Rationale: Copy number variations of the cluster of β-defensin genes have been associated with psoriasis and inflammatory bowel disease. Controversy still exists on whether the β-defensins genes determine susceptibility for chronic obstructive pulmonary disease (COPD).
Objectives: We investigated whether genomic copy number variations of the β-defensin gene cluster have a functional role in airway epithelial cells and associate with the presence of COPD.
Methods: Baseline and inflammatory induced transcript expression of DEFB4 was studied in nasal epithelial cell cultures and its effect on Pseudomonas aeruginosa inhibition was assessed. Subsequently, relevant functional cut-offs for copy numbers were used to explore associations with COPD in two independent case–control studies.
Measurements and Main Results: Copy number variation in the β-defensin encoding genes correlated with baseline mRNA DEFB4 expression levels (R2 = 0.96; P = 0.02), with a plateau effect from five copies or more. Only when higher copy numbers of β-defensin genes were present, transcription was significantly up-regulated by tumor necrosis factor-α (P < 0.0001), which resulted in better antimicrobial activity in vitro. When comparing healthy smokers with COPD patients, a copy number greater than or equal to 5 was associated with increased risk for COPD with an adjusted odds ratio of 1.8 (confidence interval, 1.1–2.8; P = 0.02), which was confirmed by a second independent case-control study.
Conclusions: Genomic copy number variation of β-defensin encoding genes has a functional role in airway epithelial cells, which may contribute to the pathogenesis of COPD.
The copy number of the β-defensin genes has been associated with psoriasis and Crohn's disease. The chromosomal 8p23 region on which β-defensin genes are located has been linked to chronic obstructive pulmonary disease, but recent genome-wide association studies have not confirmed this connection.
We show that high β-defensin genomic copy numbers determine the functional expression of β-defensin 2 in airway epithelial cells and associate with the presence of chronic obstructive pulmonary disease.
Genome-wide linkage analysis in the Boston Early-Onset COPD cohort has demonstrated significant evidence of linkage for chromosome 8p and FEV1 as marker of COPD (1, 2). However, fine mapping of the linkage peak on the chromosomal 8p23 region, on which the β-defensin cluster is located, could not further reveal significant associations between single nucleotide polymorphisms (SNP) on these genes and markers of COPD (3). Moreover, in contrast to previous small studies that reported associations between polymorphisms in the human DEFB1 gene (coding for β-defensin 1, hBD1) and COPD (4, 5), recent large genome-wide association studies for lung function and COPD did not identify chromosome 8p as an important locus for COPD susceptibility (6–8).
The lack of consistent genetic evidence for β-defensin genes contrasts with the potential role that β-defensins may play in the pathogenesis of COPD (9). DEFB1 is constitutively expressed in the human airway epithelium, whereas DEFB4 (coding for hBD2), DEFB103, and DEFB104 have a strong inducible expression pattern (10, 11). They all exert an important first-line antimicrobial activity against a broad spectrum of bacteria, fungi, and enveloped viruses that may infect or colonize the respiratory tract in COPD. Most β-defensins also act as chemoattractant for monocytes, dendritic cells, and tumor necrosis factor (TNF)-α–activated neutrophils, thereby enhancing the chronic inflammatory cascade (12–14).
The β-defensin genes DEFB4, DEFB103, DEFB104, DEFB105, and DEFB106 are part of a highly polymorphic 250-kb repeat region on chromosome 8p23 (15), whereas DEFB1 is not repeated (Figure 1). An involvement of copy number alleles might be missed in genome-wide association studies where only associations with an allele at biallelic SNPs are sought. Nevertheless, as recently demonstrated in psoriasis (16) and Crohn disease (17), copy number variation (CNV) of the β-defensin encoding genes may be more important than SNPs (3, 18, 19). We therefore investigated whether an increased copy number of β-defensin encoding genes may enhance airway inflammation and associates with the presence of COPD. Functional data were generated on upper airway epithelial cells cultured in vitro, whereas genetic associations between copy numbers at the β-defensin CNV and COPD were explored in two independent case-control studies of Belgian descent.
Monolayers of epithelial cells were generated from surgically removed nasal polyps from patients having rhinosinusitis, as described previously (20). RNA was extracted with Trizol (Life Technologies, Carlsbad, CA) and DNA with phenol chloroform iso-amyl alcohol (Life Technologies, Carlsbad, CA). A frozen vial of epithelial cells with known diploid β-defensin copy number was recultured in a T75 falcon. After reaching confluency, cells were harvested and grown in air-liquid interface according to Bals and coworkers (21).
DNA was isolated from whole blood samples using standard molecular biology procedures. The diploid copy-number of DEFB4 was determined by duplex real-time polymerase chain reaction (PCR) with the qPCR Core kit (Eurogentec, Seraing, Belgium) on an ABI 7500 system (Life Technologies, Carlsbad, CA) according to a previously established protocol by Nuytten and coworkers (22).
RNA (1 μg) was reverse transcribed into cDNA using superscript reverse transcriptase II (Life Technologies, Carlsbad, CA). Endogenous DEFB4 expression against DEFB1 expression of each sample was determined by duplex real-time PCR according to a previously established protocol (22). A delta Ct value of n equals a CNV of 2n. The variation in expression was then calculated by 2CtDEFB1-CtDEFB4. The expression levels of the different cell lines were normalized according to the DEFB4 expression seen in the cell lines that contained two copies of DEFB4.
For determination of DEFB4 or interleukin (IL)-8 expression, after stimulation with TNF-α, real-time semiquantitative PCR was performed according to a previously established protocol (23). A nontreated control was included in each experiment. The obtained ΔCt values were corrected using this negative control by subtracting the ΔCt of the transcripts in the control sample from the ΔCt value of the transcripts of the sample under study. The obtained ΔΔCt values represent the amount of transcripts of a particular gene in a sample, corrected for the amount of cDNA present in the sample and corrected for differences in basal expression in different samples. The relative copy number of the gene of interest is 2−ΔΔCt.
Two strains of Pseudomonas aeruginosa, Pa01 and a clinical isolate, were grown overnight on nonselective blood agar plates (blood agar base II) supplemented with hemine (5 mg/ml), menadione (1 mg/ml), and 5% sterile horse blood. The plates were placed at 37°C in an atmosphere consisting of 5% CO2.
Bacteria were collected from blood agar plates by a cotton swap and suspended in phosphate-buffered saline. Bacterial concentrations were determined with a spectrophotometer and the solution was diluted to a concentration of 50–500 CFU per 100 μl. TNF-α (R&D Systems, Inc., Minneapolis, MN) was diluted to a solution of 10 ng per 100 μl. Then, 100 μl of TNF-α was added to the cells growing in air-liquid interface cultures in six-well plates. The cells were incubated for 12 hours at 37°C in a humified CO2 incubator. After 12 hours of incubation with TNF-α, 50–500 CFU of bacteria were added to the cells. The initial concentration was determined by plating out 100 μl of bacterial serial dilutions. After 3 hours of incubation in a humified CO2 incubator at 37°C, the medium was removed and the bacteria were plated out on blood agar plates. The plates where incubated at 37°C for 12 hours and colonies were counted. The nasal epithelial cells were transferred in RNA later solution (Qiagen, Hilden, Germany) for RNA extraction at a later time. RNA was extracted from the cells with the Qiagen RNA extraction kit (Qiagen, Hilden, Germany). DNA was removed during RNA extraction with RNAse-free DNase (Qiagen, Hilden, Germany). The isolated RNA was stored at −70°C.
Two different and independent case–control populations were studied. The first population was recruited at the outpatient clinic of the University Hospital of Leuven, Leuven, Belgium, and consisted of 358 individuals with a minimal smoking history of at least 10 pack-years and aged over 50 years. Diagnosis of COPD was based on clinical records and prebronchodilator pulmonary function tests. Only patients with an FEV1/FVC ratio less than 0.7 and a FEV1 less than 80% predicted were finally enrolled as cases, thereby approximating a minimal requirement of Global Obstructive Lung Disease (GOLD) stage II (1). Healthy smoking controls had a FEV1/FVC ratio greater than or equal to 0.7. For both groups, subjects with a history of asthma were excluded, as were patients with other respiratory diseases affecting pulmonary function. In the replication case-control study, 107 COPD patients with GOLD stage 4 referred for lung transplantation were only included. Patients with α1-antitrypsine deficiency were excluded. Here, 150 nonrelated adult volunteers with any medical or smoking history were randomly selected and used as controls. All individuals were of Belgian descent and signed an informed consent before participation. The study was approved by the ethics committee of the University Hospital of Leuven.
All pulmonary function measurements were performed with standardized equipments (Whole body Plethysmograph, Acertys, Belgium) according to American Thoracic Society and European Respiratory Society guidelines (24). All spirometric values, such as FVC and FEV1, were prebronchodilator measurements, given as absolute values and expressed as percent predicted of reference values (25). Diffusing capacity of the lung was determined by the single breath carbon monoxide gas transfer method (Dlco) and expressed as percentage of reference values (26).
Pearson statistics and linear regression are used to correlate diploid β-defensin copy numbers with expression. Mean transcript expression levels and mean percentages of bacterial clearance are compared with unpaired and paired t tests, respectively. Patient data are summarized as frequencies (percentages) for categorical variables; means (± standard deviation) or medians (25th and 75th percentiles) for continuous variables; and compared by Pearson's chi-square test, standardized student t test, or Wilcoxon statistics, respectively. Binary logistic regression analysis was used to explore associations between diploid β-defensin copy numbers and COPD and to correct for age, sex, pack-years, and quit-years. All statistical analyses were performed using the SYSTAT package, release 7.0 (SPSS, Inc., Chicago, IL) or SAS version 9.1 (SAS Institute, Cary, NC). For all tests, two-sided P levels less than 0.05 were considered as statistical significant.
To determine the relation between diploid β-defensin copy numbers and basal corresponding transcript levels, nasal epithelial cell cultures of 48 individuals were generated. Because most defensin transcripts have basal expression levels below the detection limit of our assay (23, 27), the correlation was assessed by relating genomic diploid DEFB4 copy number with DEFB4 transcripts, which are known to be more abundantly expressed. Nasal epithelial cells of patients with two to four diploid β-defensin copies showed significantly less DEFB4 expression than epithelial cells harboring five or more β-defensin copies (P < 0.01). In the range from two to five copy numbers of the DEFB4 gene, a highly significant linear correlation could be observed with DEFB4 expression (R2 = 0.96; P = 0.02). From five copies onward, however, a plateau phase was reached in which additional copies did not result in a further significant increase of basal transcript levels (Figure 2).
Next, the influence of inflammatory signals on DEFB4 expression in cultured nasal epithelial cells was assessed as a function of the diploid β-defensin copy number. For this purpose, we selected primary cultures of nasal epithelial cells that carried either a low diploid β-defensin copy number (i.e., three or four diploid β-defensin copies), or a high copy number (i.e., eight diploid β-defensin copies) and exposed them to TNF-α, which is a common inducer of DEFB4 expression (23). IL-8 expression, which is also induced by TNF-α, was used as a positive control. After 12 hours of stimulation with TNF-α, no significant up-regulation of DEFB4 expression could be observed in nasal epithelial cells carrying a low diploid β-defensin copy number, whereas a mild to strong up-regulation of DEFB4 expression was observed in nasal epithelial cells carrying a high diploid β-defensin copy number (Figure 3A) (P < 0.0001). As expected, IL-8 expression was up-regulated in all samples, regardless of the diploid β-defensin copy number (Figure 3B) (P = 0.1).
We then investigated whether increased expression of DEFB4 also resulted in an increased antibacterial activity. Air-liquid interface cultures of nasal epithelial cells with a low diploid β-defensin copy number (primary cell lines 4-5-6) were compared with epithelial cell cultures with high diploid β-defensin copy numbers (primary cell lines 1-2-3-9) and exposed to TNF-α or phosphate-buffered saline when a differentiated epithelium was established. As previously shown, TNF-α treatment resulted in an induction of DEFB4 expression in epithelial cells carrying a high diploid β-defensin copy number, subsequently leading to improved bacterial clearance in the airway surface liquid. Indeed, when counting the amount of viable bacteria 3 hours after infecting cultured nasal epithelial cells with P aeruginosa, the cells carrying a low copy number showed no difference in clearance percentage when treated with TNF-α or left untreated (mean clearance, 40.6% and 45.3%, respectively) (P = 0.2) (Figure 4A). However, in epithelial cells carrying a high diploid copy number, an improved bacterial clearance was observed in all samples treated with TNF-α before the infection (mean clearance, 32.1% and 24.6%, respectively) (P = 0.04) (Figure 4B). Similar results were obtained with the P aeruginosa laboratory strain Pa01. Again, in cells with a low diploid β-defensin copy number, TNF-α treatment had little or no effect on bacterial clearance by cultured epithelial cells (P = 0.5) (Figure 4C). For the cells with a high diploid β-defensin copy number, TNF-α treatment before the infection resulted in a better bacterial clearance in most of the samples with the exception of cell-line 1, which did not reach statistical significance (P = 0.3) (Figure 4D). Together, the present results indicate that a higher diploid β-defensin copy number in upper airway epithelial cells increases the ability of cells to eradicate bacteria in an inflammatory environment.
Given the functional role of β-defensin copy numbers in upper airway epithelial cells, we further investigated whether β-defensin copy numbers are associated with the presence of clinical COPD. Diploid β-defensin copy numbers were determined in 204 COPD patients and compared with 154 healthy smoking controls. Demographic characteristics of cases and controls are given in Tables 1 and 2. Although all individuals were aged above 50 years and exhibited a smoking history of greater than or equal to 10 pack-years, median age and number of pack-years were found to be statistically significantly higher in the COPD group. As expected, patients with COPD presented with a statistically significantly lower mean FEV1, FVC, FEV1/FVC ratio, Dlco, and body mass index compared with the control group. Median diploid β-defensin copy numbers were found to be significantly lower in the control group (4, 4-5) compared with the COPD group (5, 4-5) (P < 0.0001). Based on the observed gene-dosage effect in airway epithelial cells, diploid β-defensin copy numbers were then categorized into three groups, ranging from diploid copy numbers associated with low expression levels (two or three gene copies) over intermediate (four copies) to high expression levels (five or more gene copies). The proportion of individuals who presented with a high diploid β-defensin copy number (five or more) was significantly higher in the COPD group compared with the controls (52% versus 38%; P = 0.008) (Figure 5A), although no significant difference could be observed for the lower and intermediate copy numbers. When finally using the cut-off of five diploid β-defensin copies to differentiate between lower and higher transcript expression, univariate analysis revealed a significant association between high diploid β-defensin copy numbers and the presence of COPD, with an odds ratio of 1.8 (confidence interval [CI], 1.2–2.7; P = 0.008). Moreover, when adjusting for other COPD-related risk factors, such as age, sex, number of pack-years, and number of years quit, high diploid β-defensin copy numbers were still significantly associated with COPD (odds ratio = 1.8; CI, 1.1–2.8; P = 0.02). Within the subgroup of patients with COPD, no significant association was found with FEV1/FVC ratio, FEV1, and Dlco, both expressed as percent predicted.
Smokers with COPD (n = 204)
Healthy Smokers (n = 154)
|Age†, yr||67 (61–74)||60 (57–65)*|
|Body mass index, kg/m2||25 ± 5||27 ± 4*|
|Pack-years†||46 (33–60)||38 (29–52)*|
|Quit-years†||2 (0–9)||3 (0–10)|
|FEV1, L||1.3 ± 0.6||3.1 ± 0.7*|
|FEV1, % predicted||47 ± 17||104 ± 13*|
|FVC, L||2.9 ± 0.8||4.2 ± 0.9*|
|FVC, % predicted||82 ± 18||107 ± 15*|
|Dlco, mm||4.3 ± 1.6||7.9 ± 1.9*|
|Dlco, % predicted||51 ± 17||87 ± 15*|
|Diploid β-defensin copy number†||5 (4–5)||4 (4–5)*|
The association was replicated in an additional and independent case-control study. Here, patients referred for lung transplantation because of end-stage COPD (GOLD IV) (n = 107) were compared with healthy nonsmoking individuals (n = 150). Similarly to our previous findings, median diploid β-defensin copy number was significantly higher in the severe COPD group compared with controls (5, 4-6 versus 4, 4-5; P < 0.0001). This was not caused by selection bias of our control sample because the latter presented with an equal diploid β-defensin copy number distribution as our former smoking controls (Figure 5). In addition, a significantly higher proportion of the patients with severe COPD had high diploid β-defensin copy numbers (five or more) compared with the control sample (74% versus 48%; P < 0.0001) (Figure 5B) with an unadjusted odds ratio of 3.1 (CI, 1.8–5.2; P < 0.0001) for having COPD.
This study demonstrates that diploid CNV in the β-defensin encoding genes correlates with baseline mRNA DEFB4 expression levels in the respiratory epithelium. Only when higher diploid copy numbers of β-defensin genes are present, DEFB4 transcription is significantly up-regulated by inflammatory signals, which resulted in a better antimicrobial activity in vitro. The finding that high diploid copy numbers of β-defensin genes associate with COPD in two independent case-control studies further indicates that β-defensins may play a dual role in the pathogenesis of COPD.
The present study is the first to show that, in addition to SNPs, copy number polymorphisms of certain genes may also confer increased risk for respiratory diseases. In particular, genomic diploid DEFB4 copy number is found to be associated with the presence of COPD, which is in line with recent reports on associations between increased genomic diploid DEFB4 copy numbers and psoriasis or Crohn disease (16, 17). Our data may also explain why current candidate gene and genome-wide associations studies in COPD, which identify susceptibility loci using SNPs as markers, have failed to detect β-defensin genes as important risk loci for disease (6–8).
Human β-defensin 2 (hBD2, encoded by DEFB4) is a highly inducible antimicrobial peptide that plays an important role in the innate immune defense at epithelial surfaces. At first sight efficient eradication of infectious agents seems to be beneficial for healthy patients and patients with COPD. Reduction of bacterial load (28) or eradication of newly acquired infections (29) in the later stages of the disease may also reduce the number of exacerbations, thereby preventing a more rapid decline of FEV1 (30). However, high expression levels of hBD2 are chemoattractant for macrophages, neutrophils, and dendritic cells, which may initiate or perpetuate a chronic inflammatory cascade typically observed in COPD (31). So far, the ambiguous role of β-defensins is not entirely understood (9), but its duality and its underlying genetic determination may certainly contribute to the observed diversity of clinical COPD phenotypes.
The β-defensin gene cluster has a genomic copy number of between 2 and 12 per diploid genome with most individuals possessing three to five copies (15). A strength of our study is that we established, based on DEFB4 transcription levels in airway epithelial cells, a relevant cut-off for diploid DEFB4 genomic copy number, which was then applied to our patient cohorts. Indeed, our in vitro data suggested that a minimal number of five copies or more are needed to induce important and functional increases in DEFB4 expression. Accordingly, five or more copies were found to be significantly associated with the presence of COPD with an odds ratio of 1.8 (CI, 1.1–2.8) after correction for other COPD-related risk factors. The odds ratio even increased to 3.1 (CI, 1.8–5.2) when considering the patients with most severe COPD referred for lung transplantation. Seventy-five percent of this highly selected patient cohort had diploid copy numbers of five or more, which largely exceeds the diploid copy number of ours and other control populations.
From two to five β-defensin copies, a linear correlation between the β-defensin copy number and DEFB4 transcripts was found. When more copies were present, a plateau effect was observed. Possibly other regulatory mechanisms, besides gene dosage, will explain this plateau. Indeed, complete genes located in CNV repeats may be under control of proteins (e.g., transcription factors) that are only transcribed from genes that are present in diploid and haploid (genes located on the X chromosome in males) state. In line of our findings, we therefore hypothesize that different target genes and repeats in the given CNV then compete for the same more finite pool of these regulatory proteins, so that from a given repeat number additional genes and repeats may not result in further linear higher expression levels.
The present study has a number of weaknesses. First, in vitro data are generated on nasal epithelial cells derived from patients with rhinosinusitis. Although different studies have shown that a continuum exist between the respiratory epithelium of the upper and lower respiratory tract (32, 33), caution should be taken when extrapolating these findings to lower airway epithelial cell functions of healthy and COPD patients. Second, diploid DEFB4 copy numbers have been reported to vary from 2 to 12 copies. In our study the maximal diploid copy number observed was eight, which is in agreement with most of the recent larger studies in which the frequency of diploid copy numbers nine or greater is below 1.5% (16, 17). As previously shown, the method we used to quantify diploid copy number differences is very sensitive and reliable with a mean standard error between replicates of 0.07 (22). Moreover, a clear plateau effect was observed in DEFB4 transcript expression when five or more genomic copies were present, suggesting that the detection of diploid copy numbers beyond eight would not greatly impact our results. Finally, patient studies with case-control design have some inherent limitations that limit the general applicability of the data. In the replication study, for instance, a nonsmoking, healthy, and significantly younger control population was used. However, smoking and age are not known to affect CNVs and when correcting in the first case-control study for the covariates age, sex, and pack-years, the significant association between diploid DEFB4 copy numbers and COPD remained unaffected. In addition, a similar frequency distribution of diploid copy numbers was obtained in both of our control populations who were of significant different age and smoking behavior.
The present study demonstrates that genomic diploid CNV of β-defensin encoding genes has a functional role in airway epithelial cells, which may contribute to the pathogenesis of COPD. Replication of our findings in larger unbiased population-based cohorts is needed to explore further the potential of genomic copy number polymorphisms, and in particular for the β-defensin CNV, as risk factor for COPD and different clinical phenotypes.
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