Culturing Keratinocytes on Biomimetic Substrates Facilitates Improved Epidermal Assembly In Vitro.

Mechanotransduction is defined as the ability of cells to sense mechanical stimuli from their surroundings and translate them into biochemical signals. Epidermal keratinocytes respond to mechanical cues by altering their proliferation, migration, and differentiation. In vitro cell culture, however, utilises tissue culture plastic, which is significantly stiffer than the in vivo environment. Current epidermal models fail to consider the effects of culturing keratinocytes on plastic prior to setting up three-dimensional cultures, so the impact of this non-physiological exposure on epidermal assembly is largely overlooked. In this study, primary keratinocytes cultured on plastic were compared with those grown on 4, 8, and 50 kPa stiff biomimetic hydrogels that have similar mechanical properties to skin. Our data show that keratinocytes cultured on biomimetic hydrogels exhibited major changes in cellular architecture, cell density, nuclear biomechanics, and mechanoprotein expression, such as specific Linker of Nucleoskeleton and Cytoskeleton (LINC) complex constituents. Mechanical conditioning of keratinocytes on 50 kPa biomimetic hydrogels improved the thickness and organisation of 3D epidermal models. In summary, the current study demonstrates that the effects of extracellular mechanics on keratinocyte cell biology are significant and therefore should be harnessed in skin research to ensure the successful production of physiologically relevant skin models.

Niacinamide mitigates SASP-related inflammation induced by environmental stressors in human epidermal keratinocytes and skin.

OBJECTIVE: To evaluate whether niacinamide (Nam) can mitigate production of inflammatory and senescence-related biomarkers induced by environmental stressors. METHODS: Human epidermal keratinocytes were exposed to UVB, urban dust, diesel exhaust and cigarette smoke extract and treated with Nam or vehicle control. Full thickness 3-D skin organotypic models were exposed to a combination of UVB and PM2.5 and treated with Nam or vehicle control. Quantitation of the SASP-related inflammatory mediators PGE2 , IL-6 and IL-8 was performed on cultured media. UVB-exposed keratinocytes treated with and without Nam were immunostained for the senescence biomarker Lamin B1 (LmnB1). Transcriptomics profiling of cigarette smoke extract effects on keratinocytes was performed. A double-blind, placebo-controlled clinical was conducted on 40 female panellists that were pretreated on back sites for two weeks with 5% Nam or vehicle and then exposed to 1.5 minimal erythemal dose (MED) solar-simulated radiation (SSR). Treated sites were compared with non-treated exposed sites for erythema and the skin surface IL-1αRA/IL-1α inflammatory biomarkers. RESULTS: Ultraviolet B induced synthesis of PGE2 , IL-8 and IL-6 and reduced LmnB1 levels in keratinocytes. Urban dust and diesel exhaust only stimulated synthesis of IL-8 whereas cigarette smoke extract only stimulated levels of PGE2 . In all exposures, treatment with Nam significantly mitigated synthesis of the inflammatory mediators and restored levels of UVB-reduced LmnB1. In the 3D skin equivalent model, Nam reduced IL-8 levels stimulated by a combination of topical PM2.5 and UV exposure. In a UV challenge clinical, pretreatment with 5% Nam reduced erythema and skin surface IL-1αRA/IL-1α inflammatory biomarkers that were induced by SSR. CONCLUSION: Since it is known that Nam has anti-inflammatory properties, we tested whether Nam can inhibit environmental stress-induced inflammation and senescence-associated secretory phenotype (SASP) biomarkers. We show Nam can reduce PGE2 , IL-6 and IL-8 levels induced by environmental stressors. Additionally, in vivo pretreatment with Nam can reduce UV-induced erythema and skin surface inflammatory biomarkers. These findings add to the body of evidence that Nam can mitigate the skin's inflammatory response elicited by environmental stressors. This supports Nam can potentially inhibit senescence and premature ageing and thereby maintain skin's functionality and appearance.

OBJECTIF: Évaluer si le niacinamide (Nam) peut atténuer la production de biomarqueurs inflammatoireset liés à la sénescence induits par les facteurs de stress environnementaux. MÉTHODES: Leskératinocytes épidermiques H uman ont été exposés aux UVB, à la poussière urbaine, aux gaz d'échappement diesel et à l'extrait de fumée de cigarette et traités avec nam ou contrôle de véhicule. Les modèles organotypic de peau 3D de pleine épaisseur ont été exposés à une combinaison d'UVB et de PM2.5 et traités avec nam ou commande de véhicule. La quantitation des médiateurs inflammatoires liés à la SASP PGE2 ,IL-6 et IL-8 a été réalisée sur des médias cultivés. Les kératinocytes exposés aux UVB traités avec et sans Nam étaient immunotachés pour le biomarqueur de sénescence Lamin B1 (LmnB1). Le profilage de transcriptomique des effets d'extrait de fumée de cigarette sur les kératinocytes a été exécuté. Un placebo contrôlé clinique à double insu a été menée sur 40 panélistes féminins qui ont été prétraités sur les sites arrière pendant deux semaines avec 5% Nam ou véhicule, puis exposés à 1,5 dose erythémique minimale (MED) rayonnement solaire simulé (SSR). Les sites traités ont été comparés à des sites exposés non traités pour l'érythème et la surface de la peau IL-1▫RA/IL-1▫ biomarqueurs inflammatoiress. RÉSULTATS: Synthèse induite par UVB des niveaux de PGE2, IL-8 et IL-6 et réduit de LmnB1 dans les kératinocytes. La poussière urbaine et les gaz d'échappement diesel n'ont stimulé que la synthèse de l'IL-8 alors que l'extrait de fumée de cigarette ne stimulait que les niveaux de PGE2 . Dans toutes les expositions, le traitement avec Nam a significativement atténué la synthèse des médiateurs inflammatoires et les niveaux restaurés de LmnB1 UVB-réduit. Dans le modèle équivalent de la peau 3D, Nam a réduit les niveaux d'IL-8 stimulés par une combinaison de PM combination of topical PM 2.5 topique et d'exposition aux UV. Dans un uv-défi clinique, prétraitement avec 5% Nam réduit érythème et la surface de la peau IL-1▫RA/IL-1▫ biomarqueurs inflammatoires qui ont été induits par SSR. CONCLUSION: Puisqu'il est connu que Nam a des propriétés anti-inflammatoires, nous avons testé si Nam peut inhiber l'inflammation induite par le stressenvironnementaltion et les biomarqueurs sécrétoires sécrétoires sécrétoires (SASP) associés à la sénescence. We montrent Nam peut réduire PGE2 ,IL-6, et IL-8 niveaux induits par les facteurs de stress environnementaux. En outre, le prétraitement in vivo avec Nam peut réduire l'érythème induit par les UV et les biomarqueurs inflammatoires de surface de la peau. Ces résultats ajoutent à l'oody bde la preuve que Nam peut atténuer la réponse inflammatoire de la peau provoquée par lesfacteurs de stress environnementaux. Cela soutient Nam peut potentiellement inhiber la sénescence et le vieillissement prématuré et ainsi maintenir la fonctionnalité de la peau et l'apparence.

Determination of in vivo dose response and allergen-specific T cells in subjects contact-sensitized to squaric acid dibutyl ester.

BACKGROUND: Squaric acid dibutyl ester (SADBE) is a known contact sensitizer, but dose-response data are not defined. OBJECTIVE: To determine the relationship between sensitization dose and contact hypersensitivity (CHS) response to SADBE in human volunteers. The study also aimed to investigate whether SADBE-reactive blood T cells could be detected using ex vivo mature dendritic cells (DCs) as antigen-presenting cells. METHOD: Forty healthy volunteers were sensitized to either 12.5, 25, 50, or 250 microg of SADBE in a 48 microL volume. This was followed by elicitation 2 weeks later with five doses (0, 0.2, 2, 20, and 200 microg in 20 microL). An additional 10 subjects received the elicitation doses without prior sensitization. Blood samples obtained after sensitization were purified into T cells and mature DCs. RESULTS: A direct relationship between sensitization dose and in vivo CHS response was observed. The SADBE dose that effectively sensitized 50% of the population (ED50) was 22 microg/cm2. Significant SADBE-specific T-cell proliferation in vitro was not observed 2 weeks after sensitization but became evident after elicitation. CONCLUSION: This study establishes the in vivo dose-response characteristics of immune reactivity to SADBE and antigen-specific T-cell reactivity.

Interactions of contact allergens with dendritic cells: opportunities and challenges for the development of novel approaches to hazard assessment.

The identification of potential skin sensitizing chemicals is a key step in the overall skin safety risk assessment process. Traditionally, predictive testing has been conducted in guinea pigs. More recently, the murine local lymph node assay (LLNA) has become the preferred test method for assessing skin sensitization potential. However, even with the significant animal welfare benefits provided by the LLNA, there is a need to develop non-animal test methods for skin sensitization. Mechanistic understanding of allergic contact dermatitis has increased substantially in recent years. For example, a number of changes are known to occur in epidermal Langerhans cells, the principal antigen-presenting dendritic cell in the skin, as a result of exposure to chemical allergens, including the internalization of surface major histocompatibility complex (MHC) class II molecules via endocytosis, the induction of tyrosine phosphorylation, the modulation of cell surface markers, and cytokine expression. The application of this knowledge to the design of predictive in vitro alternative tests provides both unique opportunities and challenges. In this review, we have focused specifically on the impact of chemical exposure on dendritic cells and the potential use of that information in the development of cell-based assays for assessing skin sensitization potential of chemicals in vitro.

Relationship of CD86 surface marker expression and cytotoxicity on dendritic cells exposed to chemical allergen.

Human peripheral blood-derived dendritic cells (DC) respond to a variety of chemical allergens by up-regulating expression of the co-stimulatory molecule CD86. It has been postulated that this measure might provide the basis for an in vitro alternative approach for the identification of skin sensitizing chemicals. We recently reported that DC, exposed in culture to the highest non-cytotoxic concentrations of various chemical allergens, displayed marginal up-regulation of membrane CD86 expression; the interpretation being that such changes were insufficiently sensitive for the purposes of hazard identification. For the work presented here, immature DC were derived from human monocytes and treated with the chemical allergens 2,4-dinitrobenzenesulfonic acid (DNBS), nickel sulfate (NiSO4), p-phenylenediamine (PPD), Bandrowski's base (BB), hydroquinone (HQ) and propyl gallate (PG) for 48 h at concentrations which induced both no to slight to moderate cytotoxicity. For comparison, DC were treated with the irritants sodium dodecyl sulfate (SDS), benzoic acid (BA), and benzalkonium chloride (BZC) at concentrations resulting in comparable levels of cytotoxicity. CD86 expression, as measured by flow cytometry, was consistently up-regulated (ranging from 162 to 386% control) on DC treated with concentrations of chemical allergens that induced approximately 10-15% cytotoxicity. The irritants BA and BZC did not induce up-regulation of CD86 expression when tested at concentrations that induced similar levels of cytotoxicity. SDS, however, up-regulated CD86 expression to 125-138% of control in 2/4 preparations when tested at concentrations which induced similar toxicity. Our results confirm that chemical allergens up-regulate CD86 expression on blood-derived DC and illustrate further that up-regulation of CD86 surface marker expression is more robust when DC are treated with concentrations of chemical allergen that induce slight to moderate cytotoxicity.

Gene expression changes in peripheral blood-derived dendritic cells following exposure to a contact allergen.

A critical step in the induction of allergic contact allergy is the activation and subsequent migration of Langerhans cells (LC), an important antigen presenting dendritic cell (DC) of the skin. As the Langerhans cells migrate, they undergo a maturation process. It has been proposed that contact allergen exposure can induce DC maturation. While changes in DC gene expression profiles induced by various maturation stimuli have been explored, there are no published reports describing genomic-scale analysis of the changes induced by chemical allergen exposure. Therefore, to explore the concept of chemical allergen-induced DC maturation and to identify genes that are regulated by exposure to allergens we examined, at the transcriptional level, the effects of exposure to a contact allergen on DC. Peripheral blood-derived DC were exposed for 24 h to either 1mM or 5 mM dinitrobenzenesulfonic acid (DNBS). Changes in gene expression were analyzed using Affymetrix U95Av2 GeneChip. Comparison of mean signal values from replicate cultures revealed 173 genes that were significantly different (P < or = 0.001) between 1 mM DNBS treated and untreated control DC and 1249 significant gene changes between 5 mM DNBS treated and control DC. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the observed transcript changes for selected genes in DC derived from a second donor. Comparison of the fold-changes in transcript levels between the two platforms and donors revealed a good correlation in both direction and magnitude. RT-PCR analysis was also used to assess the allergen specificity of a selected number of genes in DC derived from a third donor. Many of the gene expression changes were found to be induced only by exposure to the allergen, DNBS, and not by exposure to a structurally similar non-allergen, benzenesulfonic acid. A number of gene expression changes induced by allergen exposure were found to be consistent with what is known of the DC maturation process, and thus provide support for the theory of contact allergen-induced DC maturation. Additionally, it is hoped that some of the transcript changes identified through this approach will be shown to be suitable for use in the development of an in vitro predictive assay for contact sensitization.

Assessment of glycosylation-dependent cell adhesion molecule 1 as a correlate of allergen-stimulated lymph node activation.

Early changes in gene expression have been identified by cDNA microarray technology. Analysis of draining auricular lymph node tissue sampled at 48 h following exposure to the potent contact allergen 2,4-dinitrofluorobenzene (DNFB) provided examples of up- and down-regulated genes, including onzin and guanylate binding protein 2, and glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), respectively. Allergen-induced changes in these three genes were confirmed in dose-response and kinetic analyses using Northern blotting and/or reverse transcription-polymerase chain reaction techniques. The results confirmed that these genes are robust and relatively sensitive markers of early changes provoked in the lymph node by contact allergen. Upon further investigation, it was found that altered expression of the adhesion molecule GlyCAM-1 was not restricted to treatment with DNFB. Topical sensitization of mice to a chemically unrelated contact allergen, oxazolone, was also associated with a decrease in the expression of mRNA for GlyCAM-1. Supplementary experiments revealed that changes in expression of this gene are independent of the stimulation by chemical allergens of proliferative responses by draining lymph node cells. Taken together these data indicate that the expression of GlyCAM-1 is down-regulated rapidly following epicutaneous treatment of mice with chemical allergens, but that this reduction is associated primarily with changes in lymph node cell number, or some other aspect of lymph node activation, rather than proliferation.

Elucidating changes in surface marker expression of dendritic cells following chemical allergen treatment.

Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) located in lymphoid and many nonlymphoid tissues, and Langerhans cells (LC), a specialized form of DC, are found in the skin. LC play a critical role in the induction of contact dermatitis and therefore have become a focal point for the development of in vitro cell-based methods for contact sensitization testing. Because of the low abundance of skin-derived LC, methods to culture DC from peripheral blood are being used by investigators to generate LC surrogates to examine the effects of sensitizing chemicals on APC. It has been reported recently that chemical allergens can induce changes in the expression of various DC surface markers and it has been suggested that the measure of these changes in surface marker expression following allergen treatment could provide the basis for an in vitro test method to predict the contact sensitization potential of a chemical. For the work presented here, DC were differentiated from human peripheral blood mononuclear cells (PBMC-DC) in culture medium containing GM-CSF and interleukin (IL)-4 to ensure an immature phenotype or were derived from the KG-1 cell line (KG-1 DC) using a defined cytokine cocktail consisting of GM-CSF, IL-4, Flt-3/Flk-2-ligand, thrombopoietin, stem cell factor, and tumor necrosis factor-alpha (TNFalpha). Surface marker expression (HLA-DR, CD54, CD80, and CD86) on these DC was measured by flow cytometry after 48 h treatment with the known chemical allergens dinitrofluorobenzene (DNFB) and methylchloroisothiazolinone/methylisothiazolinone (MCI/MI), the irritant sodium dodecyl sulfate, lipopolysaccharide (LPS), and TNFalpha. Treatment of PBMC-DC with either MCI/MI or DNFB induced a slight upregulation of class II major histocompatibility (MHC) expression (HLA-DR), whereas LPS and TNFalpha significantly upregulated CD54 and slightly upregulated CD80 and HLA-DR expression. For KG-1 DC, only MCI/MI upregulated CD86 expression, whereas TNFalpha upregulated CD54 and slightly upregulated CD80 and CD86 expression. SDS had no effect on surface marker expression in either PBMC-DC or KG-1 DC. Changes in surface marker expression in PBMC-DC treated with chemical allergens were detected in two of five donors, suggesting a limited sensitivity of PBMC-DC under these defined isolation and culture conditions. Furthermore, we found that the presence of GM-CSF and IL-4 during chemical allergen treatment masked the ability to detect changes in surface marker expression. Our data suggest that, under these culture and treatment conditions, measurement of surface marker changes in vitro using PBMC-DC or KG-1 DC does not provide a sensitive in vitro method with sufficient dynamic range for assessing the contact sensitization potential of a chemical.

Use of a B cell marker (B220) to discriminate between allergens and irritants in the local lymph node assay.

It has been shown that exposure of mice to contact allergens induces B cell activation in the draining lymph nodes (DLN), as seen by an increase in the percentage of B220+ or IgG/IgM+ cells. We have now examined whether the measurement of the percentage of B220+ cells could be used as an alternative or supplementary endpoint for the local lymph node assay (LLNA) to differentiate between allergenic responses and those few irritants that induce low-level proliferation in the DLN. Mice were treated on the ears, daily for 3 consecutive days, with various allergens (1-chloro-2,4-dinitrobenzene, alpha-hexylcinnamaldehyde, trinitrochlorobenzene, isoeugenol, and eugenol) or irritants (benzalkonium chloride, methyl salicylate, salicylic acid, and sodium lauryl sulfate). The DLN were excised 72 h following the final topical treatment, and the cells were prepared for B220 analysis using flow cytometry. The percentage of B220+ cells in lymph nodes derived from test and vehicle-treated animals was determined for 5 allergens and 4 irritants tested in multiple experiments (n = 3 to 17). As expected, the percentage of B220+ B cells was increased with each of the allergens tested, whereas irritant treatment did not cause similar increases. Moreover, the method was reproducible. For example, the strong allergen, 1-chloro-2,4-dinitrobenzene and the weak allergen, alpha-hexylcinnamaldehyde were identified as allergens in 17 of 17 and in 12 of 13 experiments, respectively. The percentage of B220 values for each chemical treatment (41 observations for allergens; 28 observations for irritants) versus the percentage of B220 values for the concurrent vehicle controls were plotted, and a classification tree model was developed that defined a B220 test:vehicle ratio cutoff of 1.25 for discriminating between allergens (>1.25) and irritants (<1.25). Using this B220 test:vehicle ratio of 1.25 in 93% of the 69 independent observations made, the allergens and irritants tested were identified correctly. Finally, to evaluate the performance of this model in a second independent laboratory, 3 allergens and 2 irritants were tested. Each of the allergens and irritants were classified correctly using the B220 test:vehicle ratio cutoff of 1.25. These data demonstrate that analysis of B220 expression in DLN may be useful in differentiating between allergen and irritant responses induced in chemically treated mice.