Staphylococcus aureus causes aberrant epidermal lipid composition and skin barrier dysfunction

Abstract

Background: Staphylococcus (S) aureus colonization is known to cause skin barrier disruption in atopic dermatitis (AD) patients. However, it has not been studied how S. aureus induces aberrant epidermal lipid composition and skin barrier dysfunction.

Methods: Skin tape strips (STS) and swabs were obtained from 24 children with AD (6.0 ± 4.4 years) and 16 healthy children (7.0 ± 4.5 years). Lipidomic analysis of STS samples was performed by mass spectrometry. Skin levels of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA) were evaluated. The effects of MSSA and MRSA were evaluated in primary human keratinocytes (HEKs) and organotypic skin cultures.

Results: AD and organotypic skin colonized with MRSA significantly increased the proportion of lipid species with nonhydroxy fatty acid sphingosine ceramide with palmitic acid ([N-16:0 NS-CER], sphingomyelins [16:0-18:0 SM]), and lysophosphatidylcholines [16:0-18:0 LPC], but significantly reduced the proportion of corresponding very long-chain fatty acids (VLCFAs) species (C22-28) compared to the skin without S. aureus colonization. Significantly increased transepidermal water loss (TEWL) was found in MRSA-colonized AD skin. S. aureus indirectly through interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-33 inhibited expression of fatty acid elongase enzymes (ELOVL3 and ELOVL4) in HEKs. ELOVL inhibition was more pronounced by MRSA and resulted in TEWL increase in organotypic skin.

Conclusion: Aberrant skin lipid profiles and barrier dysfunction are associated with S. aureus colonization in AD patients. These effects are attributed to the inhibition of ELOVLs by S. aureus-induced IL-1β, TNF-α, IL-6, and IL-33 seen in keratinocyte models and are more prominent in MRSA than MSSA.

Keywords: Staphylococcus aureus; atopic dermatitis; ceramides; elongase; skin barrier.

FIGURE 1

Transepidermal water loss and skin lipid profiles in subjects according to the colonization of S. aureus. TEWL (A), N(C18S)-CER (B), SM (C), and LPC (D) of stratum corneum in normal subjects and non-lesional and lesional skin from patients with atopic dermatitis. TEWL was measured using a device from GPower Inc. (Seoul, Korea). Each lipid molecular species was quantified by targeted liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and normalized by sample total protein content; the data were expressed as relative percentage within each lipid subclass. Data are presented as box-and-whisker plot, with whiskers showing 10th and 90th percentile values. All data are shown as the median and interquartile range. no SA (n = 21), MSSA (n = 29), MRSA (n = 14). *p < .05, **p < .01 by the Kruskal–Wallis test with the post hoc test. LPC, lysophosphatidylcholine; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive Staphylococcus aureus; N(C18S)-CER, ceramides with C18 sphingosine as their sphingoid base and nonhydroxy fatty acids; SM, sphingomyelin; TEWL, transepidermal water loss.

FIGURE 2

Transepidermal water loss and lipid profiles in organotypic skin cultures stimulated with S. aureus. TEWL (A), N(C18S)-CER (B), SM (C), and LPC (D) in organotypic skin cultures stimulated with three different strains of MSSA or MRSA. TEWL was measured using a device from GPower Inc., Seoul, Korea (A). Each lipid molecular species was quantified by targeted liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and normalized by sample total protein content, and data were expressed as relative percentage within each lipid subclass (B, C, D). Data are presented as box-and-whisker plot, with whiskers showing minimum and maximum values. Composite data are shown from three independent experimental repetitions using three different lots of HEKs. The data are shown as the median and interquartile range. n = 8 per group. *p < .05, **p < .01, ***p < .001 by the Kruskal–Wallis test with the post hoc test. C18-NS-CER, C18 sphingosine as their sphingoid base and nonhydroxy fatty acids; LPC, lysophosphadtidylcholine; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive Staphylococcus aureus; SM, sphingomyelins; TEWL, transepidermal water loss.

FIGURE 3

Expression of ELOVLs in human primary keratinocytes and organotypic skin cultures stimulated with Staphylococci. Gene expression of ELOVLs in organotypic skin cultures stimulated with S. epidermidis (A), MSSA (B), and MRSA (C). Data are representative of three independent experimental repetitions using three different lots of HEKs S. epidermidis, MSSA, and MRSA, respectively. n = 9 per group. Immunostaining intensities of ELOVL3 and ELOVL4 in organotypic skin cultures stimulated with MRSA (D). Arrows point to ELOVL3 and ELOVL4 staining (red). Wheat germ agglutinin-conjugated FITC (green) was used to stain the cytoskeleton. DAPI stained nuclei (blue). n = 3. Scale bar = 50 μm. All data are shown as the median and interquartile range. *p < .05, **p < .01, ***p < .001 by the Kruskal–Wallis test with the post hoc test. ELOVL, elongation of very long chain fatty acids protein; MFI, mean fluorescence intensity; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive Staphylococcus aureus.

FIGURE 4

Expression of keratinocyte-derived cytokines in human primary keratinocytes stimulated with Staphylococci. HEKs were Ca2+ differentiated and stimulated with three different strains of S. epidermidis, MSSA or MRSA for 8 or 20 h. Gene expression of keratinocyte-derived cytokines in HEKs stimulated with MSSA (A) and MRSA (B) was evaluated by RT-PCR. n = 27 per group. Protein levels of TNF-α (C) and IL-1β (D) in HEKs culture media were measured by ELISA. n = 4 per group. Data are representative of three independent experimental repetitions using three different lots of HEKs. All data are shown as the median and interquartile range. *p < .05, **p < .01, ***p < .001, ****p < .0001 by the Kruskal–Wallis test with the post hoc test. MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive Staphylococcus aureus; RT-PCR, reverse transcription-polymerase chain reaction.

FIGURE 5

Expression of ELOVL3 and ELOVL4 in human primary keratinocytes stimulated with various cytokines. Gene expression of ELOVL3 and ELOVL4 was evaluated in HEKs stimulated with various concentrations of TNF-α (A), IL-1β (B), IL-6 (C), and IL-33 (D) by RT-PCR. Composite data are shown from three independent experimental repetitions using three different lots of HEKs. n = 9 per group. All data are shown as median and interquartile range. *p < .05, **p < .01, ***p < .001, ****p < .0001 by the Kruskal–Wallis test with the post hoc test. ELOVL, fatty acid elongase; MRSA, methicillin-resistant Staphylococcus aureus; RT-PCR, reverse transcription-polymerase chain reaction.

FIGURE 6

Expression of ELOVL3 and ELOVL4 in human primary keratinocytes stimulated with MRSA and neutralizing antibodies. HEKs were differentiated and preincubated with isotype control antibodies or neutralizing antibodies against TNF-α (0.1 μg/ml), IL-1β (10 ng/ml), IL-36γ (1.0 μg/ml), IL-6 (0.1 μg/ml), and IL-33 (1.0 μg/ml) separately or in combination (all same concentrations) for 3 h, followed by stimulation with MRSA (0.01 CFU/cell) for an additional 20 h. Gene expression of ELOVL3 (A and B) and ELOVL4 (C) was evaluated by RT-PCR. n = 4–6 per group. Data are representative of three independent experimental repetitions using three different lots of HEKs. All data are shown as median and interquartile range. *p < .05, **p < .01, ***p < .001 by the Kruskal–Wallis test with the post hoc test. ELOVL, fatty acid elongase; RT-PCR, reverse transcription-polymerase chain reaction.