12 research outputs found
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
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
<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
<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
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
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
Demographic and clinical characteristics of atopic dermatitis patients.
<p>Values shown are age in months (median), ratio of males versus females, percentage of methicillin-resistant <i>Staphylococcus aureus</i>, and percentage of patients colonized with α-hemolysin-producing <i>S. aureus</i>.</p>¶<p>The percentage was determined by the amount of α-hemolysin produced by <i>S. aureus</i> isolated from the patients. The amount of α-hemolysin was measured by band intensity using Multi Gauge V3.1. Scores are as follows: 0: zero; 1: up to 6000 arbitrary units (AU); 2: from 6001 to 15,000 AU; and 3: over 15,001 AU.</p
<i>S. aureus</i> EVs and soluble α-hemolysin induce production of different cytokines.
<p><b>A</b> and <b>B,</b> Pro-inflammatory cytokines in human keratinocyte culture media after treatment with soluble α-hemolysin and EVs (A) and 40 µg/ml of EVs from each strain (B). * P<0.05; ** P<0.01 versus the PBS group.</p
Comparison of indoor environmental factors between the16 children with children’s interstitial lung diseases and their 47 matched control.
a<p>Variables in the previous 1 year.</p>*<p><i>P</i><0.05.</p
Case ascertainment criteria.
<p>(A) Chest radiography showing diffuse, ill-defined ground-glass opacities and several ill-defined small nodular opacities in both lungs (in the 4 weeks after respiratory symptoms started). (B) Chest computed tomography showing diffuse centrilobular nodules in both lungs, with ground-glass opacities suggestive of peribronchiolar fibrosis (in the 4 weeks after respiratory symptoms started). (C) The most striking histological feature, interstitial thickening and fibrosis with a centrilobular distribution and relative sparing of the subpleural parenchyma (upper one-third). Original magnification ×40. (D) Interstitial fibroblasts proliferating in a pale myxoid stroma (arrows) and collapsed alveolar spaces lined by activated pneumocytes (arrowhead). Original magnification ×200.</p