Staphylococcus epidermidis isolates from atopic or healthy skin have opposite effect on skin cells: potential implication of the AHR pathway modulation

Abstract

Introduction: Staphylococcus epidermidis is a commensal bacterium ubiquitously present on human skin. This species is considered as a key member of the healthy skin microbiota, involved in the defense against pathogens, modulating the immune system, and involved in wound repair. Simultaneously, S. epidermidis is the second cause of nosocomial infections and an overgrowth of S. epidermidis has been described in skin disorders such as atopic dermatitis. Diverse isolates of S. epidermidis co-exist on the skin. Elucidating the genetic and phenotypic specificities of these species in skin health and disease is key to better understand their role in various skin conditions. Additionally, the exact mechanisms by which commensals interact with host cells is partially understood. We hypothesized that S. epidermidis isolates identified from different skin origins could play distinct roles on skin differentiation and that these effects could be mediated by the aryl hydrocarbon receptor (AhR) pathway.

Methods: For this purpose, a library of 12 strains originated from healthy skin (non-hyperseborrheic (NH) and hyperseborrheic (H) skin types) and disease skin (atopic (AD) skin type) was characterized at the genomic and phenotypic levels.

Results and discussion: Here we showed that strains from atopic lesional skin alter the epidermis structure of a 3D reconstructed skin model whereas strains from NH healthy skin do not. All strains from NH healthy skin induced AhR/OVOL1 path and produced high quantities of indole metabolites in co-culture with NHEK; especially indole-3-aldehyde (IAld) and indole-3-lactic acid (ILA); while AD strains did not induce AhR/OVOL1 path but its inhibitor STAT6 and produced the lowest levels of indoles as compared to the other strains. As a consequence, strains from AD skin altered the differentiation markers FLG and DSG1. The results presented here, on a library of 12 strains, showed that S. epidermidis originated from NH healthy skin and atopic skin have opposite effects on the epidermal cohesion and structure and that these differences could be linked to their capacity to produce metabolites, which in turn could activate AHR pathway. Our results on a specific library of strains provide new insights into how S. epidermidis may interact with the skin to promote health or disease.

Keywords: S. epidermidis; aryl hydrocarbon receptor; biofilms; indoles; inflammation; proteases; skin microbiome; skin type.

Figure 1

Phylogenetic tree of the 11 core genomes of S. epidermidis using strain core genome. The tree was generated using FastTree v2.1.11 (43).

Figure 2

All S. epidermidis strains tested have the capacity to form biofilm. The biofilms formed on polystyrene were observed after 3 days of culture by confocal microscopy, before and after 5 washes (A) and their biovolumes were measured (C). Moreover, the ATCC 12228 can form biofilm on 3D reconstructed skin 2 days after inoculation of 2.10⁵ CFU/HSE (B). For statistical comparisons, (*) indicates comparison of all versus all samples grouped by number of washes, **p <0.01.

Figure 3

Live S. epidermidis from atopic skin can have a strong protease activity compared to strains from normal and hyperseborrheic skin. Average protease activity of the 3 groups of strains against gelatin-like (A) and elastase-like (B) measured in the supernatant of S. epidermidis cultured for 20h. (C) Average, for each skin type group, of the protease EcpA mRNA levels (normalized to GyrB) expressed after a co-culture of S. epidermidis and keratinocytes for 14h. For statistical comparisons, (*) indicates comparison of all versus all samples, *p <0.05.

Figure 4

Live S. epidermidis effect on keratinocytes depends on skin type origin. (A) Average, for each skin type group of S. epidermidis, of the cytotoxicity and (B) IL-8 secretion in NHEK 2D co-culture and (C) IL-8 secretion in 3D reconstructed skin model after colonization. (D) HES images of tissues colonized with representative strains of the three skin types origins. Arrows indicate bacteria (B). For statistical comparisons, (*) indicates comparison of all versus all samples, *p <0.05, **p<0.01, ****p<0.001.

Figure 5

Live S. epidermidis can induce AhR pathway depending on skin type origin. Average of mRNA levels of AhR pathway genes CYP1A1 (A), OVOL1 (B), STAT6 (C) and average of mRNA levels of FLG1 (D) and DSG1 (E), for each skin type group of S. epidermidis. For statistical comparisons, (*) indicates comparison of all versus all samples, *p <0.05, **p<0.01, ***p<0.005, ****p<0.001.

Figure 6

S. epidermidis produce indoles depending on skin type origin. (A) Sum of indoles (tryptamine + IAld + ILA + IAA) measured in the supernatant of S. epidermidis ATCC 12228, S. epidermidis from normal, hyperseborrheic or atopic skin type; quantification of Iald (B) and ILA (C) released in the culture medium. (D) Sum of indoles measured in the co-culture medium of NHEK and S. epidermidis ATCC 12228, S. epidermidis from normal, hyperseborrheic or atopic skin type; quantification of Iald (E) and ILA (F). LB, Live bacteria; SN, supernatant. For statistical comparisons, (*) indicates comparison of all versus all samples, *p <0.05, **p<0.01, ***p<0.005.

Figure 7

(A): Reconstruction of tryptophan metabolic pathway in the 11 S. epidermidis and ATCC 12228, based on the genome sequences. Circles represent each strains: light blue ATCC 12228; green: non hyperseborrheic skin; orange: hyperseborrheic skin; dark blue: AD strains. Open circle means that the protein is not found. IAA, Indole Acetic Acid; IA, Indole-3-acetate; IAld, Indole-3-aldehyde; ILA, Indole-3-Lactic Acid. (B, C) Kynurenine metabolite quantification in the 11 S. epidermidis and the reference strain ATCC 12228 culture supernatant, after NHEK treatments with either living bacteria (LB) or bacterial supernatant (SN). Quantities are presented per group of skin type. For statistical comparisons, (*) indicates comparison of all versus all samples, *p <0.05, **p<0.01.

Figure 8

Graphical abstract. Effect of S. epidermidis on keratinocytes depends on skin type of origin of the bacteria. Strains from normal skin induce AhR/OVOL1 pathway via production of indoles while atopic strains produce low amounts of indoles and an overexpression of STAT6 resulting in a strong decrease of OVOL1 expression compared to normal and hyperseborrheic strains inductions. Moreover, strains from atopic skin induce tissue structure damages correlated with a strong protease activity and a decrease of DSG1. The thickness of the arrows represents the intensity of the induced signal, the dotted arrows represent links previously described in the literature but not verified in this study.