Mechanisims of asthma and allergic disease – 1086. Bacteria-derived extracellular vesicles as an important causative agent for asthma and COPD

Background Many bacterial components in indoor dust can evoke inflammatory pulmonary diseases. Bacteria secrete nanometer-sized vesicles into the extracellular milieu, but it remains to be determined whether bacteria-derived extracellular vesicles in indoor dust are pathophysiologically related to inflammatory pulmonary diseases. We evaluated whether extracellular vesicles (EV) in indoor air are causally related to the pathogenesis of asthma and/or emphysema. Methods EV were prepared by sequential ultrafiltration and ultracentrifugation from indoor dust collected from a bed. Innate and adaptive immune responses were evaluated after once or 4 weeks airway exposure of EV, respectively. Results Vesicles 50-200 nm in diameter were present (102.5 microgram [based on protein concentration]/g dust) in indoor dust, and inhalation of 1 microgram of these vesicles for 4 weeks caused neutrophilic pulmonary inflammation. Additionally, polymyxin B (an antagonist of endotoxin, a cell wall component of Gram-negative bacteria) reversed the inflammation induced by the dust EV. Indoor dust harbors Esherichia coli-derived vesicles; airway exposure to the vesicles for 4 weeks induced neutrophilic inflammation and emphysema, which were partially eliminated by the absence of IFN-gamma or IL-17. Interestingly, serum dust EV-reactive IgG1 levels were significantly higher in atopic children with asthma than in atopic healthy children and those with rhinitis or dermatitis. Moreover, serum dust EV-reactive IgG1 levels were also elevated in adult asthma or COPD patients than in healthy controls. Conclusions EV in indoor dust, especially derived from Gram-negative bacteria, appear to be an important causative agent in the pathogenesis of asthma and/or emphysema

Extracellular vesicles, especially derived from gram-negative bacteria, in indoor dust induce neutrophilic pulmonary inflammation associated with both Th1 and Th17 cell responses

Background Many bacterial components in indoor dust can evoke inflammatory pulmonary diseases. Bacteria secrete nanometre-sized vesicles into the extracellular milieu, but it remains to be determined whether bacteria-derived extracellular vesicles in indoor dust are pathophysiologically related to inflammatory pulmonary diseases. Objective To evaluate whether extracellular vesicles (EV) in indoor air are related to the pathogenesis of pulmonary inflammation and/or asthma. Methods Indoor dust was collected from a bed mattress in an apartment. EV were prepared by sequential ultrafiltration and ultracentrifugation. Innate and adaptive immune responses were evaluated after airway exposure of EV. Results Repeated intranasal application of indoor-dust-induced neutrophilic pulmonary inflammation accompanied by lung infiltration of both Th1 and Th17 cells. EV 50200nm in diameter were present (102.5g protein concentration/g dust) in indoor dust. These vesicles were internalized by airway epithelial cells and alveolar macrophages, and this process was blocked by treatment of polymyxin B (an antagonist of lipopolysaccharide, an outer-membrane component of Gram-negative bacteria). Intranasal application of 0.1 or 1g of these vesicles for 4weeks elicited neutrophilic pulmonary inflammation. This phenotype was accompanied by lung infiltration of both Th1 and Th17 cells, which were reversed by treatment of polymyxin B. Serum dust EV-reactive IgG1 levels were significantly higher in atopic children with asthma than in atopic healthy children and those with rhinitis or dermatitis. Conclusion & Clinical Relevance Indoor dust EV, especially derived from Gram-negative bacteria, is a possible causative agent of neutrophilic airway diseases.X112520sciescopu

Active Immunization with Extracellular Vesicles Derived from Staphylococcus aureus Effectively Protects against Staphylococcal Lung Infections, Mainly via Th1 Cell-Mediated Immunity.

Staphylococcus aureus is an important pathogenic bacterium that causes various infectious diseases. Extracellular vesicles (EVs) released from S. aureus contain bacterial proteins, nucleic acids, and lipids. These EVs can induce immune responses leading to similar symptoms as during staphylococcal infection condition and have the potential as vaccination agent. Here, we show that active immunization (vaccination) with S. aureus-derived EVs induce adaptive immunity of antibody and T cell responses. In addition, these EVs have the vaccine adjuvant ability to induce protective immunity such as the up-regulation of co-stimulatory molecules and the expression of T cell polarizing cytokines in antigen-presenting cells. Moreover, vaccination with S. aureus EVs conferred protection against lethality induced by airway challenge with lethal dose of S. aureus and also pneumonia induced by the administration of sub-lethal dose of S. aureus. These protective effects were also found in mice that were adoptively transferred with splenic T cells isolated from S. aureus EV-immunized mice, but not in serum transferred mice. Furthermore, this protective effect of S. aureus EVs was significantly reduced by the absence of interferon-gamma, but not by the absence of interleukin-17. Together, the study herein suggests that S. aureus EVs are a novel vaccine candidate against S. aureus infections, mainly via Th1 cellular response

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

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>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