There is controversy regarding whether cystic fibrosis (CF) airway epithelial cells (AECs) are intrinsically proinflammatory. The objective of the current study was to characterize the inflammatory profiles of AECs from children with CF compared with cells from healthy control subjects. We obtained AECs from healthy children (12) and children with CF (27). Biochemical and functional characteristics were assessed by stimulating cells with IFNγ, LPS, a cocktail referred to as cytomix, which consists of IFNγ, IL-1β, TNF-α, and LPS, or with human rhinovirus (HRV). Cytokine production was assessed using ELISA. Apoptotic responses to HRV infection were measured via production of single-stranded DNA. Our results indicated that CF and healthy cells exhibited similar morphology in monolayer culture. CF cells constitutively produced greater amounts of IL-6, IL-1β, and prostaglandin E2, but similar levels of IL-8 and soluble intracellular adhesion molecule–1 compared with healthy cells, and this profile was maintained through repeated passage. Stimulation with LPS or cytomix elicited similar levels of IL-8 in CF and non-CF cells. In contrast, exposure to HRV1b resulted in a marked increase in IL-8 production from CF compared with non-CF cells. CF cells also exhibited reduced apoptosis and increased viral replication compared with non-CF cells after exposure to HRV1b. We conclude that CF and healthy AECs have similar basal and stimulated expression of IL-8 in response to proinflammatory stimuli, but elevated IL-8 release in response to HRV infection. The elevated IL-8, together with dampened apoptotic responses by CF cells to HRV, could contribute to augmented airway inflammation in the setting of recurrent viral infections early in life.
In cystic fibrosis (CF), lung disease presents early in life (1, 2), and is characterized by neutrophilic inflammation in the airway (2, 3), progressive tissue damage (4), and chronic bacterial colonization in the majority of patients (4). However, the mechanisms underlying these pathological changes in the lung are not well understood.
Airway epithelial cells (AECs) are of particular interest, because they provide a physical barrier for the pulmonary microenvironment, and are actively involved in the host defenses that can contribute to airway disease (5). However, whether the airway epithelium in CF has an inherent proinflammatory capability and contributes directly to the early inflammatory responses observed in CF is still unclear. The conflicting data that have been published to date probably reflect the diverse models used to investigate the proinflammatory potential of the epithelium in CF and have included immortalized cell lines (6, 7), animal models (8, 9), ex vivo lung sections (10), or nasal polyps (11) from subjects with significant lung disease. These data have been extensively reviewed by Machen (12), leading the author to conclude that technical artifacts have precluded a definitive determination regarding whether the CF epithelium is proinflammatory. No studies, to our knowledge, have examined tissue from young children with minimal lung disease. Despite this observation, there remains a widely held belief that AECs from children with CF are inherently proinflammatory.
We used a unique, early respiratory disease surveillance program (2, 3) to obtain AECs from young children with CF using translaryngeal, nonbronchoscopic, and bronchoscopic sampling methods. AECs were also obtained from healthy control subjects, as we have described previously (13–15). The aim of the study was to test the hypothesis that AECs from children with CF are inherently proinflammatory. We compared responses of primary cell cultures from patients with CF and healthy control subjects to a variety of relevant stimuli, including human rhinovirus (HRV). Because our data are generated using AECs derived from the lower airways of young children with CF with mild airway disease, we have overcome many of the limitations of previous studies. Therefore, we believe that our data are likely a better representation of the in vivo characteristics of the CF epithelium in young children than seen in previous studies. Furthermore, because our study specifically examine epithelial responses of CF cells to HRV, a ubiquitous pathogen in the first years of life, the data have particular relevance to early mechanisms of airway inflammation. Some of the results generated from this study have been previously reported in the form of conference abstracts (16–19).
Please refer to the online supplement for full details.
Control subjects were healthy children admitted for elective surgery for nonrespiratory conditions (13–15). Children with CF were recruited during annual early surveillance visits (2, 3). Children with CF carried at least one Phe508 del allele, and 48% were homozygous. The study was approved by the Princess Margaret Hospital for Children Ethics Committee, and written consent was obtained from parents or guardians. Subject demographic data are provided in Table 1.
Phenotype (n subjects)
CF (n = 27)
Healthy Nonatopic (n = 12)
|Median age, yr (range)||4.1 (1.0–7.1)||7.2 (4.4–14.9)|
|Males, n (%)||13 (48)||5 (42)|
|Phe508del homozygous, %||48||N/A|
|Microbiology, n (%)||N/A|
|Uninfected (<102 CFU/ml)||14 (52)||—|
|Mixed oral flora only||6 (22)||—|
|Infected (>104 CFU/ml)||—||—|
|Average detectable IL-8 in BAL (mean ± SD, pg/ml)||1,141.5 ± 1,333.26||N/A|
|Average neutrophils in BAL (mean ± SD, ×103/ml)||207.1 ± 431.38||N/A|
Epithelial cells were obtained and cultured in monolayers, as previously described (13–15). Two human bronchial epithelial cell lines, 16HBE14o− and CFBE41o− (provided by Dr. Gruenert, University of California, San Francisco, California) were used to optimize experimental conditions and as comparisons with primary cells (results presented in the online supplement). The primary cell cultures were designated as healthy (pAECHNA) or CF (pAECCF).
After expansion of cultures, cells were seeded onto 12-well plates and grown in bronchial epithelial cell basal medium (BEBM; Lonza, Clonetics, Walkersville, MD) with supplements and growth factors until 90% confluence. At 24 hours before the experiment, cells were put in BEBM only, and then exposed to IFN-γ (500 U/ml), LPS (10 μg/ml), or a combination of stimuli (referred to as “cytomix” consisting of IFN-γ [500 U/ml], LPS [10 μg/ml], IL-1β [10 U/ml], and TNF-α [500 U/ml]) for up to 24 hours (20). Controls were incubated in BEBM only for the duration of the experiment. At the specified time points, supernatants and cells were collected separately and stored at −80°C.
AECs were grown in 96-well plates until 80% confluent in BEBM plus supplements and growth factors. HRV serotypes 14 and 1b (provided by Dr. Peter Wark, Hunter Medical Research Institute, Newcastle, NSW, Australia) was then added to the cells at a multiplicity of infection (MOI) of 3 and 25, and incubated at 37°C for up to 48 hours. At specified times, supernatants and cells were collected for analysis. Cell viability was assessed using a CellTiter 96 assay (Promega, Madison, WI) and a single-stranded (ss) DNA apoptosis kit (Millipore, Billerica, MA) was used to detect the percentage of apoptotic cells after viral infection, as described by the manufacturer.
Expression of IL-8 (Becton Dickinson, Biosciences, San Diego, CA), IL-1β, transforming growth factor (TGF)–β1, soluble intracellular adhesion molecule (sICAM), and epidermal growth factor (EGF) (Invitrogen, Carlsbad, CA) and prostaglandin E2 (PGE2) (R&D, Minneapolis, MN) were measured by ELISA. IL-6 was measured using a time-resolved fluorometry detection system (21) (PerkinElmer, Waltham, MA). Basal cytokine data were normalized to the number of cells, whereas IL-8 and IL-6 values measured after viral infection were normalized to viable cells.
Viral replication was determined via quantitative PCR (qPCR) using the HRV Advanced Kit (PrimerDesign Ltd., Southampton, UK). Briefly, RNA was extracted from HRV-infected cells, quantified, and reverse transcribed into cDNA using HRV-specific primer. The cDNA was then used in a Taqman qPCR reaction (Applied Biosystems, Carlsbad, CA) using HRV primer-probe mix (PrimerDesign Ltd., SouthHampton, Hants, UK) and β actin as a housekeeping gene. Serial dilution of HRV-positive control was used to generate a standard curve from which viral copy number was calculated.
Data were tested for population normality and homogeneity of variance, followed by statistical analyses using Mann-Whitney nonparametric tests. Experiments were performed in triplicate, with a minimum of five patients per experiment for each cohort. Data are presented as means (±SE), and P values less than 0.05 were considered to be significant.
At the time of admission, the 12 healthy control children had no history of chronic respiratory disease, and were free of respiratory symptoms and/or bacterial or viral infections. Of the 27 children with CF (13 males), 6 children had, on bronchoalveolar lavage (BAL), detectable mixed oral flora, two were infected with Staphylococcus aureus, two with Aspergillus fumigatus, one with Moraxella catarrhalis, one with Stenotrophomonas maltophilia and one with Staphylococcus epidermidis (Table 1). None of the children with CF had evidence of infection with Pseudomonas aeruginosa.
Primary AEC cultures from children with CF and healthy control children were established according to our published protocol (13–15), and showed no morphological differences at initial or subsequent passages (Figure 1). As seen in Figure 2, there were no significant differences in the basal release of IL-8, sICAM-1, or TGF-β1 from pAECCF and pAECHNA (P > 0.05). In contrast, levels of IL-1β, IL-6, PGE2, and EGF were significantly greater in pAECCF compared with pAECHNA (P < 0.05). In general, this trend was maintained throughout serial passage (see Figure E1 in the online supplement), although the amount of TGF-β1 in pAECCF increased after the initial passage, and then declined markedly, whereas, in pAECHNA, the amount steadily increased over successive passage. In contrast, the amount of EGF in pAECCF decreased through serial cultures, but remained constant in pAECHNA.
Exposure to LPS or cytomix increased IL-8 release in pAECCF and pAECHNA in a time-dependent manner and to a similar magnitude (Figures 3A and 3B). In contrast, exposure to IFN-γ had no consistent effect in either cell type. Measurements of IL-6 from both healthy and CF cohorts were consistently higher in response to LPS or cytomix stimulation (Figure E2) compared with nonstimulated cells in each experiment. In contrast to the baseline levels (Figure 2), there was no significant differences in IL-6 levels between pAECCF and pAECHNA after stimulation.
To determine whether inflammatory responses are specific to a particular pathogen, we exposed cells to HRV14 and HRV1b in a range of MOI or sham-infected media control, and assessed the inflammatory responses by measuring IL-8 release. We observed an elevation of IL-8 in HRV1b-infected cells compared with noninfected pAECCF (Figure 4), with a statistically significant increase at 48 hours at MOI 25 (P < 0.05). In contrast, HRV14 infection had no statistically significant effect on IL-8 release (Figure E4; P > 0.05). Analysis of pAECHNA demonstrated a similar trend with elevated IL-8 release after HRV1b (Figure 4), and only a slight increase in IL-8 after HRV14 infection (Figure E4). The same pattern of elevated IL-8 release was also observed in both cohorts for IL-6 after HRV infection (Figure E5). However, the cytokine levels produced by pAECHNA were always lower than those produced by pAECCF.
We next investigated whether there was any association between IL-8 release and cell viability, and found that, in pAECCF, the IL-8 response was significantly related to cell viability (R2 = 0.38; P < 0.05; Figure 5A), whereas viability had no effect on IL-8 release from pAECHNA (R2 = 0.03; Figure 5B). Infection of pAECCF with HRV1b induced an MOI-dependent reduction in cell viability at all time points (Figure 6), such that cell viability was reduced to approximately 16% of control values by 48 hours after infection at an MOI of 25 (P < 0.05). Similar findings were seen in pAECHNA, although the impact of infection on cell viability was significantly less than observed in pAECCF (P < 0.05). At early time points, HRV14 infection of pAECCF did not significantly reduce cell viability (Figure E6). However, at 24 and 48 hours after infection, we observed a considerable cell death after HRV14 infection at an MOI of 25 (P < 0.05). Reduced cell viability was also observed in pAECHNA after HRV14 infection (Figure E6), but there appeared to be no marked cell death, even at the highest MOI at maximum exposure time. As HRV1b appeared more pathogenic than HRV14, subsequent apoptotic and viral replication analyses were performed in primary AECs in response to HRV1b infection at MOI 25.
Because apoptosis is an important mechanism used by cells to limit viral replication, and thus its spread (22), we assessed this in response to viral infection. Despite reduced viability in response to HRV1b, pAECCF had lower levels of apoptosis compared with healthy cells (Figure 7A). Furthermore, determination of viral copy number in HRV1b-infected samples after 48-hour exposure showed a significant increase (10-fold) of HRV1b expression in pAECCF compared with healthy cells (Figure 7B; P < 0.05).
We performed the same experiments on the cell lines, and found that untreated and IFN-γ stimulated CFBE41o− and 16HBE140− cells had very low or non detectable IL-8 levels. Similar to primary cells, we observed a time dependent production of IL-8 after either LPS or cytomix stimulation with the level in CFBE41o− being significantly higher than in 16HBE14o− (Figures E7A and E7B; P < 0.05). This observation was also true for IL-6 production (Figures E7C and E7D) whereby there was consistently low IL-6 in non-stimulated and IFNγ-stimulated cells at all time points, with measurable amount detected in response to LPS or cytomix stimulation.
We also infected CFBE41o− and 16HBE14o− with HRV and compared the responses to that of primary AECs. Infection with HRV14 or HRV1b had no effect on cell viability in either cell lines (Figures E8A–E8D). In addition to different viability profiles, exposure of immortalized epithelial cell lines to HRV resulted in different cytokine profiles to the primary AECs. For example, both cell lines released greater amount of IL-8 and IL-6 after HRV14 infection than HRV1b (Figures E9A–E9D). In addition, CFBE41o− released more IL-6 than 16HBE14o− (P < 0.05), whereas IL-8 production was similar in both cell lines. However, both cell lines produced significantly lower levels of cytokines than primary AECs under basal resting and in stimulated conditions.
The mechanisms for the neutrophilic airway inflammation that is characteristic of CF from early life are poorly understood. In the present study, we investigated functional differences between AECs from healthy control subjects or children with CF at rest and in response to various inflammatory stimuli. Our data indicate that, at baseline, pAECCF and pAECHNA produced similar levels of IL-8, sICAM-1, and TGF-β1, but released greater amount of IL-6, IL-1β, PGE2, and EGF. After treatment with LPS or cytomix, IL-6 and IL-8 responses of pAECCF were similar to pAECHNA. In contrast, pAECCF released greater IL-8 and IL-6 in response to HRV infection, with a positive correlation between cell viability and IL-8 release. Furthermore, CF AECs have dampened apoptotic response to HRV infection and increased viral copy number compared with healthy cells. This suggests a possible dysregulated innate response of CF AECs to viral infection, resulting in inflammation and the potential for initiating early lung damage in CF.
Although several studies have suggested that the airway epithelium in CF is inherently proinflammatory (4, 23–25), others have suggested that inflammation is an inconsistent finding (26–29). These studies have used a variety of methods, including immortalized cell lines, nasal polyps, lung tissue sections or CF mouse models, and a variety of inflammatory stimuli. This diversity of experimental approaches is likely to be responsible for the contradictory outcomes (12). Our contrasting data obtained from primary CF cells and CF cell lines further demonstrate the importance of the experimental model. To our knowledge, ours is the first study to examine primary lower AECs from children with CF. A previous study compared inflammatory responses of CF cells using bronchial epithelial cell lines and primary AECs from individuals with and without CF (28). However, the primary cell samples were obtained from heterogenic lung transplant recipients, and autopsies and are unlikely to be representative of the CF airway epithelium before changes secondary to chronic inflammation and infection have occurred. Our study is unique with regard to a number of important factors. The children with CF included in the study were young, with similar genetic mutations, and healthy control subjects were from a similar age range. None of the children with CF had any detectable endobronchial infection with P. aeruginosa. We chose to limit our studies to children with class II CF transmembrane conductance regulator mutation to reduce genetic variability (12), and because these mutations are consistently associated with lung disease (30). However, a limitation of this approach is that we have not had an opportunity to examine whether our observations are class II mutation dependent.
To answer the main objective of this study, which was to assess whether CF airway epithelium is inherently proinflammatory, we first determined cytokine profiles in both CF and healthy epithelial cell cultures, including panels of proinflammatory (IL-8, sICAM-1, and IL-1β), anti-inflammatory (IL-6, PGE2), as well as profibrogenic (TGF-β1, EGF). Our results showed that, at an early passage, there was no marked difference in expression levels of IL-8, sICAM-1, and TGF-β1, whereas higher levels of IL-1β, IL-6, and PGE2 were observed in CF compared with healthy subjects. Overall, these data demonstrate that CF AECs are not likely to initiate a neutrophilic inflammatory milieu in unstimulated conditions.
AECs are constantly exposed to a range of stimulants, including allergens, pollutants, pathogens, and respiratory viruses, and they respond by modulating their local microenvironment through secretion of cytokines and other proinflammatory mediators. To assess whether the CF epithelium responds abnormally to inflammatory stimuli, we exposed cells to a variety of stimuli, including LPS, proinflammatory cytokines, and a common respiratory virus: HRV. Respiratory viral infections have been associated with increased disease progression (31–33); and there are now prospective data indicating that HRV is the most common viral pathogen in pulmonary exacerbations in children with CF (34). Rhinoviral infection induces the expression of a range of proinflammatory cytokines, growth factors, and adhesion molecules in airway epithelium, including IL-8 (35), epithelial neutrophil activating protein-78 (ENA-78) (36), IL-6 (37), regulated upon activation, normal T cell expressed and secreted (38), interferon gamma induced protein-10 (IP-10) (39) and intercellular adhesion molecule-1 (ICAM-1) (40). Our data indicate that, whereas pAECCF and pAECHNA have similar cytokine responses to IFN-γ, LPS, and cytomix stimulation, exposure to HRV1b resulted in a sixfold greater amount of IL-8 and IL-6. We acknowledge that the responses of some children with CF might have been modified by prior endobronchial infections; however, cytokine responses of children with and without prior infections were similar (Figure E3). Therefore, we believe that using pooled data for subsequent analyses was justified. Our observations suggest that, even though pAECCF are capable of producing cytokines that can dampen inflammation, these responses are likely to be overwhelmed, because neutrophilic inflammation driven by IL-8 is dominant, even early in life (2, 3).
In contrast to the effects of HRV1b, exposure to major serotype HRV14 had little effect on either pAECCF or pAECHNA. Further investigation showed that these responses correlated with the effect of virus on cell viability. Although exposure to HRV14 had no effect on cell viability, exposure to HRV1b resulted in a significant decrease in cell viability. These findings are in line with previous studies showing that HRV1b is more pathogenic to epithelial cells than a range of other laboratory strains of HRV (41). Given the large number of strains of HRV and the variable responses to stimulation with available laboratory strains, future studies using community strains that have been demonstrated to cause significant respiratory morbidity would provide additional, clinically relevant information regarding the inflammatory response of the CF epithelium to HRV.
Importantly, despite having a greater effect on cell viability in pAECCF, levels of apoptosis in these cells were lower than that observed for pAECHNA, suggesting that CF cells undergo necrosis (42). In addition, because apoptosis is an important innate immune mechanism to limit viral replication (22), this would explain the increased viral copy number after infection in pAECCF compared with pAECHNA. We note that our data on viral copy number in HRV1b-infected AECs are within the range reported by Gerna and colleagues (43) in nasal aspirates and BAL from young patients. Another seminal study by Message and colleagues (44) in BAL further supports our observation, with an asthmatic cohort reported to have an approximately 0.5–2 Logs greater viral load than healthy individuals. Because necrosis tends to be proinflammatory by releasing cellular contents that promote inflammation, as opposed to apoptosis, which is tightly regulated and less inflammatory (42), a vicious cycle of infection and exaggerated IL-8 release could develop after HRV infection that, in turn, amplifies and/or prolongs inflammation. Our data suggest a plausible nexus between HRV infection and AEC-initiated inflammatory cell recruitment and activation, leading to early airway inflammation, and are consistent with data suggesting slower clearance of virus, increased severity, and prolonged inflammatory responses to respiratory viral infection in CF (31, 45).
We also performed similar experiments in cell lines 16HBE14o− and CFBE41o− commonly used as in vitro models of healthy and CF cells, respectively. Our observations indicate significant qualitative and quantitative differences in the responses between cell lines and primary cells, emphasizing the limitation of using cell lines to study inflammatory responses of CF epithelium, and the need for caution when extrapolating data to epithelial cell characteristics in vivo.
In conclusion, this study describes the use of pediatric lower AECs from patients with CF to investigate responses to inflammatory stimuli and HRV. Biochemical and functional analyses suggest that dysregulated inflammatory responses to HRV and defective apoptosis could result in an amplification cycle of epithelial infection, viral replication, and IL-8 release, resulting in augmented neutrophilic airway inflammation in the CF airway. These observations provide a rationale for early intervention to reduce the inflammatory responses to recurrent respiratory viral infection to prevent lung disease in CF. Further work is required to elucidate the role of innate immune dysfunction to HRV in CF, to determine whether other common respiratory viruses elicit similar responses, and to understand whether epithelial responses to virus infection are CF transmembrane conductance regulator mutation dependent.
The authors thank Jason Terpolili, Andrea Mladinovic, and Kak-Ming Ling for technical assistance.
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