Macrophages play an important role in the pathogenesis of COPD. Macrophage polarization towards the M2 phenotype has been observed in the lung tissues of COPD patients and cigarette smokers. The molecular basis of this process remains unclear, and it has not been completely illuminated in animal models of emphysema. In our study, we combined cigarette smoke (CS) exposure with intraperitoneal injection of cigarette smoke extract (CSE) to build an emphysema model. We found by immunohistochemical staining and flow cytometry that the expression level of CD206 and the ratio of M2 to M1 macrophages was increased in emphysematous mice. We also demonstrated that decreased protein level for phosphatase and tensin homology deleted on chromosome ten (PTEN) and increased total protein levels for phosphorylation -protein kinase B (p-AKT) in the lung tissue of emphysematous mice and in CSE-treated RAW264.7 cells. In both bone marrow-derived macrophages (BMDMs) from emphysematous mice and CSE-treated RAW264.7 cells, we observed by RT-PCR that the mRNA levels of M2 macrophage-related markers and cytokines were increased. Furthermore, M1 macrophage-related markers and cytokines were decreased. Meanwhile we treated BMDMs from emphysematous mice and CSE-treated RAW264.7 cells with the phosphoinositide 3-kinase (PI3K)/Akt inhibitor (LY294002), we observed a reduction in RNA levels of M2 macrophage-related markers and cytokines. In conclusion, we confirmed that macrophage M2 polarization was induced in emphysematous mice generated by CS exposure combined with intraperitoneal injection of CSE. We also showed that M2 polarization was mediated through PTEN/PI3k/AKT pathway activation.

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BackgroundCOPD is a multi-pathogenesis disease mainly caused by smoking. A further understanding of the mechanism of smoking-related COPD might contribute to preventions and treatments of this disease in the early stages. This study was designed to identify the characteristics of M2 macrophages in COPD for a better understanding about their potential role.Materials and methodsCOPD models were built in the C57BL/6 mouse by cigarette smoke (CS) exposure combined with intraperitoneal injection of cigarette smoke extract (CSE). The modeling efficiency was evaluated by lung function and hematoxylin and eosin (H&E) staining. The number of different macrophage phenotypes was detected by immunohistochemical staining (IHS) of CD206, CD86 and CD68 on the lung tissue paraffin section. The RAW264.7 cells were polarized toward the M2 phenotype by interleukin IL-4 and confirmed by a flow cytometer. The gene expression levels of TGF-βRII, Smad2, Smad3 and Smad7 in CSE-treated M2 macrophages were detected by real-time reverse transcription polymerase chain reaction (RT-PCR). The expression levels of TGF-β/Smad pathway-related makers (TGF-βRII, p-Smad2, p-Smad3, Smad7 and TGF-β) in alveolar M2 macrophages were detected by two consecutive paraffin section IHS.ResultsThe COPD model is well established, which is confirmed by the lung function test and lung H&E staining. The whole number of macrophages and the ratio of M2/M1 phenotype are both increased (p<0.05). The level of CD206+ cells in IL-4-stimulated RAW264.7 cells is up to 93.4%, which is confirmed by a flow cytometer. The gene expression of TGF-βRII, Smad2, Smad3 and Smad7 are all enhanced (p<0.05) in CES-treated M2 macrophages, which is detected by RT-PCR. The protein levels of TGF-β/Smad pathway-related markers are all increased in alveolar M2 macrophages of the model group.ConclusionThis study found an increased deposition of alveolar M2 macrophages in the mouse COPD model and an increased expression level of TGF-β/Smad pathway in M2 macrophages, both in vitro and in vivo, induced by CSE and/or CS exposure, indicating that M2 macrophages might contribute to COPD through changing of phenotype and TGF-β/Smad pathway.

Chronic obstructive pulmonary disease (COPD) is a multi-pathogenesis chronic lung disease. The mechanisms underlying COPD have not been adequately illustrated. Many reseachers argue that microRNAs (miRs) could play a crucial role in COPD. The classic animal model of COPD is both time consuming and costly. This study proposes a novel mice COPD model and explores the role of miR-21 in COPD. A total of 50 wide-type (WT) C57BL/6 mice were separated into five euqlly-sized groups—(1) control group (CG), (2) the novel combined method group (NCM, cigarette smoke (CS) exposure for 28 days combined with cigarette smoke extract (CSE) intraperitoneal injection), (3) the short-term CS exposure group (SCSE, CS exposure for 28 days), (4) the CSE intraperitoneal injection group (CSEII, 28 days CSE intraperitoneal injection), and (5) the long-term CS exposure group (LCSE, CS exposure).The body weight gain of mice were recorded and lung function tested once the modeling was done. The pathological changes and the inflammation level by hematoxylin eosin (H&E) staining and immunohistochemical staining (IHS) on the lung tissue sections were also evaluated. The level of miR-21 in the mice lungs of the mice across all groups was detected by RT-qPCR and the effects of miR-21 knock-down in modeled mice were observed. The mice in LCSE and NCM exhibited the most severe inflammation levels and pathological and pathophysiological changes; while the changes for the mice in SCSE and CSEII were less, they remained more severe than the mice in the CG. The level of miR-21 was found to be negatively correlated with lung functions. Moreover, knocking miR-21 down from the modeled mice, ameliorated all those tested COPD-related changes. Our novel modeling method detected virtually the same changes as those detected in the classic method in WT mice, but in less time and cost. Further, it was determined that the level of miR-21 in the lungs could be an indicator of COPD severity and blocking functions of miR-21 could be a potential treatment for early stage COPD.

MicroRNAs and autophagy play important roles in chronic obstructive pulmonary disease (COPD). This study was designed to explore the role of microRNA-21 (miR-21) induced autophagy in COPD. Using the C57BL/6, miR-21-/- mice and human bronchial epithelial (16HBE) cell line, we found that in the lung tissues of mice, the level of autophagy in the COPD model group was significantly higher than that in the control group. However, compared to the COPD model, the level of autophagy was significantly lower in the miR-21-/- CSE+CS group. In the COPD model, miR-21 was overexpressed. Moreover, in human bronchial epithelial (16HBE) cells exposed to cigarette smoke extract (CSE), miR-21 expression was upregulated and autophagy was notably increased. In addition, pretreatment of 16HBE cells with miR-21 inhibitor significantly inhibited autophagy activity and decreased apoptosis, indicating that miR-21 is involved in CSE-induced autophagy and apoptosis. The results showed that miR-21 could increase autophagy and promote the apoptosis of 16HBE cells in COPD. This information contributes to our further understanding of COPD.

macrophages 5,6 . Alveolar macrophages (AM) play an indispensable role in the occurrence and development of COPD, and, at the same time, it can affect the structure of lung tissue through the initiation and degeneration of inflammation 7 .Mutative exogenous stimuli and pathological processes can change the phenotypes and functional characteristics of alveolar macrophages 8 . Since a large number of M1-type macrophage-related cytokines, such as TNF-α and IL-8, are found in bronchoalveolar lavage fluid (BAL), pulmonary macrophages from patients with COPD have been recommended to display M1 phenotypes 9 . In addition, TNF-α has been ABSTRACT INTRODUCTION In chronic obstructive pulmonary disease (COPD), macrophages play an indispensable role. In the lung tissues of COPD patients and smokers, macrophages can be observed to polarize towards M2 phenotype. The molecular mechanism of this process is unclear, and it has not been fully elucidated in COPD. METHODS We bought laboratory animals [C57BL/6 and miR-21 -/-C57BL/6(F1)] from the Jackson Laboratory. The model of COPD mice was established by cigarette smoke (CS) exposure combined with intraperitoneal injection of cigarette smoke extract (CSE). RT-PCR detected the expression levels of inflammatory factors and markers associated with M1 and M2 macrophages. The ratio of M2 macrophages to M1 macrophages was detected by immunohistochemical staining. RESULTS The level of miR-21 was increased in RAW264.7 cells intervened by CSE and in lung tissue and bone marrow-derived macrophages (BMDMs) from COPD mice. CSE can gradually over time increase the level of miR-21. The proportion of M2 macrophages to M1 macrophages had a positive correlation with miR-21. Knockdowning miR-21 can reduce lung tissue damage. CSE also increased the levels of related inflammatory factors and markers associated with M2 macrophages, and an miR-21 inhibitor can reverse this conversion. CONCLUSIONS We confirmed that CSE can lead to macrophage transformation to the M2 phenotype and an increase in the expression level of miR-21. Knockdown of the miR-21 gene could inhibit the transformation of macrophages to the M2 phenotype in COPD.

Chronic obstructive pulmonary disease (COPD) is a complex disease with multiple etiologies, while smoking is the most established one. The present study investigated the modulation of T-helper 17 (Th17) cell differentiation by the miR-21/Smad7/TGF-β pathway, and their roles in COPD. Lung tissues were obtained from lung cancer patients with or without COPD who underwent lobotomy and the levels of miR-21, TGF-β/Smad signaling molecules, RORγT, and other Th17-related cytokines were detected. Mouse COPD models were built by exposing both wild-type (WT) and miR-21−/− mice to cigarette smoke (CS) and cigarette smoke extract (CSE) intraperitoneal injection. Isolated primary CD4+ T cells were treated with either CS extract, miR-21 mimics or inhibitors, followed by measuring Th17 cells markers and the expression of TGF-β/Smad signaling molecules and RORγT. Increased levels of miR-21, Smad7, phosphorylated (p)-Smad2, p-Smad3, TGF-β, and Th17-related cytokines was detected in the lungs of COPD patients. Lung function in modeled WT mice, but not miR-21−/− ones, deteriorated and the number of inflammatory cells in the lung tissues increased compared to the control WT-mice. Moreover, primary CD4+ lymphocytes tend to differentiate into Th17 cells after the treatment with CSE or miR-21 mimics, and the expression of RORγT and the TGF-β/Smad signaling were all increased, however miR-21 inhibitors worked reversely. Our findings demonstrated that Th17 cells increased under COPD pathogenesis and was partially modulated by the miR-21/Smad7/TGF-β pathway.

BackgroundIn chronic obstructive pulmonary disease (COPD), weakness and muscle mass loss of the quadriceps muscle has been demonstrated to predict survival and mortality rates of patients. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), as a member of the TNF superfamily, has recently been identified as a key regulator of skeletal muscle wasting and metabolic dysfunction. So our aim was to study the role of TWEAK during quadriceps muscle atrophy and fiber-type transformation in COPD model rats and its possible pathway.MethodsForty-four healthy male adult Wistar rats were randomly divided into two groups: A normal control group (n = 16) and a COPD model group (n = 28). The COPD group was exposed to cigarette smoke for 90 d and injected with porcine pancreatic elastase on day 15, whereas the control group was injected with saline alone. Following treatment, weights of the quadriceps muscles were measured and hematoxylin and eosin staining was performed to identify structural changes in lung and quadriceps muscle tissue. Immunohistochemical staining was also conducted to determine the localization of TWEAK, nuclear factor (NF)-κB, muscle ring finger (MuRF)-1 and proliferator-activated coactivator (PGC)-1a proteins in the quadriceps muscle, and western blotting was used to assess the level of protein expression.ResultsCompared with controls, COPD model rats exhibited significantly lower quadriceps muscle weight (P < 0.05) accompanied by fiber atrophy and disordered fiber arrangement, a wide gap between adjacent muscle fibers, a significant reduction in nuclear number (P < 0.05) and an uneven size distribution. The proportion of fiber types was also significantly altered (P < 0.05). In addition, TWEAK expression in the quadriceps muscle of COPD model rats was significantly higher than that in control rats (P < 0.05), and was significantly associated with quadriceps atrophy and fiber-type alteration (P < 0.05). Levels of NF-κB, MuRF1 and PGC-1α expression also significantly differed between the two groups (P < 0.05).ConclusionsCollectively these data suggest that increased levels of TWEAK may lead to skeletal muscle atrophy and fiber-type alteration, which in turn may be associated with activation of the ubiquitin-proteasome pathway, involving NF-κB, MuRF1 and PGC-1α as potential regulatory factors. These preliminary results in rats suggest that TWEAK may be a therapeutic target for the treatment of muscle atrophy in COPD.