ABSTRACT
Background:
Defensins are key effector molecules of innate immunity that can contribute towards the diagnosis and monitoring of chronic obstructive pulmonary disease (COPD). The present study was conducted to investigate the role of alpha-defensins in patients with COPD by quantifying serum and sputum samples.
Methods:
A total of 180 patients were enrolled and divided into four groups, and sputum and serum values of alpha-defensins were assessed. The sensitivity, specificity, and accuracy of sputum alpha-defensin as a diagnostic biomarker were evaluated to assess its utility in diagnosing COPD.
Results:
The mean value of sputum alpha-defensins was found to be statistically significant amongst the four groups (P < 0.001). The highest levels were found in subjects with AECOPD (385.76 ± 116.62 ng/mL). Sputum alpha-defensins were found to be negatively correlated with FEV 1 values (rho = −0.31, P < 0.001).
Conclusion:
Sputum alpha-defensins can be used as a potential marker for predicting acute exacerbation of COPD. In addition, they could serve as an indicator of disease severity in COPD patients.
KEY WORDS: Alpha-defensins, biomarker, chronic obstructive pulmonary disease, exacerbation, human neutrophil peptide
INTRODUCTION
Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung condition characterised by chronic respiratory symptoms (dyspnoea, cough, sputum production, and/or exacerbations) due to abnormalities of the airways (bronchitis, bronchiolitis) and/or alveoli (emphysema) that cause persistent, often progressive, airflow obstruction.[1] It is a leading cause of morbidity and mortality worldwide with a considerable amount of economic and social burden.[2,3] Inhalation of cigarette smoke or other noxious particles leads to lung inflammation in patients with COPD. Although the precise role of respiratory infections in COPD is unclear, it can be stipulated that chronic airway inflammation caused by infections may also be an etiologic agent for the development of COPD.[4] Moreover, tracheobronchial infections are known to be a major cause of acute exacerbation in cases of COPD. The chronic inflammatory response occurring via various pathways consequently leads to the destruction of the lung parenchyma and causes interference in the normal repair and defence mechanisms of the lung.
The two most commonly used indicators of disease severity, extent, and progression in COPD are forced expiratory volume in 1 second (FEV1) and HRCT thorax. The drawback with FEV1 is that not only does it correlate poorly with symptoms and other measures of disease progression but also that it does not distinguish between causes of airflow limitation and provides no insight into identifying the extra-pulmonary manifestations of COPD. HRCT is an expensive diagnostic method with an additional component of radiation exposure. Moreover, it seldom provides information in cases of patients with chronic bronchitis, which usually affects half of the patients with COPD. In view of limitations in the utility of both spirometry and HRCT thorax, there is a need for the search of novel biomarkers in COPD that can contribute not only towards diagnosis, especially at a sub-clinical level, but also in predicting, staging, and monitoring the disease progression as well as assessing the response of therapeutic interventions.
Defensins are small cationic peptides with antimicrobial activities against both gram-negative and gram-positive bacteria, fungi, and some viruses. They play a key role as effector molecules of innate immune response and contribute to the pathogenesis of COPD by having a synergistic effect on cigarette smoking and infection-induced inflammatory reactions, which may lead to lung injury. They are divided into two sub-families in humans, namely alpha (α)-defensins and beta (β)-defensins, which have a characteristic three-dimensional fold and a 6-cysteine/3-disulphide pattern. In humans, six alpha-defensins and at least four beta-defensins are clustered in a 450-kb region on chromosome 8p23.[5] Alpha-defensins, including human neutrophil defensins 1–4, known as human neutrophil peptides or HNPs 1–4, and human enteric defensins 5–6 or HD5 and HD6 are produced by neutrophils and intestinal Paneth’s cells, whereas beta-defensins are mainly produced by epithelial cells.[6]
Alpha-defensins have multi-faceted roles in the regulation of complement activation and protease inhibitor secretion. Their accumulation in the airway secretions of patients with chronic inflammatory lung disorders has been shown to be cytotoxic towards the airway epithelial cells. The role of alpha-defensins in promoting bacterial adherence to epithelial cells in vitro further supports their contribution to the pathogenesis of COPD.[7]
Thus, it can be inferred that defensins play a key role in the pathogenesis of COPD and should be considered for COPD association studies due to their expression patterns, locations, and function. Only a few studies have reported the association of the level of alpha-defensins in the pathogenesis of COPD.[4,8,9] No such study has been reported in India so far. The association, if found, would help in the early diagnosis of COPD and timely therapeutic intervention and may help to reduce the morbidity associated with the disease. The present study was thus conducted to investigate the role of alpha-defensins as a diagnostic and severity marker in patients of COPD by quantifying the biomarker in serum and sputum samples.
METHODS
The study was conducted in the Department of Pulmonary Medicine in collaboration with the Department of Biochemistry in a Government Medical College in North India.
Study design
Case-control, cross-sectional study.
Duration of Data collection- from November 2019 to June 2021.
The optimum sample size for the present study was calculated based on a 43% positivity rate of alpha-defensin in acute exacerbation cases of COPD and a 72% positivity rate of alpha-defensins among stable COPD. Assuming a 90% confidence coefficient and 10% permissible error, the optimum sample size for the two study groups came out to be 20 cases of acute exacerbation of COPD and 80 cases of stable COPD. For these two study groups, a control group of 40 cases of patients having diseases other than COPD and 40 cases of healthy controls were also selected for reference values. Hence, the study participants were divided into four groups.
Group A: Forty healthy subjects, out of which 20 participants were healthy non-smokers, and 20 participants were healthy smokers having a smoking history of at least 10 pack years, with no signs or symptoms of any systemic disease, a normal chest X-ray, and spirometry analysis.
Group B: Forty patients as disease control, out of which 20 patients were diagnosed with bronchial asthma as per the latest Global Initiative for Asthma (GINA) guidelines[10] and 20 patients suffered from other lung diseases apart from COPD and bronchial asthma.
Group C: Eighty patients with stable COPD diagnosed as per GOLD criteria.
Group D: Twenty patients with acute exacerbation of COPD (AECOPD) diagnosed as per the GOLD criteria.[11]
Exclusion criteria
Refusal of consent
History of acute cardiovascular event 6 weeks prior to study
History of acute exacerbation of COPD 4 weeks prior to the study (except for group D)
Patients having COPD along with other co-morbid respiratory diseases, for example, COPD with pneumonia or COPD with lung cancer (for group B)
History of <10 pack years of smoking (for the smoker sub-group of group A).
All patients were included in the study after obtaining informed written consent. Patients having COPD, bronchial asthma, as well as healthy controls were subjected to spirometry. In addition, those with a history of smoking of at least 10 pack years were subjected to a chest X-ray examination. Diagnosis of COPD and assessment of severity was done according to the GOLD guidelines.[1]
Sputum and 5 mL blood samples from each participant were collected at the time of recruitment for measurement of alpha-defensin. Sputum induction with hypertonic saline was performed in patients who were unable to produce sputum. The samples were centrifuged, and the separated serum and sputum were stored individually at −80°C until analysis. The alpha-defensins level was estimated by sandwich ELISA. The kit used was Bioassay Technology Laboratory Human Defensin Alpha 1 ELISA kit with Intra-Assay: CV <8%, Inter-Assay: CV <10%, and which had a sensitivity of 2.48 ng/mL. The equipment used was an ELISA Reader-TECAN and ELISA Washer-HydroFlex-TECAN.
Statistical analysis
The statistical analysis was performed using Statistical Package for Social Science (SPSS). Continuous data were expressed as mean ± standard deviation, and non-continuous data were expressed as percentages/proportions. The data obtained were analysed using ANOVA, post-hoc test, NPar test, Kruskal–Wallis, and Mann–Whitney test as per relevance. Results on alpha-defensins in the four groups were described using mean and standard deviation (SD).
Mean values of marker alpha-defensin were compared using the analysis of variance (ANOVA) test, and pair-wise comparison between different groups was done using Student’s t-test in case of normal distribution and by using Mann–Whitney test for non-normal distribution.
The cut-off value of sputum alpha-defensin was calculated by the receiver operating characteristic (ROC) curve. The sensitivity, specificity, and accuracy of sputum alpha-defensin as a diagnostic biomarker were evaluated to assess its utility in diagnosing COPD.
RESULTS
The clinico-demographic profile of the study population is shown in Table 1. Out of a total of 180 patients, there were 131 males and 49 females, and the mean age was 47.61 ± 11.86 years. On inter-group analysis, the highest mean age was seen in AECOPD, which was 54.75 ± 9.27 years. Out of 180 study participants, 66.7% of participants were smokers, and the mean pack-years smoked was 28.50 ± 12.96 years. The mean total leucocyte count among the study participants was 10.26 ± 3.31 (×103/mm3), which was statistically different among all four groups (P = 0.001). Leucocytosis was seen in 38.3% of all the study participants, with the mean TLC count being highest in AECOPD. The mean levels of serum alpha-defensins were not found to be significantly different among all four groups (P = 0.856). However, the mean value of sputum alpha-defensins was found to be significantly different amongst the four groups (P < 0.001), as shown in Table 1. The highest levels were found in subjects with AECOPD (385.76 ± 116.62 ng/mL). The association of sputum alpha-defensins and various socio-demographic and laboratory parameters are depicted in Table 2. It was found that there is a statistically significant positive correlation between sputum alpha-defensins and parameters such as age and TLC. A pair-wise analysis of sputum alpha-defensins was done, and statistically significant higher values were observed in COPD patients (234.91 ± 212.66 ng/mL) as compared to healthy controls (133.38 ± 64.04 ng/mL) (P = 0.004). In addition, the levels were statistically higher in the AECOPD group (group D) as compared to the COPD group (group C), as shown in Table 3. The scatterplot shown in Figure 1 depicts the correlation between FEV1 (observed) and alpha-defensin (ng/mL). There was no statistically significant correlation between FEV1 (observed) and serum alpha-defensin (ng/mL) (rho = 0.01, P = 0.930). There was a moderate negative correlation between FEV1 (observed) and sputum alpha-defensin (ng/mL), and this correlation was statistically significant (rho = −0.31, P = <0.001). For every 1 unit increase in FEV1 (observed), the sputum alpha-defensin (ng/mL) decreased by 64.27 units. A moderate positive correlation between neutrophils (%) and sputum alpha-defensin (ng/mL) was noted, which was statistically significant (rho = 0.59, P = <0.001) [Figure 2]. For every 1 unit increase in neutrophils (%), the sputum alpha-defensin (ng/mL) increased by 8.40 units. A ROC curve analysis was performed mapping the sputum alpha-defensin in the diagnosis of COPD, as shown in Figure 3, and the area under the curve was found to be maximum for the group healthy versus AECOPD, which was 97.9%. As shown in Table 4, the accuracy of sputum alpha-defensins in diagnosing AECOPD from healthy patients was 97.9% at a cut-off value of 204.79 ng/mL, with a sensitivity and specificity of 100% and 90%, respectively. The accuracy of sputum alpha-defensins in diagnosing AECOPD from COPD was 85.10% at a cut-off value of 219.05 ng/mL, with a sensitivity and specificity of 100% and 61%, respectively.
Table 1.
Summary of all parameters
| Parameters | Group A | Group B | Group C Stable COPD | Group D AECOPD | P | ||
|---|---|---|---|---|---|---|---|
|
|
|
||||||
| Healthy Non-smokers | Healthy smokers | Asthma | Other lung diseases | ||||
| Mean age (years) | 36.15±11.60 | 42.70±7.76 | 43.75±12.64 | 47.90±12.90 | 50.81±10.32 | 54.75±9.27 | <0.001 |
| <40 years | 13 (65.0%) | 9 (45.0%) | 6 (30.0%) | 6 (30.0%) | 14 (17.5%) | 0 (0.0%) | <0.001 |
| 40–60 years | 7 (35.0%) | 11 (55.0%) | 13 (65.0%) | 9 (45.0%) | 50 (62.5%) | 14 (70.0%) | |
| ≥60 years | 0 (0.0%) | 0 (0.0%) | 1 (5.0%) | 5 (25.0%) | 16 (20.0%) | 6 (30.0%) | |
| Male | 12 (60.0%) | 13 (65.0%) | 12 (60.0%) | 12 (60.0%) | 66 (82.5%) | 16 (80.0%) | 0.078 |
| Female | 8 (40.0%) | 7 (35.0%) | 8 (40.0%) | 8 (40.0%) | 14 (17.5%) | 4 (20.0%) | |
| Smoking (yes) | 1 (5.0%) | 20 (100.0%) | 7 (35.0%) | 10 (50.0%) | 67 (83.8%) | 15 (75.0%) | <0.001 |
| Pack-years | 10.00±0 | 18.35±5.51 | 21.86±10.32 | 30.10±13.58 | 31.66±13.45 | 29.93±11.63 | 0.001 |
| HTN (Yes) | 0 (0.0%) | 0 (0.0%) | 12 (60.0%) | 12 (60.0%) | 38 (47.5%) | 14 (70.0%) | <0.001 |
| DM (Yes) | 0 (0.0%) | 0 (0.0%) | 3 (15.0%) | 8 (40.0%) | 21 (26.2%) | 5 (25.0%) | 0.001 |
| CAD (Yes) | 0 (0.0%) | 0 (0.0%) | 3 (15.0%) | 4 (20.0%) | 13 (16.2%) | 4 (20.0%) | 0.077 |
| TLC (×103/mm3) | 6.77±1.47 | 7.50±1.86 | 10.55±2.52 | 10.90±3.15 | 10.64±3.00 | 14.03±2.63 | <0.001 |
| Neutrophils (%) | 60.70±6.12 | 64.75±6.91 | 74.50±7.10 | 55.50±4.78 | 67.88±9.45 | 85.60±6.25 | <0.001 |
| Lymphocytes (%) | 27.50±4.89 | 25.45±5.67 | 13.25±5.80 | 33.85±3.65 | 22.88±8.67 | 9.35±5.63 | <0.001 |
| Eosinophils (%) | 3.05±1.61 | 2.70±1.45 | 6.15±2.98 | 3.40±1.88 | 2.74±1.47 | 2.65±1.35 | <0.001 |
| Serum alpha-defensin (ng/mL) | 174.49±82.36 | 125.72±74.27 | 203.00±116.58 | 142.14±59.48 | 164.29±154.76 | 155.89±58.35 | 0.117 |
| Sputum alpha-defensin (ng/mL) | 152.93±71.76 | 113.82±49.64 | 253.28±115.65 | 130.20±83.13 | 234.91±212.66 | 385.76±116.62 | <0.001 |
Table 2.
Association of sputum alpha-defensins with various socio-demographic and laboratory parameters
| Socio-demographic parameters | Sputum alpha-defensin (ng/mL) | P | |
|---|---|---|---|
| Age In years | Correlation coefficient (rho)=0.17 | 0.020 | |
| Age | <40 Years | 178.03±91.41 | 0.252 |
| 40-60 Years | 229.94±202.23 | ||
| ≥60 Years | 251.91±160.88 | ||
| Gender | Male | 227.59±180.90 | 0.265 |
| Female | 197.92±154.22 | ||
| Smoking | Yes | 231.11±197.74 | 0.664 |
| No | 196.33±110.90 | ||
| Smoking history in pack-years | Correlation coefficient (rho)=0.03 | 0.743 | |
| HTN | Yes | 244.89±178.50 | 0.020 |
| No | 200.97±169.32 | ||
| DM | Yes | 230.61±139.63 | 0.219 |
| No | 216.65±182.31 | ||
| CAD | Yes | 218.17±112.49 | 0.425 |
| No | 219.72±182.04 | ||
| Laboratory parameters | |||
| TLC (×103/mm3) | Correlation coefficient (rho)=0.38 | <0.001 | |
| Neutrophils (%) | Correlation coefficient (rho)=0.63 | <0.001 | |
| Lymphocytes (%) | Correlation coefficient (rho)=−0.61 | <0.001 | |
| Eosinophils (%) | Correlation coefficient (rho)=−0.02 | 0.798 | |
Table 3.
Pair-wise analysis of sputum alpha-defensins
| Group | Mean Sputum alpha-defensin | P | |
|---|---|---|---|
| Healthy vs other lung diseases | Group A | 133.38±64.04 | 0.007 |
| Group B | 191.74±117.33 | ||
| Healthy vs COPD | Group A | 133.38±64.04 | 0.004 |
| Group C | 234.91±212.69 | ||
| Healthy vs AECOPD | Group A | 133.38±64.04 | <0.001 |
| Group D | 385.76±116.62 | ||
| Other lung diseases vs COPD | Group B | 191.74±117.336 | 0.235 |
| Group C | 234.91±212.66 | ||
| Other lung diseases vs AECOPD | Group B | 191.74±117.336 | <0.001 |
| Group D | 385.76±116.62 | ||
| COPD vs AECOPD | Group C | 234.91±212.66 | 0.003 |
| Group D | 385.76±116.62 | ||
Figure 1.
Correlation between FEV1 (observed) and alpha-defensin in serum and sputum (ng/mL) (n = 120)
Figure 2.
Correlation between neutrophils (%) and sputum alpha-defensin (ng/mL) in (Diagnosis: COPD) (n = 80)
Figure 3.
ROC curve analysis mapping the sputum alpha-defensin in the diagnosis of COPD
Table 4.
Sensitivity and accuracy of sputum alpha-defensin in the diagnosis of COPD
| Cut-off value | Sensitivity | Specificity | Accuracy | |
|---|---|---|---|---|
| Healthy vs COPD | 180.50 | 51.30% | 80.00% | 71.00% |
| Healthy vs AECOPD | 204.79 | 100.00% | 90.00% | 97.90% |
| COPD vs AECOPD | 219.05 | 100.00% | 61.30% | 85.10% |
DISCUSSION
This study aimed to identify alpha-defensin as a potential diagnostic marker for patients of COPD by estimating its levels in serum and sputum samples. To the best of our knowledge, this was the first study done in the Indian population to examine the level of serum and sputum alpha-defensins in patients with COPD.
The results in the present study showed that sputum alpha-defensins levels were significantly higher in cases of acute exacerbation of COPD as compared to all other groups. Sputum alpha-defensins levels were also higher in COPD and other lung diseases as compared to healthy controls. Sputum alpha-defensins levels could thus be used as a marker for disease severity for patients with COPD. The results also showed that there was no significant difference in the levels of serum alpha-defensins in all groups.
Neutrophils have the capacity to accumulate in large numbers in the lungs during infection and inflammation. Although neutrophils play an important role in host defence against infection, they may also cause tissue injury and are sought to be involved in the pathogenesis of various inflammatory lung conditions.[12] In our study, leucocytosis was predominant in group D, which had a mean TLC value of 14.03 ± 2.63 (×103/mm3) (P < 0.001), and all 20 patients had predominant neutrophilia. In earlier studies, it has been seen that neutrophils are often the first cells recruited to the site of infection wherein they release effector molecules such as human neutrophilic peptides (HNPs).[13] Although HNPs exhibit direct and potent antimicrobial activities, these peptides also control infection by modulating various immune activities, including chemotaxis, phagocytosis, and cytokine induction during acute infection. However, uncontrolled inflammation can lead to tissue damage and worsen disease progression.[14] The fact that patients with acute exacerbation of COPD more frequently land up in exacerbation due to infective causes could explain the predominant leucocytosis in group D (AECOPD).
Although serum defensin levels correlate with the severity of infection, the levels may be influenced by a variety of factors such as the age of the patient, the underlying disease pathology, or any co-morbid illness. These factors may mask the true values of a serum biomarker by either falsely elevating or decreasing the levels. In the present study, there was no statistically significant difference in the value of serum alpha-defensins in the four groups. The results were in concordance with the study done in 2008 in Beijing, China, which showed no significant difference in the serum HNP1-3 level among the group of AECOPD, stable COPD, and healthy controls (P > 0.05).[10] ROC curve analysis suggested that a cut-off level of 219.05 ng/mL could be used to distinguish COPD from AECOPD with a sensitivity of 100% and specificity of 61.30%. Furthermore, a cut-off value of 180.50 ng/mL could be used to distinguish healthy controls from COPD with a sensitivity of 51.30% and specificity of 80%. It can be thus inferred from the above analysis that sputum alpha-defensins could be useful for the early prediction of cases of acute exacerbation of COPD. This may contribute to the early diagnosis of exacerbation and may improve the outcome of such patients with timely treatment strategies. However, these results could not be validated due to the paucity of available literature on levels of alpha-defensins in COPD and healthy populations.
The higher sputum alpha-defensins values could have been influenced by either infective aetiologies, increased local inflammation, and disease progression or a combination of both. The same was supported by the present study as sputum alpha-defensins were the highest in the AECOPD group (385.76 ± 116.62 ng/mL), and there was a moderate negative correlation between FEV1 (observed) and sputum alpha-defensin (ng/mL), which was statistically significant (rho = −0.31, P = <0.001). Hence, it can be assumed that sputum alpha-defensins correlated positively with the level of inflammation. Moreover, it may be proposed that sputum alpha-defensin can be used as a marker for disease severity in patients with COPD, inferring that the lower the FEV1 value, the higher the level of sputum alpha-defensin.
Defensins work as a natural antibiotic for infected cells in humans, but their role as inflammatory regulators needs to be researched further. The present study found that sputum alpha-defensins are increased in inflammatory lung conditions and can be used for differentiating these conditions from healthy controls. Moreover, the results showed that sputum alpha-defensin levels are more specific as compared to their serum counterparts. As this study is one of a kind, the results obtained in the present study can pave the way for further research in this area.
Limitations
The present study had a few limitations. A smaller number of patients in each sub-group may have affected the power of sub-group analysis. In addition, we did not analyse the pharmacological effect on alpha-defensins, which may have influenced the study results. It may be hypothesised that alpha-defensins levels could have been influenced by different ongoing treatment modalities such as long-acting beta-2-agonist, long-acting muscarinic agonist, and inhaled corticosteroids, either singly or in a combination. Alpha-defensins could have also been affected by systemic antibiotics received in patients of group D (AECOPD). Lastly, the sub-division of stable COPD patients into A, B, and E classification as per the latest GOLD guidelines was not done; thus, the correlation of alpha-defensins within these sub-groups was not studied.
CONCLUSION
Based on the study findings, it can be inferred that sputum alpha-defensins emerged as a superior marker for the studied population as compared to its serum counterpart. Specifically, the study highlights the higher value of sputum alpha-defensins in patients with acute exacerbation of COPD, which may be helpful in diagnosing these cases early. In addition, a negative correlation between sputum alpha-defensins and FEV1 indicates their potential utility as a marker for monitoring the progression of the disease. The inference extends to the hypothesis that the inflammatory regulatory role of alpha-defensins could contribute to acute exacerbations in COPD patients, particularly at higher local levels in the airways. The study calls for further research to delineate the specific point at which local alpha-defensins transition from their anti-inflammatory functions to inflammatory and tissue injury activities in the airways.
Ethics approval
The study protocol was approved by the institutional ethics committee (Letter no.: GMCH/IEC/2019/146 dated 17/12/2019).
Financial support
Nil.
Conflicts of interest
There are no conflicts of interest.
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