N-eicosapentaenoyl-ethanolamine decreases the proliferation of psoriatic keratinocytes in a reconstructed psoriatic skin model.

Psoriasis is an inflammatory skin disease that is characterized by keratinocyte hyperproliferation, abnormal epidermal differentiation and dysregulated lipid metabolism. Some lipid mediators of the N-acylethanolamines (NAEs) and monoacylglycerols (MAGs) can bind to cannabinoid (CB) receptors and are referred to as part of the endocannabinoidome. Their implication in psoriasis remains unknown. The aim of the present study was to characterize the endocannabinoid system and evaluate the effects of n-3-derived NAEs, namely N-eicosapentaenoyl-ethanolamine (EPEA), in psoriatic keratinocytes using a psoriatic skin model produced by tissue engineering, following the self-assembly method. Psoriatic skin substitutes had lower FAAH2 expression and higher MAGL, ABHD6 and ABHD12 expression compared with healthy skin substitutes. Treatments with alpha-linolenic acid (ALA) increased the levels of EPEA and 1/2-docosapentaenoyl-glycerol, showing that levels of n-3 polyunsaturated fatty acids modulate related NAE and MAG levels. Treatments of the psoriatic substitutes with 10 µM of EPEA for 7 days resulted in decreased epidermal thickness and number of Ki67 positive keratinocytes, both indicating decreased proliferation of psoriatic keratinocytes. EPEA effects on keratinocyte proliferation were inhibited by the CB1 receptor antagonist rimonabant. Exogenous EPEA also diminished some inflammatory features of psoriasis. In summary, n-3-derived NAEs can reduce the psoriatic phenotype of a reconstructed psoriatic skin model.

Gene Profiling of a 3D Psoriatic Skin Model Enriched in T Cells: Downregulation of PTPRM Promotes Keratinocyte Proliferation through Excessive ERK1/2 Signaling.

Psoriasis is a complex, immune-mediated skin disease involving a wide range of epithelial and immune cells. The underlying mechanisms that govern the epidermal defects and immunological dysfunction observed in this condition remain largely unknown. In recent years, the emergence of new, more sophisticated models has allowed the evolution of our knowledge of the pathogenesis of psoriasis. The development of psoriatic skin biomaterials that more closely mimic native psoriatic skin provides advanced preclinical models that will prove relevant in predicting clinical outcomes. In this study, we used a tissue-engineered, two-layered (dermis and epidermis) human skin substitute enriched in T cells as a biomaterial to study both the cellular and molecular mechanisms involved in psoriasis' pathogenesis. Gene profiling on microarrays revealed significant changes in the profile of genes expressed by the psoriatic skin substitutes compared with the healthy ones. Two genes, namely, PTPRM and NELL2, whose products influence the ERK1/2 signaling pathway have been identified as being deregulated in psoriatic substitutes. Deregulation of these genes supports excessive activation of the ERK1/2 pathway in psoriatic skin substitutes. Most importantly, electrophoresis mobility shift assays provided evidence that the DNA-binding properties of two downstream nuclear targets of ERK1/2, both the NF-κB and Sp1 transcription factors, are increased under psoriatic conditions. Moreover, the results obtained with the inhibition of RSK, a downstream effector of ERK1/2, supported the therapeutic potential of inhibiting this signaling pathway for psoriasis treatment. In conclusion, this two-layered human psoriatic skin substitute enriched in T cells may prove particularly useful in deciphering the mechanistic details of psoriatic pathogenesis and provide a relevant biomaterial for the study of potential therapeutic targets.

Remodeling of the Dermal Extracellular Matrix in a Tissue-Engineered Psoriatic Skin Model by n-3 Polyunsaturated Fatty Acids.

Psoriasis is an inflammatory skin disease mainly associated with an epidermal disorder. However, the involvement of the dermal extracellular matrix (ECM) composition in psoriasis is still poorly understood. This study aimed to investigate the expression of ECM components in psoriatic skin substitutes (PS-) compared with healthy skin substitutes (HS-), as well as the effect of an n-3 polyunsaturated fatty acid, namely α-linolenic acid (ALA), on the psoriatic dermal compartment (PSALA+). Liquid chromatography tandem mass spectrometry analyses revealed that the lipidome of PS- contained higher amounts of n-6 derived prostaglandins (PGE2) and lipoxygenase products (9-HODE and 15-HETE). ALA supplementation increased the levels of PGE3, 13-HOTrE, 15-HEPE, and 18-HEPE, and decreased the levels of PGE2, 15-HETE, and 9-HOPE compared with PS-, indicating that ALA modulates the dermal lipidome of psoriatic skin substitutes. Gene expression profiling showed that several genes encoding for different ECM proteins were overexpressed in PS- compared with HS-, namely COL1A1 (4.2-fold), COL1A2 (3-fold), COL3A1 (4.4-fold), COL4A1 (2.3-fold), COL4A2 (6.3-fold), COL5A1 (3.3-fold), COL5A2 (5.2-fold), and COL5A3 (4.6-fold). Moreover, the expression of collagen IV (Col IV), collagen VII (Col VII), and laminin was found to be increased in PS- compared with HS-, and to be restored with ALA (PSALA+) according to immunofluorescence staining, while only the collagen I to collagen III ratio was altered according to dot blot analyses. Linear regression analysis revealed several positive correlations, including Col III with 14-HDHA levels, fibronectin with 12-HETE and 15-HETE levels, the dermo-epidermal junction Col IV with PGF2α, 9-HODE, and 13-HODE levels, and laminin with levels of PGF2α, 9-HODE, 13-HODE, 5-HETE, 12-HETE, and 15-HETE. These results suggest that the ECM plays an underestimated role in the pathogenesis of psoriasis and that ALA supplementation can regulate the ECM composition.

Alpha-Linolenic Acid Modulates T Cell Incorporation in a 3D Tissue-Engineered Psoriatic Skin Model.

Psoriasis is an autoimmune skin disease with an increased number of leukocytes infiltrating the dermal and epidermal compartments compared with normal skin. N-3 polyunsaturated fatty acids (n-3 PUFAs) are frequently used in the clinic in order to attenuate the symptoms of psoriasis. For psoriatic patients, a supplementation of the diet with alpha-linolenic acid (ALA) reduces the activation of T cell signaling pathways, leading to a significant reduction in inflammatory cytokine secretion. However, the precise mechanism of action of n-3 PUFAs in psoriasis is still not understood. In the present study, we elucidated the bioaction of ALA on the adaptive immune component of psoriasis by using a psoriatic skin model produced with the addition of activated T cells. Healthy and psoriatic skin substitutes were produced according to the self-assembly method, using culture media supplemented with 10 µM of ALA. T cells were isolated from blood samples using a negative selection isolation method. ALA supplementation regulated the hyperproliferation and abnormal cell differentiation of psoriatic keratinocytes stimulated by T cells. Additionally, the exogenous ALA was correctly incorporated into the phospholipids of keratinocytes, which resulted in increased levels of ALA, eicosapentaenoic acid (EPA) and n-3 docosapentaenoic acid (n-3 DPA). The infiltration of T cells into the epidermis was reduced when ALA was added to the culture medium, and significant decreases in the levels of inflammatory cytokines and chemokines such as CXCL1, interleukin-6 (IL-6) and interleukin-8 (IL-8) were consequently measured in psoriatic substitutes supplemented with this n-3 PUFA. Altogether, our results showed that in this psoriatic skin model enriched with T cells, ALA exerted its beneficial effect by decreasing the quantities of inflammatory mediators released by T cells.

Development of a 3D psoriatic skin model optimized for infiltration of IL-17A producing T cells: Focus on the crosstalk between T cells and psoriatic keratinocytes.

Psoriasis is a chronic inflammatory skin disease involving several cell types, including T cells, via the IL-23/IL-17 axis. IL-17A acts on the surrounding epithelial cells thus resulting in an inflammatory feedback loop. The development of immunocompetent models that correctly recapitulate the complex phenotype of psoriasis remains challenging, which also includes both the T cell isolation and activation methods. The purpose of this work was to develop an advanced in vitro 3D psoriatic skin model that enables the study of the impact of T cells on psoriatic epithelial cells. To reach that aim, healthy and psoriatic fibroblasts and keratinocytes were used to reproduce this tissue-engineered skin model in which activated T cells, isolated beforehand from human whole blood, have been incorporated. Our study showed that isolation of T cells with the EasySep procedure, followed by activation with PMA/ionomycin, mimicked the psoriatic characteristics in an optimal manner with the production of inflammatory cytokines important in the pathogenesis of psoriasis, as well as increased expression of Ki67, S100A7, elafin and involucrin. This psoriatic model enriched in activated T cells displayed enhanced production of IL-17A, IFN-Æ´, CCL2, CXCL10, IL-1ra, IL-6 and CXCL8 compared with the healthy model and whose increased secretion was maintained over time. In addition, anti-IL17A treatment restored some psoriatic features, including epidermal thickness and basal keratinocytes proliferation, as well as a downregulation of S100A7, elafin and involucrin expression. Altogether, our study demonstrated that this model reflects a proper psoriatic inflammatory environment and is effective for the investigation of epidermal and T cell interaction over time. STATEMENT OF SIGNIFICANCE: The aim of this study was to provide an innovative 3D immunocompetent human psoriatic skin model. To our knowledge, this is the first immunocompetent model that uses skin cells from psoriatic patients to study the impact of IL-17A on pathological cells. Through the use of this model, we demonstrated that the T-cell enriched psoriatic model differs from T-cell enriched healthy model, highlighting efficient crosstalk between pathologic epithelial cells and T cells. This advanced preclinical model further mimics the original psoriatic skin and will prove relevant in predicting clinical outcomes, thereby decreasing inaccurate predictions of compound effects.

Investigation of Omega-3 Polyunsaturated Fatty Acid Biological Activity in a Tissue-Engineered Skin Model Involving Psoriatic Cells.

Clinical studies have shown that diets enriched with omega-3 (also know as n-3) polyunsaturated fatty acids could relieve the symptoms of patients with psoriasis. However, the mechanisms involved remain poorly understood. The aim of this study was to investigate the effects of α-linolenic acid (ALA) on the proliferation and differentiation of psoriatic keratinocytes in a three-dimensional skin model. Skin models featuring healthy (healthy substitute) or psoriatic (psoriatic substitute) cells were engineered by the self-assembly method of tissue engineering using a culture medium supplemented with 10 µM ALA in comparison with the regular unsupplemented medium. ALA decreased keratinocyte proliferation and improved psoriatic substitute epidermal differentiation, as measured by decreased Ki67 staining and increased protein expression of FLG and loricrin. The added ALA was notably incorporated into the epidermal phospholipids and metabolized into long-chain n-3 polyunsaturated fatty acids, mainly eicosapentaenoic acid and n-3 docosapentaenoic acid. ALA supplementation led to increased levels of eicosapentaenoic acid derivatives (15-hydroxyeicosapentaenoic acid and 18-hydroxyeicosapentaenoic acid) as well as a decrease in levels of omega-6 (also know as n-6) polyunsaturated fatty acid lipid mediators (9-hydroxyoctadecadienoic acid, 12-hydroxyeicosatetraenoic acid, and leukotriene B4). Furthermore, the signal transduction mediators extracellular signal‒regulated kinases 1 and 2 were the kinases most activated after ALA supplementation. Taken together, these results show that ALA decreases the pathologic phenotype of psoriatic substitutes by normalizing keratinocyte proliferation and differentiation in vitro.

Transcriptome Profiling Analyses in Psoriasis: A Dynamic Contribution of Keratinocytes to the Pathogenesis.

Psoriasis is an immune-mediated inflammatory skin disease with a complex etiology involving environmental and genetic factors. A better insight into related genomic alteration helps design precise therapies leading to better treatment outcome. Gene expression in psoriasis can provide relevant information about the altered expression of mRNA transcripts, thus giving new insights into the disease onset. Techniques for transcriptome analyses, such as microarray and RNA sequencing (RNA-seq), are relevant tools for the discovery of new biomarkers as well as new therapeutic targets. This review summarizes the findings related to the contribution of keratinocytes in the pathogenesis of psoriasis by an in-depth review of studies that have examined psoriatic transcriptomes in the past years. It also provides valuable information on reconstructed 3D psoriatic skin models using cells isolated from psoriatic patients for transcriptomic studies.

A Tissue-Engineered Human Psoriatic Skin Model to Investigate the Implication of cAMP in Psoriasis: Differential Impacts of Cholera Toxin and Isoproterenol on cAMP Levels of the Epidermis.

Pathological and healthy skin models were reconstructed using similar culture conditions according to well-known tissue engineering protocols. For both models, cyclic nucleotide enhancers were used as additives to promote keratinocytes' proliferation. Cholera toxin (CT) and isoproterenol (ISO), a beta-adrenergic agonist, are the most common cAMP stimulators recommended for cell culture. The aim of this study was to evaluate the impact of either CT or ISO on the pathological characteristics of the dermatosis while producing a psoriatic skin model. Healthy and psoriatic skin substitutes were produced according to the self-assembly method of tissue engineering, using culture media supplemented with either CT (10-10 M) or ISO (10-6 M). Psoriatic substitutes produced with CT exhibited a more pronounced psoriatic phenotype than those produced with ISO. Indeed, the psoriatic substitutes produced with CT had the thickest epidermis, as well as contained the most proliferating cells and the most altered expression of involucrin, filaggrin, and keratin 10. Of the four conditions under study, psoriatic substitutes produced with CT had the highest levels of cAMP and enhanced expression of adenylate cyclase 9. Taken together, these results suggest that high levels of cAMP are linked to a stronger psoriatic phenotype.

The Tissue-Engineered Human Psoriatic Skin Substitute: A Valuable In Vitro Model to Identify Genes with Altered Expression in Lesional Psoriasis.

Psoriasis is a chronic inflammatory skin disease for which no cure has emerged. Its complex etiology requires the development of an in vitro model representative of the pathology. In this study, we exploited gene profiling analyses on microarray in order to characterize and further optimize the production of a human psoriatic skin model representative of this in vivo skin disease. Various skin substitutes were produced by tissue-engineering using biopsies from normal, healthy donors, or from lesional or non-lesional skin samples from patients with psoriasis, and their gene expression profiles were examined by DNA microarray. We demonstrated that more than 3540 and 1088 genes (two-fold change) were deregulated between healthy/lesional and lesional/non-lesional psoriatic substitutes, respectively. Moreover, several genes related to lipid metabolism, such as PLA2G4E and PLA2G4C, were identified as repressed in the lesional substitutes. In conclusion, gene profiling analyses identified a list of deregulated candidate genes associated with various metabolic pathways that may contribute to the progression of psoriasis.

Synthesis and Properties of Rhomboidal Macrocyclic Subunits of Graphdiyne-Like Nanoribbons.

Rhomboidal macrocyclic subunits of graphdiyne-like nanoribbon (GDNR) bearing both alkyne and diyne units, allowing for multichannel π-conjugation, were synthesized using an oxidative Glaser-type ring closing reaction. These subunits, called the "meshes" of the nanoribbon, possess phenyl groups with decyloxy solubilizing chains on each corner. The yields of the ring closing reaction highly depend on the metal (Cu or Pd) catalyst used for the macrocyclization step. Increasing the width of the meshes from one macrocycle to two fused macrocycles resulted in a decrease of the bandgap by 0.23 eV as shown by optical spectroscopy. The optimized geometries of the meshes alongside their HOMO and LUMO orbitals were calculated using DFT calculations at the B3LYP/6-31+G** level of theory. The results showed a nearly planar conformation for both meshes with HOMO and LUMO orbitals entirely delocalized over the molecules.