Establishment of an in vitro model to study the effect of psychological stress on human skin stem cells
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Presented by: Katharina Kappler
Introduction: Even though anecdotal evidence for the impact of psychological stress on the skin exists, the molecular mechanisms how stress leads to a response in the skin are still not fully clear. One of the major stress response systems in vertebrates is the hypothalamus-piuitary-adrenal (HPA) axis that connects the central nervous system with the endocrine system and regulates the secretion of glucocorticoids, such as cortisol. Interestingly, the human skin expresses major elements of the HPA axis including corticotropin releasing factor (CRF), CRF receptors and glucocorticoids. CRF, produced in the skin in response to stressors, can bind to CRF1 receptor, which activates different pathways and regulates the expression of several genes in epidermal keratinocytes, leading to phenotypic effects, such as stimulation of proliferation, differentiation or melanogenesis. Studies have shown that CRF can stimulate the immune response regulator nuclear factor-kappa B (NF-ĸB) and the production of the cytokine IL-6 in epidermal keratinocytes. Furthermore, stress responses involve the sympathetic nervous system and the release of catecholamine neurotransmitters, such as epinephrine and norepinephrine, known to be produced in human keratinocytes. The effect of psychological stress on skin stem cells is largely unexplored. Therefore, we aimed to develop an in vitro model using skin stem cells and a psychological stress inducer along with an appropriate end point, which can be used for efficacy screening to identify modulators of stress in the skin.
Methods: The assay was established in pooled human epidermal progenitor keratinocytes (HPEKs), isolated from adult healthy donors. Cells were seeded in 6-well plates and cultured in serum-free cell culture medium without cortisol for 2 days before treatment with different stress inducers (CRF, CRF1 agonist stressin I, norepinephrine, cortisol) at various concentrations for different periods to simulate acute (6 hours) and chronic psychological stress (2 and 4 days). Supernatants were harvested at each time point and analyzed for IL-1β, IL-8 and IL-6 by ELISA to assess potential biomarkers of the stress response. After identification of the most robust biomarker and the most effective concentration for each stressor, the experiment was repeated to test for reproducibility. In addition, RNA was isolated from the cells after 2 and 4 days of treatment with 100 nM stressin I, 0.5 µM or 1 µM norepinephrine to quantify NF-ĸB gene expression by qPCR.
Results: Whereas the secretion of IL-8 and IL-1β was less pronounced and consistent, IL-6 was identified as a reliable biomarker in this model. IL-6 secretion was induced when HPEKs were incubated for 6 hours with different concentrations of the CRF1 agonist stressin I or norepinephrine. CRF only had a modest effect on IL-6 expression, whereas cortisol downregulated IL-6 levels in this setup. Under chronic stress conditions (2 or 4 days of treatment), cells showed enhanced IL-6 secretion after treatment with 100 nM stressin I or different concentrations of norepinephrine or CRF for 2 days. After 4 days, cells responded with increased IL-6 levels for all compounds tested. A repetition of the assay using the most effective concentrations of each stressor demonstrated its reproducibility. Chronic stress induced by treatment with 1 µM norepinephrine or 100 nM stressin I led to a significant increase of IL-6 secretion by up to 150% or 78%, respectively, compared to the control after 4 days of treatment. In addition, gene expression of NF-ĸB was strongly upregulated under the same conditions.
Discussion and Conclusion: Overall, the established in vitro model clearly demonstrates the function of the CRF1 agonist stressin I and norepinephrine as stress inducers to simulate psychological stress in HPEKs. IL-6, a well-known stress marker, and NF-ĸB, a transcription factor that is upregulated during stress and inflammation, have been identified as valuable biomarkers in this context. This reproducible model can be used for efficacy screening of molecules in order to identify modulators of psychological stress in the skin. Thus, it represents a valuable model for the development of a new generation of neurocosmetic active ingredients.
Methods: The assay was established in pooled human epidermal progenitor keratinocytes (HPEKs), isolated from adult healthy donors. Cells were seeded in 6-well plates and cultured in serum-free cell culture medium without cortisol for 2 days before treatment with different stress inducers (CRF, CRF1 agonist stressin I, norepinephrine, cortisol) at various concentrations for different periods to simulate acute (6 hours) and chronic psychological stress (2 and 4 days). Supernatants were harvested at each time point and analyzed for IL-1β, IL-8 and IL-6 by ELISA to assess potential biomarkers of the stress response. After identification of the most robust biomarker and the most effective concentration for each stressor, the experiment was repeated to test for reproducibility. In addition, RNA was isolated from the cells after 2 and 4 days of treatment with 100 nM stressin I, 0.5 µM or 1 µM norepinephrine to quantify NF-ĸB gene expression by qPCR.
Results: Whereas the secretion of IL-8 and IL-1β was less pronounced and consistent, IL-6 was identified as a reliable biomarker in this model. IL-6 secretion was induced when HPEKs were incubated for 6 hours with different concentrations of the CRF1 agonist stressin I or norepinephrine. CRF only had a modest effect on IL-6 expression, whereas cortisol downregulated IL-6 levels in this setup. Under chronic stress conditions (2 or 4 days of treatment), cells showed enhanced IL-6 secretion after treatment with 100 nM stressin I or different concentrations of norepinephrine or CRF for 2 days. After 4 days, cells responded with increased IL-6 levels for all compounds tested. A repetition of the assay using the most effective concentrations of each stressor demonstrated its reproducibility. Chronic stress induced by treatment with 1 µM norepinephrine or 100 nM stressin I led to a significant increase of IL-6 secretion by up to 150% or 78%, respectively, compared to the control after 4 days of treatment. In addition, gene expression of NF-ĸB was strongly upregulated under the same conditions.
Discussion and Conclusion: Overall, the established in vitro model clearly demonstrates the function of the CRF1 agonist stressin I and norepinephrine as stress inducers to simulate psychological stress in HPEKs. IL-6, a well-known stress marker, and NF-ĸB, a transcription factor that is upregulated during stress and inflammation, have been identified as valuable biomarkers in this context. This reproducible model can be used for efficacy screening of molecules in order to identify modulators of psychological stress in the skin. Thus, it represents a valuable model for the development of a new generation of neurocosmetic active ingredients.