ABSTRACT
The skin is a natural barrier between the body and the environment and is colonised by a large number of microorganisms. Here, we report a complete analysis of the response of human skin explants to microbial stimuli. Using this ex vivo model, we analysed at both the gene and protein level the response of epidermal cells to Staphylococcus epidermidis (S. epidermidis) and Pseudomonas fluorescens (P. fluorescens), which are present in the cutaneous microbiota. We showed that both bacterial species affect the structure of skin explants without penetrating the living epidermis. We showed by real-time quantitative polymerase chain reaction (qPCR) that S. epidermidis and P. fluorescens increased the levels of transcripts that encode antimicrobial peptides (AMPs), including human ß defensin (hBD)2 and hBD3, and the pro-inflammatory cytokines interleukin (IL)-1α and (IL)-1-ß, as well as IL-6. In addition, we analysed the effects of bacterial stimuli on the expression profiles of genes related to innate immunity and the inflammatory response across the epidermal layers, using laser capture microdissection (LCM) coupled to qPCR. We showed that AMP transcripts were principally upregulated in suprabasal keratinocytes. Conversely, the expression of pro-inflammatory cytokines was upregulated in the lower epidermis. These findings were confirmed by protein localisation using specific antibodies coupled to optical or electron microscopy. This work underscores the potential value of further studies that use LCM on human skin explants model to study the roles and effects of the epidermal microbiota on human skin physiology.
Subject(s)
Antimicrobial Cationic Peptides/metabolism , Cytokines/metabolism , Epidermis/microbiology , Adult , Aged , Biopsy , Epidermis/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Humans , Immunohistochemistry , In Vitro Techniques , Inflammation , Keratinocytes/microbiology , Middle Aged , Polymerase Chain Reaction , Pseudomonas fluorescens/metabolism , Staphylococcus epidermidis/metabolism , Young AdultABSTRACT
The barrier function of the skin is largely due to the stratum corneum which is essentially composed of lipids. Different external factors, such as UV irradiation, affect this skin layer and are responsible for a destabilization of the supramolecular organization of its constituted lipids. In this work, mass spectrometry and infrared spectroscopy are combined to study the correlation between the formation of oxidative compounds by UV irradiation and the lipid organization. Experiments were carried out on unsaturated lipids in film or solution form, exposed to UVA or UVB irradiation. UV exposure leads to the formation of oxygenated entities in the case of lipids with an unsaturated fatty acid moiety, resulting in a decrease in their packing which is greater when the lipids are in solution. The packing decrease is even greater following UVB irradiation.
