Extracellular vesicles derived from Staphylococcus aureus induce atopic dermatitis-like skin inflammation - PubMed (original) (raw)

Extracellular vesicles derived from Staphylococcus aureus induce atopic dermatitis-like skin inflammation

S-W Hong et al. Allergy. 2011 Mar.

Free PMC article

Abstract

Background: Recently, we found that Staphylococcus aureus produces extracellular vesicles (EV) that contain pathogenic proteins. Although S. aureus infection has been linked with atopic dermatitis (AD), the identities of the causative agents from S. aureus are controversial. We evaluated whether S. aureus-derived EV are causally related to the pathogenesis of AD.

Methods: Extracellular vesicles were isolated by the ultracentrifugation of S. aureus culture media. The EV were applied three times per week to tape-stripped mouse skin. Inflammation and immune dysfunction were evaluated 48 h after the final application in hairless mice. Extracellular vesicles-specific IgE levels were measured by ELISA in AD patients and healthy subjects.

Results: The in vitro application of S. aureus EV increased the production of pro-inflammatory mediators (IL-6, thymic stromal lymphopoietin, macrophage inflammatory protein-1α, and eotaxin) by dermal fibroblasts. The in vivo application of S. aureus EV after tape stripping caused epidermal thickening with infiltration of the dermis by mast cells and eosinophils in mice. These changes were associated with the enhanced cutaneous production of IL-4, IL-5, IFN-γ, and IL-17. Interestingly, the serum levels of S. aureus EV-specific IgE were significantly increased in AD patients relative to healthy subjects.

Conclusion: These results indicate that S. aureus EV induce AD-like inflammation in the skin and that S. aureus-derived EV are a novel diagnostic and therapeutic target for the control of AD.

© 2010 John Wiley & Sons A/S.

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Figures

Figure 1

Figure 1

Staphylococcus aureus extracellular vesicles (EV) enhance the in vitro secretion of immune and pro-inflammatory mediators by mouse dermal fibroblasts. (A) Scanning electron microscopic images that S. aureus secrete EV. Arrowheads identify S. aureus EV. (B) Levels of pro-inflammatory mediators IL-6, thymic stromal lymphopoietin, macrophage inflammatory protein-1α and eotaxin in supernatants of dermal fibroblasts after stimulation with EV and >100- and < 100-kD soluble fractions of bacterial culture media. (C) Western blotting to detect staphylococcal enterotoxin B (SEB) in EV and soluble (Sup) fractions of S. aureus culture media. (D) Levels of pro-inflammatory mediators from supernatants of dermal fibroblasts after stimulation with EV or SEB. Assays were performed in duplicate. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Figure 2

Application of Staphylococcus aureus extracellular vesicles (EV) to tape-stripped mouse skin induces atopic dermatitis-like inflammation. (A) Study protocol: application of different doses of S. aureus EV to tape-stripped mouse skin for 4 weeks (n = 5 per treatment group). (B) Skin histology [H&E staining; magnification, ×200 (upper panel) and ×400 (lower panel)]. Arrowheads identify eosinophils. (C) Histological analysis of epidermal thickness and the numbers of eosinophils and mast cells infiltrating the dermis. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Figure 3

Three-week exposure of tape-stripped mouse skin to Staphylococcus aureus extracellular vesicles (EV) induces a mixed Th1-/Th17-/Th2-type inflammatory response in the skin, and the generation of Th1 and Th17 cells in skin-draining lymph nodes (LNs). Evaluation (n = 5 per treatment group) was performed 48 h after the final application of S. aureus EV (5 μg) (performed three times a week for 3 weeks) to tape-stripped skin. *P < 0.05; **P < 0.01; ***P < 0.001. (A) Skin histology [H&E staining; magnification, ×200]. (B) Histological analysis of epidermal thickness. (C) Levels of IFN-γ and IL-17 in supernatants from S. aureus EV-treated cells from skin-draining LNs. (D) Levels of IFN-γ, IL-17, IL-4, and IL-5 in skin tissue homogenates.

Figure 4

Figure 4

Long-term exposure of tape-stripped mouse skin to Staphylococcus aureus extracellular vesicles (EV) enhances the production of IgE. Evaluation (n = 5 per treatment group) was performed 48 h after the final application of S. aureus EV (5 μg) (performed three times a week for 8 weeks) to tape-stripped skin. *P < 0.05; **P < 0.01; ***P < 0.001. (A) Skin histology [H&E staining; magnification, ×200 (upper panel) and ×400 (lower panel)]. Arrowheads identify eosinophils. (B) Histological analysis of epidermal thickness and numbers of eosinophils and mast cells infiltrating the dermis. (C) Levels of IL-17 in supernatants from S. aureus EV-treated cells from skin-draining lymph nodes. (D) Serum levels of total IgG1 and total IgE.

Figure 5

Figure 5

_Staphylococcus aureus_-derived extracellular vesicles (EV) are present on the skin of atopic dermatitis (AD) patients. This figure showed ELISA assay to detect S. aureus EV-specific proteins using anti-S. aureus EV polyclonal antibodies; lavage fluids and EV fraction of lavage fluids obtained from two AD patients have S. aureus EV-specific proteins (SA_EV, _S. aureus_-derived EV).

Figure 6

Figure 6

Staphylococcus aureus extracellular vesicles (EV)-specific IgE is elevated in atopic dermatitis (AD) patients than in age-matched healthy subjects. (A) Levels of S. aureus EV-specific (left panel) and SEB-specific (right panel) IgG1 in serum from AD patients and healthy subjects. (B) Levels of S. aureus EV-specific IgE in serum from AD patients and healthy subjects (left panel), and positive rate of elevated S. aureus EV-specific IgE in AD patients (right panel). (C) Levels of SEB-specific IgE in serum from AD patients and healthy subjects (left panel), and positive rate of elevated SEB-specific IgE in AD patients (right panel). Serum samples from AD patients (n = 30 in patients aged 6–9 years, and n = 30 in patients aged 9–16 years) and healthy subjects aged 6–16 years (n = 20); EV, _S. aureus_-derived EV; SEB, staphylococcal enterotoxin B. *P < 0.05; **P < 0.01.

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