World’s first human ex-vivo acne sebaceous gland model colonized with virulent phylotype of C. acnes
Podium 19
Presented by: Nico Forraz
INTRODUCTION: Unhealthy skin conditions such as acne-prone skin is associated with skin microbiota dysbiosis. Cutibacterium acnes (C. acnes) and Staphylococcus epidermidis (S. epidermidis) are amongst the normal skin flora components, but differences in their phylotypes, particularly C. acnes, may lead to competitive antimicrobial differences or over colonization, particularly in Sebaceous Glands (SG), and overproduction of sebum linked to acne-prone skin. Due to the lack of cosmetic strategies in association with the microbial component, we describe here our new accurate sebaceous gland model colonized with C. acnes and/or S. epidermidis.
METHODS: Human primary sebocytes were co-cultivated with 1.106 UFC/ml C. acnes Type 1A (facial acne, ATCC6919) and/or 1.105 UFC/ml S. epidermidis (ATCC12228) for 48H in our SEB4GLN optimized media without antibiotics. Bacteria and sebocyte viabilities were evaluated by numeration on geloses or with Alamar Blue reagent respectively. Lipids production was quantified by microscopy using Nile Red staining. Sebaceous glands with hair follicles were microdissected on a Leica stereomicroscope from healthy skin of the back, placed in culture with our SEB4GLN culture media on fibronectin-coated slides at the air-liquid interface and then inoculated with 1.105 UFC/ml C. acnes Type 1A and/or 1.104 UFC/ml S. epidermidis (wild strain previously isolated from a gland) sequentially. After 48 hours, supernatants were collected and seeded on specific geloses to quantify bacteria. Sebaceous glands were then fixed and labelled with antibodies to investigate morphology (MC5R) and bacterial colonization (C. acnes) together with nuclear staining (DAPI) and analysed by Z-stack confocal microscopy (Leica SP5X) to evaluate at micrometer level. 3-dimensional reconstructions were performed with all fluorescent channels using FIJI software.
RESULTS: C. acnes growth was not impacted when co-cultivated with sebocytes in our SEB4GLN medium. Phylotype IA had the tendency to stimulate sebocyte lipids production without impacting their viability. S. epidermidis overproliferated and induced sebocytes mortality which led us to decrease its inoculum concentration, its contact time and adding a rinse step after its inoculation on sebaceous glands. Control glands (after dissection and without inoculation) demonstrated a low natural level of C. acnes and S. epidermidis. No harmful effects were observed on the two inoculated strains, which validated our co-cultivation conditions. C. acnes was detected by immunofluorescence on the glands by 48 hours, alone and when co-inoculated sequentially with S. epidermidis. C. acnes was strongly located in the active areas of both sebaceous glands and the hair follicles, with a higher concentration in the central areas of glands. S.epidermidis was imaged by non-specific fluorescence and located more globally across the entire gland.
DISCUSSION AND CONCLUSIONS: We have developed an innovative model of sebaceous gland that mimics acne-prone skin with lipid overproduction and virulent phylotype C. acnes inoculation. Moreover, the specific localization of C. acnes to the active regions of glands and hair follicles is new and of great importance to allow for accurate research. This model will allow us to develop actives in skin dysbiosis conditions, but also investigate skin microbiota, which is of increasing importance in cosmetics strategies.
METHODS: Human primary sebocytes were co-cultivated with 1.106 UFC/ml C. acnes Type 1A (facial acne, ATCC6919) and/or 1.105 UFC/ml S. epidermidis (ATCC12228) for 48H in our SEB4GLN optimized media without antibiotics. Bacteria and sebocyte viabilities were evaluated by numeration on geloses or with Alamar Blue reagent respectively. Lipids production was quantified by microscopy using Nile Red staining. Sebaceous glands with hair follicles were microdissected on a Leica stereomicroscope from healthy skin of the back, placed in culture with our SEB4GLN culture media on fibronectin-coated slides at the air-liquid interface and then inoculated with 1.105 UFC/ml C. acnes Type 1A and/or 1.104 UFC/ml S. epidermidis (wild strain previously isolated from a gland) sequentially. After 48 hours, supernatants were collected and seeded on specific geloses to quantify bacteria. Sebaceous glands were then fixed and labelled with antibodies to investigate morphology (MC5R) and bacterial colonization (C. acnes) together with nuclear staining (DAPI) and analysed by Z-stack confocal microscopy (Leica SP5X) to evaluate at micrometer level. 3-dimensional reconstructions were performed with all fluorescent channels using FIJI software.
RESULTS: C. acnes growth was not impacted when co-cultivated with sebocytes in our SEB4GLN medium. Phylotype IA had the tendency to stimulate sebocyte lipids production without impacting their viability. S. epidermidis overproliferated and induced sebocytes mortality which led us to decrease its inoculum concentration, its contact time and adding a rinse step after its inoculation on sebaceous glands. Control glands (after dissection and without inoculation) demonstrated a low natural level of C. acnes and S. epidermidis. No harmful effects were observed on the two inoculated strains, which validated our co-cultivation conditions. C. acnes was detected by immunofluorescence on the glands by 48 hours, alone and when co-inoculated sequentially with S. epidermidis. C. acnes was strongly located in the active areas of both sebaceous glands and the hair follicles, with a higher concentration in the central areas of glands. S.epidermidis was imaged by non-specific fluorescence and located more globally across the entire gland.
DISCUSSION AND CONCLUSIONS: We have developed an innovative model of sebaceous gland that mimics acne-prone skin with lipid overproduction and virulent phylotype C. acnes inoculation. Moreover, the specific localization of C. acnes to the active regions of glands and hair follicles is new and of great importance to allow for accurate research. This model will allow us to develop actives in skin dysbiosis conditions, but also investigate skin microbiota, which is of increasing importance in cosmetics strategies.