Qun Wu

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

BACKGROUNDnImpaired 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.nnnOBJECTIVESnWe sought to evaluate the role of IRAK-M in IL-13-inhibited TLR2 signaling in human airway epithelial cells.nnnMETHODSnWe 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.nnnRESULTSnIRAK-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 β-defensin 2), partly through inhibiting activation of nuclear factor κB.nnnCONCLUSIONSnOur 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.

Cigarette smoke induces growth differentiation factor 15 production in human lung epithelial cells: Implication in mucin over-expression

Excessive mucus is a hallmark of chronic obstructive pulmonary disease (COPD). There is an emerging interest in the role of TGF-β signaling in the initiation and progression of COPD. Growth differentiation factor 15 (GDF15) is a divergent member of TGF-β superfamily. However, whether cigarette smoke induces airway epithelial GDF15 production and its functions in the airways have not been revealed. Therefore, we first analyzed GDF15 protein expression in airway epithelium of human COPD smokers versus normal non-smokers. We then examined the regulation and function of GDF15 in human airway epithelial cells in response to cigarette smoke exposure. We found increased GDF15 protein expression in airway epithelium (mainly in ciliated cells) of human COPD smokers compared with normal non-smokers. Furthermore, cigarette smoke exposure consistently up-regulated GDF15 expression in human airway epithelial cells. Moreover, GDF15 was shown to play a critical role in cigarette smoke-induced airway epithelial MUC5AC expression. Lastly, activation of phosphoinositide 3-kinase (PI3K) pathway was largely responsible for GDF15-induced airway epithelial MUC5AC expression. Our findings indicate that human airway epithelial cells can produce GDF15 during cigarette smoke exposure, which subsequently activates PI3K pathway to promote mucin (e.g. MUC5AC) expression. This highlights a novel role of GDF15 in regulating airway mucosal immunity (e.g. mucin) in cigarette smoke-exposed lungs.

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

BackgroundRespiratory 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.MethodsAirway 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.ResultsMp 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.ConclusionsOur 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.

A low dose of Mycoplasma pneumoniae infection enhances an established allergic inflammation in mice: the role of the prostaglandin E2 pathway.

Background Over 40% of chronic stable asthma patients have evidence of respiratory Mycoplasma pneumoniae (Mp) infection as detected by PCR, but not by serology and culture, suggesting that a low‐level Mp is involved in chronic asthma. However, the role of such a low‐level Mp infection in the regulation of allergic inflammation remains unknown.

α1-antitrypsin promotes SPLUNC1-mediated lung defense against Pseudomonas aeruginosa infection in mice

BackgroundPseudomonas aeruginosa (PA) infection is involved in various lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. However, treatment of PA infection is not very effective in part due to antibiotic resistance. α1-antitrypsin (A1AT) has been shown to reduce PA infection in humans and animals, but the underlying mechanisms remain unclear. The goal of our study is to test whether a novel endogenous host defense protein, short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is involved in the therapeutic effect of A1AT during lung PA infection.MethodSPLUNC1 knockout (KO) and littermate wild-type (WT) mice on the C57BL/6 background were intranasally infected with PA to determine the therapeutic effects of A1AT. A1AT was aerosolized to mice 2xa0hrs after the PA infection, and mice were sacrificed 24xa0hrs later. PA load and inflammation were quantified in the lung, and SPLUNC1 protein in bronchoalveolar lavage (BAL) fluid was examined by Western blot.ResultsIn WT mice, PA infection significantly increased neutrophil elastase (NE) activity, but reduced SPLUNC1 protein in BAL fluid. Notably, PA-infected mice treated with A1AT versus bovine serum albumin (BSA) demonstrated higher levels of SPLUNC1 protein expression, which are accompanied by lower levels of NE activity, lung bacterial load, and pro-inflammatory cytokine production. To determine whether A1AT therapeutic effects are dependent on SPLUNC1, lung PA load in A1AT- or BSA-treated SPLUNC1 KO mice was examined. Unlike the WT mice, A1AT treatment in SPLUNC1 KO mice had no significant impact on lung PA load and pro-inflammatory cytokine production.ConclusionA1AT reduces lung bacterial infection in mice in part by preventing NE-mediated SPLUNC1 degradation.

Trehalose-Mediated Autophagy Impairs the Anti-Viral Function of Human Primary Airway Epithelial Cells

Human rhinovirus (HRV) is the most common cause of acute exacerbations of chronic lung diseases including asthma. Impaired anti-viral IFN-λ1 production and increased HRV replication in human asthmatic airway epithelial cells may be one of the underlying mechanisms leading to asthma exacerbations. Increased autophagy has been shown in asthmatic airway epithelium, but the role of autophagy in anti-HRV response remains uncertain. Trehalose, a natural glucose disaccharide, has been recognized as an effective autophagy inducer in mammalian cells. In the current study, we used trehalose to induce autophagy in normal human primary airway epithelial cells in order to determine if autophagy directly regulates the anti-viral response against HRV. We found that trehalose-induced autophagy significantly impaired IFN-λ1 expression and increased HRV-16 load. Inhibition of autophagy via knockdown of autophagy-related gene 5 (ATG5) effectively rescued the impaired IFN-λ1 expression by trehalose and subsequently reduced HRV-16 load. Mechanistically, ATG5 protein interacted with retinoic acid-inducible gene I (RIG-I) and IFN-β promoter stimulator 1 (IPS-1), two critical molecules involved in the expression of anti-viral interferons. Our results suggest that induction of autophagy in human primary airway epithelial cells inhibits the anti-viral IFN-λ1 expression and facilitates HRV infection. Intervention of excessive autophagy in chronic lung diseases may provide a novel approach to attenuate viral infections and associated disease exacerbations.

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-CAIKKβ) 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.

α1-Antitrypsin reduces rhinovirus infection in primary human airway epithelial cells exposed to cigarette smoke.

Human rhinovirus (HRV) infections target airway epithelium and are the leading cause of acute exacerbations of COPD. Cigarette smoke (CS) increases the severity of viral infections, but there is no effective therapy for HRV infection. We determined whether α1-antitrypsin (A1AT) reduces HRV-16 infection in CS-exposed primary human airway epithelial cells. Brushed bronchial epithelial cells from normal subjects and patients diagnosed with COPD were cultured at air–liquid interface to induce mucociliary differentiation. These cells were treated with A1AT or bovine serum albumin for 2 hours and then exposed to air or whole cigarette smoke (WCS) with or without HRV-16 (5×104 50% Tissue Culture Infective Dose [TCID50]/transwell) infection for 24 hours. WCS exposure significantly increased viral load by an average of fivefold and decreased the expression of antiviral genes interferon-λ1, OAS1, and MX1. When A1AT was added to WCS-exposed cells, viral load significantly decreased by an average of 29-fold. HRV-16 infection significantly increased HRV-16 receptor intercellular adhesion molecule-1 messenger RNA expression in air-exposed cells, which was decreased by A1AT. A1AT-mediated reduction of viral load was not accompanied by increased epithelial antiviral gene expression or by inhibiting the activity of 3C protease involved in viral replication or maturation. Our findings demonstrate that A1AT treatment prevents a WCS-induced increase in viral load and for the first time suggest a therapeutic effect of A1AT on HRV infection.

Tollip SNP rs5743899 modulates human airway epithelial responses to rhinovirus infection.

Rhinovirus (RV) infection in asthma induces varying degrees of airway inflammation (e.g. neutrophils), but the underlying mechanisms remain unclear.

MUC18 Differentially Regulates Pro-Inflammatory and Anti-Viral Responses in Human Airway Epithelial Cells

OBJECTIVEnMUC18 or CD146, a transmembrane glycoprotein, is mainly expressed by endothelial cells and smooth muscle cells where it serves as a cell-cell adhesion molecule. We have found MUC18 up-regulation in airway epithelial cells of patients with asthma and chronic obstructive pulmonary disease (COPD). However, the function of MUC18 in airway epithelial cells remains unclear. In the present study, we tested the hypothesis that MUC18 exerts a pro-inflammatory function during stimulation with a viral mimic polyI:C or human rhinovirus infection.nnnMETHODSnNormal human primary airway epithelial cells were transduced with lentivirus encoding MUC18 cDNA to over-express MUC18 or with GFP (control), and treated with polyI:C or HRV for detection of pro-inflammatory cytokine IL-8 and anti-viral gene IFN-β. Additionally, we performed cell culture of human lung epithelial cell line NCIH292 cells to determine the mechanisms of MUC18 function.nnnRESULTSnWe found that MUC18 over-expression promoted IL-8 production, while it inhibited IFN-β expression following polyI:C stimulation or HRV infection. Increased phosphorylation of MUC18 serines was observed in MUC18 over-expressing cells. Reduction of MUC18 serine phosphorylation by inhibiting ERK activity was associated with less production of IL-8 following polyI:C stimulation.nnnCONCLUSIONSnOur results for the first time demonstrate MUC18s pro-inflammatory and anti-viral function in human airway epithelial cells.