Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Mar 24.
Published in final edited form as: JAMA Otolaryngol Head Neck Surg. 2015 Apr;141(4):341–349. doi: 10.1001/jamaoto.2014.3550

Sinonasal epithelial cell response to Staphylococcus aureus burden in chronic sinusitis

Michael A Kohanski 1, Andrew P Lane 1,*
PMCID: PMC4806547  NIHMSID: NIHMS771057  PMID: 25612191

Abstract

Importance

Chronic rhinosinusitis (CRS) is an inflammatory disorder of the nose and paranasal sinuses. Staphylococcus aureus is increasingly linked with CRS exacerbations. Little is known about how bacteria activate inflammatory pathways that contribute to CRS.

Objective

Here, we developed an in-vitro co-culture system to explore how infection with Staphylococcus aureus stimulates innate immune responses of sinonasal epithelial cells (SNEC).

Design, Setting, and Subjects

SNEC were collected from 13 patients during endoscopic sinus surgery and grown in culture at the air-liquid interface.

Interventions

Differentiated SNEC from control, CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP) patients were infected with Staphylococcus aureus at three different concentrations for 24-hours.

Main Outcomes and Measures

Growth of S. aureus and viability of SNEC were measured. Expression of inflammatory markers and innate immune genes were measured by RT-PCR. Basal secretion of IL-8 was determined by ELISA.

Results

Cultured SNEC from CRSsNP patients demonstrated a significant increase in expression of IL-8, and TNFα at all the tested concentrations of Staphylococcus aureus. Control or CRSwNP SNEC demonstrated a significant increase in expression only at the higher inoculum of Staphylococcus aureus. Basal secretion of inflammatory markers correlated with expression changes. No significant changes in expression were observed for the Th2 inflammatory mediators tested.

Conclusions and Relevance

In this study, we have developed a model to study early innate-immune mediated changes in sinonasal epithelial cells co-cultured at an air-liquid interface with bacteria. We also demonstrate that bacterial burden can be detected by SNEC in the absence of adaptive immune-mediated responses. CRSsNP SNECs are more sensitive to S. aureus burden than control or CRSwNP SNEC. Future studies will further develop this infection model and explore the SNEC innate immune response to bacteria.

Introduction

Chronic rhinosinusitis (CRS) is an inflammatory disorder of the nose and paranasal sinuses1 that can be broadly divided into two groups based on the presence or absence of nasal polyps (CRSwNP and CRSsNP, respectively). The persistent inflammatory response in CRS involves adaptive and innate components.2,3 The adaptive response in both forms of CRS includes a mucosal infiltrate of lymphocytes, neutrophils, and macrophages, but CRSwNP is differentiated from CRSsNP by tissue eosinophilia and a Th2-biased cytokine milieu including interleukin-4 (IL-4), IL-5 and IL-13.3,4 In addition to these leukocytes, the sinonasal mucosa also harbors resident innate immune cells such as dendritic cells and innate lymphoid cells that contribute to orchestrating the inflammatory response. Finally, sinonasal epithelial cells have a key sentinel role in host defense and in promoting inflammation through the production of antimicrobial proteins, cytokines, chemokines, and other inflammatory mediators.3,5

Recently, the epithelial cell-derived proteins thymic stromal lymphopoietin (TSLP), IL-25 and IL-33, have been demonstrated to be capable of stimulating Th-2 cytokine expression6,7. IL-33 is upregulated in epithelial cell cultures from those with recalcitrant CRSwNP5, IL-25 and IL-33 demonstrate increased expression in tissue samples from CRSwNP patients8, and TSLP may play a role in regulating the inflammatory process in CRSwNP9.

Lymphocytes, neutrophils and other immune cells are recruited to mucosal tissue through the production of cytokines, such as TNFα, which are expressed at higher levels in CRS.10 TNFα is a central mediator of inflammation and also functions as a chemoattractant for neutrophils11. As part of the innate immune response, epithelial cells are capable of secreting TNFα and other chemoattractants such as interleukin-8 in response to infectious stimuli such as viruses or intracellular bacteria.12,13

The role of bacteria in CRS remains unclear. The bacterial infection or colonization that is often seen in CRS may be the primary inflammatory stimulus or could instead occur secondary to a pre-existing inflammatory state, perhaps acting only as a disease modifier. Bacterial biofilms, which are sources of persistent bacteria and which serve as a niche for the potential rise of resistant bacteria14, have been found more often in tissue from those with CRS and may serve as reservoirs of infection in treatment resistant CRS.15 Pseudomonas aeruginosa and Staphyloccocus aureus biofilms are detected more frequently in sinonasal tissue samples from those with CRS than from those without CRS16. Colonization with S. aureus, the presence of S. aureus biofilms and intracellular Staphylococcus have been increasingly linked with CRS exacerbations suggesting that S. aureus may have a causative role in recalcitrant CRS17,18. S. aureus biofilms in those with CRS has been associated with increased expression of inflammasome genes19, and in-vitro growth of S. aureus on sinonasal tissue explants leads to increased expression of elements of the NOD-2 pro-inflammatory pathway, certain C-X-C cytokines and IL-620.

In this study, we have developed a co-culture system for examining the early interactions between sinonasal epithelial cells and live bacteria at the air-liquid interface. We demonstrate that bacterial burden can be detected by sinonasal epithelium in the absence of adaptive immune-mediated responses. This system may provide a basis for further in vitro manipulation to better tease apart the complex interactions between bacteria and the innate immune system and to better understand the role of bacterial colonization and infection in CRS.

Materials and Methods

Human Subjects

SNEC tissue was collected from the ethmoid sinuses of 13 patients during endoscopic sinus surgery and grown in culture at the air-liquid interface (ALI). Three patients were classified as having CRSwNP, 6 patients were classified as having chronic rhinosinusitis without polyps (CRSsNP), and 4 patients were classified as controls. Patients with chronic rhinosinusitis were defined by historical, endoscopic, and radiographic criteria, and by meeting the definition of the American Academy of Otolaryngology-Head and Neck Surgery Chronic Rhinosinusitis Task Force. Patients had continuous symptoms of rhinosinusitis for greater than 12 consecutive weeks which was associated with bilateral mucosal disease on computed tomography of the sinuses. Patients were classified as having CRSwNP if endoscopy confirmed the presence of bilateral polyps in the middle meatus and CRSsNP if endoscopy excluded polyps in the middle meatus.1 Control patients were defined as those without CRS who were undergoing endoscopic sinonasal surgery for DCR, CSF leak repair or removal of non-polyp nasal masses.

The mucosal tissue was transferred to phosphate buffered saline (PBS) supplemented by penicillin (100 ug/mL, Gibco, Gaithersburg, MD), streptomycin (100 µg/mL, Gibco), amphotericin B (2.5 µg/mL, Gibco), and gentamicin (50µg/mL, Gibco) and processed as described below in the SNEC culture at the air-liquid interface section.

SNEC Culture at the Air-Liquid Interface (ALI)

Sinonasal epithelial cells were grown at the air-liquid interface as previously described21. Briefly, epithelial cells were isolated from tissue samples by enzymatic degradation and grown in cell culture. Once confluent, cells were trypsinized then resuspended in Bronchial Epithelial Growth Media (BEGM) and plated onto human type IV placental collagen (Sigma, Type VI) coated 12-well Falcon filter inserts (0.4-µm pore size; Becton Dickinson, Franklin Lakes, NJ). When confluent, media was removed from above the cultures and the media below the inserts was changed to LHC Basal Medium:DMEM-H (Gibco) (50:50) containing the same concentrations of additives as BEGM with the exception that the concentration of epidermal growth factor was reduced to 0.63 ng/mL, and penicillin, gentamicin, streptomycin and amphotericin B were omitted (ALI media). Each set of SNEC cultures came from a separate patient source and was maintained at the air-liquid interface with the apical surfaces remaining free of medium for at least 3 weeks prior to study. This differentiated cell culture model, with media in the basolateral compartment and air at the apical surface, most closely resembles nasal cavity mucosa.

Co-Culture of SNEC with S. aureus of SNEC in culture

Staphylococcus aureus was obtained from ATCC (strain: ATCC 6538). S. aureus from a frozen stock was grown overnight in Luria Broth (LB) (Fisher Scientific). An aliquot was resuspended in the SNEC antibiotic free air-liquid interface media (ALI media) described above and then diluted 1:1000 into fresh ALI media or LB and grown at 37°C, 300RPM.

SNECs were grown in culture in the air-liquid interface system described above on antibiotic-free media for at least 3 weeks. S. aureus was grown at 37°C, 300RPM to a density of 107 cfu/mL (log-phase growth) in ALI media and then transferred to the apical surface of the SNEC for 1-hour at various concentrations. ALI-media alone was added to the apical surface of SNEC for 1-hour as a negative control. After 1-hour, the apical supernatant containing the bacteria was aspirated and SNEC-Staph aureus co-cultures were grown for 24-hours at 37°C, 5%CO2.

For wells used for RNA collection, or the LDH assay, after 24-hours of growth, the apical chamber was washed in 1X PBS for 10 minutes at 37°C, 5% CO2. The PBS was collected from the apical surface, and centrifuged for 1 minute at 15,000 RPM and aliquots were used to determine LDH secretion as described in the LDH assay section below. The basal ALI media was collected, spun down for 1 minute at 15,000 RPM and frozen at −80°C for later use with the ELISA assay. RNA was extracted from the SNEC as described below.

For SNEC wells used to determine S. aureus concentrations, the basal media was aspirated and 300µL of 1X PBS containing 0.1% TritonX-100 (EMD Bioscience), which will lyse the SNEC but not the S. aureus (capturing both intracellular and extracellular bacteria), was added to the apical surface and the SNEC were manually scraped off the inserts into tubes and incubated for 5 minutes at 37°C, 300RPM. Samples were then centrifuged at 10,000RPM for 1 minute, supernatant was discarded and the cell pellet was resuspended in 300µL of 1X PBS. Aliquots were then used to determine colony forming units per milliliter as described below.

Quantifying Bacterial Density

To determine bacterial density, the number of colony forming units per milliliter (cfu/mL) were measured. 100µL of culture was collected, then serially diluted in increments of 1:10 in 1x PBS (Fisher Scientific). 10µL of each dilution was spot plated in duplicate or triplicate onto LB-agar (Fisher Scientific) plates, and the plates incubated at 37°C overnight. Only dilutions that yielded between 10–100 colonies were counted, and cfu/mL values were calculated using the formula: [(#colonies)*(dilution factor)] / (volume plated in mL).

Sinonasal Epithelial Cell Death (LDH assay)

1X PBS was incubated and then collected from the apical surface of SNEC grown with and without S. aureus for 24 hours as described above. LDH release was determined using an LDH Cytotoxicity Assay Kit (Pierce; Rockford, IL) with 50µL of sample in triplicate in 96-well plates (Corning Costar 9018) according to the manufacturer instructions. Absorbance from each well was read using a microplate reader at 490nm (signal) and 680nm (background), and background was subtracted from signal (A490-A680). Negative controls (1X PBS, ALI media) and the provided LDH positive control were run in triplicate for each run.

Real Time Polymerase Chain Reaction (PCR)

Total RNA was extracted from SNEC following 24 hours of treatment in the presence or absence of S. aureus. using the RNeasy Kit by Qiagen according to the manufacturer’s directions. DNAse I (Qiagen) was used to treat RNA to remove contaminating genomic DNA. RNA concentration was determined by measuring the OD values at 260 nm. cDNA was synthesized from isolated mRNA by reverse transcribing 500 ng of RNA in a reaction volume of 20 µL using random hexamer primers (Invitrogen) and reagents from the Omniscript RT kit (Qiagen).

Real time PCR analysis was performed using the Applied Biosystems StepOnePlus machine (Foster City, CA) under standard cycling parameters for SYBR Green or Taqman per the manufacturer recommendations. For IL-8, IL-25, IL-33 and TSLP (eTable 1) the reaction mix consisted of 50 ng total RNA (IL-8, IL-25, TSLP) or 100ng total RNA (IL-33), or 5 ng total RNA (18S RNA), 10 µL of SYBR Green PCR, 1.5–5mol/L target primers, or 1.0 mol/L 18S rRNA primers, in a total volume of 20 µL. Each PCR run was accompanied by housekeeping gene 18s as an internal control, and a negative control, consisting of all components of the reaction mixture excluding target RNA. For TNFα, the reaction mixture consisted of 100ng total RNA, Taqman primers (Life Technologies - see eTable 1), and Taqman Fast Universal PCR Master Mix (Applied Biosystems) according to manufacturer recommendations. A Corresponding 18S Taqman control was also run using 5ng total RNA according to the manufacturer’s recommendations. Amplicon expression in each sample was normalized to its 18S RNA content, and the level of expression of target mRNA was determined as the delta CT (ΔCT), the difference in threshold cycles for each target and housekeeping gene. All primers were commercially synthesized by Life Technologies (eTable 1).

Enzyme Linked ImmunoSorbent Assays (ELISA)

Basal secretion of IL-8 was quantified using commercially available ELISA kits (eBioscience; San Diego, CA). Basal ALI media from SNEC from control, CRSsNP and CRSwNP patients was collected after 24 hours of growth with no staph, 103, 105, or 107 cfu/mL. Samples were centrifuged for 1 minute at 15,000 RPM and frozen at −80°C. The hIL-8 Instant ELISA kit (BMS204/3INST) was used according to manufacturer instructions. Briefly, negative control samples (SNEC grown with no bacteria) were diluted 1:10 in sample buffer, samples grown with S. aureus were diluted 1:100 in sample buffer. The assay was carried out according to manufacturer instructions using the provided pre-coated 96-well plates. Samples were read on a microplate reader at 450nm (signal) and 620nm (background). A standard curve of IL-8 provided with the kit was run in duplicate with each reaction set. Concentration of IL-8 for samples were determined by fitting to the standard curve. Mean +/−sem was calculated for control, CRSsNP and CRSwNP samples.

Statistical Analysis

Raw data from real-time PCR, cfu/mL experiments, ELISA and LDH assays were entered into a spreadsheet (Excel; Microsoft Corp, Redmond, WA). Statistical analysis was performed using a software program (Excel; Microsoft Corp, Redmond, WA). Data are expressed as mean ± SEM. Statistical significance of differences between the same populations with and without infection was determined using the Student’s paired t-test. Differences between different populations were evaluated by employing the two-tailed t-test for sample means with unequal variance. Differences were considered statistically significant at p<0.05.

Results

Growth of Staphylococcus aureus in SNEC culture media

To confirm that Staphylococcus aureus can grow in the SNEC air-liquid interface media, S. aureus was grown in fresh LB media or fresh ALI media. Colony forming units per mL were determined at various time points. Our results show that S. aureus is able to grow in ALI media (Figure 1A). Growth occurs at a slower rate and to a lower overall density. This is likely related to differences in nutrient levels between the two different types of media.

Figure 1. Growth of Staphylococcus aureus in LB and ALI media.

Figure 1

Open in a new tab

(A) S. aureus growth in ALI media (black squares)) compared to Luria Broth (open diamonds) at 37°C, 300RPM. S. aureus grows in the ALI media at a slower rate and to a lower density in ALI media compared to LB. (B) Co-culture staph growth 1 hr inoculation Shown are mean +/− s.e.m.

Co-culture of Staphylococcus aureus with human Sinonasal Epithelial Cells

We next confirmed that S. aureus will grow at the air-liquid interface of human SNECs. SNECs were grown in culture in the air-liquid interface system described in the Materials and Methods section above. S. aureus was transferred to the apical surface of the SNEC for 1-hour at concentrations of 107 cfu/mL, 105 cfu/mL, or 103 cfu/mL. These concentrations of S. aureus were chosen as there are data to suggest that there are at least 103 cfu/mL of bacteria present in an acutely infected sinus22 and to begin to explore if sinonasal epithelial cells can detect S. aureus in a concentration-dependent fashion.

After 1-hour, cfu/mL were determined for the S. aureus remaining on the apical surface, the bacteria present in the supernatant, and bacteria grown in ALI media in a culture tube. There was no difference between growth of S. aureus in a culture tube and growth of S. aureus in the supernatant (Figure 1B). After removal of the supernatant, about 10% of the S. aureus remain on the apical surface of the SNEC (Figure 1B). These data demonstrate that S. aureus remains present and can grow on the air-liquid interface which may better mimic growth of bacteria in vivo on sinus mucosa in a non-submersed system.

The density of S. aureus on the apical surface of the SNEC following the initial 1-hour inoculation with 107, 105 or 103 cfu/mL was measured following 24 hours of growth (Figure 2A). We did not observe a difference between growth of S. aureus on control SNEC compared to CRS SNEC (Figure 2A). These data show that S. aureus can grow equally well on control versus CRS SNEC.

Figure 2. S. aureus growth on SNEC and SNEC viability as a function of bacterial inoculum.

Figure 2

Open in a new tab

The apical surface of Sinonasal Epithelial Cells (SNEC) in the ALI culture system were inoculated with S. aureus at a concentration of 107 cfu/mL, 105 cfu/mL or 103 cfu/mL for 1-hr, followed by aspiration of supernatant and then growth of SNEC in culture. (A) Growth of S. aureus (cfu/mL) after 24 hours at the air-liquid interface of SNEC. No significant difference (p>0.05) between S. aureus grown on SNEC from CRS patients (black bars) versus SNEC from control patients (open bars). (B) SNEC viability as measured by apical LDH secretion (Absorbance 490nm–680nm) relative to S. aureus inoculum. No significant difference (p>0.05) in SNEC viability from CRS patients (black bars) compared to SNEC from control patients (open bars) exposed to S. aureus. Inset depicts LDH positive control, 1X PBS negative control and 1X ALI media negative control).

SNEC cell death following co-culture with S. aureus

Viability of SNECs was assessed after 24 hours of co-culture with S. aureus via LDH release from the apical surface (see Materials and Methods). LDH is released by dying mammalian cells and is used as a marker of cytotoxicity. There was an increase in LDH secretion as a function of initial bacterial loading dose compared to growth in the absence of bacteria (Figure 2B). For each initial loading dose of S. aureus, there was not a statistically significant difference (p< 0.05) in LDH secretion between CRS and control SNEC (Figure 2B). These data show that SNEC cell death increases as bacterial loading dose is increased and further suggests that there is no difference in the overall viability between control and CRS SNEC treated with bacteria.

S. aureus does not upregulate Th-2 Stimulating Cytokines

We next examined changes in expression of inflammatory mediators in SNEC from control, CRSsNP and CRSwNP patients following 24-hours of growth with S. aureus at the air-liquid interface. We measured expression of Th-2 mediators of inflammation IL-25, TSLP and IL-33 (Figure 3) as well as the pro-inflammatory cytokines, TNFα and IL-8 (Figure 4). There were no significant changes in expression (P > 0.05) for TSLP or IL-25 for control, CRSsNP, or CRSwNP SNEC co-cultured with bacteria compared to untreated SNEC (Figure 3A,B). There was not a significant difference (p >0.05) in expression of IL-33 for control SNEC or CRSwNP SNEC co-cultured with bacteria compared to untreated SNEC (Figure 3C). CRSsNP, however, did demonstrate a significant increase in IL-33 expression after inoculation with 107 cfu/mL of S. aureus (p = 0.05).

Figure 3. Expression of Th-2 stimulating cytokines by SNEC following co-culture with S. aureus.

Figure 3

Open in a new tab

Expression of (A) TSLP, (B) interleukin-25 and (C) interleukin-33 from control (black bars), CRSsNP (blue bars) or CRSwNP (red bars) SNEC co-cultured with no staph, 103, 105, or 107 cfu/mL of S. aureus for 24 hours. Expression shown as mean ΔCt (one unit of ΔCt to a 2-fold change in expression) +/− sem.

Figure 4. Expression of inflammatory cytokines TNFα and interleukin-8 by control, CRSsNP and CRSwNP SNEC following co-culture with S. aureus.

Figure 4

Open in a new tab

Expression of (A) TNFα and (B) interleukin-8 from control (black bars) SNEC co-cultured with no staph, 103, 105, or 107 cfu/mL of S. aureus for 24 hours. Expression of (C) TNFα and (D) interleukin-8 from CRSsNP (black bars) or CRSwNP (white bars) SNEC co-cultured with no staph, 103, 105, or 107 cfu/mL of S. aureus for 24 hours. Expression shown as mean ΔCt (one unit of ΔCt to a 2-fold change in expression) +/− sem. (* p-value <0.05).

CRSsNP SNEC express pro-inflammatory cytokines with a smaller burden of S. aureus

Expression of the pro-inflammatory cytokines TNFα and IL-8 were also examined to assess the ability of this system to detect changes in epithelial cell activity as a function of co-infection. TNFα and IL-8 are important mediators of inflammation and play a role in the early recruitment of neutrophils and other lymphocytes in response to bacterial infection of mucosal surfaces12,13. Up-regulation of these cytokines has been demonstrated in a submersed tissue culture model of CRS epithelial cells treated with Streptococcus pneumonia23 and varying levels of these cytokines has been demonstrated in tissue and mucus from those with CRS10. Baseline expression of TNFα and IL-8 were similar among SNEC from control, CRSsNP or CRSwNP patients (Figure 4A,C). For control SNEC, there was a trend toward increased expression of TNFα as a function of initial bacterial burden (range 6-fold to 106-fold increase), however, a significant increase in expression (p<0.05) was only seen following co-culture with 107 cfu/mL of S. aureus (Figure 4A). A similar trend of increased TNFα expression following co-culture with S. aureus was also observed with CRSwNP SNEC (Figure 4C).

CRSsNP display a significant increase in expression of TNFα after co-culture with all initial loading densities of S. aureus (Figure 4C). Furthermore, there was a significant increase (p = 0.02) in TNFα expression between CRSsNP and control SNEC following co-culture with 103 cfu/mL (5-fold difference in expression) or 105 cfu/mL (8-fold) of S. aureus. (Figure 4C).

A similar pattern of expression changes was observed for IL-8 expression with respect to control, CRSsNP and CRSwNP SNEC (Figure 4B,D). For control SNEC, there was a significant increase in expression (p <0.05) of IL-8 (range 3-fold to 46-fold) as a function of initial bacterial burden following co-culture with 105 cfu/mL or 107 cfu/mL of S. aureus (Figure 4B). Again, CRSsNP display a significant increase in expression of IL-8 after co-culture with all initial loading densities of S. aureus (Figure 4D). Furthermore, there was a significant increase (p = 0.02) in IL-8 expression between CRSsNP and control SNEC following co-culture with 103 cfu/mL (11-fold difference in expression) and a near significant increase in expression (p = 0.06) with 105 cfu/mL (6.8-fold) of S. aureus. There was no difference (p > 0.05) in expression of IL-8 between CRSwNP and control SNEC co-cultured with S. aureus.

These data demonstrate that, as has been shown in other mucosal systems, our system is capable of detecting changes in inflammation associated with bacterial infection. Furthermore, our data suggest that CRSsNP sinonasal epithelial cells are more sensitive to S. aureus burden and further suggests that CRSwNP cells behave more like controls which is consistent with more of a Th-2 type response driving CRswNP as compared to CRSsNP.

CRSsNP SNEC secrete pro-inflammatory cytokines with a smaller burden of S. aureus

To confirm that changes in expression of pro-inflammatory cytokines result in changes in secretion of cytokines, we measured secretion of Interleukin-8 into the basal compartment (See Materials and Methods). Secretion into the basal compartment was chosen as movement of cytokines in this direction in vivo would propagate the signals into the underlying stromal tissue and result in recruitment of neutrophils, macrophages and other lymphocytes. We found that secretion of Interleukin-8 into the basal compartment is significantly increased (p < 0.05) for CRSsNP SNEC from a basal secretion of 4ng/mL, to 16ng/mL with 103 cfu/mL S. aureus, to 30ng/mL with 105 cfu/mL and up to 60ng/mL with 107 cfu/mL S. aureus (Figure 5). Control and CRSwNP SNEC exhibited a significant increase (p < 0.05) in IL-8 secretion only after e xposure to 107 cfu/mL S. aureus (Figure 5). Control SNEC secreted IL-8 (62ng/mL) at a comparable level to CRSsNP (60ng/mL) at this high bacterial burden compared to CRSwNP which secreted less IL-8 (31ng/mL) (Figure 5). These data support the premise that CRSsNP SNEC are more sensitive in their response to S. aureus than control or CRSwNP SNEC.

Figure 5. Basal secretion of inflammatory cytokine IL-8 from SNEC following co-culture with S. aureus.

Figure 5

Open in a new tab

Secretion of IL-8 (ng/mL) into the basal compartment following co-culture of control (black bars), CRSsNP (blue bars) or CRSwNP (red bars) SNEC with no staph, 103, 105 or 107 cfu/mL S. aureus.

Discussion

In this study, we have developed a sinonasal epithelial cell co-culture system to study early innate-immune mediated responses of sinonasal epithelial cells that are associated with bacterial infection. We have demonstrated that sinonasal epithelial cells can detect S. aureus burden, and that sinonasal epithelial cells derived from CRSsNP patients are more sensitive to S. aureus burden than control or CRSwNP sinonasal epithelial cells. The epithelial cell response elicited by S. aureus appears to be primarily directed toward stimulating recruitment of neutrophils and lymphocytes through production of interleukin-8 and TNFα. This is consistent with the histology of CRS, which is characterized in part by neutrophil and lymphocyte infiltrates and fibrosis, with CRSwNP also having an eosinophilic component.24

In the co-culture model system, the apical surface of the differentiated epithelial cells is submerged in media containing S. aureus to allow time for the bacteria to settle. The apical liquid is then removed, and the remaining bacteria are left exposed at the air-liquid interface. Biofilm formation with Pseudomonas aeruginosa has been demonstrated at the air-liquid interface in a mouse sinonasal epithelial cell model,25 and it is possible that S. aureus forms biofilms in our system under the “harsher” conditions of the air-liquid interface. We have also observed that the final S. aureus densities are similar after 24 hours of growth (Figure 2A), implying that the inoculating density and time of exposure to higher densities of bacteria are driving the observed inflammatory changes (Fig 3–5).

Despite the fact that sinonasal epithelial cells grown in culture lack external signals from the host adaptive immune system, our results demonstrate that stimulation of sinonasal epithelial cells by S. aureus can be stratified by the clinical phenotypes of control, CRSsNP and CRSwNP. This suggests that sinonasal epithelial cells have “inflammatory” memory and that CRSsNP epithelial cell are primed to respond to lower levels of bacteria. While the individual innate immune components, such as toll-like receptors and other pattern recognition receptors, lack antigen specificity or immunologic memory, it is possible that the sinonasal epithelial cells maintain an altered but stable pattern of expression and production of innate immune mediators, perhaps as a result of chronic maladaptive inflammatory changes.

Of note, CRSwNP SNEC exposed to a high burden of S. aureus secrete significantly less IL-8 compared to control or CRSsNP SNEC (Figure 5). Our results suggest that in contrast to CRSsNP epithelial cells, CRSwNP epithelial cells are unable to mount a robust inflammatory response to bacteria, which agrees with previous research suggesting innate antimicrobial immune responses are blunted in CRSwNP, perhaps predisposing to bacterial or fungal overgrowth.26,27

Treatment of CRS exacerbations often begins with a trial of antibiotic therapy, which in theory is aimed at reducing the overall bacterial burden. In this study, CRSsNP sinonasal epithelial cells activate inflammatory pathways at a lower bacterial burden than control or CRSwNP SNEC. In an acute setting, the ability to detect bacteria at a lower threshold may be beneficial by generating an appropriate cytokine response and subsequent host inflammatory cascade that could theoretically reduce the need for extrinsic antimicrobial agents to help eradicate an infection. However, in chronic inflammatory conditions, repetitive stimulation with an irritant can lead to pathologic changes in the affected tissue as well as maladaptive inflammatory responses that sustain a chronic inflammatory state28. It is possible that as a result of chronic inflammation in patients with CRSsNP, these epithelial cells maintain the ability to detect and trigger inflammatory responses with lower bacterial burdens that results in a vicious cycle of sustained inflammation. This implies that a greater reduction of bacterial burden may be needed to alleviate bacteria-driven inflammation in CRSsNP patients. Furthermore, eradication of pathogenic bacteria may be more difficult in CRS patients where bacteria can exist in more exclusive niches (biofilms or in intracellular compartments).15,17 As such, current paradigms of antibiotic utilization which are effective in most cases of acute bacterial sinusitis in “control” patients may not effectively reduce the burden of bacteria to “sub-inflammatory” levels in bacterial CRS exacerbations.

Irrespective of the role of bacteria as a causal factor for CRS, antibiotics continue to have a central role in the treatment of active CRS29,30. While there are data to suggest that certain classes of antibiotics have direct anti-inflammatory properties31, the endpoint of antibiotic treatment is to reduce the burden of bacteria. In this study, we demonstrate that bacterial burden can be detected by sinonasal epithelial cells in the absence of adaptive immune-mediated responses. Furthermore, CRSsNP SNECs activate an inflammatory response at a lower initial inoculum of S. aureus than control or CRSwNP SNEC. This implies that a greater reduction of bacterial burden with antimicrobials may be needed to alleviate bacterially-mediated inflammation in CRSsNP patients.

Conclusions

In an air-liquid interface co-culture model system, bacterial burden can be detected by differentiated sinonasal epithelial cells in the absence of adaptive immune-mediated responses. Furthermore, CRSsNP SNECs are more sensitive to S. aureus density than control or CRSwNP SNEC. The increased sensitivity of CRSsNP sinonasal epithelial cells may be the result of stable, maladaptive changes to the innate inflammatory response pathways that help maintain a vicious cycle of sustained inflammation secondary to exposure to a bacterial irritant. Future studies will explore these ideas as well as the SNEC innate immune response to bacteria and further develop this co-culture infection model.

Supplementary Material

PCR primers

Acknowledgments

Research supported by NIH AI072502 (A.P.L.).

We would like to thank Gina Paris for her technical assistance with epithelial cell culture and qPCR techniques.

Footnotes

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Author Contributions

Conception and design by MAK and APL. Data acquisition and statistical analysis by MAK. Data analysis and interpretation by MAK and APL. Drafting and critical revision of manuscript by MAK and APL. Administrative, technical or material support by APL. Study supervision by APL.

References

  • 1.Meltzer EO, Hamilos DL, Hadley JA, et al. Rhinosinusitis: establishing definitions for clinical research and patient care. J Allergy Clin Immunol. 2004;114(6 Suppl):155–212. doi: 10.1016/j.jaci.2004.09.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Lane AP. The role of innate immunity in the pathogenesis of chronic rhinosinusitis. Curr Allergy Asthma Rep. 2009;9(3):205–212. doi: 10.1007/s11882-009-0030-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ramanathan M, Jr, Lane AP. Innate immunity of the sinonasal cavity and its role in chronic rhinosinusitis. Otolaryngol Head Neck Surg. 2007;136(3):348–356. doi: 10.1016/j.otohns.2006.11.011. [DOI] [PubMed] [Google Scholar]
  • 4.Hamilos DL, Leung DY, Wood R, et al. Evidence for distinct cytokine expression in allergic versus nonallergic chronic sinusitis. J Allergy Clin Immunol. 1995;96(4):537–544. doi: 10.1016/s0091-6749(95)70298-9. [DOI] [PubMed] [Google Scholar]
  • 5.Reh DD, Wang Y, Ramanathan M, Jr, Lane AP. Treatment-recalcitrant chronic rhinosinusitis with polyps is associated with altered epithelial cell expression of interleukin-33. Am J Rhinol Allergy. 2010;24(2):105–109. doi: 10.2500/ajra.2010.24.3446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Soumelis V, Reche PA, Kanzler H, et al. Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat Immunol. 2002;3(7):673–680. doi: 10.1038/ni805. [DOI] [PubMed] [Google Scholar]
  • 7.Mjosberg JM, Trifari S, Crellin NK, et al. Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161. Nat Immunol. 2011;12(11):1055–1062. doi: 10.1038/ni.2104. [DOI] [PubMed] [Google Scholar]
  • 8.Lam M, Hull L, McLachlan R, et al. Clinical severity and epithelial endotypes in chronic rhinosinusitis. Int Forum Allergy Rhinol. 2013;3(2):121–128. doi: 10.1002/alr.21082. [DOI] [PubMed] [Google Scholar]
  • 9.Nagarkar DR, Poposki JA, Tan BK, et al. Thymic stromal lymphopoietin activity is increased in nasal polyps of patients with chronic rhinosinusitis. J Allergy Clin Immunol. 2013;132(3):593–600 e512. doi: 10.1016/j.jaci.2013.04.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Oyer SL, Mulligan JK, Psaltis AJ, Henriquez OA, Schlosser RJ. Cytokine correlation between sinus tissue and nasal secretions among chronic rhinosinusitis and controls. Laryngoscope. 2013;123(12):E72–E78. doi: 10.1002/lary.24305. [DOI] [PubMed] [Google Scholar]
  • 11.Newton K, Dixit VM. Signaling in innate immunity and inflammation. Cold Spring Harb Perspect Biol. 2012;4(3) doi: 10.1101/cshperspect.a006049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Khair OA, Devalia JL, Abdelaziz MM, Sapsford RJ, Tarraf H, Davies RJ. Effect of Haemophilus influenzae endotoxin on the synthesis of IL-6, IL-8, TNF-alpha and expression of ICAM-1 in cultured human bronchial epithelial cells. Eur Respir J. 1994;7(12):2109–2116. doi: 10.1183/09031936.94.07122109. [DOI] [PubMed] [Google Scholar]
  • 13.Eckmann L, Kagnoff MF, Fierer J. Epithelial cells secrete the chemokine interleukin-8 in response to bacterial entry. Infect Immun. 1993;61(11):4569–4574. doi: 10.1128/iai.61.11.4569-4574.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Cohen NR, Lobritz MA, Collins JJ. Microbial persistence and the road to drug resistance. Cell Host Microbe. 2013;13(6):632–642. doi: 10.1016/j.chom.2013.05.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Suh JD, Cohen NA, Palmer JN. Biofilms in chronic rhinosinusitis. Curr Opin Otolaryngol Head Neck Surg. 2010;18(1):27–31. doi: 10.1097/MOO.0b013e328334f670. [DOI] [PubMed] [Google Scholar]
  • 16.Prince AA, Steiger JD, Khalid AN, et al. Prevalence of biofilm-forming bacteria in chronic rhinosinusitis. Am J Rhinol. 2008;22(3):239–245. doi: 10.2500/ajr.2008.22.3180. [DOI] [PubMed] [Google Scholar]
  • 17.Tan NC, Foreman A, Jardeleza C, Douglas R, Tran H, Wormald PJ. The multiplicity of Staphylococcus aureus in chronic rhinosinusitis: correlating surface biofilm and intracellular residence. Laryngoscope. 2012;122(8):1655–1660. doi: 10.1002/lary.23317. [DOI] [PubMed] [Google Scholar]
  • 18.Tan NC, Tran HB, Foreman A, Jardeleza C, Vreugde S, Wormald PJ. Identifying intracellular Staphylococcus aureus in chronic rhinosinusitis: a direct comparison of techniques. Am J Rhinol Allergy. 2012;26(6):444–449. doi: 10.2500/ajra.2012.26.3822. [DOI] [PubMed] [Google Scholar]
  • 19.Jardeleza C, Miljkovic D, Baker L, et al. Inflammasome gene expression alterations in Staphylococcus aureus biofilm-associated chronic rhinosinusitis. Rhinology. 2013;51(4):315–322. doi: 10.4193/Rhino13.045. [DOI] [PubMed] [Google Scholar]
  • 20.Cantero D, Cooksley C, Bassiouni A, Wormald PJ, Vreugde S. Staphylococcus aureus biofilm activates the nucleotide-binding oligomerization domain containing 2 (Nod2) pathway and proinflammatory factors on a human sinonasal explant model. Int Forum Allergy Rhinol. 2013;3(11):877–884. doi: 10.1002/alr.21213. [DOI] [PubMed] [Google Scholar]
  • 21.Ramanathan M, Jr, Lane AP. A comparison of experimental methods in molecular chronic rhinosinusitis research. Am J Rhinol. 2007;21(3):373–377. doi: 10.2500/ajr.2007.21.3034. [DOI] [PubMed] [Google Scholar]
  • 22.Wald ER, Milmoe GJ, Bowen A, Ledesma-Medina J, Salamon N, Bluestone CD. Acute maxillary sinusitis in children. N Engl J Med. 1981;304(13):749–754. doi: 10.1056/NEJM198103263041302. [DOI] [PubMed] [Google Scholar]
  • 23.Yoon BN, Choi NG, Lee HS, Cho KS, Roh HJ. Induction of interleukin-8 from nasal epithelial cells during bacterial infection: the role of IL-8 for neutrophil recruitment in chronic rhinosinusitis. Mediators Inflamm. 2010;2010:813610. doi: 10.1155/2010/813610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Van Bruaene N, Bachert C. Tissue remodeling in chronic rhinosinusitis. Curr Opin Allergy Clin Immunol. 2011;11(1):8–11. doi: 10.1097/ACI.0b013e32834233ef. [DOI] [PubMed] [Google Scholar]
  • 25.Woodworth BA, Tamashiro E, Bhargave G, Cohen NA, Palmer JN. An in vitro model of Pseudomonas aeruginosa biofilms on viable airway epithelial cell monolayers. Am J Rhinol. 2008;22(3):235–238. doi: 10.2500/ajr.2008.22.3178. [DOI] [PubMed] [Google Scholar]
  • 26.Kirsche H, Niederfuhr A, Deutschle T, Fuchs C, Riechelmann H. Ratio of myeloid and plasmacytoid dendritic cells and TH2 skew in CRS with nasal polyps. Allergy. 2010;65(1):24–31. doi: 10.1111/j.1398-9995.2009.02174.x. [DOI] [PubMed] [Google Scholar]
  • 27.Ramanathan M, Jr, Lee WK, Spannhake EW, Lane AP. Th2 cytokines associated with chronic rhinosinusitis with polyps down-regulate the antimicrobial immune function of human sinonasal epithelial cells. Am J Rhinol. 2008;22(2):115–121. doi: 10.2500/ajr.2008.22.3136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Galli SJ, Tsai M, Piliponsky AM. The development of allergic inflammation. Nature. 2008;454(7203):445–454. doi: 10.1038/nature07204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lund VJ. Maximal medical therapy for chronic rhinosinusitis. Otolaryngol Clin North Am. 2005;38(6):1301–1310. x. doi: 10.1016/j.otc.2005.07.003. [DOI] [PubMed] [Google Scholar]
  • 30.Mandal R, Patel N, Ferguson BJ. Role of antibiotics in sinusitis. Curr Opin Infect Dis. 2012;25(2):183–192. doi: 10.1097/QCO.0b013e328350f728. [DOI] [PubMed] [Google Scholar]
  • 31.Harvey RJ, Wallwork BD, Lund VJ. Anti-inflammatory effects of macrolides: applications in chronic rhinosinusitis. Immunol Allergy Clin North Am. 2009;29(4):689–703. doi: 10.1016/j.iac.2009.07.006. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

PCR primers
NIHMS771057-supplement-PCR_primers.pdf (76.8KB, pdf)

RESOURCES