The Effect Of Cathelicidins On The Pathological Changes Of Chronic Obstructive Pulmonary Disease

BackgroundChronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality wordwide, which has been a major and increasing global health problem. COPD is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually associated with small airway remodeling and emphysema. The mechanisms of COPD have not yet been understood, and there is still no effective therapy to cure the disease. Thus, it is important to investigate the pathogenesis of COPD.AMPs are small peptides that represent essential elements of innate immunity. AMPs include two major groups:cathelicidins and defensins. hCAP18/LL-37is the only member of human cathelicidin family. In addition to antimicrobial activities, cathelicidins have pleiotropic activities including chemotaxis, wound repair and apoptosis.Previous work demonstrated that the expression of LL-37was significantly elevated in induced sputum of COPD patients, and the expression of LL-37was positively correlated with IL-8levels, and negatively correlated with EFV1%. Further study demonstrated that LL-37was mainly located in bronchial epithelial cells, alveolar epithelial cells, inflammatory cells and fibroblasts. These results suggest that LL-37may be involved in the pathogenesis of COPD.Based on the previous findings, we found that LL-37levels in bronchial epithelial cells and alveolar epithelial cells from COPD smokers were significantly higher than healthy non-smokers and smokers, and the levels of LL-37were higher in smokers compared with non-smokers. Then we raise the questions:(1) Whether the increased levels of LL-37in small airway epithelial cells and alveolar epithelial cells are associated with the pathological changes of small airway remodeling and emphysema?(2) Whether cigarette smoke exposure is the cause for elevated expression of LL-37?(3) How epithelial-derived LL-37influences the collagen deposition and tissue remodeling?(4) Why LL-37is significantly increased in epithelium of small airways and parenchyma, the pathological changes are different in the two regions?ObjectiveIn this study, we explore the effect and mechanism of cathelicidins in the pathological changes of small airway remodeling and emphysema in COPD.Methods1. To observe the expression of LL-37in lung tissues of COPD smokers and to analyse the correlation between the expression of LL-37and the structural changes of small airway remodeling and emphysema in COPD.(1) Study subjects:Three groups of subjects were recruited for this study, including20non-smokers without COPD,22smokers without COPD, and18smokers with COPD. All subjects were patients who underwent resection of lung tumor and lung cyst and obtained at Qilu Hospital (Jinan, China). The diagnosis of COPD was made according to the guidelines of the Global Initiative for Chronic Obstructive Lung Disease.(2) Human lung tissue samples:①The pathomorphological changes, including the thickness of small airway wall, collogen deposition in the airway wall, mean linear intercept (MLI) and mean alveolar number (MAN) were measured with image-analysis system.②The expression of LL-37in lung tissues was determined by immunohistochemistry technique.③The correlation between LL-37immunoreactivity in lung tissues and structural changes of COPD was analyzed.2. To establish an experimental model for smoke-induced COPD and to evaluate the expression of cathelicidin (rCRAMP) in lung tissues and pathological changes of COPD dynamically.Male Wistar rats (200±20g) were randomly divided into cigarette smoke group (CS group) and normal control group (NC group). In CS group, rats were exposed to the smoke of10commercial, nonfilter cigarettes in ventilated whole-body smoking chambers (70cm×50cm×50cm) for30minutes each time, twice per day,6days a week for3or6months, respectively (each group consists of12animals). In NC group, animals were exposed to clean air under similar conditions (each group consists of12animals). During the6months, each rat was weighed every two weeks.(1) At the end of1-,3-and6-month of smoke exposure, the lung function was measured by body plethysmography,(2) The structural changes of small airway remodeling and emphysema including thickness of small airway wall, collogen deposition in the airway wall, mean linear intercept (MLI) and mean alveolar number (MAN) were measured with image-analysis system.(3) The expression of rCRAMP in lung tissues of rats was determined by immunohistochemistry.(4) The correlation between rCRAMP immunoreactivity in lung tissues of rats arid structural changes of COPD was analyzed.3. To investigate the role of epithelial-derived LL-37in collagen production by human lung fibroblasts.(1) Human bronchial epithelial cells (16HBE) were cultured and stimulated with CSE, and the expression of LL-37was detected by immunofluorescence staining.(2)16HBE cells were co-cultured with human lung fibroblasts (HFL-1) in transwell six-well plates, after stimulated with different concentrations of CSE, the expression of LL-37was determined by ELISA and the levels of total collagen was detected by Sircol collagen assay kit.(3) HFL-1cells were cultured in the six-well plates, after stimulated with the same concentration of CSE with step2, the expression of total collagen was detected by Sircol collagen assay kit.(4) HFL-1cells were incubated with LL-37synthesis peptide for48h, total collagen was determined by Sircol collagen assay kit and type Ⅰ and Ⅲ collagen gene were detected by RT-PCR.(5) After pretreatment with ERK inhibitor, PD98059(10uM), p38inhibitor, SB203580(10μM), JNK inhibitor, SP600125(20μM), PI3K inhibitor, LY294002(20 μM) or formyl peptide receptor-like1(FPRL1) antagonist WRW42h before LL-37stimulation, total collagen in the supernatant was assayed.(6) HFL-1cells were treated with various doses of LL-37synthesis peptide, and the levels of ERK and phosphorylated ERK (pERK) were evaluated by Western blot.(7) After pretreatment with WRW4or PD980592h before LL-37stimulation, the levels of ERK and pERK were determined by Western blot.4. To explore the heterogeneity of fibroblasts in small airways and alveoli from COPD patients.(1) The expression of vimentin, E-cadherin and CD45in fibroblasts from lung tissues was examined by double immunohistochemistry. Fibroblasts of different phenotypes were counted and the ratio was calculated in all the samples.(2) The expression of type Ⅰ collagen, type Ⅲ collagen, elastin and fibronectin in lung tissues was determined by immunohistochemistry.Results1. The expression of LL-37in lung tissues from COPD patients was associated with the pathological changes of small airway remodeling and emphysema.(1) Clinical data collection:There was no significant difference in smoking history between smokers with and without COPD, and all groups exhibited a similar age range. The forced expiratory volume of predicted (FEV1%) and FEV1/FVC of COPD smokers were significantly lower than that of healthy smokers and nonsmokers, however, thiere was no significant difference in FEV1%and FEV1/FVC between healthy smokers and healthy nonsmokers.(2) Tests on human lung tissue sections:①There were significant increases in small airway wall thickness and peribronchial collagen deposition in smokers with COPD. It was alos found that MLI was significantly increased and MAN was decreased in smokers with COPD.②LL-37in lung tissues was mainly located in epithelial cells of small airways and alveoli, inflammatory cells (including neutrophils, macrophages and lymphocytes) and fibroblasts. In non-smokers, a faint staining of LL-37was seen in lung tissues. There was a significant increase in LL-37expression in lung tissues from smokers without COPD and smokers with COPD, especially in smokers with COPD.③Among all the subjects, LL-37expression in airway epithelium and submucosa was positively correlated with airway wall thickness and collagen deposition(epithelium:r=0.62, P<0.01, r=0.73, P<0.01; submucosa:r=0.65, P<0.01; r=0.71, P<0.01). The expression of LL-37in alveoli was significantly correlated with MLI and MAN(r=0.64, P<0.01; r=-0.54, P<0.01).2. Cigarette smoke is the cause for increased levels of rCRAMP and rCRAMP levels in lung tissues of rats are associated with structural changes of small airway remodeling and emphysema.(1) The development of weight in CS group rats was significantly inhibited compared with the NC group.(2) After3-month exposure, cigarette smoke caused an increase in RL, whereas a decrease in Cdyn compared with the NC group. Following6-month cigarette smoke exposure, RL was increased by21.9%and Cdyn was decreased by23.8%. Cigrette smoke didn’t alter tidal volume (VT) of rats significantly.(3) After cigarette smoke exposure for3months, the rats shared pathological features of small airway remodeling and emphysema, including thickened airway wall thickness, increased collagen deposition in the airway wall, destruction of alveolar septum and enlarged air spaces. After6month cigarette smoke exposure, the pathological changes became more apparent. Using quantitative histomorphology techniques, there were significant increases in small airway wall thickness and peribronchial collagen deposition following6-month CS exposure. It was also found that MLI was increased after3-month cigarette smoke exposure, and MAN was significantly decreased compared with NC group.(4) The expression of rCRAMP in small airways and alveoli was significantly increased after cigarette smoke exposure. There was a progressive trend to the expression of rCRAMP in lung tissues following6-month cigarette smoke exposure.(5) There was a significantly positive correlation between rCRAMP immunoreactivity in small airway wall thickness. A similar positive correlation was found between rCRAMP immunoreactivity and the amount of collagen deposited on the airway wall. The expression of rCRAMP in the alveolus was significantly correlated with MLI andMAN.3. CSE-induced epithelial secretion of cathelicidin (LL-37) could up-regulate collagen expression in HFL-1cells via activation of FPRL1-mediated ERK pathway.(1) The exposure to CSE resulted in a substantial enhancement of LL-37staining in the cytoplasm of the16HBE cells.(2) After stimulation with CSE, the levels of LL-37in co-culture system were significantly increased and the total collagen was also elevated. In addition, LL-37neutralizing antibody significantly abolished the production of total collagen.(3) After stimulation with CSE, there is no difference in the expression of total collagen by HFL-1cells compared with the control group.(4) LL-37synthesis peptide induced a dose-dependent increase in total collagen content as well as type Ⅰ and Ⅲ collagen gene.(5) Pretreatment of cells with WRW4, a specific inhibitor of the FPRL1, could suppress LL-37-induced collagen production in a concentration-dependent manner.(6) ERK inhibitor PD98059was shown to effectively inhibit LL-37-induced collagen production. However, p38inhibitor, JNK inhibitor or PI3K inhibitor didn’t exhibit any effect on LL-37-induced collagen expression.(7) LL-37resulted in a transient phosphorylation of ERK with maximal phosphorylation observation at30min. In addition, LL-37activated ERK phosphorylaton in a dose-dependent manner, which was sensitive to the specific inhibitor PD98059.(8) ERK phosphorylation was significantly inhibited by WRW4, suggesting that LL-37-induced ERK activation was FPRL1-dependent.4. Regional fibroblast heterogeneity in lung tissues from smokers with COPD. In lung sections from non-smokers and smokers, there’s only one type of phenotype, vimentin+E-cadherin-CD45-, indicating that these fibroblasts were derived from tissue-derived mesenchymal stem cells. In small airways from smokers with COPD, rare vimentin+lung fibrobalsts coexpressed E-cadherin or CD45. However, in the alveoli, about24.3%fibroblasts are vimentin+E-cadherin+, indicating that these vimentin+fibroblasts were derived from epithelium via EMT. Nearly12.4%fibroblasts are vimenitn+CD45+, suggesting that theses fibroblasts derived from bone marrow precursors. Compared with non-smokers and smokers, the immunoreactivity of type Ⅰ collagen, type Ⅲ collagen, elastin and fibronectin in small airways from smokers with COPD was significantly increased, however, the expression of type Ⅱ collagen, type Ⅲ collagen, elastin and fibronectin in the alveolus was obviously decreased.Conclusion1. Cathelicidin (LL-37) in lung tissues from COPD patients is associated with the pathological changes of small airway remodeling and emphysema.2. Cigarette smoke is the cause for increased expression of cathelicidin (rCRAMP) in the lung tissues of rats; Cathelicidin (rCRAMP) is associated with the structural changes of small airway remodeling and emphysema in a cigarette smoke-induced COPD rat model.3. CSE induced epithelial secretion of cathelicidin (LL-37) could up-regulate collagen expression in HFL-1cells via activation of FPRL1-mediated ERK pathway, which contributes to small airway remodeling.4. Though cathelicidin (LL-37) is significantly increased in small airways and parenchyma from smokers with COPD, due to the fibroblast heterogenity in two regions, the collagen deposition in small airways and parechyma are different.