The role and mechanism of macrophage autophagy in the experimental model of chronic obstructive pulmonary disease.

INTRODUCTION: Macrophages play an important role in chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) impairs autophagy in alveolar macrophages from COPD patients, and autophagic impairment leads to reduced clearance of protein aggregates, dysfunctional mitochondria, and defective bacterial delivery to lysosomes. However, the exact function of lung macrophage autophagy in the pathogenesis of CS-induced COPD remains largely unknown. METHODS: Western blot detected the expression of autophagy-related proteins induced by CSE. The model of COPD mice was established by CS exposure combined with CSE intraperitoneal injection. Double immunofluorescence was used to measure the CD206+LC3B+ cells. The morphological changes and effects on lung function were observed. Masson staining detected the changes in collagen fibers in lung tissue. The expression levels of E-cadherinb and N-cadherinb were detected by immunohistochemistry. Western blot detected the expression of ATP6V1E1 in lung tissue. RESULTS: At 24 hours of exposure to CSE, the expression levels of LC3B (microtubule-associated protein 1A/1B-light chain 3B) and P62 (nucleoporin 62) were highest at 1% CSE and AGT5 (nucleoporin 62) at 2.5% CSE; at 48 hours, the expression levels of LC3B, P62 and AGT5 were highest at 2.5% CSE, and as the intervention time increased.CD206+LC3B+ cells were significantly higher in the COPD group. Enhanced macrophage autophagy may promote emphysema formation and aggravate lung function damage. The expression of E-cadherinb in lung tissue of the COPD group was decreased, and N-cadherinb expression was increased; the expression of E-cadherinb was increased, and N-cadherinb expression was decreased in ATG5myeΔ COPD mice. The expression of ATP6V1E1 in the lung tissue was increased in the COPD group; ATP6V1E1 expression was decreased in the lung tissues of ATG5myeΔ COPD mice. CONCLUSIONS: CSE enhanced macrophage autophagy, leads to increased lung function impairment and collagenous fiber in lung tissue, as well as promotes epithelial-mesenchymal transition, and eventually leads to small airway remodeling, which may be achieved through the ATG5/ATP6V1E1 pathway.

The effects of the miR-21/SMAD7/TGF-ß pathway on Th17 cell differentiation 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-ß 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-ß/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-ß/Smad signaling molecules and RORγT. Increased levels of miR-21, Smad7, phosphorylated (p)-Smad2, p-Smad3, TGF-ß, 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-ß/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-ß pathway.

SIRT1 attenuates endoplasmic reticulum stress and apoptosis in rat models of COPD.

The present study aimed to investigate the protective role of sirtuin 1 (SIRT1) and oxygen regulated protein 150 (ORP150) in a rat COPD model by inducing changes in ER stress and apoptosis. We separated 48 Sprague Dawley (SD) rats into four groups randomly: the control group, resveratrol group, COPD group and the resveratrol intervention group. Rats were challenged with cigarette smoke and lipopolysaccharide with resveratrol (a selective activator of SIRT1). The lung functions of the rats were measured and recorded. The expression levels of SIRT1 and ORP150 in lung tissues were examined by western blot and RTq PCR. The expression levels of the ER stress apoptosis-associated protein were determined .The apoptotic level of lung tissues was analyzed. The results suggest that SIRT1 attenuated apoptosis and ER stress in the lung tissues of rats with COPD. During this process, a positive correlation was identified between SIRT1 and ORP150.

Citrus CsACD2 Is a Target of Candidatus Liberibacter Asiaticus in Huanglongbing Disease.

Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (Las), is one of the most destructive citrus diseases worldwide, yet how Las causes HLB is poorly understood. Here we show that a Las-secreted protein, SDE15 (CLIBASIA_04025), suppresses plant immunity and promotes Las multiplication. Transgenic expression of SDE15 in Duncan grapefruit (Citrus × paradisi) suppresses the hypersensitive response induced by Xanthomonas citri ssp. citri (Xcc) and reduces the expression of immunity-related genes. SDE15 also suppresses the hypersensitive response triggered by the Xanthomonas vesicatoria effector protein AvrBsT in Nicotiana benthamiana, suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with the citrus protein CsACD2, a homolog of Arabidopsis (Arabidopsis thaliana) ACCELERATED CELL DEATH 2 (ACD2). SDE15 suppression of plant immunity is dependent on CsACD2, and overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in genome editing for novel plant resistance against devastating HLB.

Bronchial epithelial cell extracellular vesicles ameliorate epithelial-mesenchymal transition in COPD pathogenesis by alleviating M2 macrophage polarization.

Chronic obstructive pulmonary disease (COPD) is partly characterized as epithelial-mesenchymal transition (EMT)-related airflow limitation. Extracellular vesicles (EVs) play crucial roles in the crosstalk between cells, affecting many diseases including COPD. Up to now, the roles of EVs in COPD are still debated. As we found in this investigation, COPD patients have higher miR-21 level in total serum EVs. EMT occurs in lungs of COPD mice. Furthermore, bronchial epithelial cells (BEAS-2B) could generate EVs with less miR-21 when treated with cigarette smoke extract (CSE), impacting less on the M2-directed macrophage polarization than the control-EVs (PBS-treated) according to EVs miR-21 level. Furthermore, the EMT processes in BEAS-2B cells were enhanced with the M2 macrophages proportion when co-cultured. Collectively, these results demonstrate that CSE-treated BEAS-2B cells could alleviate M2 macrophages polarization by modulated EVs, and eventually relieve the EMT process of BEAS-2B cells themselves under COPD pathogenesis, revealing a novel compensatory role of them in COPD.

A Novel Murine Chronic Obstructive Pulmonary Disease Model and the Pathogenic Role of MicroRNA-21.

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.

MicroRNA-21 aggravates chronic obstructive pulmonary disease by promoting autophagy.

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.

Characteristics and potential role of M2 macrophages in COPD.

BACKGROUND: COPD 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 METHODS: COPD 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-ßRII, 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-ß/Smad pathway-related makers (TGF-ßRII, p-Smad2, p-Smad3, Smad7 and TGF-ß) in alveolar M2 macrophages were detected by two consecutive paraffin section IHS. RESULTS: The 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-ßRII, 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-ß/Smad pathway-related markers are all increased in alveolar M2 macrophages of the model group. CONCLUSION: This study found an increased deposition of alveolar M2 macrophages in the mouse COPD model and an increased expression level of TGF-ß/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-ß/Smad pathway.

Interplay of the Arabidopsis nonhost resistance gene NHO1 with bacterial virulence.

It is poorly understood why a particular plant species is resistant to the vast majority of potential pathogens that infect other plant species, a phenomenon referred to as "nonhost" resistance. Here, we show that Arabidopsis NHO1, encoding a glycerol kinase, is required for resistance to and induced by Pseudomonas syringae isolates from bean and tobacco. NHO1 is also required for resistance to the fungal pathogen Botrytis cinerea, indicating that NHO1 is not limited to bacterial resistance. Strikingly, P. s. pv. tomato DC3000, an isolate fully virulent on Arabidopsis, actively suppressed the NHO1 expression. This suppression is abolished in coi1 plants, indicating that DC3000 required an intact jasmonic acid signaling pathway in the plant to suppress NHO1 expression. Constitutive overexpression of NHO1 led to enhanced resistance to this otherwise virulent bacterium. The presence of avrB in DC3000, which activates a cultivar-specific "gene-for-gene" resistance in Arabidopsis, restored the induction of NHO1 expression. Thus, NHO1 is deployed for both general and specific resistance in Arabidopsis and targeted by the bacterium for parasitism.