We cultured human hair follicle-derived keratinocytes (FDKs) from plucked hairs. To gain insight into gene expression signatures that can distinguish atopic dermatitis from non-atopic controls without skin biopsies, we undertook a comparative study of gene expression in FDKs from adult donors with atopic dermatitis and non-atopic donors. FDK primary cultures (atopic dermatitis, n = 11; non-atopic controls, n = 7) before and after interferon gamma (IFN-γ) treatment were used for microarray analysis and quantitative RT-PCR. Comparison of FDKs from atopic and non-atopic donors indicated that the former showed activated pathways with innate immunity and decreased pathways of cell growth, as indicated by increased NLRP2 expression and decreased DKK1 expression, respectively. Treatment with IFN-γ induced the enhanced expression of IL32, IL1B, IL8, and CXCL1 in the cells from atopic donors compared to that in cells from non-atopic donors at 24 h after treatment. IL1B expression in FDKs after IFN-γ treatment correlated with IL32 expression. We hypothesized that overexpression of IL32 in hair follicle keratinocytes of patients with atopic dermatitis would lead to the excessive production of pro-IL1β and that the activation of IL1β from pro-IL1β by inflammasome complex, in which NLRP2 protein might be involved, would be augmented. This is the first report to show enhanced induction of cytokine/chemokine genes by IFN-γ in atopic dermatitis using cultured FDKs.

In the epidermis, keratinocytes are involved in physical and first-line immune protection of the host. In this study, we analyzed the molecular responses to certain contact sensitizers (2,4-dinitrochlorobenzene and NiSO(4)) and irritants (sodium dodecyl sulfate and benzalkonium chloride) in cultured human keratinocytes from the bulge region of a plucked hair follicle (bulge-derived keratinocytes [BDKs]) and compared these molecular responses to those with the human monocytic leukemia cell line, THP-1. The BDKs, individually established without invasive biopsies, showed high reactivity to these stimulants. As a primary response to the contact sensitizers, the NRF2-mediated signaling pathway was upregulated in BDKs and THP-1. The expression of IL1B and IL8 genes was not induced by the irritants but by the sensitizers in THP-1. However, the expression of the IL1B and IL8 genes was induced at higher levels by the irritants in BDKs than by the sensitizers. Many genes orchestrating keratinocyte differentiation, including ID2, were significantly upregulated in response to the sensitizers in BDKs but not those in THP-1. The use of the ID2 gene to discriminate between sensitizers and irritants might be effective as a novel marker for application during in vitro sensitization with BDKs.

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Abstract. Human keratinocyte strains derived from the bulge region of plucked human follicles were successfully established from all 43 donors (age 24-76) regardless of the age and gender. The total cell number, number of population doublings and population doubling time were similar among the strains. These bulge-derived keratinocytes, BDKs, expressed keratin family genes specific to basal cell layers of the epidermis. They also expressed CD34, one of the bulge stem cell marker genes. The growth behavior and positivity of CD34 indicate that BDKs contain stem cells. BDKs were cultured until confluency or treated with CaCl 2 to induce differentiation. Morphology and expression of keratin family genes in BDKs before and after differentiation induction with CaCl 2 were similar to those of epidermal keratinocytes obtained from skin biopsies (NHEKs). However, expression levels of keratin-10, a prickle cell layer marker, in CaCl 2 -treated BDKs were lower than those in CaCl 2 -treated NHEKs. Higher expression of integrin-·6, a basal cell layer marker, was also noted in BDKs than in NHEKs after differentiation induction. Expression of stem cell marker genes other than CD34, including CD200, Sox2 and NANOG, was about the same at confluency in both cells, but significantly higher in BDKs than NHEKs after differentiation. These results indicate that BDKs were more refractory to differentiation than NHEKs. We then examined Wnt signaling inhibitor genes, DKK-3 and WIF-1 that function as tumor suppressors. DKK-3 expression decreased in both BDKs and NHEKs after CaCl 2 -induced differentiation. Expression of WIF-1 decreased 50% in BDKs one day after CaCl 2 treatment and remained low, but was induced 1.7 times in NHEKs one day after CaCl 2 treatment and further induced thereafter (>2.5 times), suggesting that WIF-1 may be involved in maintaining the differentiation-refractory status of BDKs. Since cancer stem cells in the skin have been reported to be similar to bulge-derived stem cells, our BDK strains may be of use in studying characteristics of cancer stem cells of the epidermis.

The mechanisms involved in skin aging are not completely understood, although one of its hallmarks is the reduction of epidermal function. Aged-epidermis is in a constant state of water loss, triggering dry skin, flakiness, itchiness, inflammation and increase in skin sensitivity. Cutaneous barrier formation is a continuous and tightly regulated process involving epidermal proliferation and differentiation. A robust barrier function prevents water loss, regulates body temperature, improves host defense and protects against environmental stressors. Endogenous hyaluronic acid (HA) regulates keratinocyte proliferation, differentiation, barrier formation, cell survival, inflammation, oxidative stress, hydration and elasticity. With age, levels of endogenous HA decreases significantly, altering skin hydration and epidermal function. The goal of this study was to evaluate potential improvements in barrier function by treatment with a cosmetic formulation (HA 5 ). Human skin explants were obtained from 30 year old (young skin, YS) and 60 year old (old skin, OS) female donors and were treated or not with HA 5 for 5 days. Results showed a significant increased expression of HAS1 in OS treated with HA 5 formulation when compared to untreated OS and YS. Enhanced deposit of HA in the epidermis was also observed after HA 5 application. Treatment of OS with HA 5 restored the expression of barrier function markers (IVL, GJB4, TGM1 and TJP1) to levels similar to those observed in YS, and also had an improved histological evaluation of endogenous lipid markers. Taken together, these data suggest that the active ingredients in HA 5 (Vitis vinifera stem cell extract, marine polysaccharides and peptide complex) successfully restore endogenous HA in the epidermis, triggering a normalization of the skin barrier function. Normalization of endogenous epidermal HA levels by promoting its synthesis and preventing its degradation constitute a new approach to restore the skin's barrier function, which is a multifactorial process that involves hydration, lipid composition and keratinocyte differentiation.