Linking skin barrier improvement to underlying molecular mechanisms using a multiOMICS approach.
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Presented by: Eric Arbey
Intro :
The skin, the human body's largest organ, provides a protective barrier against multiple threats : penetration of molecules, water loss, harmful effect of environmental challenges (i.e: UV, ozone). Additionally, it enables the detection of signals such as temperature through its sensory receptors and contains immune cells enabling the detection and responses to infections. Skin barrier defects are associated with chronic inflammatory conditions such as atopic dermatitis and psoriasis. To understand the molecular mechanisms associated with improved skin barrier function, we undertook a multiOMICS approach on a human reconstructed skin model whose barrier function was modulated using a mix of lipids.
Methods :
The SkinEthicTM RHE model was grown in culture media containing either ethanol (vehicle) or a mix of lipids: free fatty acid + cholesterol + Vitamin E (=FBA mix) for 17 days.
Functional analyses of barrier properties were carried out via trans epithelial electrical resistance measurements (TEER, EVOM2 device), permeability to Bodipy®FL and caffeine, and mechanical properties of the stratum corneum.
Untargeted bottom-up proteomics, using a label free approach on a FusionTM mass spectrometer, was undertaken on total cell extracts of RHE treated with either the vehicle (ethanol) or the FBA mix. Targeted lipidomics approach on free fatty acids, free ceramides, bound ceramides, and cholesterol extracted from models was performed by mass spectrometry using a Q-TRAP instrument.
Results :
An overall improvement in skin quality and barrier function was observed after systemic FBA mix treatment. A reduction in barrier permeability to chemicals (Bodipy®FL and caffeine) through the addition of a FBA mix in the culture media was demonstrated. Moreover, TEER measurements confirmed an improved efficacy of tight junctions and cellular barrier. The stratum corneum was shown to be softened and closer to native ex-vivo stratum corneum.
Analysis of over 4000 molecular entities was performed through a targeted skin lipidomics and an untargeted proteomics approaches. Differential analysis between reconstructed skin model with low or high cellular barrier permeability revealed a very specific molecular signature. Quantities of ceramides bound to proteins were found significantly increased (2x in the low cellular barrier permeability model) with less impact on quantities of enzyme responsible for their protein binding (Tranglutaminase I) and of their synthesis . More precisely, differential analysis focusing on enzymes significantly impacted by barrier integrity change reveals a cytochrome 4F protein which was found up regulated by 2.75 fold (p=1.68e-5) in the model associated with improved barrier integrity. This Cytochrome 4F activity could overlap with eLOX3, a key enzyme in the pathway of bound ceramides.
Conclusion :
MultiOMICS analysis of a reconstructed skin model treated with the FBA mix improved barrier function and allowed us to establish a correlation between an increase in ceramides bound to proteins with improved functionals skin barrier readouts. The main known enzymes associated with lipids/ ceramides synthesis were not found to be differentially expressed. By contrast, a new cytochrome 4F was found to be up regulated by the treatment, with an activity potentially overlapping those of eLOX3, an established player in the synthesis of corneocytes bound ceramides.
The skin, the human body's largest organ, provides a protective barrier against multiple threats : penetration of molecules, water loss, harmful effect of environmental challenges (i.e: UV, ozone). Additionally, it enables the detection of signals such as temperature through its sensory receptors and contains immune cells enabling the detection and responses to infections. Skin barrier defects are associated with chronic inflammatory conditions such as atopic dermatitis and psoriasis. To understand the molecular mechanisms associated with improved skin barrier function, we undertook a multiOMICS approach on a human reconstructed skin model whose barrier function was modulated using a mix of lipids.
Methods :
The SkinEthicTM RHE model was grown in culture media containing either ethanol (vehicle) or a mix of lipids: free fatty acid + cholesterol + Vitamin E (=FBA mix) for 17 days.
Functional analyses of barrier properties were carried out via trans epithelial electrical resistance measurements (TEER, EVOM2 device), permeability to Bodipy®FL and caffeine, and mechanical properties of the stratum corneum.
Untargeted bottom-up proteomics, using a label free approach on a FusionTM mass spectrometer, was undertaken on total cell extracts of RHE treated with either the vehicle (ethanol) or the FBA mix. Targeted lipidomics approach on free fatty acids, free ceramides, bound ceramides, and cholesterol extracted from models was performed by mass spectrometry using a Q-TRAP instrument.
Results :
An overall improvement in skin quality and barrier function was observed after systemic FBA mix treatment. A reduction in barrier permeability to chemicals (Bodipy®FL and caffeine) through the addition of a FBA mix in the culture media was demonstrated. Moreover, TEER measurements confirmed an improved efficacy of tight junctions and cellular barrier. The stratum corneum was shown to be softened and closer to native ex-vivo stratum corneum.
Analysis of over 4000 molecular entities was performed through a targeted skin lipidomics and an untargeted proteomics approaches. Differential analysis between reconstructed skin model with low or high cellular barrier permeability revealed a very specific molecular signature. Quantities of ceramides bound to proteins were found significantly increased (2x in the low cellular barrier permeability model) with less impact on quantities of enzyme responsible for their protein binding (Tranglutaminase I) and of their synthesis . More precisely, differential analysis focusing on enzymes significantly impacted by barrier integrity change reveals a cytochrome 4F protein which was found up regulated by 2.75 fold (p=1.68e-5) in the model associated with improved barrier integrity. This Cytochrome 4F activity could overlap with eLOX3, a key enzyme in the pathway of bound ceramides.
Conclusion :
MultiOMICS analysis of a reconstructed skin model treated with the FBA mix improved barrier function and allowed us to establish a correlation between an increase in ceramides bound to proteins with improved functionals skin barrier readouts. The main known enzymes associated with lipids/ ceramides synthesis were not found to be differentially expressed. By contrast, a new cytochrome 4F was found to be up regulated by the treatment, with an activity potentially overlapping those of eLOX3, an established player in the synthesis of corneocytes bound ceramides.