Hong Wei Chu

Human Alveolar Epithelial Cell Injury Induced by Cigarette Smoke

Background Cigarette smoke (CS) is a highly complex mixture and many of its components are known carcinogens, mutagens, and other toxic substances. CS induces oxidative stress and cell death, and this cell toxicity plays a key role in the pathogenesis of several pulmonary diseases. Methodology/Principal Findings We studied the effect of cigarette smoke extract (CSE) in human alveolar epithelial type I-like (ATI-like) cells. These are isolated type II cells that are differentiating toward the type I cell phenotype in vitro and have lost many type II cell markers and express type I cell markers. ATI-like cells were more sensitive to CSE than alveolar type II cells, which maintained their differentiated phenotype in vitro. We observed disruption of mitochondrial membrane potential, apoptosis and necrosis that were detected by double staining with acridine orange and ethidium bromide or Hoechst 33342 and propidium iodide and TUNEL assay after treatment with CSE. We also detected caspase 3 and caspase 7 activities and lipid peroxidation. CSE induced nuclear translocation of Nrf2 and increased expression of Nrf2, HO-1, Hsp70 and Fra1. Moreover, we found that Nrf2 knockdown sensitized ATI-like cells to CSE and Nrf2 overexpression provided protection against CSE-induced cell death. We also observed that two antioxidant compounds N-acetylcysteine and trolox protected ATI-like cells against injury by CSE. Conclusions Our study indicates that Nrf2 activation is a major factor in cellular defense of the human alveolar epithelium against CSE-induced toxicity and oxidative stress. Therefore, antioxidant agents that modulate Nrf2 would be expected to restore antioxidant and detoxifying enzymes and to prevent CS-related lung injury and perhaps lessen the development of emphysema.

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.

CRISPR–Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18

Targeted knockout of genes in primary human cells using CRISPR–Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR–Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR–Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.

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.

Alteration of Lysosome Fusion and Low-grade Inflammation Mediated by Super-low-dose Endotoxin

Background: Super-low-dose endotoxemia contributes to cell stress and low-grade inflammation. Results: Super-low-dose LPS removed Tollip from late endosomes/lysosomes and blocked lysosome fusion with endosomes or autophagosomes. Tollip knock-out mice had impaired wound healing. Conclusion: Super-low-dose LPS leads to cell stress through clearing Tollip and blocking lysosome fusion. Significance: Our data reveal molecular dynamics of innate immunity regulation. Subclinical super-low-dose endotoxin LPS is a risk factor for the establishment of low-grade inflammation during the pathogenesis and progression of chronic diseases. However, the underlying mechanisms are not well understood. At the cellular level, a disruption of lysosome fusion with endosomes or autophagosomes may contribute to the potentiation of low-grade inflammation. In this study, we identified that subclinical super-low-dose endotoxin LPS can potently inhibit the process of endosome acidification and lysosome fusion with endosomes or autophagosomes in primary macrophages. Super-low-dose LPS induced the inhibitory phosphorylation of VPS34, thus leading to the disruption of endosome-lysosome fusion. This effect may depend upon the clearance and relocation of Tollip in macrophages by super-low-dose LPS. Consistent with this notion, Tollip-deficient macrophages had constitutively elevated levels of VPS34 inhibitory phosphorylation and constitutive disruption of endosome-lysosome fusion. By employing a skin excision wound-healing model, we observed that Tollip-deficient mice had significantly elevated levels of cell stress and reduced wound repair. This study reveals a novel mechanism responsible for the modulation of endosome-lysosome fusion and low-grade inflammation in innate macrophages.

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.

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

BACKGROUNDnShort 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.nnnMETHODSnHuman 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.nnnRESULTSnPam(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.nnnCONCLUSIONSnOur 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.

Low-grade inflammatory polarization of monocytes impairs wound healing.

Impaired wound healing often accompanies low‐grade inflammatory conditions, during which circulating levels of subclinical super‐low‐dose endotoxin may persist. Low‐grade inflammatory monocyte polarization may occur during chronic inflammation and deter effective wound repair. However, little is understood about the potential mechanisms of monocyte polarization by sustained insult of subclinical super‐low‐dose endotoxin. We observed that super‐low‐dose endotoxin preferentially programmes a low‐grade inflammatory monocyte state in vitro and in vivo, as represented by the elevated population of CD11b+ Ly6Chigh monocytes and sustained expression of CCR5. Mechanistically, super‐low‐dose endotoxin caused cellular stress, altered lysosome function and increased the transcription factor IRF5. TUDCA, a potent inhibitor of cellular stress, effectively blocked monocyte polarization and improved wound healing in mice injected with super‐low‐dose endotoxin. Our data revealed the polarization of low‐grade inflammatory monocytes by sustained endotoxin challenge, its underlying mechanisms and a potential intervention strategy. Copyright

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.