An Important Role of α-Hemolysin in Extracellular Vesicles on the Development of Atopic Dermatitis Induced by <i>Staphylococcus aureus</i>

<div><p>Skin barrier disruption and dermal inflammation are key phenotypes of atopic dermatitis (AD). <i>Staphylococcus aureus</i> secretes extracellular vesicles (EVs), which are involved in AD pathogenesis. Here, we evaluated the role of EVs-associated α-hemolysin derived from <i>S. aureus</i> in AD pathogenesis. α-hemolysin production from <i>S. aureus</i> was detected using western blot analyses. The cytotoxic activity of α-hemolysin on HaCaT keratinocytes was evaluated by measuring cell viability after treating cells with soluble and EVs-associated α-hemolysin. To determine the type of cell death, HaCaT keratinocytes were stained with annexin V and 7-AAD. The <i>in vivo</i> effects of α-hemolysin were evaluated by application of soluble and EV-associated α-hemolysin on the mouse skin. The present study showed that increased α-hemolysin was produced by <i>S. aureus</i> colonized on AD patients compared to healthy subjects. α-hemolysin production was also related to AD severity. In addition, EV-associated α-hemolysin was more cytotoxic to HaCaT keratinocytes than soluble α-hemolysin, and α-hemolysin-negative EVs did not induce keratinocyte death. EV-associated α-hemolysin induced necrosis, but soluble α-hemolysin induced apoptosis of keratinocytes. <i>In vivo,</i> skin barrier disruption and epidermal hyperplasia were induced by soluble and EV-associated α-hemolysin. However, AD-like dermal inflammation was only caused by EV-associated α-hemolysin. Moreover, neither skin barrier disruption nor AD-like skin inflammation was induced by α-hemolysin-negative EVs. Taken together, α-Hemolysin secreted from <i>S. aureus</i>, particularly the EV-associated form, induces both skin barrier disruption and AD-like skin inflammation, suggesting that EV-associated α-hemolysin is a novel diagnostic and therapeutic target for the control of AD.</p></div

α-Hemolysin in <i>S. aureus</i> EVs is a key factor for EVs cytotoxicity.

<p><b>A,</b> The presence of α-hemolysin in culture media, EVs-removed culture media (media-EVs), and EVs from the <i>S. aureus</i> ATCC14458 strain. <b>B,</b> Hemolytic function of soluble α-hemolysin and EVs. <b>C,</b> Viability of human keratinocytes after treatment with soluble hemolysin (5 µg/ml), <i>S. aureus</i> culture media (10 µg/ml), and EVs (20 µg/ml). <b>D,</b> α-Hemolysin in EVs from the Newman strain, α-hemolysin-deficient mutant strain, and α-hemolysin complemented strain (pHla). Human keratinocyte viability after treatment with each EVs (40 µg/ml). <b>E,</b> α-Hemolysin in EVs from randomly selected <i>S. aureus</i> from healthy controls (HC) and atopic dermatitis (AD) patients. Viability of human keratinocytes after treatment with EVs (25 µg/ml). * P<0.05; ** P<0.01 versus the PBS group.</p

<i>In vitro</i> immunogenicity of <i>S</i>. <i>aureus</i>-derived EVs (SEVs).

<p>(A) Uptake of SEVs by bone marrow-derived dendritic cells (BMDCs). BMDCs were treated with SEVs (10 μg/ml) for 24 h. BMDCs cytoplasm were stained with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate, green), nuclei with Hoechst (blue), and SEVs with DiI (red). The quantification of SEV-florescence in no-treatment and SEV-treatment group (n = 20, each group). (B) The expression of co-stimulatory molecules in BMDCs. The expression of CD80 and CD86 in BMDCs were measured 24 h after treatment with SEVs (10 μg/ml) or PBS. (C) Production of pro-inflammatory cytokines from BMDCs 24 h after SEVs treatment. BMDCs were treated with various concentrations of SEVs, and the levels of TNF-ɑ, IL-6, and IL-12 in the cell supernatants were measured by ELISA. *** indicates p< 0.001.</p

Antibody and T cell responses after SEV vaccination.

<p>For (A) and (B), SEVs (5 μg) and sham (PBS) were injected intramuscularly to mice at weekly intervals for 3 weeks (n = 10, each group). (A) The levels of SEV-reactive IgG in serum. Sera were obtained from SEV- and sham-immunized mice 7 days after each immunization and serum levels of SEV-reactive IgG were measured by ELISA. (B) SEV-specific production of IFN-γ, IL-17, and IL-4 from splenic T cells. Splenic T cells were isolated from spleens of SEV- and sham-immunized mice, and then stimulated with anti- CD3/CD28 for 72 h. The levels of IFN-γ, IL-17, and IL-4 in the cell supernatants were measured by ELISA. For (C) and (D) SEVs (5 μg) and sham (PBS) were applied intraperitoneally (IP), subcutaneously (SC), or intramuscularly (IM) at weekly intervals for 3 weeks (n = 10, each group). (C) The levels of SEV-reactive IgG in serum. Sera were obtained from SEV- and sham (PBS)-immunized mice 7 days after the last immunization. (D) SEV-specific production of IFN-γ, IL-17, and IL-4 from splenic T cells. Splenic T cells were isolated from spleen of SEV- and sham (PBS)-immunized mice, and then stimulated with anti-CD3/CD28 for 72 h. The levels of IFN-γ, IL-17 and IL-4 in the cell supernatants were measured by ELISA. * indicates p< 0.05 vs. PBS and ** indicates p< 0.01 vs. the other groups.</p

<i>S. aureus</i> on atopic dermatitis skin produces α-hemolysin.

<p><b>A,</b> Detection of α-hemolysin in culture media of <i>S. aureus</i> isolated from the skin of healthy controls (HC) and atopic dermatitis patients (AD). <b>B,</b> The percentage of α-hemolysin-producing <i>S. aureus</i> from 90 AD patients. <b>C,</b> The amount of α-hemolysin in culture media was evaluated by scoring western blot band sizes from 0 to 3. <b>D</b> and <b>E,</b> Human keratinocyte viability after treatment with 10 µg/ml <i>S. aureus</i> culture media (D) and 25 µg/ml EVs (E) for 24 hr. ** P<0.01 versus the PBS group.</p

EVs are more potent mediators of keratinocyte death compared to soluble α-hemolysin.

<p><b>A,</b> Confocal microscopy of human keratinocytes with DiI-labeled <i>S. aureus</i> EVs (red: <i>S. aureus</i> EVs, blue: nucleus). <i>S. aureus</i> EVs and nucleus are shown merged on DIC image (lower panel). Scale bar, 20 µm. <b>B,</b> α-Hemolysin in keratinocytes after treatment with identical amounts of soluble α-hemolysin and EVs. <b>C,</b> Viability of keratinocytes after treatment with each reagent. <b>D,</b> Time dependence of cell death. <b>E,</b> α-Hemolysin on intact and disrupted EVs after treatment with proteinase K. <b>F,</b> Keratinocyte viability after treatment with soluble α-hemolysin (3 µg/ml) and EVs (10 µg/ml) with the anti-α-hemolysin antibody (5% of culture media volume). * P<0.05; ** P<0.01 versus the PBS group; NS, not significant.</p

Efficacy of SEV vaccination on protection against pneumonia induced by sub-lethal dose of <i>S</i>. <i>aureus</i>.

<p>For all figures, SEVs (5 μg) and sham (PBS) were injected intramuscularly to mice at weekly intervals for 3 weeks, and then sub-lethal dose (1 × 10⁸ CFU<i>)</i> of <i>S</i>. <i>aureus</i> was applied via the oropharyngeal route one week after the last immunization. Normal: PBS-immunized and PBS-challenged mice; PBS: PBS-immunized and <i>S</i>. <i>aureus</i>-challenged mice; SEV: SEV-immunized and <i>S</i>. <i>aureus</i>-challenged mice. (A) Colony forming unit (CFU) counts from lung of SEV- and sham (PBS)-immunized mice 24 h after the <i>S</i>. <i>aureus</i> challenge (n = 10, each group). (B) Histology (left panel) and gross image (right panel) of lung from SEV- and sham (PBS)-immunized mice after the sub-lethal dose of <i>S</i>. <i>aureus</i> challenge. (C) Distribution of <i>S</i>. <i>aureus</i> before and after SEV-immunization. Cy7-labeled <i>S</i>. <i>aureus</i> was applied via the oropharyngeal route to SEV- and sham (PBS)-immunized mice. Cy7 fluorescence of whole mouse (upper panel) or lung (lower panel) was acquired by IVIS spectrum 24 h after the <i>S</i>. <i>aureus</i> challenge. (D) Bioluminescence signal in the lung tissue after Cy7-labeled <i>S</i>. <i>aureus</i> administration. The amount of the bioluminescence signal (photons/s) in the lung tissue was measured by IVIS spectrum 24h after <i>S</i>. <i>aureus</i> challenge (n = 5, each group). (E) The levels of IL-β and IL-6 in serum of SEV- and sham (PBS)-immunized mice 24 h after the <i>S</i>. <i>aureus</i> challenge (n = 10, each group). * indicates p< 0.05 vs. PBS.</p

α-Hemolysin-positive <i>S. aureus</i> EVs induces atopic dermatitis-like skin inflammation.

<p><b>A</b> and <b>B,</b> Skin alterations after treatment with 5 µg of soluble α-hemolysin and EVs (A) and 10 µg of EVs from the Newman wild-type and α-hemolysin-deficient strains (B) (n = 5 mice per group). * P<0.05; ** P<0.01; *** P<0.001 versus the PBS group; NS, not significant.</p

Efficacy of <i>S</i>. <i>aureus</i> EVs (SEVs) vaccination on protection against lethality induced by <i>S</i>. <i>aureus</i> lung infection.

<p>(A) Determination of lethal and sub-lethal doses of <i>S</i>. <i>aureus</i> in mouse pneumonia model. Survival rates in mice were evaluated after oropharyngeal application with different doses (1 × 10⁸, 2 × 10⁸, 3 × 10⁸ and 4 × 10⁸ CFU) of <i>S</i>. <i>aureus</i>. Survival was monitored every 12 h for 3 days (n = 10, each group). (B) Histologic image of mouse lung after oropharyngeal application of <i>S</i>. <i>aureus</i> (1 × 10⁸ CFU) 24 h post-infection. (C) Study protocol for SEV-immunization and challenge of the lethal dose (4 × 10⁸ CFU<i>)</i> of <i>S</i>. <i>aureus</i>. SEVs and sham (PBS) were injected intramuscularly at weekly intervals for 3 weeks, and then <i>S</i>. <i>aureus</i> was applied via oropharyngeal route one week after the last immunization. (D) Efficacy of different doses (1, 5, and 10 μg) of SEV vaccination. Survival was monitored every 12 h for 3 days (n = 10, each group). (E) Efficacy of SEV vaccination according to immunization frequency. Survival rates were monitored every 12 h for 3 days in mice immunized with SEVs (5 μg) once, twice, or three times (n = 10, each group).</p

<i>S. aureus</i> EVs induces necrotic cell death, in contrast to soluble α-hemolysin.

<p><b>A,</b> Micrographs of cell death by soluble α-hemolysin (3 µg/ml) and EVs (25 µg/ml). Pictures were taken under microscope at x 100 (upper panel) and x 200 (lower panel) magnification. Red arrows indicate ruptured cells. <b>B,</b> LDH levels in culture media after treatment with soluble α-hemolysin and EVs. <b>C,</b> HMGB-1 in culture media after treatment with soluble α-hemolysin and EVs. <b>D,</b> Flow cytometry analyses of annexin V and 7-AAD staining.</p