TLR-4 在慢性阻塞性肺病患者肺组织表达的研究

目的:观察COPD患者肺组织中TLR-4,IL-8,MUC5AC的表达,并探讨其在气道炎症、气道高分泌中的作用。方法:非COPD、COPD组男性肺癌病人各20例,取其肺叶切除后的外周肺组织,对肺组织标本行HE及AB-PAS染色,用免疫组织化学方法检测肺组织中TLR-4,IL-8,MUC5AC的表达并分析其相关性。结果:①COPD患者肺组织中TLR-4,IL-8,MUC5AC表达较对照组增高(P<0.05)。TLR-4主要在气道上皮细胞、肺巨噬细胞及血管内皮细胞表达,IL-8在气道壁、肺泡间隔、血管壁及肺组织内浸润的单核细胞、巨噬细胞、多形核白细胞均有表达,MUC5AC主要在气道上皮杯状细胞中表达。②TLR-4、IL-8表达与气道炎细胞评分成正相关(P<0.05)。TLR-4与IL-8、MUC5AC表达成正相关(P<0.05)。结论:COPD患者肺组织中TLR-4高表达可能参与了COPD的气道炎症及气道高分泌,这可能是通过增加IL-8与MUC5AC的表达来实现的。     

Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease

Excessive production of airway mucus is a cardinal feature of bronchial asthma and chronic obstructive pulmonary disease (COPD) and contributes to morbidity and mortality in these diseases. IL-13, a Th2-type cytokine, is a central mediator in the pathogenesis of bronchial asthma, including mucus overproduction. Using a genome-wide search for genes induced in airway epithelial cells in response to IL-13, we identified pendrin encoded by the SLC26A4 (PDS) gene as a molecule responsible for airway mucus production. In both asthma and COPD mouse models, pendrin was up-regulated at the apical side of airway epithelial cells in association with mucus overproduction. Pendrin induced expression of MUC5AC, a major product of mucus in asthma and COPD, in airway epithelial cells. Finally, the enforced expression of pendrin in airway epithelial cells in vivo, using a Sendai virus vector, rapidly induced mucus overproduction in the lumens of the lungs together with neutrophilic infiltration in mice. These findings collectively suggest that pendrin can induce mucus production in airway epithelial cells and may be a therapeutic target candidate for bronchial asthma and COPD.     

Effect of MMP/ADAM inhibitors on goblet cell hyperplasia in cultured human bronchial epithelial cells

While epidermal growth factor receptor (EGFR) plays a pivotal role in the repair process of epithelial cells, it is also involved in the overproduction of mucus and goblet cell hyperplasia (GCH), which occurs in chronic airway diseases such as asthma. Among the EGFR ligands, transforming growth factor (TGF)-alpha is thought to be the most important in the synthesis of mucus. Pro-TGF-alpha is cleaved to give an active form by members of the matrix metalloproteinases (MMP)/a disintegrin and metalloproteinases (ADAM) family. Thus MMP/ADAM inhibitors might prevent GCH by inhibiting transactivation of EGFR. Upon stimulation of differentiating normal human bronchial epithelial (NHBE) cells by IL-13, GCH was induced. The mucin genes MUC5AC, MUC5B, and MUC2 were upregulated whereas the expression of ciliated cell markers was greatly repressed. GM6001, a broad-spectrum inhibitor for MMP/ADAM, inhibited IL-13-induced mucin gene expression and mucus production as measured by periodic acid-Schiff staining. This was accompanied by an inhibition of TGF-alpha release. These results suggest that MMP/ADAMs play a pivotal role in the development of GCH in lung epithelial cells.     

Kaempferol Inhibits Endoplasmic Reticulum Stress-Associated Mucus Hypersecretion in Airway Epithelial Cells And Ovalbumin-Sensitized Mice

Mucus hypersecretion is an important pathological feature of chronic airway diseases, such as asthma and pulmonary diseases. MUC5AC is a major component of the mucus matrix forming family of mucins in the airways. The initiation of endoplasmic reticulum (ER)-mediated stress responses contributes to the pathogenesis of airway diseases. The present study investigated that ER stress was responsible for airway mucus production and this effect was blocked by the flavonoid kaempferol. Oral administration of ≥10 mg/kg kaempferol suppressed mucus secretion and goblet cell hyperplasia observed in the bronchial airway and lung of BALB/c mice sensitized with ovalbumin (OVA). TGF-β and tunicamycin promoted MUC5AC induction after 72 h in human bronchial airway epithelial BEAS-2B cells, which was dampened by 20 μM kaempferol. Kaempferol inhibited tunicamycin-induced ER stress of airway epithelial cells through disturbing the activation of the ER transmembrane sensor ATF6 and IRE1α. Additionally, this compound demoted the induction of ER chaperones such as GRP78 and HSP70 and the splicing of XBP-1 mRNA by tunicamycin. The in vivo study further revealed that kaempferol attenuated the induction of XBP-1 and IRE1α in epithelial tissues of OVA-challenged mice. TGF-β and tunicamycin induced TRAF2 with JNK activation and such induction was deterred by kaempferol. The inhibition of JNK activation encumbered the XBP-1 mRNA splicing and MUC5AC induction by tunicamycin and TGF-β. These results demonstrate that kaempferol alleviated asthmatic mucus hypersecretion through blocking bronchial epithelial ER stress via the inhibition of IRE1α-TRAF2-JNK activation. Therefore, kaempferol may be a potential therapeutic agent targeting mucus hypersecretion-associated pulmonary diseases.     

Epidermal Growth Factor Removal or Tyrphostin AG1478 Treatment Reduces Goblet Cells & Mucus Secretion of Epithelial Cells from Asthmatic Children Using the Air-Liquid Interface Model

Rationale

Epithelial remodelling in asthma is characterised by goblet cell hyperplasia and mucus hypersecretion for which no therapies exist. Differentiated bronchial air-liquid interface cultures from asthmatic children display high goblet cell numbers. Epidermal growth factor and its receptor have been implicated in goblet cell hyperplasia.

Objectives

We hypothesised that EGF removal or tyrphostin AG1478 treatment of differentiating air-liquid interface cultures from asthmatic children would result in a reduction of epithelial goblet cells and mucus secretion.

Methods

In Aim 1 primary bronchial epithelial cells from non-asthmatic (n = 5) and asthmatic (n = 5) children were differentiated under EGF-positive (10ng/ml EGF) and EGF-negative culture conditions for 28 days. In Aim 2, cultures from a further group of asthmatic children (n = 5) were grown under tyrphostin AG1478, a tyrosine kinase inhibitor, conditions. All cultures were analysed for epithelial resistance, markers of differentiation using immunocytochemistry, ELISA for MUC5AC mucin secretion and qPCR for MUC5AC mRNA.

Results

In cultures from asthmatic children the goblet cell number was reduced in the EGF negative group (p = 0.01). Tyrphostin AG1478 treatment of cultures from asthmatic children had significant reductions in goblet cells at 0.2μg/ml (p = 0.03) and 2μg/ml (p = 0.003) as well as mucus secretion at 2μg/ml (p = 0.04).

Conclusions

We have shown in this preliminary study that through EGF removal and tyrphostin AG1478 treatment the goblet cell number and mucus hypersecretion in differentiating air-liquid interface cultures from asthmatic children is significantly reduced. This further highlights the epidermal growth factor receptor as a potential therapeutic target to inhibit goblet cell hyperplasia and mucus hypersecretion in asthma.     

ATP induced MUC5AC release from human airways in vitro

BACKGROUND: Chronic airway diseases are often associated with marked mucus production, however, little is known about the regulation of secretory activity by locally released endogenous mediators. AIM: This investigation was performed to determine the release of MUC5AC mucin from human bronchial preparations using the purinergic agonists adenosine 5''-triphosphate (ATP) and uridine 5''-triphosphate (UTP). METHODS: Immunohistochemical and immunoradiometric assays (IRMA) were used to detect the MUC5AC mucin. Immunohistochemical analysis were performed using individual 1-13 M1 and 21 M1 MAbs recognizing a recombinant M1 mucin partially encoded by the MUC5AC gene. IRMA measurments were performed using a mixture of eight anti-M1 mucin MAbs (PM8), which included both 1-13 M1 and 21 M1 MAbs. Lysozyme and protein were also measured in the biological fluids derived from human bronchial preparations obtained from patients who had undergone surgery for lung carcinoma. RESULTS: The anti-M1 monoclonal antibodies labelled epithelial goblet cells. After challenge of human bronchial preparations with ATP, the goblet cells exhibited less staining. In contrast, UTP did not alter the immunolabelling of goblet cells. MUC5AC mucin in the bronchial fluids derived from ATP-challenged preparations was increased while UTP had no effect on release. ATP did not alter either the quantities of lysozyme or protein detected in the biological fluids. CONCLUSION: These results suggest that ATP may regulate epithelial goblet cell secretion of MUC5AC mucin from human airways in vitro.     

Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke

Mucus hypersecretion from hyperplastic airway goblet cells is a hallmark of chronic obstructive pulmonary disease (COPD). Although cigarette smoking is thought to be involved in mucus hypersecretion in COPD, the mechanism by which cigarette smoke induces mucus overproduction is unknown. Here we show that activation of epidermal growth factor receptors (EGFR) is responsible for mucin production after inhalation of cigarette smoke in airways in vitro and in vivo. In the airway epithelial cell line NCI-H292, exposure to cigarette smoke upregulated the EGFR mRNA expression and induced activation of EGFR-specific tyrosine phosphorylation, resulting in upregulation of MUC5AC mRNA and protein production, effects that were inhibited completely by selective EGFR tyrosine kinase inhibitors (BIBX1522, AG-1478) and that were decreased by antioxidants. In vivo, cigarette smoke inhalation increased MUC5AC mRNA and goblet cell production in rat airways, effects that were prevented by pretreatment with BIBX1522. These effects may explain the goblet cell hyperplasia that occurs in COPD and may provide a novel strategy for therapy in airway hypersecretory diseases.     

Effect of MMP/ADAM Inhibitors on Goblet Cell Hyperplasia in Cultured Human Bronchial Epithelial Cells

While epidermal growth factor receptor (EGFR) plays a pivotal role in the repair process of epithelial cells, it is also involved in the overproduction of mucus and goblet cell hyperplasia (GCH), which occurs in chronic airway diseases such as asthma. Among the EGFR ligands, transforming growth factor (TGF)-α is thought to be the most important in the synthesis of mucus. Pro-TGF-α is cleaved to give an active form by members of the matrix metalloproteinases (MMP)/a disintegrin and metalloproteinases (ADAM) family. Thus MMP/ADAM inhibitors might prevent GCH by inhibiting transactivation of EGFR. Upon stimulation of differentiating normal human bronchial epithelial (NHBE) cells by IL-13, GCH was induced. The mucin genes MUC5AC, MUC5B, and MUC2 were upregulated whereas the expression of ciliated cell markers was greatly repressed. GM6001, a broad-spectrum inhibitor for MMP/ADAM, inhibited IL-13-induced mucin gene expression and mucus production as measured by periodic acid-Schiff staining. This was accompanied by an inhibition of TGF-α release. These results suggest that MMP/ADAMs play a pivotal role in the development of GCH in lung epithelial cells.     

The tyrosine phosphatase, SHP-1, is involved in bronchial mucin production during oxidative stress

Mucus hypersecretion is a clinically important manifestation of chronic inflammatory airway diseases, such as asthma and Chronic obstructive pulmonary disease (COPD). Mucin production in airway epithelia is increased under conditions of oxidative stress. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 suppression is related to the development of airway inflammation and increased ROS levels. In this study, we investigated the role of SHP-1 in mucin secretion triggered by oxidative stress. Human lung mucoepidermoid H292 carcinoma cells were transfected with specific siRNA to eliminate SHP-1 gene expression. Cultured cells were treated with hydrogen peroxide (H₂O₂), and Mucin 5AC(MUC5AC) gene expression and mucin production were determined. Activation of p38 mitogen activated protein kinase (MAPK) in association with MUC5AC production was evaluated. N-acetylcysteine (NAC) was employed to determine whether antioxidants could block MUC5AC production. To establish the precise role of p38, mucin expression was observed after pre-treatment of SHP-1-depleted H292 cells with the p38 chemical blocker. We investigated the in vivo effects of oxidative stress on airway mucus production in SHP-1-deficient heterozygous (mev/+) mice. MUC5AC expression was enhanced in SHP-1 knockdown H292 cells exposed to H₂O₂, compared to that in control cells. The ratio between phosphorylated and total p38 was significantly increased in SHP-1-deficient cells under oxidative stress. Pre-treatment with NAC suppressed both MUC5AC production and p38 activation. Blockage of p38 MAPK led to suppression of MUC5AC mRNA expression. Notably, mucin production was enhanced in the airway epithelia of mev/+ mice exposed to oxidative stress. Our results clearly indicate that SHP-1 plays an important role in airway mucin production through regulating oxidative stress.     

Phosphodiesterase 4B mediates extracellular signal-regulated kinase-dependent up-regulation of mucin MUC5AC protein by Streptococcus pneumoniae by inhibiting cAMP-protein kinase A-dependent MKP-1 phosphatase pathway

Otitis media (OM) is the most common childhood bacterial infection and the major cause of conductive hearing loss in children. Mucus overproduction is a hallmark of OM. Streptococcus pneumoniae is the most common gram-positive bacterial pathogen causing OM. Among many mucin genes, MUC5AC has been found to be greatly up-regulated in the middle ear mucosa of human patients with OM. We previously reported that S. pneumoniae up-regulates MUC5AC expression in a MAPK ERK-dependent manner. We also found that MAPK phosphatase-1 (MKP-1) negatively regulates S. pneumoniae-induced ERK-dependent MUC5AC up-regulation. Therapeutic strategies for up-regulating the expression of negative regulators such as MKP-1 may have significant therapeutic potential for treating mucus overproduction in OM. However, the underlying molecular mechanism by which MKP-1 expression is negatively regulated during S. pneumoniae infection is unknown. In this study we show that phosphodiesterase 4B (PDE4B) mediates S. pneumoniae-induced MUC5AC up-regulation by inhibiting the expression of a negative regulator MKP-1, which in turn leads to enhanced MAPK ERK activation and subsequent up-regulation of MUC5AC. PDE4B inhibits MKP-1 expression in a cAMP-PKA-dependent manner. PDE4-specific inhibitor rolipram inhibits S. pneumoniae-induced MUC5AC up-regulation both in vitro and in vivo. Moreover, we show that PDE4B plays a critical role in MUC5AC induction. Finally, topical and post-infection administration of rolipram into the middle ear potently inhibited S. pneumoniae-induced MUC5AC up-regulation. Collectively, these data demonstrate that PDE4B mediates ERK-dependent up-regulation of mucin MUC5AC by S. pneumoniae by inhibiting cAMP-PKA-dependent MKP-1 pathway. This study may lead to novel therapeutic strategy for inhibiting mucus overproduction.     

Regulation of MUC5AC mucin secretion by depletion of AQP5 in SPC-A1 cells

Airway mucus is regulated by many inflammatory mediators such as ILs, TNF-alpha, EGF, PGF2alpha, LT, and so on. Recently, the relationship between membrane ion channel and mucus production has been under investigation. The present study aimed to examine whether AQP5 was involved in modulation of mucin expression and secretion in airway submucosal gland cells (SPC-A1). A recombinant plasmid (pShAQP5) containing small hairpin RNA expression cassette targeting AQP5 sequence was constructed. In pShAQP5 transiently transfected cells, ELISA showed MUC5AC synthesis and secretion were increased by 57.9% and 85.3%, respectively, on day 5 after pShAQP5 transfection. While in five stably transfected clones (shAQP5-G1, G2, G3, A2, and A5), the upregulated levels of MUC5AC mRNA were 118%, 165%, 65%, 123%, and 38%, respectively. The elevated levels of MUC5AC synthesis and secretion varied from 59-156% and 33-166%, respectively. This is the first reliable investigation of the regulation of MUC5AC mucin secretion by silencing AQP5. Further study of the regulatory mechanism between AQPs and mucins may provide new strategies for development of novel antihypersecretory drugs in airway diseases.     

JNK activation is responsible for mucus overproduction in smoke inhalation injury

Background

Increased mucus secretion is one of the important characteristics of the response to smoke inhalation injuries. We hypothesized that gel-forming mucins may contribute to the increased mucus production in a smoke inhalation injury. We investigated the role of c-Jun N-terminal kinase (JNK) in modulating smoke-induced mucus secretion.

Methods

We intubated mice and exposed them to smoke from burning cotton for 15 min. Their lungs were then isolated 4 and 24 h after inhalation injury. Three groups of mice were subjected to the smoke inhalation injury: (1) wild-type (WT) mice, (2) mice lacking JNK1 (JNK1-/- mice), and (3) WT mice administered a JNK inhibitor. The JNK inhibitor (SP-600125) was injected into the mice 1 h after injury.

Results

Smoke exposure caused an increase in the production of mucus in the airway epithelium of the mice along with an increase in MUC5AC gene and protein expression, while the expression of MUC5B was not increased compared with control. We found increased MUC5AC protein expression in the airway epithelium of the WT mice groups both 4 and 24 h after smoke inhalation injury. However, overproduction of mucus and increased MUC5AC protein expression induced by smoke inhalation was suppressed in the JNK inhibitor-treated mice and the JNK1 knockout mice. Smoke exposure did not alter the expression of MUC1 and MUC4 proteins in all 3 groups compared with control.

Conclusion

An increase in epithelial MUC5AC protein expression is associated with the overproduction of mucus in smoke inhalation injury, and that its expression is related on JNK1 signaling.     

Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop

Mucus hypersecretion and persistent airway inflammation are common features of various airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. One key question is: does the associated airway inflammation in these diseases affect mucus production? If so, what is the underlying mechanism? It appears that increased mucus secretion results from increased mucin gene expression and is also frequently accompanied by an increased number of mucous cells (goblet cell hyperplasia/metaplasia) in the airway epithelium. Many studies on mucin gene expression have been directed toward Th2 cytokines such as interleukin (IL)-4, IL-9, and IL-13 because of their known pathophysiological role in allergic airway diseases such as asthma. However, the effect of these cytokines has not been definitely linked to their direct interaction with airway epithelial cells. In our study, we treated highly differentiated cultures of primary human tracheobronchial epithelial (TBE) cells with a panel of cytokines (interleukin-1alpha, 1beta, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and tumor necrosis factor alpha). We found that IL-6 and IL-17 could stimulate the mucin genes, MUC5B and MUC5AC. The Th2 cytokines IL-4, IL-9, and IL-13 did not stimulate MUC5AC or MUC5B in our experiments. A similar stimulation of MUC5B/Muc5b expression by IL-6 and IL-17 was demonstrated in primary monkey and mouse TBE cells. Further investigation of MUC5B expression demonstrated that IL-17's effect is at least partly mediated through IL-6 by a JAK2-dependent autocrine/paracrine loop. Finally, evidence is presented to show that both IL-6 and IL-17 mediate MUC5B expression through the ERK signaling pathway.     

ITGB4 deficiency induces mucus hypersecretion by upregulating MUC5AC in RSV-infected airway epithelial cells

Respiratory syncytial virus (RSV) infection is the main cause of bronchiolitis in children. Excessive mucus secretion is one of the primary symbols in RSV related lower respiratory tract infections (RSV-related LRTI), which is closely associated with the occurrence and development of asthma in later life. Integrin β4 (ITGB4) is down-regulated in the airway epithelial cells (AECs) of asthma patients which plays a critical role in the pathogenesis of asthma. However, whether ITGB4 is involved in the pathological processes of RSV infection remains unclear. In this study, we found that decreased expression of ITGB4 was negatively correlated with the level of MUC5AC in childhood AECs following RSV infection. Moreover, ITGB4 deficiency led to mucus hypersecretion and MUC5AC overexpression in the small airway of RSV-infected mice. MUC5AC expression was upregulated by ITGB4 in HBE cells through EGFR, ERK and c-Jun pathways. EGFR inhibitors treatment inhibited mucus hypersecretion and MUC5AC overexpression in ITGB4-deficient mice after RSV infection. Together, these results demonstrated that epithelial ITGB4 deficiency induces mucus hypersecretion by upregulating the expression of MUC5AC through EGFR/ERK/c-Jun pathway, which further associated with RSV-related LRTI.     

IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo

Goblet cell metaplasia and mucus hypersecretion are important features in the pathogenesis of asthma. The cytokine IL-4 has been shown to play a role in animal models of asthma, where it induces Th2 lymphocyte differentiation and B lymphocyte IgE class switch. IL-4 has also been implicated in the differentiation of goblet cells via effects on lymphocytes and eosinophils. In this study we hypothesized that IL-4 induces airway epithelial cell mucin gene expression and mucous glycoconjugate production by direct action on these cells. In vitro, cultured airway epithelial cells (NCI-H292) expressed IL-4R constitutively, and IL-4 (10 ng/ml) induced MUC2 gene expression and mucous glycoconjugate production. In vivo, mouse airway epithelial cells expressed IL-4R constitutively, and IL-4 (250 ng) increased MUC5 gene expression and Alcian blue/periodic acid-Schiff-positive staining at 24 h; IL-4 did not increase inflammatory cell numbers in airway tissue or in bronchoalveolar lavage. TNF-alpha and IL-1beta levels in bronchoalveolar lavage were not increased in response to IL-4 instillation. These results indicate that airway epithelial cells express IL-4R constitutively and that IL-4 directly induces the differentiation of epithelium into mucous glycoconjugate-containing goblet cells.     

IL13 activates autophagy to regulate secretion in airway epithelial cells

Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.     

Nuclear erythroid 2 p45-related factor–2 Nrf2 ameliorates cigarette smoking-induced mucus overproduction in airway epithelium and mouse lungs

Background and objectiveNuclear erythroid 2 p45-related factor–2 (Nrf2) is known to play important roles in airway disorders, whereas little has been investigated about its direct role in airway mucus hypersecretion. The aim of this study is to determine whether this factor could protect pulmonary epithelium and mouse airway from cigarette-induced mucus overproduction.MethodsUsing genetic approaches, the role of Nrf2 on cigarette smoking extracts (CSE) induced MUC5AC expression was investigated in lung A549 cells. Nrf2 deficiency mice were smoked for various periods, and the airway inflammation and mucus production was characterized.ResultsAcute smoking exposure induced expression of MUC5AC and Nrf2 in both A549 cells and mouse lungs. Genetic ablation of Nrf2 augmented, whereas overexpression of this molecule ameliorated CSE-induced expression of MUC5AC. Nrf2 knockout mice, after exposure to cigarette smoking, displayed enhanced airway inflammation and mucus production.ConclusionNrf2 negatively regulated smoking-induced mucus production in vitro and in vivo, suggesting therapeutic potentials of this factor in airway diseases with hypersecreted mucus.