Life stage impact on the human skin ecosystem: lipids and the microbial community

Abstract

Sebaceous free fatty acids are metabolized by multiple skin microbes into bioactive lipid mediators termed oxylipins. This study investigated correlations between skin oxylipins and microbes on the superficial skin of pre-pubescent children (N = 36) and adults (N = 100), including pre- (N = 25) and post-menopausal females (N = 25). Lipidomics and metagenomics revealed that Malassezia restricta positively correlated with the oxylipin 9,10-DiHOME on adult skin and negatively correlated with its precursor, 9,10-EpOME, on pre-pubescent skin. Co-culturing Malassezia with keratinocytes demonstrated a link between 9,10-DiHOME and pro-inflammatory cytokines IL-1β and IL-6 production. We also observed strong correlations between other skin oxylipins and microbial taxa, highlighting life stage differences in sebum production and microbial community composition. Our findings imply a complex host-microbe communication system mediated by lipid metabolism occurring on human skin, warranting further research into its role in skin health and disease and paving the way towards novel therapeutic targets and treatments.

Fig. 1. Experimental workflow of targeted lipidomics, metagenomics, and correlational analysis of skin lipid mediator profiles, skin microbiome compositions, and in vitro mono- and co-cultures.

Microbial biomass and lipid samples were acquired from the cheek skin of pre-pubescent (GUSTO) and adult (HELIOS) subjects. Post-menopausal and pre-menopausal woman were classified based on age. Skin lipid samples were analysed using targeted LC-MS/MS. Skin microbial samples were analysed using shotgun metagenomics. Culture supernatants of keratinocyte and Malassezia monocultures and keratinocyte-Malassezia co-cultures were analysed using targeted LC-MS/MS for lipid mediators and ELISA for cytokines. Correlational analyses were performed for lipidomic/metagenomic and lipidomic/cytokine data. Figure created with BioRender.com.

Fig. 2. Concentrations of lipid mediators between pre-pubescent and adult skin are different.

Skin tape strips of the cheek from the HELIOS and GUSTO cohorts were analysed using targeted mass spectrometry, in which eight unique oxylipins and a single lipid amide were identified. Red and blue heat-map tiles indicate higher and lower lipid concentrations than the mean. The Euclidean method was used to calculate the distance measures between samples and the Ward method was used for clustering. The data was analysed in MetaboAnalyst using lipid data in pg/cm².

Fig. 3. The adult and pre-pubescent skin microbiome profiles are distinctly different compared to the skin lipid mediator profiles.

PCA plots based on the (A) nine lipid mediators or the (B) 370 microbial species. Red represents the pre-pubescent (GUSTO; N = 34) and blue represents the adult (HELIOS; N = 100) cohorts. Median relative abundance differences in the top 15 commonly abundant species between (C) pre-pubescent and adult subjects, and between (D) pre-menopausal (N = 25) and post-menopausal (N = 25) women. *Human papillomavirus type 134 was found only on one adult subject at high abundance.

Fig. 4. In vitro oxylipin production by Malassezia, human keratinocytes, and co-cultures.

A Schematic of the co-culture conditions: (I) human immortalized keratinocytes (N/TERT-1) monoculture, (II) N/TERT-1 co-cultures with four different Malassezia spp., (III) Malassezia monocultures, and (IV) mock culture only containing media. Light microscopy pictures show mono- and co-culture conditions between N/TERT-1 and M. globosa. B LC-MS/MS analysis of oxylipins in culture supernatants after 24 h of culture at 37 ^oC and 5% CO₂. Red, white, and blue colours on the heatmap indicates high, medium, and low oxylipin concentration or difference in concentration for a specific oxylipin column. To determine oxylipin concentration differences between mono- and co-cultures, the mean difference was calculated for the three biological replicates and a two-way ANOVA with post hoc Tukey’s test was performed. Comparisons with significant concentration difference are indicated in bold numerals.

Fig. 5. Correlations between cytokines produced by human keratinocytes and oxylipins in vitro.

A Concentration of cytokines produced by N/TERT-1 mono- and co-culture with M. globosa, M. restricta, M. furfur, and M. sympodialis. No cytokines were detected in Malassezia monocultures (data not shown). Red, white, and blue colours on the heatmap indicates high, medium, and low cytokine concentration or difference in concentration for a specific cytokine column. To determine cytokine concentration differences between mono- and co-cultures, the mean difference was calculated for the three biological replicates and a two-way ANOVA with post hoc Dunette’s test was performed using ‘N/TERT-1 only’ condition as control. Comparisons with significant concentration difference are indicated in bold numerals. B Correlations between specific cytokines and oxylipins. Correlations were assessed using Kendall’s Tau b with 5% false discovery rate (FDR) correction. Red indicates positive correlation while blue indicates negative correlation. Correlations with less than 5% FDR are highlighted within teal borders. The correlation strength of ± 0.30 or above is classified as strong.