In the present study, a large number of inflammatory cells and inflammatory biomarkers in induced sputum were analyzed, and their association with pulmonary function in patients with COPD were determined. Some of the observations build on previous findings and might provide new sputum inflammatory biomarkers that can be used to predict the severity of lung function decline in patients with COPD.
Studies have reported that the larger the proportion of serum eosinophils is, the higher the risk of COPD exacerbation, suggesting that the level of eosinophils in the peripheral blood could be used as a biological indicator of the prognosis of the disease. However, it was found that peripheral eosinophil counts did not accurately predict sputum eosinophil counts, especially in patients with mild-to-moderate COPD . In steroid-naïve patients with mild-to-moderate COPD, no relationship between the peripheral eosinophil count and the sputum eosinophil percentage was found, highlighting the importance of assessing the level of eosinophils in the airways . Our study focused on the relationship between local eosinophilic airway inflammation and lung function. As expected, the sputum eosinophil percentage was negatively correlated with FEV1%pred. Annette T Hastie et al. reported that a high concentration of sputum eosinophils was a better biomarker than a high concentration of blood eosinophils for the COPD patient subgroup with more severe disease, more frequent exacerbations, and a higher proportion of patients with emphysema. According to the current criteria, a high level of blood eosinophilia has been defined by a blood eosinophil threshold ≥ 2% or ≥ 300 cells/µL[24, 25]. COPD patients with high levels of blood eosinophils have been found to be more sensitive to treatment with ICS, and there was an increase in the number of exacerbations in these patients when ICS therapy was discontinued. Across studies, the incidence of high blood eosinophil levels varies significantly, ranging from 14–37% of COPD patients, while high levels of sputum eosinophils were detected in 30%~40% of COPD patients, suggesting that sputum eosinophils may an effective and more stable predictor of the response to ICS therapy in COPD patients[6, 27]. It is important for clinical trials targeting eosinophilic inflammation in COPD to consider the assessment of sputum eosinophils. However, our study showed that there were no correlations of the percentages of neutrophils, lymphocytes or monocytes in induced sputum samples with lung function decrease in patients with COPD.
CC16 is a protein secreted by Clara cells in the bronchioles and terminal bronchioles; it is specific to the lung tissue and exerts anti-inflammatory effects. Recent studies have shown that epithelial cell damage and airway remodeling result in the reduction of the level of CC16 in patients with COPD. Most previous studies have indicated that serum CC16 levels are related to functional declines in COPD patients; however, few studies have focused on sputum CC16 levels[30, 31]. We found a significantly lower level of CC16 mRNA in patients with severe and extremely severe COPD than in those with mild and moderate COPD, suggesting that that CC16 in the lower airways may contribute to the progressive anatomical damage in the end stage of COPD. Moreover, multiple regression analysis showed that the level of CC16 mRNA in the induced sputum cells was independently correlated with the decline in lung function (FEV1%pred and FEV1%FVC). The report from David Chi-Leung Lam et al. showed a relationship between low serum CC16 levels at baseline and greater lung function decline over the course of 10 years in a Chinese longitudinal cohort. In that study, they also found that the level of CC16 mRNA in endoscopic biopsies of the bronchial epithelium was also correlated with the FEV1/FVC ratio. Compared to endoscopic biopsies of bronchial epithelium, the induction of sputum production and subsequent detection of CC16 mRNA in our study was less invasive and more practical. Furthermore, based on our data, when the relative mRNA level of CC16 in the induced sputum cells was less than 1.09, it indicated that the patient may have severely limited airflow, which suggested that the CC16 mRNA level in the induced sputum cells may be a predictive biomarker for the severity of COPD.
MiRNAs are 19–22 nucleotides in length and regulate target mRNA translation or degradation. miRNAs play crucial roles in apoptosis, cell differentiation, proliferation and other biological processes . A few studies have shown that the dysregulation of several miRNAs is involved in the inflammatory response and fibrosis in patients with pulmonary disease. Based on the findings in earlier studies, we chose to detect the expression levels of MiR-155 and MiR-21 in the induced sputum cells from patients with COPD. Our study showed lower levels of miR-155 but not miR-21 in the severe and extremely severe group than in the mild and moderate group. Furthermore, miR-155 was independently positively correlated with the FEV1/FVC ratio but not FEV1%pred. Different results regarding miR-155 levels in patients with COPD have been reported in several previous studies, while in patients with eosinophilic-related lung disease, the miR-155 levels were found to be reduced[14, 32, 33]. In fact, miR-155 was reported to regulate Th cell differentiation, which causes a decrease in the level of eosinophils and the release of inflammatory cytokines, including IL-6 . The increased percentage of eosinophils but not neutrophils associated with more severe COPD in our analysis might be related to miR-155 downregulation.
In addition to the crucial findings reported above, the mRNA levels of MMP9 and LTB4R were significantly higher in the induced sputum samples from the severe and extremely severe group than in those from the mild and moderate group. Studies have shown that MMP-9, a glycosylated gelatinase, increases the deposition of extracellular matrix by mediating the accumulation and infiltration of inflammatory cells in the lung, resulting in pulmonary vascular bed remodeling and lung tissue damage in patients with COPD. Mitsunobu et al. reported that LTB4 levels were elevated in the peripheral blood or induced sputum of COPD patients, and these elevated levels could activate the inflammatory response by combining with LTB4R. The increase in LTB4R in the induced sputum samples of patients with severe and extremely severe COPD in our study provided evidence of LTB4-related inflammation. Among the inflammatory cytokines that we measured, only the reduced level of IL-β achieved statistical significance. In fact, the levels of many of the inflammatory mediators in the induced sputum supernatant were below the level of detection for commercial ELISA kits. The use of dithiothreitol to process the induced sputum samples has been reported to decrease the concentrations of many proteins, including inflammatory cytokines[36–38]. Other factors, including lysis time and temperature, affect the concentrations of the inflammatory biomarker in the induced sputum supernatant. Studies have shown that the measurement of mRNA expression may be a more sensitive method of evaluating inflammatory mediators in induced sputum[7, 39, 40]. In accordance with this, our analysis also showed that sputum cell CC16 mRNA but not protein expression in the sputum supernatant was closely and independently correlated with lung function decline (FEV1%pred and FEV1%FVC) in patients with COPD. Thus, we believe that the assessment of induced sputum cells by qRT-PCR could be a feasible and stable method for the clinical determination of the severity of COPD.
The limitations of this study include the lack of data on parameters in the serum of COPD patients; therefore, we were unable to investigate the relationships between and differences in inflammatory biomarker levels in the sputum and the blood. Future studies should be performed to determine the correlations of inflammatory mediators involved in local airway inflammation and systemic inflammation. Second, only the levels of mRNA for inflammatory biomarkers in sputum cells were measured. Studies to confirm the protein levels in sputum cells and their associations with the pulmonary function parameters are still needed. Furthermore, since the sample size was small, the results may be biased, and this sample population is not representative of the entire COPD population.