 ng/ml of IL-1α and 5 ng/ml of TNF-α (C and G); or 10 ng/ml of IL-1α, 5 ng/ml of TNF-α, and 5 ng/ml of IL6 (D and H). CK10 (A–D) and CK16 (E–H) protein expression was determined by immunohistochemistry.

Figure 6

Effect of pro-inflammatory cytokines on gene expression of inflammatory mediators by keratinocytes in skin equivalents. Skin equivalents were stimulated the last 4 days of the culture with the combination of 10 ng/ml of IL-1α, 5 ng/ml of TNF-α, and 5 ng/ml of IL-6. RNA was isolated and gene expression of TNF-α (A) and IL-8 (B) was determined by qPCR. Stimulation with the cytokine mix significantly induced TNF-α (P < 0.002) and IL-8 (P < 0.02, paired t-test).

Figure 7

Effect of IL-22 on hBD-2 protein expression in skin equivalents. Human skin equivalents were stimulated the last 4 days of the culture with 0 (A), 30 (B), 100 (C), or 300 (D) ng/ml of IL-22. hBD-2 protein expression was determined by immunohistochemistry.

Figure 8

Effect of all-trans retinoic acid (ATRA) on cytokine-induced SKALP/elafin and hBD-2 gene expression in skin equivalents. Human skin equivalents were stimulated the last 4 days of culture with a mixture of 10 ng/ml of IL-1α, 5 ng/ml of TNF-α, and 5 ng/ml of IL-6 (CM) in the presence or absence of ATRA (10⁻⁶ or 10⁻⁷ mol/L). RNA was isolated, and gene expression of SKALP/elafin (A) and hBD-2 (B) was determined by qPCR. Treatment with ATRA caused a significant decrease of cytokine-induced expression of SKALP/elafin (P < 0.05) and hBD-2 (P < 0.01, analysis of variance, repeated design). hBD-2 expression was significantly affected by both ATRA concentrations (P < 0.02) whereas SKALP/elafin expression was only affected by the highest ATRA concentration (P < 0.05, Duncan’s multiple range test).

Figure 9

Effect of all-trans retinoic acid (ATRA) on cytokine-induced SKALP/elafin and hBD-2 protein expression in skin equivalents. Human skin equivalents were stimulated for 4 days of culture with a mixture of 10 ng/ml of IL-1α, 5 ng/ml of TNF-α, and 5 ng/ml of IL-6 in the presence or absence of ATRA (10⁻⁶ or 10⁻⁷ mol/L). Protein expression of SKALP/elafin (A: no cytokines; B: cytokines; C: cytokines and ATRA) and hBD-2 (D: no cytokines; E: cytokines; F: cytokines and ATRA) was determined by immunohistochemistry. Note the reduction of SKALP/elafin and hBD-2-positive cell layers in the ATRA-treated constructs (C and F) compared to the untreated cytokine-stimulated constructs (B and E).