Use of Cytokine Mix-, Imiquimod-, and Serum-Induced Monoculture and Lipopolysaccharide- and Interferon Gamma-Treated Co-Culture to Establish In Vitro Psoriasis-like Inflammation Models

Abstract

Psoriasis (Ps), commonly perceived as a skin and joint disorder, has a complex basis and results from disturbances in the sophisticated network between skin and the immune system. This makes it difficult to properly depict the complete pathomechanism on an in vitro scale. Deciphering the complicated or even subtle modulation of intra- and intercellular factors, assisted by the implementation of in vitro human skin models, may provide the opportunity to dissect the disease background step by step. In addition to reconstructed artificial skin substitutes, which mimic the native physiological context, in vitro models are conducive to the broad “3 Rs” philosophy (reduce, refine, and replace) and represent important tools for basic and applied skin research. To meet the need for a more comprehensive in vitro Ps model, a set of various experimental conditions was applied in this study. The selection of in vitro treatment that mimicked the Ps phenotype was illustrated by analyses of discriminating biomarker genes involved in the pathogenesis of the disease, i.e., keratinocyte differentiation markers, antimicrobial peptides, chemokines, and proliferation markers. This resulted in a reproducible protocol for the use of the primary skin keratinocyte (pKC) monoculture treated with a cytokine cocktail (5MIX, i.e., interleukin (IL) 1 alpha (IL-1α), IL-17A, IL-22, oncostatin M (OSM), and tumour necrosis factor alpha (TNF-α)) at a calcium (Ca2+) concentration (i.e., 2 mM) in an applied medium, which best mirrored the in vitro Ps-like inflammatory model. In addition, based on waste skin material, the method has the potential for extensive experimentation, both in detailed molecular studies and preclinical tests.

Ursolic Acid Formulations Effectively Induce Apoptosis and Limit Inflammation in the Psoriasis Models In Vitro

“…These include the model based on the concurrent stimulation of the HaCaT culture with IL-17A, IL-22, oncostatin-M, IL-1α, and TNF-α [7]. This M5 stimulation model mimics typical features of psoriasis, including pro-inflammatory response and keratinocyte hyperproliferation, accompanied by inhibition of keratinocyte differentiation and increased expression of psoriasis-related biomarkers [21]. This model was implemented and characterized an elevated inflammatory response, evaluated by an increase in IL-6 and IL-8 production, augmentation of S100A7, S100A9, CXCL1, and CCL20 expression, and an increase in the proliferation in HaCaT cells, consistent with the psoriasis-like phenotype.…”

“…Expression of several psoriatic biomarkers, including S100A7 and S100A9, has been identified and shown to be induced in the M5 stimulation model [21]. For example, S100A7 (psoriasin) is an antimicrobial peptide, which also limits keratinocyte differentiation and induces their proliferation, mainly through IL-6 induction [38,39].…”

Challenges in Psoriasis Research: A Systematic Review of Preclinical Models

“…These KTs can come from primary cultures or from immortalized lines. Immortalized KT lines include HaCaT cells [24,[44][45][46][47] and N/TERT1, N/TERT2G [48]. Coculture monolayer consists of coculture of KTs with immune cells.…”

“…Coculture monolayer consists of coculture of KTs with immune cells. One example of this is the coculture of HaCaT cells with human leukemia monocytic cell line (THP-1) [44]. These KT-T cell coculture systems suggested that KT-T cell communication can recruit immune cells into the skin by secreting chemokines as well as regulating T-cell differentiation [49].…”

“…The addition of the cytokine mix has successfully resulted in the development of specific characteristics of psoriatic skin. For instance, Bochenska et al [44] showed how in the single cell monolayers of HaCaT cell line and primary normal human epidermal keratinocytes (NHEKs), these cytokines activate KTs and the psoriatic phenotype is obtained. Other strategies to induce psoriasis include the addition of IMQ serum-free medium (SFM) supplemented with serum from psoriatic patients [44], phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide, IFN-γ [44], and 12-O-tetradecanolylphorbol 13-acetate [33,46].…”