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

Cigarette Smoke Impairs A2A Adenosine Receptor Mediated Wound Repair through Up-regulation of Duox-1 Expression.

Cigarette smoke (CS) exposure and intrinsic factors such as the NADPH oxidases produce high levels of reactive oxygen species (ROS), ensuing inflammatory tissue injury. We previously demonstrated that CS-generated ROS, particularly hydrogen peroxide (H2O2), impaired adenosine stimulated wound repair. We hypothesized that CS exposure modulates expression of Dual oxidase 1 (Duox-1), a NADPH oxidases known to generate H2O2. To test this hypothesis, we used human bronchial epithelial cell line Nuli-1 and C57BL/6 mice. Cells were treated with 5% CS extract (CSE) for various periods of time, and mice were exposed to whole body CS for six weeks. Both CSE and CS treatment induced increased expression of Duox-1, and silencing of Doux-1 improved the rate of cell wound repair induced by CSE treatment. Nuli-1 cells pretreated with thapsigargin but not calcium ionophore exhibited increased Duox-1 mRNA expression. CSE treatment stimulated PKCα activation, which was effectively blocked by pretreatment with diphenylene iodonium, a NADPH oxidase inhibitor. Compared to control, lungs from CS-exposed mice showed a significant increase in PKCα activity and Duox-1 expression. Collectively, the data demonstrated that CS exposure upregulates expression of Duox-1 protein. This further leads to H2O2 production and PKCα activation, inhibiting A2AAR-stimulated wound repair

Long-Term Cigarette Smoke Exposure in a Mouse Model of Ciliated Epithelial Cell Function

Exposure to cigarette smoke is associated with airway epithelial mucus cell hyperplasia and a decrease in cilia and ciliated cells. Few models have addressed the long-term effects of chronic cigarette smoke exposure on ciliated epithelial cells. Our previous in vitro studies showed that cigarette smoke decreases ciliary beat frequency (CBF) via the activation of protein kinase C (PKC). We hypothesized that chronic cigarette smoke exposure in an in vivo model would decrease airway epithelial cell ciliary beating in a PKC-dependent manner. We exposed C57BL/6 mice to whole-body cigarette smoke 2 hours/day, 5 days/week for up to 1 year. Tracheal epithelial cell CBF and the number of motile cells were measured after necropsy in cut tracheal rings, using high-speed digital video microscopy. Tracheal epithelial PKC was assayed according to direct kinase activity. At 6 weeks and 3 months of smoke exposure, the baseline CBF was slightly elevated (∼ 1 Hz) versus control mice, with no change in β-agonist–stimulated CBF between control mice and cigarette smoke–exposed mice. By 6 months of smoke exposure, the baseline CBF was significantly decreased (2–3 Hz) versus control mice, and a β-agonist failed to stimulate increased CBF. The loss of β-agonist–increased CBF continued at 9 months and 12 months of smoke exposure, and the baseline CBF was significantly decreased to less than one third of the control rate. In addition to CBF, ciliated cell numbers significantly decreased in response to smoke over time, with a significant loss of tracheal ciliated cells occurring between 6 and 12 months. In parallel with the slowing of CBF, significant PKC activation from cytosol to the membrane of tracheal epithelial cells was detected in mice exposed to smoke for 6–12 months

Additional file 1: Figure S1. of MyD88 in lung resident cells governs airway inflammatory and pulmonary function responses to organic dust treatment

Lung parenchymal histopathology of experimental MyD88 bone marrow chimera (donor → recipient) mice treated once with saline or ODE. A representative murine lung section (hematoxylin and eosin stain, x10 magnification) is shown. Note that there is an absence of non-specific inflammation in saline-treated chimeric mice, and there is evidence of slight peribronchiolar and perivascular cellular cuffing following ODE treatment. Line scale is 100 μm. (PDF 699 kb