Abstract
Rationale: Cystic fibrosis (CF) is characterized by progressive pulmonary inflammation that is infection-triggered. Pseudomonas aeruginosa represents a risk factor for deterioration of lung function and reduced life expectancy.
Objectives: To assess T-cell cytokine/chemokine production in clinically stable children with CF and evaluate the association between T-cell subtypes and susceptibility for infection with P. aeruginosa.
Methods: T-cell cytokine/chemokine profiles were measured in bronchoalveolar lavage fluid (BALF) from children with CF (n = 57; 6.1 ± 5.9 yr) and non-CF control subjects (n = 18; 5.9 ± 4.3 yr). Memory responses to Aspergillus fumigatus and P. aeruginosa were monitored. High-resolution computed tomography–based Helbich score was assessed. In a prospective observational trial the association between BALF cytokine/chemokine profiles and subsequent infection with P. aeruginosa was studied.
Measurements and Main Results: Th1- (INF-γ), Th2- (IL-5, IL-13), Th17- (IL-17A), and Th17-related cytokines (IL-1β, IL-6) were significantly up-regulated in airways of patients with CF. IL-17A, IL-13, and IL-5 were significantly higher in BALF of symptomatic as compared with clinically asymptomatic patients with CF. IL-17A and IL-5 correlated with the percentage of neutrophils in BALF (r = 0.41, P < 0.05 and r = 0.46, P < 0.05, respectively). Th17- (IL-17A, IL-6, IL-1β, IL-8) and Th2-associated cytokines and chemokines (IL-5, IL-13, TARC/CCL17), but not IFN-γ levels, significantly correlated with high-resolution computed tomography changes (Helbich score; P < 0.05). P. aeruginosa– and A. fumigatus–specific T cells from patients with CF displayed significantly higher IL-5 and IL-17A mRNA expression. IL-17A and TARC/CCL17 were significantly augmented in patients that developed P. aeruginosa infection within 24 months.
Conclusions: We propose a role for Th17 and Th2 T cells in chronic inflammation in lungs of patients with CF. High concentrations of these cytokines/chemokines in CF airways precede infection with P. aeruginosa.
Progressive pulmonary inflammation is a characteristic feature in cystic fibrosis. Several investigations revealed a crucial role for T cell–mediated responses in exacerbated patients with CF. The role of T cell–mediated background inflammation in the lung of patients with CF and its impact on infection with Pseudomonas aeruginosa and lung destruction have not been elucidated.
Our data suggest a crucial role for local Th2- and Th17-mediated inflammatory responses in the lung of patients with CF. IL-17A was discovered as a positive prognostic marker for infection with P. aeruginosa.
Cystic fibrosis (CF) is an autosomal-recessive disorder caused by different mutations in the CF transmembrane regulator (CFTR) gene, coding for a protein that mainly acts as a chloride channel (1). Because CFTR is expressed on the apical site of exocrine epithelial cells (2) CF affects multiple organs, but the main life-limiting consequence is a progressive pulmonary inflammation. Inflammation in CF is mainly mediated by neutrophils present in bronchoalveolar lavage fluid (BALF), but in the submucosa, large aggregates of T cells can be found (3).
Investigations on T cell–mediated responses support the concept that CF is predominately a Th2-mediated disease (4–6). However, the discovery and characterization of Th17 cells added significantly to the understanding of T cell–mediated responses.
Th17 cells are a subset of T-helper cells that are induced by responses to transforming growth factor (TGF)-β (7) in combination with IL-6, IL-21, and IL-23 (8). They are defined by their transcription factor (RORγt) and by their cytokine/chemokine profiles, mainly IL-17A. IL-17A is a proinflammatory cytokine involved in granulopoesis, recruitment, activation and migration of neutrophils into sites of inflammation (9, 10). It leads to the induction of multiple other proinflammatory cytokines/chemokines, such as tumor necrosis factor-α, IL-6, IL-8, and granulocyte-macrophage colony–stimulating factor (11, 12), which in turn operate on other effector cells to promote and sustain inflammation. The primary mechanisms of action of Th17 cells are not yet fully understood. They seem to clear pathogens that are not adequately handled by Th1 or Th2 cells (13). In host defense against bacteria and fungi (14–17) Th17 cells have been shown to be crucial (18).
Th17 cells have been implicated in various immune-mediated diseases (19). Recently, IL-17A has also been linked to CF and in particular to pulmonary exacerbations and neutrophils (20–22). The nature of T cell–mediated background inflammation in the lung of patients with CF, its role in lung destruction, and its associations with Pseudomonas aeruginosa infection remain to be investigated.
The present study examined the role of T cell–mediated cytokine/chemokine responses in CF lung disease and assessed in a prospective observational study the association between BALF cytokine profiles and subsequent infection with P. aeruginosa.
Some of the results of these studies have been previously reported in the form of an abstract (23–26).
The protocol was approved by the local institutional review board (EK-Nr. 081/2007, 427/2008, 355/2009) and written informed consent was obtained from all patients and their parents or caregivers.
Patients were recruited from the outpatient CF clinic from the University Children’s Hospital of Vienna, Vienna, Austria. CF is being treated according to international guidelines (27) including 3-monthly visits where pharyngeal swabs and sputum was taken for microbiology. Additionally, we have introduced a bronchoscopy program where patients are invited for endoscopy on a yearly basis until they are able to produce reliable sputum samples.
BALF samples were obtained from 57 children with CF and a non-CF control group including 18 children with a history of recurrent pulmonary infections but in infection-free intervals. A detailed characteristic of these patients is shown in Table E1 in the online supplement.
Tissue samples (parenchymal and bronchial epithelial tissue) from explanted lungs were obtained from 10 patients with CF and 8 control subjects (lung fibrosis [LF] and primary pulmonary hypertension [PPH]). A detailed characteristic of these patients is shown in Table E2.
Peripheral blood samples from 14 patients with CF (n = 7 specific IgE to Aspergillus fumigatus; n = 9 P. aeruginosa colonized) without acute exacerbation and 8 healthy control subjects were taken to verify antigen-specific T-cell responses.
Clinical data on symptoms, lung function, pancreatic insufficiency, length, weight, medical treatment, signs of acute exacerbation, sensitization, symptoms of allergy or asthma, and total IgE were obtained from patient history. Microbial colonization was recorded by pharyngeal swabs or sputum (four times per year) or from BALF (once per year). A decline of lung function greater than 10%, start of an antibiotic therapy, increase in coughing, clinical signs of infection, or signs of infection in terms of C-reactive protein elevation or differential counts were considered as signs of pulmonary exacerbation (see online supplement).
For details, see online supplement.
For details, see online supplement.
Levels of IL-1β, -5, -6, -8, -13, -17A, IFN-γ, and TARC/CCL17 were measured by plate-bound multiplex ELISA (Aushon BioSystems, Billerica, MA) (see online supplement).
For details, see online supplement.
Expression levels of T-cell cytokines/chemokines (TGF-β, IL-6, IL-8, IL-17A) and transcription factors (RORc2, T-bet, GATA-3) in human lung tissue, and expression levels of antigen-specific cytokine production (IL-5, IL-17A, IFN-γ) of in vitro stimulated cells were determined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) (IQ SYBR Supermix; Bio-Rad, München, Germany) and performed on a CFX96 Thermal Cycler (Bio-Rad) (see online supplement for details and primer sequences).
For details, see online supplement.
For details, see online supplement.
To assess type and frequencies of P. aeruginosa– and A. fumigatus–specific T cells in 14 patients with CF and 7 healthy control subjects, ELISPOT analysis was performed (eBioscience, Frankfurt, Germany) (see online supplement).
For details, see online supplement.
Fifty-seven patients with CF (6.1 ± 5.9 yr; female/male, 32/25) and 18 control patients (6 ± 4.9 yr; female/male, 7/11) were enrolled. Compared with patients with CF experiencing pulmonary exacerbation (n = 16; 10 ± 7.4 yr; female/male, 10/6), clinically stable patients with CF (n = 41; 4.3 ± 4.1 yr; female/male, 15/26) were younger (P < 0.01; Mann-Whitney U test). No differences with regard to body mass index, z score for body mass index, frequency of pancreatic insufficiency, sensitization state, allergy or asthma symptoms, type of CFTR mutation, or medication with antiinflammatory drugs, DNase, bronchodilators, or inhaled corticosteroids were observed (Table 1). Thirteen patients with CF displayed specific IgE against A. fumigatus (n = 8 clinically stable). Ten patients with CF are A. fumigatus positive in BALF (n = 5 clinically stable).
| Characteristic | CF All | CF Clinically Stable | CF + Signs of Exacerbation | Control Subjects |
| N | 57 | 41 | 16 | 19 |
| Age, mean (SD), yr | 6.1 (± 5.9) | 4.3 (± 4.1) | 10.6 (± 7.4) | 6 (± 4.9) |
| Sex, female/male | 32/25 | 15/26 | 10/6 | 6/3 |
| Mutation | ||||
| ΔF508 homoz. | 29 | 22 | 7 | 0 |
| Comp. hetero. class I-III | 7 | 4 | 3 | 0 |
| Polym./CF-typic course | 12 | 8 | 4 | 0 |
| Comp. heteroz. class IV-V | 6 | 5 | 1 | 0 |
| Polym./mild course | 2 | 1 | 1 | 0 |
| Only pos. sweat test | 1 | 1 | 0 | 0 |
| Pancreatic insufficiency | 49 | 35 | 14 | 0 |
| Weight at enrollment | ||||
| Mean (SD), kg | 22.2 (± 16.8) | 17.1 (± 11) | 35.3 (± 21.8) | 23.1 (± 18) |
| Length at enrollment | ||||
| Mean (SD), m | 1.1 (± 0.3) | 1 (± 0.2) | 1.3 (± 0.4) | 1.1 (± 0.3) |
| BMI at enrollment | ||||
| Mean (SD) | 17 (± 2.5) | 16.6 (± 2) | 18 (± 3.5) | 16.3 (± 2.9) |
| Mean (SD), z score | −0.01 (± 0.1) | −0.02 (± 0.1) | 0.02 (± 0.1) | −0.002 (± 0.1) |
| Antiinflammatory drugs (LTR antagonists) | 0 | 0 | 0 | 6 |
| Daily bronchodilator use | 55 | 40 | 15 | 2 |
| DNase | 28 | 18 | 10 | 0 |
| Inhalative corticosteroids | 7 | 3 | 4 | 7 |
| Pseudomona aeruginosa pos. | 10 | 5 | 5 | 0 |
Because of basic therapy of CF-disease control patients had significantly less chronic bronchodilatator treatment and were not on DNase treatment. However, control subjects were significantly more often on leukotriene receptor antagonists and inhaled corticosteroids (Table 1).
To investigate subtypes of T cell–associated cytokines/chemokines in the lungs of patients with CF, a panel of cytokines/chemokines, including Th1-, Th2-, Th17-, and Th17-associated cytokines/chemokines was measured in BALF.
IL-17A was significantly elevated in patients with CF compared with non-CF control subjects (3.6 [6.15] vs. 0.59 [2.85]; median [IQR]; P < 0.001) (Figure 1). Moreover, the Th17-related cytokines IL-1β (12.59 [49.82] vs. 3.13 [9.91]; median [IQR]; P < 0.01) (Figure 1) and IL-6 (75.07 [116.43] vs. 13.19 [27.31]; median [IQR]; P < 0.0001) (Figure 1) were significantly higher in BALF of patients with CF as compared with non-CF control subjects. In parallel, significantly higher levels of IL-13 (1.39 [1.47] vs. 0.6 [0.48]; median [IQR]; P < 0.0001) (Figure 1) and IL-5 (CF + signs of exacerbation vs. control subjects) (1.39 [1.47] vs. 0.6 [0.48]; median [IQR]; P < 0.05) (Figure 1) were observed in CF as compared with non-CF control subjects. IL-6 and IL-8 correlated with levels of IL-17A (r = 0.59, P < 0.001; r = 0.83, P < 0.001; and r = 0.44, P < 0.05) (see Figure E1).
Figure 1. Cytokine/chemokine levels in bronchoalveolar lavage fluid of patients with cystic fibrosis (CF) differ from control subjects. Cytokine/chemokine levels of 57 patients with CF and 18 control subjects (CO) were measured by multiplex ELISA and influences of respiratory exacerbation (n = 16) were evaluated (Mann-Whitney U test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).
[More] [Minimize]To delineate exacerbation-related alterations in the local inflammatory cytokine/chemokine patterns from CF-specific background inflammation, subgroup analysis of patients with CF with and without signs of pulmonary exacerbation was performed (see Methods). Levels of IL-17A and levels of the Th2-related cytokines IL-5 and IL-13 were significantly higher in patients with CF with pulmonary exacerbation compared with patients with CF without signs of pulmonary exacerbation (Figure 1). In addition, IL-1β was higher in patients with CF with exacerbation as compared with clinically stable patients, however, not reaching significance (P = 0.07) (Figure 1). No influence of pulmonary exacerbations on the concentration of other cytokines/chemokines could be observed.
On analysis of confounding factors, A. fumigatus exposure (specific IgE to A. fumigatus [n = 13] or A. fumigatus in BALF [n = 10]) was found to have a significant influence on levels of IL-5, IL-6, IL-13, IFN-γ, and IL-17A (P < 0.01).
Th-17 cells are known to contribute to granulopoesis, recruitment, activation, and migration of neutrophils to sites of inflammation (9, 10). A significant correlation of IL-17A and neutrophil counts in BALF was observed in a subgroup of patients (n = 26, patients with CF; r = 0.41; P < 0.05) (Figure 2). In addition to IL-17A, IL-5 but none of the other cytokines that were investigated correlated with neutrophil counts in BALF (r = 0.46; P < 0.05). No correlation of IL-17A with patients’ age was detected (see Figure E2).
Figure 2. IL-17A in bronchoalveolar lavage fluid significantly correlates with the number of neutrophils in cystic fibrosis lung disease (Spearman test). Levels of IL-17A in patients with cystic fibrosis (n = 30) were measured by multiplex ELISA. The percentage of neutrophils in bronchoalveolar lavage fluid was determined by differential cell counts.
[More] [Minimize]It has recently been demonstrated that, in patients with CF, chronic infection with P. aeruginosa is associated with a Th2-type cytokine profile in the lung. However, it remained unclear whether this reflects a preexisting local cytokine milieu that predisposes to the acquisition of P. aeruginosa, or if it is solely associated with tolerance phenomena in chronic P. aeruginosa colonization (5). To address this question a prospective observational trial to assess risk cytokine/chemokine patterns for P. aeruginosa infection was conducted (Figure 3a; see Table E3 for detailed characteristics of patients). None of the patients who were assigned negative for P. aeruginosa and consequently enrolled in the prospective study were positive for P. aeruginosa within the last year before bronchoscopy and none of these patients has ever been chronically colonized with P. aeruginosa.
Figure 3. (a and b) Infection with Pseudomonas aeruginosa (PA) leads to significantly higher levels of IL-17A and TARC/CCL17. Cytokine/chemokine levels in bronchoalveolar lavage fluid were measured by multiplex ELISA and levels were compared between patients with cystic fibrosis (CF) who developed infection with P. aeruginosa (n = 14) and patients with CF who did not develop infection with P. aeruginosa (n = 24) within the observation period (24 mo) (Mann-Whitney U test; *P < 0.05).
[More] [Minimize]Fourteen out of 38 patients with CF developed infection with P. aeruginosa within the observation period. This particular subgroup displayed significantly higher levels of IL-17A (P < 0.05) and the Th2-type chemokine TARC/CCL17 (P < 0.05) as compared with patients not infected with P. aeruginosa (n = 24) during the 24 months of follow-up (Figure 3b).
Patients with IL-17A levels of the two upper quartiles corresponding to a cut off of 3.6 pg/ml had a significantly higher frequency of P. aeruginosa infection within the next 24 months as compared with those with IL-17A levels of less than 3.6 pg/ml (11 of 19 vs. 3 of 19; P < 0.05; Fisher exact test). No correlation between IL-17A and the time until infection was observed (see Figure E3). None of the other cytokines/chemokines (IL-1β, IL-5, IL-6, IL-8, IL-13, IFN-γ) were associated with a higher risk for infection with P. aeruginosa (see Figure E4).
The same analysis has also been performed for other CF-related pathogens (Haemophilus influenza, Staphylococcus aureus, Burkholderia cepacia, Candida albicans, and Streptococcus pneumoniea). In our study population, none of the other microbes seems to significantly influence cytokine levels.
Based on cross-sectional cytokine/chemokine measurements in BALF we raised the question whether these cytokine/chemokine levels are associated with structural changes in patients’ lung tissue. Therefore, computed tomography (CT) of the lung was conducted in a subgroup (n = 30). A significant correlation between the Th17-related cytokines/chemokines IL-17A, IL-1β, IL-8, and the Th2-type cytokines/chemokines IL-13, IL-5, and TARC/CCL17 with high-resolution CT–related radiologic changes, reflected in Helbich score, was observed (Figure 4). In addition, correlation of IL-17A with lung function was assessed. IL-17A negatively correlates with FEV1, maximal expiratory flow-50, and maximal expiratory flow-25 (r = −0.45, P < 0.05; r = −0.63, P < 0.001; and r = −0.46, P > 0.05, respectively) (see Figure E5).
Figure 4. Levels of Th17-associated cytokines/chemokines in bronchoalveolar lavage fluid strongly correlate with structural changes in computed tomography of patients with cystic fibrosis (Helbich score). Cytokine/chemokine levels were measured by multiplex ELISA and correlation with high-resolution computed tomography–based Helbich score was performed (Spearman test).
[More] [Minimize]To underline the in vivo importance of Th17 cells in end-stage CF lung disease expression levels of TGF-β, IL-6, IL-8, IL-17A, and the transcription factors RORc2, T-bet, and GATA-3 in parenchymal and bronchial epithelial lung tissue were assessed by real-time RT-PCR in explanted lungs. In bronchial epithelial tissue, IL-17A and IL-8 were significantly increased in patients with CF compared with control subjects (P < 0.01) (Figure 5). In CF lung disease, but not in other end-stage lung disease (LF and PPH), IL-17A expression was significantly higher in parenchymal lung tissue (P < 0.05). No differences were observed with regard to TGF-β, IL-6, RORc2, T-bet, or GATA-3.
Figure 5. IL-17A and IL-8 mRNA levels are significantly higher in lung tissue of patients with cystic fibrosis (CF) compared with control subjects. In parenchymal and bronchial epithelial lung tissue of patients with CF (n = 10) and non-CF control subjects (n = 8), expression levels of panel of T-cell cytokines/chemokines (transforming growth factor-β, IL-6, IL-8, IL-17A) and transcription factors (RORc2, T-bet, GATA-3) were compared by real-time reverse transcriptase polymerase chain reaction (Mann-Whitney U test; *P < 0.05; **P < 0.01).
[More] [Minimize]Because Th17 cells are able to induce deleterious inflammatory responses, a snapshot approach to prove the presence of these cells in lung tissue in vivo was performed using confocal microscopy. The presence of IL-17A–producing T cells and the respective receptor (IL-17RA) was assessed in cryosections of explanted lungs from CF (n = 7) and patients without CF (n = 7). IL-17A–producing T cells were detected in parenchymal lung tissue (Figure 6). Also, IL-13–producing cells were found in end-stage lung disease (see Figure E6). The IL-17A receptor was detectable in samples from both subgroups with higher expression in the CF group (Figure 6). In addition, CD45+ mononuclear cells from parenchymal lung tissue of explanted lungs were isolated and intracellular cytokine production was assessed to prove the existence of IL-17– and IL-13–producing T cells in the lung of patients with CF (see Figure E7).
Figure 6. IL-17A–producing cells are present in human lung tissue and IL-17 receptor A is up-regulated in bronchial epithelial cells of patients with cystic fibrosis (CF). Samples of explanted lungs from patients with CF (n = 7) and patients without CF (n = 7) were stained with fluorescence labeled antibodies against CD3, IL-17A, or IL-17RA. Images were acquired by a ×63 oil objective of a confocal scanning microscope. DAPI = 4′,6-diamidino-2-phenylindole; H = hematoxylin; IC = isotype control.
[More] [Minimize]To evaluate the polarization pattern of specific memory responses, antigen-specific T-cell lines to P. aeruginosa extract, a major P. aeruginosa antigen (flagellin), A. fumigatus extract, and recombinant A. fumigatus allergen 1 (asp f1) were generated from peripheral blood mononuclear cells of patients with CF and control subjects.
Again, patients with CF displayed higher expression levels of IL-17A and IL-5 as determined by real-time RT-PCR (Figure 7a; see Figure E8). Stimulation with P. aeruginosa–derived flagelin (28) provoked significantly higher levels of IL-17A and IL-5 mRNA (P < 0.01 and P < 0.05, respectively). On stimulation with P. aeruginosa extract, only IL-5 mRNA was significantly higher in patients with CF compared with control subjects (P < 0.05) (Figure 7a; see Figure E8). In response to the A. fumigatus allergen asp f1, patients with CF expressed significantly more IL-5 and IL-17A mRNA compared with control subjects (P < 0.01 and P < 0.05, respectively) (see Figure E8). Independent of the stimulation, expression levels of IFN-γ did not significantly differ between groups (Figure 7b; see Figure E8).
Figure 7. Antigen-specific T-cell responses to Pseudomonas aeruginosa in patients with cystic fibrosis (CF). (a) Messenger RNA expression levels of antigen-specific cytokine production (IL-5, IL-17A, IFN-γ) were ascertained by real-time reverse transcriptase polymerase chain reaction. (b) Frequencies of antigen-specific T cells were determined by ELISPOT and compared between patients with CF and control subjects (Mann-Whitney U test; P < 0.05). Flag = flagellin from P. aeruginosa; MC = media control; PAE = P. aeruginosa extract; PMA = phorbol 12-myristate 13-acetate; TCR = anti-CD3 antibody.
[More] [Minimize]To validate that our results reflect the frequency of antigen-specific T cells, we performed ELISPOT analysis. Patients with CF had higher frequencies of IL-17A– and IL-5–producing cells, whereas IFN-γ responses to P. aeruginosa extracts were comparable in control subjects. Antigen-specific IL-17A– and IL-5–producing cells were almost absent in control subjects (Figure 7b; see Figure E9).
Chronic inflammatory conditions occur in the lungs of patients with CF. The aim of this series of investigations was to evaluate the role of T-cell cytokine/chemokine production in the context of inflammation in clinically stable patients with CF, to define CF-specific patterns, evaluate a linkage of a certain T-cell subtype predominance and the susceptibility for P. aeruginosa infection.
We show that clinically stable patients with CF display a mixed inflammatory T-cell cytokine/chemokine profile with augmented Th1-, Th2-, and Th17-type responses. This inflammatory pattern correlates with clinical symptoms. In a prospective observational study, the Th2-type chemokine TARC/CCL17 and the Th17 cytokine IL-17A were significantly augmented in patients developing P. aeruginosa infection within the next 2 years. Moreover, we provide first evidence that in young children IL-17A is a positive prognostic marker to acquire acute P. aeruginosa infection without chronic colonization in young children with CF subsequently.
Despite the neutrophil dominance in BALF, T lymphocytes are the predominant cell type in the subepithelial bronchial tissue (29). Neutrophils, among other cells, produce IL-17A (12, 30). Support for a role of IL-17A in CF airway inflammation has been provided by several findings (20, 22, 30). Cytokines/chemokines, such as IL-1β, IL-6, or IL-8, which can be induced by IL-17A in epithelial cells or by Th17 cells (31), are augmented in sputum of P. aeruginosa–colonized patients with CF in case of pulmonary exacerbation compared with healthy control subjects (21, 32). Moreover, IL-17A–producing cells are increased in the bronchial epithelial walls of patients with CF (30). In line with our results, levels of IL-17A are reported to be increased in patients undergoing pulmonary exacerbation compared with clinically stable patients with CF not reaching statistical significance (30). In our study, differences between patients with CF undergoing pulmonary exacerbation and clinically stable patients with CF were restricted to Th2 and Th17 cytokines.
In particular, a moderate deficiency to produce the Th1-type cytokine IFN-γ has been linked (33–35) to a higher frequency of P. aeruginosa infections. CFTR−/− T cells produce increased IL-13, IL-5, and IL-17 but not IFN-γ as compared with wild-type T cells. In addition, transfer of CFTR−/− splenocytes into Rag−/− mice resulted in IgE up-regulation. We show that levels of Th2 cytokines are even more exaggerated in patients with CF undergoing pulmonary exacerbation, indicating that a cytokine/chemokine shift toward a Th2-dominant immune response is further amplified. One proposed reason for the Th2 bias is an increase in expression of the transcription factor nuclear factor of activated T cells caused by enhanced intracellular calcium-influx in CFTR-deficient lymphocytes, which could drive IL-13 production (36). This notion is also supported by a higher number of eosinophils in the lung of patients with CF (29); a high percentage of allergic sensitizations to inhalant allergens; and an increased prevalence of Th2-related disease, such as allergic bronchopulmonary aspergillosis (ABPA) (37–42). Recently, an association between P. aeruginosa colonization and ABPA has been proposed (43). This manuscript further supports this notion demonstrating an up-regulation of the Th2-type cytokine IL-5 in P. aeruginosa–specific cellular responses on P. aeruginosa exposure in vitro.
Chronic infection with P. aeruginosa is associated with a Th2-deviated cytokine profile (5). It remained unclear whether this profile precedes rather than results from P. aeruginosa infection. Our population is relatively young (mean age, 6 yr). However, there is evidence that infection with P. aeruginosa early in life correlates with worse outcome (44) and a high mortality rate (45). Interestingly, we found that those patients that subsequently acquired infection with P. aeruginosa within the next 24 months displayed significantly higher levels of IL-17A and the Th2-associated chemokine TARC/CCL17 as compared with those that remained negative for P. aeruginosa in culture. An up-regulation of the Th2-attracting chemokine TARC/CCL17 has been reported by Hartl and coworkers (5). This indicates a skewing toward a Th17-Th2–dominated immune response before infection with P. aeruginosa. On further subanalysis IL-17A turned out to be a good predictor. Stratification of patients into a “high IL-17A” and “low IL-17A” group, as defined by a cut-off level of 3.6 pg/ml determined by the median, revealed a significantly higher frequency of subsequent infection with P. aeruginosa in the “high IL-17A” group. Thus, in our study, patients with IL-17A levels greater than 3.6 pg/ml had an almost 60% chance to develop P. aeruginosa infection within the next 2 years, whereas the “low IL-17A” group had only a 16% chance to acquire P. aeruginosa infection.
To date we neither know the exact underlying pathophysiologic mechanisms that serve as responsible for this deviated cytokine/chemokine profile in patients with CF that subsequently acquire infection with P. aeruginosa, nor does it prove causality between infection and levels of IL-17A. Whether the high IL-17A profile goes along with a genetic predisposition or whether it is a consequence of earlier infections needs to be investigated. It is also possible that early infection with gram-negative pathogens, which induce a Th17 response, might lead to a vicious circle including repeated infection and triggering of Th17-associated cytokine/chemokine production. It is possible that P. aeruginosa is already present in BALF at time point of cytokine/chemokine measurement but not detectable with conventional microbiologic methods. The fact that levels of IL-17A did not correlate with time until detection of P. aeruginosa infection does not support this. Irrespective of these considerations, IL-17A may have the potential to act as a biomarker for early P. aeruginosa infections.
Reported correlations of IL-17A with IL-4 in CF (30) indicate a connection between Th17 cells and Th2 cells. Th17 cells have also been implicated in allergic responses and their ability to produce Th2-type cytokines was reported (46–48). Moreover, the identification of new T-cell subsets (Th17-Th2, Th17-Th1 [47, 49]) illustrates the complexity and plasticity of the immune system. Although it is not clear whether Th2 and Th17 cells work synergistically or antagonize each other, it seems that they cooperate and in some cases may amplify each other (50).
To assess potentially aggravating factors that relate to antigen-specific memory T-cell responses on antigen exposure, T-cell lines to A. fumigatus and P. aeruginosa extract, which both have been reported to drive Th2 responses, have been performed. Extracts and major antigens induced a Th17- and Th2-type memory response in vitro. This is of particular interest because repeated exposure to P. aeruginosa and A. fumigatus is frequently seen in patients with CF. Despite no apparent impact of A. fumigatus on life expectancy and disease progression (42) it may represent a risk factor for P. aeruginosa infection. However, P. aeruginosa colonization has been demonstrated to precede ABPA (43).
IL-17A plays a major role in recruitment, activation, and migration of neutrophils (10, 51). We also demonstrated this correlation between the number of neutrophils and IL-17A in BALF of CF (30). IL-17A–producing cells are known to be extremely potent inducers of tissue inflammation as observed in other Th17-mediated disease, such as psoriasis or rheumatoid arthritis (52–54). This is also supported by a significant correlation of IL-17A with lung function and of IL-17A, IL-1β, and IL-8 with the CT-based Helbich score, indicating a central role for Th17-mediated responses in lung destruction. In particular, the CT score in our group is very important because relative young age did not allow lung function testing in many of our patients. In line with the above-mentioned findings, the expression of the IL-17A receptor (IL17RA) on bronchial epithelial cells was higher in CF end-stage lung disease as compared with end-stage LF or PPH.
In conclusion, we propose a crucial role of IL-17–producing cells and Th2 cells in inflammation in the lung of patients with CF promoting inflammation-related destruction of lung tissue, supporting neutrophil recruitment, and defined IL-17A to be a marker preceding infection with P. aeruginosa.
| 1. | O’Sullivan BP, Freedman SD. Cystic fibrosis. Lancet 2009;373:1891–1904. |
| 2. | Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, et al.. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989;245:1066–1073. |
| 3. | Hubeau C, Lorenzato M, Couetil JP, Hubert D, Dusser D, Puchelle E, Gaillard D. Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa. Clin Exp Immunol 2001;124:69–76. |
| 4. | Dubin PJ, McAllister F, Kolls JK. Is cystic fibrosis a Th17 disease? Inflamm Res 2007;56:221–227. |
| 5. | Hartl D, Griese M, Kappler M, Zissel G, Reinhardt D, Rebhan C, Schendel DJ, Krauss-Etschmann S. Pulmonary T(h)2 response in Pseudomonas aeruginosa-infected patients with cystic fibrosis. J Allergy Clin Immunol 2006;117:204–211. |
| 6. | Wojnarowski C, Frischer T, Hofbauer E, Grabner C, Mosgoeller W, Eichler I, Ziesche R. Cytokine expression in bronchial biopsies of cystic fibrosis patients with and without acute exacerbation. Eur Respir J 1999;14:1136–1144. |
| 7. | Stockinger B, Veldhoen M, Martin B. Th17 T cells: linking innate and adaptive immunity. Semin Immunol 2007;19:353–361. |
| 8. | Ivanov II, Zhou L, Littman DR. Transcriptional regulation of Th17 cell differentiation. Semin Immunol 2007;19:409–417. |
| 9. | Jones CE, Chan K. Interleukin-17 stimulates the expression of interleukin-8, growth-related oncogene-alpha, and granulocyte-colony-stimulating factor by human airway epithelial cells. Am J Respir Cell Mol Biol 2002;26:748–753. |
| 10. | Laan M, Cui ZH, Hoshino H, Lotvall J, Sjostrand M, Gruenert DC, Skoogh BE, Linden A. Neutrophil recruitment by human IL-17 via C-X-C chemokine release in the airways. J Immunol 1999;162:2347–2352. |
| 11. | Bettelli E, Korn T, Kuchroo VK. Th17: the third member of the effector T cell trilogy. Curr Opin Immunol 2007;19:652–657. |
| 12. | Brodlie M, McKean MC, Johnson GE, Anderson AE, Hilkens CM, Fisher AJ, Corris PA, Lordan JL, Ward C. Raised interleukin-17 is immunolocalised to neutrophils in cystic fibrosis lung disease. Eur Respir J 2011;37:1378–1385. |
| 13. | Dubin PJ, Kolls JK. Th17 cytokines and mucosal immunity. Immunol Rev 2008;226:160–171. |
| 14. | Huang W, Na L, Fidel PL, Schwarzenberger P. Requirement of interleukin-17a for systemic anti-Candida albicans host defense in mice. J Infect Dis 2004;190:624–631. |
| 15. | Puel A, Cypowyj S, Bustamante J, Wright JF, Liu L, Lim HK, Migaud M, Israel L, Chrabieh M, Audry M, et al.. Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity. Science 2011;332:65–68. |
| 16. | Rudner XL, Happel KI, Young EA, Shellito JE. Interleukin-23 (IL-23)-IL-17 cytokine axis in murine Pneumocystis carinii infection. Infect Immun 2007;75:3055–3061. |
| 17. | Zielinski CE, Mele F, Aschenbrenner D, Jarrossay D, Ronchi F, Gattorno M, Monticelli S, Lanzavecchia A, Sallusto F. Pathogen-induced human Th17 cells produce IFN-gamma or IL-10 and are regulated by IL-1beta. Nature 2012;484:514–518. |
| 18. | Aujla SJ, Chan YR, Zheng M, Fei M, Askew DJ, Pociask DA, Reinhart TA, McAllister F, Edeal J, Gaus K, et al.. IL-22 mediates mucosal host defense against gram-negative bacterial pneumonia. Nat Med 2008;14:275–281. |
| 19. | Tesmer LA, Lundy SK, Sarkar S, Fox DA. Th17 cells in human disease. Immunol Rev 2008;223:87–113. |
| 20. | Dubin PJ, Kolls JK. IL-23 mediates inflammatory responses to mucoid Pseudomonas aeruginosa lung infection in mice. Am J Physiol Lung Cell Mol Physiol 2007;292:L519–L528. |
| 21. | McAllister F, Henry A, Kreindler JL, Dubin PJ, Ulrich L, Steele C, Finder JD, Pilewski JM, Carreno BM, Goldman SJ, et al.. Role of IL-17a, IL-17f, and the IL-17 receptor in regulating growth-related oncogene-alpha and granulocyte colony-stimulating factor in bronchial epithelium: implications for airway inflammation in cystic fibrosis. J Immunol 2005;175:404–412. |
| 22. | Zelante T, De Luca A, Bonifazi P, Montagnoli C, Bozza S, Moretti S, Belladonna ML, Vacca C, Conte C, Mosci P, et al.. Il-23 and the Th17 pathway promote inflammation and impair antifungal immune resistance. Eur J Immunol 2007;37:2695–2706. |
| 23. | Tiringer K. Inflammation in cystic fibrosis and end stage lung disease. Presented at the Annual Meeting of the Austrian Society of Allergy and Immunology (ÖGAI). September 15–17, 2011, Graz, Austria. |
| 24. | Tiringer K. Th17-type inflammatory responses in CF patients. PhD Symposium. June 16, 2011, Vienna, Austria. |
| 25. | Tiringer K. A Th17- and Th2-skewed cytokine profile in the lung of CF patients is a risk factor for infection with Pseudomonas aeruginosa (PA). Presented at the 35th ECFS Conference. June 6–9, 2012, Dublin, Ireland. |
| 26. | Tiringer K. An inflammatory Th17- and Th2-skewed cytokine profile in the lung of CF patients represents a risk factor for colonization with Pseudomonas aeruginosa. Presented at the World Immune Regulation Meeting (WIRM) VI. March 18–21, 2012, Davos, Switzerland. |
| 27. | Cohen-Cymberknoh M, Shoseyov D, Kerem E. Managing cystic fibrosis: strategies that increase life expectancy and improve quality of life. Am J Respir Crit Care Med 2011;183:1463–1471. |
| 28. | Campodonico VL, Llosa NJ, Grout M, Doring G, Maira-Litran T, Pier GB. Evaluation of flagella and flagellin of Pseudomonas aeruginosa as vaccines. Infect Immun 2010;78:746–755. |
| 29. | Regamey N, Tsartsali L, Hilliard TN, Fuchs O, Tan HL, Zhu J, Qiu YS, Alton EW, Jeffery PK, Bush A, et al.. Distinct patterns of inflammation in the airway lumen and bronchial mucosa of children with cystic fibrosis. Thorax 2012;67:164–170. |
| 30. | Tan HL, Regamey N, Brown S, Bush A, Lloyd CM, Davies JC. The Th17 pathway in cystic fibrosis lung disease. Am J Respir Crit Care Med 2011;184:252–258. |
| 31. | Burgler S, Ouaked N, Bassin C, Basinski TM, Mantel PY, Siegmund K, Meyer N, Akdis CA, Schmidt-Weber CB. Differentiation and functional analysis of human T(h)17 cells. J Allergy Clin Immunol 2009;123:588–595, 595 e581–587. |
| 32. | Decraene A, Willems-Widyastuti A, Kasran A, De Boeck K, Bullens DM, Dupont LJ. Elevated expression of both MMA and protein levels of IL-17a in sputum of stable cystic fibrosis patients. Respir Res 2010;11:177. |
| 33. | Brazova J, Sediva A, Pospisilova D, Vavrova V, Pohunek P, Macek M, Bartunkova J, Lauschmann H. Differential cytokine profile in children with cystic fibrosis. Clin Immunol 2005;115:210–215. |
| 34. | Moser C, Jensen PO, Kobayashi O, Hougen HP, Song Z, Rygaard J, Kharazmi A, Hb N. Improved outcome of chronic Pseudomonas aeruginosa lung infection is associated with induction of a Th1-dominated cytokine response. Clin Exp Immunol 2002;127:206–213. |
| 35. | Parker D, Cohen TS, Alhede M, Harfenist BS, Martin FJ, Prince A. Induction of type I interferon signaling by Pseudomonas aeruginosa is diminished in cystic fibrosis epithelial cells. Am J Respir Cell Mol Biol 2012;46:6–13. |
| 36. | Mueller C, Braag SA, Keeler A, Hodges C, Drumm M, Flotte TR. Lack of cystic fibrosis transmembrane conductance regulator in CD3+ lymphocytes leads to aberrant cytokine secretion and hyperinflammatory adaptive immune responses. Am J Respir Cell Mol Biol 2011;44:922–929. |
| 37. | el-Dahr JM, Fink R, Selden R, Arruda LK, Platts-Mills TA, Heymann PW. Development of immune responses to aspergillus at an early age in children with cystic fibrosis. Am J Respir Crit Care Med 1994;150:1513–1518. |
| 38. | Hemmann S, Nikolaizik WH, Schoni MH, Blaser K, Crameri R. Differential IgE recognition of recombinant Aspergillus fumigatus allergens by cystic fibrosis patients with allergic bronchopulmonary aspergillosis or aspergillus allergy. Eur J Immunol 1998;28:1155–1160. |
| 39. | Knutsen AP, Hutchinson PS, Albers GM, Consolino J, Smick J, Kurup VP. Increased sensitivity to IL-4 in cystic fibrosis patients with allergic bronchopulmonary aspergillosis. Allergy 2004;59:81–87. |
| 40. | Knutsen AP, Slavin RG. Allergic bronchopulmonary aspergillosis in asthma and cystic fibrosis. Clin Dev Immunol 2011;84:1–13. |
| 41. | Warner JO, Taylor BW, Norman AP, Soothill JF. Association of cystic fibrosis with allergy. Arch Dis Child 1976;51:507–511. |
| 42. | Wojnarowski C, Eichler I, Gartner C, Gotz M, Renner S, Koller DY, Frischer T. Sensitization to Aspergillus fumigatus and lung function in children with cystic fibrosis. Am J Respir Crit Care Med 1997;155:1902–1907. |
| 43. | Kraemer R, Delosea N, Ballinari P, Gallati S, Crameri R. Effect of allergic bronchopulmonary aspergillosis on lung function in children with cystic fibrosis. Am J Respir Crit Care Med 2006;174:1211–1220. |
| 44. | Emerson J, Rosenfeld M, McNamara S, Ramsey B, Gibson RL. Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol 2002;34:91–100. |
| 45. | Accurso FJ. Update in cystic fibrosis 2007. Am J Respir Crit Care Med 2008;177:1058–1061. |
| 46. | Cosmi L, De Palma R, Santarlasci V, Maggi L, Capone M, Frosali F, Rodolico G, Querci V, Abbate G, Angeli R, et al.. Human interleukin 17-producing cells originate from a CD161+ CD4+ T cell precursor. J Exp Med 2008;205:1903–1916. |
| 47. | Cosmi L, Maggi L, Santarlasci V, Capone M, Cardilicchia E, Frosali F, Querci V, Angeli R, Matucci A, Fambrini M, et al.. Identification of a novel subset of human circulating memory CD4(+) T cells that produce both IL-17a and IL-4. J Allergy Clin Immunol 2010;125:222–230 e221–224. |
| 48. | Raymond M, Van VQ, Wakahara K, Rubio M, Sarfati M. Lung dendritic cells induce T(h)17 cells that produce T(h)2 cytokines, express gata-3, and promote airway inflammation. J Allergy Clin Immunol 2011;128:192–201 e196. |
| 49. | Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi B, Parente E, Fili L, Ferri S, Frosali F, et al.. Phenotypic and functional features of human Th17 cells. J Exp Med 2007;204:1849–1861. |
| 50. | He R, Kim HY, Yoon J, Oyoshi MK, MacGinnitie A, Goya S, Freyschmidt EJ, Bryce P, McKenzie AN, Umetsu DT, et al.. Exaggerated IL-17 response to epicutaneous sensitization mediates airway inflammation in the absence of IL-4 and IL-13. J Allergy Clin Immunol 2009;124:761–770 e761. |
| 51. | Hellings PW, Kasran A, Liu Z, Vandekerckhove P, Wuyts A, Overbergh L, Mathieu C, Ceuppens JL. Interleukin-17 orchestrates the granulocyte influx into airways after allergen inhalation in a mouse model of allergic asthma. Am J Respir Cell Mol Biol 2003;28:42–50. |
| 52. | Lubberts E, Koenders MI, Oppers-Walgreen B, van den Bersselaar L, Coenen-de Roo CJ, Joosten LA, van den Berg WB. Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion. Arthritis Rheum 2004;50:650–659. |
| 53. | Nakae S, Nambu A, Sudo K, Iwakura Y. Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice. J Immunol 2003;171:6173–6177. |
| 54. | Wilson NJ, Boniface K, Chan JR, McKenzie BS, Blumenschein WM, Mattson JD, Basham B, Smith K, Chen T, Morel F, et al.. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol 2007;8:950–957. |
Supported by Österreichische Nationalbank (AP13846ONB), German Cystic Fibrosis Association, and Christine Kühne-Center for Allergy Research and Education.
Author Contributions: T.E., Z.S., T.F., and C.A.A. were involved in the conception, hypotheses delineation, and design of the study. G.D., M.G., S.R., E.D., E.N., R.C., A.J., Z.S., P.J., F.H., A.S., and T.F. were involved in acquisition of the data. K.T., P.F., A.T., T.E., S.G., F.H., M.K.R., W.K., and M.H. were involved in acquisition, analysis, and interpretation of the data. K.T., T.E., and S.G. were involved in writing the article. K.T., T.E., Z.S., S.G., E.D., T.F., and C.A.A. were involved in critical review of the manuscript.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201206-1150OC on January 10, 2013
Author disclosures
