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Inserm E and Department of Pediatric Pneumology, Armand Trousseau Hospital, Department of Pediatrics, Robert Debré Children's Hospital
Department of Biostatistics, Inserm U, St-Antoine Hospital, Paris
Department of Pediatrics, Georges Clémenceau Hospital, Caen
Department of Pneumology and Gastroenterology, Purpan Children's Hospital, Toulouse
Department of Pediatrics, Charles Nicolle Hospital, Rouen
Department of Pediatrics, South Hospital, Rennes, France
Children's Hospital, University of Essen, Essen, Germany
Recent evidence suggests that genetic polymorphisms that affect the production of interleukin (IL)10 may play a role in the response to pathogens in cystic fibrosis (CF). The present study was designed to investigate a possible association between alleles carried at position -1082 in the promoter region of the IL-10 gene and clinical data on 378 patients with CF. After adjustment for potential confounding variables, a significant relationship was found between the -1082GG genotype and both colonization with Aspergillus fumigatus and allergic bronchopulmonary aspergillosis. In addition, higher serum levels of IL-10 were observed in patients colonized with A. fumigatus. These results suggest that polymorphisms in the promoter region of the IL-10 gene may influence the host response to A. fumigatus in the context of CF.
One pathological feature of the lung in cystic fibrosis (CF) is the progressive development of chronic infections with a restricted spectrum of pathogens, such as Pseudomonas aeruginosa and Aspergillus fumigatus, despite the absence of immunodeficiency and the use of aggressive treatments [1]. Recently, much interest has been focused on the potential role that interleukin (IL)10 plays in the altered inflammatory and infectious lung processes in CF. Data from studies using murine models have consistently supported the view that IL-10 is an important regulator of the response to endobronchial infection with P. aeruginosa [2] and of the T-cellmediated immune response to A. fumigatus [3]. In CF, altered production of IL-10 in the lung has been reported elsewhere [4, 5]. Interestingly, results from recent studies of various inflammatory and infectious diseases support the view that variability in the production of IL-10 has a significant hereditary component in which polymorphisms in the promoter region of the IL-10 gene are implicated. A polymorphism at position -1082 has been reported to produce higher levels of IL-10 if the -1082G allele is present and lower levels of IL-10 if the -1082A allele is present [6, 7]. The purpose of the present study was to test the hypothesis that a genetically determined variation in the production of IL-10 might influence the development of chronic infection in CF. It was designed to analyze alleles carried at position -1082 in the promoter region of the IL-10 gene and serum IL-10 levels in a cohort of patients with CF.
Patients, materials, and methods.
Data on 378 patients who attended 1 of 6 major CF centers in France (Armand Trousseau Hospital and Robert Debré Children's Hospital in Paris and pediatric departments in hospitals in Caen, Rennes, Rouen, and Toulouse) and 1 CF center in Germany (Children's Hospital in Essen) were examined. The diagnosis of CF was established on the basis of the results of 2 sweat chloride tests (>60 mmol/L) and the identification of mutations in the CF transmembrane conductance regulator (CFTR) gene. The ethical committees of St. Louis Hospital (for the French CF centers) and Essen Hospital (for the German CF center) approved the study protocol. Clinical, biological, and functional data were obtained retrospectively from patients' hospital records. Patients had been followed for a mean ± SD of 11.2 ± 5.9 years before the present study. The following information was collected: sex, age at diagnosis of CF, CFTR genotype, results of pulmonary function tests (measurements of forced expiratory volume in 1 s and forced vital capacity in children 5 years old, expressed as percentages of predicted values), nutritional status (body mass index and z score), exocrine pancreatic status, and microbiological data (age at first isolation of and colonization [on the basis of at least 3 consecutive positive sputum cultures] with P. aeruginosa or A. fumigatus). Lung disease associated with A. fumigatus infection was characterized on the basis of the recommendations of the Consensus Conference of the CF Foundation and was categorized as either allergic bronchopulmonary aspergillosis (ABPA) or colonization with A. fumigatus, which was defined as having repeated positive sputum cultures for A. fumigatus and meeting at least 1 criterion for ABPA [8]. All patients were studied during a routine visit to an outpatient department when they were in a clinically stable condition.
Genomic DNA was extracted from blood samples, and the polymorphism at position -1082GA (rs1800896) of the IL-10 gene was genotyped by use of a real-time polymerase chain reaction. An allelic discrimination assay was performed by end point measurement with specific fluorescent oligonucleotides (ABI Prism 7000; Applied Biosystems). The forward (5-CCAAGACAACACTACTAAGGCTTCT-3) and reverse (5-GCTGGATAGGAGGTCCCTTACTTT-3) primer pairs and MGB probes specific for the IL-10 -1082G allele (CCTACTTCCCCCTCCCAA labeled with fluorescent FAM) and the IL-10 -1082A allele (CCCTACTTCCCCTTCCCAA labeled with fluorescent VIC) were used in amplification.
Blood samples were collected in tubes containing EDTA, to prevent further release of cytokines from blood cells. Plasma samples were stored at -80°C and were thawed immediately before measurement of IL-10. Serum IL-10 levels were determined by use of commercially available ELISA kits (R&D Systems), in accordance with the manufacturer's recommended protocols. The detection limit of the test was 30 pg/mL.
Statistical analyses were performed with R software (version 1.8.1; available at: http://www.R-project.org). Data were expressed as percentages or means ± SD. Pearson's 2 and 2 for linear trend were used for categorical data. Differences between groups were tested with the nonparametric Kruskal-Wallis test. Deviation of the expected values from Hardy-Weinberg equilibrium was also tested. The relationship between the IL-10 genotype and infection with A. fumigatus or P. aeruginosa was evaluated with proportional hazards regression models. Univariate and multivariate analyses were performed. Associations with the following patient characteristics were tested with the log likelihood ratio test: sex, circumstances of CF diagnosis (neonatal screening, neonatal ileus, and later diagnosis on the basis of clinical symptoms), birth date (cohort effect), CF center, CFTR genotype, and pancreatic status. All tests were 2-sided, and P < .05 was considered to be statistically significant.
Results.
A total of 378 patients279 from the French CF centers and 99 from the German CF centerwere included in the study, and they had a mean ± SD age at enrollment of 12.9 ± 6.1 years. A total of 204 patients (54%) were homozygous for the F508 CFTR gene mutation, 139 patients (37%) were compound heterozygous for this mutation, and 35 patients (9%) had other CFTR gene mutations. A total of 358 patients (95%) had pancreatic insufficiency. Pulmonary function data and nutritional status are shown in table 1. A total of 275 patients (73%) had a positive culture for P. aeruginosa at least once (mean ± SD age at first isolation, 5.8 ± 4.5 years), and 118 patients (31%) were colonized with P. aeruginosa (mean ± SD age at colonization, 9.0 ± 4.1 years). A total of 162 patients (43%) had at least 1 positive culture for A. fumigatus (mean ± SD age at first isolation, 10.0 ± 3.7 years). A diagnosis of ABPA was made in 27 patients (7%); all were treated with systemic corticosteroids. A total of 119 patients (31%) were colonized with A. fumigatus (mean ± SD age at colonization, 11.2 ± 3.2 years).
The relative frequencies of polymorphisms at position -1082 of the IL-10 gene in the French and German cohorts did not deviate significantly from the Hardy-Weinberg equilibrium (for the French cohort, P = .89; for the German cohort, P = .88). On the basis of these results, the cohorts were combined, and subsequent analyses were performed in the total population, in which 24.9% of patients had the -1082GG genotype, 49.7% had the -1082AG genotype, and 25.4% had the -1082AA genotype.
No influence of IL-10 genotypes could be documented for colonization with P. aeruginosa. In the total population, the hazard ratio (HR) for the -1082AA genotype was 1.00, the HR for the -1082AG genotype was 0.85 (95% confidence interval [CI], 0.541.33), and the HR for the -1082GG genotype was 0.92 (95% CI, 0.551.54) (P = .78). In the F508 homozygous group, similar findings were observed: the HR for the -1082AA genotype was 1.00, the HR for the -1082AG genotype was 0.76 (95% CI, 0.401.43), and the HR for the -1082GG genotype was 0.81 (95% CI, 0.411.58) (P = .7).
Associations were found between patients who had the -1082GG genotype and the occurrence of ABPA, in both the total population and in the F508 homozygous group (table 2). A significant association was also observed between patients who had the -1082GG genotype in the F508 homozygous group and colonization with A. fumigatus. Compared with patients who had the IL-10 -1082A allele (-1082AA/AG genotypes), patients who had the -1082GG genotype had a significantly higher risk of developing an early form of A. fumigatusrelated diseaseeither ABPA or colonization with A. fumigatus. The 50% cumulative incidence of colonization with A. fumigatus in patients who had the -1082GG genotype was reached at 12 years, compared with 16 years in patients who had the -1082A allele (HR, 1.67; P = .02). Multivariate analysis confirmed that colonization with A. fumigatus occurred earlier in patients who had the -1082GG genotype than in patients who had the -1082A allele, not only in the total population (HR, 1.67 [95% CI, 1.102.50]; P = .02), but also in the F508 homozygous group (HR, 2.02 [95% CI, 1.213.37]; P = .007).
In the total population, blood samples from 48 patients in stable clinical condition were available for IL-10 ELISA. All patients had serum IL-10 levels over the detection limit, with a median level of 340 pg/mL (range, 30826 pg/mL). Higher serum IL-10 levels (median, 495 pg/mL; range, 324784 pg/mL) were documented in patients with A. fumigatusrelated lung disease, compared with those in patients without A. fumigatusrelated lung disease (median, 249 pg/mL; range, 30826 pg/mL; P = .02, Mann-Whitney U test). In 12 patients who had measurements of serum IL-10 levels and A. fumigatusrelated lung disease, the frequency of the -1082G allele was significantly increased, with an odds ratio of 3.29 for the -1082AG genotype versus the -1082AA genotype and of 8.00 for the -1082GG genotype versus the -1082AA genotype (2 for linear trend, 3.979; P = .046).
Discussion.
A novel finding of the present study is that the IL-10 -1082GG genotype is associated with an increased occurrence of colonization with A. fumigatus and ABPA in patients with CF. Interestingly, no relationship between IL-10 genotypes and colonization with P. aeruginosa could be documented in this cohort. IL-10 has anti-inflammatory properties, such as the suppression of the synthesis of multiple pro-inflammatory cytokines and cell-mediated immunity [9]. The influence of IL-10 on the outcome of infectious diseases is variable. In some situations, IL-10 has been reported to play a beneficial role, whereas, in other instances, it has been shown to display deleterious action. In clinical bacterial infections, increased morbidity and mortality in patients with high IL-10 levels have been documented. High concentrations of IL-10 have been reported to be associated with severe outcome in preterm neonates with sepsis, pneumonia, or necrotizing enterocolitis [10]. Lehmann et al. have shown that IL-10 is associated with fatality in meningococcal disease [11]. It is now well recognized that IL-10 is necessary to counterbalance pro-inflammatory reactions, but its overexpression can be detrimental.
IL-10 has been shown to be deleterious in models of fungal infections [12]. Experimental studies have demonstrated that mice deficient in IL-10 display an increased resistance to A. fumigatus infection, and it has been suggested that blockage of IL-10 functions might result in a beneficial increase in host resistance via Th1 pathways [2]. The effect that A. fumigatus infection has on production of IL-10 is most likely influenced by host conditions. Warris et al. have investigated the release of cytokines on stimulation with A. fumigatus in healthy subjects and patients with chronic granulomatous disease [13]. Their data indicated a dysregulation between pro- and anti-inflammatory cytokines, and, in patients with chronic granulomatous disease, there was a dramatic increase in the production of IL-10, which certainly contributed to the increased susceptibility to A. fumigatus infection in these patients. A. fumigatus is a common pathogen in CF, and it is now recognized that it actively participates in lung destruction in some patients. Casaulta et al., using peripheral blood cells from patients with CF, have reported data demonstrating the ability of IL-10 to control an A. fumigatusspecific T cell response [14]. Consequently, compelling evidence from numerous studies has suggested that IL-10 is an important component of the response to A. fumigatus infection [15].
Data reported in the present study strongly suggest that heterogeneity in the IL-10 gene may influence the response to A. fumigatus infection and the occurrence of A. fumigatusrelated lung disease in patients with CF. A significant association was found between the -1082GG genotype and colonization with A. fumigatus and ABPA. This association was also documented in the subgroup of patients homozygous for the F508 CFTR gene mutation. Therefore, on the basis of results from in vitro studies indicating that the -1082G allele is associated with higher production of IL-10, it could be suggested that the level of IL-10 might influence the outcome of A. fumigatus infection in patients with CF. To provide functional information on IL-10 polymorphisms, serum IL-10 levels were evaluated in the blood from patients with CF who were in stable clinical condition. Higher serum IL-10 levels were found in the blood from patients with A. fumigatusrelated lung disease than in patients without A. fumigatusrelated lung disease. Interestingly, the frequency of the -1082G allele was significantly higher in patients with A. fumigatusrelated lung disease than in those without A. fumigatusrelated lung disease.
In conclusion, the present study provides information on polymorphisms in the IL-10 gene and the occurrence of A. fumigatusrelated lung disease in patients with CF. The results of the genetic and functional studies suggest that polymorphisms in the promoter region of IL-10 may influence how patients with CF respond to A. fumigatus infection and increase the risk for the development of chronic infection or ABPA.
Acknowledgments
We thank Josue Feingold, for helpful discussions, and Catherine Fitting, for technical assistance.
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