Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO₂} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.     

Transforming growth factor-beta1 increases airway wound repair via MMP-2 upregulation: a new pathway for epithelial wound repair?

In vivo, transforming growth factor (TGF)-beta1 and matrix metalloproteinases (MMPs) present at the site of airway injury are thought to contribute to epithelial wound repair. As TGF-beta1 can modulate MMP expression and MMPs play an important role in wound repair, we hypothesized that TGF-beta1 may enhance airway epithelial repair via MMPs secreted by epithelial cells. We evaluated the in vitro influence of TGF-beta1 on wound repair in human airway epithelial cells cultured under conditions allowing differentiation. The results showed that TGF-beta1 accelerated in vitro airway wound repair, whereas MMP inhibitors prevented this acceleration. In parallel, we examined the effect of TGF-beta1 on the expression of MMP-2 and MMP-9. TGF-beta1 induced a dramatic increase of MMP-2 expression with an increased steady-state level of MMP-2 mRNA, contrasting with a slight increase in MMP-9 expression. To confirm the role of MMP-2, we subsequently evaluated the effect of MMP-2 on in vitro airway wound repair and demonstrated that the addition of MMP-2 reproduced the acceleration of wound repair induced by TGF-beta1. These results strongly suggest that TGF-beta1 increases in vitro airway wound repair via MMP-2 upregulation. It also raises the issue of a different in vivo biological role of MMP-2 and MMP-9 depending on the cytokine microenvironment.     

Zinc modulates cytokine-induced lung epithelial cell barrier permeability

Apoptosis plays a causative role in acute lung injury in part due to epithelial cell loss. We recently reported that zinc protects the lung epithelium during inflammatory stress whereas depletion of intracellular zinc enhances extrinsic apoptosis. In this investigation, we evaluated the relationship between zinc, caspase-3, and cell-to-cell contact via proteins that form the adherens junction complex. Cell adhesion proteins are directly responsible for formation of the mechanical barrier of the lung epithelium. We hypothesized that exposure to inflammatory cytokines, in conjunction with zinc deprivation, would induce caspase-3, leading to degradation of junction proteins, loss of cell-to-cell contact, and compromised barrier function. Primary human upper airway and type I/II alveolar epithelial cultures were obtained from multiple donors and exposed to inflammatory stimuli that provoke extrinsic apoptosis in addition to depletion of intracellular zinc. We observed that zinc deprivation combined with tumor necrosis factor-alpha, interferon-gamma, and Fas receptor ligation accelerates caspase-3 activation, proteolysis of E-cadherin and beta-catenin, and cellular apoptosis, leading to increased paracellular leak across monolayers of both upper airway and alveolar lung epithelial cultures. Zinc supplementation inhibited apoptosis and paracellular leak, whereas caspase inhibition was less effective. We conclude that zinc is a vital factor in the lung epithelium that protects against death receptor-mediated apoptosis and barrier dysfunction. Furthermore, our findings suggest that although caspase-3 inhibition reduces lung epithelial apoptosis it does not prevent mechanical dysfunction. These findings facilitate future studies aimed at developing therapeutic strategies to prevent acute lung injury.     

Airway epithelial cell migration and wound repair by ATP-mediated activation of dual oxidase 1

The airway epithelium is continuously subjected to environmental pollutants, airborne pathogens, and allergens and relies on several intrinsic mechanisms to maintain barrier integrity and to promote epithelial repair processes following injury. Here, we report a critical role for dual oxidase 1 (Duox1), a newly identified NADPH oxidase homolog within the tracheobronchial epithelium, in airway epithelial cell migration and repair following injury. Activation of Duox1 during epithelial injury is mediated by cellular release of ATP, which signals through purinergic receptors expressed on the epithelial cell surface. Purinergic receptor stimulation by extracellular ATP is a critical determinant of epithelial cell migration and repair following injury and is associated with activation of extracellular signal-regulated kinases (ERK1/2) and matrix metalloproteinase-9 (MMP-9). Stimulation of these integral features of epithelial cell migration and repair processes was found to require the activation of Duox1. Our findings demonstrate a novel role for Duox1 in the tracheobronchial epithelium, in addition to its proposed role in antimicrobial host defense, by participating in epithelial repair processes to maintain epithelial integrity and barrier function in the face of environmental stress.     

Airway epithelial wounds in rhesus monkey generate ionic currents that guide cell migration to promote healing

Damage to the respiratory epithelium is one of the most critical steps to many life-threatening diseases, such as acute respiratory distress syndrome and chronic obstructive pulmonary disease. The mechanisms underlying repair of the damaged epithelium have not yet been fully elucidated. Here we provide experimental evidence suggesting a novel mechanism for wound repair: endogenous electric currents. It is known that the airway epithelium maintains a voltage difference referred to as the transepithelial potential. Using a noninvasive vibrating probe, we demonstrate that wounds in the epithelium of trachea from rhesus monkeys generate significant outward electric currents. A small slit wound produced an outward current (1.59 μA/cm(2)), which could be enhanced (nearly doubled) by the ion transport stimulator aminophylline. In addition, inhibiting cystic fibrosis transmembrane conductance regulator (CFTR) with CFTR(Inh)-172 significantly reduced wound currents (0.17 μA/cm(2)), implicating an important role of ion transporters in wound induced electric potentials. Time-lapse video microscopy showed that applied electric fields (EFs) induced robust directional migration of primary tracheobronchial epithelial cells from rhesus monkeys, towards the cathode, with a threshold of <23 mV/mm. Reversal of the field polarity induced cell migration towards the new cathode. We further demonstrate that application of an EF promoted wound healing in a monolayer wound healing assay. Our results suggest that endogenous electric currents at sites of tracheal epithelial injury may direct cell migration, which could benefit restitution of damaged airway mucosa. Manipulation of ion transport may lead to novel therapeutic approaches to repair damaged respiratory epithelium.     

Integrin-β4 regulates the dynamic changes of phenotypic characteristics in association with epithelial-mesenchymal transition (EMT) and RhoA activity in airway epithelial cells during injury and repair

Background: In airway disease such as asthma a hyperactive cellular event of epithelial-mesenchymal transition (EMT) is considered as the mechanism of pathological airway tissue remodeling after injury to the airway epithelium. And the initiation of EMT in the airways depends on the epithelial disruption involving dissolution and/or destabilization of the adhesive structures between the cells and ECM. Previously, we have shown that integrin-β4, an epithelial adhesion molecule in bronchial epithelium is an important regulator of cell proliferation and wound repair in human airway epithelial cells. Therefore, in this study we aimed to investigate whether integrin-β4 also regulates EMT phenotypes during injury and repair in airway epithelial cells of both wild type/integrin-β4^-/- mice in vivo and cultured cells treated with integrin-β4/nonsense siRNA in vitro.Methods: We induced injury to the airway epithelial cells by either repeated exposure to ozone and mechanical scratch wound, and subsequently examined the EMT-related phenotypic features in the airway epithelial cells including biomarkers expression, adhesion and cytoskeleton reorganization and cell stiffness.Results: The results show that in response to injury (ozone exposure/scratch wound) and subsequent spontaneous repair (ozone withdrawal/wound healing) both in vivo and in vitro, the airway epithelial cells underwent dynamic changes in the epithelial and mesenchymal biomarkers expression, adhesion and cytoskeleton structures as well as cell stiffness, all together exhibiting enhanced EMT phenotypic features after injury and reversal of the injury-induced effects during repair. Importantly, these injury/repair-associated EMT phenotypic changes in airway epithelial cells appeared to be dependent on integrin-β4 expression. More specifically, when integrin-β4 was deficient in mice (integrin-β4^-/-) the repair of ozone-injured airway epithelium was impaired and the recovery of ozone-enhanced EMT biomarkers expression in the airway epithelium was delayed. Similarly, in the scratch wounded airway epithelial cells with integrin-β4 knockdown, the cells were impaired in all aspects related to EMT during wound and repair including cell proliferation, wound closure rate, adhesion and cytoskeleton protein expression (vinculin and vimentin), mesenchymal-like F-actin reorganization, cell stiffness and RhoA activation.Conclusion: Taken together, these results suggested that integrin-β4 may be essential in regulating the effects of injury and repair on EMT in airway epithelial cells via influencing both the cell adhesion to ECM and cells'' physical phenotypes through RhoA signaling pathway.     

Fibroblast growth factor-9 expression in airway epithelial cells amplifies the type I interferon response and alters influenza A virus pathogenesis

Influenza A virus (IAV) preferentially infects conducting airway and alveolar epithelial cells in the lung. The outcome of these infections is impacted by the host response, including the production of various cytokines, chemokines, and growth factors. Fibroblast growth factor-9 (FGF9) is required for lung development, can display antiviral activity in vitro, and is upregulated in asymptomatic patients during early IAV infection. We therefore hypothesized that FGF9 would protect the lungs from respiratory virus infection and evaluated IAV pathogenesis in mice that overexpress FGF9 in club cells in the conducting airway epithelium (FGF9-OE mice). However, we found that FGF9-OE mice were highly susceptible to IAV and Sendai virus infection compared to control mice. FGF9-OE mice displayed elevated and persistent viral loads, increased expression of cytokines and chemokines, and increased numbers of infiltrating immune cells as early as 1 day post-infection (dpi). Gene expression analysis showed an elevated type I interferon (IFN) signature in the conducting airway epithelium and analysis of IAV tropism uncovered a dramatic shift in infection from the conducting airway epithelium to the alveolar epithelium in FGF9-OE lungs. These results demonstrate that FGF9 signaling primes the conducting airway epithelium to rapidly induce a localized IFN and proinflammatory cytokine response during viral infection. Although this response protects the airway epithelial cells from IAV infection, it allows for early and enhanced infection of the alveolar epithelium, ultimately leading to increased morbidity and mortality. Our study illuminates a novel role for FGF9 in regulating respiratory virus infection and pathogenesis.     

H(2)O(2) inhibits alveolar epithelial wound repair in vitro by induction of apoptosis

Reactive oxygen species (ROS) are released into the alveolar space and contribute to alveolar epithelial damage in patients with acute lung injury. However, the role of ROS in alveolar repair is not known. We studied the effect of ROS in our in vitro wound healing model using either human A549 alveolar epithelial cells or primary distal lung epithelial cells. We found that H(2)O(2) inhibited alveolar epithelial repair in a concentration-dependent manner. At similar concentrations, H(2)O(2) also induced apoptosis, an effect seen particularly at the edge of the wound, leading us to hypothesize that apoptosis contributes to H(2)O(2)-induced inhibition of wound repair. To learn the role of apoptosis, we blocked caspases with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (zVAD). In the presence of H(2)O(2), zVAD inhibited apoptosis, particularly at the wound edge and, most importantly, maintained alveolar epithelial wound repair. In H(2)O(2)-exposed cells, zVAD also maintained cell viability as judged by improved cell spreading and/or migration at the wound edge and by a more normal mitochondrial potential difference compared with cells not treated with zVAD. In conclusion, H(2)O(2) inhibits alveolar epithelial wound repair in large part by induction of apoptosis. Inhibition of apoptosis can maintain wound repair and cell viability in the face of ROS. Inhibiting apoptosis may be a promising new approach to improve repair of the alveolar epithelium in patients with acute lung injury.     

Activation of MMP-9 by human lung epithelial cells in response to the cystic fibrosis-associated pathogen Burkholderia cenocepacia reduced wound healing in vitro

Burkholderia cepacia complex is a group of bacterial pathogens that cause opportunistic infections in cystic fibrosis (CF). The most virulent of these is Burkholderia cenocepacia. Matrix metalloproteinases (MMPs) are upregulated in CF patients. The aim of this work was to examine the role of MMPs in the pathogenesis of B. cepacia complex, which has not been explored to date. Real-time PCR analysis showed that B. cenocepacia infection upregulated MMP-2 and MMP-9 genes in the CF lung cell line CFBE41o- within 1 h, whereas MMP-2, -7, and -9 genes were upregulated in the non-CF lung cell line 16HBE14o-. Conditioned media from both cell lines showed increased MMP-9 activation following B. cenocepacia infection. Conditioned media from B. cenocepacia-infected cells significantly reduced the rate of wound healing in confluent lung epithelia (P < 0.05), in contrast to conditioned media from Pseudomonas aeruginosa-infected cells, which showed predominant MMP-2 activation. Treatment of control conditioned media from both cell lines with the MMP activator 4-aminophenylmercuric acetate (APMA) also resulted in clear activation of MMP-9 and to a much lesser extent MMP-2. APMA treatment of control media also delayed the repair of wound healing in confluent epithelial cells. Furthermore, specific inhibition of MMP-9 in medium from cells exposed to B. cenocepacia completely reversed the delay in wound repair. These data suggest that MMP-9 plays a role in the reduced epithelial repair observed in response to B. cenocepacia infection and that its activation following B. cenocepacia infection contributes to the pathogenesis of this virulent pathogen.     

Cigarette Smoke Modulates Repair and Innate Immunity following Injury to Airway Epithelial Cells

Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding. Upon mechanical injury CS caused a delayed recovery in the epithelial barrier integrity and wound closure. Furthermore CS enhanced innate immune responses, as demonstrated by increased expression of the antimicrobial protein RNase 7. These differential effects on epithelial repair and innate immunity were both mediated by CS-induced oxidative stress. Overall, our findings demonstrate modulation of wound repair and innate immune responses of injured airway epithelial cells that may contribute to COPD development and progression.     

Repair and regeneration of the airway epithelium

Despite an efficient defence system, the airway surface epithelium, in permanent contact with the external milieu, is frequently injured by inhaled pollutants, microorganisms and viruses. The response of the airway surface epithelium to an acute injury includes a succession of cellular events varying from the loss of the surface epithelium integrity to partial shedding of the epithelium or even to complete denudation of the basement membrane. The epithelium has then to repair and regenerate to restore its functions, through several mechanisms including basal cell spreading and migration, followed by proliferation and differentiation of epithelial cells. The cellular and molecular factors involved in wound repair and epithelial regeneration are closely interacting and imply extracellular matrix proteins, matrix metalloproteinases (MMPs) and their inhibitors as well as cytokines and growth factors secreted by airway epithelial and mesenchymal cells. The development of in vitro and in vivo models of airway epithelium wound repair allowed the study of the spatio-temporal modulation of these factors during the different steps of epithelial repair and regeneration. In this context, several studies have demonstrated that the matrix and secretory environment are markedly involved in these mechanisms and that their dysregulation may induce remodelling of the airway mucosa. A better knowledge of the mechanisms involved in airway epithelium regeneration may pave the way to regenerative therapeutics allowing the reconstitution of a functional airway epithelium in numerous respiratory diseases such as asthma, chronic obstructive pulmonary diseases, cystic fibrosis and bronchiolitis.     

Hog barn dust slows airway epithelial cell migration in vitro through a PKCalpha-dependent mechanism

Agricultural work and other occupational exposures are responsible for approximately 15% of chronic obstructive pulmonary disease (COPD). COPD involves airway remodeling in response to chronic lung inflammatory events and altered airway repair mechanisms. However, the effect of agricultural dust exposure on signaling pathways that regulate airway injury and repair has not been well characterized. A key step in this process is migration of airway cells to restore epithelial integrity. We have previously shown that agents that activate the critical regulatory enzyme protein kinase C (PKC) slow cell migration during wound repair. Based on this observation and direct kinase measurements that demonstrate that dust extract from hog confinement barns (HDE) specifically activates the PKC isoforms PKCalpha and PKCepsilon, we hypothesized that HDE would slow wound closure time in airway epithelial cells. We utilized the human bronchial epithelial cell line BEAS-2B and transfected BEAS-2B cell lines that express dominant negative (DN) forms of PKC isoforms to demonstrate that HDE slows wound closure in BEAS-2B and PKCepsilon DN cell lines. However, in PKCalpha DN cells, wound closure following HDE treatment is not significantly different than media-treated cells. These results suggest that the PKCalpha isoform is an important regulator of cell migration in response to agricultural dust exposure.     

Interleukin-1beta augments in vitro alveolar epithelial repair

Biologically active interleukin (IL)-1beta is present in the pulmonary edema fluid obtained from patients with acute lung injury and has been implicated as an important early mediator of nonpulmonary epithelial wound repair. Therefore, we tested the hypothesis that IL-1beta would enhance wound repair in cultured monolayers from rat alveolar epithelial type II cells. IL-1beta (20 ng/ml) increased the rate of in vitro alveolar epithelial repair by 118 +/- 11% compared with that in serum-free medium control cells (P < 0.01). IL-1beta induced cell spreading and migration at the edge of the wound but not proliferation. Neutralizing antibodies to epidermal growth factor (EGF) and transforming growth factor-alpha or inhibition of the EGF receptor by tyrphostin AG-1478 or genistein inhibited IL-1beta-induced alveolar epithelial repair, indicating that IL-1beta enhances in vitro alveolar epithelial repair by an EGF- or transforming growth factor-alpha-dependent mechanism. Moreover, the mitogen-activated protein kinase pathway is involved in IL-1beta-induced alveolar epithelial repair because inhibition of extracellular signal-regulated kinase activation by PD-98059 inhibited IL-1beta-induced alveolar epithelial repair. In conclusion, IL-1beta augments in vitro alveolar epithelial repair, indicating a possible novel role for IL-1beta in the early repair process of the alveolar epithelium in acute lung injury.     

Delay of airway epithelial wound repair in COPD is associated with airflow obstruction severity

Background

Airway epithelium integrity is essential to maintain its role of mechanical and functional barrier. Recurrent epithelial injuries require a complex mechanism of repair to restore its integrity. In chronic obstructive pulmonary disease (COPD), an abnormal airway epithelial repair may participate in airway remodeling. The objective was to determine if airway epithelial wound repair of airway epithelium is abnormal in COPD.

Methods

Patients scheduled for lung resection were prospectively recruited. Demographic, clinical data and pulmonary function tests results were recorded. Emphysema was visually scored and histological remodeling features were noted. Primary bronchial epithelial cells (BEC) were extracted and cultured for wound closure assay. We determined the mean speed of wound closure (MSWC) and cell proliferation index, matrix metalloprotease (MMP)-2, MMP-9 and cytokines levels in supernatants of BEC 18 hours after cell wounding. In a subset of patients, bronchiolar epithelial cells were also cultured for wound closure assay for MSWC analyze.

Results

13 COPD and 7 non COPD patients were included. The severity of airflow obstruction and the severity of emphysema were associated with a lower MSWC in BEC (p = 0.01, 95% CI [0.15-0.80]; p = 0.04, 95% CI [−0.77;-0.03] respectively). Cell proliferation index was decreased in COPD patients (19 ± 6% in COPD vs 27 ± 3% in non COPD, p = 0.04). The severity of COPD was associated with a lower level of MMP-2 (7.8 ± 2 10⁵ AU in COPD GOLD D vs 12.8 ± 0.13 10⁵ AU in COPD GOLD A, p = 0.04) and a lower level of IL-4 (p = 0.03, 95% CI [0.09;0.87]). Moreover, higher levels of IL-4 and IL-2 were associated with a higher MSWC (p = 0.01, 95% CI [0.17;0.89] and p = 0.02, 95% CI [0.09;0.87] respectively). Clinical characteristics and smoking history were not associated with MSWC, cell proliferation index or MMP and cytokines levels. Finally, we showed an association of the MSWC of bronchial and corresponding bronchiolar epithelial cells obtained from the same patients (p = 0.02, 95% CI [0.12;0.89]).

Conclusion

Our results showed an abnormal bronchial epithelial wound closure process in severe COPD. Further studies are needed to elucidate the contribution and the regulation of this mechanism in the complex pathophysiology of COPD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0151-9) contains supplementary material, which is available to authorized users.     

Mathematical modeling of airway epithelial wound closure during cyclic mechanical strain.

The repair of airway epithelium after injury is crucial in restoring epithelial barrier integrity. Because the airways are stretched and compressed due to changes in both circumferential and longitudinal dimensions during respiration and may be overdistended during mechanical ventilation, we investigated the effect of cyclic strain on the repair of epithelial wounds. Both cyclic elongation and compression significantly slowed repair, with compression having the greatest effect. We developed a mathematical model of the mechanisms involved in airway epithelial cell wound closure. The model focuses on the differences in spreading, migration, and proliferation with cyclic strain by using separate parameters for each process and incorporating a time delay for the mitotic component. Numerical solutions of model equations determine the shape of the diffusive wave solutions of cell density that correspond to the influx of cells into the wound during the initial phase of reepithelialization. Model simulations were compared with experimental measurements of cell density and the rate of wound closure, and parameters were determined based on measurements from airway epithelial cells from several different sources. The contributions of spreading, migration, and mitosis were investigated both numerically and experimentally by using cytochalasin D to inhibit cell motility and mitomycin C to inhibit proliferation.     

Keratinocyte growth factor accelerates wound closure in airway epithelium during cyclic mechanical strain.

The airway epithelium may be damaged by inhalation of noxious agents, in response to pathogens, or during endotracheal intubation and mechanical ventilation. Maintenance of an intact epithelium is important for lung fluid balance, and the loss of epithelium may stimulate inflammatory responses. Epithelial repair in the airways following injury must occur on a substrate that undergoes cyclic elongation and compression during respiration. We have previously shown that cyclic mechanical strain inhibits wound closure in the airway epithelium (Savla and Waters, 1998b). In this study, we investigated the stimulation of epithelial wound closure by keratinocyte growth factor (KGF) in vitro and the mechanisms by which KGF overcomes the inhibition due to mechanical strain. Primary cultures of normal human bronchial epithelial cells (NHBE) and a cell line of human airway epithelial cells, Calu 3, were grown on Silastic membranes, and a wound was scraped across the well. The wells were then exposed to cyclic strain using the Flexercell Strain Unit, and wound closure was measured. While cyclic elongation (20% maximum) and cyclic compression (approximately 2%) both inhibited wound closure in untreated wells, treatment with KGF (50 ng/ml) significantly accelerated wound closure and overcame the inhibition due to cyclic strain. Since wound closure involves cell spreading, migration, and proliferation, we investigated the effect of cyclic strain on cell area, cell-cell distance, and cell velocity at the wound edge. While the cell area increased in unstretched monolayers, the cell area of monolayers in compressed regions decreased significantly. Treatment with KGF increased the cell area in both cyclically elongated and compressed cells. Also, when cells were treated with KGF, cell velocity was significantly increased in both static and cyclically strained monolayers, and cyclic strain did not inhibit cell migration. These results suggest that KGF is an important factor in epithelial repair that is capable of overcoming the inhibition of repair due to physiological levels of cyclic strain.     

MyD88 in lung resident cells governs airway inflammatory and pulmonary function responses to organic dust treatment

Inhalation of organic dusts within agriculture environments contributes to the development and/or severity of airway diseases, including asthma and chronic bronchitis. MyD88 KO (knockout) mice are nearly completely protected against the inflammatory and bronchoconstriction effects induced by acute organic dust extract (ODE) treatments. However, the contribution of MyD88 in lung epithelial cell responses remains unclear. In the present study, we first addressed whether ODE-induced changes in epithelial cell responses were MyD88-dependent by quantitating ciliary beat frequency and cell migration following wounding by electric cell-substrate impedance sensing. We demonstrate that the normative ciliary beat slowing response to ODE is delayed in MyD88 KO tracheal epithelial cells as compared to wild type (WT) control. Similarly, the normative ODE-induced slowing of cell migration in response to wound repair was aberrant in MyD88 KO cells. Next, we created MyD88 bone marrow chimera mice to investigate the relative contribution of MyD88-dependent signaling in lung resident (predominately epithelial cells) versus hematopoietic cells. Importantly, we demonstrate that ODE-induced airway hyperresponsiveness is MyD88-dependent in lung resident cells, whereas MyD88 action in hematopoietic cells is mainly responsible for ODE-induced TNF-α release. MyD88 signaling in lung resident and hematopoietic cells are necessary for ODE-induced IL-6 and neutrophil chemoattractant (CXCL1 and CXCL2) release and neutrophil influx. Collectively, these findings underscore an important role for MyD88 in lung resident cells for regulating ciliary motility, wound repair and inflammatory responses to ODE, and moreover, show that airway hyperresponsiveness appears uncoupled from airway inflammatory consequences to organic dust challenge in terms of MyD88 involvement.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0272-9) contains supplementary material, which is available to authorized users.