Staphylococcus aureus-derived extracellular vesicles induce neutrophilic pulmonary inflammation via both Th1 and Th17 cell responses

Authors


  • Edited by: Angela Haczku

Correspondence

Yoon-Keun Kim MD, PhD and Yong Song Gho PhD, Department of Life Science, Pohang University of Science and Technology (POSTECH), Pohang, Korea.

Tel.: +82-54-279-2125

Fax: +82-54-279-8449

E-mail: juinea@postech.ac.kr; ysgho@postech.ac.kr

Abstract

Background

Recent evidence indicates that Staphylococcus aureus, one of the most important human pathogens, secretes vesicles into the extracellular milieu.

Objective

To evaluate whether inhalation of S. aureus-derived extracellular vesicles (EV) is causally related to the pathogenesis of inflammatory pulmonary diseases.

Methods

Staphylococcus aureus EV were prepared by sequential ultrafiltration and ultracentrifugation. The innate immune response was evaluated in vitro after the application of EV to airway epithelial cells and alveolar macrophages. In vivo innate and adaptive immune responses were evaluated after airway exposure to EV. Adjuvant effects of EV on the development of hypersensitivity to inhaled allergens were also evaluated after airway sensitization with S. aureus EV and ovalbumin (OVA).

Results

Staphylococcus aureus and S. aureus EV were detected in house dust. Alveolar macrophages produced both tumor necrosis α (TNF-α) and interleukin 6 (IL-6) after in vitro stimulation with S. aureus EV, whereas airway epithelial cells produced only IL-6. Repeated airway exposure to S. aureus EV induced both Th1 and Th17 cell responses and neutrophilic pulmonary inflammation, mainly via a Toll-like receptor 2 (TLR2)-dependent mechanism. In terms of adjuvant effects, airway sensitization with S. aureus EV and OVA resulted in neutrophilic pulmonary inflammation after OVA challenge alone. This phenotype was partly reversed by the absence of interferon γ (IFN-γ) or IL-17.

Conclusion

Staphylococcus aureus EV can induce Th1 and Th17 neutrophilic pulmonary inflammation, mainly in a TLR2-dependent manner. Additionally, S. aureus EV enhance the development of airway hypersensitivity to inhaled allergens.

Asthma is a chronic inflammatory disease characterized by inflammation in the airways and airway hyper-responsiveness [1]. Asthma is caused by overwhelming activation of the adaptive immune system against inhaled allergens [2]. The Th2 hypothesis may explain asthma pathogenesis because the asthma phenotype, which includes eosinophilic inflammation and increased IgE, is in accordance with the phenotype of the Th2 cell response. However, much evidence indicates that a significant proportion of asthmatic and chronic obstructive pulmonary disease (COPD) patients are characterized by neutrophilic inflammation and increased interferon γ (IFN-γ) and interleukin 17 (IL-17) levels [3, 4]. Although these phenotypes are obvious in severe and corticosteroid-resistant patients, the pathogenesis of Th1 and Th17 neutrophilic inflammation in asthma and COPD patients is unclear.

Airborne allergens are important causative agents of asthma. House dust mite-derived allergens demonstrate protease activity and induce a Th2 cytokine–mediated immune response. For example, Der p1, a house dust mite-derived allergen, can stimulate mast cells and eosinophils and elicit a Th2-type immune response [5, 6]. However, many allergens are immunologically inert. For example, the body maintains tolerance to inhaled innocuous antigens [7]. Nevertheless, airway inflammation can be caused by such antigens, and it remains unclear why some individuals develop airway inflammation to inhaled allergens, while others do not. One possible explanation for hypersensitivity to inhaled allergens is disruption of airway tolerance by pathogen-associated molecular patterns (PAMP). Studies have shown that airway exposure to allergens contaminated with low-dose lipopolysaccharide (LPS) induces Th2 immune responses, whereas high-dose LPS induces both Th1 and Th17 immune responses [3, 8]. Viral PAMP also induce hypersensitivity to inhaled allergens; allergen plus high-dose double-stranded RNA (dsRNA) induce a Th1 cell response, while low-dose dsRNA elicits both Th2 and Th17 cell responses [9, 10].

Staphylococcus aureus is a Gram-positive bacterium and an important pathogen that mediates both community-acquired and nosocomial infections [11, 12]. Many reports have demonstrated interesting links between S. aureus and allergic diseases, such as asthma and atopic dermatitis. For example, S. aureus colonized skin lesions in 70–90% of atopic dermatitis patients [13]. Asthma severity is associated with S. aureus [14], and S. aureus-derived molecules, such as lipoteichoic acid (LTA) and peptidoglycan (PGN), can induce airway inflammation [15, 16]. Recently, we demonstrated that S. aureus secretes vesicles into the extracellular milieu; these are termed ‘extracellular vesicles’ (EV) [17]. We also showed that S. aureus-derived EV can induce atopic dermatitis-like skin inflammation [18].

Here, we hypothesized that S. aureus-derived EV in house dust are an important causative agent underlying the development of inflammation pulmonary diseases, such as asthma and/or COPD. To test this, we evaluated pulmonary inflammation and associated immunologic mechanisms induced by airway exposure to S. aureus-derived EV. We also evaluated whether S. aureus-derived EV enhance the development of airway hypersensitivity to inhaled allergens.

Methods

Mice

Wild-type (WT) C57BL/6 and BALB/c mice and IL-4R−/− and IFN-γ−/− mice (BALB/c background) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). IL-17−/− (BALB/c background) mice were gifted from Y-C Sung (POSTECH, Pohang, Korea). Toll-like receptor 2 (TLR2) −/− mice (C57BL/6 background) were purchased from Oriental BioService (Kyoto, Japan). Mice were bred in specific pathogen-free facilities at POSTECH, and all live animal experiments were approved by the Pohang University Science and Technology (POSTECH) Ethics Committee.

Reagents

The innocuous allergen ovalbumin (OVA) grade V was purchased from Sigma-Aldrich (St. Louis, MO, USA). Endotoxin in OVA was removed by DetoxiGel Endotoxin Removing Gel (Thermo, Rockford, IL, USA). LTA and PGN were purchased from InvivoGen (San Diego, CA, USA).

Isolation of S. aureus EV

Staphylococcus aureus-derived EV were obtained as described previously [17]. Briefly, S. aureus (ATCC14458) was cultured in nutrient broth (Merck, Darmstadt, Germany) at 37°C to an OD600 of 1.0. Bacteria were harvested by centrifugation and filtration. The filtrate was concentrated by ultrafiltration using a 100-kD hollow-fiber membrane (Amersham Biosciences, Uppsala, Sweden). The resulting concentrated filtrate was filtered to remove any remaining cells, and extracellular vesicles were isolated by ultracentrifugation at 150 000 g for 3 h. Protein concentrations of the S. aureus-derived vesicles were measured using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Hereafter, the S. aureus-derived vesicle dose refers to the quantity of S. aureus-derived vesicle proteins. Silkworm Larva Plasma assay (Wako Pure Chemical Industries Ltd., Osaka, Japan) to determine the PGN amount in EV was performed according to manufacturer's instruction.

Evaluation of in vitro innate immune response by S. aureus-derived EV

An airway epithelial cell line (A549 cells) and alveolar macrophage cell line (MH-S cells) were seeded (1 × 105/well) onto 24-well plates at 37°C. These cells were stimulated with various amounts of LTA (1, 10 μg/ml), PGN (3, 30 μg/ml), and S. aureus EV (0.1, 1, and 10 μg/ml). After 24 h (for A549) or 15 h (for MH-S) of stimulation, culture supernatants were collected and cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA).

To evaluate in vitro innate immune responses induced by S. aureus-derived EV in TLR2−/− mice, peritoneal macrophages were isolated as described previously [19]. Briefly, mice were injected intraperitoneally with thioglycollate medium. Four days later, peritoneal fluid was collected and cells were isolated. Cells were seeded (2 × 105/well) onto 24-well plates and incubated for 2 h. Plates were then washed twice to remove nonadherent cells. After an overnight incubation, peritoneal macrophages were incubated with stimulants and supernatants were collected after 15 h.

Confocal microscopy imaging

A549 and MH-S cells were seeded onto a gelatin-coated coverslip and administered 5 μg/ml of Vybrant DiO (Invitrogen, Eugene, OR, USA)-labeled S. aureus EV. After 5 h of this procedure, coverslips were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. Then, to label cell membrane, coverslips were stained with Vybrant DiI (Invitrogen). Coverslips were then stained with Hoechst and mounted on a glass slide. Images were obtained using FluoView 1000 (Olympus).

Analysis of house dust

House dust was collected from a westernized house, dissolved in sterile PBS, and inoculated on Luria–Bertani (LB) agar spread plates. Plates were incubated at 37°C for 24 h, and colonies were picked and analyzed by biochemical assessment (Samkwang Medical Laboratories, Seoul, Korea). To detect EV in house dust, bacteria were removed from dust-dissolved PBS by centrifugation and the supernatants were ultracentrifuged at 150 000 g for 3 h. Pellets were collected as the dust EV fraction. Protein levels in dust EV fractions were measured by Bradford assay. The levels of S. aureus EV-related proteins in house dust were determined using a polyclonal anti-S. aureus EV antibody, as described previously [18].

In vivo evaluation of the effects of S. aureus EV on the development of innate and adaptive immune responses

To evaluate the innate immune response to S. aureus EV, 0.1 or 1 μg of EV in 30 μl PBS was administered intranasally to mice once or twice, and then inflammation over time was measured. To evaluate the adaptive immune response, S. aureus EV were applied intranasally to mouse airways twice per week for three weeks. Immunologic parameters and inflammation were evaluated 6 and 24 h after the last application, respectively.

In vivo evaluation of the adjuvant effects of S. aureus EV on the development of hypersensitivity to inhaled allergens

A noneosinophilic asthma model induced by sensitization with LPS-contaminated allergens was generated, as described previously [3]. In the present study, S. aureus EV were used as a substitute for LPS. Briefly, 6-week-old mice were sensitized intranasally with 75 μg LPS-depleted OVA plus S. aureus EV (0.1 or 1 μg) on days 0, 1, 2, and 7 and then challenged with 50 μg OVA alone on days 14, 15, 21, and 22. Immunologic analysis was conducted 6 h after the allergen challenge on day 21. Lung inflammation was evaluated 24 h after the last allergen challenge on day 22.

Evaluation of lung inflammation

Cellularity in bronchoalveolar lavage (BAL) fluid was analyzed as described previously. [3] Briefly, after counting the total number of cells in BAL samples, the cell pellet was diluted with 200 μL PBS. In total, 300 inflammatory cells were counted and the BAL differential cell count was determined by Diff-Quik staining. Inflammatory cells were classified as macrophages, lymphocytes, neutrophils, or eosinophils.

Evaluation of T-cell responses

Single cells were isolated from lung tissues. For the re-stimulation response, lung cells were incubated (4 × 106 /ml) in 96-well plates at 37°C with S. aureus EV (0.1 μg/ml) or medium only. After 72-h incubation, culture supernatant was collected and cytokine levels therein were determined. For CD3 and CD28 antibody stimulation, isolated lung cells were incubated in plates coated with anti-CD3 and anti-CD28 antibodies (1 μg/ml each; eBioscience, San Diego, CA) at 37°C. After 12-h incubation, culture supernatant was collected and cytokine levels were determined.

Measurement of cytokine and antibody levels

Cytokine levels from BAL fluids or culture supernatants were measured by ELISA, according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Serum levels of total IgG1, IgG2a, and IgE antibodies were measured by ELISA according to the manufacturer's instructions (Bethyl Laboratory Inc., Montgomery, TX, USA).

Statistical analysis

For multiple comparisons among groups, an analysis of variance (anova) was used, followed by unpaired t-tests or Wilcoxon rank sum tests for pairwise comparisons. An anova linearity test was used to test for trends. The statistical significance was set a priori at < 0.05.

Results

S. aureus-derived EV in house dust

To evaluate the presence of S. aureus, house dust was cultured on LB agar plates. After biochemical analysis, S. aureus, S. hominis, Micrococcus lylae, Pseudomonas stutzeri, and three unidentified bacilli were identified in house dust (Fig. 1A). Next, we evaluated the presence of S. aureus-derived EV in house dust. The levels of S aureus-derived EV were measured by ELISA using polyclonal antibodies to S. aureus EV [18]. Our present data indicated that 25 and 4 ng of S. aureus EV were present in 1 μg of the dust EV fraction and 1 μg of dust, respectively (Fig. 1B). Taken together, these results suggest that S. aureus-derived EV are present in dust from a westernized house.

Figure 1.

Staphylococcus aureus and S. aureus extracellular vesicles (EV) in house dust. (A) House dust from a westernized house was inoculated on a Luria–Bertani (LB) agar plate. After incubation for 24 h, bacterial colonies were obtained and then biochemically analyzed. (B) S. aureus EV-related proteins in house dust and the isolated EV fraction were quantified by enzyme-linked immunosorbent assay (ELISA).

Induction of pro-inflammatory mediator production from airway epithelial cells and alveolar macrophages by S. aureus EV in vitro

Airway epithelial cells and alveolar macrophages are the first line of defense against pathogenic nanoparticles. We examined whether S. aureus EV were internalized by these cells and whether this induced pro-inflammatory mediator production. Using confocal microscopy, S. aureus EV were detected in the cytoplasm of both airway epithelial cells (A549 cells) and alveolar macrophages (MH-S cells) (Fig. 2A). The production of both tumor necrosis factor α (TNF-α) and IL-6 by alveolar macrophages was enhanced by stimulation with S. aureus EV as well as with LTA and PGN in a dose-dependent manner; however, TNF-α was not produced by airway epithelial cells after stimulation with S. aureus EV, LTA, or PGN, whereas IL-6 was enhanced by only S. aureus EV, but not by LTA and PGN (Fig. 2B). Staphylococcal enterotoxin B is a well-known exotoxin produced by S. aureus. We evaluated the production of TNF-α and IL-6 after the stimulation with this toxin. This study showed that the production of these cytokines from alveolar macrophages (MH-S cells) was not enhanced by the stimulation with staphylococcal enterotoxin B (Fig. S1). Taken together, these findings suggest that S. aureus EV can be internalized into both airway epithelial cells and alveolar macrophages and that the production of pro-inflammatory mediators by these two cells is distinct after the stimulation with S. aureus EV.

Figure 2.

In vitro and in vivo innate immune response induced by Staphylococcus aureus extracellular vesicles (EV). (A) Confocal microscopic imaging showed that S. aureus EV is internalized by airway epithelial cells (A549) and alveolar macrophages (MH-S). Left and middle panels show green-colored EV on DIC images. Right panel shows merged image (green: S. aureus EV, blue: nucleus, red: cell membrane). S. aureus EV were detected in cytoplasm. (B) Levels of pro-inflammatory cytokines (tumor necrosis factor α [TNF-α] and interleukin 6 [IL-6]) in supernatants of A549 cells and MH-S cells after stimulation with lipoteichoic acid (LTA), peptidoglycan (PGN), and S. aureus EV. (C) The number of inflammatory cells in bronchoalveolar lavage (BAL) fluids at different time points after the inhalation of S. aureus EV. (D) TNF-α, IL-12, and IL-6 levels in BAL fluids at different time points after the inhalation of S. aureus EV. Number means concentration (μg/ml). Data are expressed as mean ± SEM; *< 0.05; **< 0.01; ***< 0.001 vs the basal level.

Innate immune responses in the lung induced by airway exposure to S. aureus EV

To evaluate in vivo innate immune responses by S. aureus EV, different doses of S. aureus EV were applied to mouse airways. The number of inflammatory cells in BAL fluid was enhanced after exposure to EV; inflammatory cells were significantly increased by exposure to 1 μg EV as compared with 0.1 μg EV (Fig. 2C). Similarly, the production of pro-inflammatory and immunomodulatory mediators, such as TNF-α, IL-12 (a Th1-polarizing cytokine), and IL-6 (Th17-polarizing), was significantly enhanced after the application of S. aureus EV in a dose-dependent manner (Fig. 2D). However, the production of IL-4 (a Th2-polarizing cytokine) was not enhanced by the inhalation of S. aureus EV (data not shown). These results show that short-term inhalation of S. aureus EV induces the production of pro-inflammatory mediators, such as TNF-α and Th1- and Th17-polarizing cytokines, in a dose-dependent manner.

Pulmonary inflammation caused by repeated inhalation of S. aureus EV

To evaluate whether repeated airway exposure to S. aureus EV induces pulmonary inflammation, S. aureus EV were administered intranasally to mouse airways twice per week for three weeks, and phenotypes were evaluated 24 h after the last application (Fig. S3A). Lung infiltration of inflammatory cells, especially neutrophils, was enhanced by S. aureus EV to a significantly greater degree than by PBS, in a dose-dependent manner (Fig. 3A). Histological analysis of lung tissues also showed that both peribronchiolar and perivascular infiltration of inflammatory cells was enhanced by exposure to S. aureus EV (Fig. 3B). In terms of antibody production, repeated exposure to EV increased IgG1 production in a dose-dependent manner, whereas it did not enhance IgG2a or IgE production (Fig. S3B). Taken together, these findings suggest that repeated airway exposure to S. aureus EV induces neutrophilic pulmonary inflammation in a dose-dependent manner.

Figure 3.

Both Th1 and Th17 cell responses are induced by repeated inhalation of Staphylococcus aureus extracellular vesicles (EV). BALB/c wild-type mice (= 5 per group) were exposed to S. aureus EV twice per week for 3 weeks. (A) Bronchoalveolar lavage (BAL) cellularity 24 h after the final S. aureus EV exposure on day 15.*< 0.05; **< 0.01; ***< 0.001 vs the phosphate-buffered saline (PBS) group. (B) Representative lung histology 24 h after the final S. aureus EV exposure on day 15. (hematoxylin & eosin [H&E] stain, ×100). (C) Interferon γ (IFN-γ), interleukin 17 (IL-17), and IL-4 levels in BAL fluids 6 h after the final EV application.*< 0.05 by an analysis of variance (anova) linearity test. (D) IFN-γ, IL-17, and IL-4 levels in culture supernatants 72 h after the stimulation of lung cells with S. aureus EV (0.1 μg/ml).*< 0.05; **< 0.05 by anova linearity test. Data are expressed as mean ± SEM.

Production of T-cell subset cytokines by repeated inhalation of S. aureus EV

To characterize T-cell responses induced by repeated inhalation of S. aureus EV, immunologic parameters were measured 6 h after the final S. aureus EV inhalation (Fig. S3A). IFN-γ (Th1 cytokine) and IL-17 (Th17 cytokine) levels were increased by the inhalation of S. aureus EV in a dose-dependent manner, whereas BAL IL-4 levels were not (Fig. 3C). To evaluate T-cell-related cytokine production in the lungs induced by S. aureus EV inhalation, isolated lung cells were stimulated with S. aureus EV (0.1 μg/ml) and then the levels of T-cell-derived cytokines were determined. Consistent with BAL cytokine levels, the production of both Th1 (IFN-γ) and Th17 (IL-17) cytokines was enhanced in mice exposed to S. aureus EV as compared with those exposed to PBS, in a dose-dependent manner; however, BAL IL-4 levels were not (Fig. 3D). Taken together, these results suggest that repeated inhalation of S. aureus EV induces both Th1 and Th17 immune responses.

Adjuvant effects of S. aureus EV on the development of airway hypersensitivity to inhaled allergens

Inhalation of allergens contaminated with PAMP breaks airway tolerance to inhaled allergens [3, 9, 10]. Because S. aureus EV and allergens can easily be inhaled simultaneously, we evaluated the adjuvant effects of S. aureus EV on the development of immune responses to inhaled allergens. To test this, mice were sensitized intranasally with an allergen (OVA) and S. aureus EV and then challenged with OVA alone as shown in Fig. S4A. BAL cellularity 24 h after the last OVA challenge showed that lung infiltration of inflammatory cells, especially macrophages and neutrophils, was significantly increased in mice sensitized with OVA plus 0.1 or 1 μg EV as compared with those sensitized with PBS or OVA alone (Fig. 4A). Similar to BAL cellularity, histological analysis of lung tissues showed that both peribronchiolar and perivascular infiltration of inflammatory cells was enhanced in OVA + EV (0.1 or 1 μg)-sensitized mice compared to OVA- or PBS-sensitized mice (Fig. 4B). Serum IgG1 levels were also significantly increased in the former as compared with the latter groups, whereas IgG2a and IgE levels were similar in all groups (Fig. S4B). When cytokine levels in BAL fluids were evaluated 6 h after OVA challenge, IL-17 levels were significantly increased in OVA + EV (0.1 or 1 μg)-sensitized mice as compared with OVA- or PBS-sensitized mice, IFN-γ levels were enhanced in only OVA + EV (1 μg)-exposed mice as compared with the other groups, and IL-4 levels were similar among all groups (Fig. 4C). Additionally, to evaluate cytokine production by lung T cells, isolated lung cells were stimulated with anti-CD3 and anti-CD28 antibodies. IL-17 levels in culture supernatants were enhanced in OVA + EV (0.1 or 1 μg)-sensitized mice to a greater degree than in OVA- or PBS-sensitized mice. IFN-γ levels were enhanced only in the OVA + EV (1 μg)-sensitized mice vs the other groups. Lastly, IL-4 levels were similar among all groups (Fig. 4D). Collectively, these results suggest that inhalation of S. aureus EV with allergens disrupts airway tolerance to inhaled allergens, which is characterized by Th17 and/or Th1 cell responses and noneosinophilic inflammation.

Figure 4.

Sensitization with Staphylococcus aureus extracellular vesicles (EV) and allergens induces both Th1 and Th17 cell responses and noneosinophilic pulmonary inflammation. BALB/c wild-type mice (= 5 each group) were sensitized intranasally with ovalbumin (OVA, 75 μg) plus S. aureus EV (0.1 or 1 μg) on days 0, 1, and 7 and then challenged with OVA (50 μg) on days 14, 15, 21, and 22. (A) Bronchoalveolar lavage (BAL) cellularity 24 h after the final OVA challenge. OVA/OVA: sensitized with OVA alone, EV0.1/OVA and EV1/OVA: sensitized with OVA plus 0.1 and 1 μg S. aureus EV, respectively. (B) Representative lung histology (hematoxylin & eosin [H&E] stain, ×200). (C) Interferon γ (IFN-γ), interleukin 17 (IL-17), and IL-4 levels in BAL fluids 6 h after OVA challenge on day 21. (D) IFN-γ, IL-17, and IL-4 levels in culture supernatants 12 h after the stimulation of lung cells with anti-CD3 and anti-CD28 antibodies. Data are expressed as mean ± SEM; *< 0.05; **< 0.01 vs OVA/OVA group; ***< 0.05 by an analysis of variance (anova) linearity test.

Role of IFN-γ, IL-4, and IL-17 in the development of allergic inflammation induced by sensitization with allergen plus S. aureus EV

To delineate the roles of Th1, Th2, and Th17 cytokines in the development of allergic inflammation induced by sensitization with S. aureus EV, IFN-γ-, IL-4Rα-, and IL-17-deficient mice were sensitized intranasally with OVA plus EV (1 μg) and then challenged with OVA alone (Fig. S4A). When we evaluated phenotypes 24 h after the last OVA challenge, lung infiltration of inflammatory cells was partly reversed by the absence of IFN-γ or IL-17, but was unaffected by the absence of IL-4Rα (Fig. 5A). Histological analysis showed that both peribronchiolar and perivascular infiltration of inflammatory cells was decreased in OVA + EV-exposed IFN-γ−/−and IL-17−/− mice compared to WT and IL-4Rα−/− mice sensitized in the same manner (Fig. 5B). In terms of antibody production, serum OVA-specific IgG1 levels were significantly decreased in OVA + EV-sensitized IFN-γ−/−, IL-4Rα−/−, and IL-17−/− mice compared to WT mice sensitized in the same manner; however, OVA-specific IgG2a levels were significantly increased in OVA + EV-sensitized IL-4Rα−/− mice compared to IFN-γ−/−, IL-17−/−, and WT mice sensitized in the same manner (Fig. 5C). Taken together, these results suggest that pulmonary inflammation induced by allergens plus S. aureus EV is dependent on both the Th1 and Th17 cell responses, irrespective of allergen-specific antibody production.

Figure 5.

Pulmonary inflammation induced by sensitization with allergens and Staphylococcus aureus extracellular vesicles (EV) is dependent on both interferon γ (IFN-γ) and interleukin 17 (IL-17). IFN-γ−/−, IL4Rα−/−, IL-17−/−, and wild-type (WT) (all BALB/c background) mice (= 5 each) were sensitized intranasally with ovalbumin (OVA, 75 μg) plus S. aureus EV (1 μg) on days 0, 1, and 7 and then challenged with OVA (50 μg) on days 14, 15, 21, and 22. (A) Bronchoalveolar lavage (BAL) cellularity 24 h after the final OVA challenge. (B) Representative lung histology (hematoxylin & eosin [H&E] stain, ×200). (C) Serum OVA-specific IgG1 and IgG2a levels. Data are expressed as mean ± SEM; *< 0.05; **< 0.01; ***< 0.001.

TLR2-dependency of S. aureus EV-induced innate immune responses

Staphylococcus aureus is recognized by TLR2 in host cells [20]. S. aureus EV were found to harbor LTA, which is a TLR2 ligand [21] (Fig. S2). Thus, we examined the role of TLR2 in the recognition of S. aureus EV using TLR2−/− mice. Peritoneal macrophages were isolated from TLR2−/− and WT mice and then stimulated with S. aureus EV and LTA. IL-6 and TNF-α production by peritoneal macrophages after the stimulation with S. aureus EV was almost completely abolished by the absence of TLR2. In addition, IL-6 and TNF-α production after the stimulation with LTA was completely blocked in the absence of TLR2 (Fig. 6A). Next, to evaluate in vivo innate immune responses after airway exposure to S. aureus EV in TLR2−/− and WT mice, S. aureus EV were twice administered intranasally to mouse airways. Lung infiltration of inflammatory cells, especially macrophages and neutrophils, was significantly lower in S. aureus EV-exposed TLR2−/−mice than in WT mice exposed in the same manner (Fig. 6B). Additionally, the production of pro-inflammatory and immunomodulatory mediators, such as TNF-α and IL-6, was significantly enhanced after S. aureus EV inhalation in WT mice, whereas this enhanced production was abolished by the absence of TLR2 (Fig. 6C). Collectively, these results suggest that inhalation of S. aureus EV induces pulmonary inflammation and the production of pro-inflammatory and immunomodulatory mediators in a principally TLR2-dependent manner.

Figure 6.

Pulmonary inflammation and production of pro-inflammatory and/or immune modulating mediators induced by Staphylococcus aureus extracellular vesicles (EV) are dependent on Toll-like receptor 2 (TLR2). (A) Peritoneal macrophages isolated from TLR2−/−mice were stimulated with 1 μg of lipoteichoic acid (LTA) and S. aureus EV. After the stimulation for 15 h, culture supernatants were collected and tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) levels were measured by enzyme-linked immunosorbent assay (ELISA). For (B) and (C), TLR2−/−and wild-type (WT) (all C57BL/6 background) mice (= 5 each) were exposed intranasally to S. aureus EV (1 μg) twice and their phenotypes were evaluated 24 h after the final application. (B) Bronchoalveolar lavage (BAL) cellularity. (C) The levels of TNF-α and IL-6 in BAL fluids. Data are expressed as mean ± SEM; *< 0.05; **< 0.01.

Discussion

The role of infectious agents in the etiology of many inflammatory diseases is increasingly being recognized. S. aureus colonizes the skin and nasopharynx and is important in the development or aggravation of allergic diseases, such as atopic dermatitis and/or asthma. Recent evidence demonstrates that S. aureus produces EV that contain many pathogenesis-related molecules [17]. Here, we aimed to elucidate the relationship between S. aureus-derived EV and the development of inflammatory pulmonary diseases. Our data suggest that S. aureus EV in an indoor environment can induce neutrophilic pulmonary inflammation and enhance airway hypersensitivity to inhaled allergens.

Many environmental factors and pathogens are associated with allergic diseases [12, 22]. S. aureus colonization of the skin and nasopharynx has been suggested to be a major risk factor for many diseases [23, 24]. However, the relationship between nasopharyngeal colonization by S. aureus and asthma has been reported not to be significant [14, 25]. The current study showed that S. aureus and S. aureus EV are present in indoor dust, indicating that S. aureus in house dust produces EV. These findings led us to speculate that EV derived from S. aureus that is present in the indoor environment, rather than S. aureus colonization of the nasopharynx, is important in the development of pulmonary inflammation.

Recent evidence has demonstrated that bacteria-derived EV can act as a pathogenic agent for the development of inflammatory diseases [18, 26, 27]. In addition, many reports have shown that EV from many pathogenic bacteria modulate the host immune response and induce inflammation [28-30]. The first line of defense against inhaled pathogenic particles is airway epithelial cells and alveolar macrophages. The present study showed that S. aureus EV can be internalized by these cells, leading to the production of pro-inflammatory mediators, including TNF-α, and pulmonary inflammation after airway exposure to S. aureus EV. Moreover, the production of these pro-inflammatory mediators and pulmonary inflammation were found to be dependent on TLR2 signaling. Collectively, these findings suggest that airway exposure to S. aureus EV induces innate immune dysfunction, mainly via a TLR2-dependent mechanism.

Adaptive immune responses play an important role in the pathogenesis of allergic diseases. Growing evidence indicates that Th1 and Th17 as well as Th2 cell responses are important for the development of asthma [31-34]. Both Th1 and Th17 cell responses have been found to be related to the development of neutrophilic inflammation [3, 35]. The present study showed that repeated inhalation of S. aureus EV induced neutrophilic pulmonary inflammation, which was associated with enhanced production of both Th1- and Th17-related cytokines. IL-12 and IL-6 are well-known mediators of Th1 and Th17 polarization, respectively [36, 37]. The current study showed that inhalation of S. aureus EV enhanced the production of both IL-12 and IL-6. Together, these findings suggest that inhalation of S. aureus EV induces both Th1 and Th17 cell responses, partly via up-regulation of Th1- and Th17-polarizing cytokines.

Much evidence has been shown that the production of IgG1 and IgE antibodies is related to IL-4 and that IgG2a production is up-regulated by IFN-γ, whereas down-regulated by IL-4 [38, 39]. However, some evidence suggests that the production of IgG1 is dependent on IFN-γ and IL-17 [40, 41]. The present study showed that the production of IgG1 enhanced by S. aureus EV exposure was reversed by the absence of IFN-γ or IL-17. In contrast, the production of IgG2a after exposure to S. aureus EV was enhanced in the absence of IL-4R, although this antibody production was not enhanced in WT mice. To sum up, these findings suggest that the production of IgG1 enhanced by S. aureus EV is dependent on both Th1 and Th17 cell responses.

The immune system maintains tolerance to inert antigens. However, much evidence suggests that simultaneous inhalation of innocent antigens (allergens) and adjuvants, such as bacteria- or virus-derived PAMP, induces airway hypersensitivity to those allergens [3, 10]. Our data suggest that sensitization by allergens plus S. aureus EV induced allergen-induced neutrophilic pulmonary inflammation as well as the infiltration of mononuclear cells, which was associated with both Th1 and Th17 cell responses. Moreover, the infiltration of neutrophils and mononuclear cells into airways was found to be dependent on both IFN-γ and IL-17. These results suggest that Th1 and Th17 cell responses induced by S. aureus EV are important in chemotaxis of inflammatory cells. Given the presence of S. aureus EV in indoor environments, these data suggest that inhalation of allergens contaminated with S. aureus EV may be an important mechanism in the development of airway hypersensitivity to inhaled allergens.

In summary, this is to our knowledge the first report that inhalation of S. aureus EV induces both Th1 and Th17 cell responses and neutrophilic pulmonary inflammation. In addition, we found that inhalation of S. aureus EV enhances airway immune responses to inhaled allergens, characterized by both Th1 and Th17 cell responses and noneosinophilic pulmonary inflammation. Moreover, our data suggest that immune responses induced by S. aureus EV are principally dependent on TLR2 signaling. Thus, we suggest that S. aureus EV in indoor environments may be an important causative agent of pulmonary inflammatory diseases.

Acknowledgments

We thank Jee-In Lim and Chae-Min Kim for their providing secretarial assistance and members of the POSTECH animal facility for their experimental expertise. This study was supported by the National Research Foundation of Korea Grant funded by the Korean Government (No. 2011-0000879). M.H.J. was supported by the World Class University (WCU) program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R31-10105).

Conflict of interest

The authors declare that there are no conflicts of interest.

Author contributions

M.K. and S.H. designed and did experiments, analyzed and interpreted results, and wrote the manuscript; E.C., W.L., and Y.K did animal experiments; S.J. and M.J. designed experiments and wrote the manuscript; Y.G. and Y.K. directed the study and wrote the manuscript.

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