Evaluation of Anti-inflammatory and Atrophogenic Effects of Glucocorticoids on Reconstructed Human Skin

Abstract

Topical glucocorticoids (GCs) are extensively used in the treatment of inflammatory skin diseases. However, their long-term use is often accompanied by severe and eventually irreversible adverse effects, with atrophy being the most important limitation. Currently, most non-clinical studies involve animal testing, so the results are not always representative of the situation in humans. The aim of this project was to establish an in vitro test protocol for the evaluation of the anti-inflammatory and atrophic potential of topically applied GCs in reconstructed human skin. Initial studies with fibroblasts and keratinocytes confirmed the anti-inflammatory and atrophogenic effects of GCs, as evidenced by decreased cytokine production and collagen mRNA expression. In non-pretreated reconstructed human skin (EpiDermFT™), the topical application of GCs for seven days strongly reduced the secretion of interleukin (IL)-6. GC-induced skin atrophy, known to appear only after prolonged treatment, was not detected by the analysis of epidermal thickness and collagen mRNA expression. However, reproducible epidermal inflammation was established for the first time in reconstructed human skin. Topical treatment with tumour necrosis factor (TNF) increased IL-6 release and strongly reduced epidermal thickness accompanied by severe parakeratosis. GC treatment of reconstructed human skin reduced IL-6 levels and completely resolved parakeratosis, leading to the normalisation of epidermal thickness. These induced inflammatory conditions mimic more closely the clinical situations in which GCs are used, and therefore appear to be more suitable for future investigations for the establishment of a human-based in vitro test protocol for evaluating wanted and unwanted GC effects.

Calcipotriol counteracts betamethasone-induced decrease in extracellular matrix components related to skin atrophy

“…The effects of glucocorticoids on extracellular matrix components have been studied in monolayer cultures of human dermal fibroblasts by several groups, however, with rather divergent results [1, 28, 33]. In one study, treatment with glucocorticoids for one to five days showed no effect on COL1A1 and COL3A1 mRNA expression while HAS2 mRNA expression and HA production were significantly suppressed [1].…”

Tailored dendritic core-multishell nanocarriers for efficient dermal drug delivery: A systematic top-down approach from synthesis to preclinical testing

“…During the last 10 years, in vitro skin models have emerged as valuable tools for skin absorption studies, and have proved suitable for drug efficiency testing as demonstrated with glucocorticoid studies [25,26]. Hence, to evaluate the biocompatibility and efficiency of DXM loaded CMS nanocarriers in a more complex system, an inflammatory skin disease model was established.…”

“…Rodent skin however demonstrates major anatomical and physiological differences relative to human skin, something that skin models aim to overcome [19][20][21]. Reconstructed human skin has been used to model atopic dermatitis [22], ichthyosis vulgaris [23] and non-melanoma skin cancer in vitro [24], and to assess nanoparticle-mediated delivery [12] and therapeutic efficacies [25,26] .…”

TLR2/1 and sphingosine 1-phosphate modulate inflammation, myofibroblast differentiation and cell migration in fibroblasts

“…Primary cultures of normal human dermal fibroblasts were isolated from human juvenile foreskin and cultured as described [21,22]. To reduce donor-specific properties, cells were pooled from at least three donors.…”

“…Primers (synthesized by TIB Molbiol, Berlin, Germany) with the following sequences were used: COL1A1, COL3A1, HMBS, IL-8, MMP1, TLR1-10 and YWHAZ as published previously [21,23]; ACTA, 5′-TGGGCTCTGTAAGGCCGGCT-3′ and 5′-TCACCC CCTGATGTCTGGGACG-3′; IFN-α, 5′-GTGAGGAAATACTTCCAAAGAATC AC-3′ and 5′-TCTCATGATTTCTGCTCTGACAA-3′; IFN-β, 5′-CAGCAATTTT CAGTGTCAGAAGC-3′ and 5′-TCATCCTGTCCTTGAGGCAGT-3′; IL-1α, 5′-CGCCAATGACTCAGAGGAAGA-3′ and 5′-AGGGCGTCATTCAGGATGAA-3′; IL-1β, 5′-TGGAGCAACAAGTGGTGT-3′ and 5′-TTGGGATCTACACTCT CCAGC-3′; IL-6, 5′-CACAGACAGCCACTCACCTC-3′ and 5′-TTTTCTGCCA GTGCCTCTTT-3′; S1PR1, 5′-CACCGTGCTGCCGCTCTACC-3′ and 5′-GCAG CGCCAGCGACTTCTCA-3′; S1PR2, 5′-GCGCCATTGTGGTGGAAAA-3′ and 5′-CATTGCCGAGTGGAACTTGCT-3′; S1PR3, 5′-GGTGATTGTGGTGAGC GTGTT-3′ and 5′-AGGCCACATCAATGAGGAAGA-3′; S1PR4, 5′-GTGGTG CTGGAGAACTTGCT-3′ and 5′-GGTCACTCAGCGTGATGTTC-3′; S1PR5, 5′-GCTTGCTCCACTGTCTTGC-3′ and 5′-GCGCGTAGAGTGCACAGA-3′; TNF, 5′-CCCAGGGACCTCTCTCTAATCA-3′ and 5′-GCTACAGGCTTGTCAC TCGG-3′. Fold difference in gene expression was normalized to the housekeeping genes, HMBS and YWHAZ, which showed the most constant level of expression in preliminary investigations in which four different housekeeping genes (i.e.…”