3D Model of Human Epidermis Exposed to Staphylococci Reveals Keratinocyte Responses Unique to Atopy-Associated S. aureus Challenge

Keratinocytes are crucial for skin barrier function, but their responses to different bacteria are not well understood. Here, we present a high-throughput analysis of the transcriptome of reconstituted human epidermis (Figure S1) exposed to two ubiquitous yet distinctly different bacterial skin colonizers: the prototypical commensal Staphylococcus epidermidis and the opportunistic pathogenic Staphylococcus aureus [1].

Using NGS of human epidermal-like tissue from three independent experiments, we observed a partially common, yet overall highly divergent, transcriptome program in keratinocytes in response to these bacteria (Figure 1A). Several genes, such as those involved in keratinization and the formation of the epidermal barrier (e.g., LCE2D) or immune response (e.g., IL20), were shared between keratinocytes treated with S. epidermidis and S. aureus, suggesting converging mechanisms in how keratinocytes respond to Staphylococci (see Figure S2 for a comparison of S. epidermidis or S. aureus vs. the vehicle control).

However, a higher number of genes were altered in keratinocytes by S. aureus compared to S. epidermidis (Figure 1B), with the majority of genes showing downregulation in response to S. aureus (Figure 1A). These downregulated genes were functionally enriched for several interdependent processes, including the formation of the cornified envelope, water homeostasis, and lipid metabolism (Figure 1C). Importantly, housekeeping genes were not downregulated by S. aureus, indicating that the reconstituted epidermis maintained its essential biological properties.

Skin colonization by S. aureus is commonly associated with atopic dermatitis (AD), where the bacteria can exacerbate disease symptoms [2]. Among the top 30 genes most significantly altered by S. aureus in the epidermal model, those associated with lipid metabolism and/or lipid signaling—referred to here as lipid-related (LR) genes—were predominant (Figure 1A purple).

Given the known genetic variation between S. aureus isolates from the skin of AD individuals [3, 4], we investigated whether the keratinocyte response is S. aureus strain-dependent. The majority of S. aureus isolates from AD skin are methicillin-sensitive (MSSA) [3]. All tested MSSA strains similarly reduced PSAPL1, DGAT2, PLA2G2F, HSD11B1, SMPD3, and ELOVL3 expression in keratinocytes, suggesting that the global alterations in LR gene expression were specific to the species of S. aureus rather than its strains (Figure 2A).

In contrast to live bacteria, S. aureus-conditioned media did not affect the lipid-associated cutaneous transcriptome, indicating that S. aureus does not act merely as a source of secretory stimulating factors.

We also demonstrate a lack of association between the extent of biofilm formation by S. aureus and changes in LR gene expression (Figure 2A and Figure S3). Similarly, our findings suggest that typical virulence factors, such as serine protease V8 and cysteine proteases (staphopains), which are disabled in the sspABC and agr mutants, are not required for the alteration of LR gene expression profiles in keratinocytes (Figure 2A and Figure S3).

Finally, although the in vitro 3D epidermis model is a simplified representation compared to the multicellular changes observed in AD, the genes regulated by S. aureus in keratinocytes were found by RT-qPCR to be relevant to the global features of epidermal alterations in AD. Notably, their reduced expression was observed in AD and partially correlated with S. aureus skin colonization (Figure 2B).

The lipid barrier in the epidermis of patients with AD undergoes significant changes, characterized by a decrease in long-chain ceramides, sphingomyelins, and lysophosphatidylcholines compared to that in healthy individuals [5, 6]. Our unbiased approach identified global transcriptome changes in an epidermal model challenged with Staphylococci, consistent with previously demonstrated S. aureus-driven changes in keratinocyte ELOVL3 expression [2]. Additionally, we uncovered LR genes not previously associated with AD or S. aureus skin colonization in AD patients, such as PSAPL1, SMPD3, and HSD11B1 (Figures 1 and 2), which may serve as potential diagnostic markers for AD.

Together, these data suggest that S. aureus contributes to skin pathologies such as AD potentially by influencing lipid content, structure, and/or lipid signaling in the epidermis through the regulation of relevant genes.

P.B., J.C.: conceptualization; P.B., M.K., A.M.: methodology; P.B., M.C., M.T., A.M.: investigation; J.C., P.B.: writing – original draft; P.B., M.C., M.T., A.M., M.K., M.P., P.S., M.M., J.C.: review and editing; J.C.: funding acquisition; M.P., P.S., M.M.: resources; M.K., M.P., and J.C.: supervision.

The authors declare no conflicts of interest.