Cigarette Smoke Induces Human Epidermal Receptor 2-Dependent Changes in Epithelial Permeability.

The airway epithelium constitutes a protective barrier against inhaled insults, such as viruses, bacteria, and toxic fumes, including cigarette smoke (CS). Maintenance of bronchial epithelial integrity is central for airway health, and defective epithelial barrier function contributes to the pathogenesis of CS-mediated diseases, such as chronic obstructive pulmonary disease. Although CS has been shown to increase epithelial permeability, current understanding of the mechanisms involved in CS-induced epithelial barrier disruption remains incomplete. We have previously identified that the receptor tyrosine kinase human epidermal receptor (HER) 2 growth factor is activated by the ligand neuregulin-1 and increases epithelial permeability in models of inflammatory acute lung injury. We hypothesized that CS activates HER2 and that CS-mediated changes in barrier function would be HER2 dependent in airway epithelial cells. We determined that HER2 was activated in whole lung, as well as isolated epithelial cells, from smokers, and that acute CS exposure resulted in HER2 activation in cultured bronchial epithelial cells. Mechanistic studies determined that CS-mediated HER2 activation is independent of neuregulin-1 but required upstream activation of the epidermal growth factor receptor. HER2 was required for CS-induced epithelial permeability as knockdown of HER2 blocked increases in permeability after CS. CS caused an increase in IL-6 production by epithelial cells that was dependent on HER2-mediated extracellular signal-regulated kinases (Erk) activation. Finally, blockade of IL-6 attenuated CS-induced epithelial permeability. Our data indicate that CS activates pulmonary epithelial HER2 and that HER2 is a central mediator of CS-induced epithelial barrier dysfunction.

HER2 activation results in ß-catenin-dependent changes in pulmonary epithelial permeability.

The receptor tyrosine kinase human epidermal growth factor receptor-2 (HER2) is known to regulate pulmonary epithelial barrier function; however, the mechanisms behind this effect remain unidentified. We hypothesized that HER2 signaling alters the epithelial barrier through an interaction with the adherens junction (AJ) protein ß-catenin, leading to dissolution of the AJ. In quiescent pulmonary epithelial cells, HER2 and ß-catenin colocalized along the lateral intercellular junction. HER2 activation by the ligand neuregulin-1 was associated with tyrosine phosphorylation of ß-catenin, dissociation of ß-catenin from E-cadherin, and decreased E-cadherin-mediated cell adhesion. All effects were blocked with the HER2 inhibitor lapatinib. ß-Catenin knockdown using shRNA significantly attenuated neuregulin-1-induced decreases in pulmonary epithelial resistance in vitro. Our data indicate that HER2 interacts with ß-catenin, leading to dissolution of the AJ, decreased cell-cell adhesion, and disruption of the pulmonary epithelial barrier.

IL-13 dampens human airway epithelial innate immunity through induction of IL-1 receptor-associated kinase M.

BACKGROUND: Impaired airway mucosal immunity can contribute to increased respiratory tract infections in asthmatic patients, but the involved molecular mechanisms have not been fully clarified. Airway epithelial cells serve as the first line of respiratory mucosal defense to eliminate inhaled pathogens through various mechanisms, including Toll-like receptor (TLR) pathways. Our previous studies suggest that impaired TLR2 function in T(H)2 cytokine-exposed airways might decrease immune responses to pathogens and subsequently exacerbate allergic inflammation. IL-1 receptor-associated kinase M (IRAK-M) negatively regulates TLR signaling. However, IRAK-M expression in airway epithelium from asthmatic patients and its functions under a T(H)2 cytokine milieu remain unclear. OBJECTIVES: We sought to evaluate the role of IRAK-M in IL-13-inhibited TLR2 signaling in human airway epithelial cells. METHODS: We examined IRAK-M protein expression in epithelia from asthmatic patients versus that in normal airway epithelia. Moreover, IRAK-M regulation and function in modulating innate immunity (eg, TLR2 signaling) were investigated in cultured human airway epithelial cells with or without IL-13 stimulation. RESULTS: IRAK-M protein levels were increased in asthmatic airway epithelium. Furthermore, in primary human airway epithelial cells, IL-13 consistently upregulated IRAK-M expression, largely through activation of phosphoinositide 3-kinase pathway. Specifically, phosphoinositide 3-kinase activation led to c-Jun binding to human IRAK-M gene promoter and IRAK-M upregulation. Functionally, IL-13-induced IRAK-M suppressed airway epithelial TLR2 signaling activation (eg, TLR2 and human ß-defensin 2), partly through inhibiting activation of nuclear factor κB. CONCLUSIONS: Our data indicate that epithelial IRAK-M overexpression in T(H)2 cytokine-exposed airways inhibits TLR2 signaling, providing a novel mechanism for the increased susceptibility of infections in asthmatic patients.

SPLUNC1 deficiency enhances airway eosinophilic inflammation in mice.

Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is enriched in normal airway lining fluid, but is significantly reduced in airway epithelium exposed to a Th2 cytokine milieu. The role of SPLUNC1 in modulating airway allergic inflammation (e.g., eosinophils) remains unknown. We used SPLUNC1 knockout (KO) and littermate wild-type (C57BL/6 background) mice and recombinant SPLUNC1 protein to determine the impact of SPLUNC1 on airway allergic/eosinophilic inflammation, and to investigate the underlying mechanisms. An acute ovalbumin (OVA) sensitization and challenge protocol was used to induce murine airway allergic inflammation (e.g., eosinophils, eotaxin-2, and Th2 cytokines). Our results showed that SPLUNC1 in the bronchoalveolar lavage fluid of OVA-challenged wild-type mice was significantly reduced (P < 0.05), which was negatively correlated with levels of lung eosinophilic inflammation. Moreover, SPLUNC1 KO mice demonstrated significantly higher numbers of eosinophils in the lung after OVA challenges than did wild-type mice. Alveolar macrophages isolated from OVA-challenged SPLUNC1 KO versus wild-type mice had higher concentrations of baseline eotaxin-2 that was amplified by LPS (a known risk factor for exacerbating asthma). Human recombinant SPLUNC1 protein was applied to alveolar macrophages to study the regulation of eotaxin-2 in the context of Th2 cytokine and LPS stimulation. Recombinant SPLUNC1 protein attenuated LPS-induced eotaxin-2 production in Th2 cytokine-pretreated murine macrophages. These findings demonstrate that SPLUNC1 inhibits airway eosinophilic inflammation in allergic mice, in part by reducing eotaxin-2 production in alveolar macrophages.

Up-regulation of MUC18 in airway epithelial cells by IL-13: implications in bacterial adherence.

Airway bacterial infections are a major problem in lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Increased Th2 cytokines, such as IL-13, are observed in lung diseases and may contribute to bacterial infections. How Th2 cytokines affect bacterial infection remains unknown. MUC18, an adhesion molecule shown to be involved in the pathogenesis of malignant melanoma, has been recently identified in airway epithelial cells of patients with COPD. We investigated MUC18 regulation by IL-13 and the role of MUC18 in bacterial adherence to epithelial cells. Human airway tissues, brushed bronchial epithelial cells from normal subjects and subjects with asthma, and epithelial cell lines (e.g., HEK293 cells) were used to study the regulation of MUC18 by IL-13 and the involvement of MUC18 in bacterial (e.g., Mycoplasma pneumoniae [Mp] and nontypeable Haemophilus influenzae [NTHi]) adherence to epithelial cells. Asthmatic bronchial epithelium expressed higher levels of MUC18 than normal bronchial epithelium. IL-13 increased MUC18 in cultured bronchial epithelial cells from normal subjects and particularly from subjects with asthma. IL-13-induced MUC18 expression may be modulated in part through transcription factor specificity protein 1. Overexpression of human MUC18 in HEK293 cells increased cell-associated Mp and NTHi levels. Moreover, MUC18 was shown to directly interact with Mp and NTHi. These results for the first time show that an allergic airway milieu (e.g., IL-13) increases MUC18 expression, which may contribute to increased bacterial infection/colonization in asthma and other lung diseases.

SPLUNC1 regulation in airway epithelial cells: role of Toll-like receptor 2 signaling.

BACKGROUND: Respiratory infections including Mycoplasma pneumoniae (Mp) contribute to various chronic lung diseases. We have shown that mouse short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein was able to inhibit Mp growth. Further, airway epithelial cells increased SPLUNC1 expression upon Mp infection. However, the mechanisms underlying SPLUNC1 regulation remain unknown. In the current study, we investigated if SPLUNC1 production following Mp infection is regulated through Toll-like receptor 2 (TLR2) signaling. METHODS: Airway epithelial cell cultures were utilized to reveal the contribution of TLR2 signaling including NF-κB to SPLUNC1 production upon bacterial infection and TLR2 agonist stimulation. RESULTS: Mp and TLR2 agonist Pam3CSK4 increased SPLUNC1 expression in tracheal epithelial cells from wild type, but not TLR2(-/-) BALB/c mice. RNA interference (short-hairpin RNA) of TLR2 in normal human bronchial epithelial cells under air-liquid interface cultures significantly reduced SPLUNC1 levels in Mp-infected or Pam3CSK4-treated cells. Inhibition and activation of NF-κB pathway decreased and increased SPLUNC1 production in airway epithelial cells, respectively. CONCLUSIONS: Our data for the first time suggest that airway epithelial TLR2 signaling is pivotal in mycoplasma-induced SPLUNC1 production, thus improving our understanding of the aberrant SPLUNC1 expression in airways of patients suffering from chronic lung diseases with bacterial infections.

Hypolipidaemic and antioxidant effect of Enicostemma littorale Blume aqueous extract in cholesterol fed rats.

Enicostemma littorale aqueous extract (1.5 g/100 g body weight/day, p.o.) was administered to rats along with hypercholesterolaemic diet for 6 weeks and the hypolipidaemic and antioxidant effect was evaluated. Feeding cholesterol increased serum cholesterol, serum triglycerides, LDL, VLDL and decreased HDL levels as compared to normal diet fed rats. Enicostemma littorale treatment increased HDL levels and decreased serum cholesterol, triglyceride, LDL, VLDL, LDL/HDL ratio. In addition, treatment with the extract showed a decrease in activities of erythrocyte catalase, superoxide dismutase and lipid peroxidation levels, with an increase in reduced glutathione levels as compared to cholesterol fed untreated rats. Liver and kidney cholesterol levels and triglyceride levels were also decreased in Enicostemma littorale treated rats. Hepatic HMG CoA reductase activity was significantly reduced in the extract treated hypercholesterolemic rats. Lovastatin was used as a reference drug. The hypolipidaemic and antioxidant effect of Enicostemma littorale aqueous extract in cholesterol fed rats is being reported for the first time.

Airway epithelial NF-κB activation promotes Mycoplasma pneumoniae clearance in mice.

BACKGROUND/OBJECTIVE: Respiratory infections including atypical bacteria Mycoplasma pneumoniae (Mp) contribute to the pathobiology of asthma and chronic obstructive pulmonary disease (COPD). Mp infection mainly targets airway epithelium and activates various signaling pathways such as nuclear factor κB (NF-κB). We have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1) serves as a novel host defense protein and is up-regulated upon Mp infection through NF-κB activation in cultured human and mouse primary airway epithelial cells. However, the in vivo role of airway epithelial NF-κB activation in host defense against Mp infection has not been investigated. In the current study, we investigated the effects of in vivo airway epithelial NF-κB activation on lung Mp clearance and its association with airway epithelial SPLUNC1 expression. METHODOLOGY/MAIN RESULTS: Non-antimicrobial tetracycline analog 9-t-butyl doxycycline (9-TB) was initially optimized in mouse primary tracheal epithelial cell culture, and then utilized to induce in vivo airway epithelial specific NF-κB activation in conditional NF-κB transgenic mice (CC10-(CA)IKKß) with or without Mp infection. Lung Mp load and inflammation were evaluated, and airway epithelial SPLUNC1 protein was examined by immunohistochemistry. We found that 9-TB treatment in NF-κB transgene positive (Tg+), but not transgene negative (Tg-) mice significantly reduced lung Mp load. Moreover, 9-TB increased airway epithelial SPLUNC1 protein expression in NF-κB Tg+ mice. CONCLUSION: By using the non-antimicrobial 9-TB, our study demonstrates that in vivo airway epithelial NF-κB activation promotes lung bacterial clearance, which is accompanied by increased epithelial SPLUNC1 expression.

MAPK/AP-1 activation mediates TLR2 agonist-induced SPLUNC1 expression in human lung epithelial cells.

BACKGROUND: Short Palate Lung and Nasal epithelium Clone 1 (SPLUNC1) is a newly described host defense protein, primarily expressed in large airway epithelial cells. Reduced SPLUNC1 has been reported in allergic and cigarette smoke-exposed airways. We found that Mycoplasma pneumoniae increases SPLUNC1 in airway epithelium in part via activating TLR2-NF-κB pathway. However, the contribution of additional signaling pathways to TLR2-mediated SPLUNC1 expression remains unclear. In the present study, we investigated if TLR2-induced mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) signaling regulates SPLUNC1 expression in human lung epithelial cells. METHODS: Human lung epithelial NCI-H292 cells were stimulated with a TLR2 agonist Palmitoyl (3)-Cys-Ser-Lys (4)-OH (Pam(3)CSK(4)). MAPK/AP-1 activation and its role in SPLUNC1 regulation were investigated by Western blot, c-Jun activation assay, chromatin immunoprecipitation (ChIP) and real-time PCR. SPLUNC1 promoter activity was assessed by a luciferase reporter assay. RESULTS: Pam(3)CSK(4) increased SPLUNC1 expression in NCI-H292 cells in a dose- and time-dependent manner, and enhanced SPLUNC1 promoter activity. Pam(3)CSK(4)-treated cells demonstrated activated MAPK and c-Jun compared to untreated cells. ChIP assay indicated increased c-Jun binding to the SPLUNC1 promoter following Pam(3)CSK(4) stimulation. Inhibition of ERK1/2 significantly reduced Pam(3)CSK(4)-mediated c-Jun activation and SPLUNC1 expression. CONCLUSIONS: Our results for the first time demonstrate that TLR2-mediated MAPK/AP-1 activation up-regulates lung epithelial SPLUNC1 expression at the transcriptional level. Understanding SPLUNC1 gene regulation should provide more specific therapeutic targets to restore deficient SPLUNC1 production in diseased airways.

Cigarette smoke decreases MARCO expression in macrophages: implication in Mycoplasma pneumoniae infection.

Bacterial infections including Mycoplasma pneumoniae (Mp) are a major cause of exacerbations in chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) is the leading cause of COPD, and affects the function of alveolar macrophages that act as the first line of defense against the invading respiratory pathogens. Macrophages express a transmembrane receptor called macrophage receptor with collagenous structure (MARCO) that is involved in the clearance of microorganisms. Whether CS down-regulates MARCO and eventually decreases the clearance of Mp has not been investigated. We utilized human monocytic cell line (THP-1)-derived macrophages to examine the effects of CS extract (CSE) on MARCO expression and Mp growth. Specifically, macrophages were pre-exposed to CSE for 6 h, and then infected with or without Mp for 2 h. MARCO was examined at both mRNA and protein levels by using real-time PCR and immunofluorescent staining, respectively. Mp in the supernatants was quantified by quantitative culture. In addition, a neutralizing MARCO antibody was added to macrophages to test if blockade of MARCO impaired Mp clearance. We found that CSE significantly decreased MARCO expression in a dose-dependant manner at 6 h post-CSE. Mp levels in CSE-treated cells were higher than those in non-CSE-treated cells, indicating a decreased pathogen clearance. Additionally, neutralizing MARCO in macrophages markedly increased Mp levels. Our results indicate that cigarette smoke exposure down-regulates MARCO expression in macrophages, which may be in part responsible for impaired bacterial (e.g., Mp) clearance.

Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells.

Airway epithelial cells are constantly exposed to environmental insults such as air pollution or tobacco smoke that may contain high levels of reactive nitrogen and reactive oxygen species. Previous work from our laboratory demonstrated that the reactive oxygen species (ROS), hydrogen peroxide (H(2)O(2)), specifically activates neutral sphingomyelinase 2 (nSMase2) to generate ceramide and induce apoptosis in airway epithelial cells. In the current study we examine the biological consequence of exposure of human airway epithelial (HAE) cells to reactive nitrogen species (RNS). Similar to ROS, we hypothesized that RNS may modulate ceramide levels in HAE cells and induce apoptosis. We found that nitric oxide (NO) exposure via the NO donor papa-NONOate, failed to induce apoptosis in HAE cells. However, when papa-NONOate was combined with a superoxide anion donor (DMNQ) to generate peroxynitrite (ONOO(-)), apoptosis was observed. Similarly pure ONOO(-)-induced apoptosis, and ONOO(-)-induced apoptosis was associated with an increase in cellular ceramide levels. Pretreatment with the antioxidant glutathione did not prevent ONOO(-)-induced apoptosis, but did prevent H(2)O(2)-induced apoptosis. Analysis of the ceramide generating enzymes revealed a differential response by the oxidants. We confirmed our findings that H(2)O(2) specifically activated a neutral sphingomyelinase (nSMase2). However, ONOO(-) exposure did not affect neutral sphingomyelinase activity; rather, ONOO(-) specifically activated an acidic sphingomyelinase (aSMase). The specificity of each enzyme was confirmed using siRNA to knockdown both nSMase2 and aSMase. Silencing nSMase2 prevented H(2)O(2)-induced apoptosis, but had no effect on ONOO(-)-induced apoptosis. On the other hand, silencing of aSMase markedly impaired ONOO(-)-induced apoptosis, but did not affect H(2)O(2)-induced apoptosis. These findings support our hypothesis that ROS and RNS modulate ceramide levels to induce apoptosis in HAE cells. However, we found that different oxidants modulate different enzymes of the ceramide generating machinery to induce apoptosis in airway epithelial cells. These findings add to the complexity of how oxidative stress promotes lung cell injury.

Nitric oxide-enhanced caspase-3 and acidic sphingomyelinase interaction: a novel mechanism by which airway epithelial cells escape ceramide-induced apoptosis.

Reactive nitrogen species (RNS) are implicated in the pathophysiology of inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease. The molecular mechanisms and signaling events involved in lung cell injury by RNS are still poorly understood. In the current study, we observe a novel anti-apoptotic response to nitric oxide (NO) exposure (via the NO donors 3-morpholine-syndnonimine (SIN1) or papa-NONOate) of human airway epithelial (HAE) cells. NO exposure via the NO donors increased cellular ceramide levels via ceramide synthase but did not trigger an apoptotic response. Rather, exposure to the NO donors promoted an increase in the protein-protein interaction between acidic sphingomyelinase (aSMase) and caspase-3, with aSMase sequestering caspase-3 and preventing its cleavage. In contrast, when aSMase was silenced in HAE cells or was knocked out in mice, an increase in cleaved caspase-3 was observed. This elevated caspase-3 cleavage was further augmented upon NO exposure (via SIN1 or papa-NONOate) of HAE cells and could be prevented by an inhibitor to ceramide synthase. These results demonstrate a novel mechanism of NO modulation of apoptosis, in which HAE cells exposed to NO via an NO donor induces ceramide generation via ceramide synthase. However, this ceramide induction does not lead to apoptosis unless aSMase is knocked down, allowing the release of caspase-3, its activation and execution of apoptosis.

Function and regulation of SPLUNC1 protein in Mycoplasma infection and allergic inflammation.

Respiratory infections, including Mycoplasma pneumoniae (Mp), contribute to asthma pathobiology. To date, the mechanisms underlying the increased susceptibility of asthmatics to airway Mp infection remain unclear. Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is a recently described large airway epithelial cell-derived molecule that was predicted to exert host defense activities. However, SPLUNC1 function and regulation in an infectious or allergic milieu are still unknown. We determined host defense and anti-inflammatory functions of SPLUNC1 protein in Mp infection and the regulation of SPLUNC1 by Mp and allergic inflammation (e.g., IL-13). SPLUNC1 function was examined in Mp or human airway epithelial cell cultures by using SPLUNC1 recombinant protein, overexpression and RNA interference. Human and mouse bronchial epithelial SPLUNC1 was examined using immunostaining, Western blotting, ELISA, laser capture microdissection, and real-time PCR. Mouse models of Mp infection and allergic inflammation and air-liquid interface cultures of normal human primary bronchial epithelial cells were used to study SPLUNC1 regulation by Mp and IL-13. We found that: 1) SPLUNC1 protein decreased Mp levels and inhibited epithelial IL-8 production induced by Mp-derived lipoproteins; 2) normal human and mouse large airway epithelial cells expressed high levels of SPLUNC1; and 3) although Mp infection increased SPLUNC1, IL-13 significantly decreased SPLUNC1 expression and Mp clearance. Our results suggest that SPLUNC1 serves as a novel host defense protein against Mp and that an allergic setting markedly reduces SPLUNC1 expression, which may in part contribute to the persistent nature of bacterial infections in allergic airways.