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Tissue resident cells differentiate S. aureus from S. epidermidis via IL-1β following barrier disruption in healthy human skin

  • Julia C. Lang,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliations AIMES-Center for the Advancement of Integrated Medical and Engineering Sciences, Karolinska Institutet and KTH Royal Institute of Technology, Stockholm, Sweden, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

  • Andreas Brutscher,

    Roles Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing

    Affiliations AIMES-Center for the Advancement of Integrated Medical and Engineering Sciences, Karolinska Institutet and KTH Royal Institute of Technology, Stockholm, Sweden, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

  • Marcus Ehrström,

    Roles Resources, Writing – review & editing

    Affiliation Nordiska Kliniken, Stockholm, Sweden

  • Keira Melican

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    keira.melican@ki.se

    Affiliations AIMES-Center for the Advancement of Integrated Medical and Engineering Sciences, Karolinska Institutet and KTH Royal Institute of Technology, Stockholm, Sweden, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

Abstract

The Staphylococcus sp. are a dominant part of the human skin microbiome and present across the body. Staphylococcus epidermidis is a ubiquitous skin commensal, while S. aureus is thought to colonize at least 30% of the population. S. aureus are not only colonizers but a leading cause of skin and soft tissue infections and a critical healthcare concern. To understand how healthy human skin may differentiate commensal bacteria, such as S. epidermidis, from the potential pathogen methicillin-resistant S. aureus (MRSA), we use ex vivo human skin models that allow us to study this host-bacterial interaction in the most clinically relevant environment. Our work highlights the role of the outer stratum corneum as a protective physical barrier against invasion by colonizing Staphylococci. We show how the structural cells of the skin can internalize and respond to different Staphylococci with increasing sensitivity. In intact human skin, a discriminatory IL-1β response was identified, while disruption of the protective stratum corneum triggered an increased and more diverse immune response. We identified and localized tissue resident Langerhans cells (LCs) as a potential source of IL-1β and go on to show a dose-dependent response of MUTZ-LCs to S. aureus but not S. epidermidis. This suggests an important role of LCs in sensing and discriminating between bacteria in healthy human skin, particularly in intact skin and provides a detailed snapshot of how human skin differentiates between friend and potential foe. With the rise in antibiotic resistance, understanding the innate immune response of healthy skin may help us find ways to enhance or manipulate these natural defenses to prevent invasive infection.

Author summary

How healthy human skin can differentiate between commensal bacteria that live harmoniously on our skin and those bacteria that have the potential to cause invasive infection, is of great interest. Here we use ex vivo human skin models to show how the outer layer of human skin resists bacterial invasion and that disruption of this layer leads to increased infection and inflammation. We show how colonization of both intact and disrupted skin by Staphylococcus aureus causes an increased IL-1β signaling compared to the commensal S. epidermidis. We show that tissue resident Langerhans cells are well positioned throughout the skin epidermis and that MUTZ-LCs can differentiate commensal from pathogen via IL-1β. This work identifies one of the subtle, yet effective, pathways by which human skin differentiates commensals and pathogens. Understanding these mechanisms could lead to potential new treatment and prevention targets, mechanisms by which we can enhance innate immunity.

Introduction

Human skin, our largest organ, forms the first physical and immunological defense against bacteria. Paradoxically, our skin is also covered by a highly diverse and dynamic microbiome which is believed to play a direct role in protecting us from invasive bacterial pathogens [1]. The Staphylococcus sp. are a prominent feature of the human skin microbiome, with S. epidermidis, the most common coagulase negative Staphylococci (CoNS), comprising over 90% of the skin resident aerobic flora [2]. Around 20% of the human population are also colonized with the potentially pathogenic S. aureus, with another 30% being transient carriers [3]. S. aureus is responsible for 76% of all skin and soft tissue infections and numerous invasive infections [4,5]. Methicillin resistant S. aureus (MRSA) causes the highest proportion of deaths related to antibiotic resistant bacteria in the developed world [6,7]. An ongoing challenge in research is to understand how these bacteria can be both a harmless commensal and a potentially deadly pathogen. In this work we sought to understand how healthy human skin recognizes and responds to colonizing Staphylococcus, and how skin may differentiate between S. epidermidis and S. aureus. Understanding how our skin protects us in the vast majority of cases we encounter S. aureus will give greater clarity into our first line of defense and open the possibilities for enhancing or manipulating our innate protection when needed.

For many years, studying host-pathogen interaction between Staphylococcus sp. and skin has been performed using mouse models [810]. While adding great knowledge to the field, it is becoming increasingly evident that the human skin response to Staphylococcus differs significantly from mouse. Mice are not natural hosts for human Staphylococcus isolates [11] and the skin of rodents and humans have dramatically different structure and immunological function [12]. Additionally, elements of Staphylococcus pathogenesis have been shown to be human specific, including the interaction of Staphylococcal toxins with human neutrophils, and between Staphylococcus and human langerin, the receptor of skin resident Langerhans cells [13,14]. These factors highlight the importance of studying host-pathogen interactions in the most clinically relevant microenvironment, human skin [15,16]. We have developed several human skin models to address host-pathogen interaction. We previously showed in a xenografted model, where healthy human skin is grafted onto immunocompromised mice, that bacterial colonization patterns of MRSA and the inflammatory response of human skin differed significantly from previous mouse models [17]. In this current work, we have optimized explant human skin models to differentiate the response of healthy human skin to S. epidermidis vs MRSA. We combine this with cell culture to understand the role of each cell type in this early response. An advantage of our adult human skin models is that they contain the full complement of tissue resident immune cells and here we focus on Langerhans cells (LCs) and their ability to differentiate S. epidermidis vs MRSA. These skin resident immune cells are often described as a bridging element between innate and adaptive immunity and play an important role in activating specific T-cell responses [18].

Our results reveal the critical role of the human skin outer layer, the stratum corneum, in providing a physical barrier to bacterial invasion. We also demonstrate how the skin structural cells can internalize and respond to bacteria, with an increasing responsiveness as we move into deeper layers of the skin. We then delve further into the different inflammatory response to MRSA and S. epidermidis and identify Langerhans cells as a potential driver of this differentiation. This work offers a high-resolution understanding of the early phases of human skin colonization and the differentiation between friend and foe.

Results

An intact stratum corneum forms the first line of defense against Staphylococcus sp.

We previously showed in an in vivo xenograft model that the human stratum corneum is an important barrier to MRSA invasion [17]. Here we study the importance of an intact stratum corneum during colonization by commensal vs pathogenic bacteria. To do this we optimized an ex vivo tissue explant model using biopsies of fresh, healthy, human skin from multiple donors. The integrity and morphology of the skin model was validated after 72 h of ex vivo culture (S1 Fig). These full thickness biopsies contain an intact stratum corneum atop the epidermal and dermal layers. The surface of the skin was topically inoculated with ~1.6x106 CFU/cm2 S. aureus USA300 (MRSA) [19], or S. epidermidis 1457 [20]. This inoculation dose was correlated with the reported density of the human skin microbiota [21]. Following 48 h of colonization, there was significant differences in the ability of S. epidermidis or MRSA to colonize the skin, with significant higher MRSA CFUs (Fig 1A). Individual variability between donors was seen, as expected when using adult skin donors with inevitable variances in microbiota and prior exposure. In our xenograft model, we had shown an IL-8 response to skin colonization with MRSA [17] and we therefore tested for potential inflammatory differences between colonization by MRSA or S. epidermidis. No significant difference in either IL-8 (Fig 1B) or an additional inflammatory marker CXCL12 (S2A Fig) were seen between colonization with MRSA or S. epidermidis. The time frames of this ex vivo model were significantly shorter than those reported for the xenograft and we therefore evaluated the localization of bacteria at this 48 h timepoint. Confocal microscopy revealed that the majority of S. aureus USA300 expressing GFP were found adhered to the intact stratum corneum (Fig 1C). This is of importance as this outer layer is lacking from mouse models and often tape stripped away in other human models [2224]. No significant bacterial penetration into the tissue was seen, correlating to our previous work [17]. We did however locate occasional intracellular bacteria in nucleated cells of the upper epidermal layers (Fig 1D). This data reinforced the important role of the stratum corneum as the key initial binding site for Staphylococcus sp. on the skin, and the integrity of the skin barrier to protect against tissue invasion but also suggested a role for intracellular bacteria during skin colonization.

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Fig 1. Inflammatory response to colonizing Staphylococcus sp. in human skin.

(A) CFU counts of S. epidermidis 1457 and S. aureus USA300 48 h after epicutaneous colonization of ex vivo human skin. Data show means ± SD. Dots represent individual data points, colours differentiate skin donors (n = 6). Dotted line depicts inoculum. Significance determined by one-way ANOVA with Tukey’s correction for multiple comparisons. Significance is denoted by *** p ≤ 0.001 or **** p ≤ 0.0001. (B) IL-8 ELISA, as x-fold change over uninfected controls. Dots represent individual data points, colours differentiate skin donors (n = 6). Significance determined by unpaired t-test with Welch’s correction. ns = not significant (C) Representative immunofluorescence staining of cytokeratin-10 (orange), f-actin (white), nuclei (blue) in skin model topically colonized with S. aureus USA300 GFP (green) for 48 h. Scale bars = 50μm. N = 3. (D) Insert of area in (C) showing intracellular S. aureus USA300 GFP.

https://doi.org/10.1371/journal.ppat.1012056.g001

Staphylococci can be internalized by skin structural cells with differential inflammatory responses

The intracellular localization of Staphylococcus may be a means to penetrate into deeper tissue, but it has also been suggested that intracellular localization occurs in chronic wounds and recurrent infection [2527]. To investigate the role of intracellular bacteria in human skin colonization, we studied the ability of S. epidermidis and MRSA to internalize in the key structural cells of the human skin. We included primary isolated human corneocytes, human keratinocytes (HEKa) and dermal fibroblasts (HDFn). We challenged the cells with an MOI of 1:100 of both S. epidermidis or MRSA for 3 h before gentamicin-lysostaphin treatment. The cells were further incubated until 24 h p.i. Intracellular CFU was determined after 3 h (Fig 2A) and IL-8 expression after 24 h (Fig 2B). Corneocytes demonstrated no intracellular uptake or invasion of either S. epidermidis or MRSA (Fig 2A) and no expression of IL-8 was detected. As corneocytes are anuclear cells, often considered dead, the lack of response is not unexpected. In line with their abilities and functions as non-professional phagocytes, keratinocytes and fibroblasts harbored intracellular S. epidermidis and MRSA (Fig 2A). Both keratinocytes and fibroblasts contained significantly more MRSA than S. epidermidis, while the number of intracellular MRSA and S. epidermidis were significantly higher in the fibroblasts than in keratinocytes (Fig 2A). Keratinocytes displayed a low IL-8 response to both S. epidermidis and MRSA compared to untreated cells while fibroblasts displayed a significantly higher response to MRSA as compared to S. epidermidis or untreated cells (Fig 2B). The cytotoxicity of infected keratinocytes is comparable between MRSA and S. epidermidis (S3A Fig), while there was increased cytotoxicity of MRSA vs S. epidermidis in infected fibroblasts (S3B Fig). Microscopy analysis of cells infected with GFP expressing MRSA or S. epidermidis for 3 h validated these results indicating no intracellular uptake of MRSA into corneocytes (S4 Fig), while keratinocytes and fibroblasts showed distinct patterns for intracellular MRSA and S. epidermidis (Fig 2C). The keratinocytes infection was heterogenous with individual cells harboring numerous clustered MRSA, suggestive of a phagosomal localization [28]. Fibroblasts were more homogenously infected with intracellular bacteria spreading throughout the cell body (Fig 2C).

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Fig 2. Intracellular Staphylococcus sp. induce an IL-8 response in keratinocytes and fibroblasts, but not corneocytes.

(A) Intracellular CFU counts of S. epidermidis 1457 (filled circles) and S. aureus USA300 (empty circles) 3 h p.i. of tape-stripped corneocytes, human keratinocytes (HEKa) and fibroblasts (HDFn). Bars represent means ± SD, dots show individual data points (corneocytes and fibroblasts n = 3; keratinocytes n = 5; 3 technical replicates). Dotted line = initial inoculum. Significance was determined by a two-way ANOVA with Tukey’s correction for multiple comparisons. Significance is denoted by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 or **** p ≤ 0.0001. (B) IL-8 response measured by ELISA in cell culture supernatants of S. epidermidis 1457 or S. aureus USA300 infected keratinocytes and fibroblasts. Dot plot shows individual data points (fibroblasts n = 3; keratinocytes n = 5; 3 technical replicates each). Significance was determined by a two-way ANOVA with Tukey’s correction for multiple comparisons. Significance is denoted by ** p ≤ 0.01 or **** p ≤ 0.0001. Only significant differences are annotated for clarity. (C) Microscopic detection of intracellular S. aureus GFP and S. epidermidis GFP in cell monolayers of human keratinocytes (HEKa) or fibroblasts (HDFn); uninfected CTR vs 3h infection. Immunostaining of f-actin (red) and nuclei (blue) in HEKa and HDFn. Scale bars = 50μm.

https://doi.org/10.1371/journal.ppat.1012056.g002

This data demonstrates that while corneocytes are the key binding target in early skin colonization, these cells do not internalize our Staphylococcus bacteria or initiate an apparent inflammatory response. Keratinocytes and fibroblasts could both internalize Staphylococcus and generate an IL-8 inflammatory signal. Fibroblasts demonstrated a greater inflammatory response in general as well as an increased discriminatory potential between S. epidermidis and MRSA. This suggests a striated tissue response from the structural cells, graduating from non-responsive corneocytes on the outer skin surface through the keratinocyte-rich epidermis to the most responsive fibroblasts located in the skin dermis.

Disruption of the stratum corneum enhances S. aureus colonization and tissue inflammatory response

These data highlighted differences in the skin layers, with the outer most layers being non-responsive to Staphylococcus, but the structural cells located in the deeper tissue more capable of discriminating MRSA from S. epidermidis. Recent work has shown that MRSA, via V8 protease, can lead to an urge to itch skin [29]. Scratching leads to minor disruptions in the outer layers of skin and we wanted to study if these minor disruptions would trigger a more substantial inflammatory skin response. We altered our tissue model by superficially scratching the surface of the human skin before bacterial colonization (S5 Fig). This barrier disruption led to significantly higher loads of MRSA compared to S. epidermidis, with noted variation between individual skin donors (Fig 3A). Inoculation of scratched skin with MRSA led to significantly higher expression of IL-8 than inoculation with S. epidermidis (Fig 3B). Scratched skin inoculated with either MRSA or S. epidermidis had increased IL-8 compared to their donor matched non-infected controls, and for MRSA also a significant increase compared to unscratched skin (S6 Fig). Expression of CXCL12 was however not significantly altered, indicating a specificity in the IL-8 response and demonstrating differences from other recently reported mouse models [30] (S2B Fig). This data further supports the role of the stratum corneum in protecting the tissue, showing how minor disruptions in this layer leads to increasing bacterial loads as well as inflammatory signaling, particularly for MRSA.

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Fig 3. Stratum corneum disruption results in increased inflammatory response to S. aureus colonization.

(A) CFU counts of S. epidermidis 1457 and S. aureus USA300 48 h after epicutaneous colonization of biopsies with disrupted stratum corneum (s.c.). Bars = means ± SD, dots show individual data points (n = 7, skin donors indicated by colors). Dotted line depicts inoculum. Significance determined by one-way ANOVA with Tukey’s correction for multiple comparisons. Significance denoted by **** p ≤ 0.0001. (B) IL-8 response displayed as x-fold change over uninfected controls measured by ELISA in homogenates of skin biopsies with disrupted s.c. inoculated with S. epidermidis 1457 or S. aureus USA300. Dot plot shows individual data points (n = 7, skin donors indicated by colors). Significance determined by unpaired t test with Welch’s correction. Significance denoted by * p ≤ 0.05. (C-D) Cytokine expression displayed as x-fold change from uninfected controls in colonized skin explants with an intact s.c. (C) or disrupted s.c. (D) measured by LEGENDplex bead-based immunoassay. The box plots represent interquartile range (IQR) with the median shown as black line. Dots show individual data points (n = 6, 2–3 individual skin donors). Statistical significance determined by two-way ANOVA with Šídák’s correction for multiple comparisons and denoted by ** p ≤ 0. 01 and **** p ≤ 0. 0001. n.d. = not detected. Only significant differences are annotated for clarity. (E) Selected cytokines on a radar plot shown as mean x-fold changes over uninfected controls. (F) Spearman correlation between CFUs and IL-1β, IL-17A and IL-12p70 measured in human skin biopsies 48 h p.i. Significance for each cytokine compared to the CFU/ml denoted by ** p ≤ 0. 01 and **** p ≤ 0. 0001.

https://doi.org/10.1371/journal.ppat.1012056.g003

To further evaluate the inflammatory differences in scratched and unscratched skin we expanded our analysis to a panel of inflammatory cytokines including IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1, IL-6, CXCL8 (IL-8), IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33. In intact, unscratched skin, only IL-17A and IL-1β were increased above the non-infected controls (Fig 3C). Interestingly, IL-1β expression was significantly higher in MRSA inoculated tissue compared to S. epidermidis (Fig 3C). In tissues with a disrupted, scratched stratum corneum, the cytokine responses were increased for most factors (Fig 3D). Both IL-1β and IL-17A showed significantly different expression when colonized by MRSA compared to S. epidermidis (Fig 3D). For better visualization, we created a radar plot of the mean x-fold changes for the most significantly upregulated factors, IL-1β, MCP-1, CXCL8 (IL-8), IL-12p70 and IL-17A (Fig 3E). This showed the IL-1β, IL-17A and IL-12p70 dominance of the skin response to MRSA which is enhanced 30x in the case of IL-1β following barrier disruption. A significant positive correlation between bacterial CFUs and IL-1β, IL-17A and IL-12p70 concentrations in individual biopsy was found (Figs 3F and S7). IL-17A has long been reported to have an important role in cutaneous and mucosal host defense to control the microbiota and was found to be involved in defense against MRSA skin infection in mice [8]. IL-12p70 is crucial to polarize naïve T cells towards a Th1 phenotype, but also has been described to induce Tregs and other immunosuppressive cells [3134]. IL-1β, the most strongly upregulated cytokine, has been described to promote neutrophil recruitment [10,35] and to modulate the expression of tight junction proteins [36].

This data demonstrates immunological differences in how healthy human skin responds to MRSA and S. epidermidis and the significant effect of barrier dysfunction on the amplitude of these responses. A key responsive factor identified in the human skin was IL-1β.

Langerhans cells (MUTZ-LC) do not internalize Staphylococcus but differentiate S. aureus from S. epidermidis via IL-1β

We then sought to investigate which cell types may be involved in the IL-1β response in both intact and scratched skin. Many cell types have been implicated in IL-1β signaling including keratinocytes, mast cell and Langerhans cells (LCs) [3739]. Our human skin model contains the full repertoire of tissue resident immune cells including LCs. To localize LCs in the tissue we performed confocal imaging of separated epidermal sheets. We identified the distinctive dendrite-like appearance of the LCs in the epidermis positively labelled with langerin (CD207) (Fig 4A). The cell bodies were located close to the epidermal, dermal basal membrane (Fig 4A and 4C), while the cell projections reached the outer surface of the skin (Fig 4B). This localization suggested a possible role for these cells in early identification of bacteria in both intact and disrupted skin. To study the individual role of the LCs in our tissue response to S. epidermidis and MRSA we differentiated a MUTZ-3 cell line into a LC phenotype. Successful differentiation of the cells was confirmed by analysis of langerin, CD1a and HLA-DR expression (S8 Fig). We visually confirmed langerin expression and dendritic like morphology of the differentiated cells as previously described [40,41] (Fig 4D). To study the ability of the MUTZ-LCs to internalize and respond to MRSA or S. epidermidis we infected these cells with both bacteria at MOIs of 5, 10 or 50. After 3 h of exposure we treated with gentamicin-lysostaphin. The cells were then incubated further to 24 h. No intracellular CFUs were found after 3 h, demonstrating a limited capacity for MUTZ-LCs to internalize Staphylococci (S9 Fig). Despite the lack of bacterial uptake, we saw a significant, dose dependent release of IL-1β in the cell culture media 24 h after MRSA exposure (Fig 4E). Exposure to S. epidermidis resulted in limited IL-1β release (Fig 4E). We also investigated the IL-1β response of the other major cell types in human skin, keratinocytes, and fibroblasts. Keratinocytes, demonstrated an ability to produce IL-1β and differentiate S. aureus and S. epidermidis (Fig 4F), while fibroblasts IL-1β was below the detection limit (Fig 4G). Cytotoxicity data for keratinocytes, fibroblasts and MUTZ-LC infection indicated that the significant differences in IL-1β expression are not directly correlated to cell death (S10 Fig). Previous work in the field identified a role for the S. aureus α-toxin in stimulating myeloid cells to produce IL-1β and modulate immune tolerance in neonate mouse skin [42]. To test the role of this toxin we infected keratinocytes with a S. aureus USA300 lacking expression of α-toxin from the Nebraska Transposon Mutant Library (NE1354, JE2 hla::ΦNΣ) (S. aureus USA300 JE2 Δhla), compared to its isogenic parental strain USA300 JE2. Infection of keratinocytes with a S. aureus USA300 Δhla did not show significantly decreased IL-1β (S11A Fig). Cytotoxicity of the cells was not significantly affected by α-toxin, nor was the IL-8 inflammatory response (S11B, S11C and S11D Fig). This data suggests that in this human cell model, α-toxin expression is not the driving factor for IL-1β differentiation.

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Fig 4. Skin resident Langerhans cells are located throughout the epidermis and MUTZ-LCs can differentiate S. aureus from S. epidermidis.

(A) Orthogonal projection of an epidermal sheet close to the basal membrane. Immunofluorescent staining of f-actin (red), nuclei (blue) and langerin (green). Scale bar = 20μm. Arrow indicates Langerhans cell (green) (B) Orthogonal projection of an epidermal sheet close to the stratum corneum. Immunofluorescent staining of f-actin (red), nuclei (blue) and langerin (green). Scale bar = 20μm. Arrows indicate overlap of Langerhans cells projections with keratinocytes cell walls. Scale bar = 20μm. (C) XZ projection of an epidermal sheet showing a Langerhans cell body at the base of the epidermis with projections extending to the skin surface. F-actin (red), nuclei (blue) and langerin (green). Scale bar = 20μm. (D) Maximum projection of MUTZ-LCs. F-actin (red), nuclei (blue) and langerin (green). Scale bar = 25μm. (E) IL-1β measured by ELISA in MUTZ-LCs supernatants at 24h p.i. after 3 h infection with S. epidermidis 1457 or S. aureus USA300. Bars represent means ± SD, dots show individual data points (n = 3–6 experimental repeats, with 2 technical replicates). Statistical significance determined by two-way ANOVA with Tukey’s correction for multiple comparisons. Statistical significance denoted by ** p ≤ 0.01 or **** p ≤ 0.0001. Only significant differences are annotated for clarity. (F) IL-1β measured by ELISA in keratinocyte (HEKa) supernatants at 24h p.i. after 3 h infection with S. epidermidis 1457 or S. aureus USA300. Bars represent means ± SD, dots show individual data points (n = 3 experimental repeats, with 2 technical replicates). Statistical significance determined by two-way ANOVA with Tukey’s correction for multiple comparisons. Statistical significance denoted by **** p ≤ 0.0001. Only significant differences are annotated for clarity. (G) IL-1β measured by ELISA in fibroblast (HDFn) supernatants at 24h p.i. after 3 h infection with S. epidermidis 1457 or S. aureus USA300. Bars represent means ± SD, dots show individual data points (n = 3 experimental repeats). Detection limit of ELISA shown by dotted line.

https://doi.org/10.1371/journal.ppat.1012056.g004

Taken together, our data indicated than the IL-1β expression patterns we saw in the human skin potentially correlates to MUTZ-LC exposure. IL-1β was the only cytokine differentially upregulated in intact skin exposed to MRSA vs S. epidermidis, which could be explained by the Langerhans cells projections at the skin surface. Scratched skin, in which there would be increased exposure to both LCs and keratinocytes, correlated to the higher IL-1β and significant differentiation between MRSA and S. epidermidis. This data suggests that LCs can differentiate MRSA from S. epidermidis and that these cells could play a key role in the IL-1β response we identified in situ in the intact human skin model.

Discussion

Our work in healthy human skin advances our understanding of the first line of the human skin immune response to colonizing Staphylococci. We highlight the critical role of the stratum corneum in the initial protection to bacteria and shows how even minor disruption of this layer increases bacterial colonization and the inflammatory response. We show a potential role for intracellular bacteria during skin colonization and how the skin structural cells have differing sensitivities to staphylococci. We identify IL-1β in both scratched and unscratched skin and show how it is a key differentiator in the human skin response to the commensal S. epidermidis compared to the potentially pathogenic MRSA. Our cell data suggests tissue resident Langerhans cells are a source of the IL-1β and demonstrates their ability to differentiate MRSA from S. epidermidis.

The new connections between epicutaneous S. aureus and itch shown by the Chiu lab [29] further highlights the relevance of our models as we seek to understand the early phases of host-bacterial interaction. While colonization of an intact stratum corneum showed very little immune response in the skin, transient surface colonization by MRSA may induce itch and skin damage that opens a window for the bacteria to infect deeper layers of the skin and trigger a more pronounced immune response. We demonstrate that the corneocytes in the stratum corneum do not internalize Staphylococci, but the underlying non-professional phagocytic keratinocytes and fibroblast cells can become intracellularly infected and have an increasing, graduated responsiveness. This finding is in line with others, showing that keratinocytes in stratum granulosum and stratum spinosum but also fibroblasts can harbor intracellular bacteria [1,43]. This intracellular niche provides a hiding place from the host immune defense as well as many antibiotics [44] but both cell types have intracellular defense mechanisms to eliminate S. aureus [4547]. Skin diseases such as atopic dermatitis, diabetic foot ulcers (DFUs), chronic non-healing wounds and recurrent infections, are all associated with intracellular S. aureus [4852]. Our work shows that intracellular Staphylococci can occur very early in colonization even without apparent barrier breach.

Our screening for inflammatory signals in response to Staphylococcal colonization revealed an increased expression of IL-1β, IL-17A and IL-12p70 for MRSA. The finding that IL-1β is a dominant skin response to particularly MRSA led us to the intricate localization of LCs throughout the skin epidermis, showing cell projections reaching all the way to the skin surface. Our data suggests a limited ability of these cells to phagocytose Staphylococci nor did we note any intracellular bacteria in LCs in situ. Internalization of microbes into LCs has long been suggested as a mechanism for antigen presentation to T cells and has been shown for other microbes, but may not be as essential for Staphylococci [18,53,54]. We show a clear IL-1β response of MUTZ-LCs to a short bacterial exposure with a strong preference to MRSA. Keratinocytes also produce a differentiating IL-1β response, which fits well with previous work in mouse models which demonstrated that IL-1β was generated by S. aureus intradermal infection but not epicutanous [55]. While others have suggested that a majority of IL-1β is secreted by infiltrating neutrophils [10], this cannot be the case in our ex vivo model, as no infiltrating immune cells are present. Our work does suggest however that IL-1β may already be an important inflammatory signal prior to the arrival of inflammatory cells. Together with previous data, our work suggests that the Langerhans cell projections we identify at the skin surface may initiate the first IL-1β differentiating response to S. aureus vs S. epidermidis. Barrier breach, either via scratching or injury then exposes keratinocytes and other cell types to bacteria, leading to further inflammation and immune cell recruitment. The role of Langerhans cells and particularly the subtle difference between our findings and previous mouse based studies [8,55] fits well with innovative work from the van Sorge lab which demonstrated how the langerin receptor of human LCs binds the wall teichoic acid (WTA) of MRSA but less to S. epidermidis [13]. They showed a clear difference in the interaction of MRSA with human langerin as opposed to the murine receptor [13]. Our work further strengthens these findings to human LCs in situ with an IL-1β dominant differentiation response to human tissue colonization by MRSA vs S. epidermidis. A limitation to this current study is that we have not been able to define which cells produced the IL-1β in situ but we are aiming to address this in future work. Further extensions will include evaluation of bacterial factors of interest such as PSMα [55] and the aforementioned WTA structures [56].

Our study highlights the role of the stratum corneum in skin colonization by Staphylococci. Tissue resident immune cells are important in the early identification and response to different bacterial species, differentiating commensals from pathogens. Further evaluation of the critical role of epidermal LCs in the context of staphylococcal human skin colonization may help reveal whether this is a key pathway to generating skin immunological tolerance to commensal vs potentially pathogenic bacteria.

Material and methods

Ethics statement

Healthy human skin was obtained as surgical excess from elective plastic surgery. Written informed consent was obtained and all procedures were performed according to Swedish National guidelines and approved by the Regionala etikprövningsnämd I Stockholm (ethical approval no: 2015/432-31 and 2023-00567-02).

Cell lines

The human epidermal keratinocyte cell line (HEKa) (Gibco, Thermo Fisher Scientific Cat# C0055C) was cultured in EpiLife medium (Gibco, Thermo Fisher Scientific Cat# MEPI500CA) supplemented with human keratinocyte growth supplement (Gibco, Thermo Fisher Scientific Cat# S0015) at 37°C with 5% CO2. The human dermal fibroblasts cell line (HDFn) (Gibco, Thermo Fisher Scientific Cat# C0045C) was cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (Gibco, Thermo Fisher Scientific Cat# 31330038) supplemented with L-glutamine (Thermo Fisher Scientific Cat# A2916801), 15 mM HEPES and 5% charcoal-stripped fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific Cat# 12676029) at 37°C with 5% CO2. The MUTZ-3 cell line (DSMZ, no ACC 295) was cultured in Minimum Essential Medium Alpha (MEMα) (Gibco, Thermo Fisher Scientific Cat# 22571020) supplemented with 20% charcoal-stripped FBS, 10% medium conditioned by the 5637 cell line (ATCC no. HTB-9), 1x GlutaMAX supplement (Gibco, Thermo Fisher Scientific Cat# A1286001) and 1x antibiotic-antimycotic (Gibco, Thermo Fisher Scientific Cat# 15240096) at 37°C with 5% CO2. All cells were handled and maintained according to the supplier recommendations and following standard cell culture procedures.

Bacteria

The bacterial strains are used in this study are listed in Table 1.

For all experiments, bacteria were grown in Tryptic Soy Broth (TSB) (Sigma) at 37°C with 180 rpm shaking or on Tryptic Soy Agar (TSA) plates (Sigma) at 37°C overnight. S. aureus USA300 GFP was grown in presence of 10μg/ml chloramphenicol. S. aureus USA300 JE2 Δhla was grown in presence of 5μg/ml erythromycin. For infections or inoculations, the bacteria were picked from a colony and grown overnight. On the day of the experiment, a fresh culture was re-inoculated and grown to a density of OD600 = 0.3. Bacteria were washed twice in 1xPBS before use. Alternatively, frozen stock bacterial suspensions of ≈1x109 CFU/ml in PBS were used.

Human skin tissue

Healthy human skin was obtained as surgical excess from elective plastic surgery. Written informed consent was obtained from all participants, and all procedures were approved by the Regionala etikprövningsnämd I Stockholm (approval no: 2015/432-31 and 2023-00567-02). Donors underwent standard surgical preparation which included topical decontamination of the skin with Chlorhexidine. Skin was stored at 4°C until use and cleaned again with 70% ethanol before biopsy preparation.

Ex vivo human skin culture and bacterial colonization

The explant human skin model was prepared and cultured as previously described by Schulz et al. 2019 [17]. Briefly, 10 mm full thickness skin biopsies were prepared with a biopsy punch (Agnthos) and placed into a 12 well transwell insert (Sarstedt), supported with CO2 independent media (Gibco, Thermo Fisher Scientific Cat# 18045088) supplemented with 10% FBS and 1% GlutaMax (Thermo Fisher Scientific). To seal the edges of the explants, hydrogel rings were prepared from a hydrogel band aid using 11- and 7-mm punches and attached to the top of the explants. Overnight the prepared biopsies stabilized at 37°C in the incubator before topical inoculation with 2 μl of bacteria adjusted to 6.5x108 CFU/ml in 1xPBS (1.3x106 CFU/biopsy). Biopsies treated with 1xPBS were used as negative control. For the ex vivo colonization experiment with disrupted stratum corneum, the epidermis of biopsies was carefully scratched with a needle before colonization with bacteria. The applied liquid was allowed to completely evaporate before the explants were incubated at 37°C for 48 h. Culture medium was changed every 24 h. After 48 h the biopsies were homogenized in 500 μl PBS supplemented with 1x HALT protease inhibitor (ThermoScientific, Thermo Fisher Scientific Cat# 87785) using FastPrep lysing matrix A tubes at 6 m/s for 3 x 20 s in a Fast-Prep-24 homogenizer (MP Biomedicals). Serial dilutions of the homogenates were plated on TSA and incubated at 37°C to determine viable CFUs. The remaining skin homogenates were centrifuged at 16000 x g and collected supernatants were stored at -20°C for further analysis. Some biopsies were fixed overnight with 4% paraformaldehyde at 4°C, washed with PBS and used for staining and imaging purposes.

Dermal/Epidermal sheet separation

To separate the epidermal sheet from the dermis of ex vivo human skin biopsies, the samples were incubated in dispase II (Gibco, Thermo Fisher Scientific Cat# 17105041) diluted to 1.8U/ml in CO2 independent medium supplemented with 10% FBS at 4°C overnight. Using sterile tweezers, the epidermis was carefully peeled off the dermis the following day and used for microscopic analysis as described below.

HEKa and HDFn bacterial infection

Cells were grown to 80% confluency, enzymatically harvested, and seeded in a 48-well plate (Corning) at a density of 2x105 cells/ml for HEKa and 1.6x105 cells/ml for HDFn in 250μl appropriate cell culture media. The following day, the attached cells were infected with the different strains at noted multiplicity of infection (MOI). Uninfected cells were used as negative control. After 3 h incubation at 37°C and 5% CO2, the extracellular bacteria were removed in all samples by adding 250μl appropriate cell culture medium supplemented with 100 μg/ml gentamicin and 5 μg/ml lysostaphin (Sigma-Aldrich Cat# L7386) (gentamicin-lysostaphin protection assay) for 30 min at 37°C and 5% CO2. The 3 h post infection (p.i.) samples were washed with 1xPBS, lysed with 0.1% Triton X-100 and serial dilutions were plated on TSA to determine intracellular colony forming units (CFUs). 21 h after removing extracellular bacteria, the supernatants from the remaining samples were collected, centrifuged at 9400 x g for 15 min and stored at -20°C for further analysis.

MUTZ-3 bacterial infection

To differentiate MUTZ-3 cells into a Langerhans cell phenotype (MUTZ-LC) 2x105 cells/ml MUTZ-3 were exposed to 100ng/ml GM-CSF (PeproTech Cat# 300–03), 10ng/ml TGFβ (PeproTech Cat# 100–21) and 2.5ng/ml TNFα (PeproTech Cat# 300-01A) for 9–10 days in a 12-well plate (1 ml/well). The media was change on day 4 and 8 by and antibiotic-antimycotic was removed on day 8. For the infection assay, MUTZ-LCs were seeded at a density of 2x105 cells/ml in Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco, Thermo Fisher Scientific Cat# 12440053) supplemented with 10% charcoal-stripped FBS in a 24-well plate (500 μl/well) and infected with S. aureus USA300 or S. epidermidis 1457 at different MOIs. As for HEKa and HDFn, cell culture supernatants were collected at 3 h p.i. and the infection was stopped using 100 μg/ml gentamicin and 5 μg/ml lysostaphin for 30 min. 24 h p.i. the supernatants were collected again. All supernatants were stored at -20°C for cytokine detection. In addition, intracellular CFUs were determined in cell lysates at 3 h p.i. by plating serial dilutions on TSA.

Primary corneocyte sampling and bacterial inoculation

After thorough cleaning human skin tissue with 70% ethanol, primary corneocytes were collected using D100 D-squame sampling discs (Clinical & Derm, Thermo Fisher Scientific Cat# NC9714282). Circles with Ø10 mm were punched out and placed into a 48-well plate (Corning). The attached corneocytes were infected with 250μl bacterial suspension at 2x107 CFU/ml in CO2 independent media supplemented with 10% FBS and 1% GlutaMax (Thermo Fisher Scientific). After 3h incubation at 37°C and 5% CO2, the supernatant was collected, and extracellular bacteria were removed by adding 250μl medium supplemented with 100 μg/ml gentamicin and at least 5 μg/ml lysostaphin (gentamicin-lysostaphin protection assay). After 30 min incubation, cells were washed with 1xPBS and intracellular CFUs were determined by lysing with 0.3% Triton x-100 for 10 min and plating serial dilutions on TSA. The collected supernatants were centrifuged at 9400 x g for 15 min and stored at -20°C for further analysis. For microscopy, some infected corneocytes were fixed in 4% PFA for 5 min at RT before staining.

Corneocyte staining for autofluorescence

To visualize the freshly insolated and infected corneocytes (described above) we used 0.4% trypan blue solution to visualize the red autofluorescence of corneocytes following a protocol by Mills et al 2022 [59].

Cytotoxicity assay

To measure the potential cytotoxic effects of the infection with the different staphylococcal strains on the cells, a lactate dehydrogenase (LDH)-assay was performed using the CytoTox 96 Non-Radioactive Cytotoxicity Kit according to the manufacturer’s recommendations (Promega). With this colorimetric assay the amount of LDH released into the culture supernatant by infected and untreated control cells was measured and cell death was calculated as following:

The maximum release was achieved by adding 1% (V/V) Triton X-100 to untreated control cells.

Cytokine detection

Interleukin-8 (IL-8) concentrations were determined with the IL-8/CXCL8 DuoSet ELISA kit (R&D Systems, Cat# DY208), CXCL12 with the CXCL12/SDF-1 DuoSet ELISA kit (R&D Systems, Cat# DY350) and IL-1β concentrations were determined with the ELISA MAX Deluxe Set Human IL-1β (BioLegend, Cat# 437004) in 96-well half area Costar assay plates (Corning) according to manufacturer’s protocol with halved volumes. For IL-8 detection, cell culture supernatants from HEKa and HDFn were used at a 10-fold dilution, human skin homogenate supernatants at a 20-fold dilution, and corneocyte supernatants undiluted. For IL-1β detection, cell culture supernatants were used without dilution. Corrected optical density after colorimetric reaction at OD = 450 nm (corrected with OD = 570 nm) was measured with plate reader Tecan Infinite M1000 pro and analyzed with Excel version 16.79.1 and GraphPad Prism version 10. The panel of cytokines was measured using the LEGENDplex Human Inflammation Panel 1 (13-plex) (BioLegend, Cat# 740809) kit, detecting human IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1, IL-6, CXCL8 (IL-8), IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33. The assay was performed following manufacturer instructions.

FACS Analysis of MUTZ-LC

Following differentiation of MUTZ-3 into MUTZ-LCs, the differentiation was validated by the measurement of Langerhans cell specific cell surface markers including langerin, CD1a and HLA-DR. MUTZ-LCs were fixed in 4% PFA for 10 min at RT and washed times in 1xPBS followed by blocking with cold 1xPBs supplemented with 10% FBS. Cells were stained with the primary antibodies anti-hu langerin/CD207 clone 929F3.1 (Novus Biologicals, Cat# DDX0362P-100), anti-hu CD1a clone SK9 (BioLegend, Cat# 344902) or anti-hu HLA-DR clone LN3 (BioLegend, Cat# 327002) and the secondary antibodies Alexa Fluor 488 anti-rat IgG (Invitrogen, Thermo Fisher Scientific Cat# A-11006), Alexa Fluor 555 anti-mouse IgG (Invitrogen, Thermo Fisher Scientific Cat# A-31570) or Alexa Fluor 594 anti-mouse IgG (Invitrogen, Thermo Fisher Scientific Cat# A32744). Cell counts were acquired using a Sony ID7000 spectral cell analyzer, data analyzed using FlowJo 10.9.0 and GraphPad Prism 10.

Microscopy

Fixed (4% paraformaldehyde) human skin samples were embedded in OCT matrix (Tissutek, Saktura) and 5 or 10 μm sections were sliced with an CryoStar NX70 cryostat (Thermo Scientific) and collected on SuperFrost Plus microscopy slides (Thermo Scientific). For H&E staining, the sections were stained in hematoxylin and erythrosin B according to the manufacturer’s protocol and imaged using the Zeiss Axioplan. For immunohistochemistry, the sections were blocked with 0.1% Triton X-100 and 10% FBS in 1xPBS overnight at 4°C and stained with the following reagents and antibodies: NucBlue Fixed Cell ReadyProbes Reagent (DAPI) (Invitrogen), Alexa Fluor 647 Phalloidin 1:500 (Invitrogen, Thermo Fisher Scientific Cat# A22287), anti-cytokeratin-10 1:400 (Invitrogen, clone DE-K10, Thermo Fisher Scientific Cat# MA5-13705), Alexa Fluor 555 anti-mouse IgG 1:1000 (Invitrogen). For imaging of the cell monolayers (Fig 2), cells were handled as described above for infections but were seeded in an 8-well slide chamber (ibidi 190711/6) on a permanox plastic Nunc Microscope Slide (Thermo Scientific, Cat# 160005). The cells were fixed with 4% PFA for 5 min at room temperature. Blocking and staining was performed as described above. After fixing the epidermal sheets for 2 h in 4% PFA at 4°C, 0.5% Triton X-100 in TBS was used to permeabilize 45 min at 4°C. The sheets were blocked with 0.1% Triton X-100 and 10% FBS in 1xPBS for 2 h at 37°C and stained with the following reagents and antibodies: NucBlue Fixed Cell ReadyProbes Reagent (DAPI) (Invitrogen, Thermo Fisher Scientific Cat# R37606), Alexa Fluor 647 Phalloidin 1:500 (Invitrogen), anti-CD207/langerin 1:400 (novus Biologicals, 929F3.01), Alexa Fluor 488 anti-rat IgG 1:500 (Invitrogen). The prepared microscopy samples were mounted in Dako fluorescence medium (Agilent Technologies) and imaged with a Zeiss LSM980-Airy2 (Fig 1) or a Leica Stellaris 5 X (Figs 2 and 4) Confocal Laser Scanning Microscope (CLSM). Fixed MUTZ-LCs were stained for nuclei, f-actin and langerin using the same antibodies as described above mounted and imaged. Image analysis was performed with Zeiss Zen lite (Version: 3.99.02000), LAS X Office (Version: 1.4.5 27713) or ImarisViewer (Version 10.1.0).

Statistical analysis

Statistical tests used were unpaired t test with Welch’s correction to compare two groups, and one-way or two-way ANOVA with Tukey’s or Šídák’s correction for multiple comparisons. The analysis was performed with GraphPad Prism 10 (GraphPad Software, Inc. La Jolla, CA). Statistical significance was indicated with * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001) or **** (p ≤ 0.0001) or ns (not significant). Details of statistical tests used and the n numbers for each experiment can be found in the figure legends.

Supporting information

S1 Fig. Tissue integrity of ex vivo model.

H&E staining of human skin cross sections 0 h and 72 h after ex vivo culture Scale bar = 50μm.

https://doi.org/10.1371/journal.ppat.1012056.s001

(TIF)

S2 Fig. CXCL12 in ex vivo human skin.

ELISA CXCL12 of intact (A) and disrupted (B) human skin homogenates 48h after colonization with S. epidermidis 1457 and S. aureus USA300, displayed as x-fold change over uninfected controls. Dots represent individual data points, colours differentiate skin donors (n = 2–3). Significance determined by unpaired t-test with Welch’s correction.

https://doi.org/10.1371/journal.ppat.1012056.s002

(TIF)

S3 Fig. Cytotoxicity of infected keratinocytes and fibroblasts.

Cytotoxicity of keratinocytes (A) and fibroblasts (B) infected for 3 h with S. epidermidis 1457 or S. aureus USA300, measured after 24 h by LDH assay. Bars = means ± SD; dots represent individual data points (n = 3, with 2 technical replicates). Significance determined by ordinary one-way ANOVA with Tukey’s correction for multiple comparisons. Significance is denoted by * p ≤ 0.05, *** p ≤ 0.001 or **** p ≤ 0.0001.

https://doi.org/10.1371/journal.ppat.1012056.s003

(TIF)

S4 Fig. No intracellular S. aureus in human corneocytes.

Microscopic detection of intracellular S. aureus GFP in cell monolayers of tape-stripped human corneocytes; uninfected CTR vs 3h infection. Autofluorescence staining of corneocytes (red). Scale bars = 50μm.

https://doi.org/10.1371/journal.ppat.1012056.s004

(TIF)

S5 Fig. Disruption of stratum corneum.

Representative microscopy image of disrupted stratum corneum by scratching the surface of human skin biopsies. Immunofluorescent staining of keratinocytes in stratum spinosum and granulosum expressing cytokeratin-10 (orange), f-actin (white), nuclei (blue). Scale bar = 50μm.

https://doi.org/10.1371/journal.ppat.1012056.s005

(TIF)

S6 Fig. IL-8 response of unscratched intact vs. scratch disrupted human skin colonized with Staphylococcus sp.

Comparison of data shown individually in Figs 1 and 3. ELISA IL-8 of human skin homogenates 48 h after colonization with S. epidermidis 1457 and S. aureus USA300, displayed as x-fold change over uninfected controls. Dots represent individual data points, colours differentiate skin donors (n = 6–7). Significance determined by unpaired t-test with Welch’s correction and Holm-Šídák’s multiple comparison test. Significance is denoted by * p ≤ 0.05.

https://doi.org/10.1371/journal.ppat.1012056.s006

(TIF)

S7 Fig. Correlation between number of colonizing Staphylococcus sp. and skin cytokine response.

Correlation between CFU/ml and concentration of IL-1β, IL-17A and IL-12p70 in human skin biopsies 48h p.i. A two tailed Spearman’s rank-order correlation shows a positive relationship between CFUs and IL-1β (r = 0.7112, p<0.0001), IL-17A (r = 0.5390, p = 0.0010) and IL-12p70 (r = 0.4958, p = 0.0029).

https://doi.org/10.1371/journal.ppat.1012056.s007

(TIF)

S8 Fig. Confirmation of MUTZ_LC differentiation.

Histograms of stained (red) MUTZ-LCs expressing langerin (CD207), CD1a and HLA-DR vs unstained MUTZ-LCs (grey) analyzed by FACS.

https://doi.org/10.1371/journal.ppat.1012056.s008

(TIF)

S9 Fig. Intracellular Staphylococcus sp. in MUTZ-LC.

Intracellular CFU counts of S. epidermidis 1457 and S. aureus USA300 3 h p.i. of MUTZ-LC. Dots show individual data points (n = 3; 2 technical replicates).

https://doi.org/10.1371/journal.ppat.1012056.s009

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S10 Fig. Cytotoxicity of infected cell lines.

Cytotoxicity of MUTZ-LC (A), fibroblasts (B) and keratinocytes (C) infected for 3h with S. epidermidis 1457 or S. aureus USA300 measured after 24 h by LDH assay. Bars = means ± SD; dots represent individual data points (n = 3, with 2 technical replicates). Significance determined by Ordinary one-way ANOVA with Tukey’s correction for multiple comparisons. Significance is denoted by * p ≤ 0.05.

https://doi.org/10.1371/journal.ppat.1012056.s010

(TIF)

S11 Fig. Comparison between S. aureus USA300 JE2 Δhla and S. aureus USA300 JE2 in keratinocytes.

(A) IL-1β measured by ELISA in HEKa supernatants at 24 h p.i. after 3 h infection with S. aureus USA300 JE2 Δhla or S. aureus USA300 JE2. Bars represent means ± SD, dots show individual data points (n = 3, with 2 technical replicates). (B) Cytotoxicity of keratinocytes infected for 3h with S. aureus USA300 JE2 Δhla or S. aureus USA300 JE2, measured after 24 h by LDH assay. Bars = means ± SD; dots represent individual data points (n = 3, with 2 technical replicates). (C) IL-8 measured by ELISA in HEKa supernatants at 24 h p.i. after 3 h infection with MOI100 S. aureus USA300 JE2 Δhla or S. aureus USA300 JE2. Bars represent means ± SD, dots show individual data points (n = 3, with 2 technical replicates). (D) Cytotoxicity of keratinocytes infected for 3 h with MOI100 S. aureus USA300 JE2 Δhla or S. aureus USA300 JE2, measured after 24 h by LDH assay. Bars = means ± SD; dots represent individual data points (n = 3, with 2 technical replicates).

https://doi.org/10.1371/journal.ppat.1012056.s011

(TIF)

Acknowledgments

The authors thank Mariam Shahata and Tianqi Zhang for technical assistance and support, Professor Liv Eidsmo for valuable discussions and staff at Nordiska Kliniken for facilitating sample collection. Imaging data was collected at the BIC facility, Karolinska Institutet.

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