ORIGINAL ARTICLE
Staphylococcus aureus impairs the airway epithelial barrier in vitro Zacki Malik, MBBS (Hons), Eugene Roscioli, PhD, Jae Murphy, MBBS, Judy Ou, MBBS, Ahmed Bassiouni, MD, Peter-John Wormald, MD and Sarah Vreugde, MD, PhD
Background: Chronic rhinosinusitis (CRS) is a cluster of disorders that result in sinonasal mucosal inflammation. Staphylococcus aureus (S. aureus) is associated with severe and recalcitrant CRS. The purpose of our study was to investigate the effect of S. aureus on respiratory epithelial barrier structure and function. Methods: Conditioned media from S. aureus reference strains (American Type Culture Collection [ATCC] 13565, 14458, and 25923) was applied to air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs) and transepithelial electrical resistance (TEER) was measured to assess cell-to-cell integrity. Electron microscopy was used to gauge the ciliated area and tight junctions (TJs). Additionally, the expression of the TJ protein zona occludens-1 (ZO-1) was examined via immunofluorescence. Statistical analysis was performed using analysis of variance (ANOVA) with pairwise Bonferroni-adjusted t tests. Results: Secreted products applied to ALI cultures from S. aureus strain 13565 caused a concentration-dependent decline in electrical impedance compared to controls and reference strains 14458 and 25923 (p < 0.001). Electron mi-
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hronic rhinosinusitis (CRS) is an inflammatory disease that primarily involves the nasal and paranasal sinus mucosa. Affected individuals have a severely reduced quality of life, with a disease burden similar to sufferers of back pain and chronic heart disease.1 CRS is highly prevalent, with an economic impact estimated to be $8.6 billion an-
Department of Surgery–Otorhinolaryngology Head and Neck Surgery, The Queen Elizabeth Hospital, and the University of Adelaide, Adelaide, SA, Australia Correspondence to: Sarah Vreugde, MD, PhD, Department of Otorhinolaryngology Head and Neck Surgery, The Queen Elizabeth Hospital, 28 Woodville Rd, Woodville South, South Australia 5011, Australia; e-mail: [email protected] Funding sources for the study: National Health and Medical Research Council, Australia. Potential conflict of interest: None provided. Received: 8 January 2015; Revised: 4 February 2015; Accepted: 8 February 2015 DOI: 10.1002/alr.21517 View this article online at wileyonlinelibrary.com.
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croscopy showed a distinct separation between adjacent cells apically, in the region of TJs. The ciliated area was not affected; however, ZO-1 expression became discontinuous in HNECs exposed to the 13565 strain’s conditioned media. Conclusion: Conditioned media of the S. aureus strain 13565 damages the airway epithelium by disrupting the TJs between primary HNECs grown at an ALI. These findings suggest that strain-specific S. aureus–secreted product(s) compromise epithelial barrier function, which may constitute 1 of the roles played by S. aureus in the pathophysiology of recalcitrant CRS. Further research is required to C 2015 ARSuncover the relevant molecular mechanisms. AAOA, LLC.
Key Words: air-liquid interface; chronic rhinosinusitis; epithelium; electric impedance; tight junctions How to Cite this Article: Malik Z, Roscioli E, Murphy J, et al. Staphylococcus aureus impairs the airway epithelial barrier in vitro. Int Forum Allergy Rhinol. 2015;5:551–556.
nually in the United States alone,2 yet the etiopathogenesis remains elusive. Disease-related defects in the epithelium have been implicated in chronic conditions including asthma,3 inflammatory bowel disease,4 and atopic dermatitis.5 Recently, a damaged epithelial barrier has been implicated in the pathogenesis of CRS.6 Breaches in the epithelial barrier may increase allergen load, restrict mucociliary clearance, and stimulate cells of the adaptive immune response, contributing to the inflammation.6, 7 The respiratory epithelium protects our airways by creating a barrier, secreting antimicrobial factors, and performing mucociliary clearance.8 Adjacent cells of the respiratory epithelium form tight junctions (TJs) to maintain cell-to-cell proximity. These adhesion complexes near the cell apices help restrict paracellular access to submucosal tissue and maintain planar tissue architecture for smooth mucus flow.9 The paracellular barrier is thought to be composed of 2 physiologic components, one a collection of charge-selective small pores and the other being larger,
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less-discriminatory interruptions.10 Pathogens can directly cleave TJ proteins or indirectly modify the cytoskeleton via signal transduction.11 This increased permeability can expose submucosal tissue to antigens and pathogens, as described in the intestinal epithelium of Crohn’s patients.11 Zona occludens-1 (ZO-1) is an integral protein of the TJ complex that anchors neighboring cell cytoplasms.12 It is central to TJ function and has a well-defined role, making it an attractive target for research. Many bacteria have been implicated in the pathogenesis of CRS, but none more so than Staphylococcus aureus (S. aureus). S. aureus is associated with recalcitrant disease,13 antibiotic treatment failure,14 and poor postoperative outcomes.15 S. aureus may employ a number of mechanisms to evade the immune system, including biofilm formation.13 There is also evidence that S. aureus enterotoxins impact ciliary structure and function.16 However, little else is known about S. aureus–derived virulence factors that target innate defenses in the respiratory tract. The aim of this study is to investigate the effect of S. aureus on the barrier function of primary human nasal epithelial cells (HNECs) grown at an air-liquid interface (ALI) in vitro. Conditioned media from different S. aureus strains were tested for their impact on cell-to-cell integrity, TJs, and ciliation.
Materials and methods Cells and cell culture Ethical approval for nasal brushings from healthy volunteers was granted from the Queen Elizabeth Hospital Human Ethics Committee and only consented patients were included in the study. Exclusion criteria included active smoking, age less than 18 years, systemic diseases, and signs or symptoms of allergic rhinitis or CRS. HNEC harvesting and culture was carried out as specified in the supplementary information.
ALI culture HNECs were maintained at an ALI following the PneumaCultTM ALI culture method (StemCell Technologies, Victoria, Australia). Briefly, cells were expanded to 80% confluence and then propagated for a further 24 hours in PneumaCultTM ALI induction media. After induction, cells were detached, seeded at a density of 1 × 105 cells per well onto collagen-coated culture supports perforated with 0.4-μm pores (BD Biosciences, San Jose, CA), and cultured in PneumaCultTM Expansion Media until the cells completely covered the membrane surface. Expansion media was then removed and replaced with PneumaCultTM Maintenance Media in the basal chamber only, exposing the apical cell surface to the atmosphere. Regular examinations were made to assess the integrity of the cell layer.
Preparing microbial conditioned media Three American Type Culture Collection (ATCC) reference strains of S. aureus were used in the study: strain 13565 produces enterotoxin-A, strain 14458 produces enterotoxin-B, and strain 25923 is biofilm-forming.17 Sterile conditioned media was collected from each strain, as detailed in the supplementary information. Conditioned media for each strain was diluted with PneumacultTM Complete Basal Medium (StemCell Technologies, Victoria, Australia) to concentrations of 25%, 50%, and 75%. Each treatment was applied to the apical chamber so as to most closely recreate the conditions of airway infection. A single 12-well ALI plate was used for the 9 S. aureus conditioned media treatments—3 strains at 3 concentrations—and 3 controls. The “no cells” control was an ALI membrane that contained no cells and used no conditioned media in the apical fluid. Similarly, the “cells only” control used no conditioned media in the apical fluid but this time had cells on the ALI membrane. Finally, the “non-enriched media” control used sterile Luria broth that was put through the same process as the infected Luria broth then applied to the cells at a concentration of 75%.
Transepithelial electrical resistance Differentiated airway epithelial cells resist the passage of electrical current after the formation of TJ complexes at the apicolateral margin of adjacent cells. To determine transepithelial electrical resistance (TEER) imparted by epithelial cells maintained at an ALI, cultures were assessed using the EVOM voltmeter system (World Precision Instruments, Sarasota, FL) that passes electric current across the cell monolayer using STX2 “chopstick” electrodes (World Precision Instruments, Sarasota, FL). Briefly, 100 μL of media was added to the apical chamber of ALI cultures to form an electrical circuit across the cell monolayer and into the basal chamber. Cultures were maintained at 37°C during the measurement period using a heating platform. TEER values, in Ohms per square centimeter (.cm2 ), were derived by subtracting the resistance of the culture support then multiplying this value by the support’s surface area, as described.18 We conducted 3 separate experiments using cells from unique donors. Measurements were taken at 0, 0.5, 1, 2, 4, 6, 12, 24, and 48 hours after treatment application. We found that between the 12-hour and 24-hour measurements all the samples containing cells, including controls, declined in resistance below their time zero reading. An outside factor such as time since media change or resubmerging cells previously at ALI appears to have interfered with the normal cellular attachment. Hence, the 12-hour time point was determined to be that which carried the least outside interference and allowed the greatest window for treatment effect.
Electron microscopy sample preparation Samples from 1 experiment were fixed after TEER measurements and separated for scanning electron microscopy
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(SEM) and transmission electron microscopy (TEM) analyses as detailed in the supplementary information.
Ciliated area measurement An FEI Quanta 450 (FEI, Hillsboro, OR) was used to view samples from a single experiment at ×1000, at which point cilia were visible. Each sample had 5 images taken at random for blinded analysis of ciliated area, in a technique adapted from Tamashiro et al.19 Briefly, an image of each sample was imported into PhotoShop CS2 (Adobe, San Jose, CA), where a grid of 100 equal squares was overlaid. Scores of 0, 0.5, or 1 were given to each square—0 implying no cilia and 1 implying complete cilia coverage—then aggregated to form a percent value that was representative of the total area covered by cilia.
Immunofluorescence for ZO-1 in ALI cultures S. aureus conditioned media was applied for 48 hours in the same manner as the TEER experiment prior to staining for ZO-1. Samples from 1 experiment were fixed and fluorescently stained as described in the supplementary information.
a resistance measure of 85%, whereas S. aureus 13565 decreased resistance to 61% and 48% for the concentrations of 50% and 75% (%conditioned media of total media applied), respectively (Fig. 1). Both reductions in resistance were highly significant compared to non-enriched media control (p < 0.001). S. aureus 14458 conditioned media at 75% concentration was the only other treatment to give a resistance reading below that of the non-enriched media control, with a decline to 82%. However, this reduction did not reach statistical significance.
S. aureus 13565 disrupts TJs In control cells, the “kissing point” of adjacent cell membranes represents an intact TJ, determined with TEM (Fig. 2A, B).21 In samples treated with 75% S. aureus 13565 conditioned media there is an apparent separation of TJs (Fig. 2C, D). Hallmark features of cell death, including altered cytoplasmic electron density or nuclear chromatin arrangement, were not observed.22
Ciliated area was maintained Bacterial culture for DNA extraction S. aureus ATCC 13565, 14458, and 25923 were cultured in nutrient broth at 37°C overnight. Lysostaphin (SigmaAldrich, St. Louis, MO) was used at 1 in 100 dilution at 37°C for 1 hour to lyse the bacteria. Genomic DNA extraction was carried out using DNeasy Blood & Tissue Kit (Qiagen, GmbH, Hilden, Germany), as per the manufacturer’s instructions. The DNA concentration was determined using a Spectrophotometer Nanodrop 2000 (Thermo Scientific, Waltham, MA).
Detection of target toxin genes A Taq polymerase chain reaction (PCR) Kit (New England Biolabs, Beverly, MA) was used to amplify SEA, SEB, hla, hlb, hld, and 16S genes,20 according to the manufacturer’s instructions and as detailed in the supplementary information.
Statistical analysis Analysis of variance (ANOVA) with post hoc pairwise Bonferroni-adjusted t tests were used for statistical analysis. R statistical software (R Project for Statistical Computing; http://www.r-project.org) and the IPython Notebook (IPython; http://ipython.org) were used.
Results S. aureus 13565 decreases TEER S. aureus 13565 conditioned media caused a concentrationdependent reduction in TEER. This occurred across 3 experiments with unique cell donors. Twelve hours after treatment application, the non-enriched media control exhibited
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The ciliated area was measured using SEM to determine whether ciliary structures were affected by S. aureus 13565 conditioned media. As shown in Figure 3, there was no significant difference in ciliated area compared with control cells.
ZO-1 expression changes in S. aureus 13565–treated samples Immunofluorescent staining was used to determine ZO-1 expression in S. aureus 13565 conditioned media–treated samples. Untreated cells stained consistently for ZO-1 localized to membrane boundaries, with an average of 1 cell nucleus, stained with 4 ,6-diamidino-2-phenylindole (DAPI), per ZO-1 ring (Fig. 4A). The staining pattern was altered at the 2 higher concentrations of S. aureus 13565 conditioned media. In particular, the ZO-1 rings were larger, averaging 2 to 3 cell nuclei per loop. Faint or discontinuous regions of fluorescence can also be seen intermittently (Fig. 4C, D).
Toxin genes for the ATCC 13565, 14458, and 25923 strains To determine candidate toxins responsible for the observed effect, the presence of common S. aureus toxin genes in the genomic DNA of the strains used was tested by PCR. Previous work has already shown the ATCC 13565 strain to produce enterotoxin-A (SEA) and the 14458 strain to produce enterotoxin-B (SEB),23 and the current PCR analysis confirmed the presence of respective genes in corresponding strains. All strains tested carried the hemolysin hla, hlb, hld genes20 (Fig. 5).
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FIGURE 1. S. aureus strain 13565 decreased TEER, a measure of cell-to-cell attachment. A concentration-dependent decline in TEER was caused by S. aureus 13565 conditioned media (conditioned media as a percentage of total media) but not by the 14458 or 25923 strains. The no-treatment control had a resistance measure of 85%, whereas at 50% and 75% concentrations the S. aureus 13565 conditioned media had a resistance of 61% and 48%, respectively. This graph shows resistance 12 hours after treatment, across 3 unique cell donors and experiments. Resistance measures are shown as a proportion of those at time zero, prior to any treatments. Data represents mean ± SD; ***p < 0.001. ANOVA with Bonferroni-adjusted t tests. ANOVA = analysis of variance; TEER = transepithelial electrical resistance.
Discussion This study examined the effect of S. aureus–secreted products on the barrier function of primary HNECs in vitro. The S. aureus 14458 and 25923 strains did not have detrimental effects on TJ structure or function. Contrastingly, strain 13565 conditioned media caused a concentrationdependent decline in TEER measurements. This decrease in resistance represents separation of cells, allowing greater ion conductance across the epithelial layer.24 Electron microscopy imaging confirmed this by showing separation of cells at the level of TJs. Together with a scattered, discontinuous ZO-1 immunolocalization, these findings support the hypothesis that a specific virulence factor of S. aureus, expressed by the 13565 strain but not by the 14458 and 25923 strains, could be targeting the mucosal barrier at the level of the junctional complexes. S. aureus effects on TJs have been modeled in other tissue types, including keratinocytes25 and enterocytes.26 Both instances showed altered ZO-1 expression although neither used strain 13565, and there were major differences in experimental design, such as the use of live infections as opposed to secreted products used in the present study. Different S. aureus strains are known to carry and express a
broad range of virulence factors through which they infect organ systems with differing efficiency.27 Our data shows that the 13565, 14458, and 25923 strains carry several hemolysin genes and enterotoxin A (13565) or enterotoxin B (14458) genes. Strain 14458, a known enterotoxin B and hemolysin A–producing reference strain,23, 28 and strain 25923, a biofilm-forming reference strain,29 showed no effects significantly different to controls. The 13565 strain that severely compromised the TJ structure in our study is known to produce enterotoxin A24 and hemolysin B.30 Enterotoxins are superantigens that can be identified in the tissue of almost 50% of CRS patients.31 They can bypass the specific antibody-antigen complex between major histocompatibility complex class II molecules and the receptors on T helper cells,31 thus stimulating a nonspecific T cell expansion and related inflammatory response. Staphylococcal enterotoxins can induce a proinflammatory reaction when applied to HNECs,32 but they have not been shown to affect junctional complexes. S. aureus hemolysin A has been shown to disrupt the junctional integrity of intestinal epithelial cells; however, the effect on HNECs has not been tested.33 Bacterial toxins that have been found to disrupt junctional complexes are commonly proteases.34, 35 S. aureus strains are known to produce 10 major secreted
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FIGURE 4. S. aureus 13565 caused discontinuous ZO-1 expression. Analyzing the TJ protein ZO-1 with immunofluorescence showed a regular, continuous pattern of expression in control cells (A) and S. aureus 13565 conditioned media at 25% concentration (B). At 50% (C) and 75% (D) concentrations, a discontinuous pattern was seen with larger TJ loops: 2 to 3 nuclei per TJ ring on average, compared to 1 nuclei per loop in the control sample. TJ = tight junction; ZO-1 = zona occludens-1. FIGURE 2. Disrupted tight junctions on transmission electron microscopy. Tight junctions are important for barrier function and can be seen as cytoplasmic “kisses” between adjacent cell cytoplasms (A, B, arrows). They were intact in the controls and S. aureus 13565 conditioned media at 25% concentration (A, B) but disrupted at 75% concentration (C, D).
FIGURE 5. Toxin genes in S. aureus ATCC 13565, 14458, and 25923 strains. PCR on genomic DNA shows the presence of the SEA gene in the 13565 strain, the SEB gene in the 14458 strain and different hemolysin genes in all strains tested. ATCC = American Type Culture Collection; PCR = polymerase chain reaction; SEA = staphylococcal enterotoxin type A; SEB = staphylococcal enterotoxin type B.
FIGURE 3. Ciliated area was not affected by S. aureus 13565. (A, B) Control HNECs grown at ALI, subjected to control media. SEM (A) and TEM (B) images showed cells to form structurally normal cilia. (C-D) HNECs grown at ALI, subjected to 13565 conditioned media (75%). Measurements of the proportion of the surface area covered by cilia showed no significant difference between samples exposed to S. aureus 13565 conditioned media (p > 0.05). Wide variability in ciliated area can be seen between regions of the same sample. ALI = air-liquid interface; HNEC = human nasal epithelial cell; SEM = scanning electron microscopy; TEM = transmission electron microscopy.
proteases that mediate virulence mechanisms.36 Further studies are needed to determine the identity of the S. aureus 13565 secreted product responsible for TJ disruption in our study. TJs have the important function of keeping epithelial cells of the upper respiratory tract closely apposed to
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restrict paracellular access to submucosal tissue. Increased permeability can allow the entrance of environmental allergens and pathogens.6 A higher allergenic load coming into contact with cells of adaptive immunity could act as a catalyst for the chronic, abnormal immune response central to CRS pathogenesis.7 Both allergy and S. aureus biofilms are associated with the Th2 inflammation that is typical for CRS patients who present with nasal polyps.37 Patients with nasal polyps were shown, in a tissue culture and explant model, to have reduced ZO-1 expression under certain inflammatory cytokine environments.6 It is plausible that unrestricted passage of allergens and bacterial products including superantigens into submucosal tissue could cause greater sensitization and the skewing toward Th2 inflammation and allergy. Future research opportunities could further elucidate whether S. aureus strain 13565 is found commonly in the sinuses of recalcitrant CRS sufferers. Until now, most research on S. aureus in CRS has considered the species as a whole rather than delineating between strains. Perhaps only certain strains are causative in CRS pathogenesis, with
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others merely commensals. Correlating with the presence of polyposis may also help to determine the likelihood of our Th2 inflammatory hypothesis described previously in the discussion.
ability to impair the respiratory epithelial barrier structure and function of primary HNECs in vitro. This virulence factor is novel in the context of human sinonasal epithelium and could suggest a mechanism for how S. aureus overcomes innate barrier protection in CRS.
Conclusion S. aureus infection is commonly implicated in CRS pathogenesis. Much work has shown an association between S. aureus and poorer disease outcomes but has not proved a causative role for the pathogen. Our data shows that secreted products from 1 particular strain—13565—have the
Acknowledgments We acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at Adelaide Microscopy, The University of Adelaide.
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Staphylococcus aureus impairs the airway epithelial barrier in vitro Zacki Malik, MBBS (Hons), Eugene Roscioli, PhD, Jae Murphy, MBBS, Judy Ou, MBBS, Ahmed Bassiouni, MD, Peter-John Wormald, MD and Sarah Vreugde, MD, PhD
Background: Chronic rhinosinusitis (CRS) is a cluster of disorders that result in sinonasal mucosal inflammation. Staphylococcus aureus (S. aureus) is associated with severe and recalcitrant CRS. The purpose of our study was to investigate the effect of S. aureus on respiratory epithelial barrier structure and function. Methods: Conditioned media from S. aureus reference strains (American Type Culture Collection [ATCC] 13565, 14458, and 25923) was applied to air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs) and transepithelial electrical resistance (TEER) was measured to assess cell-to-cell integrity. Electron microscopy was used to gauge the ciliated area and tight junctions (TJs). Additionally, the expression of the TJ protein zona occludens-1 (ZO-1) was examined via immunofluorescence. Statistical analysis was performed using analysis of variance (ANOVA) with pairwise Bonferroni-adjusted t tests. Results: Secreted products applied to ALI cultures from S. aureus strain 13565 caused a concentration-dependent decline in electrical impedance compared to controls and reference strains 14458 and 25923 (p < 0.001). Electron mi-
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hronic rhinosinusitis (CRS) is an inflammatory disease that primarily involves the nasal and paranasal sinus mucosa. Affected individuals have a severely reduced quality of life, with a disease burden similar to sufferers of back pain and chronic heart disease.1 CRS is highly prevalent, with an economic impact estimated to be $8.6 billion an-
Department of Surgery–Otorhinolaryngology Head and Neck Surgery, The Queen Elizabeth Hospital, and the University of Adelaide, Adelaide, SA, Australia Correspondence to: Sarah Vreugde, MD, PhD, Department of Otorhinolaryngology Head and Neck Surgery, The Queen Elizabeth Hospital, 28 Woodville Rd, Woodville South, South Australia 5011, Australia; e-mail: [email protected] Funding sources for the study: National Health and Medical Research Council, Australia. Potential conflict of interest: None provided. Received: 8 January 2015; Revised: 4 February 2015; Accepted: 8 February 2015 DOI: 10.1002/alr.21517 View this article online at wileyonlinelibrary.com.
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croscopy showed a distinct separation between adjacent cells apically, in the region of TJs. The ciliated area was not affected; however, ZO-1 expression became discontinuous in HNECs exposed to the 13565 strain’s conditioned media. Conclusion: Conditioned media of the S. aureus strain 13565 damages the airway epithelium by disrupting the TJs between primary HNECs grown at an ALI. These findings suggest that strain-specific S. aureus–secreted product(s) compromise epithelial barrier function, which may constitute 1 of the roles played by S. aureus in the pathophysiology of recalcitrant CRS. Further research is required to C 2015 ARSuncover the relevant molecular mechanisms. AAOA, LLC.
Key Words: air-liquid interface; chronic rhinosinusitis; epithelium; electric impedance; tight junctions How to Cite this Article: Malik Z, Roscioli E, Murphy J, et al. Staphylococcus aureus impairs the airway epithelial barrier in vitro. Int Forum Allergy Rhinol. 2015;5:551–556.
nually in the United States alone,2 yet the etiopathogenesis remains elusive. Disease-related defects in the epithelium have been implicated in chronic conditions including asthma,3 inflammatory bowel disease,4 and atopic dermatitis.5 Recently, a damaged epithelial barrier has been implicated in the pathogenesis of CRS.6 Breaches in the epithelial barrier may increase allergen load, restrict mucociliary clearance, and stimulate cells of the adaptive immune response, contributing to the inflammation.6, 7 The respiratory epithelium protects our airways by creating a barrier, secreting antimicrobial factors, and performing mucociliary clearance.8 Adjacent cells of the respiratory epithelium form tight junctions (TJs) to maintain cell-to-cell proximity. These adhesion complexes near the cell apices help restrict paracellular access to submucosal tissue and maintain planar tissue architecture for smooth mucus flow.9 The paracellular barrier is thought to be composed of 2 physiologic components, one a collection of charge-selective small pores and the other being larger,
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less-discriminatory interruptions.10 Pathogens can directly cleave TJ proteins or indirectly modify the cytoskeleton via signal transduction.11 This increased permeability can expose submucosal tissue to antigens and pathogens, as described in the intestinal epithelium of Crohn’s patients.11 Zona occludens-1 (ZO-1) is an integral protein of the TJ complex that anchors neighboring cell cytoplasms.12 It is central to TJ function and has a well-defined role, making it an attractive target for research. Many bacteria have been implicated in the pathogenesis of CRS, but none more so than Staphylococcus aureus (S. aureus). S. aureus is associated with recalcitrant disease,13 antibiotic treatment failure,14 and poor postoperative outcomes.15 S. aureus may employ a number of mechanisms to evade the immune system, including biofilm formation.13 There is also evidence that S. aureus enterotoxins impact ciliary structure and function.16 However, little else is known about S. aureus–derived virulence factors that target innate defenses in the respiratory tract. The aim of this study is to investigate the effect of S. aureus on the barrier function of primary human nasal epithelial cells (HNECs) grown at an air-liquid interface (ALI) in vitro. Conditioned media from different S. aureus strains were tested for their impact on cell-to-cell integrity, TJs, and ciliation.
Materials and methods Cells and cell culture Ethical approval for nasal brushings from healthy volunteers was granted from the Queen Elizabeth Hospital Human Ethics Committee and only consented patients were included in the study. Exclusion criteria included active smoking, age less than 18 years, systemic diseases, and signs or symptoms of allergic rhinitis or CRS. HNEC harvesting and culture was carried out as specified in the supplementary information.
ALI culture HNECs were maintained at an ALI following the PneumaCultTM ALI culture method (StemCell Technologies, Victoria, Australia). Briefly, cells were expanded to 80% confluence and then propagated for a further 24 hours in PneumaCultTM ALI induction media. After induction, cells were detached, seeded at a density of 1 × 105 cells per well onto collagen-coated culture supports perforated with 0.4-μm pores (BD Biosciences, San Jose, CA), and cultured in PneumaCultTM Expansion Media until the cells completely covered the membrane surface. Expansion media was then removed and replaced with PneumaCultTM Maintenance Media in the basal chamber only, exposing the apical cell surface to the atmosphere. Regular examinations were made to assess the integrity of the cell layer.
Preparing microbial conditioned media Three American Type Culture Collection (ATCC) reference strains of S. aureus were used in the study: strain 13565 produces enterotoxin-A, strain 14458 produces enterotoxin-B, and strain 25923 is biofilm-forming.17 Sterile conditioned media was collected from each strain, as detailed in the supplementary information. Conditioned media for each strain was diluted with PneumacultTM Complete Basal Medium (StemCell Technologies, Victoria, Australia) to concentrations of 25%, 50%, and 75%. Each treatment was applied to the apical chamber so as to most closely recreate the conditions of airway infection. A single 12-well ALI plate was used for the 9 S. aureus conditioned media treatments—3 strains at 3 concentrations—and 3 controls. The “no cells” control was an ALI membrane that contained no cells and used no conditioned media in the apical fluid. Similarly, the “cells only” control used no conditioned media in the apical fluid but this time had cells on the ALI membrane. Finally, the “non-enriched media” control used sterile Luria broth that was put through the same process as the infected Luria broth then applied to the cells at a concentration of 75%.
Transepithelial electrical resistance Differentiated airway epithelial cells resist the passage of electrical current after the formation of TJ complexes at the apicolateral margin of adjacent cells. To determine transepithelial electrical resistance (TEER) imparted by epithelial cells maintained at an ALI, cultures were assessed using the EVOM voltmeter system (World Precision Instruments, Sarasota, FL) that passes electric current across the cell monolayer using STX2 “chopstick” electrodes (World Precision Instruments, Sarasota, FL). Briefly, 100 μL of media was added to the apical chamber of ALI cultures to form an electrical circuit across the cell monolayer and into the basal chamber. Cultures were maintained at 37°C during the measurement period using a heating platform. TEER values, in Ohms per square centimeter (.cm2 ), were derived by subtracting the resistance of the culture support then multiplying this value by the support’s surface area, as described.18 We conducted 3 separate experiments using cells from unique donors. Measurements were taken at 0, 0.5, 1, 2, 4, 6, 12, 24, and 48 hours after treatment application. We found that between the 12-hour and 24-hour measurements all the samples containing cells, including controls, declined in resistance below their time zero reading. An outside factor such as time since media change or resubmerging cells previously at ALI appears to have interfered with the normal cellular attachment. Hence, the 12-hour time point was determined to be that which carried the least outside interference and allowed the greatest window for treatment effect.
Electron microscopy sample preparation Samples from 1 experiment were fixed after TEER measurements and separated for scanning electron microscopy
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(SEM) and transmission electron microscopy (TEM) analyses as detailed in the supplementary information.
Ciliated area measurement An FEI Quanta 450 (FEI, Hillsboro, OR) was used to view samples from a single experiment at ×1000, at which point cilia were visible. Each sample had 5 images taken at random for blinded analysis of ciliated area, in a technique adapted from Tamashiro et al.19 Briefly, an image of each sample was imported into PhotoShop CS2 (Adobe, San Jose, CA), where a grid of 100 equal squares was overlaid. Scores of 0, 0.5, or 1 were given to each square—0 implying no cilia and 1 implying complete cilia coverage—then aggregated to form a percent value that was representative of the total area covered by cilia.
Immunofluorescence for ZO-1 in ALI cultures S. aureus conditioned media was applied for 48 hours in the same manner as the TEER experiment prior to staining for ZO-1. Samples from 1 experiment were fixed and fluorescently stained as described in the supplementary information.
a resistance measure of 85%, whereas S. aureus 13565 decreased resistance to 61% and 48% for the concentrations of 50% and 75% (%conditioned media of total media applied), respectively (Fig. 1). Both reductions in resistance were highly significant compared to non-enriched media control (p < 0.001). S. aureus 14458 conditioned media at 75% concentration was the only other treatment to give a resistance reading below that of the non-enriched media control, with a decline to 82%. However, this reduction did not reach statistical significance.
S. aureus 13565 disrupts TJs In control cells, the “kissing point” of adjacent cell membranes represents an intact TJ, determined with TEM (Fig. 2A, B).21 In samples treated with 75% S. aureus 13565 conditioned media there is an apparent separation of TJs (Fig. 2C, D). Hallmark features of cell death, including altered cytoplasmic electron density or nuclear chromatin arrangement, were not observed.22
Ciliated area was maintained Bacterial culture for DNA extraction S. aureus ATCC 13565, 14458, and 25923 were cultured in nutrient broth at 37°C overnight. Lysostaphin (SigmaAldrich, St. Louis, MO) was used at 1 in 100 dilution at 37°C for 1 hour to lyse the bacteria. Genomic DNA extraction was carried out using DNeasy Blood & Tissue Kit (Qiagen, GmbH, Hilden, Germany), as per the manufacturer’s instructions. The DNA concentration was determined using a Spectrophotometer Nanodrop 2000 (Thermo Scientific, Waltham, MA).
Detection of target toxin genes A Taq polymerase chain reaction (PCR) Kit (New England Biolabs, Beverly, MA) was used to amplify SEA, SEB, hla, hlb, hld, and 16S genes,20 according to the manufacturer’s instructions and as detailed in the supplementary information.
Statistical analysis Analysis of variance (ANOVA) with post hoc pairwise Bonferroni-adjusted t tests were used for statistical analysis. R statistical software (R Project for Statistical Computing; http://www.r-project.org) and the IPython Notebook (IPython; http://ipython.org) were used.
Results S. aureus 13565 decreases TEER S. aureus 13565 conditioned media caused a concentrationdependent reduction in TEER. This occurred across 3 experiments with unique cell donors. Twelve hours after treatment application, the non-enriched media control exhibited
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The ciliated area was measured using SEM to determine whether ciliary structures were affected by S. aureus 13565 conditioned media. As shown in Figure 3, there was no significant difference in ciliated area compared with control cells.
ZO-1 expression changes in S. aureus 13565–treated samples Immunofluorescent staining was used to determine ZO-1 expression in S. aureus 13565 conditioned media–treated samples. Untreated cells stained consistently for ZO-1 localized to membrane boundaries, with an average of 1 cell nucleus, stained with 4 ,6-diamidino-2-phenylindole (DAPI), per ZO-1 ring (Fig. 4A). The staining pattern was altered at the 2 higher concentrations of S. aureus 13565 conditioned media. In particular, the ZO-1 rings were larger, averaging 2 to 3 cell nuclei per loop. Faint or discontinuous regions of fluorescence can also be seen intermittently (Fig. 4C, D).
Toxin genes for the ATCC 13565, 14458, and 25923 strains To determine candidate toxins responsible for the observed effect, the presence of common S. aureus toxin genes in the genomic DNA of the strains used was tested by PCR. Previous work has already shown the ATCC 13565 strain to produce enterotoxin-A (SEA) and the 14458 strain to produce enterotoxin-B (SEB),23 and the current PCR analysis confirmed the presence of respective genes in corresponding strains. All strains tested carried the hemolysin hla, hlb, hld genes20 (Fig. 5).
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FIGURE 1. S. aureus strain 13565 decreased TEER, a measure of cell-to-cell attachment. A concentration-dependent decline in TEER was caused by S. aureus 13565 conditioned media (conditioned media as a percentage of total media) but not by the 14458 or 25923 strains. The no-treatment control had a resistance measure of 85%, whereas at 50% and 75% concentrations the S. aureus 13565 conditioned media had a resistance of 61% and 48%, respectively. This graph shows resistance 12 hours after treatment, across 3 unique cell donors and experiments. Resistance measures are shown as a proportion of those at time zero, prior to any treatments. Data represents mean ± SD; ***p < 0.001. ANOVA with Bonferroni-adjusted t tests. ANOVA = analysis of variance; TEER = transepithelial electrical resistance.
Discussion This study examined the effect of S. aureus–secreted products on the barrier function of primary HNECs in vitro. The S. aureus 14458 and 25923 strains did not have detrimental effects on TJ structure or function. Contrastingly, strain 13565 conditioned media caused a concentrationdependent decline in TEER measurements. This decrease in resistance represents separation of cells, allowing greater ion conductance across the epithelial layer.24 Electron microscopy imaging confirmed this by showing separation of cells at the level of TJs. Together with a scattered, discontinuous ZO-1 immunolocalization, these findings support the hypothesis that a specific virulence factor of S. aureus, expressed by the 13565 strain but not by the 14458 and 25923 strains, could be targeting the mucosal barrier at the level of the junctional complexes. S. aureus effects on TJs have been modeled in other tissue types, including keratinocytes25 and enterocytes.26 Both instances showed altered ZO-1 expression although neither used strain 13565, and there were major differences in experimental design, such as the use of live infections as opposed to secreted products used in the present study. Different S. aureus strains are known to carry and express a
broad range of virulence factors through which they infect organ systems with differing efficiency.27 Our data shows that the 13565, 14458, and 25923 strains carry several hemolysin genes and enterotoxin A (13565) or enterotoxin B (14458) genes. Strain 14458, a known enterotoxin B and hemolysin A–producing reference strain,23, 28 and strain 25923, a biofilm-forming reference strain,29 showed no effects significantly different to controls. The 13565 strain that severely compromised the TJ structure in our study is known to produce enterotoxin A24 and hemolysin B.30 Enterotoxins are superantigens that can be identified in the tissue of almost 50% of CRS patients.31 They can bypass the specific antibody-antigen complex between major histocompatibility complex class II molecules and the receptors on T helper cells,31 thus stimulating a nonspecific T cell expansion and related inflammatory response. Staphylococcal enterotoxins can induce a proinflammatory reaction when applied to HNECs,32 but they have not been shown to affect junctional complexes. S. aureus hemolysin A has been shown to disrupt the junctional integrity of intestinal epithelial cells; however, the effect on HNECs has not been tested.33 Bacterial toxins that have been found to disrupt junctional complexes are commonly proteases.34, 35 S. aureus strains are known to produce 10 major secreted
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FIGURE 4. S. aureus 13565 caused discontinuous ZO-1 expression. Analyzing the TJ protein ZO-1 with immunofluorescence showed a regular, continuous pattern of expression in control cells (A) and S. aureus 13565 conditioned media at 25% concentration (B). At 50% (C) and 75% (D) concentrations, a discontinuous pattern was seen with larger TJ loops: 2 to 3 nuclei per TJ ring on average, compared to 1 nuclei per loop in the control sample. TJ = tight junction; ZO-1 = zona occludens-1. FIGURE 2. Disrupted tight junctions on transmission electron microscopy. Tight junctions are important for barrier function and can be seen as cytoplasmic “kisses” between adjacent cell cytoplasms (A, B, arrows). They were intact in the controls and S. aureus 13565 conditioned media at 25% concentration (A, B) but disrupted at 75% concentration (C, D).
FIGURE 5. Toxin genes in S. aureus ATCC 13565, 14458, and 25923 strains. PCR on genomic DNA shows the presence of the SEA gene in the 13565 strain, the SEB gene in the 14458 strain and different hemolysin genes in all strains tested. ATCC = American Type Culture Collection; PCR = polymerase chain reaction; SEA = staphylococcal enterotoxin type A; SEB = staphylococcal enterotoxin type B.
FIGURE 3. Ciliated area was not affected by S. aureus 13565. (A, B) Control HNECs grown at ALI, subjected to control media. SEM (A) and TEM (B) images showed cells to form structurally normal cilia. (C-D) HNECs grown at ALI, subjected to 13565 conditioned media (75%). Measurements of the proportion of the surface area covered by cilia showed no significant difference between samples exposed to S. aureus 13565 conditioned media (p > 0.05). Wide variability in ciliated area can be seen between regions of the same sample. ALI = air-liquid interface; HNEC = human nasal epithelial cell; SEM = scanning electron microscopy; TEM = transmission electron microscopy.
proteases that mediate virulence mechanisms.36 Further studies are needed to determine the identity of the S. aureus 13565 secreted product responsible for TJ disruption in our study. TJs have the important function of keeping epithelial cells of the upper respiratory tract closely apposed to
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restrict paracellular access to submucosal tissue. Increased permeability can allow the entrance of environmental allergens and pathogens.6 A higher allergenic load coming into contact with cells of adaptive immunity could act as a catalyst for the chronic, abnormal immune response central to CRS pathogenesis.7 Both allergy and S. aureus biofilms are associated with the Th2 inflammation that is typical for CRS patients who present with nasal polyps.37 Patients with nasal polyps were shown, in a tissue culture and explant model, to have reduced ZO-1 expression under certain inflammatory cytokine environments.6 It is plausible that unrestricted passage of allergens and bacterial products including superantigens into submucosal tissue could cause greater sensitization and the skewing toward Th2 inflammation and allergy. Future research opportunities could further elucidate whether S. aureus strain 13565 is found commonly in the sinuses of recalcitrant CRS sufferers. Until now, most research on S. aureus in CRS has considered the species as a whole rather than delineating between strains. Perhaps only certain strains are causative in CRS pathogenesis, with
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others merely commensals. Correlating with the presence of polyposis may also help to determine the likelihood of our Th2 inflammatory hypothesis described previously in the discussion.
ability to impair the respiratory epithelial barrier structure and function of primary HNECs in vitro. This virulence factor is novel in the context of human sinonasal epithelium and could suggest a mechanism for how S. aureus overcomes innate barrier protection in CRS.
Conclusion S. aureus infection is commonly implicated in CRS pathogenesis. Much work has shown an association between S. aureus and poorer disease outcomes but has not proved a causative role for the pathogen. Our data shows that secreted products from 1 particular strain—13565—have the
Acknowledgments We acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at Adelaide Microscopy, The University of Adelaide.
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